ROCK Inhibitor Enhances The Growth And Migration Of BRAF Mutant Skin Melanoma Cells

Background:Melanoma is due to the melanin cells in the skin base layer undergo malignant transformation,the microcysts in normal melanocytes in the nevus or patch of the skin mucosa have the possibility of malignant transformation,genetic and environmental factors such as ultraviolet irradiation are the cause.In recent years,the incidence and mortality of melanoma is on the rise.While the five-year survival rate of early melanoma after surgical removal of is very high,the survival rate of patients with advanced metastatic melanoma is very low.Many reports mentioned that abnormalities in the Rho family signal pathway can be detected when the melanomas occur,in recent years Rho protein and effector is the development of new cancer drug target.Rho related protein kinase(ROCK)is a serine threonine kinase belonging to the AGC(PKA/PKG/PKC)family,which regulates the shape and migration of cells by acting on the cytoskeleton.It also plays an important regulatory role in cell survival,proliferation and apoptosis through multiple downstream targets.The two known ROCK subtypes of mammals,ROCK1 and ROCK2,have an overall homology of 62%and kinase domain homology of 92%.Because ROCK affects a variety of biological processes,ROCK inhibitors have been tested for clinical effects,including on cancer.ROCK inhibitor Fasudil is approved for clinical treatment of cerebral vasospasm in Japan and China.Although the targeted therapy or immune treatment bringing some light to the melanoma patients,there is still no satisfactory treatment because of there are many variations of melanoma.It is reported that the inhibition of ROCK inhibited the migration,invasion,proliferation and survival of melanoma cell lines in vitro,and Y-27632 as the most widely used ROCK inhibitors have been reported many times that it can inhibit melanoma cells.However,the occurrence of melanoma development involving regulation of multiple genes and signaling pathways.ROCK inhibitors,although playing a very important role in the occurrence of melanoma development,its target effect obviously but the mechanism is unknown,so it cannot be used clinically.The specific drug inhibitor specificity inhibiting ROCK protein has been applied in clinic to treat cardiovascular disease and cerebralvasospasm.Many reports show that discovered the Rho family signaling pathways reduce in the building of malignant tumor in vitro model.Protein involved in RhoA signalling pathways increased significantly in a wide variety of tumor.In addition,it has been reported that increased expression of Rho signal pathway related proteins contributes to metastatic behavior in certain cancers.As is known to all,Rho protein and its effect factors has the important functions of adjusting the cytoskeleton,it is the important targets for new anticancer drug.Therapies based on targeted Rho protein have been shown to reduce tumor progression and promote tumor cell death.It has been reported in vivo and in vitro all proving that using of small molecule inhibitor Y-27632,a highly specific drug inhibitor of ROCK protein inhibited the occurrence of tumors in a group of skin and eye melanoma cell lines and blocked the formation of melanoma in rodent models.In early stage of the experiment we also found the cells form changed after Y-27632 treatment of melanoma,tabular pseudopodia reduce while filiform pseudopodia increase at the same time.However,there are many pathological types of malignant tumors,even the immunohistochemical results of the same clinical classification of malignant tumors are varied,it can’t generalize.It also reports that ROCK inhibitors Y-27632 can increase the SW480 colon cancer cell migration.In addition,RND3,a ROCK inhibitor,has been shown to enhance melanoma cell migration.These literatures show that ROCK inhibitor has different functions in different melanoma cells,but the mechanism of its action remains to be further studied.Therefore,in our study,we further explored the mechanism of ROCK inhibitor Y-27632 on melanoma cells by exploring the effect of ROCK inhibitor on melanoma cells of different classifications,so as to provide guidance for the clinical application of related drugs in melanoma.Part I:Biological effects of Rho-related kinase inhibitors on human melanoma cellResearch Purpose:To study the