Calotropis procera extract induces apoptosis and cell cycle arrest at G2/M phase in human skin melanoma (SK-MEL-2) cells.

Calotropis procera (family: Asclepiadaceae) contains cardiac glycosides which are cytotoxic to cancer cells. The extracts of C. procera have been reported to be cytotoxic to many cancer cell lines and this is the first report against the human skin melanoma cells (SK-MEL-2). The SK-MEL-2 cells treated with C. procera methanolic extract (CPME) were analysed for growth inhibition and apoptosis. The exposure of phosphatidylserine in apoptotic SK-MEL-2 was analysed by using the Annexin-V FITC flow cytometry method. In CPME-treated SK-MEL-2 cells, 19.6% of apoptotic and 58.3% dead cells were observed. The 15.97% and 15.85% of early apoptotic cells were found at 20 µg/mL of the ouabain and paclitaxel, respectively. Active caspases, nuclear degradation confirmed apoptotic SK-MEL-2 cells in time- and dose-dependent manner. The cell cycle analysis shows that CPME treated cells halt at G2/M phase. Significant cytotoxic activity of CPME against SK-MEL-2 may be attributed to its high cardenolide content.

Comparative evaluation of the cytotoxic and apoptotic potential of Poecilocerus pictus and Calotropis gigantea.

Calotropis gigantea, the giant milkweed, is traditionally used for the treatment of cancer and in Ayurvedic medicine as an anti-helminthic, anti-pyretic, and anti-malarial agent. Poecilocerus pictus, an orthopteran insect, feeds on C. gigantea and both are known to possess cardiac glycosides. The increasing reports on the specific cytotoxicity of cardiac glycosides on human tumor cell lines led us to attempt characterization and comparative evaluation of cardenolides in both the insect and plant extracts for their anti-tumor and apoptotic potential.Chemical characterization using high-performance thin layer chromatography, ultraviolet and infrared spectra analysis confirmed the presence of cardiac glycosides, but differences in the components of the insect extract were indicative of biotransformation. The cytotoxicity studies revealed a more potent trend for the insect extract compared with the plant extract on A549 and COLO205 cells. There was a considerably lesser measure of toxicity on WI38 cells and peripheral blood lymphocytes, whereas B16F1 remained unaffected by both extracts. DNA ploidy analysis on COLO205 indicated that both extracts induced dose-dependent apoptosis. Therefore, both the insect and the plant extracts differentiate between human cancer cells and normal cells and exhibit species specificity. Further investigations are necessary to establish these extracts as promising lead candidates for anti-neoplastic activity.

Development and characterization of new cell line BPH22 from midgut epithelial cells of Poekilocerus pictus (Fabricius, 1775).

Poekilocerus pictus is a painted grasshopper, which feeds on Calotropis sp. containing the cardiac glycosides. A new cell line BPH22 is developed from midgut of P. pictus to study its unique physiology and biochemistry. Initially, the Graces insect medium is used with 10% (v/v) fetal bovine serum. After four passages, the serum quantity was reduced up to 0%.The lag phase in growth curve was 5 d and log phase is up to 10-11 d. The addition of Calotropis extract in Graces medium enhanced the growth of cells. The Calotropis extract with Graces medium altered the morphology of cells of BPH22. The amplification and sequencing of 16S rRNA gene confirmed the origin and purity of the BPH22.

Cytotoxic compounds from Poecilocerus pictus feeding on Calotropis gigantea.

Poecilocerus pictus, an aposematic insect, feeds on Calotropis gigantea (Asclepiadaceae) and sequesters cardiac glycosides which are used in chemical defense. The aim was to characterize the components of the methanolic extracts of P. pictus and C. gigantea and investigate preferential cytotoxicity of the insect extract, if any, on human cancer cell lines. Comparative chemical characterization by HPTLC, UV and IR studies revealed the presence of cardenolides in both the extracts and biotransformation of some of the ingested cardenolides in the insect extract. Cytotoxicity testing of the insect extract on human and murine normal and cancer cell lines indicated a time and dose dependent inhibition of A549 and COLO205 with a 48 hour IC50 of < 5 microg/ml, but no effect on normal cell line W138 or murine cell lines. Lymphotoxicity and genotoxicity experiments on human peripheral blood lymphocytes tested negative. DNA ploidy analysis on COLO205 indicated 36% apoptosis induction by 10 microg/ ml of insect extract. In brief, our studies established that the methanolic extract of P. pictus contained ingested cardenolides, of which some were biotransformed. Within a range of concentrations, P. pictus extract suppressed viability in A549 and COLO205 cells but not in normal cells, lymphocytes or murine cells. It induced apoptosis in COLO205. The insect extract discriminated human cancer from normal cells, inhibited cancer cell viability specifically and may represent a vital lead for a chemotherapeutic drug.