Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions

Radiation Oncology

Dirk Vordermark¹  Helge Taubert³  Harish Kommera⁴  Goran N Kaluđerović⁴  Reinhard Paschke⁴  Matthias Kappler²  Henri Wichmann¹  Jacqueline Keßler¹  Sarina Passin¹  Martin P Zschornak¹  Matthias Bache¹ 

[1] Department of Radiotherapy, Martin-Luther-University Halle-Wittenberg, Dryanderstr. 4, 06110 Halle, Germany;Department of Oral and Maxillofacial Plastic Surgery, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Str. 40, 06120 Halle, Germany;Div. Molecular Urology, Clinic of Urology, University Hospital Erlangen, Erlangen, Germany and Nikolaus-Fiebiger-Center for Molecular Medicine, Friedrich Alexander University Erlangen-Nürnberg, Germany;Biozentrum, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 22, 06120 Halle, Germany

关键词: irradiation;    hypoxia;    glioma cells;    betulinc acid;   

Others  :  1223904
DOI  :  10.1186/1748-717X-6-111

 received in 2011-05-25, accepted in 2011-09-09,  发布年份 2011

【 摘 要 】

Background

Betulinic acid (BA) is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas), the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy) may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied.

Methods

In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays) and protein expression was examined with Western blot analyses.

Results

Under normoxic conditions, a half maximal inhibitory concentration (IC₅₀) of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively) and U343MG cells (p < 0.05 and p = 0.01, respectively). The combination of BA with radiotherapy resulted in an additive effect in the U343MG cell line under normoxic and hypoxic conditions. Weak radiation enhancement was observed in U251MG cell line after treatment with BA under normoxic conditions. Furthermore, under hypoxic conditions, the incubation with BA resulted in increased radiation enhancement. The enhancement factor, at an irradiation dose of 15 Gy after treatment with 10 or 15 μM BA, was 2.20 (p = 0.02) and 4.50 (p = 0.03), respectively. Incubation with BA led to decreased cell migration, cleavage of PARP and decreased expression levels of survivin in both cell lines. Additionally, BA treatment resulted in a reduction of HIF-1α protein under hypoxic conditions.

Conclusion

Our results suggest that BA is capable of improving the effects of tumor therapy in human malignant glioma cells, particularly under hypoxic conditions. Further investigations are necessary to characterize its potential as a radiosensitizer.

