Anti-tumor Activity Evaluation And Anti-SK-N-SH Cell Mechanism Exploration Of Piperlongumine Analogs

Malignant tumor is a frequently occured disease and a serious threat to human health.“New cases of cancer about 14 million in annual year” said in 2012 World Cancer report.New cases in China accounted for 22%,the death accounted for 26%,more than a quarter of the global death.So it is extremely important for cancer treatment.At present,the method of cancer treatments are surgical treatment,chemotherapy and radiotherapy.Cytotoxicity-based chemotherapy is an important method of tumor therapy.However,the effect of most anti-tumor compounds on tumor cells seems no obvious selectivity.When tumor tissue was targeted,the normal tissue was often suffered as well.That's why most anti-tumor drugs produces lots of adverse reactions.Piperlongumine is a naturally occurred compound firstly found in the root of Piper longum L.,which can improve the level of ROS(reactive oxygen species)in tumor cells which could cause the death of tumor cells,while the damage to normal cells is little.Thus,Piperlongumine has become a research hotspot in recent years.However,low solubility and the unclear mechanism may be the limit for further application of Piperlongumine.Our team synthesized 51 Piperlongumine analogs formerly,so here in my targets were activity evaluation of those newly synthesized analogs,and mechanism exploration of the most potent analog.A brief of my research is as follows:The first step is the activity screen.MTT assay was used to screen the anti-tumor activity of 51 Piperlongumine analogs in vitro.The tumor cell lines include SGC79901,MGC803,EJ,EC109 and SK-N-SH.Two compounds' IC50 value were less than 1 ?M in 48 hours MTT assays on 5 cell lines,eleven compounds were between 1 and 5 ?M and three compounds were between 5 to 20 ?M.Notably compound PL-C21 has the best overall activity on 5 cell lines,and the most sensltive cell is SK-N-SH.IC50 of PL-C21 to SK-N-SH is 280 nM,which is 10 times better than Piperlongumine.The second step is the anti-tumor mechanism of compound PL-C21.The apoptotic bodies were observed by staining,Cell cycles,apoptosis,reactive oxygen species and mitochondrial membrane potential were detected by flow cytometry.Proteins related with ROS were detected by Western Blot assay.Staining experiments observed nuclear shrinkage and crescent-shaped nuclear membrane in early apoptotic cells.While nuclear rupture and round apoptotic bodies in different size were observed in late apoptotic cells.Compound PL-C21 induced G2/M phase block,ROS(reactive oxygen species)increase,MMP(mitochondrial membrane potential)decrease and cell apoptosis of SK-N-SH cells were detected by Flow Cytometry,and could be reversed by the addition of NAC(Acetylcysteine,ROS inhibitor).We also found that with the increasing concentration of PL-C21,pro-apoptotic protein Bax,Bad expression increased,and active-Caspase3,active-Caspase9 expression increased The expression of Active-PAPR of caspase3 substrate was increased.Intracellular ROS levels and p38 protein phosphorylation level were partially reversed by the addition of SB62005(a p38 MAPK signal pathway specific inhibitor)to SK-N-SH.Above all,the most potent compound PL-C21 could induce apoptosis,G2/M phase block,ROS increase,MMP decrease,activate mitochondrial internal apoptosis pathway and MAPK pathway.