Mevastatin Inhibits Proliferation Of NSCLC And Enhances The Effects Of Chemotherapy And Radiotherapy On NSCLC

Lung cancer is the number one cause of cancer-related mortality in the world .The mortality and morbility of lung cancer are increasing every year. Non small cell lung cancer(NSCLC) accounts for approximately 80% of all kinds of lung cancer cases, which is not sensitivity to chemotherapy and radiotherapy , and the 5-year survival rate for NSCLC remains lower than 15%.Rescent studies have demonstrated that cancer cell require more cholesterol than normal cell. Inhibiting synthesis of cholesterol may be an effective method in prevention and treatment of cancer. Mevastatin ,one kind of 3-Hydroxy-3-methylgutaryl CoA(HMG- CoA) reductase inhibitors is an agent that inhibits the rate-limiting step of the mevalonate(MVA) pathway.MVA is the precursor in the biosynthesis of isoprenoid compounds including cholesterol, dolichol and ubiquinone.Furthermore, mevalonate-derived prenyl groups enable precise cellular localization and function of many proteins such as Ras and Rho proteins. It is essential in maintaining cellular membrane structure and integrity. Isoprenoid compounds involve in cell proliferation and cell signal transduction and cell invasion. Rescent studies showed that HMG- CoA reductase inhibitors(statins) have been shown to inhibit proliferation and to induce apoptosis in a variety of tumor cells(Caco-2 cell line,HL-60 cell line ), resulting in retardation of tumor growth, and/or inhibition of the metastatic process. statins have also been demonstrated to potentiate the antitumor effects of some cytokines and chemotherapeutics and radiotherapy without increasing the side effect to normal orgnizations.but the molecular mechanisms of these function are not clare.In the present study, we examined whether mevastatin could inhibited the proliferation of NSCLC cells and enhance the effect of chemotherapy and radiotherapy in NSCLC, in order to look for a new method for the treatment of NSCLC.This study has been devided into four parts: Part 1 The inhibitory effects of mevastatin on the proliferation of NSCLC Objective: To investigate the inhibitory effects of mevastatin on the proliferation of NSCLC.Mothods: 1. MTT assay was performed in vitro to evaluate the proliferation inhibition effects of mevastatin on NSCLC. 2. The cell cycle distribution were evaluated with flow cytometer. 3.The mRNA expression of p21 was measured with reverse transcription-polymerase chain reaction. 4.The expression of p21 protein were test with flow cytometer .Results: 1.Mevastatin inhibited the proliferation of NSCLC in a time-dependent and concentration-depended manner. The IC50 value was 50μmol/L .2.Cell cycle analysis showed that A549 and NCI-H520 cells underwent G0/G1 phase arrest under exposure to mevastatin . After incubation with mevastatin25-100μmol/L ,the percentages of A549 and NCI-H520 cells in G0/G1 phase were 44.46%,68.15%,71.16%and 48.48%,70.24%,82.86% respectively ,they were significantly higher than group control(P<0.05,P<0.01). 3.The growth-inhibitory effect and cell cycle arresting of mevastatin in A549 and NCI-H520 cells could be effectively reversed by the addition of mevalonate, 4. Mevastatin caused no change in expression of P21 mRNA and total P21 protein(p>0.05).Concomitantly,P21protein localized on cellular membrane decreased (P<0.05,P<0.01).Conclusions: 1.Mevastatin inhibited cell proliferation of NSCLC cell lines in a time-dependent and concentration-depended manner. 2.Mevastatin suppressed proliferation by inducing G0/G1 phase arrest.3. Mevastatin exert growth inhibitory effect and cell cycle arresting by inhibiting mevalonate synthesis.Its mechanisms involved in blocking the isoprenylation of p21 protein.Part 2 The effects of mevastatin on apoptosis in NSCLC cells Objective: To investigate the apoptosis induction effect of mevastatin against human non-small cell lung cancer(NSCLC) cell lines and to explore the mechanisms . Methods: 1.The apoptosis induction effects of A549 and NCI-H520 cells were studied by flow cytometry, electron microscope observation and acridine orange/ ethidium bromide (AO/EB) double fluorescent dye staining.2.The mRNA expression of XIAP and Survivin was measured with reverse transcription-polymerase chain reaction. 3.The protein expression of Survivin was assayed by Western blot analysisResults: 1. The apoptotic cells detected by acridine orange/ ethidium bromide (AO/EB) double fluorescent dye staining was found after treatment with mevastatin.The color change of A549 and NCI-H520 cell was observed under a fluorescent microscope. The number of apoptotic cells increased in a time-dependent and concentration-depended manner after incubation with 25-100μmol/L mevastatin. The percentage of apoptotic A549 and NCI-H520 cells were8.17%,21.83%,29.53% and 18.58%,21.67%,36.39% respectively, were significantly higher than group control (P<0.05,P<0.01).2 Electron microscope apoptotic change was observed in A549 and NCI-H520 cells after treatmet with 50-100μmol/L mevastatin at 48 hours: pro-apoptosis cell has cavitations and blebbing; advanced apoptosis cell has obvious cytoplasm condensation,nuclcer fragmentation,chromatin compaction and segregation. 