1. Studyof in vitro cytotoxicity andanticancer activity of paniculatin and its synthetic derivatives againstgastric cancer cell lines and cell isolated from NE patient samples to screeneffective lead compounds.Stomach, esophageal cancer celllines like H314, SCC25, AGS, HS746T, FLO-1, OE-33, etc. along with normal cells will becultured and maintained. The lead compounds willbe screened against those cell lines for invitro cytotoxicity using MTT assay.
The IC50 anddose-response curve will be generated to screen for most effective drugchoices. Change in cell cycle and apoptosis willbe assessed in best effective drug choice in most responsive cancer cell lines.Furtherchange of oncogenic phenotypes of these cells will be evaluated through colonyforming ability, anchorage independent growth by soft agar assay andmigration/invasion by Boyden chamber assay.
finally Activation of the transcriptionfactor HIF-1? assessed by Western blot and by quantifying mRNA expressionlevels of VEGF, a target gene of HIF-1?. Autophagy was monitored by Westernblot for the conversion of LC3-I to LC3-II and by confocal microscopy Objective2: Investigation of intracellular molecular targets and anticancer mechanism ofthe lead compounds at cell signaling The molecular mechanism offunctioning of the compounds will be investigated by assessing survival andapoptotic signaling of NF-kB, TGF-b, ERK1/2, PI-3/AKT etc. The intracellulartargets of the compound will be assessed through up/down regulation of key moleculeslike P53, PTEN, KLF2, Caspase-3, 8, 9, BCL, BAX etc. (depending upon the cancercell types).
Objective3: In Vivo study oftoxicity-histocompatibility of the compounds in vital organs and assessment ofantitumor activity in tumor bearing mouse Normal mice will be injected i.p, i.v and orallyin different acute and chronic doses and histopathological section staining oftheir vital organs like heart, liver, kidney, brain etc. will be undertaken toassess toxicity. Furthermore, function of these organs will be evaluated bymeasuring levels of serum glutamic oxaloacetic transaminase (SGOT), serumglutamic-pyruvic transaminase (SGPT), cretinine, acetylcholinesterase etc.Finally, preferred dose modality will be used in tumor bearing mice andregression of tumor will be assessed compared to vehicle treated control.