| Cancer is still one of the leading causes of death worldwide for the health of human beings, especially in low- and middle-income countries. The caner occurrences is growing year by year, according to WHO, death from cancer are projected to rise to over 11 million in 2030. Among over 200 chemotherapeutics drugs used in clinical, most of them were cytotoxic drugs. They will bring about series of serious side effects as they exhibit their anti-tumor effects, side effects plus to drug resistance issue become the main reasons to limit the application of chemotherapy drugs. So, look for high efficiency and low toxicity of new non-cytotoxic chemotherapy drugs become the major trend of developing anti-tumor drugs.Tetrazanbigen (TNBG) was a creatively designed and synthesized leading compound with sterolic structure; it was also authorized by State Intellectual Property Office of the People's Republic of China. Founded by NSFC(NO: 30371612, 30772595), we found that TNBG exhibited good anti-tumor effects in solid tumor cell line, and TNBG showed no cross-resistance with many other anti-tumor drugs in vitro. We also found that TNBG could inhibited tumor growth and prolonged the survival time of tumor-bearing mice and rabbits significantly and tumor lipid accumulation was observed in tumor cells other than in normal cells specifically. Through gene chip, proteomics and other related molecular biology technologies. We speculated that TNBG induced lipid accumulation from two ways. TNBG on the one hand could increase the synthesis of triglyceride, cholesterol and other lipid substance; on the other hand, TNBG could inhibit the transportation of lipid substance out of the tumor cells, so lipid accumulation was observed in tumor cells. So we put forward a new viewpoint that TNBG was a non-cytotoxic anti-tumor agent with the Peroxisome Proliferate Activatived Receptorγ(PPARγ), the Sterol Regulatory Element Binding Proteins (SREBPs), the Microsomal Triglyceride Transfer Protein (MTTP), et al, as its targets. The size exclusion chromatograph-HPLC (SEC-HPLC) results showed that TNBG could combind with PPARγreceptor specifically (Kd=1000nM).Based on the preliminary results, in this article, the main experiments and results are as follows:1 In vitro experiment, MTT assay was introduced to analysis the anti-tumor effects of TNBG in vitro. It was found that TNBG exhibited its anti-proliferation activities in a concentration-dependent manner. The IC50 values were between 2-9μg/ml among the six tumor cell lines, and the lowest IC50 was 2.13±0.65μg/ml (n=3) on human hepatoma cell QGY-7701. The results indicated that TNBG was a potent, broad-spectrum anti-tumor agent.2 In vivo experiment, we constructed a xenografts animal model of nude mice bearing QGY-7701 cells. According to the results, we found:①The inhibition rate of TNBG was 87.02% after treated with TNBG in a dose of 1.5mg/kg for 5 weeks consecutively.②There were no significant differences (P>0.05) on the immune function, the liver function, the kidney function and the blood lipid between the TNBG treated group and the control group.③Lipid sepicifc SudanⅢstaining showed that there was no lipid accumulation in the sections of heart, liver, spleen, lung, kidney, brain, aorta and tumor from the control group excepted in the tumor cells of TNBG treated group.④Stable signs of vital, normal activities of eating, defecation and other normal activities were observed during the TNBG treated time. The results all together indicated that TNBG could induced lipid accumulation in tumor cells to result in the death of tumor cells specifically while little side effects was observed in vivo, the anti-tumor effect of TNBG was tumor target.3 RNA interfering technology was used to establish the cell model of PPARγinhibition in human hepatocellular carcinoma QGY-7701 cells. Through Real Time PCR (RT-PCR) method, the expression of PPARγwas decreased by 80% in the form of mRNA; Through Western blot method, the expression of PPARγwas decreased by 71% in the form of protein. The best interfering sequence for PPARγwas"TCTTCCGGAGAACAATCAG". The cells after transfected by the plasmid of which carrid the best sequence was named as"psiHIVU6-PPARγ-QGY". This part of paper supplied the cell model for study of the anti-tumor mechanism of TNBG, and also the cell model for research new drugs based on PPARγtarget.4 The experiment of lipid accumulation in vitro.