Akalesh Kumar Verma

Changes in glutathione, oxidative stress and mitochondrial membrane potential in apoptosis involving the anticancer activity of cantharidin isolated from redheaded blister beetles, epicauta hirticornis.

The present work describes the anticancer activity of cantharidin isolated from red-headed blister beetles, Epicauta hirticornis and its possible mode of action involving induction of apoptosis, oxidative stress and decrease in glutathione against murine ascites Daltons lymphoma. The structure of isolated compound was confirmed as cantharidin by X-ray diffraction method. Cantharidin treatment showed potent anticancer activity with an increase in life span (~ 87%) of tumor-bearing mice. Cantharidin treatment induced apoptosis in Daltons lymphoma cells and also caused an oxidative stress due to generation of reactive oxygen species (ROS) and an increase in lipid peroxidation. The observed canthardin-mediated decrease in glutathione and glutathione related enzymes activities in the tumor cells may weaken the cellular antioxidant system. Moreover, cantharidin treatment also caused a significant decrease in mitochondrial cytochrome c and simultaneous increase in cytosolic cytochrome c which ultimately facilitates activation of caspase 9 and 3 to augment mitochondrial apoptotic pathway causing cancer cell death. Based on the present findings, it may be suggested that cantharidin-mediated anticancer activity could be due to decrease in the protective ability of cancer cells by ROS and subsequent activation of effecter caspases leading to apoptotic cell death.

Cantharidin-Mediated Ultrastructural and Biochemical Changes in Mitochondria Lead to Apoptosis and Necrosis in Murine Dalton's Lymphoma

Cantharidin, a type of terpenoid, is the blistering agent of blister beetles frequently used in traditional medicine. The isolation and anticancer activity of cantharidin from blister beetles, Mylabris cichorii has been recently reported by us. This study deals with changes in mitochondrial structure and function and understanding their significance in the underlying mechanism(s) in cantharidin-mediated antitumor effects in Daltons lymphoma (DL) bearing mice. Cantharidin treatment caused the appearance of abnormal mitochondrial features which included roundish mitochondria with thickened membranes, irregularity in cristae, and appearance of small and large size vacuoles in mitochondria of DL cells. Cantharidin treatment resulted in a decrease in mitochondrial reduced glutathione, succinate dehydrogenase activity, mitochondrial membrane potential, and induced apoptosis and necrosis in DL cells. The decrease/release of mitochondrial cytochrome c were also observed after cantharidin treatment. Flow cytometry-based cell cycle analysis showed a time-dependent accumulation of the sub-G0 population of DL cells, thus, confirming the involvement of apoptosis in tumor cells in cantharidin-mediated antitumor activity. These finding signify that the apoptosis induced by cantharidin in DL cells should involve mitochondrial-dependent pathways. It is suggested that these cantharidin-mediated changes in mitochondria may play a crucial role in its antitumor activity.

Antitumor effect of blister beetles: an ethno-medicinal practice in Karbi community and its experimental evaluation against a murine malignant tumor model.

ETHNOPHARMACOLOGICAL IMPORTANCE The blister beetles Epicauta hirticornis and Mylabris cichorii are used as a folk medicine by the Karbi tribe in Karbi Anglong district of Assam, India for the treatment of different human ailments, including cancer cases. AIM OF THE STUDY It includes field survey related to zoo-therapeutic aspects of two blister beetles in Karbi community, isolation of bio-active compound and evaluation of its antitumor potential with possible mode of action against murine Ehrlich ascites carcinoma (EAC). MATERIALS AND METHODS The main bio-active compound of blister beetles was isolated from ethyl acetate extract and the structure was confirmed as cantharidin using NMR, IR, Mass and X-ray diffractometer. The effect of cantharidin on apoptosis, necrosis, autophagy and the apoptosis related signaling pathways were determined using different bioassays, including cell cycle analysis, mitochondrial membrane potential, western blot analysis of cytochrome c, caspases 9, 3/7 assays, and lactate dehydrogenase (LDH) assay. RESULTS Cantharidin induced apoptosis, necrosis and autophagy cell death in EAC cells. The decrease in mitochondrial membrane potential was observed, which may help to release cytochrome c from mitochondria to cytosol. Cantharidin treatment caused up-regulation of caspases 9 and -3/7 and a decrease in LDH activity in EAC cells. CONCLUSION The major bioactive compound of these blister beetles is cantharidin which induces severe apoptosis in EAC cells involving mitochondrial intrinsic pathway. Cantharidin-mediated inhibition of LDH activity may lead to short supply of NAD(+) and cut off energy and anabolic supply to cancer cells.

