Combination Anti-cancer Effect Of C-Met-Targeting And Anticancer Agents On Malignant Colon Cells

Colorectal cancer is the third most commonly diagnosed cancer in theworld with almost half of the people who die of this cancer in developedcountries. Over one million new cases of colorectal cancer were clinicallydiagnosed, and that this type of cancer killed more than600,000people eachyear. Aberrant c-Met expression in colon cancer cells has been discovered topromote colon tumor proliferation and metastasis. Moreover, serum levels of(HGF) which is the high affinity ligand have been detected in colorectalcancer patients, supporting that c-Met is also a target for treatment of coloncancer. A number of c-Met/HGF inhibitors/antagonists have been evaluated incolorectal cancers. However, there is no data available to evaluatecombination efficiency of targeting c-Met with chemotherapy or radiotherapyin colon cancers. In previous study, we have found that c-Met played animportant role in colon cancer cells proliferation. In the present study, wefurther explored the promise via siRNA-mediated knockdown of c-Met,mimicking targeted therapy, in colon cancers plus either chemotherapy orradiotherapy. Our particularly interest will be focused on exploring thecombination effects of c-Met targeted therapy and anticancer agents.Objective:1To further explore the importance of c-Met in colon cancer cellproliferation and migration.2To explore the combination anti-cancer effects of c-Met-targeting and anti-cancer agents.Methods：1Construction of tet-inducible c-Met knockdown in colon cancer cellsSW6202SW620cells transfected with scrambled shRNA or c-Met-shRNA were treated with different concentrations of doxycycline for72h. After that,cells were subjected to Western blot assay and immune-fluorescenceobservation to evaluate the efficiency of doxycycline to induce c-MetshRNA.3SW620cells and cells transfected with scramble shRNA and c-Met-shRNAwere treated with400nM doxycycline for72h, and then the cell migrationwas assessed using24-well polycarbonate membrane inserts system. Themigrated cells were stained with Hoechst33342and observed byfluorescent microscope.4SW620cells and cells transfected with scramble shRNA and c-Met-shRNAwere plated in6-well plated and treated with400nM doxycycline for72h,and then cells were plated in96-well and treated with differentchemotherapeutic agents, including5-FU, cisplatin, taxol and Irinotecan.After treatment for48h, cell proliferation was assessed using Alamar blueassay and colony formation assay.Results:1Inducible c-Met-shRNA SW620cell lines were established.2Addition of100-1000nM doxycycline in culture medium induced RNAiwith significant reduction of c-Met protein and mRNA being detected,while no c-Met knockdown was detected in control cells expressingscrambled shRNA. Inducible c-Met knockdown is remarkably sensitive toswitch concentrations in a dose-dependent manner with completelyknockdown in400nM doxycycline.3Transwell migration assay was used to evaluate whether c-Met knockdownaffect cell migration. Cells were treated with or without DOX (400nM) for3d, and then allowed to migrate for12h. Accompanied by inhibition on c-Met by addition of doxycycline, the number of cells that migrated to lowerside of the chamber was significantly reduced. Meanwhile, in SW620orscramble group, no significant alteration in migration ability wasdemonstrated.4Conditional c-Met knockdown by doxycycline was confirmed by confocal microscopy observation. The addition of400nM doxycycline in culturemedium induced RNAi with significant reduction of c-Met protein, whileno c-Met knockdown was detected in control cells expressing scrambledshRNAs or SW620cells.5Cells were treated with5-Fu (5μM), taxol (2.5nM) with or withoutDOX(400nM) for3d. Subsequently, cell proliferation was quantified bycolony formation assay. Comparing with SW620-scrambled cells, additionof doxycycline in cell culture medium decreased the colony-forming unitsof SW620-shRNA cells which were inhibited by5-Fu and taxol.6Alamar-Blue results suggested that c-Met knockdown enhanced the anti-proliferation effect of5-Fu on SW620cells.Conclusion:1Inducible c-Met knockdown system was successfully established inSW620colon cancer cells.2c-Met played an important role in migration of SW620cells3Knockdown of c-Met was suggested to enhance anti-proliferationcapability of5-FU.