[en] One of the recently emerging anticancer strategies is the use of natural dietary compounds, such as sulforaphane, a cancer-chemopreventive isothiocyanate found in broccoli. Based on the growing evidence, sulforaphane acts through molecular mechanisms that interfere with multiple oncogenic pathways in diverse tumor cell types. Herein, we investigated the anticancer effects of bioavailable concentrations of sulforaphane in ovarian carcinoma cell line A2780 and its two derivatives, adriamycin-resistant A2780/ADR and cisplatin-resistant A2780/CP cell lines. Since tumor microenvironment is characterized by reduced oxygenation that induces aggressive tumor phenotype (such as increased invasiveness and resistance to chemotherapy), we evaluated the effects of sulforaphane in ovarian cancer cells exposed to hypoxia (2% O2). Using the cell-based reporter assay, we identified several oncogenic pathways modulated by sulforaphane in hypoxia by activating anticancer responses (p53, ARE, IRF-1, Pax-6 and XRE) and suppressing responses supporting tumor progression (AP-1 and HIF-1). We further showed that sulforaphane decreases the level of HIF-1alpha protein without affecting its transcription and stability. It can also diminish transcription and protein level of the HIF-1 target, CA IX, which protects tumor cells from hypoxia-induced pH imbalance and facilitates their migration/invasion. Accordingly, sulforaphane treatment leads to diminished pH regulation and reduced migration of ovarian carcinoma cells. These effects occur in all three ovarian cell lines suggesting that sulforaphane can overcome the chemoresistance of cancer cells. This offers a path potentially exploitable in sensitizing resistant cancer cells to therapy, and opens a window for the combined treatments of sulforaphane either with conventional chemotherapy, natural compounds, or with other small molecules.
Harris AL: Hypoxia - a key regulatory factor in tumor growth. Nat Rev Cancer 2: 38-47, 2002.
Semenza GL: Hypoxia-inducible factors: mediators of cancer progression and targets for cancer therapy. Trends Pharmacol Sci 33: 207-214, 2012.
Brown JM and Wilson WR: Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer 4: 437-447, 2004
Birner P, Schindl M, Obermair A., Breitenecker G and Oberhuber G: Expression of hypoxia-inducible factor lalpha in epithelial ovarian tumors: its impact on prognosis and on response to chemotherapy. Clin Cancer Res 7 1661-1668, 2001.
Rohwer N and Cramer T Hypoxia-mediated drug resistance: novel insights on the functional interaction of HIFs and cell death pathways. Drug Resist Updat 14: 191-201, 2011.
Myzak MC, Dashwood WM, Orner G.A., Ho E and Dashwood RH: Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Ape-minus mice. FASEB J 20: 506-508, 2006.
Chaudhuri D, Orsulic S and Ashok BT Antiproliferative activity of sulforaphane in Akt-overexpressing ovarian cancer cells. Mol Cancer Ther 6: 334-345, 2007
Bryant CS, Kumar S, Chamala S., Shah J, Pal J, Haider M., Seward S, Qazi AM, Morris R, Semaan A, et al.: Sulforaphane induces cell cycle arrest by protecting RB-E2F-1 complex in epithelial ovarian cancer cells. Mol Cancer 9: 47, 2010.
Chang CC, Hung CM, Yang Y.R., Lee MJ and Hsu YC: Sulforaphane induced cell cycle arrest in the G2/M phase via the blockade of cyclin B1/CDC2 in human ovarian cancer cells. J Ovarian Res 6: 41, 2013.
Zhou C, Poulton EJ, Grün F, Bammler T.K., Blumberg B., Thummel KE and Eaton DL: The dietary isothiocyanate sulforaphane is an antagonist of the human steroid and xenobiotic nuclear receptor. Mol Pharmacol 71: 220-229, 2007.
