Study On The Anti-tumor Activity In Vitro Of GMZ-1, A Podophyllotoxin Derivative

Objective: To study the anti-tumor effect in vitro of GMZ-1 which is a novel podophyllotoxin derivative, and the mechanism of its anti-tumor effect. Methods:1 The anti-tumor activity in vitro of GMZ-1 was measured by MTT assayMTT assay was used to measure the IC50 values of GMZ-1 on several tumor cell lines, including human cervical cancer cells (Hela), non-small-cell lung carcinoma cells(A549), human breast carcinoma cells (MCF-7), human liver cancer cells(HepG-2) , human ovarian carcinoma cells (SKOV3), human stomach carcinoma cells (BGC-823,MGC-803), human oral squamous carcinoma cells (KB), human oral squamous carcinoma cells resistance to VCR(KBV200), human erythroleukaemia cells (K562), human erythro- leukaemia cells resistance to ADM, and one normal human cell line, human hypertrophic scar fibroblast (FB).2 Detection of apoptosis of tumor cells induced by GMZ-1Giemsa staining and Heochst 33342 staining were used to observe the morphological changes of K562/A02 cells after treated with GMZ-1; agarose gel electrophoresis was used to observe the effect of GMZ-1 on DNA of KBV200 cells; the apoptotic rate of K562/A02 cells after treated with GMZ-1 was measured by flow cytometry(FCM).3 The effect of cell cycle of K562/A02 cells after treated with GMZ-1 was analyzed by FCM.4 The mRNA expressions of p21,p53,Bax,Caspase-3,PCNA,CyclinA,CyclinB1,Mdr-1 in K562/A02 cells after treated with GMZ-1 were semi-quantified by reverse transcription PCR (RT-PCR).5 The protein expressions of Caspase-3 and Mdr in K562/A02 after treated with GMZ-1 were detected by Western-blot. 6 Effect of GMZ-1 on microtubule of HepG-2 cellsImmunofluorescence was used to observe the morphological changes of microtubule in HepG-2 cells after treated with GMZ-1 and the inhibition ratio of GMZ-1 on microtubule was measured by fluorescent semi-quantitation.7 Toxicity indexs in vivo of GMZ-1 was detected initially by Acute Toxicity Tests.Results:1 The anti-tumor activity in vitro of GMZ-1 The results of MTT assay showed that GMZ-1 revealed a very intense cytotoxic activity towards many carcinoma cell lines, the range of IC50 values was 0.066 to 0.27μmol/L, which was much bertter than VP-16(range of IC50 values: 1.06μmol/L～14.1μmol/L). GMZ-1 was also more sensitive to multidrug resistant tumor cells than VP-16. What is more, GMZ-1 had low cytotoxic towards normal human cells like FB. According to the growth curve of K562/A02 cells, we found that GMZ-1 inhibited growth of tumor cells in a dose-dependent manner.2 GMZ-1 induced apoptosis of tumor cells.After treated with GMZ-1, the typical features of apoptosis such as nucleus shrinkage and splitting, chromatin condensation and deeply dyeing were observed by Giemsa and Heochst 33342 staining in K562/A02 cells. Typical DNA fragmentation pattern (DNA ladder) of apoptosis was observed by Agarose gel electrophoresis in KBV200 cells. FCM analysis indicated that the apoptotic rate of K562/A02 cells accreted consequently with the working concentration of GMZ-1 uprised.3 Effect of GMZ-1 on cell cycle of K562/A02 cellsAfter treated with GMZ-1 for 12h, 24h and 36h, the cell cycles of K562/A02 cells were all arrested at G2+M phase.4 Regulating expressions of p21, p53, Bax, Caspase-3, PCNA, CyclinA, CyclinB1, Mdr-1 in K562/A02 cellsThe results of RT-PCR showed that GMZ-1 could up-regulate the mRNA expressions of p21, p53, Bax, CyclinA, and down-regulate the mRNA expressions of CyclinB1, PCNA, Mdr-1, then induced apoptosis of tumor cells, inhibited proliferations of tumor cells and reversed multidrug resistance.5 Effect on protein expressions of Caspase-3 and Mdr in K562/A02 cellsThe Western blotting experiments showed that GMZ-1 could decrease protein expression of Mdr.6 Immunofluorescence was used to study the effect of GMZ-1 on microtubule of HepG-2 cellsThe microtubule was bushy, reticulodromous, fluorescent brightly and spatial in normal HepG-2 cells, while in the cells after treated with GMZ-1 it was discrete, astroid and fluorescent darkly. Like vincristine, the effect of GMZ-1 on microtubule polymerizing was inhibition, not promoting. The IC50 value of inhibitting microtubule polymerizing was 0.066±0.007μmol/L,a little difference compared with podophyllotoxin(0.071±0.007μmol/L).7 Toxicity indexs of GMZ-1The reactions of peritoneal injection with GMZ-1 were slow response, scrunch, quiescence, diarrhea and blockage in intestine. The LD50 value was 97.90±19.21mg/kg (95% confidence interval).Conclusions: GMZ-1 showed a powerful anti-tumor activity against many human tumor cell lines in vitro. The IC50 values were all about at 0.10μmol/L , 10~100 times more than that of VP-16. Inducing apoptosis might be a main mode of actions of GMZ-1, meanwhile, GMZ-1 could interfere cell cycles of tumor cells, and then inhibit proliferation of tumor cells. The mechanisms of these pharmacological effects were regulating apoptotic gene and protein expressions, regulating PCNA and cyclins, regulating expressions of multidrug resistance related gene and protein. GMZ-1 showed a little adverse reaction in intestine or stomach, other adverse effects were planed to study later. In a word, GMZ-1 was a new anti-tumor compound with high activity, high selectivity, definite mechanism. It was worth to study further more to get an overall estimated index.