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Proteasome inhibition with bortezomib suppresses growth and induces apoptosis in osteosarcoma

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Posted 11 Aug 2011 — by James Street
Category bortezomid, Human osteosarcoma research, in vitro, Molecular Osteosarcoma Studies, Mouse Osteosarcoma Studies, Osteosarcoma

Issue

International Journal of Cancer

International Journal of Cancer

Volume 127, Issue 1, pages 67–76, 1 July 2010

  1. Yuriy Shapovalov,
  2. David Benavidez,
  3. Daniel Zuch,
  4. Roman A. Eliseev,†

Article first published online: 5 NOV 2009

DOI: 10.1002/ijc.25024

Keywords:

  • osteosarcoma;
  • proteasome inhibition;
  • bortezomib;
  • apoptosis;
  • Runx2

Abstract

Osteosarcomas are primary bone tumors of osteoblastic origin that mostly affect adolescent patients. These tumors are highly aggressive and metastatic. Previous reports indicate that gain of function of a key osteoblastic differentiation factor, Runx2, leads to growth inhibition in osteosarcoma. We have previously established that Runx2 transcriptionally regulates expression of a major proapoptotic factor, Bax. Runx2 is regulated via proteasomal degradation, and proteasome inhibition has a stimulatory effect on Runx2. In this study, we hypothesized that proteasome inhibition will induce Runx2 and Runx2-dependent Bax expression sensitizing osteosarcoma cells to apoptosis. Our data showed that a proteasome inhibitor, bortezomib, increased Runx2 and Bax in osteosarcoma cells. In vitro, bortezomib suppressed growth and induced apoptosis in osteosarcoma cells but not in nonmalignant osteoblasts. Experiments involving intratibial tumor xenografts in nude mice demonstrated significant tumor regression in bortezomib-treated animals. Immunohistochemical studies revealed that bortezomib inhibited cell proliferation and induced apoptosis in osteosarcoma xenografts. These effects correlated with increased immunoreactivity for Runx2 and Bax. In summary, our results indicate that bortezomib suppresses growth and induces apoptosis in osteosarcoma in vitro and in vivo suggesting that proteasome inhibition may be effective as an adjuvant to current treatment regimens for these tumors. Published 2009 UICC. This article is a US Government work and, as such, is in the public domain in the United States of America.

Osteosarcoma is a devastating primary bone cancer of osteoblastic origin that affects children and young adults.1, 2 It is highly aggressive, and has a propensity for early distant spread with metastases involving the lung in more than 80% of cases.3, 4 At present, the treatment of osteosarcoma includes chemotherapy with doxorubicin/adriamycin or methotrexate and large-scale surgery; however, current chemotherapeutic regimens do not significantly increase the postsurgical 5-year survival rate of 50–60%.5 There is, therefore a need for new treatment modalities in osteosarcoma that would complement current treatments and improve overall survival.

According to clinico-pathological data, 80% of osteosarcomas exhibit undifferentiated phenotype6 and express low amounts of bone-specific osteoblastic markers, such as osteocalcin.7, 8 Expression of osteocalcin and many other osteoblastic differentiation markers is regulated by the bone-specific member of the Runx family of transcription factors, Runx2.9 Runx factors are the key mediators of the TGFβ- and BMP-dependent signaling.10 Runx1, Runx2 and Runx3 regulate cell growth and differentiation in hematopoietic,11 skeletal,12 and nerve and gut13 tissues, respectively. During malignant transformation, function of the Runx family factors is frequently disrupted. Runx1 is mutated and present as a nonfunctional fusion protein in hematologic malignancies, such as acute myeloid leukemia14; Runx2 function is suppressed in osteosarcoma15; and Runx3 is mutated in gastric cancers.16 The fact that structurally and functionally similar Runx factors are inactivated in various types of cancer suggests that they may act as tumor suppressors. However, various reports also indicate an oncogenic role of Runx factors in different tumors as reviewed by Blyth et al.17 As regards with osteosarcoma, expression of a late differentiation marker, osteocalcin, is significantly decreased in these tumors7, 8 indicating that Runx2 function is compromised. Moreover, Thomas et al. have recently established that Runx2 activity is suppressed in all studied osteosarcoma cell lines and Runx2 protein levels are dramatically reduced in phenotypically aggressive osteosarcoma cell lines, such as 143B, U2OS, G292 and some others.15 We and others have shown that gain of Runx2 function inhibits proliferation and induces apoptosis in osteosarcoma cells.15, 18 We have also identified a possible mechanism of tumor suppressor action of Runx2. We showed that Runx2 transcriptionally activates expression of a major proapoptotic gene, Bax,19 thus decreasing the Bcl2 to Bax ratio and sensitizing cells to apoptosis.18 Therefore treatments that induce Runx2 may be effective in suppression of tumor growth in osteosarcoma.

Runx2 is known to be regulated via proteasomal degradation.20–22 Recent studies by Mukherjee et al.23 and Giuliani et al.24 have demonstrated that in osteoblasts, proteasome inhibition induces Runx2 protein level and activity. We therefore hypothesized that in osteosarcoma, proteasome inhibition will induce Runx2 and Bax leading to growth inhibition and induction of apoptosis. We studied the effect of a clinically approved proteasome inhibitor, bortezomib (Velcade®), in osteosarcoma cell lines in vitro and in orthotopic osteosarcoma xenografts in mice.

Material and Methods

Materials

Most chemicals were obtained from Sigma (St. Louis, MO) unless otherwise noted. Cell culture media, media ingredients and antibiotics were from Invitrogen (Carlsbad, CA). Bortezomib was from Millennium Pharmaceuticals (Cambridge, MA). Oligonucleotides were custom made by IDT (Coralville, IA). Primary antibodies for Runx2 (mouse monoclonal) and ubiquitin (rabbit polyclonal) were from Santa Cruz (Santa Cruz, CA), for Bax (rabbit monoclonal) from Epitomics (Burlingame, CA), and for β-actin (mouse monoclonal) from Sigma. The Ki-67/active caspase-3 antibody mix for immunohistochemistry was from Biocare (Concord, CA). Secondary antibodies, Precision Plus molecular weight markers, dry milk and buffer ingredients for immunoblotting were from Bio-Rad (Hercules, CA). Secondary antibodies for immunohistochemistry were from Rockland (Gilbertsville, PA). Retroviral shRNA vectors against Runx2 and a control vector, pSM2c, were from Open Biosystems (Huntsville, AL). FuGENE® HD transfection reagent was from Roche (Basel, Switzerland)

Cell culture and treatment

Human fetal osteoblasts transformed with SV40 T antigen, hFOB 1.19, and osteosarcoma cells, HOS and 143B, were obtained from ATCC (Manassas, VA). Luciferase-expressing 143B-luc cells were a kind gift of Dr. T.C. He. OS187 cells were a kind gift of Dr. R. Gorlick. HFOB cells were cultured in DMEM/F12 medium at 37°C. HOS, 143B, 143B-luc and OS187 cells were cultured in DMEM medium at 37°C. All media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin mixture. Seeding densities were kept constant and adjusted for different plating surface areas. All cells were cultured for 24 hr before treatments. Either bortezomib dissolved in PBS or PBS alone was added to cell media, and various assays were performed at indicated time-points as described below. Subsets of 143B cells were infected with either pSM2c control retrovirus or with 1 of 4 retroviruses carrying different shRNAs against Runx2. Stable clones were selected for 2 weeks using puromycin at 2 μg ml−1. Runx2 expression was assayed with immunoblotting and the 2 clones showing strongest knock-down of Runx2 were chosen for our study along with the stable pSM2c-transfected control.

Cellular growth assay

Cells plated on 6-well plates at indicated time-points were trypsinized, resuspended in 1 ml of PBS and counted using Cellometer® automated cell counter from Nexcelom Bioscience (Lawrence, MA).

Real-time RT-PCR

Total cellular RNA was isolated using RNeasy Mini Kit by Qiagen (Valencia, CA) and reverse transcribed into cDNA using iScript cDNA synthesis kit by Bio-Rad according to the manufacturer’s instructions. One microgram of cDNA was subjected to real-time PCR using following sets of primers: Runx2 (5-CCG GAA TGC CTC TGC TGT TAT GA-3′ and 5′-ACT GAG GCG GTC AGA GAA CAA ACT-3′), alkaline phosphatase (5′-TGC AGT ACG AGC TGA ACA GGA ACA-3′ and 5′-TCC ACC AAA TGT GAA GAC GTG GGA-3′), Bax (5′- CAC CAG CTC TGA GCA GAT CAT GAA G -3′ and 5′- GCG GCA ATC ATC CTC TGC AG -3′) and GAPDH (5′-GAG TCA ACG GAT TTG GTC GT-3′ and 5′-GAC AAG CTT CCC GTT CTC AG-3′). Real-time PCR was performed using the RotorGene real-time DNA amplification system (Qiagen). SYBR Green reagent produced by Abgene (Rockford, IL) was used to monitor DNA synthesis. The expression of proteins of interest was normalized to the expression of GAPDH.

Dual luciferase promoter-reporter assay

Cells plated on 12-well plates were transfected with the 6xOSE-luc firefly luciferase reporter9 at 0.5 μg per well. The renilla luciferase promoter-less reporter, pRL-TK, produced by Promega (Madison, WI), was cotransfected at 0.05 μg per well as a reference. FuGene HD reagent by Roche was used for transfections. After 24 hr cells were lyzed and firefly and renilla luciferase activities were measured using a Dual Luciferase Reporter Assay System (Promega) in an Optocomp 1 luminometer according to the manufacturer’s instructions. The firefly luciferase signal was normalized to the renilla luciferase signal and expressed as relative luminescence units (RLU).

Western blotting

Cells were lyzed, and the protein concentration in lysates was measured using the Bradford assay. Twenty five to forty micrograms of total protein per sample was mixed 1:1 with 2xLaemmli buffer; boiled and subjected to electrophoresis using NuPage precast 4–12% gradient polyacrylamide gels (Invitrogen) followed by electroblotting onto polyvinylidene difluoride membranes (Bio-Rad). Blots were blocked in 5% dry milk dissolved in PBS containing 0.01% of Tween-20 (PBST), probed with primary antibody resuspended in 2.5% dry milk dissolved in PBST at 2 (Runx2), 1 (ubiquitin) or 0.5 (Bax) μg ml−1, incubated with horseradish peroxidase-conjugated secondary antibody resuspended in 2.5% dry milk dissolved in PBST at 0.2 μg ml−1, developed using SuperSignal WestPico chemiluminescent substrate produced by Thermo Scientific (Rockford, IL), and photographed. To verify equal loading, blots were stripped in Re-Blot Plus stripping buffer produced by Millipore (Billerica, MA), reprobed with anti-β-actin antibody resuspended in 2.5% dry milk in PBST at 0.5 μg ml−1 and developed as described above. Band intensities were measured using densitometry and Adobe® software.

Apoptotic morphology and nuclear condensation assay

Cells plated on 24-well plates were treated with bortezomib as indicated in the text. At 24 and 48 hr thereafter, cellular morphology was examined using Zeiss AxioVert inverted microscope under visible light illumination. A nuclear fluorescent stain, Hoechst 33342, was added to the media at 1 μM. Fluorescent nuclear signal was visualized using the above microscope under UV light illumination.

