Archive for the ‘Dog Osteosarcoma’ Category

One lucky dog: Cancer treatment saves pooch’s leg

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Posted 21 Nov 2011 — by James Street
Category Dog Osteosarcoma, radiation

By VIMAL PATEL
vimal.patel@theeagle.com

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S.Villanueva
Veterinary technician Autumn Brown prepares Rowdy, a dog suffering from a cancerous tumor in his leg, for surgery Friday at the Texas Institute for Preclinical Studies on the Texas A&M campus.

 

An 8-year-old Great Pyrenees named Rowdy was the recipient Friday of a cutting-edge surgery at Texas A&M to treat bone cancer.

Veterinarians used a specially designed microdrill to poke into the 81-pound dog’s front right leg to inject radioactive cancer-fighting isotopes directly into the tumor.

The osteosarcoma tumor generally has a 90 percent fatality rate, and the current standard of care is to amputate the leg and follow with chemotherapy.

But removing a leg from a pooch Rowdy’s size can be limiting and painful, and long-term success isn’t guaranteed, vets said at the Texas Institute for Preclinical Studies on Raymond Stotzer Parkway.

So owners were reluctant to amputate, instead searching for relief and control, if not a cure, of the tumor.

“We’re just trying to give these dogs a better quality of life for as long as we can,” said Theresa Fossum, director of TIPS.

The procedure is known as liquid brachytherapy. The surgeon used a tiny drill with a diameter of .017 of an inch that was designed by Houston-based Valco Instruments

Rowdy, who was unconscious during the surgery with his tongue hanging from his mouth, was lucky, Fossum said, because his owners, the Cordts family from San Antonio, noticed a lump on his leg and quickly sought treatment.

Osteosarcoma is the same malignant bone tumor seen in human children, said Mark Lenox, director of imaging for TIPS. It is the most common cancerous bone tumor in youth, and it usually appears during adolescence, tending to occur in the shin, thigh and upper arm.

“So we’re treating dogs and that’s good — they’re getting leading-edge treatment,” Lenox said. “But what we’re really targeting is how to cure this in children.”

Mitaplatin, a potent fusion of cisplatin and the orphan drug dichloroacetate

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Posted 11 Aug 2011 — by James Street
Category Cisplatin, DCA (Dichloroacetate), Dog Osteosarcoma, Glucose, Human osteosarcoma research, Molecular, Molecular Osteosarcoma Studies, Osteosarcoma, Warburg Hypothesis
  1. Shanta Dhara and
  2. Stephen J. Lipparda,b,1

+ Author Affiliations


  1. aDepartment of Chemistry and

  2. bKoch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139

  1. Contributed by Stephen J. Lippard, October 29, 2009 (received for review August 30, 2009)

Abstract

The unique glycolytic metabolism of most solid tumors, known as the Warburg effect, is associated with resistance to apoptosis that enables cancer cells to survive. Dichloroacetate (DCA) is an anticancer agent that can reverse the Warburg effect by inhibiting a key enzyme in cancer cells, pyruvate dehydrogenase kinase (PDK), that is required for the process. DCA is currently not approved for cancer treatment in the USA. Here, we present the synthesis, characterization, and anticancer properties of c,t,c-[Pt(NH3)2(O2CHCl2)2Cl2], mitaplatin, in which two DCA units are appended to the axial positions of a six-coordinate Pt(IV) center. The negative intracellular redox potential reduces the platinum to release cisplatin, a Pt(II) compound, and two equivalents of DCA. By a unique mechanism, mitaplatin thereby attacks both nuclear DNA with cisplatin and mitochondria with DCA selectively in cancer cells. The cytotoxicity of mitaplatin in a variety of cancer cell lines equals or exceeds that of all known Pt(IV) compounds and is comparable to that of cisplatin. Mitaplatin alters the mitochondrial membrane potential gradient (Δψm) of cancer cells, promoting apoptosis by releasing cytochrome c and translocating apoptosis inducing factor from mitochondria to the nucleus. Cisplatin formed upon cellular reduction of mitaplatin enters the nucleus and targets DNA to form 1,2-intrastrand d(GpG) cross-links characteristic of its own potency as an anticancer drug. These properties of mitaplatin are manifest in its ability to selectively kill cancer cells cocultured with normal fibroblasts and to partially overcome cisplatin resistance.

Normal cells typically use mitochondrial oxidative phosphorylation to metabolize glucose and switch over to glycolysis only when there is little or no oxygen, producing lactate as a byproduct. Cancer cells avidly consume glucose for energy by glycolysis to survive in the hypoxic environment of malignant lesions (1), a phenomenon known as the Warburg effect (2). The dependence of cancer cells on glycolysis comes not only from oxygen deprivation, but also partly from their inability to synthesize ATP in response to the mitochondrial membrane potential gradient (Δψm) (3). This unique glucose metabolic pathway of cancer cells has identified the mitochondrion as a prime target for cancer therapy (4–7). In addition, cancer cells develop the ability to avoid apoptosis by various pathways that ignore the command to commit cellular suicide (8, 9). Compounds that trigger apoptosis through selective action on mitochondrial target sites of cancer cells bypass defective upstream mechanisms and trigger apoptosis in tumor cells that are otherwise resistant (10).

Dichloroacetate (DCA) is used in humans to treat lactic acidosis (11). DCA inhibits the activity of pyruvate dehydrogenase kinase (PDK), thereby stimulating the mitochondrial enzyme pyruvate dehydrogenase (PDH). When turned off, PDH no longer converts pyruvate to acetyl-CoA required for mitochondrial respiration and glucose-dependent oxidative phosphorylation (12). DCA thus shifts cellular metabolism from glycolysis to glucose oxidation, decreasing Δψm (13) and helping to open mitochondrial transition pores (MTPs). This metabolic switch facilitates translocation of proapoptotic mediators like cytochrome c (cyt c) and apoptosis inducing factor (AIF), both of which stimulate apoptosis. DCA thereby drives cancer cells to commit suicide by apoptosis (13). Unlike most other anticancer agents, DCA does not appear to have any deleterious effect on normal cells. DCA reverses mitochondrial changes in a wide range of cancers, making malignant cells more vulnerable to normal cell death programs (14). Being an orphan drug, DCA is both nonpatentable and readily available, but it is not yet approved for use in cancer therapy (15). There is substantial preclinical evidence from both in vitro and in vivo models that DCA might be useful to treat cancer in humans, and a translation to early- phase clinical trials would be of interest (16–18). Funding for such trials would be a challenge because DCA is a generic drug. However, because it withdraws cancer cells from a state of apoptosis resistance, DCA is an attractive sensitizer that could be given concurrently with chemotherapy or radiation therapy. Alternatively, a formulation could be synthesized that incorporates DCA.

