Emodin Sensitizes Cervical Cancer Cells to Vinblastine by Inducing Apoptosis and Mitotic Death.

In recent years, studies on the effects of combining novel plant compounds with cytostatics used in cancer therapy have received considerable attention. Since emodin sensitizes tumor cells to chemotherapeutics, we evaluated changes in cervical cancer cells after its combination with the antimitotic drug vinblastine. Cellular changes were demonstrated using optical, fluorescence, confocal and electron microscopy. Cell viability was assessed by MTT assay. The level of apoptosis, caspase 3/7, Bcl-2 protein, ROS, mitochondrial membrane depolarization, cell cycle and degree of DNA damage were analyzed by flow cytometry. The microscopic image showed indicators characteristic for emodin- and vinblastine-induced mitotic catastrophe, i.e., multinucleated cells, giant cells, cells with micronuclei, and abnormal mitotic figures. These compounds also increased blocking of cells in the G2/M phase, and the generated ROS induced swelling and mitochondrial damage. This translated into the growth of apoptotic cells with active caspase 3/7 and inactivation of Bcl-2 protein and active ATM kinase. Emodin potentiated the cytotoxic effect of vinblastine, increasing oxidative stress, mitotic catastrophe and apoptosis. Preliminary studies show that the combined action of both compounds, may constitute an interesting form of anticancer therapy.

The Multidirectional Effect of Azelastine Hydrochloride on Cervical Cancer Cells.

A major cause of cancer cell resistance to chemotherapeutics is the blocking of apoptosis and induction of autophagy in the context of cell adaptation and survival. Therefore, new compounds are being sought, also among drugs that are commonly used in other therapies. Due to the involvement of histamine in the regulation of processes occurring during the development of many types of cancer, antihistamines are now receiving special attention. Our study concerned the identification of new mechanisms of action of azelastine hydrochloride, used in antiallergic treatment. The study was performed on HeLa cells treated with different concentrations of azelastine (15-90 µM). Cell cycle, level of autophagy (LC3 protein activity) and apoptosis (annexin V assay), activity of caspase 3/7, anti-apoptotic protein of Bcl-2 family, ROS concentration, measurement of mitochondrial membrane potential (Δψm), and level of phosphorylated H2A.X in response to DSB were evaluated by cytometric method. Cellular changes were also demonstrated at the level of transmission electron microscopy and optical and fluorescence microscopy. Lysosomal enzyme activities-cathepsin D and L and cell viability (MTT assay) were assessed spectrophotometrically. Results: Azelastine in concentrations of 15-25 µM induced degradation processes, vacuolization, increase in cathepsin D and L activity, and LC3 protein activation. By increasing ROS, it also caused DNA damage and blocked cells in the S phase of the cell cycle. At the concentrations of 45-90 µM, azelastine clearly promoted apoptosis by activation of caspase 3/7 and inactivation of Bcl-2 protein. Fragmentation of cell nucleus was confirmed by DAPI staining. Changes were also found in the endoplasmic reticulum and mitochondria, whose damage was confirmed by staining with rhodamine 123 and in the MTT test. Azelastine decreased the mitotic index and induced mitotic catastrophe. Studies demonstrated the multidirectional effects of azelastine on HeLa cells, including anti-proliferative, cytotoxic, autophagic, and apoptotic properties, which were the predominant mechanism of death. The revealed novel properties of azelastine may be practically used in anti-cancer therapy in the future.