Betulinyl Sulfamates as Anticancer Agents and Radiosensitizers in Human Breast Cancer Cells

Abstract

Betulinic acid (BA), a natural compound of birch bark, is cytotoxic for many tumors. Recently, a betulinyl sulfamate was described that inhibits carbonic anhydrases (CA), such as CAIX, an attractive target for tumor-selective therapy strategies in hypoxic cancer cells. Data on combined CAIX inhibition with radiotherapy are rare. In the human breast cancer cell lines MDA-MB231 and MCF7, the effects of BA and betulinyl sulfamates on cellular and radiobiological behavior under normoxia and hypoxia were evaluated. The two most effective betulinyl sulfamates CAI 1 and CAI 3 demonstrated a 1.8–2.8-fold higher cytotoxicity than BA under normoxia in breast cancer cells, with IC50 values between 11.1 and 18.1 µM. BA exhibits its strongest cytotoxicity with IC50 values of 8.2 and 16.4 µM under hypoxia. All three substances show a dose-dependent increase in apoptosis, inhibition of migration, and inhibition of hypoxia-induced gene expression. In combination with irradiation, betulinyl sulfamates act as radiosensitizers, with DMF10 values of 1.47 (CAI 1) and 1.75 (CAI 3) under hypoxia in MDA-MB231 cells. BA showed additive effects in combination with irradiation. Taken together; our results suggest that BA and betulinyl sulfamates seem to be attractive substances to combine with radiotherapy; particularly for hypoxic breast cancer.

Cellular and radiobiological effects of carbonic anhydrase IX in human breast cancer cells

“…2 and 3). These effects are in line with previous reports that also demonstrated reductions in CA9 mRNA and CAIX protein level in tumor cells in response to treatment with RNA interference or CAI U104 (10,21,22,(34)(35)(36). CAI-induced changes were attributed to the possible degradation or internalization of CAIX (21,36).…”

“…Initial studies of a colorectal carcinoma xenograft model revealed that CAIX inhibition with different CAIX inhibitors (CAI: Acetazolamide, DH348 or 11c) or CA9 knockdown by shRNA delayed tumor growth and radiosensitized tumor cells (16,17). These findings corroborate other studies showing that inhibition of carbonic anhydrases with different CAIs radiosensitized different tumor cell lines (21,35,42). This effect may be due to the reduced acidification of the extracellular environment (16,17) or the intracellular acidification ( Fig.…”

“…This study of rat prostate cancer cells confirmed the intracellular acidification, anti-proliferative and pro-apoptotic effects of CAIX inhibition with CAI U104 (29). In addition, recently, our previous results confirmed CAI-induced apoptosis for betulinyl sulfamates in MDA-MB-231 and MCF7 cells using different methods (21,40). A further study detected CAI-induced apoptosis in different cancer cell lines (41).…”

“…In the present study, inhibition of CAIX was performed by two alternative strategies, namely treatment with CA9 siRNA or exposure to the CAIX/CAXII selective inhibitor U104, which is currently being tested in a phase I clinical trial in patients with advanced solid tumors (19). Our previous study indicated that betulin 3,28-disulfamate, which is described as a CAI by Winum et al (20), radiosensitized MDA-MB-231 breast cancer cells (21). Recently, CAI FC9403A but not CAI S4 showed synergistic effects with irradiation in MDA-MB-231 breast cancer spheroids (22).…”

“…Sulforhodamine B assay. The SRB assay was performed as previously described (21). MDA-MB-231 and MCF-7 cells were seeded in 96-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) and treated with CA9 siRNA or CAI U104 (10, 100 and 250 µM) under hypoxic conditions for 72 h. The cells were then fixed, washed and dyed with 0.4% sulforhodamine, and absorbance was measured at 540 nm using a GENios microplate reader (Tecan Group AG, Männedorf, Switzerland).…”