effects of Rho-related kinase inhibitors Y-27632 and Fasudil on the migration and proliferation of human skin melanoma cells(UACC257,UACC62,MeWo,M14),mouse melanoma cells(B16F1),and human melanoma cells(HEM).Research Method:1.Melanoma cell culture and drug treatment:all melanoma cells and primary melanocytes(HEM)were cultured in Dulbecco improved Eagle medium,in which 10%fetal bovine serum and 0.1%penicillin/streptomycin were added.Y-27632 dissolved in distilled water,10 mm solution,treatment concentration of Y-27632 is 10μM.2.Melanoma cell migration and proliferation:approximately 7.5×104 cells/ml of melanoma cells were resuscitated in a 96-well plate,add 10 μM Y-27632 or 10 μM phosphate buffer,cultured in a incubator,and cell count kit-8(CCK8)was used to measure absorbance at a specified time to detect cell proliferation.3.Wound healing test:Melanoma cells were resuscitated on a 6-well plate and grew to 100%confluence.The cells were removed with a linear scratch at the tip of a sterile straw and treated with phosphate buffer,Y-27632 or Fasudil.The process of scratch healing was photographed with a digital camera.The wound healing percentage is the healing area divided by the total scratch area.4.Transwell assay:To test the effect of Y-27632 on the migration of melanoma cells,cells(UACC257)were resuspended in DMEM medium of 0.1%serum at a density of 1×105 cells/ml and added to the upper chamber.500 μL DMEM medium containing 10%phosphate buffer was added to the lower chamber.Incubate at 37 degrees Celsius for 24 hours.After removing the unmigrated cells on the filter membrane,the cells that had migrated across the membrane were fixed with 4%paraformaldehyde(sigma-aldrich)and stained with 0.1%crystal violet(sigma-aldrich)after washing.The number of cells migrating to the inferior lumen was recorded in 6 randomly selected high-power microscope fields.Research Results:Different melanoma cell lines were treated with ROCK inhibitors(Y-27632,Fasudil),and the proliferation and migration of different melanoma cells were observed by cell culture,drug treatment,scratch test,CCK8 test and migration test.We found that ROCK inhibitor can promote the growth and migration of melanoma cells(UACC257,UACC62).It can also inhibit the growth and migration of melanoma cells(B16).Therefore,the effect of ROCK inhibitor is cell-specific.Through literature review,we noticed the existence of BRAF mutation in melanoma cells.Through the detection of a series of cells(MeWo,M14,HEM),we found that ROCK inhibitor promoted the growth and migration of BRAF mutant melanoma cells.Research Conclusion:In vitro experiments,ROCK inhibitor can promote the growth and migration of melanoma cells(UACC257 and UACC62)and inhibit the growth and migration of melanoma cells(B16).Thus,the role of ROCK inhibitors is cell-specific.Through the detection of a series of cells(MeWo,M14,HEM),we found that ROCK inhibitor promoted the growth and migration of BRAF mutant melanoma cells.Therefore,the effect of ROCK:nhibitor on melanoma cells depends largely on cell type.It is suggested that ROCK inhibitor should pay attention to avoid BRAF mutated melanoma patients in clinical application.Part Ⅱ:Effect of Rho-related kinase inhibitor Y-27632 on human melanoma cell lines with BRAF mutationResearch Purpose:To investigate the mechanism of Rho-related kinase inhibitor Y-27632 on human melanoma cell lines with BRAF mutationResearch Method:Small interfering RNA was used to block the expression of ROCK1 and ROCK2 in melanoma cells,and PCR was used to verify the effective down-regulation of ROCK1 and ROCK2 in melanoma cells.Scratch test and CCK8 proliferation test were conducted on melanoma cells which were knocked out by ROCK,and it was found that both ROCK 1 and ROCK2,and ROCK 1/ROCK2 were down-regulated at the same time,which could promote the growth of melanoma cells.Confirmed for melanoma cells after a series of the promoting effect is caused by the ROCK inhibitors,we use protein immunoblot method,tested after joining ROCK inhibitor Y-27632,changes in a series of protein of PI3K/AKT/mTOR pathway and the RAF/MEK/ERK pathway,so as to screen out the effect proteins of ROCK inhibitors that promoting or inhibiting melanoma cells:ERK and AKT.Then,we cultured UACC257 melanoma cells under the action of Y-27632 and AKT or BRAF or ERK pathway inhibitors and conducted a series of experiments