Berti E, Bartsch H and Gerhäuser C: Inhibition of angiogenesis and endothelial cell functions are novel sulforaphane-mediated mechanisms in chemoprevention. Mol Cancer Ther 5: 575-585, 2006.
Yao H, Wang H, Zhang Z., Jiang BH, Luo J and Shi X: Sulforaphane inhibited expression of hypoxia-inducible factor-lalpha in human tongue squamous cancer cells and prostate cancer cells. Int J Cancer 123: 1255-1261, 2008.
Jeong JK, Moon MH, Seo J.S., Seol JW, Lee YJ and Park SY Sulforaphane blocks hypoxia-mediated resistance to TRAIL-induced tumor cell death. Mol Med Rep 4: 325-330, 2011.
Kaelin WG Jr and Ratcliffe PJ: Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol Cell 30: 393-402, 2008.
Lendahl U, Lee KL, Yang H and Poellinger L Generating specificity and diversity in the transcriptional response to hypoxia. Nat Rev Genet 10: 821-832, 2009
Pijnenborg JM, Wijnakker M, Hagelstein J., Delvoux B and Groothuis PG: Hypoxia contributes to development of recurrent endometrial carcinoma. Int J Gynecol Cancer 17 897-904, 2007
Liang D, Ma Y, Liu J., Trope CG, Holm R, Nesland JM and Suo Z: The hypoxic microenvironment upgrades stem-like properties of ovarian cancer cells. BMC Cancer 12: 201, 2012.
Williams E, Martin S, Moss R., Durrant L and Deen S: Co-expression of VEGF and CA9 in ovarian high-grade serous carcinoma and relationship to survival. Virchows Arch 461: 33-39, 2012.
Sedlák J, Sedláková O, Hlavcák P, Hunáková L, Bizik J, Grofová M and Chorváth B: Cell surface phenotype and increased penetration of human multidrug-resistant ovarian carcinoma cells into in vitro collagen-fibroblasts matrix. Neoplasma 43: 389-395, 1996.
Bodo J, Chovancova J, Hunakova L and Sedlak J: Enhanced sensitivity of human ovarian carcinoma cell lines A2780 and A2780/CP to the combination of cisplatin and synthetic isothiocyanate ethyl 4-isothiocyanatobutanoate. Neoplasma 52: 510-516, 2005
Svastová E, Huliková A, Rafajová M, Zat'ovicová M, Gibadulinová A, Casini A, Cecchi A, Scozzafava A, Supuran CT, Pastorek J, et al.: Hypoxia activates the capacity of tumor-associated carbonic anhydrase IX to acidify extracellular pH. FEB S Lett 577: 439-445, 2004
Chen H, Landen CN, Li Y, Alvarez RD and Tollefsbol TO: Enhancement of cisplatin-mediated apoptosis in ovarian cancer cells through potentiating G2/M arrest and p21 upregulation by combinatorial epigallocatechin gallate and sulforaphane. J Oncol 2013: 872957, 2013.
Comerford KM, Cummins EP and Taylor CT c-JunNH2-terminal kinase activation contributes to hypoxia-inducible factor lalpha-dependent P-glycoprotein expression in hypoxia. Cancer Res 64: 9057-9061, 2013.
An J, Liu H, Magyar C.E., Guo Y., Veena MS, Srivatsan ES, Huang J and Rettig MB: Hyperactivated JNK is a therapeutic target in pVHL-deficient renal cell carcinoma. Cancer Res 73: 1374-1385, 2013.
Gillies RJ and Gatenby RA: Hypoxia and adaptive landscapes in the evolution of carcinogenesis. Cancer Metastasis Rev 26: 311-317, 2007
Sibhatu MB, Smitherman PK, Townsend AJ and Morrow CS: Expression of MRP1 and GSTP1-1 modulate the acute cellular response to treatment with the chemopreventive isothiocyanate, sulforaphane. Carcinogenesis 29: 807-815, 2008.
