Effects of hypoxia on human cancer cell line chemosensitivity

BMC Cancer

Sara Strese¹  Mårten Fryknäs¹  Rolf Larsson¹  Joachim Gullbo¹ 

[1] Clinical Pharmacology, Department of Medical Sciences, Uppsala University, Akademiska Sjukhuset, 751 85 Uppsala, Sweden

关键词: Drug resistance;    Hypoxic incubator;    FMCA;    Cancer cell lines;    Anoxia;    Hypoxia;    Chemotherapy;   

Others  :  1079659
DOI  :  10.1186/1471-2407-13-331

 received in 2013-03-14, accepted in 2013-06-28,  发布年份 2013

【 摘 要 】

Background

Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditions of normoxia, hypoxia and anoxia.

Methods

A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA).

Results

Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs.

Conclusions

A panel of standard cytotoxic agents was tested against five different human cancer cell lines cultivated at normoxic, hypoxic and anoxic conditions. Results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia than hypoxia.

【 授权许可】

   
2013 Strese et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Durand RE, Raleigh JA: Identification of nonproliferating but viable hypoxic tumor cells in vivo. Cancer Res 1998, 58(16):3547-3550.
• [2]Evans SM, Joiner B, Jenkins WT, Laughlin KM, Lord EM, Koch CJ: Identification of hypoxia in cells and tissues of epigastric 9L rat glioma using EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide]. Br J Cancer 1995, 72(4):875-882.
• [3]Partridge SE, Aquino-Parsons C, Luo C, Green A, Olive PL: A pilot study comparing intratumoral oxygenation using the comet assay following 2.5% and 5% carbogen and 100% oxygen. Int J Radiat Oncol Biol Phys 2001, 49(2):575-580.
• [4]Tannock IF: The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Br J Cancer 1968, 22(2):258-273.
• [5]Evans SM, Hahn SM, Magarelli DP, Koch CJ: Hypoxic heterogeneity in human tumors: EF5 binding, vasculature, necrosis, and proliferation. Am J Clin Oncol 2001, 24(5):467-472.
• [6]Hanahan D, Folkman J: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996, 86(3):353-364.
• [7]Zetter BR: Angiogenesis and tumor metastasis. Annu Rev Med 1998, 49:407-424.
• [8]Brahimi-Horn MC, Chiche J, Pouyssegur J: Hypoxia and cancer. J Mol Med 2007, 85(12):1301-1307.
• [9]Hicklin DJ, Ellis LM: Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 2005, 23(5):1011-1027.
• [10]Zhou J, Schmid T, Schnitzer S, Brune B: Tumor hypoxia and cancer progression. Cancer Lett 2006, 237(1):10-21.
• [11]Vander Heiden MG, Cantley LC, Thompson CB: Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 2009, 324(5930):1029-1033.
• [12]Cardone RA, Casavola V, Reshkin SJ: The role of disturbed pH dynamics and the Na+/H + exchanger in metastasis. Nat Rev Cancer 2005, 5(10):786-795.
• [13]Bogenrieder T, Herlyn M: Axis of evil: molecular mechanisms of cancer metastasis. Oncogene 2003, 22(42):6524-6536.
• [14]Grau C, Khalil AA, Nordsmark M, Horsman MR, Overgaard J: The relationship between carbon monoxide breathing, tumour oxygenation and local tumour control in the C3H mammary carcinoma in vivo. Br J Cancer 1994, 69(1):50-57.
• [15]Brizel DM, Dodge RK, Clough RW, Dewhirst MW: Oxygenation of head and neck cancer: changes during radiotherapy and impact on treatment outcome. Radiother Oncol 1999, 53(2):113-117.
• [16]Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, Vaupel P: Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res 1996, 56(19):4509-4515.
• [17]Bodner G, Schocke MF, Rachbauer F, Seppi K, Peer S, Fierlinger A, Sununu T, Jaschke WR: Differentiation of malignant and benign musculoskeletal tumors: combined color and power Doppler US and spectral wave analysis. Radiology 2002, 223(2):410-416.
• [18]Brown JM, Giaccia AJ: The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res 1998, 58(7):1408-1416.
• [19]Graeber TG, Osmanian C, Jacks T, Housman DE, Koch CJ, Lowe SW, Giaccia AJ: Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature 1996, 379(6560):88-91.
• [20]Brown JM, Wilson WR: Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer 2004, 4(6):437-447.
