Induced Apoptosis On K562/A02 By New Podophyllotoxin Derivatives CWH-2 And Its Molecular Mechanism

Objective:The paper studied novel podophyllotoxin derivative CWH-2 mediated K562/A02 which had the stronger anti-aeschynomenous and multidrug resistance(MDR) tumor cells activity in vitro, and further studied its mechanism, provided experiment basis of structure optimization for podophyllotoxin derivatives.Methods:1 CWH- 2 anti-tumor activeness in vitro by MTT assayIn a series of experiments, eight tumorigenic human cells, including A549, human cervical cancer cells(Hela), human ovarian carcinoma cells(SKOV3)、SH-SY5Y、human breast carcinoma cells(MCF-7), human liver cancer cell line(Hep G-2), human erythroleukaemia cells(K562) and multidrug-resistant human erythroleukaemia cells induced by Doxorubicin(K562/A02). Moreover, two normal human cells, including Hacat and human hypertrophic scar fibroblast(FB) were chosen to determine the data of IC50 by MTT assay. More then, analysis of CWH- 2 anti-tumor activeness mediated K562 / A02 cell and draw K562 and K562 / A02 cell growth curve.2 Effect of CWH-2 on the tumor cell morphology changes in the structure were observed by Giemsa staining and Hoechst33342 staining, in order to study CWH- 2 effect on promoting apoptosis and its mechanism.3 The DNA ladder was revealed by agarose gel electrophoresis.4 Flow cytometry analysis the cell cycle stste and apoptosis rate of K562 and K562 / A02 after treated different dose of CWH- 2.5 The m RNA expression of mdr-1,p53,bax,caspase-3,PCNAand TopoⅡα in K562 and K562/A02 cells after treated different dose of CWH – 2 was detected by semi-quantified by reverse transcription PCR(RT-PCR).Objective:1 Inhibition of tumor growth in vitro by CWH-2.The results of MTT assay showed that CWH-2 had favourable activity to defense many tumor cells and the IC50 values were from 0.39~8.63μmol/L, this data has Significantly higher antitumor activity than the lated clinical antitumor drug VP- 16(3.74 ~ 67.44 u mol/L). The determination of parent leukemia cell lines and the resistant strains showed, CWH- 2 had the potential of multi-drug resistance. The effect of CWH – 2 to kill normal Hacat cells and Fb had significantly decreased, IC50 was 224.38±3.83μmol/L and 60.93±1.05μmol/L.This result displayed CWH-2 has the ability to choose cells to kill. What’s more, the IC50 of CWH-2 was 0.53±0.04μmol/L, and the IC50 of resistant strains was 21.81±0.92μmol/L by MTT assay. The results of cell growth curve also showed that CWH-2 had inhibitory activity to the multi-drug resistant tumor cells(K562/A02) and their parental cells(K562) in dose-dependent manner.2 Inhibition of tumor growth in vivo by CWH-2.The effect of CWH-2 on tumor growth was studied by mice transplanted entity-sarcoma S180 model. Intraperitoneal injection of CWH-2(20mg/kg and 30mg/kg) successively in 6 days, followed with tumor cells inoculation. Mouse S180 sarcoma was sensitive to CWH-2, and the inhibition ratios of treated groups(CWH-2, 20mg/kg and 30mg/kg) were 58.39% and 64.48%(P<0.01) compared with untreated control. Curative doses of CWH-2 were well tolerated with low or no systemic toxicity in terms of body weight loss in contrast to the administration of VP-16.3 Inducing apoptosis by CWH-2.Giemsa and Hoechest33342 staining showed that CWH-2 evoked typical apoptotic features such as nuclear membrane shrinkage, chromatin condensation, deep dyeing. Typical apoptosis body is observed in high dose group. Agarose gel electrophoresis showed typical DNA fragmentation pattern(DNA ladder) and confirmed the apoptosis induced by different concentrations of Cwh-2. Flow cytometric analysis indicated that the apoptosis rate caused by CWH-2 was in a dose and time dependent manner. The results from three parts(morphology, biochemistry, and flow cytometry) confirmed that CWH- 2 can promote leukemia K562 / how A02 resistant strains to apoptosis.4 Effect of CWH-2 on cell cycle.After treatment with 3μmol/L, 8μmol/L, 12μmol/L of CWH-2 for 24 h and 48 h, flow cytometry studies revealed that the number of cells in S phase was blocked.5 Regulating expression of mdr-1、topoⅡα, p53, bax,caspase-3 and PCNA m RNA by CWH-2The expression of mdr-1 、 topo Ⅱ α, p53, bax,caspase-3 and PCNA m RNA in K562 and(or) K562/A02 cells exposed to CHW-2 was investigated by RT-PCR. The results showed that the expression of PCNA m RNA was descreased in K562, but p53, caspase-3, bax and topoⅡα m RNA levels increased, meanwhile the expression of mdr-1 and PCNA m RNA decreased, and p53, caspase-3, bax and topoⅡα was increased.Conclusions:Compared with the positive control drug, CWH-2 showed stronger anti-tumor activity against various human tumor cell lines than positive control(etoposide). And CWH-2 had inhibitory activity to the multi-drug resistant tumor cells(K562/A02) in vitro but VP-16 had no inhibitory activity. It also had a distinguished inhibitive effect to mice sarcoma S180 in vivo. Anti-tumor mechanism of CWH-2 may be related with the following channels: it could upregulate p53, caspase-3, bax, topoⅡα and downregulate PCNA and mdr-1.Therefore, as a low toxicity and independent intellectual property rights of new anti-MDR-tumor compound, CWH-2 has much better prospects.