Rudolf E, Andelová H and Cervinka M: Activation of several concurrent proapoptic pathways by sulforaphane in human colon cancer cells SW62Q Food Chem Toxicol 47 2366-2373, 2009
Horiuchi A, Hayashi T, Kikuchi N., Hayashi A, Fuseya C, Shiozawa T and Konishi I: Hypoxia upregulates ovarian cancer invasiveness via the binding of HIF-1α to a hypoxia-induced, methylation-free hypoxia response element of S100A4 gene. Int J Cancer 131: 1755-1767, 2012.
Selvendiran K, Bratasz A, Kuppusamy M.L., Tazi MF, Rivera BK and Kuppusamy P: Hypoxia induces chemoresistance in ovarian cancer cells by activation of signal transducer and activator of transcription 3. Int J Cancer 125: 2198-2204, 2009
Milane L, Duan Z and Amiji M: Role of hypoxia and glycolysis in the development of multi-drug resistance in human tumor cells and the establishment of an orthotopic multi-drug resistant tumor model in nude mice using hypoxic pre-conditioning. Cancer Cell Int 11: 3, 2011.
Shimogai R, Kigawa J, Itamochi H., Iba T, Kanamori Y, Oishi T., Shimada M, Sato S, Kawaguchi W, Sato S, et al.: Expression of hypoxia-inducible factor lalpha gene affects the outcome in patients with ovarian cancer. Int J Gynecol Cancer 18: 499-505, 2008.
Wong C, Wellman TL and Lounsbury KM: VEGF and HIF-1alpha expression are increased in advanced stages of epithelial ovarian cancer. Gynecol Oncol 91: 513-517, 2003.
Kim K, Park WY, Kim J.Y., Sol MY, Shin DH, Park do Y, Lee CH, Lee JH and Choi KU: Prognostic relevance of the expression of CA IX, GLUT-1, and VEGF in ovarian epithelial cancers. Korean J Pathol 46: 532-540, 2012.
Cheng JC, Klausen C and Leung PC: Hypoxia-inducible factor 1 alpha mediates epidermal growth factor-induced down-regulation of E-cadherin expression and cell invasion in human ovarian cancer cells. Cancer Lett 329: 197-206, 2013.
Hynninen P, Vaskivuo L, Saarnio J., Haapasalo H, Kivelä J, Pastoreková S, Pastorek J, Waheed A, Sly WS, Puistola U, et al.: Expression of transmembrane carbonic anhydrases IX and XII in ovarian tumours. Histopathology 49: 594-602, 2006.
Jeon YK, Yoo DR, Jang Y.H., Jang SY and Nam MJ: Sulforaphane induces apoptosis in human hepatic cancer cells through inhibition of 6-phosphofructo-2-kinase/fructose-2, 6-bi phosphatase mediated by hypoxia inducible factor-1-dependent pathway. Biochim Biophys Acta 1814: 1340-1348, 2011.
Sermeus A and Michiels C: Reciprocal influence of the p53 and the hypoxic pathways. Cell Death Dis 2: e164, 2011.
Chew YC, Adhikary G, Wilson G.M., Xu W and Eckert RL: Sulforaphane induction of p21(Cipl) cyclin-dependent kinase inhibitor expression requires p53 and Sp1 transcription factors and is p53-dependent. J Biol Chem 287: 16168-16178, 2012.
Kolamunne RT, Dias IH, Vernallis A.B., Grant MM and Griffiths HR Nrf2 activation supports cell survival during hypoxia and hypoxia/ reoxygenation in cardiomyoblasts; the roles of reactive oxygen and nitrogen species. Redox Biol 1: 418-426, 2013.
Kensler TW, Egner PA, Agyeman A.S., Visvanathan K., Groopman JD, Chen JG, Chen TY, Fahey JW and Talalay P: Keapl-nrf2 signaling: a target for cancer prevention by sulforaphane. Top Curr Chem 329: 163-177, 2013.