Caspase-3 activity assay

Caspase-3 activity in cell lysates was measured using fluorogenic substrate cleavage assay.25 The reaction mixture contained caspase-3 fluorogenic substrate Ac-DEVD-AMC produced by Calbiochem (San Diego, CA) resuspended at 20 μM in PBS and 10 μg of the cell lysate. The reactions were loaded into 96-well plates and incubated at 37°C for 30 min. Fluorescence of the -AMC tag cut off by active caspase-3 was measured at 440 nm (excitation 380 nm) using a Hitachi plate reader.

In vivo osteosarcoma xenograft model

To model osteosarcoma in vivo we used human osteosarcoma 143B cells expressing luciferase (143B-luc).26 Cells were grown to confluency and resuspended in sterile Hanks buffered saline (HBSS) at 5 × 106 per ml. Ten microliters of the cell suspension containing 5 × 104 cells were injected orthotopically into the medullar cavity of right tibiae of 5-week-old immunocompromised nude Nu/Nu mice (Crl:Nu/Nu-FoxN1nu) purchased from Charles River Laboratories (Cambridge, MA). The injection was performed using a Hamilton syringe into the opening in tibial tuberosity pre-made with a 25-gauge needle. Left tibias were sham injected. Tumor growth was monitored longitudinally using bioluminescence imaging (BLI). For bioluminescent imaging the mice were anesthetized and injected intraperitoneally with 100 μl of luciferin substrate. Bioluminescence was measured using a Xenogen imager. A total of 16 mice were injected of which 12 developed tumors as detected with BLI.

Treatment of mice with bortezomib

On Day 8 after the injection of tumor cells, 12 tumor-bearing mice were randomly divided in 2 groups—the control group (−Bzm) and the treated group (+Bzm). Animals in the treated group received 1 mg kg−1 of bortezomib dissolved in sterile PBS intraperitoneally (IP) every 3 days for 3 weeks. Mice in the control group received sterile PBS IP injections. All animals were sacrificed on Day 28 after the injection of tumor cells, and both hind limbs were excised. Tumor volume was calculated from the 2D caliper measurements using the following formula: tumor volume = length × (width)2 × π/6.27

Immunohistochemistry

Both tumor-bearing and sham-injected tibias were fixed in 10% neutral buffered formalin for 3 days, decalcified in 14% EDTA for 10 days, and subsequently embedded in paraffin. Serial 4 μm sections were cut and mounted on glass slides. Sections were either stained with H&E or processed for immunohistochemistry as follows: sections were deparaffinized using xylene and ethanol, rehydrated and permeabilized with 0.1% Triton in a TRIS-citrate retrieval buffer at 95°C for 15 min. To block endogenous peroxidase activity, sections were treated with methanol/1% H2O2 for 30 min. After the wash, sections were blocked with TBS/0.1% triton/10% normal serum for 1 hr, followed by incubation with the primary antibody for 1 hr. The immunolabeling performed included Ki-67/active caspase-3 double labeling, Runx2 and Bax. After subsequent rinse in PBS, sections were incubated with the corresponding HRP-conjugated secondary antibody, washed and developed with DAB (Ki-67, Runx2 and Bax) and/or Vulcan Red (active caspase-3) substrates. After the development reaction had been terminated, sections were rinsed, dehydrated, mounted and counterstained with either hematoxylin (Ki-67/active caspase-3 and Bax) or FastGreen (Runx2) stains.

In situ proliferation and apoptosis assay

To assess the effect of bortezomib on cell proliferation and apoptosis in xenografted osteosarcoma tumors in mice, formalin-fixed, paraffin-embedded tissue sections were probed with the Ki-67/active caspase-3 antibody cocktail. The tissue sections were visualized using a Zeiss Axioplan light microscope equipped with a DAGE video camera that was connected to a Ludl XYZ motorized stage, a color monitor, and a personal computer, and the number of Ki-67 and active caspase-3 positive cells was counted. Quantitative data were obtained utilizing a software program Stereologer 1.3.1 developed by Systems Planning and Analysis (Alexandria, VA), which provided us with an unbiased optical dissector approach for accurate cell counting. The entire tissue section was chosen as a reference area. In addition, spacing of 1,100 μm for Ki-67 and 400 μm for active caspase-3 was established as an optimal interval that would allow us to sample the population of positively stained cells within the reference area. Placement of counting grids provided us with random areas to be analyzed. Results were obtained as the number of positively stained cells per field of view. The treated group was compared to the control group and the results were expressed as the fold change over control (active caspase-3) or percent of control (Ki-67).

Quantitative analysis of Runx2 and Bax immunostaining

Tissue sections probed for Runx2 or Bax were visualized using Zeiss Axioscop 40 microscope and 40× objective. The random fields (10 per section) were photographed under constant illumination and exposure using a high resolution digital camera attached to the microscope. The images were blindly analyzed for nuclear Runx2 or cytosolic Bax staining intensities using the ImageJ software. The treated group was compared to the control group and the results were expressed as the fold change over control.

Statistical analysis

Experiments were repeated 3 to 5 times. Mean values and standard deviations were calculated, and the statistical significance was determined using a Student’s t-test or ANOVA. Data with p < 0.05 were considered statistically significant.

Results

Characterization of osteosarcoma cell lines, HOS, 143B and OS187

The majority of osteosarcomas are aggressive and metastatic.6 Human osteosarcoma cell lines, 143B and OS187, have been reported to have an aggressive and invasive phenotype and form tumors and metastases when injected into mice.15, 26, 28 We therefore chose 143B and OS187 cell lines for our study as representatives of an aggressive osteosarcoma. The 143B cells are Ki-Ras-transformed derivatives of HOS cells.26 For this reason, HOS cell line was used as a nontransformed control for 143B cells. HOS cells show limited tumorigenic and metastatic potential.26 OS187 cells were included to expand our study and to exclude a possibility of cell-line specific effects in related HOS and 143B cells. OS187 cell line was produced directly from an osteosarcoma patient specimen.29 It has been described as an aggressive cell line that forms tumors when injected orthotopically into mouse bone and is capable of lung metastasis.28 Immortalized human osteoblastic cell line, hFOB,30 was included as a nonmalignant control. HFOB cells are transformed with a temperature-sensitive SV40 T large antigen which is active at 33°C leading to unrestricted cell growth, but inactivated at 37°C.30 To avoid potential artifacts caused by active SV40 T large antigen, we used hFOB cells at a nonpermissive temperature of 37°C. These conditions allow adequate growth of hFOB cells and have been used in previous studies.31

To assess the phenotype of HOS, 143B and OS187 cells, we first measured their cell growth rate in comparison to hFOB cells. As shown in Figure 1a, the growth rate of HOS cells was slightly higher than the growth rate of nonmalignant hFOB osteoblasts while the growth rate of 143B and OS187 cells was significantly increased. The 143B and OS187 cells had similar doubling time of ∼24 hr. Next, we measured expression of osteoblastic differentiation markers, Runx2 and alkaline phosphatase (ALP) using real-time RT-PCR approach. Figure 1b (top panel) shows that all studied osteosarcoma cells expressed Runx2 confirming their commitment to osteoblastic lineage. Runx2 mRNA levels in HOS and 143B cells were higher whereas in OS187 cells they were 50% lower than in nonmalignant controls. ALP mRNA level (Fig. 1b, bottom panel) in HOS cells was similar to that in hFOB cells while in 143B and OS187 cells, it was significantly decreased, confirming their undifferentiated phenotype. Western blot analysis demonstrated that total Runx2 protein levels were slightly decreased in HOS cells and significantly downregulated in 143B and OS187 cells when compared to hFOB cells (Fig. 1c, top blot). The most pronounced decrease was observed in the faster migrating MASN isoform of Runx2 (lower band) while the slower migrating MRIPV isoform (upper band) was less affected.32 We then assessed Runx2 transcriptional activity using the 6xOSE-luc promoter-reporter construct.9 The basal activity of this reporter in the absence of forced expression of Runx2 was relatively low and ranged from 1,000 to 7,000 RLU or 5- to 20-fold over the background shown as a dotted line in Figure 1d. Figure 1d shows that 6xOSE-luc activity was significantly decreased in all studied osteosarcoma cells when compared to nonmalignant hFOB cells. In our previous work, we have described a regulatory role of Runx2 in Bax expression.18 We therefore investigated whether Runx2 protein levels in the studied cells correlate with Bax protein levels. Figure 1c (middle blot) shows that Bax protein levels correlated with Runx2 levels and were slightly decreased in HOS cells and significantly decreased in 143B and OS187 cells when compared to hFOB cells. In summary, these data indicate that tumorigenic and metastatic osteosarcoma cells, 143B and OS187, have a highly proliferative and undifferentiated phenotype whereas less aggressive HOS cells are less proliferative and more differentiated. Runx2 protein levels correlate with Bax levels and inversely correlate with proliferative capacity in the studied cells.

Figure 1. Cell growth and expression of differentiation markers in osteosarcoma cells and in immortalized non-malignant osteoblasts. (a) Cell growth assay. HFOB, HOS, 143B and OS187 cells were seeded in 6-well plates at a density of 200,000 per well. After 24 or 48 hr cells were harvested and counted using an automated cell counter; (b) Assay of mRNA expression of osteoblastic differentiation markers. Real-time RT-PCR analysis was performed to measure relative expression of Runx2 (top panel) and alkaline phosphatase (ALP, bottom panel) as described in Methods; (c) Assay of protein expression of Runx2 and Bax. Immunoblotting was performed to assess Runx2 (top blot) protein levels in cell lysates. Blots were then reprobed for Bax (middle blot) and for β-actin to verify equal loading (bottom blot). Blots are representatives of 3; (d) Assay of Runx2 transcriptional activity. Dual luciferase assay and the 6xOSE-luc reporter were used to measure Runx2 transcriptional activity in the studied cells. Dotted line represents a Background signal. Data in (a), (b), and (d) are Means ± SD (n = 3 to 5). Asterisk (*) in (d) indicates p < 0.05 when compared to the levels found in hFOB cells.

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Effect of proteasome inhibition with bortezomib on Runx2 and Bax in osteosarcoma cells

Our data in Figure 1 illustrate a discrepancy between mRNA and protein levels of Runx2 in the studied osteosarcoma cells. Relative to hFOB cells, in HOS cells Runx2 mRNA was at 260% while Runx2 protein measured with densitometry was at 80%; in 143B cells Runx2 mRNA was at 150% while Runx2 protein was at 46%; and in OS187 cells Runx2 mRNA was at 50% while Runx2 protein was at 38%. This discrepancy between mRNA and protein levels likely indicates increased Runx2 protein degradation. Runx2 is known to be regulated by proteasome20–22; and proteasome inhibition increases Runx2 levels and function.23, 24 We therefore studied effects of a proteasome inhibitor, bortezomib, on Runx2 protein level and function in HOS, 143B and OS187 osteosarcoma cells. For our experiments we chose a concentration of bortezomib of 25 nM because, according to the pharmacokinetic study by Attar et al.,33 this is a physiologically relevant intermediate plasma concentration of bortezomib in patients after a bolus injection. Figure 2a (top blot) shows that 24-hr incubation with bortezomib at 25 nM increased the amount of ubiquitinated proteins confirming the inhibitory effect of bortezomib on proteasomes in the studied cells. Runx2 protein levels were also significantly increased in bortezomib-treated cells (middle blot). Runx2 transcriptional activity as measured with the 6xOSE-luc reporter, was upregulated in bortezomib-treated cells with the most pronounced effect observed in OS187 cells (Fig. 2b). As has been reported by our group before,18 Runx2 regulates Bax expression. Therefore, we examined whether the bortezomib-mediated increase in Runx2 protein level and function correlates with Bax expression. Bax expression in bortezomib-treated osteosarcoma cells was measured using real-time RT-PCR and showed, on average, a 3-fold induction when compared to cells treated with vehicle control (Fig. 2c). The above experiments demonstrate that proteasome inhibition with bortezomib increases Runx2 protein level and activity as well as Bax expression in the studied osteosarcoma cells.