Platinum(II) compounds are used in 50% of all cancer therapies (19). Among these, cisplatin, carboplatin, and oxaliplatin have Food and Drug Administration approval and are in the clinic worldwide (20, 21). The use of platinum(II) drugs, cisplatin in particular, to treat malignancies is limited because of side effects and acquired resistance (22). Resistance can emerge from failure to execute apoptosis despite initiation of the apoptotic cascade caused by either the predominance of anti-apoptotic factors or defects in downstream effectors. Cisplatin resistance in ovarian carcinoma cells is associated with a reduced apoptotic response (23). To overcome tumor cell resistance and toxicity to normal tissues, we have been exploring strategies to target platinum constructs to cancer cells. Our tactic has been to employ substitutionally inert platinum(IV) compounds (24), which serve as prodrugs and release clinically effective levels of platinum(II) compounds, such as cisplatin, following cellular uptake (25–27). Appropriately designed platinum(IV) complexes are less likely to be deactivated before reaching their cancer cell destination target. The activity of platinum(IV) complexes generally involves reduction with loss of the axial ligands, affording an active platinum(II) complex that readily binds to DNA. Satraplatin is one such Trojan horse platinum(IV) compound that is currently under investigation for the treatment of patients with advanced prostate cancer (28).

We therefore designed a Pt(IV) compound (mitaplatin, 1) having two DCA moieties (Fig. 1) in the axial positions. We hypothesized that DCA released inside the cells by reduction of the platinum would simultaneously alter mitochondrial metabolism and deliver a dose of cisplatin (Fig. 1). Mitaplatin was thereby expected to have dual killing modes toward cancer cells, one in which cisplatin interacts with its key target, nuclear DNA, and the other, DCA released upon reduction, following a pathway to induce mitochondria-dependent apoptosis by mitochondrial membrane depolarization and efflux of proapoptotic mediators. Here, we describe the synthesis, characterization, and dual-action cell killing ability of mitaplatin as well as its remarkable ability to selectively destroy cancer cells in a coculture with normal fibroblasts.

Fig. 1.

Chemical structures and mechanism of action of mitaplatin (1). After crossing the plasma membrane, mitaplatin becomes reduced to release of the active drugs cisplatin and DCA. DCA inhibits mitochondrial PDK, which leads to PDH activation and increased glucose oxidation by promoting influx of acetyl-CoA into the mitochondria. DCA decreases the mitochondrial membrane potential (Δψm). Opening of the Δψm-sensitive mitochondrial transition pores (MTPs) leads to efflux of cyt c and AIF. Cisplatin formed in the reduction process interacts with its key target, nuclear DNA.

Results and Discussion

Synthesis and Characterization of Mitaplatin (1).

Mitaplatin (1), a formulation of DCA, was prepared by reaction of c,c,t-[Pt(NH3)2Cl2(OH)2] with dichloroacetic anhydride in >50% yield. Its formation was evidenced by disappearance of the O–H stretching band of the starting compound and the presence of a C=O stretch at 1651 cm−1 in the infrared spectra. The structure was confirmed by 1H, 13C, and 195Pt NMR spectroscopy, by HRMS, and by elemental analysis. ESI-HRMS (M–H) Calcd. = 554.8145, Found = 554.8138. 1H NMR (DMSO-d6) δ 7.95 (s, 2H), 6.52 (br, 6H); 13C NMR (DMSO-d6) δ 170.41, 65.27; 195Pt NMR (DMSO-d6): δ = 1205.28 ppm. Anal: Calcd for C4H8Cl6N2O4Pt: C, 8.64; H, 1.45; N, 5.04. Found: C, 8.13; H, 1.65; N, 4.88. Mitaplatin is redox-active and displays an irreversible reduction revealed by cyclic voltammetric analysis. The Pt(IV)/Pt(II) couple is near −0.173 V vs. Ag/AgCl at pH 7.4 and the value at pH 6.0 is −0.152 vs. Ag/AgCl. Voltammograms are given in Figs. S1 and S2. These reduction potentials indicate that mitaplatin will be readily reduced in cells. The cathodic reduction potential depends on the electron-withdrawing power and the bulkiness of the axial and auxiliary ligands. The low reduction potential of mitaplatin is influenced by the presence of chlorine atoms from the DCA molecules near the platinum center (29).

In Vitro Cellular Cytotoxicity Assays.

The ability of mitaplatin to promote cell death was evaluated by the MTT assay and the results were compared against those for cisplatin or DCA using NTera-2, HeLa, U2OS, A549, and MCF-7 cancer cells as well as MRC-5 normal fibroblasts (Fig. S3). Results are presented in Table 1. Mitaplatin has an IC50 value of 0.051 μM, comparable to that of cisplatin (IC50, 0.043 μM), in cisplatin-sensitive testicular NTera-2 cells and is more toxic than DCA alone. In U2OS osteosarcoma cells, cisplatin has an IC50 of 3.9 μM, whereas that of mitaplatin is 6.4 μM. Similarly, in HeLa cervical cancer cells, comparable IC50 values for mitaplatin and cisplatin were observed, 2.0 and 1.20 μM, respectively. Control experiments with the well known platinum(IV) compound c,c,t-[Pt(NH3)2Cl2(O2CCH3)2], revealed it to be several-fold less active than mitaplatin in all cells (Table S1). Mitaplatin was also established to have cytotoxicity comparable to that of cisplatin in the NCI/DTP 60 cell line growth inhibition assay, exceeding almost all known Pt(IV) compounds. This enhanced potency of mitaplatin is consistent with the expected dual killing mechanism.