2.Flow cytometry showed that the apoptosis rate of A549 and NCI-H520 cells were significantly higher than group control(P<0.05).The apoptosis rate increased in a concentration-depended manner. Inducing apoptosis effect of mevastatin could also be reversed by the addition of mevalonate.Abvious sub-G1 was observed (another evidence of apoptosis )after treatment with mevastatin in A549 and NCI-H520 cells. 3.After treatment of NSCLC with25-100μmol/L mevastatin for 48hours, the mRNA and protein expression of survivin was reduced in a concentration-depended manner.4. After treatment of NSCLC with 25-100μmol/L mevastatin for 48hours, the mRNA expression of XIAP was reduced in a concentration-depended manner.Conclusion: 1. There were apoptosis changes in flow cytometer and morphologic electron microscope after treatment with mevastatin in NSCLC cells.Mevastatin could induce apoptosis in A549 and NCI-H520. 2.The mechanisms involved in down-regulation the expression of XIAP and Survivin. 3.Inducing apoptosis effect of mevastatin could be reversed by the addition of mevalonate,demorstreated that mevastatin induced apoptosis in NCI-H520 and A549 cell line throuth mevalonate passway. Part 3 The inhibitory effects of mevastatin on invasion and adherence in NSCLC cellsObjective: To investigate the inhibitory effects of mevastatin on invasion and adherence in NSCLC cell lines and to explore the mechanisms .Methods: 1.The inhibitory effects of mevastatin on adherence were determined by adherence test.2 The inhibitory effects of mevastatin on invasion were determined by transwell assay. 3. The protein expression of MMPand NF-κB was assayed by Western blot analysis.Results:1 The adherence effects were inhibited by mevastatin in NSCLC cell lines.The adherence rates of A549 and NCI-H520 cells were significantly decreased after treatment with mevastatin in concentration- depended manner. 2.Mevastatin inhibited invasion of NSCLC in a concentration-depended manner. After treatment with 25-100μmol/L mevas- tatin for 48 hours, the mean number of transwelled A549 cells were 123.68,67.20 and 29.41 respectivedly, and the mean number of transwelled NCI-H520 cells were 136.36,58.80 and 27.97 respectivedly. Both of them were lower than that in group control (P<0.01). The invasion and adherence inhibitory effect could also be reversed by the addition of mevalonate. 3. After treatment of NSCLC with 25-100μmol/L mevastatin for 48 hours, the protein expression of MMP-2,MMP-9,NF-κB(p65)were reduced in a concentration-depended manner.Conclusion: 1. The invasion and adherence effects were inhibited by mevastatin in NSCLC cell lines 2.The mechanisms were involved in down-regulation the protein expression of MMP-2,MMP-9,NF-κB(p65). Part 4 The sensitizasion effects and mechanism of mevastatin on chemotherapy and radiotherapy in NSCLCObjective: To investigate the sensitizasion effects and mechanisms of mevastatin on chemotherapy and radiotherapy in NSCLC.Methods: 1. The growth inhibitory effects of mevastatin used alone or in the combination with DDP,VP16 and radiotherapy were mearsured with MTT assay in NSCLC. The Q value was calculated to judge the synergistic effect. 2. The apoptosis of cancer cells were studied by flow cytometry after treatment with mevastatin alone or in the combination with chemotherapy and radiotherapy. 3. The protein expression of P53 was assayed by Western blot analysis.Results: 1. Comparing group radiotherapy alone with group mevastatin combining radiotherapy, the inhibitory rate in the combined group was higher than group control in a time-dependent and concentration-depended manner. 2. After treatment with 25μmol/L mevastatin combined 10Gy radiotherapy for 48 hours in A549 and NCI-H520 cells , the apoptosis rate were 41.27% and 49.19% respectivedly, the apoptosis rate in combined group was higher than that in control group(P<0.01). 3. DDP inhibited proliferation of NSCLC in a time-dependent and concentration-depended manner. 4. Mevastatin combined with DDP increased the inhibitory effect on the proliferation of NSCLC, and Q value is larger than 1.15, synergistic effect between the two drugs was found. 5.VP16 inhibited proliferation of NSCLC in a time-dependent and concentration-depended manner. 6.Mevastatin combined with VP16 increased the inhibitory effect on the proliferation of NSCLC, and Q value is larger than 1.15, synergistic effect between the two drugs was found. 7. After treatment with mevastatin combined DDP for 48 hours in NSCLC ,the apoptosis rate were 55.27% and 42.93%, the apoptosis rate in combined group was higher than that in group control (P<0.01).8.The protein expression of P53 in combined group were significantly increased than in group control in a concentration-depended manner.Conclusions: 1. Mevastatin increased the effect of radiotherapy, which demonstrated that there was a synergistic effect between mevastatin and radiotherapy , its mechanisms involved in induction of apoptosis and up-regulation the protein expression of P53. 2. Mevastatin could increase the inhibitory effect of DDP and VP16 on the growth of NSCLC, which demonstrated that there was a synergistic effect between mevastatin and chemotherapy. Its mechanisms involved in induction of apoptosis and up-regulation the protein expression of P53.