The Inhibitory Effect Of Metformin On Prostate Cancer

Objective To study the influence of IL-6on the proliferation,migration and viability of prostate cancer cell, to get a further knowledge ofprostate cancer recurrence and the occurrence of castration-resistantprostate cancer, to explore the action mechanism of IL-6on prostate cancerdevelopment；to observe the influence of metformin on the migration,multiplication and cell cycle of LNCaP-IL-6， a constructed IL-6overexpressing prostate cancer cell line, to further explore the relevantinhibitory mechanisms of prostate cancer by metformin.Method （1） IL-6over-expression LNCaP stable cell line(LNCaP-IL-6) and LNCaP control cell line（LNCaP-ctr） were onstructedby lentiviral transfection,then GFP (green fluorescent protein) was detectedby laser scanning confocal microscope to determine the transfectionefficiency;cell morphology was observed under inverted microscope；IL-6expression level in the supernatant of LNCaP cell medium was detected byELISA，LNCaP-ctr and LNCaP-IL-6cell viability was analysed by MTTassay after treated with bicalutamide for different time periods；Twist andE-cadherin proteins expression level in both LNCaP-ctr and LNCaP-IL-6 cells were detected by western blot，and their mRNA expression level weredetected by RT-PCR.（2）Different concentrations of metformin wereadded in the culture medium，the viability of LNCaP-IL-6and LNCaP-ctrcells were measured by MTT assay，the most suitable concentration ofmetformin was choosed for cell treatment.FCM（flowcytometry）was usedto analyse the alteration of LNCaP-ctr and LNCaP-IL-6cell cycles.Wond-healing assay was used to measure LNCaP-IL-6cell migration withor without metformin treatment,then Twist and E-cadherin proteinexpression level in both groups was measured by Western blot，and theirmRNA expression level was measured by RT-PCR.Results (1)IL-6over expression LNCaP stable cell line（LNCaP-IL-6） and control LNCaP cell line（LNCaP-ctr） wereconstructed successfully，GFP was expressed in both cell lines as expected，which was detected by laser scanning confocal microscope， themorphology of LNCaP-IL-6presented to be spindle-like or asteroid，withcell junctions reduced significantly and cell distributed chaotically. IL-6secretion level in LNCaP-IL-6cell culture medium for24h was5802±128.09pg/mL，much more than that of LNCaP-ctr and nomal LNCaPcell groups，which was just1.14±0.19pg/mL and1.98±0.84pg/mL. Therewas a significant proliferative advantage for LNCaP-IL-6cells whilecompared with LNCaP-ctr cells （P＜0.05）. With the treatment ofbicalutamide，LNCaP-IL-6cell line was presented more survivable than LNCaP-ctr cell line（P＜0.05）.Twist protein and mRNA expression levelsin LNCaP-IL-6cell line were much higher than in LNCaP-ctr cell line（P＜0.05），and E-cadherin protein and mRNA expression levels inLNCaP-IL-6cell line were much lower than in LNCaP-ctr cell line（P＜0.05）.（2）Metformin has a less inhibitory effect on LNCaP-IL-6cell linewhen compared with LNCaP-ctr cell line，the survival rate of LNCaP-IL-6group is60%higher than LNCaP-ctr group，when compared with eachother，the difference between two groups was statistically significant，P＜0.05.2mM metformin was chose as the optimal concentration for celltreatment according to MTT results；FMC results reflected that cell cyclesof both cell lines were arrested in S phase.With2mM metfomin treatment，LNCaP-IL-6cell migration was greatly inhibited. Twist protein and mRNAexpression levels in metformin treated LNCaP-IL-6cell were lower than inuntreated LNCaP-IL-6cell（P＜0.05），and E-cadherin protein and mRNAexpression levels in the former were higher than in the latter（P＜0.05）.Conclusion （1）IL-6enhance LNCaP cell proliferation andmigration ability,and promote LNCaP cell viability against bicalutamide，a kind of antiandrogen drug， and reduce LNCaP cell E-cadherinexpression,an important adhesion molecular on cell surface，by increasingTwist protein expression level.（2）Metformin could arrest cell cycle，forwhich reason the proliferation of prostate cancer cell was inhibited，thedown-regulation of Twist and up-regulation of E-cadherin induced by metformin，which could obstruct cell migration and EMT，may be one ofthe important mechanisms by which metformin suppress prostate cancer.