- Email: [email protected]
Potentiation of Mitomycin C Tumoricidal Activity for Transitional Cell Carcinoma by Histone Deacetylase Inhibitors In Vitro
Potentiation of Mitomycin C Tumoricidal Activity for Transitional Cell Carcinoma by Histone Deacetylase Inhibitors In Vitro Abdalla Ali Deb, Shandra S. Wilson, Kyle O. Rove, Binod Kumar, Sweaty Koul, Douglas D. Lim, Randall B. Meacham and Hari K. Koul*,† From the Program in Urosciences, Division of Urology, Department of Surgery, University of Colorado School of Medicine, Aurora, Colorado
Abbreviations and Acronyms BCG ⫽ bacillus Calmette-Guérin CI ⫽ combination index FACS ⫽ fluorescence activated cell sorting HDAC ⫽ histone deacetylase MMC ⫽ mitomycin C MTT ⫽ 2,5-diphenyltetrazolium bromide PI ⫽ propidium iodide VPA ⫽ valproic acid Submitted for publication March 18, 2011. Supported by the University of Colorado School of Medicine, Department of Surgery Chair commitment and Academic Enrichment funds (HKK), and Academic Enrichment seed grant fund (SSW and HKK). * Correspondence and requests for reprints: Division of Urology, Department of Surgery, Anschutz Medical Campus, RC-2, 12700 East 19th Ave., MS C-317, Aurora, Colorado 80045 (telephone: 303-724-6300; FAX: 303-724-6330; e-mail: [email protected]). † Financial interest and/or other relationship with Novartis.
Purpose: Histone deacetylase inhibitors represent promising cancer treatments since they offer improved access to target DNA/protein complexes by cytotoxic agents. We hypothesized that histone deacetylase inhibitors would be most effective when combined with DNA damaging agents such as mitomycin C. Valproic acid is a safe, affordable histone deacetylase inhibitor. We examined the effect of the combination of valproic acid and mitomycin C on human bladder cancer cells in vitro and compared this to the effect of valproic acid or mitomycin C alone on the cells. Materials and Methods: We used HTB5 and HTB9 cells derived from low and high grade bladder tumors, respectively. HTB5 and HTB9 cells were grown in modified Eagle’s and RPMI medium, respectively. Cell growth and proliferation were measured by standard methods. Apoptosis was evaluated microscopically after dual staining of cells with annexin V-fluorescein isothiocyanate/propidium iodide. The change in protein expression was analyzed by Western blot. Results: Treatment of HTB5 and HTB9 bladder cancer cells for 24 to 72 hours with valproic acid and mitomycin C resulted in concentration and time dependent decreases in viability and proliferation. HTB9 cells showed marked sensitivity to mitomycin C with a 48-hour 50% median inhibitory concentration of 1 g. Cells were less sensitive to valproic acid alone with a 48-hour 50% median inhibitory concentration of 2.5 mM. The chromatin structure relaxation induced by valproic acid pretreatment sensitized the bladder cancer cell lines, augmenting the cytotoxic action of mitomycin C. Valproic acid potentiated the induction of cell death by mitomycin C in each cell line in synergistic fashion. The effect of combining the 2 drugs was greater than the sum effect of each drug alone. Conclusions: Results indicate that the valproic acid and mitomycin C combination is effective, likely due to synergistic mechanisms. Animal model validation is needed but early results suggest promising intravesical treatments for superficial bladder cancer. Key Words: urinary bladder; carcinoma, transitional cell; mitomycin; valproic acid; epigenesis, genetic
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IN 1955 Bateman first described intravesical chemotherapy with thiotepa for superficial bladder cancer confined to the mucosa and submucosa (clinical stages Tis, Ta and T1).1 Since that time, the introduction of
other chemotherapies such as MMC, valrubicin and doxorubicin, and BCG immunotherapy have dramatically lowered rates of recurrence (chemotherapy and BCG) and progression (BCG) when used in conjunction with
0022-5347/11/1866-2426/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 2426-2433, December 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.07.107
AND
RESEARCH, INC.
MITOMYCIN C TUMORICIDAL ACTIVITY FOR TRANSITIONAL CELL CANCER
transurethral resection of bladder tumors.2,3 While much research has focused on optimal treatment strategies to minimize the recurrence rate and progression to muscle invasive or metastatic disease, not all patients necessarily respond to these programs and they may ultimately require radical cystectomy with urinary diversion.4 Such aggressive intervention may still be avoidable in some patients if newer programs of chemotherapy and immunotherapy, and combination therapies can be devised that show increased benefit. MMC, isolated from Streptomyces caespitosus, is an alkylating agent that results in damaging DNA cross-links that inhibit the DNA replication apparatus.5 MMC can be administered as a single instillation after transurethral resection of bladder tumors or as once weekly dosing for 6 weeks. A recent metaanalysis showed that MMC is inferior to BCG with respect to the recurrence rate in individuals at high risk and MMC remains a second line option for patients who are unsuitable candidates for BCG or in whom BCG fails.6 Also, while the recurrence rate decreases with MMC therapy, the long-term course, including progression and overall survival, is not affected. Since in some patients side effects develop to first line BCG therapy, including dysuria, fever, malaise and rarely sepsis, improving the results of more tolerated intravesical chemotherapies such as MMC is drastically needed. Recently efforts to identify new targets in underlying cancer biology have focused on epigenetic changes, including DNA methylation and histone acetylation, which regulate gene transcription.7,8 Histone acetyltransferases and HDAC are responsible for adding and removing acetyl groups to histones, promoting less or more interaction between histones and DNA. HDAC inhibitors represent a group of compounds that inhibit the native activity of HDAC in vivo, promoting an open chromatin configuration that is hypothesized to be more accessible to DNA targeting agents. VPA (2-propyl-pentanoic acid), a member of the HDAC inhibitor family, affects chromatin remodeling and alters gene transcription.9 –11 These drugs alone induce cyclin dependent kinase inhibitors, leading to cell cycle arrest. VPA also shows significant in vitro and in vivo antitumor activity against neuroblastoma, glioma, leukemia, breast cancer, prostate cancer and bladder cancer cells. By inhibiting HDAC activity numerous nonhistone proteins may also be modified by acetylation, highlighting the potential effect that VPA may have on other molecular processes.12,13 We investigated the potential role of VPA in enhancing MMC cytotoxicity in the 2 well characterized bladder cell lines HTB9 and HTB5, which were derived from invasive tumors. HTB9 was derived from a superficial, grade 2 transitional cell carci-
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noma and HTB5 was derived from a muscle invasive, grade 4 transitional cell carcinoma. We hypothesized that VPA, by opening up the chromatin structure, would potentiate the tumoricidal activity and DNA damage caused by MMC in these representative cell lines.
