Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells

Abstract

Selective inhibition with sulphonamides of carbonic anhydrase (CA) IX reduces cell proliferation and induces apoptosis in human cancer cells. The effect on CA IX expression of seven previously synthesised sulphonamide inhibitors, with high affinity for CA IX, as well as their effect on the proliferation/apoptosis of cancer/normal cell lines was investigated. Two normal and three human cancer cell lines were used. Treatment resulted in dose- and time-dependent inhibition of the growth of various cancer cell lines. One compound showed remarkably high toxicity towards CA IX-positive HeLa cells. The mechanisms of apoptosis induction were determined with Annexin-V and AO/EB staining, cleaved caspases (caspase-3, caspase-8, caspase-9) and cleaved PARP activation, reactive oxygen species production (ROS), mitochondrial membrane potential (MMP), intracellular pH (pHi), extracellular pH (pHe), lactate level and cell cycle analysis. The autophagy induction mechanisms were also investigated. The modulation of apoptotic and autophagic genes (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin and LC3) was measured using real time PCR. The positive staining using γ-H2AX and AO/EB dye, showed increased cleaved caspase-3, caspase-8, caspase-9, increased ROS production, MMP and enhanced mRNA expression of apoptotic genes, suggesting that anticancer effects are also exerted through its apoptosis-inducing properties. Our results show that such sulphonamides might have the potential as new leads for detailed investigations against CA IX-positive cervical cancers.

Progress in the development of human carbonic anhydrase inhibitors and their pharmacological applications: Where are we today?

“…Moreover, compound 382 also induced ROS production and arrested the Sub-G1 phase cell cycle in HeLa cells. Overall, these extensive studies indicated promising anticancer activity of compound 380 which may appear beneficial to develop this molecule as a potent chemotherapeutic agent in future 337.…”

Inhibitory activity against carbonic anhydrase IX and XII as a candidate selection criterion in the development of new anticancer agents

“…Disappointingly, a large fraction of publications on the subject reported compounds' cytotoxicity data obtained in normoxic conditions thus making the involvement of hCA IX and XII debateable [30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45] . Although such testing can be relevant in some cases, we consider these conditions irrelevant as long as antiproliferative activity of hCA inhibitors is concerned.…”

Assessment of the antiproliferative and apoptotic roles of sulfonamide carbonic anhydrase IX inhibitors in HeLa cancer cell line

“…Increasing ROS and NRF-2 levels are indicative of increased oxidative stress in these cells. NRF-2 is an important transcription factor regulating the gene sequence of antioxidant enzymes that protects the cells from increased free radicals via activation of the antioxidant systems 14 , 15 . In our study, the increase in NRF-2 confirms the increase in intracellular ROS in HeLa cells after administration of sulfonamide A1 .…”

“…In a previous study, we have reported the synthesis and inhibitory activity against carbonic anhydrase isoforms I, II, IX, and XII of new sulfonamide derivatives 13 . Furthermore, their cytotoxic effects were examined on several cancer cell lines as well as normal cells 14 , 15 . In this study, six different synthesised imine and amine sulfonamide derivatives with documented CA IX inhibitor activity 13 have been tested in terms of their cytotoxic effects in cancer cells (HT-29, HeLa and MDA-MB-231), and in normal cells (PNT1A, HEK-293).…”