Isoliquiritigenin Induces Caspase-Dependent Apoptosis via Downregulation of HPV16 E6 Expression in Cervical Cancer Ca Ski Cells

Abstract

Flavonoids have antitumoral properties and may be attractive candidates as anticancer therapy. Isoliquiritigenin which is a constituent of licorice (Glycyrrhiza inflata), a plant commonly used in traditional Uyghur medicine in Xinjiang, China, was studied for antiproliferative and apoptotic activity in human cervical cancer cells, Ca Ski, SiHa, HeLa, and C-33A. Its molecular mechanism of action was specifically examined in Ca Ski cells. Isoliquiritigenin decreased cell viability, induced cell accumulation in G2/M and morphological and biochemical features of apoptosis in the four cancer cell lines. In Ca Ski cells, isoliquiritigenin led to a downregulation of HPV16 E6 expression associated with an increase of p53 and p21 levels, enhanced expression of Bax and decreased expression of Bcl-2 and Bid proform triggering dissipation of the mitochondrial membrane potential, released cytochrome c to the cytosol followed by activation of caspase cascade with cleavage of caspase-9, caspase-3, and PARP. Caspase-8 was also cleaved. Moreover treatment with a pan-caspase inhibitor prevented apoptosis. As Ca Ski cells are representative of carcinoma naturally occurring in the cervix, our results suggest a potential benefit of isoliquiritigenin for cervical cancer prevention and treatment.

Key words

Leguminosae - Glycyrrhiza inflata - isoliquiritigenin - apoptosis - HPV16 E6 - cervix carcinoma