METHODS Chemicals and Reagents MMC (Fisher BioReagents, Fair Lawn, New Jersey) was dissolved in dimethyl sulfoxide solution to a concentration of 2 mg/ml. VPA and MTT were obtained from SigmaAldrich®. Primary antibodies, including pro-caspase 9, and cyclin A and B1, were obtained from Cell Signaling Technology®.
Cell Lines and Culture Conditions The HTB9 and HTB5 human bladder cancer cell lines were obtained from ATCC®. All media and additives were obtained from Invitrogen™. HTB9 was maintained in RPMI media supplemented with 10% fetal bovine serum, 1% sodium pyruvate and 1% streptomycin. HTB5 was maintained in modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% sodium pyruvate and 1% streptomycin. Cells were incubated in a humidified environment at 37C with 5% CO2.
Cell Proliferation and Viability Measurement Cell proliferation was measured using the MTT dye assay, as described previously.14 HTB9 and HTB5 were seeded at a density of 1 ⫻ 104 cells per well in 96-well plates and incubated overnight. Cells were treated with various concentrations of VPA (500 M and 1 mM) and MMC (100, 250 and 500 ng), and 4 combinations of VPA (500 M and 1 mM) and MMC (100 and 250 ng) for 24, 48 and 72, hours followed by the addition of MTT dye. As indicated, cells were pretreated overnight with VPA before MMC treatment. Absorbance was measured at 550 nm using a microplate reader. For the viability assay all cell lines were seeded in a 24-well plate at 2 ⫻ 105 cells per well and treated as described, followed by the addition of 0.4% crystal violet in 0.2 M citrate buffer for 30 minutes.15 After washing excess dye with water the incorporated crystal violet was made soluble with 2-ethoxyethanol. Absorbance was measured at 600 nm.
Cell Cycle Analysis After treatments with various concentrations of VPA and MMC for 48 hours cells were trypsinized, washed twice with phosphate buffered saline and counted. Approximately 1 ⫻ 106 cells were stained with PI and analyzed for cell cycle arrest using an FC 500 FACS device (Beckman Coulter®) at the Core Facility, Anschutz Medical Campus at our institution. The relative breakdown of cells in different phases of the cell cycle was determined by ModFit LT™ 3.0.
Determinations Cellular apoptosis. We analyzed cellular apoptosis using the Vibrant™ Apoptosis Assay Kit, as recommended by the manufacturer. Briefly, cells were grown and treated as
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mentioned. Cells were washed and incubated with YO-PRO®-1 and PI according to manufacturer instructions. They were visualized by fluorescence microscopy, in addition to FACS analysis.
ergy with a CI of approximately 0.4 to 0.8 for HTB9 cells (fig. 3). CI was not calculated for the HTB5 cell line.
Protein expression using Western blot. After treatments with VPA and/or MMC for the desired periods the cells were lysed in radio-immunoprecipitation assay buffer and resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were transferred to a polyvinylidene difluoride membrane, followed by incubation with appropriate antibodies. Antibody binding was detected with an enhanced chemiluminescence substrate.
MMC-VPA Combination Induced S/G2-M cell cycle arrest. When cells were treated with a combination of VPA (500 M or 1 mM) and MMC (100 or 250 ng) for 48 hours we found that a significant number of cells were arrested in the S and G2-M phases of the cell cycle compared to that of untreated cells, or cells monotreated with VPA or MMC. Figure 4, a and b shows the results. Of control cultures 63% and 61% of cells were in G0-G1 phase, 25% and 29% were in S phase, and 13% and 9% were in G2-M phase at 48 hours in the HTB9 and HTB5 cell lines, respectively. However, combination treatments consisting of 500 M VPA with 250 ng MMC and 1 mM VPA with 250 ng MMC resulted in an increase in the number of cells in S phase to 76% and 75% in HTB9 cultures, and 59% and 49% in HTB5 cultures at 48 hours. Similarly the relative number of cells in G2-M phase also increased to 45% and 50% in HTB9 cells, and to 90% and 80% in HTB5 cells, respectively, after 48 hours of combination therapy with 500 M VPA and 100 ng MMC or 1 mM VPA and 100 ng MMC. Western blot analysis indicated increased cyclin B1 and A expression in the combination treatment groups compared to that in the untreated groups and the groups monotreated with MMC or VPA (fig. 4, c). These data provide strong evidence for cell cycle arrest induced by MMC combined with VPA and in turn for cell growth inhibition. However, decreased cell growth may also have been attributable to apoptotic cell death, in addition to cell growth inhibition. Hence, we also investigated whether combination treatment induced apoptosis in these bladder cancer cell lines.
Statistical Analysis Unless specified otherwise results are shown as the mean ⫾ SD of data from at least 4 independent experiments done in triplicate. Statistical significance was determined by the 2-tailed equal variance Student t test with p ⱕ0.05 considered significant. To determine the synergistic, additive or antagonistic effects of the drug combinations we used CalcuSyn (Biosoft®). Synergism was defined as values greater than the expected additive effect of the drugs. The median effect plot of drug activities and the CI, which was used to signify the synergistic, additive or antagonistic effect, were determined according to Chou and Talalay.16 CI less than 1 indicates synergistic effects of the 2 drugs, CI equal to 1 indicates additive effects and CI greater than 1 indicates antagonistic effects. We used a general equation for the dose effect, fa/fu ⫽ (D/Dm)M, where D represents the drug dose, Dm represents the median effect dose, signifying potency, fa represents the fraction affected by the dose, fu represents the fraction unaffected (fu ⫽ 1 ⫺ fa) and M represents the exponent signifying the shape of the dose-effect curve.