to further verify the relationship between ROCK inhibitor and AKT,ERK and BRAF and how the ROCK inhibitor exerts different effects on different melanoma cells by influencing the three above.Research Results:In B16F1 cells,Y-27632 had no significant effect on the ATK/mTOR pathway,and early ERK activity was indeed significantly reduced after Y-27632 treatment.In Me Wo of human melanoma cells with wild-type BRAF gene,no significant changes in AKT activity were found,while ERK activity decreased.In UACC257 cells,the ATK activity after Y-27632 treatment,especially the phosphorylation ATK activity at Thr 308 and Ser 473,was significantly increased,while the ERK activity was not significantly changed.In UACC62 human melanoma cells with BRAF mutant gene,the activity of phosphorylated AKT was significantly increased,while the activity of ERK also was moderately increased.UACC257 melanoma cells were cultured under the action of Y-27632 and AKT or BRAF or ERK pathway inhibitor,and it was found that AKT or BRAF or ERK pathway inhibitor completely inhibited the effect of Y-27632 on the enhancement of the growth of melanoma cells.In addition,when UACC257 cells performed wound healing experiments under the combined action of Y-27632 and other inhibitors in vitro,we found that AKT inhibitor partially blocked the ability of Y-27632 to enhance wound healing of melanoma cells.Both BRAF inhibitor PLX4032 and ERK inhibitor Sorafenib can inhibit RAF/ERK pathway,and these two inhibitors can completely block the enhanced effect of Y-27632 on the wound healing of melanoma cells.Research Conclusion:The results of the activity test showed that the down-regulation of ROCK1/2 activity in melanoma cells could promote the growth and migration of melanoma cells.Through protein western blot,it was finally found that ROCK inhibitor promoted the growth and migration of BRAF mutant melanoma cells by activating ERK and AKT pathways.Thus,ROCK inhibitors enhance the growth and migration of BRAF mutant melanoma cells by activating AKT and ERK,while inhibit the growth and migration of BRAF wild-type melanoma cells by inhibiting the ERK pathway.This has not been reported before.This part provides a theoretical basis for ROCK inhibitor treatment of BRAF wild-type melanoma.Part Ⅲ:The effect of Y-27632 on tumorigenic ability of human melanoma cell line(UACC257、MeWo)Research Purpose:To further investigate the role of Y-27632 in the development of melanoma through in vivo experiments.Research Methods:Nude mice aged 8 weeks were randomly divided into control group(PBS group only,3 mice)or drug treatment group(Y-27632 were added to PBS,3 mice).One million UACC257/MeWo cells were subcutaneously injected into the back skin of mice,and each mouse was injected 6 times.The experimental group received intraperitoneal injection of PBS(10 mg/kg)containing Y-27632,3 times a week for 2 weeks.The control group received intraperitoneal injection of PBS(10 mg/kg),3 times a week for 2 weeks.Mice were sacrificed 3 weeks after transplantation,tumors were collected and weighed.Research Results:In vivo experiment of UACC257 melanoma cells,the tumorigenesis rate of melanoma was 100%in the nude mouse model after intraperitoneal injection of Y-27632,while the control group was about 80%.The average weight and volume of tumors in the experimental group were larger than those in the control group.In vivo experiments of Me Wo melanoma cells,the tumorigenesis rate of both components were about 100%.In the nude mouse model after intraperitoneal injection of Y-27632,the average weight and volume of the tumor in the experimental group were smaller than those in the control group.Statistical analysis was performed after repeating the experiment for three times(P<0.01).Research Conclusion:In vitro administration of ROCK inhibitor significantly increased the growth rate of BRAF mutant melanoma and inhibited the growth rate of BRAF wild-type melanoma in nude mouse models.Therefore,we believe that the effect of ROCK inhibitor on melanoma cells depends largely on the cell type,suggesting that ROCK inhibitor’s role as a therapeutic tool may be tumor-specific or patient-specific,Our results suggest that ROCK inhibitor should be paid attention to avoid BRAF mutated melanoma patients in clinical application,and provide a theoretical basis for ROCK inhibitor treatment of BRAF wild-type melanoma.