Combination of Betulinic Acid Fragments and Carbonic Anhydrase Inhibitors-A New Drug Targeting Approach.

Human carbonic anhydrase IX (hCA IX) is a zinc(II)-dependent metalloenzyme that plays a critical role in the conversion of carbon dioxide and water to protons and bicarbonate. It is a membrane-bound protein with an extracellular catalytic center that is predominantly overexpressed in solid hypoxic tumors. Sulfamates and sulfonamides, for example acetazolamide (AZA), have been used to inhibit hCA IX in order to improve the response to solid hypoxic tumors. In the present study, we propose a new drug targeting approach by attaching the natural cytotoxic substances betulin and betulinic acid (BA) via a linker to sulfonamides. The conjugate was designed with different spacer lengths to accumulate at the target site of hCA IX. Computational and cell biological studies suggest that the length of the linker may influence hCA IX inhibition. Cytotoxicity tests of the newly synthesized bifunctional conjugates 3, 5, and 9 show effective cytotoxicity in the range of 6.4 and 30.1 µM in 2D and 3D tumor models. The hCA IX inhibition constants of this conjugates, measured using an in vitro enzyme assay with p-nitrophenyl acetate, were determined in a low µM-range, and all compounds reveal a significant inhibition of hypoxia-induced CA activity in a cell-based assay using the Wilbur-Anderson method. In addition, the cells respond with G1 increase and apoptosis induction. Overall, the dual strategy to produce cytotoxic tumor therapeutics that inhibit tumor-associated hCA IX was successfully implemented.

MSBA-S - A pentacyclic sulfamate as a new option for radiotherapy of human breast cancer cells.

Many pentacyclic triterpenoids show anti-cancer and anti-inflammatory properties. Recently, we detected a pronounced cytotoxicity and radiosensitivity of two betulinyl sulfamates in human breast cancer cells. Besides betulinic acid scaffold (BSBA-S), we synthesized several new sulfamate-coupled scaffolds from oleanolic acid (OSBA-S), ursolic acid (USBA-S), platanic acid (PSBA-S) and maslinic acid (MSBA-S). Highest cytotoxicity was monitored in breast cancer cell lines after MSBA-S treatment showing in SRB assays IC50 values between 3.7 µM and 5.8 µM. Other sulfamate/triterpene conjugates, however, were less cytotoxic holding IC50 values between 6.6 µM and >50 µM, respectively. MSBA-S-treated breast cancer cells displayed significantly reduced clonogenic survival and an increased rate of apoptosis as compared to the other conjugates. In addition, MSBA-S in combination with irradiation resulted in effects on radiosensitivity in MDA-MB-231 cells (DMF10 = 1.14). In particular, ROS formation was strongly assessed in MSBA-S-treated breast cancer cells. Our findings suggest that the sulfamate derivative of maslinic acid MSBA-S might be a new option for the radiation therapy in breast cancer cells.

Betulin Sulfonamides as Carbonic Anhydrase Inhibitors and Anticancer Agents in Breast Cancer Cells.

Hypoxia-regulated protein carbonic anhydrase IX (CA IX) is up-regulated in different tumor entities and correlated with poor prognosis in breast cancer patients. Due to the radio- and chemotherapy resistance of solid hypoxic tumors, derivatives of betulinic acid (BA), a natural compound with anticancer properties, seem to be promising to benefit these cancer patients. We synthesized new betulin sulfonamides and determined their cytotoxicity in different breast cancer cell lines. Additionally, we investigated their effects on clonogenic survival, cell death, extracellular pH, HIF-1α, CA IX and CA XII protein levels and radiosensitivity. Our study revealed that cytotoxicity increased after treatment with the betulin sulfonamides compared to BA or their precursors, especially in triple-negative breast cancer (TNBC) cells. CA IX activity as well as CA IX and CA XII protein levels were reduced by the betulin sulfonamides. We observed elevated inhibitory efficiency against protumorigenic processes such as proliferation and clonogenic survival and the promotion of cell death and radiosensitivity compared to the precursor derivatives. In particular, TNBC cells showed benefit from the addition of sulfonamides onto BA and revealed that betulin sulfonamides are promising compounds to treat more aggressive breast cancers, or are at the same level against less aggressive breast cancer cells.