Table 1.

Cell killing ability of mitaplatin

Mitaplatin Promotes Apoptosis in Cancer Cells.

To investigate the ability of mitaplatin to promote apoptosis in cancer cells by a mitochondrial-regulated mechanism, changes in the mitochondrial transmembrane potential (Δψm) of cancerous NTera-2 and healthy normal fibroblast cells before and after mitaplatin treatment were investigated by two assays. Mitochondrial attack is associated with a drop in Δψm. For this reason Δψm is an important parameter of mitochondrial function and has been used to monitor mitochondrial death. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) is a lipophilic cationic dye which, depending on Δψm, accumulates as a green monomer in the cytoplasm or as red-emitting aggregates in hyperpolarized mitochondria of cancer cell (30). The negative charge established by the mitochondrial membrane potential allows the lipophilic dye, bearing a delocalized positive charge, to enter mitochondria where it accumulates. When a critical concentration is exceeded, J-aggregates form, which fluoresce red. In apoptotic cells, Δψm collapses, and JC-1 cannot accumulate in mitochondria. In these cells, JC-1 remains in the cytoplasm in a green fluorescent monomeric form. Control NTera-2 cells exhibited heterogeneous staining of the cytoplasm with both red and green fluorescence in the same cells (Fig. 2A). Treatment of these cells with mitaplatin for 4 h decreased the red fluorescence. Mitochondrial membrane depolarization was detected by a shift in fluorescence emission of JC-1 from red to green. There was no significant effect of mitaplatin on Δψm of normal fibroblasts or of cisplatin on Δψm of NTera-2 cells. The detailed results are given in Fig. S4.

Fig. 2.

Disruption of mitochondrial function and induction of apoptosis in cancer cells by mitaplatin. (A) Changes in the mitochondrial membrane potential as revealed by the JC-1 assay. Treatment with 100 μM mitaplatin dramatically caused the collapse of mitochondrial membrane potentials in NTera-2 cells. In live cells, JC-1 exists either as a green fluorescent monomer at depolarized membrane potentials (positive to −100 mV) or as an orange-red fluorescent J-aggregate at hyperpolarized membrane potentials (negative to −140 mV). The shift in membrane charge was observed by disappearance of fluorescent red-orange-stained mitochondria (large negative Δψm) and an increase in fluorescent green-stained mitochondria (loss of Δψm). (B) Reversal of mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM) assay. Mitaplatin significantly depolarized the NTera-2 cells but had no effect on the healthy normal fibroblast cells. Mitochondria were stained with mitotracker red. (C) Cytochrome c release visualized by fluorescence microscopy. Immunolocalization of cytochrome c (green) and mitochondrial morphology (red) shown in untreated NTera-2 cells and in mitaplatin treated NTera-2 cells. Cells were grown for 24 h on glass coverslips and treated with mitaplatin, fixed after treatment, and immunostained with anti-cytochrome c monoclonal antibodies. Mitochondria were stained with mitotracker red. (D) Translocation of AIF in mitaplatin treated cells. Staining of AIF (Ab) and nuclei (Hoechst) in NTera-2 cells before and after 12 h treatment with mitaplatin. Arrows indicate cells with particularly evident presence of AIF in the nucleus.

To investigate whether DCA released from mitaplatin can restore the hyperpolarization of cancer cells to the level of normal cells, we carried out a TMRM assay (31). All cancer cell lines have significantly more hyperpolarized Δψm compared to normal cells and therefore exhibit increased fluorescence of the Δψm-sensitive positive dye tetramethyl rhodamine methyl ester, TMRM. Incubation of the NTera-2 cells with mitaplatin for 48 h reversed the hyperpolarization and returned the Δψm to the level of normal cells (Fig. 2B). In contrast, mitaplatin did not alter the Δψm of the normal fibroblasts. Because dichloroacetate activates pyruvate dehydrogenase, which increases delivery of pyruvate into mitochondria, DCA released upon reduction of mitaplatin (Fig. 1) increased glucose oxidation, depolarizing the mitochondria and returning the membrane potential to levels of the noncancer cells.

Mitochondrial cyt c, which functions as an electron carrier in the respiratory chain, translocates to the cytosol in cells undergoing apoptosis, where it participates in activation of apoptotic proteins (32). The mechanism responsible for this process is unknown. Cyt c release from mitochondria is an early event in the apoptotic process induced by mitaplatin treatment in NTera-2 cells, as visualized by using a FITC-conjugated antibody for the protein and fluorescence microscopy. The cytosol from untreated cells showed no detectable cyt c (Fig. 2C). In contrast, cytosolic cyt c accumulated significantly after 4 h of treatment with mitaplatin.

AIF is a proapoptotic mitochondrial protein (33). Like cyt c, AIF is a bifunctional protein having both electron transfer and apoptogenic functions. AIF is released from mitochondria and translocated to nuclei, stimulating chromatin condensation and DNA fragmentation. We were interested to determine whether mitochondrial outer membrane permeabilization by mitaplatin induces apoptosis only by release of caspase-dependent factors, such as cyt c, or whether caspase-independent processes, such as that mediated by AIF, might be operative. We therefore investigated the location of AIF in the mitaplatin-treated cells. As shown in Fig. 2D, mitaplatin treatment led to translocation of AIF from the mitochondria to nuclei of NTera-2 cells.

To quantify mitaplatin-induced apoptosis in cancer cells, an annexin-V assay was performed by using flow cytometry. With this analysis, we determined the percentage of apoptotic cells at 48 h after exposure to mitaplatin, cisplatin, or DCA. Apoptosis was detected in cancerous U2OS, HeLa, and A549 cells with 10 μM mitaplatin and cisplatin. Cisplatin at 10 μM concentration evoked apoptosis in normal MRC-5 cells whereas mitaplatin did not produce any detectable apoptosis with these normal cells (Table 2).

Table 2.

Quantification of apoptosis induced by mitaplatin, cisplatin, and DCA using an annexin V assay

Visualization of Pt-1,2-d(GpG) Adduct Formation by Mitaplatin.

