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.

SESN2 Knockdown Increases Betulinic Acid-Induced Radiosensitivity of Hypoxic Breast Cancer Cells.

Betulinic acid (BA) is a natural compound well known for its anti-inflammatory, anti-viral, anti-bacterial, anti-malarial effects and anti-tumor properties. Its enhanced cytotoxicity in tumor cells and induction of cell death in various cancer entities qualifies BA as an interesting candidate for novel treatment concepts. Our analyses showed enhanced cytotoxicity and radiosensitization under hypoxic conditions in human breast cancer cells. So far, the underlying mechanisms are unknown. Therefore, we investigated the BA-treated human breast cancer cell lines MDA-MB-231 and MCF-7 under normoxic and hypoxic conditions based on microarray technology. Hypoxia and BA regulated a variety of genes in both breast cancer cell lines. KEGG pathway analysis identified an enrichment of the p53 pathway in MCF-7 cells (wtp53) under hypoxia. In MDA-MB-231 cells (mtp53) an additional BA incubation was required to activate the p53 signaling pathway. Fourteen down-regulated and up-regulated genes of the p53 pathway were selected for further validation via qRT-PCR in a panel of five breast cancer cell lines. The stress-induced gene Sestrin-2 (SESN2) was identified as one of the most strongly up-regulated genes after BA treatment. Knockdown of SESN2 enhanced BA-induced ROS production, DNA damage, radiosensitivity and reduced autophagy in breast cancer cells. Our results identified SESN2 as an important target to enhance the radiobiological and anti-tumor effects of BA on breast cancer cells.

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.

Evaluation of the Betulinic Acid-Cisplatin conjugate APC and its precursor DE9B for the treatment of human malignant glioma.

Despite the existence of multimodal therapy concepts, glioblastoma remains a tumor type with one of the worst prognoses. In particular, the poor prognosis is due to the lack of therapeutic efficacy of chemical agents and irradiation in hypoxic tumor areas. New therapeutic strategies could improve the treatment of glioblastoma. In this study, we investigated the therapeutic efficacy of a conjugate of cisplatin (DDP), a widely used chemotherapeutic agent, and betulinic acid (BA), a natural product from plane tree bark, in glioblastoma cells under different oxygen conditions. We investigated the effects of the BA-DDP conjugate κN',N''-{3-acetyloxy-BA-28-[2-(2-aminoethyl)aminoethyl]amide} dichlorido platinum(II) (APC) and its precursor 3-acetyloxy-BA-28-[2-(2-aminoethyl)aminoethyl]amide (DE9B) on cytotoxicity, cell growth, apoptosis, migration and radiosensitivity compared to BA or DDP alone under different oxygen conditions. Based on the EC50 values, the precursor DE9B exhibited the strongest cytotoxic effects of the analyzed chemotherapeutic agents. The BA-DDP conjugate APC achieved a moderate cytotoxic effect in glioma cells. Both of the newly developed agents induced cell growth delay, apoptosis and inhibition of migration. Furthermore, additive effects could be achieved in combination with irradiation. In contrast to those of BA and DDP, the cell biological effects of APC and DE9B were not influenced by the oxygen concentration. In this study, the linking of BA and DDP did not produce a compound with additive therapeutic effects on glioblastoma cell lines in vitro. Nevertheless, the results of this study suggest that the precursor DE9B is an effective BA derivative for the treatment of glioblastoma in vitro.

Radiosensitization and a Less Aggressive Phenotype of Human Malignant Glioma Cells Expressing Isocitrate Dehydrogenase 1 (IDH1) Mutant Protein: Dissecting the Mechanisms.

The presence of an isocitrate dehydrogenase 1 (IDH1) mutation is associated with a less aggressive phenotype, increased sensitivity to radiation, and increased overall survival in patients with diffuse glioma. Based on in vitro experimentations in malignant glioma cell lines, the consequences on cellular processes of IDH1R132H expression were analyzed. The results revealed that IDH1R132H expression enhanced the radiation induced accumulation of residual γH2AX foci and decreased the amount of glutathione (GSH) independent of the oxygen status. In addition, expression of the mutant IDH1 caused a significant increase of cell stiffness and induced an altered organization of the cytoskeleton, which has been shown to reinforce cell stiffness. Furthermore, IDH1R132H expression decreased the expression of vimentin, an important component of the cytoskeleton and regulator of the cell stiffness. The results emphasize the important role of mutant IDH1 in treatment of patients with diffuse gliomas especially in response to radiation. Hence, detection of the genetic status of IDH1 before therapy massively expands the utility of immunohistochemistry to accurately distinguish patients with a less aggressive and radiosensitive IDH1-mutant diffuse glioma suitable for radiotherapy from those with a more aggressive IDH1-wildtype diffuse glioma who might benefit from an individually intensified therapy comprising radiotherapy and alternative medical treatments.