①TNBG could increase the lipid accumulation of QGY-7701 cells significantly through Oil red O staining after treated with TNBG in a qualitative way. The lipid accumulation induced by TNBG was in a time- and concentration- dependent manner. We also observed that the growth of TNBG was inhibited obviously.②MTT assay was introduced again to detect the anti- tumor effect of TNBG on"psiHIVU6-PPARγ-QGY"cells. The results showed that the IC50 value to"psiHIVU6-PPARγ-QGY"cells was 3.21±0.10μg/ml (n=3), the IC50 value to QGY-7701 cells was 2.13±0.65μg/ml (n=3), and there was significant difference between the two IC50 values (P<0.05). The results indicated that as the lipid accumulation was decreased by PPARγinhibition, the in vitro anti-tumor activity was decreased at the same time.③Automatic biochemical analysis was employed to analysis the components of lipid substances in a quantitative way. (1) Compared with the lipid substances in QGY-7701 cells without TNBG treated, the triglyceride (TG) was 346.43 nmol/mg which was increased by 69.12%, the total cholesterol (TC) was 38.99 nmol/mg which was increased by 63.00%, the free fatty acid (FFA) was 159.52 nmol/mg which was decreased by 28.82%, the total lipid substance(counted for the TG, TC, and FFA) was increased by 20.33% after treated with TNBG (the concentration was 2.0μg/ml) in QGY-7701 cells for 72h. (2) Compared with the lipid substances in QGY-7701 cells with TNBG treated, the TG was 306.64 nmol/mg, the decreased TG accouted for 28.10% of the TG induced by TNBG in QGY-7701 cells; the TC was 35.44 nmol/mg, the decreased TC accouted for 23.56% of the TC induced by TNBG in QGY-7701 cells; the FFA was 168.04 nmol/mg, the increased FFA accouted for 13.19% of the FFA induced by TNBG in QGY-7701 cells, the total lipid substances was decreased by 37.80% after treated with TNBG (the concentration was 2.0μg/ml) in"psiHIVU6-PPARγ-QGY"cells for 72h. The results indicated that PPARγpathway was a very important way by which TNBG induce the lipid accumulation. (3) Compared with the lipid substances in QGY-7701 cells induced by TNBG, the total lipid substances induced by TNBG was increased by 62.20% after treated with TNBG in"psiHIVU6-PPARγ- QGY"cells for 72h. The results showed that there were other rather important pathways by which TNBG induced lipid accumulation. All the results indicated that PPARγplays an important role in the lipid accumclation induced by TNBG. Apart from PPARγ, there were other pathways or targets such as SREBPs, MTTP, et al, which may responsible for the lipid accumulation induced by TNBG. The above results indicated that the lipid accumulation induced by TNBG was the reason of anti-tumor activities from qualitative and quantitative ways. The possible mechanism of lipid accumulation was resulted from SREBPs and PPARγactivation, MTTP inhibition and so on. 5 Flow cytometry (FCM) was employed to analysis the cell cycle distribution and apoptosis. The results showed that the cell cycle of QGY-7701 was arrested in S phase and the apoptosis induction was observed with TNBG treated for 72h in QGY-7701 and"psiHIVU6- PPARγ-QGY"cells. After the PPARγexpression was inhibited, TNBG showed a decrease potent of cell cycle arrest and apoptosis induction. The results showed that TNBG induced cell cycle arrest and apoptosis in a PPARγdependent manner.In summary, TNBG showed good anti-tumor activities while low side effects in vitro and in vivo experiments. The mechanism responsible for its lipotoxicity anti-tumor effect may be was the lipid accumulation induced by TNBG which finally leaded to the cell cycle arrest and apoptosis in a PPARγdependent manner, the other pathwasys or targets such as SREBPs activation, MTTP inhibition and so on would act as its targets to induce lipid accumulation. TNBG was a multi-targets, lipotoxicity, and non-cytotoxicity anti-tumor leading compound.
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