Because the anticancer activity of cisplatin derives from the formation of intrastrand 1,2-d(GpG) cross-links on nuclear DNA (34), we investigated whether cisplatin released by reduction of mitaplatin leads to this signature event by using a monoclonal antibody R-C18 (35) specific for this adduct (SI Text). After 12 h incubation of NTera-2 cells with mitaplatin, formation of 1,2-d(GpG) intrastrand cross-links was observed by antibody-derived green fluorescence in the nuclei of these cells (Fig. S5). These results confirm that mitaplatin has dual cell-killing modes involving DCA, which destroys mitochondrial function, and cisplatin, which simultaneously impedes DNA-mediated processes in the nucleus.

Selective Killing of Cancer Cells by Mitaplatin.

One of the main obstacles to cancer therapy is the inability to successfully target cancer cells, while not harming normal cells. Even when therapeutic agents are delivered locally to a primary tumor, systemic toxicities still arise. Modern medicine desperately needs anti-cancer molecules that kill cancer cells and leave healthy cells alone. Most cancer therapies today are very toxic to tumor and healthy cells alike, and the patient can succumb to treatment rather than the disease. Furthermore, cells develop a resistance to external agents, so chemotherapy may only work for a short time period. Because DCA has selective toxicity toward cancer cells by targeting PDK, we investigated whether mitaplatin would also display specificity for cancer. We therefore treated a coculture of normal fibroblasts and cancerous NTera-2 cells with mitaplatin, cisplatin, or a mixture of one equivalent of cisplatin and two equivalents of DCA, the stoichiometric composition released upon intracellular mitaplatin reduction. The morphology of these cells at different time points was examined by using bright field microscopy (Fig. 3A). The two types of cells in the coculture are clearly visible because of differences in their morphology. In the coculture, cisplatin and the mixture of cisplatin and DCA killed both the fibroblasts and NTera-2 cancer cells, whereas mitaplatin selectively killed the cancer cells. The results obtained in this study provide compelling evidence that mitaplatin can selectively kill cancer cells, leaving normal cells untouched.

Fig. 3.

Mitaplatin selectively induced apoptosis of human cancer cells and not normal cells. (A) Treatment of a coculture of normal fibroblast cells (elongated) and NTera-2 cells (round) with cisplatin, mitaplatin, and a mixture of one equivalent of cisplatin and two equivalents of DCA. (B) Selective killing of cancerous A549 (round) cells by mitaplatin in a coculture with normal MRC-5 (elongated) cells assayed using LIVE/DEAD staining. After mitaplatin or cisplatin exposure for 24 h, cells were stained with calcein AM (green fluorescence) and ethidium homodimer-1 (red fluorescence) to differentiate between live and dead cells, respectively.

To verify that preferential cancer cell killing occurs with mitaplatin, this study was further extended to a coculture of human lung cancer A549 cells and normal human lung fibroblasts MRC-5.

The selective killing of normal cells by mitaplatin was demonstrated by using a LIVE/DEAD viability assay, which allowed for the simultaneous determination of live and dead cells in a coculture by labeling live cells with calcein AM dye, which fluoresces only when cleaved by intracellular esterase enzymes, and ethidium heterodimer (EthD-1), which only enters dead cells with disrupted cell membranes (Fig. 3B). Fig. 3B confirms that, unlike cisplatin, a conventional chemotherapeutic agent, mitaplatin selectively induced cell death in human cancer A549 cells, but not in normal MRC-5 cells under the similar treatment conditions. To address whether the selective killing of cancer cells by mitaplatin might be a consequence of its selective uptake, we measured the nuclear and cytosolic concentrations of platinum by atomic absorption spectroscopy (AAS) after mitaplatin or cisplatin treatment of normal and cancer cells. Cytosolic and nuclear extracts were prepared from normal MRC-5 and cancerous A549 cells after incubation with 10 μM mitaplatin or cisplatin for 24 h. Platinum concentrations determined by AAS (Table S2) confirm the uptake of mitaplatin by both cells types.

Mitaplatin Action on Cisplatin-Resistant Cells.

Although very little is known about the effects of cisplatin on the mitochondria of tumor cells (36), a recent study showed that it might have direct impact on mitochondria in head and neck cancer (37). Mitochondrial defects are associated with the cisplatin resistance phenotype (38), and several hypotheses have been suggested to explain this observation. A more negative membrane potential might promote translocation of the active, cationic form of cisplatin from the cytoplasm to mitochondria, thus diminishing platination of nuclear DNA. This effect would suggest that a combination of cisplatin with a mitochondrial targeting moiety would be an attractive therapeutic strategy for attacking cisplatin-resistant tumors. We therefore studied a pair of cisplatin sensitive A2780 and resistant A2780/CP70 ovarian cancer cells (Table 1 and Fig. S6). As controls we used cisplatin and c,c,t-[Pt(NH3)2Cl2(O2CCH3)2]. The cells displayed a low level of resistance to mitaplatin (IC50 for A2780, 1.1 μM; IC50 for A2780/CP70, 3.34 μM) compared to cisplatin (corresponding IC50 values of 0.56 and 6.0 μM). Results for the A2780/CP70 cells indicate that DCA plays a role in making cisplatin-resistant cells susceptible toward mitaplatin treatment. A2780/CP70 cells were much more resistant to the control platinum(IV) compound c,c,t-[Pt(NH3)2Cl2(O2CCH3)2]. These data suggest mitaplatin as a promising candidate for further development in the treatment of cisplatin-resistant cells.

Summary.

In conclusion, mitaplatin displays a dual-killing mode that can only be effective in cancer cells. The platinum center interacts with its own target, nuclear DNA, and DCA released upon reduction attacks mitochondria. These results support the utility of mechanisms targeting cancer cell-specific pathways as an avenue for developing selective anticancer agents. Mitaplatin offers a formulation for future studies incorporating the orphan drug DCA to further its use in the clinic.