Figure 2. Proteasome inhibition with bortezomib restores Runx2 and Bax in osteosarcoma cells. Cells were seeded at the same density, cultured for 24 hr, and then treated with bortezomib at 25 nM for another 24 hr. Cells were collected and total RNA or protein was extracted. (a) Effect of proteasome inhibition on Runx2 protein level. Immunoblotting was performed to assess ubiquitin (top blot) protein levels in cell lysates. Blots were re-probed for Runx2 (middle blot) and for β-actin (bottom blot). Blots are representatives of 3; (b) Effect of proteasome inhibition on Runx2 activity. Dual luciferase assay and the 6xOSE-luc reporter were used to measure Runx2 transcriptional activity in the studied cells; (c) Effect of proteasome inhibition on Bax expression. Bax mRNA levels were measured using real-time RT-PCR analysis and normalized to GAPDH mRNA levels. Data in (b) and (c) are Means ± SD (n = 4). Asterisk (*) indicates p < 0.05 when compared to the levels found in corresponding control cells (−Bzm).

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Proteasome inhibition selectively suppresses growth and induces apoptosis in osteosarcoma cells

As mentioned above, Runx2 has been shown to function as a tumor suppressor in osteosarcoma. Therefore, by restoring Runx2 protein level and activity, bortezomib may exert an inhibitory effect on osteosarcoma cells. In addition, as a general proteasome inhibitor, bortezomib may affect other molecular pathways regulated by proteasome and thus confer an additional suppression of cell proliferation and functioning. To determine the effect of bortezomib in the studied cells, we performed a dose-response and time-course analysis by evaluating growth rates of nonmalignant hFOB cells and of HOS, 143B and OS187 osteosarcoma cells in vitro. Figure 3a shows that increasing doses of bortezomib did not have any inhibitory effect on the growth of hFOB cells. However, in HOS, 143B and OS187 osteosarcoma cell lines bortezomib suppressed cell growth in a dose-dependent manner. To expand our study and confirm the effect of proteasome inhibition, we used another proteasome inhibitor, MG132, and found that after 24 hr it decreased osteosarcoma cell number on average by 70% and hFOB cell number by 30% (Data not shown). Because MG132 is not an FDA approved compound and because it shows higher toxicity in nonmalignant hFOB cells, we continued our studies using only bortezomib. Bortezomib-treated osteosarcoma cells exhibited morphological features of apoptosis, such as shrinkage and rounding of cell bodies along with condensation of chromatin (Fig. 3b). A specific apoptotic marker protease, caspase-3, was activated in bortezomib-treated osteosarcoma cells (Fig. 3c). These data indicate that proteasome inhibition with bortezomib selectively suppresses growth and induces apoptosis in osteosarcoma cells but not in nonmalignant control cells in culture.

Figure 3. Bortezomib suppresses growth and induces apoptosis in osteosarcoma cells. (a) Effect of proteasome inhibition on growth of osteosarcoma cells. Cells were plated at similar density in 6-well plates and after 24-hr incubation, were treated with the indicated doses of bortezomib or with PBS as a vehicle control. After 24 and 48 hr, cells were harvested and counted in an automated cell counter; (b) Bortezomib-treated osteosarcoma cells show apoptotic morphology. Cells cultured for 24 hr were treated with 25 nM bortezomib (+Bzm) or PBS (−Bzm) for another 24 hr. Cellular morphology was examined under the microscope and photographed (top panels). Chromatin condensation and nuclear shrinkage was detected by staining the cells with Hoechst33342 and visualizing them under the UV illumination using a fluorescence microscope (lower panels). Panels are representative of 12; (c) Activation of caspase-3 in bortezomib-treated osteosarcoma cells. Cells cultured for 24 hr were treated with 25 nM bortezomib (+Bzm) or PBS (−Bzm) for another 24 hr. Cells were lyzed and caspase-3 activity in cell lysates was measured using a fluorogenic substrate, Ac-DEVD-AMC, as described in detail in Methods. Data in (a) and (c) are Means ± SD (n = 4–6). Asterisk (*) indicates p < 0.05 when compared to vehicle control-treated cells (−Bzm). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Knock-down of Runx2 abolishes the inhibitory effect of bortezomib in osteosarcoma cells

By inhibiting protein degradation, bortezomib can have a wide range of effects on various cell signaling systems other than Runx2. To evaluate a specific contribution of Runx2 in the inhibitory effect of bortezomib on osteosarcoma cells, we performed a stable knock-down of Runx2 in 143B cells using 2 different retroviral shRNA constructs. We then treated these cells as well as control cells transfected with the empty pSM2c retroviral vector with bortezomib at 25 nM for 24 hr. Figure 4a shows that cells transfected with either shRNA1 or shRNA2 against Runx2 contained ∼30% of the amount of Runx2 found in cells transfected with the control vector (pSM). Moreover, while treatment with bortezomib significantly increased Runx2 protein levels in control transfectants (Fig. 4a, lane 2), it did not increase Runx2 protein levels in shRNA-transfected cells (Fig. 4a, lane 4 and 6). Bax expression, as measured with real-time RT-PCR correlated with Runx2 protein levels and significantly increased in bortezomib-treated control transfectants but remained unchanged in bortezomib-treated shRNA1- and 2-transfected cells. Figure 4c shows that the knock-down of Runx2 abolished the inhibitory effect of bortezomib in 143B osteosarcoma cells. These results demonstrate that the inhibitory effect of bortezomib in osteosarcoma cells is at least partially dependent on Runx2.

Figure 4. Knockdown of Runx2 decreases the effect of bortezomib in osteosarcoma cells. The 143B cells stably transfected with either a control vector, pSM2c (pSM), or 2 different shRNA constructs against Runx2, shRNA1 and 2, were cultured for 24 hr, treated with bortezomib at 25 nM for 24 hr and harvested. (a) Knock-down of Runx2 in 143B cells. Runx2 protein levels in stably transfected cells were assayed with immunoblotting. Bortezomib induced Runx2 levels in control cells but not in shRNA1- or 2-transfected cells. Blots were re-probed for β-actin to verify equal loading. Blots are representative of 3; (b) Effect of proteasome inhibition on Bax expression in control and Runx2 shRNA-transfected cells. Bax expression was assayed with real-time RT-PCR; (c) Effect of proteasome inhibition on growth of control and Runx2 shRNA-transfected cells. Cells were counted in an automated cell counter. Data in (b) and (c) are Means ± SD (n = 4). Asterisk (*) indicates p < 0.05 when compared to vehicle control-treated cells (−Bzm).

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Inhibition of proteasome with bortezomib suppresses growth of osteosarcoma xenografts in vivo

To study the effect of bortezomib on osteosarcoma development in vivo, we performed xenografting of osteosarcoma cells into nude Nu/Nu mice. Luciferase-expressing 143B (143B-luc) cells26 were injected orthotopically into right tibiae of mice. Development of xenografted tumors was followed longitudinally using bioluminescent imaging (BLI). Figure 5a demonstrates the presence of a strong bioluminescent signal in tumor-bearing mice (top right panel) and absence of a signal in uninjected animals (top left panel). By Day 8, tumor-injected tibiae in 12 out of 16 animals developed consistent BLI signal which increased on average 4-fold when compared to the signal at Day 1, indicating successful engraftment of the tumors. Tumor engraftment was later confirmed histologically (Fig. 5a, lower panels). At Day 8, we divided animals in 2 groups so that each group had equal average BLI signal, and started intraperitoneal PBS (control group) or bortezomib (treatment group) injections at a dose of 1 mg kg−1 every 3 days. In the absence of detailed pharmacokinetic studies of bortezomib in mice, it is difficult to correlate the in vivo and in vitro doses. Previously bortezomib was used at 0.5–2 mg kg−1 in mouse models and gave consistent responses without any noticeable toxicity.34, 35 We, therefore, chose an intermediate dose of 1 mg ml−1 for our animal studies. We have not noticed any toxic effects, i.e., weight loss, in our treated animals. Animals were sacrificed at Day 28, and tumor sizes were determined. Figure 5b shows that bortezomib had a pronounced inhibitory effect on growth of osteosarcoma xenografts in mice. The average size of osteosarcoma tumors in the bortezomib-treated group was only 30% of that in the control group.

Figure 5. Osteosarcoma xenograft model and effect of bortezomib on tumor progression in vivo. Athymic nude mice were injected intratibially with 143B-luc cells at Day 0 and development of tumors was monitored by measuring bioluminescence. At Day 8, mice were divided in 2 groups and received either PBS or bortezomib at 1 mg kg−1 every 3 days. Mice were sacrificed at Day 28. (a) Engrafted tumors emit strong bioluminescent signal (top panels). Bioluminescent imaging (BLI) was performed before (top left panel) and 8 days after (top right panel) the injection of tumor cells. Histochemical analysis confirms tumor engraftment (bottom panels). Both tumor-bearing (bottom right panel) and non-tumor bearing (bottom left panel) limbs were excised, processed for histochemistry and stained with H&E. Femoral heads (marked with open arrowheads) are shown for orientation. When compared to normal tibiae (marked with solid arrowhead), 143B-luc injected tibiae develop tumors (marked with “X”). The panels are representative of 6; (b) Bortezomib suppresses growth of osteosarcoma in vivo. At sacrifice tumors were measured with calipers and tumor sizes calculated as described in Methods; (c) Bortezomib inhibits proliferation and induces apoptosis in osteosarcoma xenografts. Shown is the representative H&E staining (left panel) and immunostaining for Ki-67/active caspase-3 (right panel) of bortezomib-treated tumors. White arrows in the left panel and black arrows in the right panel mark apoptotic cells. The yellow arrow in the right panel marks proliferating, Ki-67-positive cells; (d) Runx2 (top panels) and Bax (bottom panels) immunostaining intensities are increased in tumor samples from bortezomib-treated animals. Tissue sections from osteosarcoma xenografts in mice were probed for Runx2 or Bax and photographed as described in Methods. Data in (b) are Means ± SD (n = 6). Asterisk (*) indicates p < 0.05 when compared to the vehicle control-treated group (−Bzm).