• [21]Roninson IB: The role of the MDR1 (P-glycoprotein) gene in multidrug resistance in vitro and in vivo. Biochem Pharmacol 1992, 43(1):95-102.
• [22]Wartenberg M, Ling FC, Muschen M, Klein F, Acker H, Gassmann M, Petrat K, Putz V, Hescheler J, Sauer H: Regulation of the multidrug resistance transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) and reactive oxygen species. FASEB J 2003, 17(3):503-505.
• [23]Matzow T, Cowen RL, Williams KJ, Telfer BA, Flint PJ, Southgate TD, Saunders MP: Hypoxia-targeted over-expression of carboxylesterase as a means of increasing tumour sensitivity to irinotecan (CPT-11). J Gene Med 2007, 9(4):244-252.
• [24]Erler JT, Cawthorne CJ, Williams KJ, Koritzinsky M, Wouters BG, Wilson C, Miller C, Demonacos C, Stratford IJ, Dive C: Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance. Mol Cell Biol 2004, 24(7):2875-2889.
• [25]Lash GE, Fitzpatrick TE, Graham CH: Effect of hypoxia on cellular adhesion to vitronectin and fibronectin. Biochem Biophys Res Commun 2001, 287(3):622-629.
• [26]Kim JY, Kim YJ, Lee S, Park JH: The critical role of ERK in death resistance and invasiveness of hypoxia-selected glioblastoma cells. BMC Cancer 2009, 9:27. BioMed Central Full Text
• [27]Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P, Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshert E: Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 1998, 394(6692):485-490.
• [28]Griguer CE, Oliva CR, Kelley EE, Giles GI, Lancaster JR Jr, Gillespie GY: Xanthine oxidase-dependent regulation of hypoxia-inducible factor in cancer cells. Cancer Res 2006, 66(4):2257-2263.
• [29]Baumann RP, Penketh PG, Seow HA, Shyam K, Sartorelli AC: Generation of oxygen deficiency in cell culture using a two-enzyme system to evaluate agents targeting hypoxic tumor cells. Radiat Res 2008, 170(5):651-660.
• [30]Minchinton AI, Tannock IF: Drug penetration in solid tumours. Nat Rev Cancer 2006, 6(8):583-592.
• [31]Karlsson H, Fryknas M, Larsson R, Nygren P: Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system. Exp Cell Res 2012, 318(13):1577-1585.
• [32]Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA: Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 2010, 148(1):3-15.
• [33]Danielsen T, Skoyum R, Rofstad EK: Hypoxia-induced changes in radiation sensitivity in human melanoma cells: importance of oxygen-regulated proteins, adenylate energy charge and cell cycle distribution. Radiother Oncol 1997, 44(2):177-182.
• [34]Yang SJ, Pyen J, Lee I, Lee H, Kim Y, Kim T: Cobalt chloride-induced apoptosis and extracellular signal-regulated protein kinase 1/2 activation in rat C6 glioma cells. J Biochem Mol Biol 2004, 37(4):480-486.
• [35]Koch S, Mayer F, Honecker F, Schittenhelm M, Bokemeyer C: Efficacy of cytotoxic agents used in the treatment of testicular germ cell tumours under normoxic and hypoxic conditions in vitro. Br J Cancer 2003, 89(11):2133-2139.
• [36]Hay MP, Gamage SA, Kovacs MS, Pruijn FB, Anderson RF, Patterson AV, Wilson WR, Brown JM, Denny WA: Structure-activity relationships of 1,2,4-benzotriazine 1,4-dioxides as hypoxia-selective analogues of tirapazamine. J Med Chem 2003, 46(1):169-182.
• [37]Sakata K, Kwok TT, Murphy BJ, Laderoute KR, Gordon GR, Sutherland RM: Hypoxia-induced drug resistance: comparison to P-glycoprotein-associated drug resistance. Br J Cancer 1991, 64(5):809-814.
• [38]Rohwer N, Dame C, Haugstetter A, Wiedenmann B, Detjen K, Schmitt CA, Cramer T: Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB. PLoS One 2010, 5(8):e12038.
• [39]Lee K, Zhang H, Qian DZ, Rey S, Liu JO, Semenza GL: Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization. Proc Natl Acad Sci USA 2009, 106(42):17910-17915.
• [40]Roccaro AM, Hideshima T, Raje N, Kumar S, Ishitsuka K, Yasui H, Shiraishi N, Ribatti D, Nico B, Vacca A, Dammacco F, Richardson PG, Anderson KC: Bortezomib mediates antiangiogenesis in multiple myeloma via direct and indirect effects on endothelial cells. Cancer Res 2006, 66(1):184-191.
• [41]Shin DH, Chun YS, Lee DS, Huang LE, Park JW: Bortezomib inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated repression of hypoxia-inducible factor-1. Blood 2008, 111(6):3131-3136.