Homology Modeling and characterization of Phosphoenolpyruvate Carboxykinase (PEPCK) from Schistosoma japonicum

Phosphoenolpyruvate carboxykinase (PEPCK), a carboxylase enzyme is present in all living organisms. It catalyzes metal-nucleotide coupled reversible decarboxylation and phosphorylation between phosphoenolpyruvate (PEP) and oxaloacetate (OAA) depending on the system and the availability of the intermediates. In fungi, plants and in most bacteria, production of PEP from OAA by PEPCK is the key step during gluconeogenesis. In healthy human cytosolic PEPCK enzyme is present only during glucose starvation; cytosolic PEPCK rapidly disappears on the replenishment of glucose due to hormonal control of the transcription of the cytosolic PEPCK- gene. In some parasitic helminthes like Ascaris suum, nematodes such as Haemonchus contortus, PEPCK carry out the reverse reaction to produce OAA from PEP. In Trypanosoma cruzi and all species of the genus Leishmania, this enzyme is very active even in the presence of high level of carbohydrate. There is a significant functional difference between parasitic PEPCK and mammalian host PEPCK. These differences between the mammalian host and the parasite enzyme strongly support the belief that PEPCK should be further investigated as a possible target for selective chemotherapeutic agents. The experimental 3D structure of PEPCK of Schistosoma japonicum is not available in protein data bank. Therefore, based on the knowledge of the best template (3DT7), model of 3D structure of Schistosoma japonicum PEPCK was prepared using Modeller v9.10 software and processed in to energy minimization, Ramachandran plot analysis, quality assessment and characterization.

Dietary Ascorbic Acid-Mediated Augmentation of Antitumor Activity and Protection Against Toxicities Induced by Cis-Diamminedichloroplatinum-(II) in Dalton’s Lymphoma-Bearing Mice

Abstract: Cis -Diamminedichloroplatinum-(II) (CDDP) commonly known as cisplatin is considered as a major anticancer drug against a broad spectrum of malignancies. This study evaluates the modulatory effect of dietary ascorbic acid (AA) on the therapeutic efficacy of CDDP against murine ascites Dalton’s lymphoma (DL) and some tissue toxicities in tumor-bearing mice. As compared to CDDP alone, the combination treatment with ascorbic acid (AA) plus CDDP showed better therapeutic efficacy against murine ascites Dalton’s lymphoma. DL cells treated with CDDP showed the appearance of apoptotic features involving fragmentation of nucleus into discrete masses and plasma membrane blebbing. As compared to CDDP alone, combination treatment caused an increase in the number of apoptotic DL cells. Reduced glutathione (GSH) level was noted to decrease in DL cells while it increased in kidney after combination treatment. Blood haemoglobin (Hb), red blood cells (RBCs) and white blood cells (eosinophils, basophils and lymphocytes) were also decreased after CDDP treatment while overall betterment in hematological parameters was noted after combination treatment. The analysis of renal function tests (RFT) and liver function tests (LFT) suggest an improvement against CDDP-induced liver and kidney toxicities after combination treatment. The decrease in GSH levels particularly in DL cells and an increase in kidney and liver after combination treatment may have a role in the antitumor activity and decrease in CDDP-induced toxicity in the tumor-bearing host. Improvement in the LFT, RFT and hematological toxicities after combination treatment may have a beneficial effect in the improved survival of tumor-bearing mice.