【 授权许可】

   
2011 Bache et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Sheline GE: Radiation therapy of brain tumors. Cancer 1977, 39:873-881.
• [2]Eiznhamer DA, Xu ZQ: Betulinic acid: a promising anticancer candidate. IDrugs 2004, 7:(4): 359-373.
• [3]Alakurtti S, Mäkelä T, Koskimies S, Yli-Kauhaluoma J: Pharmacological properties of the ubiquitous natural product betulin. Eur J Pharm Sci 2006, 29(1):1-13.
• [4]Kessler JH, Mullauer FB, de Roo GM, Medema JP: Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer types. Cancer Lett 2007, 251(1):132-145.
• [5]Pisha E, Chai H, Lee IS, Chagwedera TE, arnsworth NR, Cordell GA, Beecher CW, Fong HH, Kinghorn AD, Brown DM, et al.: Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat Med 1995, 1(10):1046-1051.
• [6]Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E, Gambacorti-Passerini C, Formelli F: Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett 2002, 175(1):17-25.
• [7]Sawada N, Kataoka K, Kondo K, Arimochi H, Fujino H, Takahashi Y, Miyoshi T, Kuwahara T, Monden Y, Ohnishi Y: Betulinic acid augments the inhibitory effects of vincristine on growth and lung metastasis of B16F10 melanoma cells in mice. Br J Cancer 2004, 90(8):1672-1678.
• [8]Fulda S, Friesen C, Los M, Scaffidi C, Mier W, Benedict M, Nuñez G, Krammer PH, Peter ME, Debatin KM: Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res 1997, 57(21):4956-4964.
• [9]Sandberg F, Dutschewska H, Christov V, Spassov S: Spondianthus preussii var. glaber Engler: Pharmacological screening and occurrence of triterpenes. Acta Pharm Suec 1987, 24:253-256.
• [10]Fulda S, Jeremias I, Steiner HH, Pietsch T, Debatin KM: Betulinic acid: a new cytotoxic agent against malignant brain-tumor cells. Int J Cancer 1999, 82(3):435-441.
• [11]Kasperczyk H, La Ferla-Brühl K, Westhoff MA, Behrend L, Zwacka RM, Debatin KM, Fulda S: Betulinic acid as new activator of NF-kappaB: molecular mechanisms and implications for cancer therapy. Oncogene 2005, 24(46):6945-6956.
• [12]Rabi T, Shukla S, Gupta S: Betulinic acid suppresses constitutive and TNFalpha-induced NF-kappaB activation and induces apoptosis in human prostate carcinoma PC-3 cells. Mol Carcinog 2008, 47(12):964-973.
• [13]Mullauer FB, Kessler JH, Medema JP: Betulinic acid induces cytochrome c release and apoptosis in a Bax/Bak-independent, permeability transition pore dependent fashion. Apoptosis 2009, 14(2):191-202.
• [14]Kwon HJ, Shim JS, Kim JH, Cho HY, Yum YN, Kim SH, Yu J: Betulinic acid inhibits growth factor-induced in vitro angiogenesis via the modulation of mitochondrial function in endothelial cells. Jpn J Cancer Res 2002, 93(4):417-425.
• [15]Rzeski W, Stepulak A, Szymański M, Sifringer M, Kaczor J, Wejksza K, Zdzisińska B, Kandefer-Szerszeń M: Betulinic acid decreases expression of bcl-2 and cyclin D1, inhibits proliferation, migration and induces apoptosis in cancer cells. Naunyn Schmiedebergs Arch Pharmacol 2006, 374(1):11-20.
• [16]Chintharlapalli S, Papineni S, Ramaiah SK, Safe S: Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Res 2007, 67(6):2816-2823.
• [17]Wick W, Grimmel C, Wagenknecht B, Dichgans J, Weller M: Betulinic acid-induced apoptosis in glioma cells: A sequential requirement for new protein synthesis, formation of reactive oxygen species, and caspase processing. J Pharmacol Exp Ther 1999, 289(3):1306-1312.
• [18]Tan Y, Yu R, Pezzuto JM: Betulinic acid-induced programmed cell death in human melanoma cells involves mitogen-activated protein kinase activation. Clin Cancer Res 2003, 9(7):2866-2875.
• [19]Ganguly A, Das B, Roy A, Sen N, Dasgupta SB, Mukhopadhayay S, Majumder HK: Betulinic acid, a catalytic inhibitor of topoisomerase I, inhibits reactive oxygen species-mediated apoptotic topoisomerase I-DNA cleavable complex formation in prostate cancer cells but does not affect the process of cell death. Cancer Res 2007, 67(24):11848-11858.
• [20]Collingridge DR, Piepmeier JM, Rockwell S, Knisely JP: Polarographic measurements of oxygen tension in human glioma and surrounding peritumoral brain tissue. Radiother Oncol 1999, 53(2):127-131.
• [21]Jensen RL: Hypoxia in the tumorigenesis of gliomas and as a potential target for therapeutic measures. Neurosurg Focus 2006, 20(4):E24.
• [22]Flynn JR, Wang L, Gillespie DL, Stoddard GJ, Reid JK, Owens J, Ellsworth GB, Salzman KL, Kinney AY, Jensen RL: Hypoxia-regulated protein expression, patient characteristics, and preoperative imaging as predictors of survival in adults with glioblastoma multiforme. Cancer 2008, 113(5):1032-1042.