RESULTS VPA-MMC Synergy in Bladder Cancer Cell Lines We first examined the effect of varying doses of VPA and MMC on cell proliferation and viability in bladder cancer cell lines. Figures 1 and 2 show the results. HTB9 and HTB5 cells showed decreased proliferation and viability with the combination of VPA and MMC. The combination of 1 mM VPA and 250 ng MMC showed the maximum synergistic effect (decreased cell proliferation and viability) in each cell line (figs. 1, c and f, and 2, c and f). For combination studies the dose ranges were selected to approximate the 50% median inhibitory concentration of VPA and MMC observed from proliferation and growth data. Using MTT methods in 96-well plates cell proliferation was assessed 48 hours after treatments and these values were used to evaluate CI to determine whether the activities of the 2 drugs were synergistic (CI less than 1) or not synergistic (CI 1 or greater). VPA-MMC treatment showed considerable synergism over the dose ranges tested with fair SD. Results revealed significant syn-
Induced apoptosis. Other investigations of MMC indicated that apoptosis induction is an important element of its anticancer efficacy. To further evaluate the mechanism of VPA/MMC synergy we assessed pro-caspase 9 levels and FACS using annexin V-PI fluorescent dyes. Microscopic visualization of cells stained with annexin V-PI revealed an increased apoptotic response to combination therapy compared to monotreatment with either drug (fig. 5, a). Flow cytometry analysis with annexin V-PI also demonstrated the same pattern of apoptosis in the 2 cell lines (fig. 5, c and d). Furthermore, the MMC and VPA combination increased relative apoptosis in each cell line, that is a 3.1 and 4.21fold decrease in pro-caspase 9 over that in controls in HTB9 and HTB5 cells, respectively (fig. 5, b).
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Figure. 1. VPA and MMC decreased cell proliferation. HTB9 cells were cultured and treated with varying concentrations of VPA (a) or MMC (b), or combination (c) for different intervals. HTB5 cells were seeded and treated with varying concentrations of VPA (d) or MMC (e), or combination (f) for different intervals. After defined time point decreased cell proliferation was determined by decreased MTT dye. Each experiment was done in triplicate. Bars indicate SD. H, hours. nG, ng.
DISCUSSION The current study shows that nontoxic doses of VPA significantly enhanced the tumoricidal activity of MMC in human bladder cancer cell lines. The chemosensitization effect of VPA in human bladder tumors was found in superficial and muscle invasive bladder cancer cell lines. Previous studies from our laboratory revealed the cytotoxic effect of VPA on bladder cancer cells.8 In the current study we determined whether the HDAC inhibitor VPA could have an additive or even a synergistic role in combination with MMC, a drug used clinically to treat superficial bladder cancer. HDAC inhibitors, including VPA, vorinistat, MS275, FK228, sodium phenylbutyrate and others, have shown promising, significant in vitro and in vivo activity against various tumor model sys-
tems.17,18 The low toxicity of HDAC inhibitors to normal cells compared to tumor cells is a hallmark of this class of drug.19 These HDAC inhibitors directly cause histone hyperacetylation, resulting in a consequent increase in tumor suppressor gene reexpression. This explains part of the anticancer effect of these agents. However, combining HDAC inhibitors with cytotoxic therapy is a rational progression in this area since the HDAC inhibitor offers improved access to the target DNA/protein complex by cytotoxic agents.8 Several preclinical reports show that HDAC inhibitors synergize with cytotoxic agents such as DNA topoisomerase II inhibitors, taxanes and mitomycin as well as biological agents such as imatinib and gemtuzumab.20 Even some HDAC inhibitors, such as trichostatin A, selectively sensitize melanoma cell lines to ionizing radiation.21
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Figure. 2. VPA and MMC decreased cell viability. HTB9 cells were cultured and treated with varying concentrations of VPA (a) or MMC (b), or combination (c) for different intervals. HTB5 cells were seeded and treated with varying concentrations of VPA (d) or MMC (e), or combination (f) for different intervals. After defined time point decreased cell viability was determined as decreased cell number by adding crystal violet. Each experiment was done in triplicate. Bars indicate SD. H, hours. nG, ng.
These studies reveal the potential impact that HDAC inhibitors can have across many types of cancer. The data presented enhance our understanding of the VPA mechanism of action. Previous studies
showed that VPA enhances the effects of epirubicin for solid cancer while the combination of 5-AZA-2=deoxycytidine and VPA showed an overall 22% response rate for leukemia.22 In 2006 Friedmann et al reported the synergistic effect of VPA and MMC for
Figure. 3. CI of VPA-MMC combination (⫻). Cells were cultured and treated with 500 M or 1 mM VPA (⫹A), and 100, 250 or 500 ng MMC (circles, B). At 48 hours cell viability was measured. CI was determined for dose effect of VPA and MMC alone or combined (a) and median effect of VPA-MMC combination (b). Graph indicates synergism of combination since CI was between 0.4 and 0.8.
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Figure. 4. VPA-MMC combination increased S/G2/M cell cycle arrest. HTB9 (a) and HTB5 (b) cells were cultured and treated with 500 M or 1 mM VPA, 100 or 250 ng (nG) MMC alone, or VPA-MMC combination. At 48 hours cell cycle was analyzed. Bars indicate SD. At 48 hours of treatment proteins were isolated and Western blot was done against cyclin A and B1 (c). Each experiment was done in triplicate. Lane 1, untreated control. Lane 2, 1 mM VPA. Lane 3, 100 ng MMC. Lane 4, 250 ng MMC. Lane 5, 1 mM VPA plus 100 ng MMC. Lane 6, 1 mM VPA plus 250 ng MMC.
colon cancer.23 Importantly most of these compounds are not effective against cancers when used alone. For example, epirubicin is not effective for melanoma or colorectal cancer. This supports the case for developing multidrug therapies, including combination with HDAC inhibitors, such as VPA with MMC, so that mechanistically 1 drug cooperatively enhances the tumoricidal activity of the other,24 as we currently report. We are aware of only 2 studies of HDAC inhibitors in conjunction with MMC. One study was done with sodium butyrate combined with MMC for melanoma and the other showed a synergistic decrease in the viability of colorectal adenocarcinoma cell lines treated with MMC and VPA.21,23 However, the exact mechanism of the synergism between these 2 drugs remains unknown. Management for nonmuscle invasive bladder cancer still largely depends on epirubicin and MMC therapy while BCG is the first line choice for intermediate to high risk bladder tumors, including carcinoma in situ. Drug toxicity, adverse reactions and drug intolerance remain major impediments to continued treatment with BCG or MMC. Thus, including an HDAC inhibitor such as VPA in an MMC
treatment regimen could be an alternative for treating transitional cell carcinoma of the bladder with better efficacy and decreased side effects. In this study we found that combining VPA with MMC caused a synergistic decrease in the viability of superficial and muscle invasive bladder cancer cell lines. Also, these 2 drugs together induced apoptosis in these in vitro models of bladder cancer. Furthermore, since VPA has a known, favorable side effect profile and a long history of use in humans, it represents a good candidate for combination treatment, particularly for bladder cancer, since when agents are administered intravesically, systemic absorption should be decreased. This emphasizes the need for further in vivo investigation of VPA to develop safe dosing regimens in combination with MMC for further consideration in a clinical trial in patients with treatment naïve, recurrent or resistant superficial bladder cancer phenotypes.
