Studies On Tumor Cell Line Activities Of Deoxypodophyllotoxin And Its Mechanism Of Action

Objective: The paper studied Deoxypodophyllotoxin (DPPT) with tumorcells, and discuss its function mechanism initially.Methods:1DPPT has the stronger anti-tumor activeness by MTT and SRB assay invitro.In a series of experiments, twelve tumorigenic human cells, includinghuman oral squamous carcinoma cell (KB), human colon carcinoma cells(LOVO), human cervical cancer cells (Hela), human ovarian carcinoma cellline (SKVO3), human hepatic cancer cells (HepG-2)，human pulmonarycancer cells (A549), human erythroleukaemia cell line (K562) and two normalhuman cell line, including human vascular endothelial cells (VEC) and humanosteoblasts cell (MC-3T3) were chosen to determine the cytotoxic activity ofDPPT.2Effect of DPPT on the tumor cell morphology changes in the structure wereobserved by optical and fluorescence microscope.3The DNA ladder was revealed by agarose gel electrophoresis.4Apoptotic rate and the cell cycle state of KB cell line treated by DPPT for12h and24h were detected by flow cytometry (FCM) by turns.5The mRNA expression of p53、bcl-2、bax、caspase-3、p21、PCNA、cyclinA、cyclinB in KB cell line was semi-quantified by reverse transcription PCR(RT-PCR).6The protein expression of PCNA in KB was detected byimmunohistochemistry and Western-blot.Results:1Effect of DPPT on the proliferation of cancer cells.The results of MTT assay showed that DPPT had favourable activity to many adherent tumor cells especially to human oral squamous carcinoma cell(KB) and the IC50values were from5.6to2.9nmol/L. The GI50of DPPTvalues were from4.0to1.2nmol/L by SRB assay. What’s more, the DPPT haslow toxicity to normal human vascular endothelial cell line (VEC) and humanosteoblasts cell (MC-3T3). The indicated that it had selectivity between tumorcell line and normal cell line.2Inducing apoptosis by DPPTIn the Giemsa and Hoechest33342staining experiments, the orphologicalchanges of cells was more and more obvious with the increasing concentrationof DPPT and it leaded to the appearance of apoptotic bodies in different size inthe end. Agarose gel electrophoresis showed typical DNA fragmentationpattern and confirmed the apoptosis induced by DPPT. The results of flowcytometric analysis showed that the percentage of apoptotic cells wasincreasing in dose and time dependent manner. All of these results confirmedthat DPPT could indce the apoptosis of KB.3Effect of DPPT on the cell cycle.KB cell lines which were treated with DPPT (8nmol/L,24h) could blockcell phase at G2+M, and by using fluorescence microscopy, it was observedthat DPPT in this concentration could destroy the structure of microtubuleseriously.4Regulating expression of p53、p21、 caspase-3、 bax、 bcl-2、 cyclinA、cyclinB、pcna mRNA and PCNA by DPPT.The results of RT-PCR showed that after exposed to DPPT for24h, thelevels of p53mRNA, p21mRNA，caspase-3mRNA, bax mRNA and cyclinAwere increased, meanwhile the expression of bcl-2mRNA and cyclinB1mRNA were decreased in a dose dependent way. The data were significantdifference when compared to vehicle.The expression of PCNA was examined by immunohistochemistry andwestern blot. It was found that with the increasing dosage of DPPT, thecoloring of PCNA was light gradually when contrast to untreated group, and atthe same time the content of PCNA was reduced in a dose dependent manner. This suggested that the property which DPPT could overcome multi-drugresistance may be associated with the expression of PCNA.These results indicated that DPPT could inhibit the proliferation of manykinds of tumor cells including multi-drug resistance tumor cells, and themechanism may be related to regulating the expression of genes whichconcerned with apoptosis, decreasing the level of its expression productPCNA, blocking the cell cycle, and destroy the cytoskeleton.