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To elucidate a possible mechanisms of bortezomib-induced inhibition of osteosarcoma tumor growth, we performed histochemical studies and immunostaining of formalin-fixed paraffin-embedded tumor sections. Cytological analysis of H&E stained sections revealed presence of cells exhibiting apoptotic morphology in bortezomib-treated group, such as shrinkage and rounding of cell bodies along with condensation and fragmentation of nuclei (Fig. 5c, left panel, marked by white arrows). To assess changes in tumor cell proliferation and confirm induction of apoptosis in the bortezomib-treated group in vivo, sections were subjected to double immunostaining for Ki-67 and the active form of caspase-3 respectively (Fig. 5c, right panel). The assay showed decreased number of cells positive for Ki-67 (brown staining marked by yellow arrow) and increased presence of cells positive for active caspase-3 (pink staining marked by black arrows). To determine whether these effects of bortezomib correlated with the increase in Runx2 and Bax levels in xenografted tumors, tumor sections were probed with Runx2- and Bax-specific antibodies (Fig. 5d). The assay showed that both Runx2 (top panels) and Bax (bottom panels) staining intensities were significantly increased in tumors from the bortezomib-treated group. Quantitative analysis of immunostaining for Ki-67, active caspase-3, Runx2 and Bax demonstrated that Ki-67 immunoreactivity decreased by 60% (Fig. 6a); active caspase-3 immunoreactivity increased 3-fold (Fig. 6b); Runx2 immunoreactivity increased 2.7-fold (Fig. 6c) and Bax immunoreactivity increased 2-fold (Fig. 6d) in the bortezomib-treated animals when compared to the control animals. Taken together, these experiments indicate that proteasome inhibition with bortezomib suppresses growth, induces apoptosis, and increases Runx2 and Bax levels in xenografted osteosarcoma tumors in mice.

Figure 6. Bortezomib inhibits proliferation, induces apoptosis, and increases Runx2 and Bax levels in osteosarcoma xenografts in mice. Quantitative analysis of Ki-67 (a), active caspase-3 (b), Runx2 (c) and Bax (d) immunostaining in osteosarcoma xenografts. The number of Ki-67 positive or active caspase-3 positive cells was determined using stereological approach and Runx2 and Bax staining intensities were measured as described in Methods. Immunostaining in the control group (−Bzm) was compared to the immunostaining in the treated group (+Bzm) and results were plotted. Data are Means ± SD (n = 6). Asterisk (*) indicates p < 0.05 when compared to the vehicle control-treated group (−Bzm).

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Discussion

A summary of our key findings is as follows: (i) when compared to nonmalignant hFOB cells, osteosarcoma cell lines, HOS, 143B and OS187 have decreased protein levels of Runx2 and Bax as well as suppressed Runx2 activity; (ii) proteasome inhibition with bortezomib restores Runx2 level and function and increases Bax expression in these osteosarcoma cells; (iii) bortezomib induces apoptosis in HOS, 143B and OS187 cells in vitro; and (iv) bortezomib induces tumor regression and apoptosis in orthotopic osteosarcoma xenografts in mice that correlates with increased Runx2 and Bax levels. On the basis of these findings, we conclude that bortezomib may be effective when used as a therapeutic adjunct in management of osteosarcoma and that its effect may be related to restoration of the level and function of Runx2, which acts as a tumor suppressor in this type of malignancy.

These data complement our previous findings that gain of Runx2 function in osteosarcoma cells sensitizes these cells to apoptosis via transcriptional upregulation of a major proapoptotic factor, Bax.19 Our results also provide an explanation for a recently described suppression of Runx2 level and function in a variety of osteosarcoma cell lines.15 Increased proteasomal degradation may play a major role in this suppression of Runx2 and, as suggested by our findings, inhibition of proteasome may effectively restore Runx2 level and function in osteosarcoma cells. Proteasomal degradation has been previously implicated in the regulation of Runx220–22, 36 however its role in down-regulation of Runx2 level in cancerous cells has not been described. The mechanism underlying the observed increased degradation of Runx2 in osteosarcoma is a subject of future studies and is currently under investigation by our group. Targeting for proteasomal degradation by Smurf1 or by Cyclin D1/Cdk4 has been shown to destabilize Runx2 and suppress Runx2 protein levels.20, 21 We have found that Smurf1 is significantly upregulated in the studied osteosarcoma cell lines and that siRNA-mediated inhibition of Smurf1 restores Runx2 levels in these cells (data not shown) implicating Smurf1 as a ubiquitin ligase responsible for Runx2 degradation in osteosarcoma. Overall, our data confirm previous findings15, 18 that Runx2 can act as a tumor suppressor in osteosarcoma and suggest that in such instances osteosarcoma cells develop mechanisms to suppress Runx2 leading to arrested differentiation and desensitization to pro-apoptotic signals. It should be noted that substantial evidence has also been accumulated on the oncogenic role of Runx2 and other 2 Runx factors in cancer. As suggested in an elegant review by Blyth et al., this ambiguous role of Runx factors in cancer may be due to their dependence on various co-factors, presence of functional p53 protein, and other regulatory signals.17 The fact that Runx2 increases the potential for bone metastasis in breast and prostate cancer cells37, 38 may be a tissue specific effect. Active Runx2 might be necessary for breast and prostate cancer cells to mimic bone-like phenotype and to home effectively in bone, while the described pro-apoptotic function of Runx2 may be suppressed via some mechanism that is yet to be elucidated.

Our most clinically relevant finding is that bortezomib, a proteasome inhibitor approved for clinical use and proven effective in treating lymphoma and multiple myeloma,39 may be equally effective in suppressing osteosarcoma. Our data indicate that bortezomib selectively induces apoptosis in osteosarcoma cells in vitro and in osteosarcoma xenografts in vivo. Inhibition of proteasome may have an effect on a wide range of cellular mechanisms. However, regardless of the pathways involved, suppression of tumor growth and selective induction of apoptosis by bortezomib in osteosarcoma is a promising and potentially important finding. It suggests a novel treatment option for this devastating disease. Pre- or postoperative bortezomib regimen may be an effective complement to current chemotherapies.

Acknowledgements

The 143B-luc cells were a kind gift of Dr. T.C. He of the University of Chicago to Dr. E. Sampson. OS187 cells were a kind gift of Dr. R. Gorlick of the Memorial Sloan-Kettering Cancer Center. The authors thank R. Tierney, L. MacMahon and Dr. L. Flick of the University of Rochester for their help with immunohistochemistry; Dr. E. Sampson of the University of Rochester for his help with osteosarcoma xenograft model and shRNA experiments; and Dr. D. Hicks, Dr. R. Rosier and Dr. R. O’Keefe of the University of Rochester for their expertise and fruitful discussions.

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Kaempferol (found in Ginkgo Biloba) induced apoptosis via endoplasmic reticulum stress and mitochondria-dependent pathway in human osteosarcoma U-2 OS cells

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Posted 21 Jul 2011 — by James Street
Category Ginkgo Biloba (Kaempferol), Human osteosarcoma research, Molecular Osteosarcoma Studies, Mouse Osteosarcoma Studies, Osteosarcoma
Mol Nutr Food Res. 2010 Nov;54(11):1585-95.

Source

Department of Biological Science and Technology, China Medical University, Taichung, Taiwan.

Abstract

Kaempferol is a natural flavonoid. Previous studies have reported that kaempferol has anti-proliferation activities and induces apoptosis in many cancer cell lines. However, there are no reports on human osteosarcoma. In this study, we investigate the anti-cancer effects and molecular mechanisms of kaempferol in human osteosarcoma cells. Our results demonstrate that kaempferol significantly reduces cell viabilities of U-2 OS, HOB and 143B cells, especially U-2 OS cells in a dose-dependent manner, but exerts low cytotoxicity on human fetal osteoblast progenitor hFOB cells. Comet assay, DAPI staining and DNA gel electrophoresis confirm the effects of DNA damage and apoptosis in U-2 OS cells. Flow cytometry detects the increase of cytoplasmic Ca(2+) levels and the decrease of mitochondria membrane potential. Western blotting and fluorogenic enzymatic assay show that kaempferol treatment influences the time-dependent expression of proteins involved in the endoplasmic reticulum stress pathway and mitochondrial signaling pathway. In addition, pretreating cells with caspase inhibitors, BAPTA or calpeptin before exposure to kaempferol increases cell viabilities. The anti-cancer effects of kaempferol in vivo are evaluated in BALB/c(nu/nu) mice inoculated with U-2 OS cells, and the results indicate inhibition of tumor growth. In conclusion, kaempferol inhibits human osteosarcoma cells in vivo and in vitro.

Ketoprofen in topical formulation decreases the matrix metalloproteinase-2 expression and pulmonary metastatic incidence in nude mice with osteosarcoma

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Posted 08 Jun 2011 — by James Street
Category Chemotherapy, Ketoprofen, Local Recurrence, Lung Metastases, Metastases, metastases, Mouse Osteosarcoma Studies
  1. Setsuya Kamei1,2,
  2. Kenshi Sakayama1,
  3. Shinta Tamashiro2,
  4. Junichi Aizawa1,2,
  5. Joji Miyawaki1,2,
  6. Tatsuhiko Miyazaki3,
  7. Haruyasu Yamamoto1,
  8. Yoshiaki Norimatsu2,
  9. Hiroshi Masuno2,*

Article first published online: 22 DEC 2008

DOI: 10.1002/jor.20832

Keywords:

  • ketoprofen;
  • osteosarcoma;
  • pulmonary metastasis;
  • matrix metalloproteinase-2

Abstract

The aim of this study was to investigate whether ketoprofen (KP) in topical formulation affected the tumor growth and pulmonary metastasis of LM8 cells, which were inoculated subcutaneously into the back space of male nude mice. At 7 days after inoculation, the tumor was treated topically for 3 weeks with either a KP-containing patch (KP group) or a placebo-containing patch (placebo group). The pulmonary metastatic incidence was 100% in the placebo group and 60% in the KP group. The tumor mass of the KP group without pulmonary metastasis, termed the KP/metastasis(−) group, was smaller than that of the placebo group. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor (VEGF) was performed. The tumors of the KP/metastasis(−) group contained fewer PCNA-positive cells and many more TUNEL-positive cells in comparison to the placebo group. In the placebo group, MMP-2 and VEGF were extensively expressed within the tumor, whereas in the KP/metastasis(−) group the expression of these two proteins was very low. In conclusion, the topical treatment of osteosarcoma with KP decreased the expression of MMP-2 and VEGF, thus resulting in the suppression of tumor growth and pulmonary metastasis. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 909–915, 2009

Effect of ascorbic acid, lysine, proline and green tea extract on human osteosarcoma cell line MNNG-HOS xenografts in nude mice Evaluation of tumor growth and immunohistochemistry 01

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Posted 19 May 2011 — by James Street
Category Lung Metastases, Metastases, Mouse Osteosarcoma Studies

Abstract

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling MMPs, VEGF, Ki-67 (proliferative protein), and constitutents of ECM play a critical role in angiogenecontaining lysine, proline, arginine, ascorbic acid, and green tea extract (NM) on the growth of tumors induced by implanting human osteosarcoma MNNG in athymic nude mice and the expression of MMPs, VEGF, Ki-67 and fibroenectin in these tumors, as well as the production of mucin (by PAS staining). We also investigated the effect of the supplemented diet on serum ascorbic acid, total protein content, alkaline phosphatase activity, and liver enzymes Athymic male nude mice (n=12) were inoculated with 3×106 osteosarcoma cells MNNG-HOS and randomy divided into group A (fed a regular diet) and group B (fed a regular diet supplemented with 0.5% NM). Four weeks later, the mice were sacrificed. Results showed that NM inhibited the growth and reduced the size of tumors in nude mice. Histological evaluation revealed increased mitotic index, MMP-9, and VEGF secretion in the control group tissues. Results demonstrate that the nutrient mixture of lysine, proline, arginine, ascorbic acid, and green tea extract tested strongly suppressed the growth of tumors without adverse effects in nude mice, suggesting potential as an anticancer agent.