CONCLUSIONS VPA synergized the cytotoxicity of MMC in 2 bladder cancer cell lines at doses that are clinically
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Figure. 5. VPA enhanced apoptosis when added to MMC. Cells were stained with annexin V-PI and visualized under fluorescence microscopy (a). nG, ng. Reduced from ⫻40 ⫻ ⫻10 ⫽ ⫻400. At 48 hours proteins were isolated and active caspase-9 was measured (b). Relative fold increase in apoptosis of HTB9 and HTB5 cells treated with VPA alone, MMC alone or VPA-MMC combination for 48 hours (c and d). YO-PRO-1 and PI florescent dye was added and cells were washed with phosphate buffered saline. FACS analysis was done to determine apoptotic cell death rate. Each experiment was done in triplicate. Bars indicate SD.
achievable in humans. This effect was noted with S/G2-M cell cycle arrest and increased apoptosis. Also, a pattern of benefit from combination treatment with MMC and VPA was seen in the HTB5 muscle invasive bladder cancer cell line, indicating improved efficacy of anticancer activity over that of MMC alone. Since the 2 drugs are used clinically in different settings, additional study of this multidrug
regimen is needed to confirm its positive therapeutic benefit for bladder cancer.
ACKNOWLEDGMENTS Dr. Cheol Yong Yoon, Seoul National University, South Korea, assisted with the combination index plot.
REFERENCES 1. Bateman JC: Chemotherapy of solid tumors with trimethylene thiophosphoramide. New Engl J Med 1955; 252: 879. 2. Morales A, Eidinger D and Bruce AW: Intracavitary bacillus Calmette-Guerin in the treat-
ment of superficial bladder tumors. J Urol 2002; 167: 891.
of 3703 patients from 11 randomized trials. J Clin Epidemiol 2000; 53: 676.
3. Huncharek M, Geschwind JF, Witherspoon B et al: Intravesical chemotherapy prophylaxis in primary superficial bladder cancer: a meta-analysis
4. Lamm DL, Blumenstein BA, Crawford ED et al: A randomized trial of intravesical doxorubicin and immunotherapy with bacille Calmette-Guerin for
MITOMYCIN C TUMORICIDAL ACTIVITY FOR TRANSITIONAL CELL CANCER
transitional-cell carcinoma of the bladder. N Engl J Med 1991; 325: 1205. 5. Iyer VN and Szybalski W: A molecular mechanism of mitomycin action: linking of complementary DNA strands. Proc Natl Acad Sci U S A 1963; 50: 355. 6. Shelley MD, Mason MD and Kynaston H: Intravesical therapy for superficial bladder cancer: a systematic review of randomised trials and metaanalyses. Cancer Treat Rev 2010; 36: 195. 7. Frew AJ, Johnstone RW and Bolden JE: Enhancing the apoptotic and therapeutic effects of HDAC inhibitors. Cancer Lett 2009; 280: 125. 8. Byun S, Kim FJ, Khandrika L et al: Differential effects of valproic acid on growth, proliferation and metastasis in HTB5 and HTB9 bladder cancer cell lines. Cancer Lett 2009; 281: 196. 9. Johanssen CU: Mechanisms of action of valproate: a commentary. Neurochem Int 2000; 37: 103. 10. Cinatl J Jr, Kotchetkov R, Blaheta R et al: Induction of differentiation and suppression of malignant phenotype of human neuroblastoma BE(2)-C cells by valproic acid: enhancement by combination with interferon-a. Int J Oncol 2002; 20: 97. 11. Yuan PX, Huang LD, Jiang YM et al: The mood stabilizer valproic acid activates mitogen-activated protein kinases and promotes neurite growth. J Biol Chem 2001; 276: 31674.
12. Bacon CL, Gallagher HC, Haughey JC et al: Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase. J Neurochem 2002; 83: 12. 13. Kamitani H, Taniura S, Watanabe K et al: Histone acetylation may suppress human glioma cell proliferation when p21 WAF/Cip1 and gelsolin are induced. Neuro-oncol 2002; 4: 95. 14. Kumar B, Joshi J, Kumar A et al: Radiosensitization by diospyrin diethylether in MCF-7 breast carcinoma cell line. Mol Cell Biochem 2007; 304: 287. 15. Barton KN, Paielli D, Zhang Y et al: Second generation replication-competent oncolytic adenovirus armed with improved suicide genes and ADP gene demonstrates greater efficacy without increased toxicity. Mol Ther 2006; 13: 347. 16. Chou TC and Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 27. 17. Sambucetti LC, Fischer DD, Zabludoff S et al: Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects. J Biol Chem 1999; 274: 34940. 18. Kosugi H, Towatari M, Hatano S et al: Histone deacetylase inhibitors are the potent inducer/
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enhancer of differentiation in acute myeloid leukemia: a new approach to anti-leukemia therapy. Leukemia 1999; 13: 1316. 19. Marchion DC, Bicaku E, Daud AI et al: Sequencespecific potentiation of topoisomerase II inhibitors by the histone deacetylase inhibitor suberoylanilide hydroxamic acid. J Cell Biochem 2004; 92: 223. 20. Carraway HE and Gore SD: Addition of histone deacetylase inhibitors in combination therapy. J Clin Oncol 2007; 25: 1955. 21. Cunneen TS, Conway RM and Madigan MC: In vitro effects of histone deacetylase inhibitors and mitomycin C on tenon capsule fibroblasts and conjunctival melanoma cells. Arch Ophthalmol 2009; 127: 414. 22. Garcia-Manero G, Kantarjian HM, Sanchez-Gonzalez B et al: Phase 1/2 study of the combination of 5-aza-2=-deoxycytidine with valproic acid in patients with leukemia. Blood 2006; 108: 3271. 23. Friedmann I, Atmaca A, Chow KU et al: Synergistic effects of valproic acid and mitomycin C in adenocarcinoma cell lines and fresh tumor cells of patients with colon cancer. J Chemother 2006; 18: 415. 24. Rosenthal MA, Green M, Wiernik P et al: Epirubicin has modest single-agent activity in head and neck cancer but limited activity in metastatic melanoma and colorectal cancer: Phase II studies by the eastern cooperative oncology group. Am J Clin Oncol 1998; 21: 509.
