Studies On The Antitumor Activities And Mechanisms Of A Noval Synthesizd Gambogic Acid Derivative, NG-18

NG-18 (N-(2-ethoxyethyl)gambogamide), a noval compound, is structurally modified from the gambogic acid (GA) which is the major active ingredient of gamboges by the Duan Wenhu group, Phytochemistry Department of Shanghai Institute of Materia Medica. The present study focused on evaluating the antitumor effect of NG-18 in vitro, and then the mechanism of apoptosis induced by NG-18 in HL-60 cells was investigated preliminary.MTT assay for suspend cells and SRB assay for attached cells were employed to evaluate the cytotoxic effects of NG-18 against 19 human tumor cell lines (including two human leukemia cell lines, two lung adenocarcinoma cell lines, three gastric adenocarcinoma cell lines, three colorectal carcinoma cell lines, three breast carcinoma cell lines, two ovarian carcinoma cell lines, one cervical adenocarcinoma cell line and three hepatocellular carcinoma cell lines) in vitro. The cell proliferation assay clarified its potent cytotoxic effects on various tumor cell lines. The mean IC₅₀ values of NG-18 and its leading compound GA were 0.86μM and 1.24μM respectively. According to these results, we found that NG-18 had stronger inhibitory effect than its leading compound GA against those cell lines in vitro. Next, PI staining-FACS analysis, Annexin VFITC binding assay and DNA fragmentation were employed.to observe apoptosis inducing capability of NG-18 on HL-60 cells. The results indicated that NG-18 possessed strong capability to induce apoptosis in HL-60 cells. The apoptosis induced by NG-18 was in time and does dependent manners. By the assay of PI staining-FACS analysis, we found that treatment with-0.5μM NG-18 for 6 h or 1μM NG-18 for 3 h, the apoptosis was induced significantly. DNA fragmentation assay showed that treatment with 0.5μM NG-18 for 6 h, DNA fragment could be detected obviously. But the cell cycle, distribution failed to respond to NG-18.We investigated the mechanism of apoptosis induced by NG-18 preliminary. Firstly, alkaline signal cell gel electrophoresis techniqtie was employed to test whether nuclear DNA was damaged in the early apoptotic cells. The result indicated that the apoptosis induced by NG-18 was not result from the damage of nuclear DNA. At the beginning and in the process of apoptosis, a big protease family, Caspases, play a critical role in it. Caspases (Cystein-containing, aspartat-specific proteases) are cysterine proteases in essence and exist as inactive proenzymes in healthy cells. Using Western Blot assay, we found that the initiator Caspases, Caspase-8 and Caspase-9, in extrinsic and intrinsic apoptosis pathways respectively, could be activated almost at the same time in the apoptosis induced by NG-18. This result indicated that both extrinsic and intrinsic apoptosis pathways took part in the apoptosis induced by NG-18 in HL-60 cells. Next, Cells were pretreated with pan-Caspase inhibitor Z-VAD-FMK, and then exposed with NG-18. The result indicated that Z-VAD-FMK could inhibit apoptosis induced by NG-18 completely. Other Caspase inhibitors such as Z-VDVAD-FMK for Caspase-2, Z-LEHD-FMK for Caspase-9 and Z-IETD-FMK for Caspase-8 were used to treat with cells in the same way. The results showed that except Caspase-8 inhibitor Z-IETD-FMK, Z-VDVAD-FMK and Z-LEHD-FMK could not inhibit the apoptosis induced by NG-18. Z-IETD-FMK could inhibit NG-18 induced apoptosis almost by 66.7ï¼…. These results indicated that the apoptosis induced by NG-18 was in a Caspase-dependent manner in which Caspase-8 acted as a key executor. Integrating the results, we drew a conclusion: both extrinsic and intrinsic apoptosis pathways took part in the apoptosis induced by NG-18 in HL-60 cells, but the extrinsic pathway was the main apoptotic pathway. In the process of apoptosis induced by NG-18 in HL-60 cells, Bcl-2, Bax and Bid which belonged to Bcl-2 protein family also contributed to the apoptosis.In conclusion, this study identifies that NG-18 exerts significant antitumor activity in vitro and it can induce apoptosis in tumor cells. Nevertheless, it doesn't cause evident change on cell cycle distribution. The apoptosis induced by NG-18 in HL-60 cells is in a Caspase-dependent manner in which Caspase-8 plays a very important role. Some Bcl-2 family proteins also take part in the apoptosis.