Insight Into Cellceutix Corporation’s Breakthrough Cancer Compound

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Posted 03 May 2011 — by James Street
Category Cat osteosarcoma, Dog Osteosarcoma, genetic research, Mouse Osteosarcoma Studies, Proteomics

May 03, 2011 06:30 ET

BEVERLY, MA–(Marketwire – May 3, 2011) – Cellceutix Corporation (OTCQB: CTIX), a biopharmaceutical company focused on discovering and developing small molecule drugs to treat unmet medical conditions including drug-resistant cancers, today provided additional insight into the uniqueness of Kevetrin™, its flagship cancer compound in development. Kevetrin™ is a proprietary novel molecule which has a distinct advantage over other compounds in development, or drugs in current use. The advantage lies in its mode of action. Kevetrin not only activates p53 in a non-genotoxic manner, it also acts as a double-edged sword in killing tumor cells. Kevetrin activates both transcription-dependent and transcription-independent pathways to promote apoptosis (programmed cell death). Kevetrin also alters E3 processivity of MDM2. Monoubiquitination of p53 by Kevetrin not only stabilizes both wild type and mutant p53, but also induces apoptosis in mutant p53. Kevetrin showed potent efficacy in many mutant and wild type tumor xenograft models, thus Kevetrin demonstrated effectiveness in a wide range of tumor types. To the best of our knowledge, no other compound has shown such potent efficacy in tumors of varied p53 status.

The development of cancer is a multistage process driven by a progressive accumulation of mutations and epigenetic abnormalities in multiple genes that have highly diverse functions. Today’s commonly used drugs (e.g. trastuzumab, gefitinib and imatinib) target either specific molecules (such as HER2 for trastuzumab and EGFR for gefitinib) or functions as a multikinase inhibitor (imatinib). This approach is based on the observation that a tumor cell, despite its plethora of genetic alterations, can seemingly exhibit dependence on a single oncogenic pathway or protein for its sustained proliferation and/or survival, termed oncogene addiction. These agents target specific oncogenes in human cancer and causes cell death. However, the clinical responses in most of the cases are relatively short-lived. This is most clearly illustrated in the case of erlotinib where clinical response typically averages only 6-9 months in duration and is almost invariably followed by disease progression. Thus clinical experience with molecular targeted agents shows that cancers can escape a given state of oncogene addiction through mutations in other genes and pathways.

The activation (or reactivation) of p53 is a promising strategy for cancer treatment. Restoration of p53 tumor suppressor pathways triggers massive apoptosis through the intrinsic mitochondrial mediated pathway of apoptosis. This approach has the capacity to treat a wide range of tumors, but demonstrating success has been elusive to researchers. MDM2, an ubiquitin ligase for p53, plays a central role in the stability of p53. Nutlins and RITA compounds inhibit the interaction between p53 and MDM2. Both compounds induce apoptosis in the tumor, but are limited to normal or wild type p53. Additionally, Nutlin has been shown to be genotoxic. Other compounds, e.g., CP-31398, PRIMA-1, ellipticine, target mutant p53 only. Mutant p53 is a complex target since it is not one protein, but rather an extensive array of proteins with different properties that limit the range of tumors treated by these compounds. In addition, the clinical use of ellipticine has been limited by toxic side effects.

Based on these scientific parameters, Kevetrin has the unique ability to target tumors independent of p53 status and induce apoptosis thereby controlling tumor growth in a wide range tumor types, setting it apart from today’s therapies and other compounds in development.

About Cellceutix

Cellceutix Corporation is a preclinical cancer, anti-inflammatory and autism drug developer. Cellceutix owns the rights to eight drug compounds, including Kevetrin, which it is developing as a treatment for certain cancers, KM-133, for the treatment of psoriasis, and KM-391, for the treatment of autism. More information is available on the Cellceutix web site at www.cellceutix.com.

This Press Release contains forward-looking statements that are based on our current expectations, beliefs and assumptions about the industry and markets in which Cellceutix Corporation operates. Such forward-looking statements involve known and unknown risks, uncertainties, and other factors that may cause Cellceutix’s actual results to be materially different from any future results expressed or implied by these statements. Actual results may differ materially from what is expressed in these statements, and no assurance can be given that Cellceutix can successfully implement its core business strategy and improve future earnings.

The factors that may cause Cellceutix’s actual results to differ from its forward-looking statements include: Cellceutix’s current critical need for additional cash to sustain existing operations and meet ongoing existing obligations and capital requirements; Cellceutix’s ability to implement its new product development and commercialization, enter into clinical trials, expand the intellectual property portfolio, and receive regulatory approvals in a timely and cost-effective manner. All forward-looking statements are also expressly qualified in their entirety by the cautionary statements included in Cellceutix’s SEC filings, including its quarterly reports on Form 10-Q and its annual report on Form 10-K.

Kevetrin, KM-133, and KM-391 have not been studied in humans at this time. The Company’s positive results in animal studies do not necessarily guarantee success in humans, though they may form the basis for beginning Phase 1 trials.

New study finds compounds show promise in blocking STAT3 signaling as treatment for osteosarcoma

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Posted 12 Apr 2011 — by James Street
Category CURCUMIN, genetic research, Metastases, Molecular Osteosarcoma Studies, Mouse Osteosarcoma Studies, Nutrition and Cancer, Osteosarcoma, STAT3

Contact: Erin Pope
Erin.Pope@NationwideChildrens.org
614-355-0495
Nationwide Children’s Hospital

A study appearing in the journal Investigational New Drugs and conducted by researchers at Nationwide Children’s Hospital, discovered that two new small molecule inhibitors are showing promise in blocking STAT3, a protein linked to the most common malignant bone tumor, osteosarcoma. These small molecule inhibitors – one derived from a portion of the turmeric spice – may serve as a new, non-toxic treatment for these deadly tumors.

Osteosarcoma is aggressive and its treatment outlook has not changed significantly over the last 20 years. Treatment consists of a combination of toxic chemotherapy and aggressive surgical resection. Yet, despite these options, patients have at most a 50-to-60 percent five-year disease-free survival rate.

“The outcome for patients with advanced or metastatic osteosarcoma continues to be dismal, emphasizing the need for new therapies,” said the study’s lead author Jaiyuh Lin, PhD, principal investigator in the Center for Childhood Cancer in The Research Institute at Nationwide Children’s Hospital. “Directly targeting STAT3 signaling represents a potential therapeutic approach to treating this type of cancer.”

STAT3 is a member of a protein family that plays a role in relaying signals from cytokines and growth factors. The abnormal activation of STAT proteins is becoming more commonly associated with unrestricted cell growth and transformation of normal cells into malignant cells. Abnormal STAT3 activation has been seen in human and canine osteosarcoma cell lines and shows cancer-causing-capabilities in cultured cells and mouse models.

“Recent experiments aimed at blocking STAT3 signaling have been successful, resulting in the inhibition of growth and the induction of death in tumors,” said Dr. Lin, also a faculty member at The Ohio State University College of Medicine. “They have also shown that blocking STAT3 in normal cells is neither harmful nor toxic.”

Dr. Lin and his team evaluated two newly developed compounds, LLL12 and FLLL32, to determine their ability to inhibit STAT3 activity in human osteosarcoma cells. FLLL32 is derived from the dietary agent curcumin, the principal compound in the popular Indian spice turmeric.

Findings showed that both agents were able to inhibit STAT3 activity and suppressed tumor growth in the mouse model that was developed using human osteosarcoma cells, and primary osteosarcoma xenograft provided by Nationwide Children’s Hospital scientist, Peter Houghton, PhD, directly from a patient.

“This study suggests that LLL12 and FLLL32 should be suitable for targeting osteosarcoma and possibly certain types of cancer cells with persistently activated STAT3,” said Dr. Lin. “This approach deserves further exploration as a potential treatment of osteosarcoma.”

CLINICAL AND PHARMACOKINETIC EVIDENCE OF A LIFE-THREATENING INTERACTION BETWEEN METHOTREXATE AND KETOPROFEN

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Posted 21 Jan 2011 — by James Street
Category Chemotherapy, Drug Interactions, Mouse Osteosarcoma Studies

References and further reading may be available for this article. To view references and further reading you must purchase this article.

Antoine Thyss, Johanna Kubar, Gerard Milano, Moise Namer and Maurice Schneider

Centre Antoine-Lacassagne, Nice, France

Available online 11 September 2003.

Abstract

Co-administration of ketoprofen was found in 4 of 118 cycles of high-dose methotrexate (MTX) analysed retrospectively in thirty-six patients. All 4 cycles were characterised by severe MTX toxicity, which was fatal in three cases. Simultaneous administration of ketoprofen was associated with prolonged and striking enhancement of serum MTX levels. There were no abnormalities in MTX kinetics or evidence of MTX toxicity when ketoprofen was given at least 12 h after completion of high-dose MTX infusion. The kidney may be the site of drug interaction. This high-risk association between MTX toxicity and ketoprofen may also apply to other non-steroidal anti-inflammatory drugs.
Article Outline

• References

The Lancet
Volume 327, Issue 8475, 1 February 1986, Pages 256-258

Inhibitory effects of 22-oxa-calcitriol and all-trans retinoic acid on the growth of a canine osteosarcoma derived cell-line in vivo and its pulmonary metastasis in vivo

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Posted 21 Jan 2011 — by James Street
Category Dog Osteosarcoma, Lung Metastases, Molecular Osteosarcoma Studies, Mouse Osteosarcoma Studies, Osteosarcoma, Osteosarcoma Outcomes
Author(s): Barroga EF, Kadosawa T, Okumura M, Fujinaga T
Source: RESEARCH IN VETERINARY SCIENCE    Volume: 68    Issue: 1    Pages: 79-87    Published: FEB 2000
Times Cited: 7     References: 23
Abstract: Pulmonary metastasis is a major cause of death and a major obstacle to the successful treatment of canine osteosarcoma. However, the residual capacity of the neoplasia for differentiation and its susceptibility to undergo apoptosis may be used to suppress its growth and metastatic properties. The highly metastasizing POS (HMPOS) canine osteosarcoma cell line which preferentially metastasize to the lungs was used to test the possible efficacy of 22-oxa-calcitriol (OCT) and all-trans retinoic acid (ATRA) to inhibit growth and pulmonary metastasis of the subcutaneously grown osteosarcoma in nude mice. Treatments in vitro, morphologically elongated and increased alkaline phosphatase activity and staining of cells. Tumour growth in vivo was inhibited significantly and the combination treatment of OCT and ATRA (OCT + ATRA) exerted a synergistic and stronger suppression at concentration of 1.0 mu g kg(-1) body weight when given subcutaneously three times a week for 5 weeks. The subcutaneous rumours of the control mice consisted of osteoblast-like cells and isolated chondroblast-like cells, but formed several areas of osteoid and increased amount of collagen tissue in all treated mice. Pinpoint macrometastatic nodules developed only in all control mice. Micrometastatic nodule developed only in two of six mice treated with ATRA. However, nodule size and number, and lung wet weight were all reduced significantly. Metastasis were not seen in the mice treated with OCT or OCT + ATRA. This study demonstrated that inhibition of growth and pulmonary metastasis was induced by subcutaneous treatment with these drugs and suggest that both its differentiating and apoptotic inducing activities may be responsible for the antitumour effects. These drugs may be useful in the clinic as an adjunct for the treatment of canine osteosarcoma. (C) 2000 Harcourt Publishers Ltd.
Document Type: Article
Language: English
Reprint Address: Barroga, EF (reprint author), Hokkaido Univ, Grad Sch Vet Med, Dept Vet Clin Sci, Lab Vet Surg, Sapporo, Hokkaido 0600818 Japan
Addresses:
1. Hokkaido Univ, Grad Sch Vet Med, Dept Vet Clin Sci, Lab Vet Surg, Sapporo, Hokkaido 0600818 Japan
Publisher: W B SAUNDERS CO LTD, 24-28 OVAL RD, LONDON NW1 7DX, ENGLAND
Subject Category: Veterinary Sciences
IDS Number: 287TP
ISSN: 0034-5288

Rapamycin Inhibits Ezrin-Mediated Metastatic Behavior in a Murine Model of Osteosarcoma

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Posted 21 Jan 2011 — by James Street
Category Local Recurrence, Lung Metastases, Metastases, Molecular Osteosarcoma Studies, Mouse Osteosarcoma Studies, Osteosarcoma

1. Xiaolin Wan,
2. Arnulfo Mendoza,
3. Chand Khanna, and
4. Lee J. Helman

+ Author Affiliations

1.
Molecular Oncology Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland

1. Requests for reprints:
Lee J. Helman, Molecular Oncology Section, Pediatric Oncology Branch, Building 10, Room 1 West-3750, National Cancer Institute, NIH, Bethesda, MD 20892-1106. Phone: 301-496-4257; Fax: 301-480-4318; E-mail: helmanl@nih.gov.