Abbreviations and Acronyms BCG ⫽ bacillus Calmette-Guérin CI ⫽ combination index FACS ⫽ fluorescence activated cell sorting HDAC ⫽ histone deacetylase MMC ⫽ mitomycin C MTT ⫽ 2,5-diphenyltetrazolium bromide PI ⫽ propidium iodide VPA ⫽ valproic acid Submitted for publication March 18, 2011. Supported by the University of Colorado School of Medicine, Department of Surgery Chair commitment and Academic Enrichment funds (HKK), and Academic Enrichment seed grant fund (SSW and HKK). * Correspondence and requests for reprints: Division of Urology, Department of Surgery, Anschutz Medical Campus, RC-2, 12700 East 19th Ave., MS C-317, Aurora, Colorado 80045 (telephone: 303-724-6300; FAX: 303-724-6330; e-mail: [email protected]). † Financial interest and/or other relationship with Novartis.
Purpose: Histone deacetylase inhibitors represent promising cancer treatments since they offer improved access to target DNA/protein complexes by cytotoxic agents. We hypothesized that histone deacetylase inhibitors would be most effective when combined with DNA damaging agents such as mitomycin C. Valproic acid is a safe, affordable histone deacetylase inhibitor. We examined the effect of the combination of valproic acid and mitomycin C on human bladder cancer cells in vitro and compared this to the effect of valproic acid or mitomycin C alone on the cells. Materials and Methods: We used HTB5 and HTB9 cells derived from low and high grade bladder tumors, respectively. HTB5 and HTB9 cells were grown in modified Eagle’s and RPMI medium, respectively. Cell growth and proliferation were measured by standard methods. Apoptosis was evaluated microscopically after dual staining of cells with annexin V-fluorescein isothiocyanate/propidium iodide. The change in protein expression was analyzed by Western blot. Results: Treatment of HTB5 and HTB9 bladder cancer cells for 24 to 72 hours with valproic acid and mitomycin C resulted in concentration and time dependent decreases in viability and proliferation. HTB9 cells showed marked sensitivity to mitomycin C with a 48-hour 50% median inhibitory concentration of 1 g. Cells were less sensitive to valproic acid alone with a 48-hour 50% median inhibitory concentration of 2.5 mM. The chromatin structure relaxation induced by valproic acid pretreatment sensitized the bladder cancer cell lines, augmenting the cytotoxic action of mitomycin C. Valproic acid potentiated the induction of cell death by mitomycin C in each cell line in synergistic fashion. The effect of combining the 2 drugs was greater than the sum effect of each drug alone. Conclusions: Results indicate that the valproic acid and mitomycin C combination is effective, likely due to synergistic mechanisms. Animal model validation is needed but early results suggest promising intravesical treatments for superficial bladder cancer. Key Words: urinary bladder; carcinoma, transitional cell; mitomycin; valproic acid; epigenesis, genetic
2426
www.jurology.com
IN 1955 Bateman first described intravesical chemotherapy with thiotepa for superficial bladder cancer confined to the mucosa and submucosa (clinical stages Tis, Ta and T1).1 Since that time, the introduction of
other chemotherapies such as MMC, valrubicin and doxorubicin, and BCG immunotherapy have dramatically lowered rates of recurrence (chemotherapy and BCG) and progression (BCG) when used in conjunction with
0022-5347/11/1866-2426/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 2426-2433, December 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.07.107
AND
RESEARCH, INC.
MITOMYCIN C TUMORICIDAL ACTIVITY FOR TRANSITIONAL CELL CANCER
transurethral resection of bladder tumors.2,3 While much research has focused on optimal treatment strategies to minimize the recurrence rate and progression to muscle invasive or metastatic disease, not all patients necessarily respond to these programs and they may ultimately require radical cystectomy with urinary diversion.4 Such aggressive intervention may still be avoidable in some patients if newer programs of chemotherapy and immunotherapy, and combination therapies can be devised that show increased benefit. MMC, isolated from Streptomyces caespitosus, is an alkylating agent that results in damaging DNA cross-links that inhibit the DNA replication apparatus.5 MMC can be administered as a single instillation after transurethral resection of bladder tumors or as once weekly dosing for 6 weeks. A recent metaanalysis showed that MMC is inferior to BCG with respect to the recurrence rate in individuals at high risk and MMC remains a second line option for patients who are unsuitable candidates for BCG or in whom BCG fails.6 Also, while the recurrence rate decreases with MMC therapy, the long-term course, including progression and overall survival, is not affected. Since in some patients side effects develop to first line BCG therapy, including dysuria, fever, malaise and rarely sepsis, improving the results of more tolerated intravesical chemotherapies such as MMC is drastically needed. Recently efforts to identify new targets in underlying cancer biology have focused on epigenetic changes, including DNA methylation and histone acetylation, which regulate gene transcription.7,8 Histone acetyltransferases and HDAC are responsible for adding and removing acetyl groups to histones, promoting less or more interaction between histones and DNA. HDAC inhibitors represent a group of compounds that inhibit the native activity of HDAC in vivo, promoting an open chromatin configuration that is hypothesized to be more accessible to DNA targeting agents. VPA (2-propyl-pentanoic acid), a member of the HDAC inhibitor family, affects chromatin remodeling and alters gene transcription.9 –11 These drugs alone induce cyclin dependent kinase inhibitors, leading to cell cycle arrest. VPA also shows significant in vitro and in vivo antitumor activity against neuroblastoma, glioma, leukemia, breast cancer, prostate cancer and bladder cancer cells. By inhibiting HDAC activity numerous nonhistone proteins may also be modified by acetylation, highlighting the potential effect that VPA may have on other molecular processes.12,13 We investigated the potential role of VPA in enhancing MMC cytotoxicity in the 2 well characterized bladder cell lines HTB9 and HTB5, which were derived from invasive tumors. HTB9 was derived from a superficial, grade 2 transitional cell carci-
2427
noma and HTB5 was derived from a muscle invasive, grade 4 transitional cell carcinoma. We hypothesized that VPA, by opening up the chromatin structure, would potentiate the tumoricidal activity and DNA damage caused by MMC in these representative cell lines.