Materials and Methods

The complexes cis-[Pt(NH3)2Cl2] (39) and c,c,t-[Pt(NH3)2Cl2(OH)2] (40) were synthesized as described. Distilled water was purified by passage through a Millipore Milli-Q Biocel water purification system (18.2 MΩ) containing a 0.22-μm filter. Anti-cytochrome c (Ab-1) sheep polyclonal antibody was procured from Calbiochem. Alexa Fluor 488-labeled secondary antibody donkey anti-(sheep IgG) was obtained from Invitrogen for cytochrome c detection. For AIF detection, we used a rabbit polyclonal IgG antibody from Santa Cruz Biotechnology, Inc. Alexa Fluor 546-labeled secondary antibody goat anti-(rabbit IgG) was purchased from Invitrogen. The detection of the cisplatin 1,2-d(GpG) intrastrand adduct was carried out using a monoclonal adduct-specific antibody R-C18 which was kindly provided by Jürgen Thomale (University of Duisburg-Essen). FITC labeled secondary antibody rabbit anti-(rat Ig) was obtained from Invitrogen. Specific adhesion slides for immunofluoresecence were purchased from Squarix Biotechnology. JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbo-cyanine iodide) was obtained from Cayman Chemicals. 1H, 13C, and 195Pt NMR spectra were recorded on a Bruker AVANCE-400 NMR spectrometer with a Spectro Spin superconducting magnet in the Massachusetts Institute of Technology Department of Chemistry Instrumentation Facility (MIT DCIF). Atomic absorption spectroscopic measurements were taken on a Perkin-Elmer AAnalyst 300 spectrometer. HRMS analysis was carried out on a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer in the MIT DCIF. Fluorescence imaging studies were performed with an Axiovert 200M inverted epifluorescence microscope (Zeiss) equipped with an EM-CCD digital camera C9100 (Hamamatsu). An X-Cite 120 metal-halide lamp (EXFO) was used as the light source. The microscope was operated with Volocity software (Improvision).

Synthesis of Mitaplatin c,c,t-[Pt(NH3)2Cl2(O2CCHCl2)2] (1).

To a solution of c,c,t-[Pt(NH3)2Cl2(OH)2] (0.2 g, 0.6 mmol) in DMF (5 mL) was added dichloroacetic anhydride (0.28 g, 1.5 mmol) and the reaction mixture was stirred at room temperature for 4 h. Diethyl ether was added to the mixture to precipitate a light yellow solid, which was washed several times with diethyl ether and dried. Mitaplatin (1) was isolated in 55% (0.29 g) yield. IR (KBr): νmax 3178, 3076, 3012, 1651, 1568, 1435, 1333, 1214, 1103, 1021, 819, 789, 723, 666, 582 cm−1; ESI-HRMS (M–H) Calcd. = 554.8145, Found = 554.8138. 1H NMR (DMSO-d6) δ 7.95 (s, 2H), 6.52 (br, 6H); 13C NMR (DMSO-d6) δ 170.41, 65.27; 195Pt NMR (DMSO-d6): δ = 1205.28 ppm. Anal: Calcd for C4H8Cl6N2O4Pt: C, 8.64; H, 1.45; N, 5.04. Found: C, 8.13; H, 1.65; N, 4.88.

Detection of Cyt c and AIF.

NTera-2 cells were seeded on microscope coverslips (1 cm) at a confluence of 1,600 cells per slip and incubated overnight at 37 °C in DMEM. The medium was changed and mitaplatin was added to a final concentration of 100 μM. The cells were incubated for 4, 12, or 24 h at 37 °C. The medium was then removed and the cells were incubated with fixing solution for 1 h at room temperature followed by three washes with PBS (pH 7.4). Cells were then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 1 h, then washed twice with PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min, followed by six washes with PBS. Cells were then rinsed with blocking buffer (PBS, 0.1% goat serum, 0.075% glycin), incubated for 1 h at 37 °C with the anti-cytochrome c [anti-cytochrome c (Ab-1) sheep pAb, Calbiochem] antibody or AIF [AIF (H-300), Santa Cruz Biotechnology Inc.] antibody, both diluted 1:50 in blocking buffer, washed twice with blocking buffer, and incubated at 37 °C with Alexa Fluor 488 donkey anti-(sheep IgG) (Invitrogen) antibody for cyt c release and Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) antibody for AIF (dilution 1:50 in blocking buffer) for 1 h. After two washes with blocking buffer and four washes with water, Mitrotacker Red for cyt c release and Hoechst bis-benzamide for AIF release were used to stain mitochondria and nuclei, respectively. Microscope coverslips were mounted on microscope slides using mounting solution for imaging.

JC-1 Assay.

GM61869 and NTera-2 cells were cultured on cover slips to a density of 1 × 106 cells/mL and incubated overnight at 37 °C. Cells were then treated with 100 μM mitaplatin for 4 and 48 h at 37 °C. A solution of JC-1 reagent (Cayman Chemicals; 10 μg/mL in DMEM) was added and incubation was carried out at 37 °C for 30 min. The cells were washed with PBS five times, fixed in 4% paraformaldehyde, and mounted onto glass slides using the procedure described above.

TMRM Assay.

Analysis of mitochondrial membrane potential (Δψm) was carried out by using TMRM. A similar procedure as mentioned above for the JC-1 assay was followed. Before fixing the cells, they were treated with 2 μM TMRM for 30 min at 37 °C.

LIVE/DEAD Assay.

In vitro selective killing was performed using the LIVE/DEAD Viability/Cytoxicity Assay (Molecular Probes). A549 and MRC-5 cells were cultured on sterile glass coverslips as subconfluent monolayers for 24 h at 37 °C in 5% CO2 and grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were then treated with 100 μM cisplatin or mitapltin for 24 h at 37 °C in 5% CO2. The cells were washed with Dulbecco’s PBS (D-PBS) to remove serum esterase activity generally present in serum-supplemented growth media before the assay. Calcein AM (4 mM in anhydrous dimethyl sulfoxide, DMSO) and EthD-1 (2 mM in DMSO/water, 1:4 vol/vol) were added to PBS (1:1,000 ratio) to produce a LIVE/DEAD working solution as recommended by the manufacturer. The samples were first washed in three changes of PBS and 100 μL LIVE/DEAD working solution was added on the coverslip and incubated at room temperature for 30 min. Subsequently, the samples were placed in PBS before being examined with a fluorescence microscope.