• References

• 1 Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010; 127: 2893-2917
• 2 Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, Snijders PJF, Meijer CJLM. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348: 518-527
• 3 Garland SM. Human papillomavirus update with a particular focus on cervical disease. Pathology 2002; 34: 213-224
• 4 Prétet JL, Jacquard AC, Carcopino X, Charlot JF, Bouhour D, Kantelip B, Soubeyrand B, Leocmach Y, Mougin C, Riethmuller D. Human papillomavirus (HPV) genotype distribution in invasive cervical cancers in France: EDITH study. Int J Cancer 2008; 122: 428-432
• 5 Da Rocha AB, Lopes RM, Schwartsmann G. Natural products in anticancer therapy. Curr Opin Pharmacol 2001; 1: 364-369
• 6 Vaya J, Belinky PA, Aviram M. Antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward LDL oxidation. Free Radic Biol Med 1997; 23: 302-313
• 7 Chan SC, Chang YS, Wang JP, Chen SC, Kuo SC. Three new flavonoids and antiallergic, anti-inflammatory constituents from the heartwood of Dalbergia odorifera . Planta Med 1998; 64: 153-158
• 8 Tawata M, Aida K, Noguchi T, Ozaki Y, Kume S, Sasaki H, Chin M, Onaya T. Anti-platelet action of isoliquiritigenin, an aldose reductase inhibitor in licorice. Eur J Pharmacol 1992; 212: 87-92
• 9 Maggiolini M, Statti G, Vivacqua A, Gabriele S, Rago V, Loizzo M, Menichini F, Amdò S. Estrogenic and antiproliferative activities of isoliquiritigenin in MCF7 breast cancer cells. J Steroid Biochem Mol Biol 2002; 82: 315-322
• 10 Hsu YL, Kuo PL, Lin CC. Isoliquiritigenin induces apoptosis and cell cycle arrest through p 53-dependent pathway in Hep G2 cells. Life Sci 2005; 77: 279-292
• 11 Jung JI, Lim SS, Choi HJ, Cho HJ, Shin HK, Kim EJ, Chung WY, Park KK, Park JHY. Isoliquiritigenin induces apoptosis by depolarizing mitochondrial membranes in prostate cancer cells. J Nutr Biochem 2006; 17: 689-696
• 12 Hsu YL, Kuo PL, Chiang LC, Lin CC. Isoliquiritigenin inhibits the proliferation and induces the apoptosis of human non-small cell lung cancer a549 cells. Clin Exp Pharmacol Physiol 2004; 31: 414-418
• 13 Kwon GT, Cho HJ, Chung WY, Park KK, Moon A, Park JHY. Isoliquiritigenin inhibits migration and invasion of prostate cancer cells: possible mediation by decreased JNK/AP-1 signaling. J Nutr Biochem 2009; 20: 663-676
• 14 Ii T, Satomi Y, Katoh D, Shimada J, Baba M, Okuyama T, Nishino H, Kitamura N. Induction of cell cycle arrest and p 21(CIP1/WAF1) expression in human lung cancer cells by isoliquiritigenin. Cancer Lett 2004; 207: 27-35
• 15 Iwashita K, Kobori M, Yamaki K, Tsushida T. Flavonoids inhibit cell growth and induce apoptosis in B16 melanoma 4A5 cells. Biosci Biotechnol Biochem 2000; 64: 1813-1820
• 16 Zhou GS, Song LJ, Yang B. Isoliquiritigenin inhibits proliferation and induces apoptosis of U87 human glioma cells in vitro . Mol Med Rep 2013; 7: 531-536
• 17 Chen X, Wu Y, Jiang Y, Zhou Y, Wang Y, Yao Y, Yi C, Gou L, Yang J. Isoliquiritigenin inhibits the growth of multiple myeloma via blocking IL-6 signaling. J Mol Med 2012; 90: 1311-1319
• 18 Kim D, Ramachandran S, Baek S, Kwon SH, Kwon KY, Cha SD, Bae I, Cho CH. Induction of growth inhibition and apoptosis in human uterine leiomyoma cells by isoliquiritigenin. Reprod Sci 2008; 15: 552-558
• 19 Auyeung KKW, Ko JKS. Novel herbal flavonoids promote apoptosis but differentially induce cell cycle arrest in human colon cancer cell. Invest New Drugs 2010; 28: 1-13
• 20 Hsu YL, Chia CC, Chen PJ, Huang SE, Huang SC, Kuo PL. Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p 53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells. Mol Nutr Food Res 2009; 53: 826-835
• 21 Yuan X, Zhang B, Gan L, Wang ZH, Yu BC, Liu LL, Zheng QS, Wang ZP. Involvement of the mitochondrion-dependent and the endoplasmic reticulum stress-signaling pathways in isoliquiritigenin-induced apoptosis of HeLa cell. Biomed Environ Sci 2013; 26: 268-276
• 22 Zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002; 2: 342-350
• 23 Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p 53. Cell 1993; 75: 495-505
• 24 Filippova M, Song H, Connolly JL, Dermody TS, Duerksen-Hughes PJ. The human papillomavirus 16 E6 protein binds to tumor necrosis factor (TNF) R1 and protects cells from TNF-induced apoptosis. J Biol Chem 2002; 277: 21730-21739
• 25 Filippova M, Parkhurst L, Duerksen-Hughes PJ. The human papillomavirus 16 E6 protein binds to Fas-associated death domain and protects cells from Fas-triggered apoptosis. J Biol Chem 2004; 279: 25729-25744
• 26 Goodwin EC, DiMaio D. Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways. Proc Natl Acad Sci U S A 2000; 97: 12513-12518
• 27 Vogt M, Butz K, Dymalla S, Semzow J, Hoppe-Seyler F. Inhibition of Bax activity is crucial for the antiapoptotic function of the human papillomavirus E6 oncoprotein. Oncogene 2006; 25: 4009-4015
• 28 Hamada K, Sakaue M, Alemany R, Zhang WW, Horio Y, Roth JA, Mitchell MF. Adenovirus-mediated transfer of HPV 16 E6/E7 antisense RNA to human cervical cancer cells. Gynecol Oncol 1996; 63: 219-227
• 29 Pattillo RA, Hussa RO, Story MT, Ruckert AC, Shalaby MR, Mattingly RF. Tumor antigen and human chorionic gonadotropin in CaSki cells: a new epidermoid cervical cancer cell line. Science 1977; 196: 1456-1458
• 30 Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ, Muñoz N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999; 189: 12-19
• 31 Bernard B, Fest T, Prétet JL, Mougin C. Staurosporine-induced apoptosis of HPV positive and negative human cervical cancer cells from different points in the cell cycle. Cell Death Differ 2001; 8: 234-244