METHODS Chemicals and Reagents MMC (Fisher BioReagents, Fair Lawn, New Jersey) was dissolved in dimethyl sulfoxide solution to a concentration of 2 mg/ml. VPA and MTT were obtained from SigmaAldrich®. Primary antibodies, including pro-caspase 9, and cyclin A and B1, were obtained from Cell Signaling Technology®.
Cell Lines and Culture Conditions The HTB9 and HTB5 human bladder cancer cell lines were obtained from ATCC®. All media and additives were obtained from Invitrogen™. HTB9 was maintained in RPMI media supplemented with 10% fetal bovine serum, 1% sodium pyruvate and 1% streptomycin. HTB5 was maintained in modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% sodium pyruvate and 1% streptomycin. Cells were incubated in a humidified environment at 37C with 5% CO2.
Cell Proliferation and Viability Measurement Cell proliferation was measured using the MTT dye assay, as described previously.14 HTB9 and HTB5 were seeded at a density of 1 ⫻ 104 cells per well in 96-well plates and incubated overnight. Cells were treated with various concentrations of VPA (500 M and 1 mM) and MMC (100, 250 and 500 ng), and 4 combinations of VPA (500 M and 1 mM) and MMC (100 and 250 ng) for 24, 48 and 72, hours followed by the addition of MTT dye. As indicated, cells were pretreated overnight with VPA before MMC treatment. Absorbance was measured at 550 nm using a microplate reader. For the viability assay all cell lines were seeded in a 24-well plate at 2 ⫻ 105 cells per well and treated as described, followed by the addition of 0.4% crystal violet in 0.2 M citrate buffer for 30 minutes.15 After washing excess dye with water the incorporated crystal violet was made soluble with 2-ethoxyethanol. Absorbance was measured at 600 nm.
Cell Cycle Analysis After treatments with various concentrations of VPA and MMC for 48 hours cells were trypsinized, washed twice with phosphate buffered saline and counted. Approximately 1 ⫻ 106 cells were stained with PI and analyzed for cell cycle arrest using an FC 500 FACS device (Beckman Coulter®) at the Core Facility, Anschutz Medical Campus at our institution. The relative breakdown of cells in different phases of the cell cycle was determined by ModFit LT™ 3.0.
Determinations Cellular apoptosis. We analyzed cellular apoptosis using the Vibrant™ Apoptosis Assay Kit, as recommended by the manufacturer. Briefly, cells were grown and treated as
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mentioned. Cells were washed and incubated with YO-PRO®-1 and PI according to manufacturer instructions. They were visualized by fluorescence microscopy, in addition to FACS analysis.
ergy with a CI of approximately 0.4 to 0.8 for HTB9 cells (fig. 3). CI was not calculated for the HTB5 cell line.
Protein expression using Western blot. After treatments with VPA and/or MMC for the desired periods the cells were lysed in radio-immunoprecipitation assay buffer and resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands were transferred to a polyvinylidene difluoride membrane, followed by incubation with appropriate antibodies. Antibody binding was detected with an enhanced chemiluminescence substrate.
MMC-VPA Combination Induced S/G2-M cell cycle arrest. When cells were treated with a combination of VPA (500 M or 1 mM) and MMC (100 or 250 ng) for 48 hours we found that a significant number of cells were arrested in the S and G2-M phases of the cell cycle compared to that of untreated cells, or cells monotreated with VPA or MMC. Figure 4, a and b shows the results. Of control cultures 63% and 61% of cells were in G0-G1 phase, 25% and 29% were in S phase, and 13% and 9% were in G2-M phase at 48 hours in the HTB9 and HTB5 cell lines, respectively. However, combination treatments consisting of 500 M VPA with 250 ng MMC and 1 mM VPA with 250 ng MMC resulted in an increase in the number of cells in S phase to 76% and 75% in HTB9 cultures, and 59% and 49% in HTB5 cultures at 48 hours. Similarly the relative number of cells in G2-M phase also increased to 45% and 50% in HTB9 cells, and to 90% and 80% in HTB5 cells, respectively, after 48 hours of combination therapy with 500 M VPA and 100 ng MMC or 1 mM VPA and 100 ng MMC. Western blot analysis indicated increased cyclin B1 and A expression in the combination treatment groups compared to that in the untreated groups and the groups monotreated with MMC or VPA (fig. 4, c). These data provide strong evidence for cell cycle arrest induced by MMC combined with VPA and in turn for cell growth inhibition. However, decreased cell growth may also have been attributable to apoptotic cell death, in addition to cell growth inhibition. Hence, we also investigated whether combination treatment induced apoptosis in these bladder cancer cell lines.
Statistical Analysis Unless specified otherwise results are shown as the mean ⫾ SD of data from at least 4 independent experiments done in triplicate. Statistical significance was determined by the 2-tailed equal variance Student t test with p ⱕ0.05 considered significant. To determine the synergistic, additive or antagonistic effects of the drug combinations we used CalcuSyn (Biosoft®). Synergism was defined as values greater than the expected additive effect of the drugs. The median effect plot of drug activities and the CI, which was used to signify the synergistic, additive or antagonistic effect, were determined according to Chou and Talalay.16 CI less than 1 indicates synergistic effects of the 2 drugs, CI equal to 1 indicates additive effects and CI greater than 1 indicates antagonistic effects. We used a general equation for the dose effect, fa/fu ⫽ (D/Dm)M, where D represents the drug dose, Dm represents the median effect dose, signifying potency, fa represents the fraction affected by the dose, fu represents the fraction unaffected (fu ⫽ 1 ⫺ fa) and M represents the exponent signifying the shape of the dose-effect curve.
RESULTS VPA-MMC Synergy in Bladder Cancer Cell Lines We first examined the effect of varying doses of VPA and MMC on cell proliferation and viability in bladder cancer cell lines. Figures 1 and 2 show the results. HTB9 and HTB5 cells showed decreased proliferation and viability with the combination of VPA and MMC. The combination of 1 mM VPA and 250 ng MMC showed the maximum synergistic effect (decreased cell proliferation and viability) in each cell line (figs. 1, c and f, and 2, c and f). For combination studies the dose ranges were selected to approximate the 50% median inhibitory concentration of VPA and MMC observed from proliferation and growth data. Using MTT methods in 96-well plates cell proliferation was assessed 48 hours after treatments and these values were used to evaluate CI to determine whether the activities of the 2 drugs were synergistic (CI less than 1) or not synergistic (CI 1 or greater). VPA-MMC treatment showed considerable synergism over the dose ranges tested with fair SD. Results revealed significant syn-
Induced apoptosis. Other investigations of MMC indicated that apoptosis induction is an important element of its anticancer efficacy. To further evaluate the mechanism of VPA/MMC synergy we assessed pro-caspase 9 levels and FACS using annexin V-PI fluorescent dyes. Microscopic visualization of cells stained with annexin V-PI revealed an increased apoptotic response to combination therapy compared to monotreatment with either drug (fig. 5, a). Flow cytometry analysis with annexin V-PI also demonstrated the same pattern of apoptosis in the 2 cell lines (fig. 5, c and d). Furthermore, the MMC and VPA combination increased relative apoptosis in each cell line, that is a 3.1 and 4.21fold decrease in pro-caspase 9 over that in controls in HTB9 and HTB5 cells, respectively (fig. 5, b).
