Studies On Anti-mdr Tumor Cells Activities Of CIP-36 And Its Mechanism Of Action

Objective:The paper studied novel podophyllotoxin derivative CIP-36 which had the stronger anti-aeschynomenous and multidrug resistance (MDR) tumor cells activity in vitro, and further studied the inhibitory action and characteristic of CIP-36 to MDR and aeschynomenous tumor cells and animal transplant tumor raise in vivo, and discuss its function mechanism initially.Methods:1 CIP-36 has the stronger anti-tumor activeness by MTT and SRB assay in vitro.In a series of experiments, twelve tumorigenic human cells, including human oral squamous carcinoma cell (KB), mdr human oral squamous carcinoma cell (KBv200), human cervical cancer cells (Hela), human ovarian carcinoma cells (SKOV3), human stomach carcinoma cells (BGC-823), human breast carcinoma cells (MCF-7), human colon carcinoma cell line (LoVo), human osteosarcoma cells (HOS), human erythroleukaemia cells (K562) and multidrug-resistant human erythroleukaemia cells induced by Doxorubicin (K562/AO2), human liver cancer cell line (SMMC-7721, HepG-2) and two normal human cells, including human vascular endothelial cells (VEC) and human hypertrophic scar fibroblast (FB) were chosen to determine the cytotoxic activity of CIP-36.2 Effect of CIP-36 on the tumor cell morphology changes in the structure were observed by optical and fluorescence microscope.3 The DNA ladder was revealed by agarose gel electrophoresis.4 Apoptotic rate and the cell cycle state of K562/AO2 cells treated by CIP-36 for 6h,12h and 24h were detected by flow cytometry (FCM) by turns.5 The mRNA expression of p53, bcl-2, bax, caspase-3, p21, topoⅡα, topoⅡβ, PCNA, cyclinA, cyclinBl and mdr-1 in K562 and(or) K562/AO2 cells was semi-quantified by reverse transcription PCR (RT-PCR).6 The protein expression of P-gp in K562/AO2 was detected by immunohistochemistry and Western-blot.7 Effect of CIP-36 on topoisomeraseⅡαactivity was detected by topoisomeraseⅡDNA cleavage assay.8 Antitumor activity of CIP-36 in vivo was observed on animal transplant tumor model.Results:1 Effect of CIP-36 on the proliferation of cancer cells.The results of MTT assay showed that CIP-36 had favourable activity to many adherent tumor cells especially to human oral squamous carcinoma cell (KB) and the IC50 values were from 1.41 to 5.96μmol/L. The GI50 of CIP-36 on K562 and K562/A02 was 1.02 and 3.34μmol/L by SRB assay. What's more, CIP-36 had low toxicity to normal human vascular endothelial cells (VEC) and fibroblasts (FB). This indicated that it had selectivity between tumor cells and normal cells. The results of cell growth curve also showed that CIP-36 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 CIP-36.The effect of CIP-36 on tumor growth was studied by KunMing mice transplanted entity-sarcoma S180 model. Intraperitoneal injection of CIP-36 (25mg/kg and 50mg/kg) successively in 5 days, followed with tumor cells inoculation. Mouse S180 sarcoma was sensitive to CIP-36, and the inhibition ratios of treated groups (CIP-36,25mg/kg and 50mg/kg) were 55.11%and 62.72%(P<0.01) compared with untreated control. Curative doses of CIP-36 were well tolerated with low or no systemic toxicity in terms of body weight loss in contrast to the administration of etoposide.3 Inducing apoptosis by CIP-36.Giemsa and Hoechest33342 staining showed that CIP-36 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 CIP-36. Flow cytometric analysis indicated that the apoptosis rate caused by CIP-36 was in a dose and time dependent manner.4 Effect of CIP-36 on cell cycle.After treatment with 1μmol/L～4μmol/L of CIP-36 for 6h,12h and 24h, flow cytometry studies revealed that the number of cells in S phase was higher than other phases at 6 and 12h. Then CIP-36 arrested the cell cycle at S/G2+M phase after 24h.5 Regulating expression of p53, p21, bcl-2, bax, caspase-3, topoⅡα, topoⅡβ, PCNA, cyclinA, cyclinB1 mdr-1 mRNA and p-gp by CIP-36.The expression of p53, caspase-3, bax, p21, bcl-2, topoⅡα, topoⅡβ, PCNA, cyclinA, cyclinB1 and mdr-1 mRNA in K562 and (or) K562/AO2 cells exposed to CIP-36 was investigated by RT-PCR. The results showed that p53, caspase-3, bax, p21, topoⅡαand cyclinA mRNA levels increased, meanwhile the expression of bcl-2, PCNA, cyclinB1 and mdr-1 mRNA decreased. But topoⅡβmRNA was indifference compared with the control. Immunohistochemistry and Western blotting experiments showed apoptosis induced by CIP-36 was associated with the decreased of P-gp.6 Effect of CIP-36 on the topoisomeraseⅡαactivity.The inhibitory activity of CIP-36 as a TOPOⅡpoison is derived from stabilizing a normally transient DNA-topoisomeraseⅡα-drug complex. The same concentration of CIP-36 inhibited topoisomeraseⅡαactivity was higher than the positive control etoposide (VP-16).Conclusions:Compared with the positive control drug, CIP-36 showed stronger anti-tumor activity against various human tumor cell lines than positive control (etoposide). And CIP-36 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 CIP-36 may be related with the following channels:it could upregulate p53, caspase-3, p21, bax, topoⅡα cyclinA and downregulate bcl-2, PCNA, cyclinB1 and mdr-1; CIP-36 could act as an anti-MDR-tumor agent by inhibited TopoⅡαactivity and P-gp expression.Therefore, as a low toxicity and independent intellectual property rights of new anti-MDR-tumor compound, CIP-36 has much better prospects.