• [23]Jung GR, Kim KJ, Choi CH, Lee TB, Han SI, Han HK, Lim SC: Effect of betulinic acid on anticancer drug-resistant colon cancer cells. Basic Clin Pharmacol Toxicol 2007, 101(4):277-285.
• [24]Selzer E, Pimentel E, Wacheck V, Schlegel W, Pehamberger H, Jansen B, Kodym R: Effects of betulinic acid alone and in combination with irradiation in human melanoma cells. J Invest Dermatol 2000, 114(5):935-940.
• [25]Eder-Czembirek C, Erovic BM, Czembirek C, Brunner M, Selzer E, Pötter R, Thurnher D: Betulinic Acid a Radiosensitizer in Head and Neck Squamous Cell Carcinoma Cell Lines. Strahlenther Onkol 2010, 186:143-148.
• [26]Kappler M, Rot S, Taubert H, Greither T, Bartel F, Dellas K, Hänsgen G, Trott KR, Bache M: The effects of knockdown of wild-type survivin, survivin-2B or survivin-delta3 on the radiosensitization in a soft tissue sarcoma cells in vitro under different oxygen conditions. Cancer Gene Ther 2007, 14(12):994-1001.
• [27]Hahnel A, Wichmann H, Kappler M, Kotzsch M, Vordermark D, Taubert H, Bache M: Effects of osteopontin inhibition on radiosensitivity of MDA-MB-231 breast cancer cells. Radiat Oncol 2010, 5:82. BioMed Central Full Text
• [28]Jeremias I, Steiner HH, Benner A, Debatin KM, Herold-Mende C: Cell death induction by betulinic acid, ceramide and TRAIL in primary glioblastoma multiforme cells. Acta Neurochir 2004, 146(7):721-729.
• [29]Qiu L, Wang Q, Di W, Jiang Q, Schefeller E, Derby S, Wanebo H, Yan B, Wan Y: Transient activation of EGFR/AKT cell survival pathway and expression of survivin contribute to reduced sensitivity of human melanoma cells to betulinic acid. Int J Oncol 2005, 27(3):823-830.
• [30]Yoon JJ, Lee YJ, Kim JS, Kang DG, Lee HS: Betulinic acid inhibits high glucose-induced vascular smooth muscle cells proliferation and migration. J Cell Biochem 2010, 111(6):1501-1511.
• [31]Fulda S, Debatin KM: Sensitization for anticancer drug-induced apoptosis by betulinic Acid. Neoplasia 2005, 7(2):162-170.
• [32]Li Y, He K, Huang Y, Zheng D, Gao C, Cui L, Jin YH: Betulin induces mitochondrial cytochrome c release associated apoptosis in human cancer cells. Mol Carcinog 2010, 49(7):630-640.
• [33]Thurnher D, Turhani D, Pelzmann M, Wannemacher B, Knerer B, Formanek M, Wacheck V, Selzer E: Betulinic acid: a new cytotoxic compound against malignant head and neck cancer cells. Head Neck 2003, 25(9):732-740.
• [34]Parrondo R, de las Pozas A, Reiner T, Rai P, Perez-Stable C: NF-kappaB activation enhances cell death by antimitotic drugs in human prostate cancer cells. Mol Cancer 2010, 9:182. BioMed Central Full Text
• [35]Eder-Czembirek C, Czembirek C, Erovic BM, Selzer E, Turhani D, Vormittag L, Thurnher D: Combination of betulinic acid with cisplatin--different cytotoxic effects in two head and neck cancer cell lines. Oncol Rep 2005, 14(3):667-671.
• [36]Shu HK, Kim MM, Chen P, Furman F, Julin CM, Israel MA: The intrinsic radioresistance of glioblastoma-derived cell lines is associated with a failure of p53 to induce p21(BAX) expression. Proc Natl Acad Sci USA 1998, 95:14453-14458.
• [37]Bassi C, Mello SS, Cardoso RS, Godoy PD, Fachin AL, Junta CM, Sandrin-Garcia P, Carlotti CG, Falcão RP, Donadi EA, Passos GA, Sakamoto-Hojo ET: Transcriptional changes in U343 MG-a glioblastoma cell line exposed to ionizing radiation. Hum Exp Toxicol 2008, 27(12):919-929.
• [38]Kessler J, Hahnel A, Wichmann H, Rot S, Kappler M, Bache M, Vordermark D: HIF-1α inhibition by siRNA or chetomin in human malignant glioma cells: effects on hypoxic radioresistance and monitoring via CA9 expression. BMC Cancer 2010, 10:605. BioMed Central Full Text
• [39]Karna E, Szoka L, Palka JA: Betulinic acid inhibits the expression of hypoxia-inducible factor 1alpha and vascular endothelial growth factor in human endometrial adenocarcinoma cells. Mol Cell Biochem 2010, 340:(1-2):15-20.
• [40]Staab A, Loeffler J, Said HM, Diehlmann D, Katzer A, Beyer M, Fleischer M, Schwab F, Baier K, Einsele H, Flentje M, Vordermark D: Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells. BMC Cancer 2007, 7:213. BioMed Central Full Text
• [41]Staab A, Fleischer M, Loeffler J, Said HM, Katzer A, Plathow C, Einsele H, Flentje M, Vordermark D: Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro. Strahlenther Onkol 2011, 187(4):252-259.
• [42]Chen L, Feng P, Li S, Long D, Cheng J, Lu Y, Zhou D: Effect of hypoxia-inducible factor-1alpha silencing on the sensitivity of human brain glioma cells to doxorubicin and etoposide. Neurochem Res 2009, 34(5):984-990.