Chan WK, Yao G, Gu YZ and Bradfield CA: Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation. J Biol Chem 274: 12115-12123, 1999
Takacova M, Holotnakova T, Vondracek J., Machala M, Pencikova K, Gradin K., Poellinger L, Pastorek J, Pastorekova S and Kopacek J: Role of aryl hydrocarbon receptor in modulation of the expression of the hypoxia marker carbonic anhydrase IX. Biochem J 419: 419-425, 2009
Cavalli LR, Riggins RB, Wang A, Clarke R and Haddad BR: Frequent loss of heterozygosity at the interferon regulatory factor-1 gene locus in breast cancer. Breast Cancer Res Treat 121: 227-231, 2010.
Wiczk A, Hofman D, Konopa G and Herman-Antosiewicz A: Sulforaphane, a cruciferous vegetable-derived isothiocyanate, inhibits protein synthesis in human prostate cancer cells. Biochim Biophys Acta 1823: 1295-1305, 2012.
Wykoff CC, Beasley NJ, Watson P.H., Turner KJ, Pastorek J, Sibtain A, Wilson G.D., Turley H., Talks KL, Maxwell PH, et al.: Hypoxia-inducible expression of tumor-associated carbonic anhy-drases. Cancer Res 60: 7075-7083, 2000.
Kaluz S, Kaluzová M, Opavsky R, Pastoreková S, Gibadulinová A, Dequiedt F, Kettmann R and Pastorek J: Transcriptional regulation of the MN/CA 9 gene coding for the tumor-associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element. J Biol Chem 274: 32588-32595, 1999.
Kaluzová M, Kaluz S, Lerman MI and Stanbridge EJ: DNA damage is a prerequisite for p53-mediated proteasomal degradation of HIF-lalpha in hypoxic cells and downregulation of the hypoxia marker carbonic anhydrase IX. Mol Cell Biol 24: 5757-5766, 2004
Pastorek J, Pastoreková S, Callebaut I, Mornon JP, Zelník V, Opavsky R, Zat'ovicová M, Liao S, Porteteile D, Stanbridge EJ, et al.: Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene 9: 2877-2888, 1994
Ditte P Dequiedt F, Svastova E, Hulikova A, Ohradanova-Repic A, Zatovicova M, Csaderova L, Kopacek J, Supuran CT, Pastorekova S, et al. Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors. Cancer Res 71: 7558-7567, 2011.
Fang JS, Gillies RD and Gatenby RA: Adaptation to hypoxia and acidosis in carcinogenesis and tumor progression. Semin Cancer Biol 330-337, 2008.
Gatenby RA and Gillies RJ: A microenvironmental model of carcinogenesis. Nat Rev Cancer 8: 56-61, 2008.
Sedlakova O, Svastova E, Takacova M., Kopacek J, Pastorek J and Pastorekova S: Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating machinery in tumors. Front Physiol 4: 400, 2014
Jakubikova J, Cervi D, Ooi M., Kim K, Nahar S, Klippel S., Cholujova D, Leiba M, Daley JF, Delmore J, et al.: Anti-tumor activity and signaling events triggered by the isothiocyanates, sulforaphane and phenethyl isothiocyanate, in multiple myeloma. Haematologica 96: 1170-1179, 2011.
Kaminski BM, Weigert A, Brüne B, Schumacher M., Wenzel U, Steinhilber D, Stein J and Ulrich S: Sulforaphane potentiates oxaliplatin-induced cell growth inhibition in colorectal cancer cells via induction of different modes of cell death. Cancer Chemother Pharmacol 67: 1167-1178, 2011.
Mokhtari RB, Kumar S, Islam S.S., Yazdanpanah M., Adeli K, Cutz E and Yeger H: Combination of carbonic anhydrase inhibitor, acetazolamide, and sulforaphane, reduces the viability and growth of bronchial carcinoid cell lines. BMC Cancer 13: 378, 2013.