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Figure. 1. VPA and MMC decreased cell proliferation. HTB9 cells were cultured and treated with varying concentrations of VPA (a) or MMC (b), or combination (c) for different intervals. HTB5 cells were seeded and treated with varying concentrations of VPA (d) or MMC (e), or combination (f) for different intervals. After defined time point decreased cell proliferation was determined by decreased MTT dye. Each experiment was done in triplicate. Bars indicate SD. H, hours. nG, ng.
DISCUSSION The current study shows that nontoxic doses of VPA significantly enhanced the tumoricidal activity of MMC in human bladder cancer cell lines. The chemosensitization effect of VPA in human bladder tumors was found in superficial and muscle invasive bladder cancer cell lines. Previous studies from our laboratory revealed the cytotoxic effect of VPA on bladder cancer cells.8 In the current study we determined whether the HDAC inhibitor VPA could have an additive or even a synergistic role in combination with MMC, a drug used clinically to treat superficial bladder cancer. HDAC inhibitors, including VPA, vorinistat, MS275, FK228, sodium phenylbutyrate and others, have shown promising, significant in vitro and in vivo activity against various tumor model sys-
tems.17,18 The low toxicity of HDAC inhibitors to normal cells compared to tumor cells is a hallmark of this class of drug.19 These HDAC inhibitors directly cause histone hyperacetylation, resulting in a consequent increase in tumor suppressor gene reexpression. This explains part of the anticancer effect of these agents. However, combining HDAC inhibitors with cytotoxic therapy is a rational progression in this area since the HDAC inhibitor offers improved access to the target DNA/protein complex by cytotoxic agents.8 Several preclinical reports show that HDAC inhibitors synergize with cytotoxic agents such as DNA topoisomerase II inhibitors, taxanes and mitomycin as well as biological agents such as imatinib and gemtuzumab.20 Even some HDAC inhibitors, such as trichostatin A, selectively sensitize melanoma cell lines to ionizing radiation.21
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Figure. 2. VPA and MMC decreased cell viability. HTB9 cells were cultured and treated with varying concentrations of VPA (a) or MMC (b), or combination (c) for different intervals. HTB5 cells were seeded and treated with varying concentrations of VPA (d) or MMC (e), or combination (f) for different intervals. After defined time point decreased cell viability was determined as decreased cell number by adding crystal violet. Each experiment was done in triplicate. Bars indicate SD. H, hours. nG, ng.
These studies reveal the potential impact that HDAC inhibitors can have across many types of cancer. The data presented enhance our understanding of the VPA mechanism of action. Previous studies
showed that VPA enhances the effects of epirubicin for solid cancer while the combination of 5-AZA-2=deoxycytidine and VPA showed an overall 22% response rate for leukemia.22 In 2006 Friedmann et al reported the synergistic effect of VPA and MMC for
Figure. 3. CI of VPA-MMC combination (⫻). Cells were cultured and treated with 500 M or 1 mM VPA (⫹A), and 100, 250 or 500 ng MMC (circles, B). At 48 hours cell viability was measured. CI was determined for dose effect of VPA and MMC alone or combined (a) and median effect of VPA-MMC combination (b). Graph indicates synergism of combination since CI was between 0.4 and 0.8.
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Figure. 4. VPA-MMC combination increased S/G2/M cell cycle arrest. HTB9 (a) and HTB5 (b) cells were cultured and treated with 500 M or 1 mM VPA, 100 or 250 ng (nG) MMC alone, or VPA-MMC combination. At 48 hours cell cycle was analyzed. Bars indicate SD. At 48 hours of treatment proteins were isolated and Western blot was done against cyclin A and B1 (c). Each experiment was done in triplicate. Lane 1, untreated control. Lane 2, 1 mM VPA. Lane 3, 100 ng MMC. Lane 4, 250 ng MMC. Lane 5, 1 mM VPA plus 100 ng MMC. Lane 6, 1 mM VPA plus 250 ng MMC.
colon cancer.23 Importantly most of these compounds are not effective against cancers when used alone. For example, epirubicin is not effective for melanoma or colorectal cancer. This supports the case for developing multidrug therapies, including combination with HDAC inhibitors, such as VPA with MMC, so that mechanistically 1 drug cooperatively enhances the tumoricidal activity of the other,24 as we currently report. We are aware of only 2 studies of HDAC inhibitors in conjunction with MMC. One study was done with sodium butyrate combined with MMC for melanoma and the other showed a synergistic decrease in the viability of colorectal adenocarcinoma cell lines treated with MMC and VPA.21,23 However, the exact mechanism of the synergism between these 2 drugs remains unknown. Management for nonmuscle invasive bladder cancer still largely depends on epirubicin and MMC therapy while BCG is the first line choice for intermediate to high risk bladder tumors, including carcinoma in situ. Drug toxicity, adverse reactions and drug intolerance remain major impediments to continued treatment with BCG or MMC. Thus, including an HDAC inhibitor such as VPA in an MMC
treatment regimen could be an alternative for treating transitional cell carcinoma of the bladder with better efficacy and decreased side effects. In this study we found that combining VPA with MMC caused a synergistic decrease in the viability of superficial and muscle invasive bladder cancer cell lines. Also, these 2 drugs together induced apoptosis in these in vitro models of bladder cancer. Furthermore, since VPA has a known, favorable side effect profile and a long history of use in humans, it represents a good candidate for combination treatment, particularly for bladder cancer, since when agents are administered intravesically, systemic absorption should be decreased. This emphasizes the need for further in vivo investigation of VPA to develop safe dosing regimens in combination with MMC for further consideration in a clinical trial in patients with treatment naïve, recurrent or resistant superficial bladder cancer phenotypes.