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Abstract

Osteosarcoma is the most frequent primary malignant tumor of bone with a high propensity for metastasis. We have previously showed that ezrin expression is necessary for metastatic behavior in a murine model of osteosarcoma (K7M2). In this study, we found that a mechanism of ezrin-related metastatic behavior is linked to an Akt-dependent mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (S6K1)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) pathway. Suppression of ezrin expression either by antisense transfection or by small interfering RNAs or disruption of ezrin function by transfection of a dominant-negative ezrin-T567A mutant led to decreased expression and decreased phosphorylation of both S6K1 and 4E-BP1. Proteosomal inhibition by MG132 reversed antisense-mediated decrease of S6K1 and 4E-BP1 protein expression, but failed to affect the effect of ezrin on phosphorylation of S6K1 and 4E-BP1. Blockade of the mTOR pathway with rapamycin or its analog, cell cycle inhibitor-779 led to significant inhibition of experimental lung metastasis in vivo. These results suggest that blocking the mTOR/S6K1/4E-BP1 pathway may be an appropriate target for strategies to reduce tumor cell metastasis.

* ezrin
* S6K1
* 4E-BP1
* metastasis
* rapamycin

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Introduction

Ezrin is a member of the ezrin/radixin/moesin (ERM) family of proteins that link the cell membrane to the actin cytoskeleton and are involved in cytoskeletal organization (1, 2) . Ezrin is believed to be involved in intracellular signal transduction that is related to cell migration and metastasis because ezrin is reported to be a substrate for tyrosine kinases (3, 4) and binds adhesion molecules such as CD43, CD44, intercellular adhesion molecule-1, and intercellular adhesion molecule-2 (5–7) . Of note, high levels of CD44 seem to be dependent on ezrin expression and are associated with invasion and metastatic behavior of tumor cells (8). The discovery that merlin/schwannomin, the neurofibromatosis-2–associated tumor-suppressor protein, is related to the ezrin/radixin/moesin family has provided additional insights into the relationship between ezrin and tumorigenesis (9). Recently, we have found ezrin expression in murine osteosarcoma and rhabdomyosarcoma to be necessary for metastatic behavior (10–12) . Suppression of ezrin protein expression by antisense transfection or stable expression of short hairpin RNA, or disruption of ezrin function by transfection of a dominant-negative ezrin significantly reduced the metastatic behavior in both murine models and was associated with decreased Akt and mitogen-activated protein kinase (MAPK) activity (11, 12) . However, the specific mechanism or mechanisms by which ezrin mediates the metastatic process remains to be elucidated.

Rapamycin and analogues such as cell cycle inhibitor-779 (CCI-779) are currently undergoing clinical and preclinical evaluations as an anticancer agent. The anticancer property of rapamycin is attributed to the inhibition of mammalian target of rapamycin (mTOR) signaling pathway, which controls mRNA translation and cell proliferation. Rapamycin binds to the FK506 binding protein (FKBP-12), and this complex interacts with mTOR. This interaction inhibits mTOR kinase activity and subsequently decreases phosphorylation and activation of p70 ribosomal protein S6 kinase (p70S6K, S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) that play fundamental roles in ribosome biogenesis and cap-dependent translation, respectively (13, 14) . A previous study has found that the ability of rapamycin to inhibit metastatic tumor growth and angiogenesis in in vivo mouse models is linked to reduced translational production of vascular endothelial growth factor vascular endothelial growth factor and to blockage of vascular endothelial growth factor–induced endothelial cell signaling (15). Most recently, investigators have showed that rapamycin can reverse resistance to doxorubicin in a mouse model of an Akt-driven aggressive lymphoma (16). These data suggest that blockade of the mTOR pathway might also have an inhibitory effect on both resistance to cytotoxic therapy as well as tumor metastasis.

In this study, we linked ezrin-related metastatic behavior to activation of S6K1 and 4E-BP1 signaling. We found that antisense-mediated and small interfering RNA (siRNA)–induced reduction of ezrin expression or disruption of ezrin function by transfection of a dominant-negative ezrin-T567A mutant led to decreased expression and decreased phosphorylation of both S6K1 and 4E-BP1. Proteosomal inhibition by MG132 reversed antisense-mediated decrease of S6K1 and 4E-BP1 protein expression, but failed to affect the effect of ezrin on phosphorylation of S6K1 and 4E-BP1. Finally treatment with rapamycin and its analogue, CCI-779 led to a significant inhibition of experimental metastasis in vivo. These results indicate that blockade of the mTOR/S6K1/4E-BP1 pathway by rapamycin could be of potential therapeutic benefit for reducing tumor cell metastasis in osteosarcoma.
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Materials and Methods

Cell Culture. The K12, K7M2 murine OSA cell lines, ezrin-antisense clones 13, 1.46 and 1.52 cells, and dominant-negative ezrin (T567A mutant) clones T567A-GFP-7, and T567A-GFP-8 as well as empty-GFP clones empty-GFP-2.5, empty-GFP-2.7 cells generated from K7M2 have been previously described (13, 14) . These cells were maintained in DMEM containing 10% fetal bovine serum, l-glutamine (2 mmol/L), penicillin (100 units/mL), and streptomycin (100 units/mL, BioSource International Inc, Camarillo, CA) at 37°C in a humidified CO2 incubator.

Antibodies and Reagents. Anti-ezrin monoclonal antibody was purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies to phospho-S6K1 (Thr389), S6K1, phospho-4E-BP1 (Thr37), 4E-BP1, phospho-Akt (Ser473), Akt, phospho-4E-BP1 (Ser2448), phospho-p44/42 MAPK, and p44/42 MAPK were purchased from Cell Signaling Technology Inc. (Beverly, MA). Anti-actin antibody was from Amersham Pharmacia Biotech (Piscataway, NJ). MG132 was purchased from Calbiochem (San Diego, CA). U0126 was purchased from Promega Corp. (Madison, WI). LY294002 was purchased from Sigma Chemical Co.. Rapamycin was purchased from LC Laboratories (Woburn, MA). CCI-779 was obtained from Developmental Therapeutics Program, National Cancer Institute (Bethesda, MD) and Wyeth Laboratories (Philadelphia, PA).

Transfection. Myc-tagged, activated Akt, dominant-negative Akt (Akt K179M), and empty vector (pUSE) were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). K7M2 cells were transfected with dominant-negative Akt and empty vector by using electroperation in a gene Pulsar (0.22 kV/cm; capacitance, 960 μF; Bio-Rad, Richmond, CA). After selection in medium containing G418, single clones were isolated and expanded. Ezrin-antisense clones 1.46 and 1.52 cells were transfected with activated Akt and empty vector by using electroperation in a Bio-Rad gene Pulsar (0.22 kV/cm; capacitance, 960 μF). After 72 hours, these cells were harvested and subjected to Western blot analysis.

Western Blot Analysis. Confluent cells were lysed in lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 150 mmol/L sodium chloride, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pryophosphate, 1 mmol/L β-glycerolphosphate, 1 mmol/L sodium orthovanadate, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin). Protein lysates (20-50 μg per lane), as determined by Bio-Rad protein assay, were separated in 10% to 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). Membranes were blocked with 5% nonfat dried milk in TBS-T (20 mmol/L Tris-HCl, pH 7.5, 8 g/l of sodium chloride, 0.1% Tween 20) and then incubated with primary antibodies. Horseradish peroxidase conjugated anti-rabbit IgG (Cell Signaling) was used as secondary antibody. Protein was visualized using enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).

S6K1 Assay. S6K1 activity was determined in vitro as described previously (17).

Gene Silencing with siRNAs. We obtained annealed, 21-bp siRNA duplexes from Dharmacon Research Inc. (Lafayette, CO). The target sequence for ezrin was 5′-AAGGAAUCCUUAGCGAUGAGA-3′, corresponding to position 440 to 460 in the human ezrin mRNA. A siRNA targeting a nonspecific sequence was purchased from Dharmacon Research Inc. and served as a negative control. We applied siRNA duplexes at a final concentration of 100 nmol/L using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA).

In vivo Experimental Metastasis Assay. Four- to 5-week-old female beige severe combined immunodeficient (SCID) mice (Charles River Laboratories, Wilmington, MA) were inoculated with 1 × 106 K7M2 cells per mouse via the tail vein and then randomly assigned to treatment groups. Mice were treated i.p. daily × 5 days for 5 to 6 consecutive weeks with 5 mg/kg rapamycin, 5 mg/kg CCI-779, 20 mg/kg CCI-779, or vehicle alone. All mice underwent complete necropsy for confirmation of pulmonary metastases. All animal work was done with the approval of the Animal Care and Use Committee of the National Cancer Institute.

Lung Histopathology. Lung tissue was fixed in 10% formalin and embedded in paraffin. Paraffin sections (5 μm) were stained with H&E.
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Results

Reduction of S6K1 and 4E-BP-1 Phosphorylation by Suppression of Ezrin Expression. Previous experiments using cDNA microarray and Northern blot analysis have showed that the highly metastatic K7M2 cell line has much higher ezrin expression compared with the less metastatic K12 cell line (10). To study the specific role of ezrin and ezrin-mediated signaling pathways, we generated stable K7M2 clones expressing antisense ezrin cDNA (11). The expression levels of ezrin protein in G418-resistant clones were analyzed by Western blotting using a monoclonal ezrin antibody. Both 1.46 and 1.52 clones transfected with antisense vector showed a marked decrease of ezrin protein expression, whereas clone 13 had a minimal reduction in ezrin expression compared with K7M2 cells ( Fig. 1A ).
Figure 1.
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Figure 1.

