Paul B. Tchounwou

Vernonia calvoana Shows Promise towards the Treatment of Ovarian Cancer

The treatment for ovarian cancers includes chemotherapies which use drugs such as cisplatin, paclitaxel, carboplatin, platinum, taxanes, or their combination, and other molecular target therapies. However, these current therapies are often accompanied with side effects. Vernonia calvoana (VC) is a valuable edible medicinal plant that is widespread in West Africa. In vitro data in our lab demonstrated that VC crude extract inhibits human ovarian cancer cells in a dose-dependent manner, suggesting its antitumor activity. From the VC crude extract, we have generated 10 fractions and VC fraction 7 (F7) appears to show the highest antitumor activity towards ovarian cancer cells. However, the mechanisms by which VC F7 exerts its antitumor activity in cancer cells remain largely unknown. We hypothesized that VC F7 inhibits cell proliferation and induces DNA damage and cell cycle arrest in ovarian cells through oxidative stress. To test our hypothesis, we extracted and fractionated VC leaves. The effects of VC F7 were tested in OVCAR-3 cells. Viability was assessed by the means of MTS assay. Cell morphology was analyzed by acridine orange and propidium iodide (AO/PI) dye using a fluorescent microscope. Oxidative stress biomarkers were evaluated by the means of lipid peroxidation, catalase, and glutathione peroxidase assays, respectively. The degree of DNA damage was assessed by comet assay. Cell cycle distribution was assessed by flow cytometry. Data generated from the MTS assay demonstrated that VC F7 inhibits the growth of OVCAR-3 cells in a dose-dependent manner, showing a gradual increase in the loss of viability in VC F7-treated cells. Data obtained from the AO/PI dye assessment revealed morphological alterations and exhibited characteristics such as loss of cellular membrane integrity, cell shrinkage, cell membrane damage, organelle breakdown, and detachment from the culture plate. We observed a significant increase (p < 0.05) in the levels of malondialdhyde (MDA) production in treated cells compared to the control. A gradual decrease in both catalase and glutathione peroxidase activities were observed in the treated cells compared to the control. Data obtained from the comet assay showed a significant increase (p < 0.05) in the percentages of DNA cleavage and comet tail length. The results of the flow cytometry analysis indicated VC F7 treatment caused cell cycle arrest at the S-phase checkpoint. Taken together, our results demonstrate that VC F7 exerts its anticancer activity by inhibiting cell proliferation, inducing DNA damage, and causing cell cycle arrest through oxidative stress in OVAR-3 cells. This finding suggests that VC F7 may be a potential alternative dietary agent for the prevention and/or treatment of ovarian cancer.

p53 as a unique target of action of cisplatin in acute leukaemia cells

AbstractAcute promyelocytic leukaemia (APL) occurs in approximately 10% of acute myeloid leukaemia patients. Arsenic trioxide (ATO) has been for APL chemotherapy, but recently several ATO‐resistant cases have been reported worldwide. Cisplatin (CDDP) enhances the toxicity of ATO in ovarian, lung cancer, chronic myelogenous leukaemia, and HL‐60 cells. Hence, the goal of this study was to investigate a novel target of CDDP action in APL cells, as an alternate option for the treatment of ATO‐resistant APL patients. We applied biochemical, molecular, confocal microscopy and advanced gene editing (CRISPR‐Cas9) techniques to elucidate the novel target of CDDP action and its functional mechanism in APL cells. Our main findings revealed that CDDP activated p53 in APL cells through stress signals catalysed by ATM and ATR protein kinases, CHK1 and CHK2 phosphorylation at Ser 345 and Thr68 residues, and downregulation and dissociation of MDM2‐DAXX‐HAUSP complex. Our functional studies confirmed that CDDP‐induced repression of MDM2‐DAXX‐HAUSP complex was significantly reversed in both nutilin‐3‐treated KG1a and p53‐knockdown NB4 cells. Our findings also showed that CDDP stimulated an increased number of promyelocytes with dense granules, activated p53 expression, and downregulated MDM2 in liver and bone marrow of APL mice. Principal conclusion of our study highlights a novel mode of action of CDDP targeting p53 expression which may provide a basis for designing new anti‐leukaemic compounds for treatment of APL patients.