Cytotoxicity Of The Novel Indole Derivatives And Their Effects On Apoptotic Signalings In Human Cancer Cells

Cancer, also known as malignant tumor, is one of the major killers to human health and life in modern society. With the development of medical technology, life span expectancy of the patients has been extended, but cancer can’t be prevented or cured fundamentally. Plenty of scholars have been always developing new anti-cancer drugs and hope to find some that can not only have good cancer-fighting properties but also minimize the toxic effect on the organisms, which is the ideal mode of treating cancer. Sdudies have found that some indole compounds have good anticancer activity, so The extraction and synthesis of this kind of compounds has become a research hotspot.It has been well documented that p53can’t functionproperly in many cancer cells,partly due to the inactivation of it by MDM2.The interaction of p53and MDM2has been thoroughly elucidated and in recent years many small-molecule inhibitors of the MDM2-p53interaction were discovered based on the structural characteristics of the suppressive sites.One of the widely used inhibitors isMI-219,which is a indolecompound and can almost completely imitate the four key residues in the complex of p53-MDM2with strong affinity and excellent pharmacodynamics. A series of novel indole derivatives of MI-219were synthesized by Dr Hu Wenhao’s Lab, and this study was conducted to screen the anti-proliferative activities of these compounds against multiple human cancer cell lines.Compounds A.001and N1424603,which showed good apoptotic properties, were chosen for further investigation of the effects of these compounds on the apoptotic signalings in the Bel-7402human liver cancer cells.1. Anti-proliferative roles of the novel indole derivatives in human cancer cells linesA total of17compounds were screened for their apoptotic activities in5human cancer cell lines using MTT assays.There were several compounds, such as compounds A.001and N1424603, showing anti-proliferative activities in a dose-dependent manner in one or more cell lines.A.001and N1424603were chosen for the subsequent studies on the effects of them on the apoptotic signalings in Bel-7402liver cancer cells. 2. Apoptosis roles of A.001and N1424603in Bel-7402human hepatocarcinoma cellsAnnexinV/PI double staining method was used to detect the apoptosis of Bel-7402after treatmentwith A.001or N1424603.The results showed that both A.001and N1424603could induce apoptosis in a dose-dependent manner, suggesting that the anti-proliferative effects of these two compounds mentioned above were attributed to their pro-apoptotic preperties.3. Effect of A.001and N1424603on cell cycle distribution in Bel-7402human hepatocarcinoma cellsApart from inducing apoptosis,many anticancer drugs inhibit proliferation by blocking cell cycle.So we used PI staining to detect cell cycle distribution.We found that the two compounds couldaffect the distribution of cell cycle. A.001arrested Bel-7402cell at S phase at low concentration and G1phase at high concentration, whereasN1424603, when the concentration increased,arrested cells in G2phase.N1424603also led to an increase, following a reduction of thecells in S phase.It reducedcells in G1phase at a high concentration.The results revealed that the two compounds could inhibit the proliferation of the cancer cells by affecting cell cycle.4. The effect of A.001and N1424603on the CDKIP21,p27and p53have been shown to arrest cell cycle,so we examined the effect of A.001and N1424603on their expressions by western blotting method.The data showed that A.001couldup-regulate the expression of p21and p27,but had no effect on p53and p-p53;N1424603could up-regulate the expression of p21and down-regulate the expression of p27, but hadno effect on p53and p-p53.5. Effects of A.001and N1424603on MAPK pathwayMAPK pathway plays an important role in affecting cell proliferation and apoptosis.So we examined the effects of two compoundson this pathway.The results showed that A.001had no effect on the expressions of ERK,p38and JNK,but down-regulated p-ERK expression and up-regulated p-p38and p-JNK expressions in a concentration-related way.Similarly, N1424603also had no effect on the expressions of ERK,p38and JNK,but could affect the active forms of these proteins.These results suggested that the compoundsinduced cell cytotoxicity possibly related to alterations of MAPK signalings.