Suppression of ezrin expression leads to reduced S6K1 and 4E-BP1 phosphorylation and expression. A, ezrin expression was analyzed by Western blotting in K12, K7M2, and ezrin antisense transfectant cells using anti-ezrin antibody. B, S6K1 phosphorylation and expression were determined by Western blot analysis in K12, K7M2, and ezrin-antisense transfected cell lines. C, S6K1 activity was measured in K12, K7M2, and ezrin-antisense transfected cell lines using a peptide substrate (AKRRRLSSLRA) as described. Columns, mean; bars, SE. D, 4E-BP1 phosphorylation and expression were determined by Western blot analysis in K12, K7M2, and ezrin-antisense transfected cell lines. E, K7M2 cells plated in six-well plates were transfected with 100 nmol/L control or ezrin siRNA using LipofectAMINE 2000. After 5 days, cells were lysed in lysis buffer and then subjected to Western blot analysis. Blotting with antibody against actin was used to confirm equal loading of the protein. Similar results were achieved in three independent experiments.

We sought to determine whether S6K1 and 4E-BP-1, two major downstream targets of mTOR that play critical roles in translation regulation, were involved in ezrin-mediated metastatic signaling because our previous data showed ezrin-mediated effects on Akt activity. We first examined the phosphorylation and expression status of S6K1 and 4E-BP-1 in K12 and K7M2 cell lines. Our data revealed that S6K1 and 4E-BP-1 are both more highly phosphorylated and expressed in K7M2 cells compared with K12 cells ( Fig. 1B and D). Furthermore, S6K1 activity, as evaluated by in vitro kinase assays, is significantly elevated in K7M2 cells compared with K12 cells ( Fig. 1C). Down-regulation of ezrin expression decreased phosphorylation and expression of S6K1 and 4E-BP-1 as well as S6K1 activity ( Fig. 1B-D). To further test the effects of ezrin on the regulation of S6K1 and 4E-BP1, we next targeted ezrin by siRNA. Inhibition of ezrin expression by siRNA led to decreased phosphorylation of S6K and 4E-BP1, with minimal reduction of S6K1 and 4E-BP1 expression ( Fig. 1E). These data suggest that S6K1 and 4E-BP1 are associated with ezrin-mediated signaling.

Proteosomal Inhibition Reverses Ezrin-Antisense–Mediated Suppression of S6K1 and 4E-BP1 Expression, but Does Not Affect S6K1 and 4E-BP1 Phosphorylation. We sought to determine whether the observed decrease in S6K1 activity and 4E-BP1 phosphorylation was a direct result of decreased protein expression or an independent activity of ezrin suppression. We therefore treated K7M2 antisense clones with MG132, a proteasome inhibitor, to block protein degradation. As shown in Fig. 2 , exposure of cells to MG132 (50 μmol/L) for 6 hours reversed the decrease in S6K1 and 4E-BP1 protein expression induced by ezrin-antisense transfection but failed to affect S6K1 and 4E-BP1 phosphorylation. Similar results were also observed after 6 hours of treatment with doses as low as 2 μmol/L MG132 (data not shown). Neither 2 nor 50 μmol/L had any significant affect on cell viability as determined by visual inspection and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (data not shown). These data suggest that suppression of S6K1 and 4E-BP1 expression by ezrin-antisense transfection is mediated via enhanced degradation of S6K1 and 4E-BP1 proteins through a proteasome-dependent pathway, and that down-regulation of S6K1 and 4E-BP1 phosphorylation induced by ezrin-antisense transfection is an independent effect of ezrin down-regulation.
Figure 2.
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Figure 2.

Proteosomal inhibition with MG132 reverses ezrin anti-sense–mediated reduction of S6K1 and 4E-BP1 expressions but does not affect S6K1 and 4E-BP1 phosphorylation. 13, 1.46, and 1.52 cells were treated with MG132 (50 mmol/L) for 6 hours and then lysed in lysis buffer for Western blot analysis of S6K1 and 4E-BP1 phosphorylation and expression. Similar results were achieved in three independent experiments.

Disruption of Ezrin Function by Transfection of Dominant-Negative Ezrin (T567A) Inhibited Akt, S6K1, and 4E-BP1 Phosphorylation. Our previous data showed that disruption of ezrin function by stable transfection of dominant-negative ezrin (T567A) completely inhibited experimental metastases in mice (11). To further determine whether the role of functional ezrin in K7M2 cell metastasis is also linked to Akt/mTOR signaling pathway, we analyzed both phosphorylation and expression of Akt, S6K1 and 4E-BP1 in cell lines transfected with dominant-negative ezrin (T567A) or empty vector. As shown in Fig. 3A , Akt, S6K1 and 4E-BP1 phosphorylation were inhibited in dominant-negative ezrin (T567A) clones compared with empty vector clones. In addition, stable transfection of dominant-negative ezrin (T567A) led to reduction of S6K1 and 4E-BP1 protein expression but failed to influence the levels of Akt protein. These data are consistent with our findings in ezrin-antisense transfection cells ( Fig. 1). We have previously shown phosphorylation and activity of p44/42 MAPK are reduced when ezrin protein expression is suppressed by stable transfection of ezrin antisense (11). However, p44/42 MAPK phosphorylation is increased, not decreased, in cells expressing ezrin-T567A ( Fig. 3), whereas the total expression of p44/42 MAPK is not significantly changed in these clone cells ( Fig. 3A). Thus, phosphorylation of ezrin-T567 residue seems to be a crucial site for ezrin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway, but not for ezrin-mediated activation of the p44/42 MAPK signaling pathway.
Figure 3.
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Figure 3.

Disruption of ezrin function resulted in decreased phosphorylation of Akt, S6K1, and 4E-BP1 (A) and ezrin-associated phosphorylation of S6K1 and 4E-BP1 is rapamycin sensitive (B). A, phosphorylation and expression of Akt, S6K1, 4E-BP1, and p44/42 MAPK were analyzed by Western blotting in empty vector-GFP and dominant-negative ezrin (T567A) clones. B, confluent cells were treated with rapamycin (100 nmol/L), LY294002 (10 μmol/L), or U0126 (10 μmol/L) for 1 hour, lysed, and then subjected to Western blot analysis of Akt, S6K1, and 4E-BP1 phosphorylation and expression. Similar results were achieved in two separate experiments.

Ezrin-Linked S6K1 and 4E-BP1 Phosphorylation Is Rapamycin Sensitive. To further determine whether the ezrin-linked S6K1 and 4E-BP1 phosphorylation is mTOR dependent, K7M2 cells were treated with an mTOR inhibitor rapamycin, as well as a PI3K inhibitor LY294002, and a MAPK inhibitor U0126, for 1 hour. As shown in Fig. 3B, rapamycin inhibited S6K1 and 4E-BP1 phosphorylation but did not affect Akt phosphorylation. LY294002 completely inhibited not only Akt phosphorylation but also S6K1 and 4E-BP1 phosphorylation. U0126 affected neither Akt phosphorylation nor S6K1 and 4E-BP1 phosphorylation. These inhibitors failed to alter the expression of Akt, S6K1, and 4E-BP1. Taken together, these data suggest that the ezrin-related S6K1 and 4E-BP1 phosphorylation in K7M2 cells is rapamycin sensitive and downstream of PI3K.

Suppression of Experimental Metastasis in the K7M2 Murine Osteosarcoma Model by Rapamycin and Its Analogue CCI-779. To determine the potential effect of mTOR inhibition on experimental metastases in the K7M2 murine osteosarcoma model, we evaluated the effect of rapamycin and its analogue CCI-779. Mice were treated i.p. daily × 5 days every week for 5 to 6 weeks with 5 mg/kg rapamycin, 5 mg/kg CCI-779, 20 mg/kg CCI-779, or vehicle alone. Treatment with rapamycin and CCI-779 significantly prolonged the survival (morbidity associated with pulmonary metastasis) of SCID beige mice ( Fig. 4A ). In the control group, only 25% (2 of 8) mice survived beyond 38 days, whereas 100% (9 of 9) in both 5 mg/kg rapamycin- and 20 mg/kg CCI-779–treated groups were alive at 38 days. Two of 9 mice treated with 5 mg/kg CCI-779 died, but no gross pulmonary metastasis were detected in the two dead mice. At necropsy, no evidence of tumor was observed. Therefore, the cause of death in these mice is uncertain. All eight mice in the control group after 38 days of injection developed multiple lung metastases, whereas 1 of 7 mice in the 5 mg/kg CCI-779–treated group and 2 of 9 mice in the 5 mg/kg rapamycin–treated group developed single lung metastases. We failed to detect gross lung metastasis in any mouse treated with 20 mg/kg CCI-779 ( Fig. 4B). Histopathologic examination of H&E-stained sections of lungs was done in all animals. In contrast to multiple, large pulmonary metastases seen in all control-treated mice ( Fig. 4C, left), we found single small micrometastases in 6 of 9 mice treated with 5 mg/kg rapamycin (not shown), 6 of 9 mice treated with 5 mg/kg CCI-779 ( Fig. 4C, middle, micrometastasis indicated by arrow), and in only 2 of 9 mice treated at 20 mg/kg CCI-779 ( Fig. 4C, right, indicate normal lung only).
Figure 4.
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Figure 4.

Impact of rapamycin and its analogue CCI-779 on survival (A) and experimental lung metastases (B) in tumor-inoculated SCID beige mice. K7M2 cells (1 × 106 per mouse) were injected into the tail vein of SCID beige mice. These mice were treated i.p. daily × 5 in each of 5 to 6 consecutive weeks with 5 mg/kg rapamycin, 5 mg/kg CCI-779, 20 mg/kg CCI-779, or vehicle alone. All mice underwent complete necropsy and confirmation of metastases. C, representative H&E-stained lung sections (40× power). Left, the entire field is composed of tumor nodules. Middle, note only small microscopic tumor nodule (arrow). Right, no tumor nodules are noted, only normal lung. Bar, 100 μm.
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Discussion

Ezrin is known to be involved in a variety of cellular functions, such as cell cytoskeletal organization, cell motility, and morphogenesis. The high levels of ezrin expression in cell lines of endometrial (18), colorectal (19), and pancreatic carcinoma with high metastatic potential (20) have suggested that its expression has been associated with events that may promote tumor progression and metastasis. Consistent with that, our recent studies found that high expression of ezrin in K7M2 murine osteosarcoma cells is associated with highly metastatic behavior (10). Suppression of ezrin protein by antisense transfection and disruption of ezrin function significantly reduced lung metastases in two distinct mouse tumor models (11, 12) , providing an excellent experimental model to investigate the mechanisms of ezrin-mediated metastasis. In this report, we show that both blockade of ezrin expression either by antisense transfection or by siRNA and disruption of ezrin function by stable transfection of dominant-negative ezrin (T567A) led to inhibition of S6K1 and 4E-BP1 phosphorylation ( Figs. 1B, D, and E and 3A), which both lie downstream of mTOR and play fundamental roles in ribosome biogenesis and cap-dependent translation, respectively (21, 22) . These results indicate that ezrin signaling is involved in regulating mRNA translation and provide, for the first time, a linkage between ezrin and mTOR signaling.