Annexin-V Assay.

Flow cytometry with a Vybrant Apoptosis Assay kit (annexin V conjugated to allophycocyanin, Invitrogen) was used to determine whether treatment specifically induces apoptosis. Briefly, 5 × 105 cells for each cell line were seeded into six-well tissue culture plates and incubated overnight to 60–70% confluence under standard growth conditions. Media for the cell lines were then replaced with fresh growth media with and without a 10 μM dose of cisplatin, mitaplatin, and a 20 μM dose of DCA. Treatment groups for each cell line were replicated three times. The cells were then incubated for 48 h at 37 °C and harvested with 0.25% trypsin-EDTA. Cells were washed with PBS and subsequently stained by annexin V as per the manufacturer’s protocol. Flow cytometry was performed on a BD LSR II flow cytometer (BD Biosciences) and data were analyzed on BD FACSDiva (BD Biosciences).

Acknowledgments

This work was supported by the National Cancer Institute Grant CA034992 (to S.J.L.) and the Koch Institute for Integrative Cancer Research (S.D.).

Footnotes

  • 1To whom correspondence should be addressed at:
    Department of Chemistry, Room 18–498, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307.

    E-mail: lippard@mit.edu

  • Author contributions: S.D. and S.J.L. designed research; S.D. performed research; S.D. and S.J.L. analyzed data; and S.D. and S.J.L. wrote the paper.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0912276106/DCSupplemental.

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UPenn Initiates Canine Osteosarcoma Study with Advaxis HER2

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Posted 08 Jul 2011 — by James Street
Category Dog Osteosarcoma, genetic, HER2/neu

press release

July 7, 2011, 2:45 p.m. EDT

 

PRINCETON, N.J., Jul 07, 2011 (BUSINESS WIRE) — Advaxis, Inc. ADXS +2.63% , a leader in developing the next generation of immunotherapies for cancer and infectious diseases, announces that the first dog has entered a dose-ranging in canine osteosarcoma at the University of Pennsylvania School of Veterinary Medicine.

Canine Osteosarcoma is a cancer of long (leg) bones that is a leading killer of large dogs over the age of 10 years. Standard treatment is amputation immediately after diagnosis, followed by chemotherapy. Invariably, however, the cancer metastasizes to the lungs. With chemotherapy, dogs survive about 18 months compared to 6-12 months, without treatment. The HER2 antigen is believed to be present in up to 50% of osteosarcoma. ADXS-HER2 creates an immune attack on cells expressing this antigen and has been developed to treat human breast cancer. The Company plans to file an IND later this year for this indication.

In 2010, Advaxis contracted with the University of Pennsylvania School of Veterinary Medicine to conduct a canine clinical program to determine the safety and efficacy of ADXS-HER2 in osteosarcoma. Positive results may lead to research in humans, as well.

“There is an especially high unmet need for safe, effective and reasonably priced cancer therapy in the companion animal market,” commented Thomas A Moore, Chairman and CEO of Advaxis, Inc. “This early study gets us started.”

About the Canine Osteosarcoma Trial

The study will be under the direction of Dr. Nicola Mason, an assistant professor at the University of Pennsylvania School of Veterinary Medicine. Only dogs with a histological diagnosis of osteosarcoma and evidence of expression of HER2/neu by malignant cells will be eligible for enrollment.

All dogs will receive 4 weeks of carboplatin therapy. Four weeks after the last carboplatin dose, dogs will receive ADXS-HER2 once every three weeks for a total of 3 doses. Group 1 (3 dogs) will receive 1×10(8) CFU per dose, Group 2 (3 dogs) will each receive 5×10(8) CFU per dose and Group 3 (3 dogs) will receive 1×10(9) CFU per dose. Additional dogs may be added to a Group to gather more data should if a potentially dose limiting toxicities, be observed. Therefore 9-18 dogs may be treated in the initial study.

About the University of Pennsylvania School of Veterinary Medicine

Penn’s School of Veterinary Medicine is one of the world’s premier veterinary schools. Founded in 1884, the School was built on the concept of Many Species, One Medicine(TM). The birthplace of veterinary specialties, the School serves a distinctly diverse array of animal patients, from pets to horses to farm animals at our two campuses. In Philadelphia, on Penn’s campus, are the Matthew J. Ryan Veterinary Hospital for companion animals, as well as classrooms, laboratories and the School’s administrative offices. The large-animal facility, New Bolton Center, in Kennett Square, Pa., encompasses hospital facilities for the care of horses and food animals as well as diagnostic laboratories serving the agriculture industry. The School has successfully integrated scholarship and scientific discovery with all aspects of veterinary medical education.

About Advaxis Incorporated

Advaxis is a biotechnology company developing proprietary, live but attenuated Listeria monocytogenes (Listeria) vaccines that deliver engineered tumor antigens, which stimulate multiple, simultaneous immunological mechanisms to fight cancer. Today, the Company has fifteen (15) distinct, cancer-fighting constructs in various stages of development, directly and through strategic collaborations with such recognized sites of excellence as the City of Hope, the Roswell Park Cancer Institute, the National Cancer Institute, the University of Pittsburgh, Cancer Research — UK, the University of British Columbia and the Department of Homeland Security. Please visit the Company’s portals: advaxis.com | facebook | twitter | LinkedIn

Forward-Looking Statements

Certain statements contained in this press release are forward-looking statements that involve risks and uncertainties. The statements contained herein that are not purely historical are forward looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934, as amended. Forward-looking statements deal with the Company’s current plans, intentions, beliefs and expectations and statements of future economic performance. Forward-looking statements involve known and unknown risks and uncertainties that may cause the Company’s actual results in future periods to differ materially from what is currently anticipated. Factors that could cause or contribute to such differences include those discussed from time to time in reports filed by the Company with the Securities and Exchange Commission. The Company cannot guarantee its future results, levels of activity, performance or achievements.

