Effects Of Survivin SiRNA On Proliferation Of Human Lung Cancer Cell Lines And Chemosensitivity Of Paclitaxel

Lung cancer is a malignant tumor with the highest incidence and mortality and has an upward trend in the incidence in the world. At present, the main therapy of lung cancer is surgery, combined with chemotherapy and radiotherapy. However, most patients discovered at late stage lost the opportunity to receive operating. Serious tolerance of cancer cells to chemotherapy or radiotherapy resulted in poor prognosis in many cases. It is still a puzzle to cure lung cancer, so seeking a new therapeutic strategy has an important significance.RNA interference (RNAi), a new technique developed in the late 2000s, has a powerful ability to silence gene expression. It is capable of triggering a certain post-transcriptional regulation program, resulting in the degradation of specific mRNA and the inhibition of the corresponding gene. RNAi, characterized by specificity, high efficiency and durability, has been widely used in various fields, such as tumor gene therapy, antivirus infection, filter of gene drug and so on. The key to RNAi, as other gene therapy, is how to select its target gene.Survivin, a novel inhibitor of apoptosis protein (IAP), is characterized by a unique structure with a single baculovirus IAP repeat and no zinc binding domain known as RING finger, and by a selective distribution in most common human cancers but not in terminally differentiated adult tissues. It can not only inhibite cell apoptosis, but also regulate cell proliferation and play an important role in generation of blood vessels. Survivin expression has been shown to be associated with carcinogenesis, cancer progression, drug resistance and poor prognosis. Inhibition of survivin expression and /or function in tumor cells triggers apoptosis as well as a defect in cell division. Thus, survivin is considered to be a promising target for cancer prevention and therapeutics.Paclitaxel (taxol) is the best natural anticancer drug introduced into medicalpractice during last several decades. It is currently used in the treatment of solid tumors, including lung cancer. Enhanced microtubule polymerization has been suggested to be responsible for the antitumor activity of the drug. Though useful, paclitaxel is clearly not ideal, because of its aqueous insolubility, deleterious side effects and susceptibility to several mechanisms of drug resistance. Overexpression of P-gp (P-glycoprotein) and alterations in the tubulin gene are frequently reported causes of paclitaxel-resistance. However, this evidence cannot yet fully explain the mechanisms responsible for acquired paclitaxel-resistance in human cancer. Recently , the relationship between overexpression of survivin and paclitaxel-resistance has been revealed, but relative research is rare in lung cancer.Thus, we designed this study to assess the growth-inhibiting effects of paclitaxel and silencing of survivin by RNAi on human lung cancer cell lines in vitro, especially to describe the role of survivin in mediating resistance to paclitaxel chemotherapy and the possibility of targeting of survivin as a strategy of chemosensitization in lung cancer. The present work has important clinical implications since it could provide rational laboratory basis for the design of combined therapies to enhance the responsiveness of lung cancer to paclitaxel and decrease paclitaxel dosage.Part I Growth-inhibiting effects of paclitaxel on human lungcancer cell linesObjective: To assess the growth-inhibiting effects of paclitaxel on human lung cancer cell lines with different p53 status in vitro.Method: 3 human lung cancer cell lines A549 (p53 wild-type), H322 (p53 mutant-type) and H1299 (p53 absence-type) were exposed to paclitaxel at different concentration or different treating time, then the growth inhibiting effects of the cell line were determined by MTT assay, the change of the cell cycle andapoptosis were analyzed with flow cytometry and the protein expressions of acetyl-tubulin and p53 were detected by Western blot analysis. Fluorescence microscopy was used to observe the nucleolus change stained by Hoechst33342.Results: The growth inhibition effects of paclitaxel on the three cells was time dependent (P<0.05). The IC₅₀(50% inhibitory concentration) of paclitaxel at 72h was 38.01nmol/L, 184.76nmol/L, 62.26nmol/L for A549, H322 and H1299 cells respectively, and at 96h was 9.2nmol/L, 29.78nmol/L, 37.55nmol/L for those three cells in-order. Paclitaxel of different concentration had different effect on each lung cancer cell line (P<0.05). At the concentration-dependent experiment, the proportion of apoptosis was highest at 10nmol/L paclitaxel, while the percentage of G₂/M phase increased in accordance with raise of the paclitaxel concentration, when paclitaxel concentration changed from 0.1nmol/L to 100nmol/L (P＜0.05). At the time-dependent experiment, the inducement of apoptosis was time dependent (P<0.05) and the percentage of G₂/M phase was highest at 12h when paclitaxel was 10nmol/L. When treated by 1000nmol/L paclitaxel, A549 showed the time-dependent G₂/M arrest (P<0.05) and H322 and H1299 had the highest G₂/M arrest at 24h. Lengthening paclitaxel treatments (48h or more) resulted in appearance of polyploidy cells in H1299. The two concentrations could induce the time-dependent apoptosis, but the higher one caused apoptosis later than the lower one did. Apoptosis had no relationship with G₂/M arrest (P>0.05). The typical apoptotic cells with condensed and fragmented chromatin could be observed in cells treated by paclitaxel under fluorescence microscopy. Paclitacxel up-regulated