Recent studies reported that S6K1 and 4E-BP1 also are regulated through the PI3K/Akt-signaling pathway (23). These studies raise the possibility of a direct signaling pathway from PI3K/Akt to mTOR. Our previous studies show that the inhibition of ezrin expression resulted in markedly reduced Akt phosphorylation and activity (11). Both S6K1 and 4E-BP1 phosphorylation were completely inhibited by the PI3K inhibitor LY294002 in K7M2 cells ( Fig. 3B). Furthermore, stable transfection of the dominant-negative Akt (K179M mutant) into K7M2 cells led to reduction of Akt phosphorylation as well as S6K1 and 4E-BP1 phosphorylation (data not shown). On the other hand, transient transfection of activated Akt into ezrin-antisense clones 1.46 and 1.52 cells led to up-regulation of S6K1 and 4E-BP1 phosphorylation (data not shown). These data are consistent with a PI3K/Akt/mTOR pathway in these cells. Phosphorylation of ezrin at T567 has been identified to play an important role in its conformational activation. Inactive, cytosolic ezrin, in a closed conformation through head-to-tail interaction between the amino- and carboxyl-terminal domains, requires phosphorylation at residue T567 and interaction with phosphatidylinositol 4,5-bisphosphate to cause unfolding, translocation to the plasma membrane, and cross-linking between integral membrane proteins and cytoskeleton (24–27) . Disruption of ezrin function by transfection of ezrin-T567A mutant significantly reduced lung metastases in two distinct mouse tumor models (11, 12) . In this study, we found that transfection of ezrin-T567A mutant into K7M2 cells not only inhibited Akt phosphorylation but also inhibited S6K1 and 4E-BP1 phosphorylation ( Fig. 3), which is consistent with our findings in ezrin-antisense transfected cells ( Fig. 1). As noted, ezrin has been found to directly bind PI3K (28). Thus, ezrin-mediated regulation of mTOR targets S6K1 and 4E-BP1 seems to be indirect through a direct interaction of ezrin with PI3K or phosphatidylinositol 4,5-bisphosphate leading to sequential activation of PI3K/Akt/mTOR signaling cascades.

The role of Akt in the regulation of mTOR activation is complex. Although Ser2448 in mTOR has been identified to be a direct phosphorylation target of Akt (29), substitution of Ser2448 by alanine failed to alter the ability of mTOR to activate S6K1 (30). We examined the phosphorylation of serine residue 2448 of mTOR in ezrin-antisense transfected cell lines. Down-regulation of ezrin failed to affect mTOR phosphorylation on Ser2448 (data not shown). Recent study has shown that phosphorylation of Ser2448 does not seem to modulate in vitro 4E-BP1 phosphorylation by mTOR (31). Moreover, mutation of Ser2035 in mTOR inhibited the abilities of mTOR to phosphorylate S6K1 and 4E-BP1 in vitro (32). Furthermore, in our study ezrin-associated phosphorylation of S6K1 and 4E-BP1 is rapamycin sensitive, suggesting that these observed ezrin effects occur through a mTOR signaling pathway. However, the specific mechanism remains to be elucidated.

To further determine the functional significance of ezrin-regulated mTOR/S6K1/4E-BP1 pathways, we studied the effect of mTOR inhibition on in vitro and in vivo metastatic pathways. Suppression of S6K1 and 4E-BP1 by rapamycin led to decreased K7M2 cell migration and invasion compared with untreated cells (data not shown). Treatment of tumor-inoculated SCID beige mice with rapamycin and CCI-779 resulted in prolonged survival and inhibition of pulmonary metastasis ( Fig. 4A and B). These results suggest that mTOR/ S6K1/4E-BP1 pathways play an important role in ezrin-mediated metastatic behavior. Recently, rapamycin has been reported to inhibit metastatic tumor growth in other murine models (15, 33) . Thus, inhibition of the mTOR/S6K1/4E-BP1 pathway by rapamycin or other inhibitors may be worthy of clinical evaluation as an antimetastatic intervention. The challenge will be to develop schedules of rapamycin and its analogues that can be chronically administered without causing significant immunosuppression.
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Acknowledgments

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Sung-Hyeok Hong for help in performing the invasion experiments, and Sally Hausman and Ed Sausville for the supply of CCI-779.
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Footnotes

* Received August 30, 2004.
* Revision received December 13, 2004.
* Accepted January 6, 2005.

* ©2005 American Association for Cancer Research.

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The anticancer effects of vitamin K

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Posted 20 Nov 2010 — by James Street
Category Alternative Therapies, Mouse Osteosarcoma Studies, Natural Therapies, Nutrition and Cancer

Alternative Medicine Review,  August, 2003  by Davis W. Lamson,  Steven M. Plaza
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The proto-oncogene bcl-2 has been shown to protect against apoptosis. The addition of bcl-2 to cell cultures (2B4 and FL5.12) countered the cell death imposed by the oxidative burst from K3. Bcl-2 was not able to decrease the ROS produced by K3, as measured by cyanide-resistant oxygen consumption, yet it was able to inhibit dose-related killing of the cell lines from 50 [micro]M-200 [micro]M of K3. These results suggest that ROS, acting as second messengers, signal downstream transcription factors, such as nuclear factor-kappa B (NF-kB), Fos/Jun, and others that may be protected by the antioxidant-governing activities of Bcl-2. (93) The transcription factor NF-kB is involved in stress-induced FasL expression. Fas is one of the important death receptors in the tumor necrosis factor superfamily. The gene encoding the ligand for Fas, designated as FasL, activates Fas by trimerization of the receptor. Activation-induced cell death is commonly mediated by the Fas/FasL system, and menadione induces Fas as well as FasL expression. Capriccio et al (94) found that a functional Fas/ FasL system was needed in order to induce apoptosis. Experiments showed that mutant leukemia cell lines lacking a functional Fas ligand were resistant to FasL killing by menadione. It was also found that mice lacking functional FasL or expression of Fas were also resistant to the cytotoxic effects of K3.

Vitamin K3 has been shown to inhibit growth and induce apoptosis of stomach cancer cells in a dose-dependent fashion. The inhibition was found to be due to sulfhydryl arylation of critical cystine residues that mediated protein tyrosine phosphorylation. The inhibition of cell growth by K3 induced tyrosine phosphorylation of hepatocyte growth factor receptors (c-met) and epidermal growth factor receptors (EGFR), which in turn activated the RAS signaling pathway. The addition of vitamin K3 also created a sustained phosphorylation of extracellular signal-regulated kinase (ERK), part of the mitogen-activated protein kinase (MAPK) superfamily associated with cellular signal transduction, proliferation, and apoptosis. Vitamin K3 is thought to induce both protein tyrosine kinase activation from the receptor pathway and inhibit ERK protein tyrosine phosphatases (that dephosphorylate activated kinases). ERK phosphorylation has been found to be critical to not only growth factor-induced cell proliferation, but also to K3-mediated cell death. It is thought that K3 can act without ligand binding through the inhibition of protein tyrosine phosphatases. (95)

Cell Cycle Arrest

The ability of vitamin K to induce cell cycle arrest and cell death may also be explained by the inhibition of protein kinases in association with a cyclin-dependent mechanism. Cyclins are regulatory proteins of the cell cycle that activate cellular maturation-promoting factors. Cyclins complex and modulate the protein kinase catalytic subunit of proteins such as p34CDC2, known alternately as cyclin dependent kinasel (CDK1). This protein kinase is a member of the serine/threonine protein kinase family. The designation “CDC2″ refers to “cell division cycle 2″ at the G1 to S and G2 to M transitions. Cyclins, such as cyclin B1, have no inherent enzymatic activity; rather, they act by means of cyclin-dependent kinases that phosphorylate serine and threonine residues on kinase cell cycle regulators. For example, cyclin B1 complexes with p34CDC2, forming the maturation-promoting factor, which in turn is essential in G1/S and G2/M transitions in the cell cycle. Phosphorylation and dephosphorylation act as on-and-off switches for the cell cycle. Dephosphorylation of p34CDC2 increases its activity.

K3 can inhibit CDKs. such as CDK1 (p34CDC2) (100 [micro]M for 1 hour) by hyperphosphorylating the protein. (45,96) The addition of menadione to malignant cell culture has been shown to inhibit the cell cycle at the G1/S and S/G2 phases. Concentrations in the 25-100 [micro]M range have been found to delay S/G2 in a dose-dependent manner. (45) Cell division cycle 25 (CDC25) are protein-tyrosine phosphatases critical for cell cycle progression. This family of CDC25 phosphatases is responsible for the activation of cyclin-dependent kinase CDC2 through the removal of two phosphate groups. CDC25A, required for the progression from G1 to S, has been found to be inactivated by vitamin K3, and the loss of enzymatic activity was due to modification of the active site. (97)

The addition of vitamin K3 to HepG2 cells hyperphosphorylated the CDC2 kinase, inactivating the enzyme and inhibiting the cell cycle. (98) It has been proposed that menadione modifies the active sites of the CDC25 dual specificity protein phosphatases and reduces or even abolishes the dephosphorylating activity of the enzyme. Vitamin K3 binds to active sulfhydryl groups of cysteine residues at active p34CDC2 sites. (45) This action stems from binding to the catalytic domain of CDC25 phosphatase. K3 also decreased protein-tyrosine phosphatase by 2- to 3-fold (45) and suppressed the expression of proliferating cell antigen as well as cyclin B in S phase. (99)

Vitamin K2 has also been shown to work at the level of the cell cycle, acting on cyclins to inhibit the cell cycle and initiate differentiation. It is a powerful inducer of differentiation in a number of myeloid leukemia cell lines in various stages of maturation. The mechanism of differentiation by K2 differs from retinoic acid. Vitamin K2 has not been found to bind retinoic acid receptors (RAR) alpha, beta, or gamma, or retinoid X receptor (RXR) alpha receptors. (68) This work with vitamin K2 implies there is an undiscovered nuclear receptor or mechanism for differentiation.

Researchers have proposed that the p21 gene may act with vitamin K2 as an additional factor in cellular differentiation. Previously it was thought that tumor suppressor genes such as p53 and BRCA1 induce the expression of the p21 gene. It was demonstrated that vitamin K2 can also stimulate p21 in a p53-independent manner. (100) (K2 was also shown to be unable to induce p53 in MG-63 human osteosarcoma cells, while inducing p21 gene.) MG-63 cells, shown to lack the p53 gene, were inhibited by vitamin K2 at high concentrations between [10.sup.-7] and [10.sup.-5] M/L. The elevated levels of p21 resulted in the differentiation of osteosarcoma cells.

The action of vitamin K2 in cell cycle arrest acts at the G1/S transition. When K2 transcriptionally activates the p21 protein, it complexes and inhibits the phosphorylation of G1 cyclin-dependent kinases in the cell cycle. This results in the arrest of cells in the G0/G1 phase of the cell cycle.

Conclusion

Vitamin K, in all its various forms, has been shown to have anticancer effects. Vitamin K cancer research has focused on two basic mechanisms to explain these effects. The older mechanism relies on an oxidative effect produced by the one-electron cycling of vitamin K3 that surpasses the oxidative capacity of the cancer cell, leading to death. Other mechanisms have been proposed due to the anticancer effect of vitamin K forms that either do not readily cycle (K1 and K2) or that are at levels that do not initiate cycling. These clues to another mechanism have led researchers to discover an alternative mechanism of action that acts at the level of protein kinases and phosphatases. Vitamin K has been found to act on proteins such as myc and fos, which in turn leads to growth arrest and death. Cell cycle arrest has also been found to be initiated by phosphatases at the level of cyclins, which are critical in the cell cycle.

Acknowledgements

The authors wish to thank Richard and Jileen Russell and the Smiling Dog Foundation for a grant supporting this project; Bastyr University for grant administration; and the Complementary Cancer Research Center for partial support.

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