Preliminary Investigation On The Relationship Between The Expression Of ASPPs And Chemotherapeutic Reaction Of Tumor Cells

Objective: Innate or acquired resistance to chemotherapeutic agents is a main cause of failure of therapy and relapse of cancer. It was shown that blocking of apoptosis is responsible for resistance of cancer cells to DNA injuring chemotherapeutic agents. p53 is an important tumor suppression gene regulating cell cycle arrest and apoptosis induced by DNA injuring chemotherapeutic agents. Apoptosis-stimulating proteins of p53 (ASPPs) are regulatory proteins of p53, ASPP1 and ASPP2 stimulate proapoptic function of p53, and iASPP inhibit the function. In our previous experiments apoptosis of non-small cell lung cancer line A549 and melanoma cell line A375P might be induced by cisplatin (CDDP) and pirarubicin (THP), the expression of ASPP2 was upregulated, the expression of iASSP was downregulated in treated cells. In this experiment the expression of ASPP2 and iASPP of CDDP- or THP-treated A549 and SMMC-7721 cells was measured, nuclear factor–κB(NF-κB) of A549 was analyzed to explore the role of ASPPs family and their interaction with NF-κB in chemotherapy of cancer.Methods: Human hepatoma cell line SMMC-7721 was treated with cisplatin (CDDP), Pirarubicin (THP),β-elemene(β-Ele) or Vinblastine (VLB). The smallest effitive concentrations of different DNA-damaging agents and the growth arrest of SMMC-7721 cell lines were detected by MTT assay and then calculation the proliferation inhibition rate of SMMC-7721 under four concentrations of the agents. To examine the role of ASPP in this tumor cell, the expression level of ASPP2 mRNA was measured with semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and iASPP protein was detected with Western Blot in SMMC-7721 cell line. The translocation of NF-κB was analyzed with immunostaining assay. Finally all the data were processed in SPSS software. Means was denoted with means±SD, and analyzed by variance analysis (ANOVA) statistical methods according to the features of different data. P<0.05 has statistical significance.Results: 1) The results of MTT assay showed thatβ-Ele, THP, or VLB has significant inhibitory effect on the proliferation of SMMC-7721 cells. CDDP showed weak effect of proliferation suppression at common concentration (3μg/ml). In theβ-Ele treatment group, the inhibitory effect strengthened along with the concentration increasing (P<0.01). There were little difference in the suppression rates of THP orβ-ele between higher dose group and the lower dose in these two treatments. 2) The analysis of RT- PCR products indicated that the mRNA expression of ASPP2 in SMMC-7721 cell line treated with CDDP or THP for 24h were higher than that of the control groups(P=0.01, P<0.01 respectively). However,there was no significant difference between theβ-Ele or VLB treated groups and the control group in SMMC-7721 cell line(P=0.185). 3) Western Blot assays showed that the iASPP protein expression was downregulated in SMMC-7721 and A549 treated with THP for 24h, but the proteins expression of iASPP in SMMC-7721 cell line showed no significant difference between the control group and theβ-Ele, CDDP treatment groups. 4)Immunostaining assay showed that THP treatment influence the trans- location, staining of A549 was darker than that of control A549.Conclusion: THP andβ-Ele have obviously inhibitory effect on the proliferation of SMMC-7721 and A549 cells. Upregulated ASPP2 expression and/or downregulated iASPP expression are possibly the important mechanism of THP anticancer. However, CDDP is not sensitive to SMMC-7721. There is little difference of iASPP expression between the CDDP group and the control group. The iASPP may be associated with drug resistance. The study of A549 cell line indicated that NF-κB is activated by THP. The interactions and their mechanism between ASPPs and NF-κB in regulation of tumor chemoreaction will be defined.