CONCLUSIONS VPA synergized the cytotoxicity of MMC in 2 bladder cancer cell lines at doses that are clinically
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Figure. 5. VPA enhanced apoptosis when added to MMC. Cells were stained with annexin V-PI and visualized under fluorescence microscopy (a). nG, ng. Reduced from ⫻40 ⫻ ⫻10 ⫽ ⫻400. At 48 hours proteins were isolated and active caspase-9 was measured (b). Relative fold increase in apoptosis of HTB9 and HTB5 cells treated with VPA alone, MMC alone or VPA-MMC combination for 48 hours (c and d). YO-PRO-1 and PI florescent dye was added and cells were washed with phosphate buffered saline. FACS analysis was done to determine apoptotic cell death rate. Each experiment was done in triplicate. Bars indicate SD.
achievable in humans. This effect was noted with S/G2-M cell cycle arrest and increased apoptosis. Also, a pattern of benefit from combination treatment with MMC and VPA was seen in the HTB5 muscle invasive bladder cancer cell line, indicating improved efficacy of anticancer activity over that of MMC alone. Since the 2 drugs are used clinically in different settings, additional study of this multidrug
regimen is needed to confirm its positive therapeutic benefit for bladder cancer.
ACKNOWLEDGMENTS Dr. Cheol Yong Yoon, Seoul National University, South Korea, assisted with the combination index plot.
REFERENCES 1. Bateman JC: Chemotherapy of solid tumors with trimethylene thiophosphoramide. New Engl J Med 1955; 252: 879. 2. Morales A, Eidinger D and Bruce AW: Intracavitary bacillus Calmette-Guerin in the treat-
ment of superficial bladder tumors. J Urol 2002; 167: 891.
of 3703 patients from 11 randomized trials. J Clin Epidemiol 2000; 53: 676.
3. Huncharek M, Geschwind JF, Witherspoon B et al: Intravesical chemotherapy prophylaxis in primary superficial bladder cancer: a meta-analysis
4. Lamm DL, Blumenstein BA, Crawford ED et al: A randomized trial of intravesical doxorubicin and immunotherapy with bacille Calmette-Guerin for
MITOMYCIN C TUMORICIDAL ACTIVITY FOR TRANSITIONAL CELL CANCER
transitional-cell carcinoma of the bladder. N Engl J Med 1991; 325: 1205. 5. Iyer VN and Szybalski W: A molecular mechanism of mitomycin action: linking of complementary DNA strands. Proc Natl Acad Sci U S A 1963; 50: 355. 6. Shelley MD, Mason MD and Kynaston H: Intravesical therapy for superficial bladder cancer: a systematic review of randomised trials and metaanalyses. Cancer Treat Rev 2010; 36: 195. 7. Frew AJ, Johnstone RW and Bolden JE: Enhancing the apoptotic and therapeutic effects of HDAC inhibitors. Cancer Lett 2009; 280: 125. 8. Byun S, Kim FJ, Khandrika L et al: Differential effects of valproic acid on growth, proliferation and metastasis in HTB5 and HTB9 bladder cancer cell lines. Cancer Lett 2009; 281: 196. 9. Johanssen CU: Mechanisms of action of valproate: a commentary. Neurochem Int 2000; 37: 103. 10. Cinatl J Jr, Kotchetkov R, Blaheta R et al: Induction of differentiation and suppression of malignant phenotype of human neuroblastoma BE(2)-C cells by valproic acid: enhancement by combination with interferon-a. Int J Oncol 2002; 20: 97. 11. Yuan PX, Huang LD, Jiang YM et al: The mood stabilizer valproic acid activates mitogen-activated protein kinases and promotes neurite growth. J Biol Chem 2001; 276: 31674.
12. Bacon CL, Gallagher HC, Haughey JC et al: Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase. J Neurochem 2002; 83: 12. 13. Kamitani H, Taniura S, Watanabe K et al: Histone acetylation may suppress human glioma cell proliferation when p21 WAF/Cip1 and gelsolin are induced. Neuro-oncol 2002; 4: 95. 14. Kumar B, Joshi J, Kumar A et al: Radiosensitization by diospyrin diethylether in MCF-7 breast carcinoma cell line. Mol Cell Biochem 2007; 304: 287. 15. Barton KN, Paielli D, Zhang Y et al: Second generation replication-competent oncolytic adenovirus armed with improved suicide genes and ADP gene demonstrates greater efficacy without increased toxicity. Mol Ther 2006; 13: 347. 16. Chou TC and Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 27. 17. Sambucetti LC, Fischer DD, Zabludoff S et al: Histone deacetylase inhibition selectively alters the activity and expression of cell cycle proteins leading to specific chromatin acetylation and antiproliferative effects. J Biol Chem 1999; 274: 34940. 18. Kosugi H, Towatari M, Hatano S et al: Histone deacetylase inhibitors are the potent inducer/
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enhancer of differentiation in acute myeloid leukemia: a new approach to anti-leukemia therapy. Leukemia 1999; 13: 1316. 19. Marchion DC, Bicaku E, Daud AI et al: Sequencespecific potentiation of topoisomerase II inhibitors by the histone deacetylase inhibitor suberoylanilide hydroxamic acid. J Cell Biochem 2004; 92: 223. 20. Carraway HE and Gore SD: Addition of histone deacetylase inhibitors in combination therapy. J Clin Oncol 2007; 25: 1955. 21. Cunneen TS, Conway RM and Madigan MC: In vitro effects of histone deacetylase inhibitors and mitomycin C on tenon capsule fibroblasts and conjunctival melanoma cells. Arch Ophthalmol 2009; 127: 414. 22. Garcia-Manero G, Kantarjian HM, Sanchez-Gonzalez B et al: Phase 1/2 study of the combination of 5-aza-2=-deoxycytidine with valproic acid in patients with leukemia. Blood 2006; 108: 3271. 23. Friedmann I, Atmaca A, Chow KU et al: Synergistic effects of valproic acid and mitomycin C in adenocarcinoma cell lines and fresh tumor cells of patients with colon cancer. J Chemother 2006; 18: 415. 24. Rosenthal MA, Green M, Wiernik P et al: Epirubicin has modest single-agent activity in head and neck cancer but limited activity in metastatic melanoma and colorectal cancer: Phase II studies by the eastern cooperative oncology group. Am J Clin Oncol 1998; 21: 509.