acetyl-tubulin and wild-type p53 protein expression in time- and concentration- dependence, while had no influence on mutant-p53 protein.Conclusion: Paclitaxel could inhibit the growth of the three human lung cancer cell lines with different p53 status in vitro in time-dependent manner. Different concentration paclitaxel also has different effect on each lung cancer cell line. It is capable of inducing apoptosis and G₂/M arrest but the late two have norelationship between each other. Different concentration paclitaxel might work with different mechanism. Low concentration paclitaxel could also inhibit effectively the growth of lung cancer cells by prolonging treatment. Our data also indicates that paclitaxel could up-regulate wild-p53 protein expression and induce both p53-dependent and p53-independent apoptosis pathways. p53 may play a role in the effect of high concentration paclitaxel on the cycle arrest of lung cancer cells, but has not significant effect on the chemosensitivity of those lung cancer cells to paclitaxel.Part II Growth-inhibiting effects of survivin siRNA on humanlung cancer cell linesObjective: To assess the growth-inhibiting effects of survivin-specific siRNA on human lung cancer cell lines in vitro.Methods: A549 and H1299 cells were transfected with chemosynthetic survivin siRNA by using RNAi technique. The effect of siRNA on cell proliferation, cell cycle, and apoptosis was analyzed by MTT assay and flow cytometry respectively. The expression change of survivin mRNA was detected by RT-PCR. The protein expression levels of survivin, p53, p21 and PARP were evaluated by Western blot.Results: Endogenous survivin levels were lower in A549 cells than in H322 and H1299 cells. The mRNA and protein expressions of survivin in cells transfected by the chemosynthetic siRNA were significantly lower than those of untransfected cells (P<0.01), but there were no significant difference between blank control group and non-silencing siRNA group (P>0.05). Survivin siRNA could inhibit significantly the growth and proliferation of both A549 cell with wild-type p53 alleles and H1299 cell without p53 allele in concentrationdependence (P<0.05), and produce a increase in the G₁ fraction and a decrease in the S fraction, but only induce some apoptosis in A549 cells. Transfection resulted in PARP cleaving and up-regulated the expressions of p21 and p53 protein.Conclusion: There may be a inhibitory effect on each other between survivin and p53. Silencing survivin gene by the RNAi technology could significantly decrease the expression of survivin gene. Our data suggest that survivin-specific siRNA could be a selective treatment to kill lung cancer cells regardless of the presence or absence of wild-type p53 alleles.Part IIIGrowth-inhibiting effects of paclitaxel combined with survivin-specific siRNA on human lung cancer cell linesObjective: To investigate if survivin play a role in resistance to paclitaxel and the growth-inhibiting effects of survivin-specific siRNA combined with paclitaxel on human lung cancer cell lines in vitro.Methods: 3 human lung cancer cell lines A549, H322 and H1299 were exposed to paclitaxel at different concentration or different treating time, then the protein expressions of survivin, p-Erk (phosphor-Erk) and PARP were detected by Western blot analysis and the change of the cell cycle and apoptosis were analyzed with flow cytometry. To determine if survivin-specific siRNA could enhance the responsiveness of lung cancer to paclitaxel, we established four different treatment groups using A549 cell lines: control, survivin siRNA, paclitaxel and survivin siRNA+paclitaxel. The effects on cell proliferation, cell cycle and apoptosis was analyzed by MTT assay and flow cytometry respectively. The protein expression levels of survivin, p53, p21 and PARP were evaluated by Western blot experiments.Results: Western blot experiments to test early time points after paclitaxelResults: Western blot experiments to test early time points after paclitaxel treatment indicated that survivin induction by paclitaxel was an early event, as early as 4h, and extending treatment time beyond 24h (48h or more) diminished the effect of paclitaxel on survivin induction in comparison with earlier time points. The change of survivin expression by paclitaxel was coincident with paclitaxei-mediated G₂/M arrest, and the coherence was independent of p53 status. Erk/p-Erk had a high expression in lung cancer cells, but its central role in paclitaxel effects was tissue and/or cell line specific. More importantly, inhibition survivin with siRNA not only silence endogenous survivin, but also silence paclitaxel-induced survivin in A549 cells. A combination of survivin-specific siRNA and a low concentration of paclitaxel (10nmol/L) significantly inhibited the proliferation of A549 cells (P＜0. 05) and moderately increased apoptosis rate compared to either treatment alone. Treatment with survivin-specific siRNA and paclitaxel also produced a increase in the G₁ and G₂/M fraction and a decrease in the S fraction and resulted in a more cleaved PARP and upregulated the protein expressions of p21 and p53.Conclusion: This observation suggests that survivin expression plays a critical role in cell viability and that induction of survivin by paclitaxel is a potential drug resistance factor leading to cell survival. The mitotic survival pathway may be at least one means involving survivin by which cancer cells counteract paclitaxel induced apoptosis following drug treatment. Inhibition of paclitaxel mediated induction of survivin by siRNA significantly enhances the chemosensitivity of lung cancer cells to paclitaxel.