Effects Of Metformin And Cisplatin On Human Lung Cancer Xenograft In Nude Mice

Objective: Lung cancer is one of the malignant tumors which is thebiggest threat to human life，and its incidence and mortality are growing fast.Many countries reported that the incidence and mortality of lung cancersignificantly increased in the past50years, and the incidence and mortality oflung cancer was the number one killer of all malignant tumors in male patients,and number two killer in female patients. Although there are a variety oftreatments, the overall5-year survival rate still has no obvious change. Lungcancer is the leading cause of death worldwide. Each year over one millionpeople, including850,000men and330,000women died of lung cancer. InChina, the annual incidence of lung cancer is thirty-five per ten thousandpeople, and80%-90%of the patients is non-small cell lung cancer (NSCLC).Diabetes is also a common and frequently-occurring disease, and type2diabetes accounts for the vast majority of diabetes. The population of type2diabetics with malignant tumor is growing rapidly. Type2diabetics have ahigher chance to develop various malignant tumors than the generalpopulation. Metformin is a double guanidine oral hypoglycemic drug, which isextensively prescribed for type2diabetics with insulin resistance with arecommended dosage of BMI＞25kg/m2by the American DiabetesAssociation(ADA) and the European Association for the Study of Diabetes(EASD). Metformin can inhibit glycogen dysplasia and decomposition, reducethe high output of hepatic glycogen, delay the absorption of glucose in the gut,and delay the beta cell of pancreas islet failure. Studies of recent yearsindicated that metformin could reduce the risk of diabetics to developmalignant tumors, inhibit tumor cell growth, and enhance the effect ofchemotherapy drugs. The antitumor effect of metformins has receivedattention by the academy of the diabetes and cancer research. Clinical studies demonstrated that diabetics taking metformin significantly decreasedincidence of cancer, which has opened the prologue of metformin in cancerresearch, and gradually become a "bright spot" study. The mechanism thatmetformin reduces the incidence of cancer may include:①Metformin directlyinhibits tumor through ATM-LKB1-AMPK-mTOR pathway;②Metforminreduces insulin levels and indirectly inhibit malignant tumor;③Metformininduces cell cycle stagnation and apoptosis, and reduces the growth of factorsignals;④Metformin reduces the levels of cell cycle protein;⑤Metformincauses inhibition of proto-oncogenes HER2;⑥Metformin hasanti-inflammatory effects;⑦Metformin enhances improvement of T cells’memory, and⑧Metformin may have other effects. Cisplatin is a commonlyused platinum anticancer drug. This study is designed to evaluate the effects ofmetformin in combination with commonly used cisplatin on tumor growth,Survivin, MMP-2, VEGF-C and VEGFR-3, expressions in lung cancerxenograft on nude mice， to explore the molecular mechanism and topotentially develop new effective drugs for the treatment of lung caner.Methods: Thirty-three4-to5-week-old male nude mice were bred underspecific conditions. Human lung cancer A549cells in the logarithmic phasewere inoculated into the left axillary subcutaneous of nude mice to establishhuman lung cancer xenograft study model. When the tumor diameter reachedan average of4mm, one nude mouse was removed because of the smallesttumor volume developed. The rest32nude mice were randomly divided intometformin-treated, cisplatin-treated, metformin combined withcisplatin-treated and untreated control groups. The nude mice in thecisplatin-treated group were injected once with cisplatin with a dose of5mg/kg/week and were allowed to take sterile distilled water through mouth.The nude mice in the metformin-treated group were administrated withmetformin with a dose of200mg/kg/d by intragastric administration and wereallowed to inject with equivalence physiological saline. The nude mice in themetformin combined with cisplatin-treated group were administrated withmetformin200mg/kg/d by intragastric administration and were allosed to inject with equivalent of cisplatin injection. The control group was treatedwith sterile distilled water, and at the same time injected intraperitoneally withphysiological saline. Forty-two days after administration, all the nude micewere sacrificed and tumor tissues were sampled. Expression of Survivin,MMP-2, VEGF-C, and VEGFR-3proteins in the tumor tissues were detectedby immumohistochemical method. The average integral optical density valuewas calculated by Buaa true color pathological image analysis system. TheSurvivin,MMP-2,VEGF-C and VEGFR-3mRNA expression in the tumortissue were detected with florescent real-time quantitative PCR.Results: The integral optical density of Survivin, MMP-2, VEGF-C andVEGFR-3protein immunohistochemical staining slices were successfullydetected with the pathological image analysis system. Expression levels ofSurvivin, MMP-2, VEGF-C and VEGFR-3protein of cisplatin-treated andmetformin combined with cisplatin-treated groups were lower (respectively, P<0.05) than those in the control group. Compared with the control group, theexpression levels of MMP-2protein of the metformin-treated group were alsoreduced(P<0.05). Compared with the metformin-treated group andcisplatin-treated group, the expression levels of survivin, MMP-2, VEGF-Cand VEGFR-3protein of the metformin combined with cisplatin-treated groupwere reduced (respectively,P<0.05).The Survivin, MMP-2, VEGF-C and VEGFR-3mRNA expression weredetected by fluorescent real-time PCR method. Compared with the controlgroup, statistical analysis showed that the expression levels of MMP-2ofmetformin-treated group were reduced (P<0.05). Compared with the controlgroup, the expression levels of Survivin, MMP-2, VEGF-C and VEGFR-3mRNA of the cisplatin-treated group and the metformin combined withcisplatin-treated group were reduced (respectively, P<0.05). Compared withthe metformin-treated group and the cisplatin-treated group, the expressionlevels of Survivin, MMP-2, VEGF-C and VEGFR-3mRNA of the metformincombined with cisplatin-treated group were reduced (respectively, P<0.05).Conclusion: Using metformin could inhibit MMP-2expression. Using cisplation or metformin combined with cisplatin can inhibit the expression ofSurvivin, MMP-2, VEGF-C and VEGFR-3. Combination drug of metforminand cisplation can enhance the anti-tumor efficacy.