• 32 Bernard B, Prétet JL, Charlot JF, Mougin C. Human papillomaviruses type 16+ and 18+ cervical carcinoma cells are sensitive to staurosporine-mediated apoptosis. Biol Cell 2003; 95: 17-26
• 33 Charlot JF, Prétet JL, Haughey C, Mougin C. Mitochondrial translocation of p 53 and mitochondrial membrane potential (Delta Psi m) dissipation are early events in staurosporine-induced apoptosis of wild type and mutated p 53 epithelial cells. Apoptosis 2004; 9: 333-343
• 34 Charlot JF, Nicolier M, Prétet JL, Mougin C. Modulation of p 53 transcriptional activity by PRIMA-1 and Pifithrin-alpha on staurosporine-induced apoptosis of wild-type and mutated p 53 epithelial cells. Apoptosis 2006; 11: 813-827
• 35 Nicolier M, Decrion-Barthod AZ, Launay S, Prétet JL, Mougin C. Spatiotemporal activation of caspase-dependent and -independent pathways in staurosporine-induced apoptosis of p 53 wt and p 53 mt human cervical carcinoma cells. Biol Cell 2009; 101: 455-467
• 36 Kroemer G, El-Deiry WS, Golstein P, Peter ME, Vaux D, Vandenabeele P, Zhivotovsky B, Blagosklonny MV, Malorni W, Knight RA, Piacentini M, Nagata S, Melino G. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death. Cell Death Differ 2005; 12 (Suppl. 02) 1463-1467
• 37 Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 2006; 25: 4798-4811
• 38 Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilization in cell death. Physiol Rev 2007; 87: 99-163
• 39 Vousden KH, Lu X. Live or let die: the cellʼs response to p 53. Nat Rev Cancer 2002; 2: 594-604
• 40 Ohtsuka T, Ryu H, Minamishima YA, Macip S, Sagara J, Nakayama KI, Aaronson SA, Lee SW. ASC is a Bax adaptor and regulates the p 53-Bax mitochondrial apoptosis pathway. Nat Cell Biol 2004; 6: 121-128
• 41 Wu Y, Mehew JW, Heckman CA, Arcinas M, Boxer LM. Negative regulation of bcl-2 expression by p 53 in hematopoietic cells. Oncogene 2001; 20: 240-251
• 42 Tomita Y, Marchenko N, Erster S, Nemajerova A, Dehner A, Klein C, Pan H, Kessler H, Pancoska P, Moll UM. WT p 53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization. J Biol Chem 2006; 281: 8600-8606
• 43 Leu JI, Dumont P, Hafey M, Murphy ME, George DL. Mitochondrial p 53 activates Bak and causes disruption of a Bak-Mcl1 complex. Nat Cell Biol 2004; 6: 443-450
• 44 Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Pancoska P, Moll UM. p 53 has a direct apoptogenic role at the mitochondria. Mol Cell 2003; 11: 577-590
• 45 Garnett TO, Filippova M, Duerksen-Hughes PJ. Accelerated degradation of FADD and procaspase 8 in cells expressing human papilloma virus 16 E6 impairs TRAIL-mediated apoptosis. Cell Death Differ 2006; 13: 1915-1926
• 46 Bennett M, Macdonald K, Chan SW, Luzio JP, Simari R, Weissberg P. Cell surface trafficking of Fas: a rapid mechanism of p 53-mediated apoptosis. Science 1998; 282: 290-293
• 47 Jong JE, Jeong KW, Shin H, Hwang LR, Lee D, Seo T. Human papillomavirus type 16 E6 protein inhibits DNA fragmentation via interaction with DNA fragmentation factor 40. Cancer Lett 2012; 324: 109-117
• 48 Kalantari M, Calleja-Macias IE, Tewari D, Hagmar B, Lie K, Barrera-Saldana HA, Wiley DJ, Bernard HU. Conserved methylation patterns of human papillomavirus type 16 DNA in asymptomatic infection and cervical neoplasia. J Virol 2004; 78: 12762-12772
• 49 Bhattacharjee B, Sengupta S. CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2. Virology 2006; 354: 280-285
• 50 Ding DC, Chiang MH, Lai HC, Hsiung CA, Hsieh CY, Chu TY. Methylation of the long control region of HPV16 is related to the severity of cervical neoplasia. Eur J Obstet Gynecol Reprod Biol 2009; 147: 215-220
• 51 Jacquin E, Baraquin A, Ramanah R, Carcopino X, Morel A, Valmary-Degano S, Bravo I, de Sanjose S, Riethmuller D, Mougin C, Prétet JL. Methylation of human papillomavirus type 16 E2BS#1, E2BS#2 and Sp1 binding site CpGs in cervical cancer samples determined by high resolution melting PCR. J Clin Microbiol 2013; 51: 3207-3215
• 52 Zorko BA, Pérez LB, De Blanco EJC. Effects of ILTG on DAPK1 promoter methylation in colon and leukemia cancer cell lines. Anticancer Res 2010; 30: 3945-3950
• 53 Orlikova B, Schnekenburger M, Zloh M, Golais F, Diederich M, Tasdemir D. Natural chalcones as dual inhibitors of HDACs and NF-κB. Oncol Rep 2012; 28: 797-805
• 54 Nair A, Venkatraman M, Maliekal TT, Nair B, Karunagaran D. NF-kappaB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix. Oncogene 2003; 22: 50-58
• 55 Branca M, Giorgi C, Ciotti M, Santini D, Di Bonito L, Costa S, Benedetto A, Bonifacio D, Di Bonito P, Paba P, Accardi L, Mariani L, Ruutu M, Syrjänen S, Favalli C, Syrjänen K. Upregulation of telomerase (hTERT) is related to the grade of cervical intraepithelial neoplasia, but is not an independent predictor of high-risk human papillomavirus, virus persistence, or disease outcome in cervical cancer. Diagn Cytopathol 2006; 34: 739-748
• 56 Xu M, Katzenellenbogen RA, Grandori C, Galloway DA. NFX1 plays a role in human papillomavirus type 16 E6 activation of NFkappaB activity. J Virol 2010; 84: 11461-11469
• 57 Lin Z, Bazzaro M, Wang MC, Chan KC, Peng S, Roden RBS. Combination of proteasome and HDAC inhibitors for uterine cervical cancer treatment. Clin Cancer Res 2009; 15: 570-577
• 58 Hong-zhi L, Mamat A, Ablise M, Niyaz U, Turdi R. Synthesis and characterisation of isoliquiritigenin derivatives. Chin J New Drugs 2009; 19: 1882-1886
• 59 Saunier M, Monnier-Benoit S, Mauny F, Dalstein V, Briolat J, Riethmuller D, Kantelip B, Schwarz E, Mougin C, Prétet JL. Analysis of human papillomavirus type 16 (HPV16) DNA load and physical state for identification of HPV16-infected women with high-grade lesions or cervical carcinoma. J Clin Microbiol 2008; 46: 3678-3685