SOURCE: Advaxis Incorporated

        Advaxis Incorporated
        Conrad F. Mir, 609-452-9813
        Executive Director
        mir@advaxis.com
        or
        Advaxis Incorporated
        Diana Moore
        Analyst
        dmoore@advaxis.com

Veterinary oncology chief named chairman of small animal clinical sciences

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Posted 05 Jul 2011 — by James Street
Category Cat osteosarcoma, Dog Osteosarcoma, genetic, vaccine
Filed under Announcements, InsideUF (Campus), Top Stories on Tuesday, July 5, 2011.

GAINESVILLE, Fla. — Rowan Milner, the Hill’s Associate Professor of Oncology at the University of Florida College of Veterinary Medicine, has been named the new chairman of the college’s department of small animal clinical sciences following a national search.

Milner, who also serves as chief of the oncology service for the UF Veterinary Hospitals, will succeed Colin Burrows in the position following Burrows’ retirement after nearly 30 years of service. Milner’s appointment was effective July 1.

“As chair, Dr. Milner will assume overall responsibilities for faculty recruitment, mentoring and promotion,” said Glen Hoffsis, the college’s dean. “He will also be responsible for budget management, leadership in research and veterinary and graduate student education.”

Milner will work closely with the hospital’s chief of staff to continue provide high-quality clinical service to the nearly 20,000 small animal patients that are treated annually at UF.

“Dr. Milner also will work with the scientific community of the Health Science Center, practicing veterinarians from Florida and other constituents of the college and our hospital,” Hoffsis said.

Dually board-certified in veterinary internal medicine and veterinary oncology, Milner received his early academic training from the University of Pretoria in South Africa. His research interests include osteosarcoma, melanoma vaccine, stereotactic radiosurgery, targeted radiotherapy and tumor suppressor genes.

He joined UF’s faculty in 2001 and has twice received Clinician of the Year awards from UF veterinary students. In recognition of his development of a promising new melanoma vaccine and for other research, Milner was named Clinical Researcher of the Year by the Florida Kennel Club in 2007. In 2011, he won the Pfizer Award for Research Excellence and in 2009 he received a faculty enhancement opportunity award from the Office of the Provost at UF.

-30-

Credits

Contact
Sarah Carey, careysk@ufl.edu, 352-294-4242

Zoledronic Acid Inhibits Vasculogenic Mimicry in Murine Osteosarcoma Cell Line In Vitro

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Posted 30 Jun 2011 — by James Street
Category Dog Osteosarcoma, Human osteosarcoma research, in vitro, Local Recurrence, Lung Metastases, Osteosarcoma, Zoledronic Acid

To study the effects of zoledronic acid (ZA) on the vasculogenic mimicry of osteosarcoma cells in vitro.

Methods: A Three-dimensional culture of LM8 osteosarcoma cells on a type I collagen matrix was used to investigate whether osteosarcoma cells can develop vasculogenic mimicry, and to determine the effects of ZA on this process. In addition, the cellular ultrastructural changes were observed using scanning electron microscopy and laser confocal microscopy.

The effects of ZA on the translocation of RhoA protein from the cytosol to the membrane in LM8 cells were measured via immunoblotting.

Results: ZA inhibited the development of vasculogenic mimicry by the LM8 osteosarcoma cells, decreased microvilli formation on the cell surface, and disrupted the F-actin cytoskeleton. ZA prevented translocation of RhoA protein from the cytosol to the membrane in LM8 cells.

Conclusions: ZA can impair RhoA membrane localization in LM8 cells, causing obvious changes in the ultrastructure of osteosarcoma cells and induce cell apoptosis, which may be one of the underlying mechanisms by which the agent inhibits the development of vasculogenic mimicry by the LM8 cells.

Author: Dehao FuXianfeng HeShuhua YangWeihua XuTao LinXiaobo Feng
Credits/Source: BMC Musculoskeletal Disorders 2011, 12:146

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.

VEGFA gene amplification associated with poor prognosis in osteosarcoma

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Posted 19 Apr 2011 — by James Street
Category Antiagiogenesis, genetic, genetic research, genetic research, Human osteosarcoma research, Local Recurrence, Lung Metastases, Metastases, Osteosarcoma, Osteosarcoma Outcomes
Posted April 18, 2011

Yang J. Cancer. 2011;doi:10.1002/cncr.26116.

Results from an analysis of tissue sections from 58 patients with osteosarcoma show that the vascular endothelial growth factor pathway genes, including VEGFA, are amplified in osteosarcoma.

VEGFA amplification is a poor prognostic factor for tumor-free survival, as well as an important mechanism for elevated VEGFA protein expression.

Researchers collected clinicopathologic data and formalin-fixed, paraffin-embedded tissue sections from 58 patients treated for primary, conventional, central osteosarcoma at the Tianjin Medical University Cancer Institute and Hospital in Tianjin, China. To identify the altered pathways, researchers analyzed recurrent amplified and deleted genes using the Kyoto Encyclopedia of Genes and Genomes.

Researchers identified 33 key pathways with multiple component genes altered at the chromosome level. These pathways included the VEGF signaling, mammalian target of rapamycin, cellular adhesion molecule, adherens junction, Wnt and hedgehog signaling pathways. The VEGF pathway was most frequent, with 13 amplified genes, including VEGFA.

“Abundant” expression of VEGFA was noted in 74.1% of patients with osteosarcoma. Further analysis showed that VEGFA gene amplification had a strong correlation with abundant VEGFA protein expression, which researchers said suggests that VEGFA gene amplification contributes to the elevated expression of VEGFA and vascular features in osteosarcoma, and poorer survival. A survival analysis on patients with VEGFA gene amplification showed that DFS rates were lower in this group.

Researchers then stratified patients into low and high VEGFA groups. Those in the high group had both VEGFA gene amplification and positive VEGFA protein expression. Kaplan-Meier analysis showed that tumor-free survival rates were worse for patients in the high VEGFA group (P=.037).

Yang and colleagues said the pattern of overall copy number alterations from their microarray-based comparative genomic hybridization dataset was surprisingly similar to that of patients evaluated in Canada and Norway, which suggests that issues of small sample size frequently associated with most cancer types may not be so serious for osteosarcoma.