• [42]Duyndam MC, van Berkel MP, Dorsman JC, Rockx DA, Pinedo HM, Boven E: Cisplatin and doxorubicin repress Vascular Endothelial Growth Factor expression and differentially down-regulate Hypoxia-inducible Factor I activity in human ovarian cancer cells. Biochem Pharmacol 2007, 74(2):191-201.
• [43]Zhang H, Qian DZ, Tan YS, Lee K, Gao P, Ren YR, Rey S, Hammers H, Chang D, Pili R, Dang CV, Liu JO, Semenza GL: Digoxin and other cardiac glycosides inhibit HIF-1alpha synthesis and block tumor growth. Proc Natl Acad Sci USA 2008, 105(50):19579-19586.
• [44]Escuin D, Kline ER, Giannakakou P: Both microtubule-stabilizing and microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accumulation and activity by disrupting microtubule function. Cancer Res 2005, 65(19):9021-9028.
• [45]Forde JC, Perry AS, Brennan K, Martin LM, Lawler MP, Lynch TH, Hollywood D, Marignol L: Docetaxel maintains its cytotoxic activity under hypoxic conditions in prostate cancer cells. Urol Oncol 2010, 30:912-919.
• [46]Kennedy KA, Siegfried JM, Sartorelli AC, Tritton TR: Effects of anthracyclines on oxygenated and hypoxic tumor cells. Cancer Res 1983, 43(1):54-59.
• [47]Sermeus A, Cosse JP, Crespin M, Mainfroid V, de Longueville F, Ninane N, Raes M, Remacle J, Michiels C: Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity. Mol Cancer 2008, 7:27.
• [48]Sullivan R, Graham CH: Hypoxia prevents etoposide-induced DNA damage in cancer cells through a mechanism involving hypoxia-inducible factor 1. Mol Cancer Ther 2009, 8(6):1702-1713.
• [49]Onozuka H, Tsuchihara K, Esumi H: Hypoglycemic/hypoxic condition in vitro mimicking the tumor microenvironment markedly reduced the efficacy of anticancer drugs. Cancer Sci 2011, 102(5):975-982.
• [50]Kasuya K, Tsuchida A, Nagakawa Y, Suzuki M, Abe Y, Itoi T, Serizawa H, Nagao T, Shimazu M, Aoki T: Hypoxia-inducible factor-1alpha expression and gemcitabine chemotherapy for pancreatic cancer. Oncol Rep 2011, 26(6):1399-1406.
• [51]Kamiyama H, Takano S, Tsuboi K, Matsumura A: Anti-angiogenic effects of SN38 (active metabolite of irinotecan): inhibition of hypoxia-inducible factor 1 alpha (HIF-1alpha)/vascular endothelial growth factor (VEGF) expression of glioma and growth of endothelial cells. J Cancer Res Clin Oncol 2005, 131(4):205-213.
• [52]de Wilt JH, Manusama ER, van Tiel ST, van Ijken MG, ten Hagen TL, Eggermont AM: Prerequisites for effective isolated limb perfusion using tumour necrosis factor alpha and melphalan in rats. Br J Cancer 1999, 80(1–2):161-166.
• [53]Hu Y, Kirito K, Yoshida K, Mitsumori T, Nakajima K, Nozaki Y, Hamanaka S, Nagashima T, Kunitama M, Sakoe K, Komatsu N: Inhibition of hypoxia-inducible factor-1 function enhances the sensitivity of multiple myeloma cells to melphalan. Mol Cancer Ther 2009, 8(8):2329-2338.
• [54]Teicher BA, Lazo JS, Sartorelli AC: Classification of antineoplastic agents by their selective toxicities toward oxygenated and hypoxic tumor cells. Cancer Res 1981, 41(1):73-81.
• [55]Hudson CC, Liu M, Chiang GG, Otterness DM, Loomis DC, Kaper F, Giaccia AJ, Abraham RT: Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin. Mol Cell Biol 2002, 22(20):7004-7014.
• [56]Mayerhofer M, Valent P, Sperr WR, Griffin JD, Sillaber C: BCR/ABL induces expression of vascular endothelial growth factor and its transcriptional activator, hypoxia inducible factor-1alpha, through a pathway involving phosphoinositide 3-kinase and the mammalian target of rapamycin. Blood 2002, 100(10):3767-3775.
• [57]Pencreach E, Guerin E, Nicolet C, Lelong-Rebel I, Voegeli AC, Oudet P, Larsen AK, Gaub MP, Guenot D: Marked activity of irinotecan and rapamycin combination toward colon cancer cells in vivo and in vitro is mediated through cooperative modulation of the mammalian target of rapamycin/hypoxia-inducible factor-1alpha axis. Clin Cancer Res 2009, 15(4):1297-1307.
• [58]Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, Wilhelm S, Lynch M, Carter C: Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 2006, 66(24):11851-11858.
• [59]D’Amato RJ, Loughnan MS, Flynn E, Folkman J: Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci USA 1994, 91(9):4082-4085.