Combined 3-O-acetylbetulin treatment and carbonic anhydrase IX inhibition results in additive effects on human breast cancer cells.

Hypoxia plays a key role in tumor progression and resistance to radiotherapy. Expression of the transmembrane-tethered enzyme carbonic anhydrase IX (CA IX) is strongly induced by hypoxia. High CA IX expression levels correlate with poor prognosis in cancer patients. Previously, we showed that the downregulation of CA IX expression by siRNA interference and the inhibition of CA IX activity results in increased cytotoxicity, inhibition of migration and radiosensitization of hypoxic cancer cells. Betulinic acid (BA) is a natural compound derived from birch bark. It has shown promising anti-tumor effects due to its cancer cell specific cytotoxic properties. We have shown that BA inhibits the HIF-1α pathway, resulting in apoptosis, inhibition of migration and enhanced cytotoxicity of breast cancer cells. In this study, we investigate the effects of the novel betulin derivative 3-O-acetylbetulin (3-AC) and carbonic anhydrase inhibitors (CAI) octyl disulfamate (OCT) or 4-(3-[4-fluorophenyl]ureido)benzenesulfonamide (SLC-0111), on cellular and radiobiological parameters in MDA-MB-231 and MCF-7 cells. Treatment with 3-AC or OCT alone only caused moderate cytotoxicity, reduction in cell migration, ROS production and DNA damage. However, the combined treatment with 3-AC and CAI strongly enhanced radiosensitivity, increased cytotoxicity, inhibited cell motility and enhanced DNA damage. Our findings suggest that the combination of two bioactive drugs 3-AC and a CAI, such as OCT or SLC-0111, could be a promising therapeutic approach for targeting hypoxic tumor cells.

In vitro assessment of triterpenoids NVX-207 and betulinyl-bis-sulfamate as a topical treatment for equine skin cancer.

Equine sarcoid (ES) is the most prevalent skin tumor in equids worldwide. Additionally, aging grey horses frequently suffer from equine malignant melanoma (EMM). Current local therapies targeting these skin tumors remain challenging. Therefore, more feasible topical treatment options should be considered. In order to develop a topical therapy against ES and EMM, betulinyl-bis-sulfamate and NVX-207, derivatives of the naturally occurring betulin and betulinic acid, respectively, were evaluated for their antiproliferative (crystal violet staining assay), cytotoxic (MTS assay) and apoptotic (AnnexinV staining, cell cycle investigations) effects on primary ES cells, EMM cells and equine dermal fibroblasts in vitro. The more potent derivative was assessed for its in vitro penetration and permeation on isolated equine skin within 30 min and 24 h using Franz-type diffusion cells and HPLC analysis. Betulinyl-bis-sulfamate and NVX-207 inhibited the proliferation and metabolism in ES cells, EMM cells and fibroblasts significantly (p < 0.001) in a time- and dose-dependent manner. NVX-207 had superior anticancer effects compared to betulinyl-bis-sulfamate. Both compounds led to the externalization of phosphatidylserines on the cell membrane and DNA fragmentation, demonstrating that the effective mode of action was apoptosis. After 48 h of treatment with NVX-207, the number of necrotic cells was less than 2% in all cell types. Detected amounts of NVX-207 in the different skin layers exceeded the half-maximal inhibitory concentrations calculated by far. Even though data obtained in vitro are auspicious, the results are not unconditionally applicable to the clinical situation. Consequently, in vivo studies are required to address the antitumoral effects of topically applied NVX-207 in ES and EMM patients.