Man’s best friend: A joint tumor marker in man and dog

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Posted 18 Apr 2011 — by James Street
Category Dog Osteosarcoma, genetic, Tumor biomarkers
Published: Monday, April 18, 2011 – 09:05 in Biology & Nature

Despite steadily improving methods for its diagnosis and treatment, cancer still represents one of the most frequent causes of death in humans. What is less well known is that this also holds true for pets such as dogs. Each year, an estimated 4,000 dogs in Austria develop cancer and about half the dogs over 10 years old die because they develop a carcinoma that is biologically similar to a human tumour. CEA is one of the most important markers for tumours. It is found in high concentrations in cancer patients and is thought to have a signalling function in tumour cells, which it effects via a specific receptor molecule, the CEA receptor. Jenson-Jarolim’s work now shows that CEA itself is constructed extremely differently in dogs and humans: the antigen represents a particularly heterogeneous and complex system of different families of molecules. In contrast, however, the CEA receptor is essentially identical in the two species. The scientists explain the finding by proposing that the CEA receptor is a very old molecule in evolutionary terms and that because of its biological importance it has remained practically unchanged in the two species.

Subsequent work will address the nature of the molecules that bind to the receptor in human breast cancer or in cancer of the milk glands in dogs. The hope is that the knowledge can be exploited for new therapeutic approaches. Jensen-Jarolim is excited by the prospect. “Because dogs have shorter life-spans than humans, similar processes place on a shorter time-scale. This means that research in dogs gives faster results. By means of comparative research on the two species – so-called comparative medicine – it might be possible to develop a new generation of diagnostic and therapeutic procedures much, much faster. And these may be applicable both to humans and to animals.”

Source: University of Veterinary Medicine — Vienna

Genome Advance of the Month: Sequencing Insights Into Multiple Myeloma

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Posted 16 Apr 2011 — by James Street
Category genetic, genetic research, genetic research, Targeted Cancer Therapy, Understanding Cancer
March 2011
By Jonathan Gitlin, Ph.D.
Science Policy Analyst
Drawing of a Myeloma cell (abnormal plasma cell) making M proteins. M proteins are antibodies created by a Myeloma cell. Photo courtesy of NCI

Cancer is a genetic disease. Period.

Some inherited forms of cancer run in families, but genomic mistakes accumulated during life — such as those from exposure to chemicals in cigarette smoke, or from a stray bit of cosmic radiation, or random mistakes in DNA duplication — are the cause of most tumors.

That’s why the National Human Genome Research Institute (NHGRI) partnered with the National Cancer Institute (NCI) in 2005 to launch The Cancer Genome Atlas (TCGA), a large-scale effort to map the disease-causing genetic changes in the most widely recognized cancers, of which there are more than 200 types.

Even as the work of TCGA progresses, other research teams from around the world use genome-wide analyses to hunt down the genetic roots of cancer. For the March Genome Advance of the Month, NHGRI has selected a study that shows how the power of sequencing technology has generated an important discovery, even when studying a relatively small number of patients, in this case only 38. TCGA studies, on the other hand, include 500 patients in the analyses of each tumor type.

The study, published in the March 24, 2011 issue of the journal Nature, describes how a nationwide team of researchers organized by Todd R. Golub, M.D., director of the cancer program at The Eli and Edythe L. Broad Institute in Cambridge, Mass., made several new discoveries about the genetic causes of multiple myeloma, a currently incurable form of cancer that affects a type of immune cell called a B lymphocyte. More than 20,000 Americans develop multiple myeloma each year; about half that number die from the disease annually.

The researchers used whole-genome sequencing in 23 patients and whole-exome sequencing, where just the protein-coding regions of the human genome are sequenced, in 16 patients. One patient was studied with both techniques. The researchers compared the sequence of the DNA in each patient’s tumor to their normal DNA. By comparing tumor DNA to normal DNA, the researchers discovered around 2.9 mutations for every million bases of DNA. This resulted in nearly 7,500 point mutations across the genome, 35 of which change amino acids in proteins, as well as 21 chromosomal rearrangements that affect regions that code for proteins. The mutations were many times more likely to happen to C or G bases (rather than As or Ts), and less common in regions that code for proteins (exons) compared to introns or regions between genes. The researchers found that ten genes in particular showed significant rates of mutations, including six that had never previously been linked to cancer.

One particular mutation, in an enzyme called BRAF, may quickly lead to clinical treatment with existing medications that inhibit BRAF; however, only a small subset of multiple myeloma patients had this mutation. Also, in nearly half of the patients, the researchers discovered mutations in genes involved in processing RNA, the translation of RNA into protein, and the subsequent folding of proteins, suggesting many possible targets for future therapies.

The results suggest, the researchers wrote, that sequencing studies will produce “new insights into cancer not anticipated by existing knowledge.” Genomics has the potential to have a positive impact on many areas of medicine, but the most immediate are likely to be achieved in more accurately diagnosing an illness, in pharmacogenomics (tailoring drug treatment to a patient’s DNA) and in cancer care.

Read the study: Initial genome sequencing and analysis of multiple myeloma. Nature, March 24, 2011

In March there were also other interesting developments in the field, grouped and summarized below.

Clinical Advances:

Evolutionary biology:

Prospective identification of tumorigenic osteosarcoma cancer stem cells in OS99-1 cells based on high aldehyde dehydrogenase activity

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Posted 14 Apr 2011 — by James Street
Category Dog Osteosarcoma, Human osteosarcoma research, Stem Cell Research

Lin Wang

Paul Park

Huina Zhang

Frank La Marca

Chia-Ying Lin

Article first published online: 22 MAR 2010

Abstract

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines, of which a significantly higher ALDH activity was present in the OS99-1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99-1 cells were grown in NOD/SCID mice, we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDHbr cells) from the OS99-1 xenografts were much less frequent, averaging 3% of the entire tumor population, compared to those isolated directly from the OS99-1 cell line. ALDHbr cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDHlo cells), generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDHbr cells illustrated the stem cell characteristics of self-renewal, the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A, Nanog and Sox-2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma-initiating cells and may potentially have therapeutic implications for human osteosarcoma.

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