• [60]Bauer KS, Dixon SC, Figg WD: Inhibition of angiogenesis by thalidomide requires metabolic activation, which is species-dependent. Biochem Pharmacol 1998, 55(11):1827-1834.
• [61]Brown JM: The hypoxic cell: a target for selective cancer therapy–eighteenth Bruce F Cain Memorial Award lecture. Cancer Res 1999, 59(23):5863-5870.
• [62]Marcu L, Olver I: Tirapazamine: from bench to clinical trials. Curr Clin Pharmacol 2006, 1(1):71-79.
• [63]Reddy SB, Williamson SK: Tirapazamine: a novel agent targeting hypoxic tumor cells. Expert Opin Investig Drugs 2009, 18(1):77-87.
• [64]Rapisarda A, Uranchimeg B, Scudiero DA, Selby M, Sausville EA, Shoemaker RH, Melillo G: Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. Cancer Res 2002, 62(15):4316-4324.
• [65]Kummar S, Raffeld M, Juwara L, Horneffer Y, Strassberger A, Allen D, Steinberg SM, Rapisarda A, Spencer SD, Figg WD, Chen X, Turkbey IB, Choyke P, Murgo AJ, Doroshow JH, Melillo G: Multihistology, target-driven pilot trial of oral topotecan as an inhibitor of hypoxia-inducible factor-1alpha in advanced solid tumors. Clin Cancer Res 2011, 17(15):5123-5131.
• [66]Liu L, Ning X, Sun L, Zhang H, Shi Y, Guo C, Han S, Liu J, Sun S, Han Z, Wu K, Fan D: Hypoxia-inducible factor-1 alpha contributes to hypoxia-induced chemoresistance in gastric cancer. Cancer Sci 2008, 99(1):121-128.
• [67]Lindhagen E, Nygren P, Larsson R: The fluorometric microculture cytotoxicity assay. Nat Protoc 2008, 3(8):1364-1369.
• [68]Larsson R, Kristensen J, Sandberg C, Nygren P: Laboratory determination of chemotherapeutic drug resistance in tumor cells from patients with leukemia, using a fluorometric microculture cytotoxicity assay (FMCA). Int J Cancer 1992, 50(2):177-185.
• [69]Lamb J, Crawford ED, Peck D, Modell JW, Blat IC, Wrobel MJ, Lerner J, Brunet JP, Subramanian A, Ross KN, Reich M, Hieronymus H, Wei G, Armstrong SA, Haggarty SJ, Clemons PA, Wei R, Carr SA, Lander ES, Golub TR: The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science 2006, 313(5795):1929-1935.
• [70]Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005, 102(43):15545-15550.
• [71]da Huang W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009, 4(1):44-57.
• [72]Elvidge GP, Glenny L, Appelhoff RJ, Ratcliffe PJ, Ragoussis J, Gleadle JM: Concordant regulation of gene expression by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: the role of HIF-1alpha, HIF-2alpha, and other pathways. J Biol Chem 2006, 281(22):15215-15226.
• [73]Vaupel P, Thews O, Hoeckel M: Treatment resistance of solid tumors: role of hypoxia and anemia. Med Oncol 2001, 18(4):243-259.
• [74]Rohwer N, Cramer T: Hypoxia-mediated drug resistance: novel insights on the functional interaction of HIFs and cell death pathways. Drug Resist Updat 2011, 14(3):191-201.
• [75]Schwartz HS: Pharmacology of mitomycin C. III. In vitro metabolism by rat liver. J Pharmacol Exp Ther 1962, 136:250-258.
• [76]Iwai A, Fujii Y, Kawakami S, Takazawa R, Kageyama Y, Yoshida MA, Kihara K: Down-regulation of vascular endothelial growth factor in renal cell carcinoma cells by glucocorticoids. Mol Cell Endocrinol 2004, 226(1–2):11-17.
• [77]Tochizawa S, Masumori N, Yanai Y, Ohmoto Y, Yabuuchi Y, Tsukamoto T: Antitumor effects of a combination of interferon-alpha and sorafenib on human renal carcinoma cell lines. Biomed Res 2008, 29(6):271-278.
• [78]Lee TH, Seng S, Sekine M, Hinton C, Fu Y, Avraham HK, Avraham S: Vascular endothelial growth factor mediates intracrine survival in human breast carcinoma cells through internally expressed VEGFR1/FLT1. PLoS Med 2007, 4(6):e186.
• [79]Lee JH, Shim JW, Choi YJ, Heo K, Yang K: The combination of sorafenib and radiation preferentially inhibits breast cancer stem cells by suppressing HIF-1alpha expression. Oncol Rep 2013, 29(3):917-924.
• [80]Spannuth WA, Nick AM, Jennings NB, Armaiz-Pena GN, Mangala LS, Danes CG, Lin YG, Merritt WM, Thaker PH, Kamat AA, Han LY, Tonra JR, Coleman RL, Ellis LM, Sood AK: Functional significance of VEGFR-2 on ovarian cancer cells. Int J Cancer 2009, 124(5):1045-1053.