| Purpose:Colorectal cancer(CRC)is one of most common tumors over the world,the morbidity and mortality rates are both high.However,the mechanism of CRC carcinogenesis and progression remains to be understood.Increasing evidence showed that Rho/Rho kinase(ROCK)were commonly activated in several tumor types and are critical for proliferation and invasion of cancer cells.Recently,ROCK1 was demonstrated to play crucial roles in modulating mitochondrial dynamics and cell apoptosis.ROCK1/2 inhibitors exhibit strong tumor suppressing activity in various cancer types.RKI-1447,a novel small molecule inhibitor of ROCK1 and ROCK2,was demonstrated to inhibit tumor cell growth in breast cancer and clear cell renal cell carcinoma.However,the underlying molecular mechanisms of anti-cancer properties of RKI-1447 in CRC are not fully understood.In this study,we focused on evaluating the anti-cancer effect of RKI-1447 on colorectal carcinoma apoptosis and cell growth,and investigating the molecular mechanism of RKI-1447 caused cell growth delay.We aimed to clarify the role of metabolic reprogramming and Mitochondria-ER crosstalk in RKI-1447 induced CRC cell apoptosis.We proposed that validating the mechanistic working model would provide novel biomarker and therapeutic strategy for CRC early diagnosis and therapy.Methods:1.HCT-8?HCT-116?SW480 and DLD1 cell were cultured in vitro and treated with a gradient concentration of RKI-1447(0,10,20,40,80,160,320 ?M).Cell viability of each group was detected by Tetrazolium bromide colorimetric(MTT)method;2.Colony formation assay was applied to evaluate cell growth of HCT-8 and HCT-116 cells with different concentrations of RKI-1447(0,20,40,80 ?M);3.The cell apoptosis of HCT-8 and HCT-116 cells treated with or without RKI-1447 were detected by flow cytometry.FITC-conjugated Annexin V was used to detect the presence of apoptosis;4.Western blot were utilized to detect expression of injury related indicators of PARP-1 and caspase-3 in HCT-8 and HCT-116 cells;and analyze the indicated proteins as OPA1,OMA1,p-eIF2? and CHOP expression change;5.XF96 seahorse cellular bioenergetics analyzer was applied to detect the overall oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)of cells treated with or without RKI-1447(0?20?40?80 ?M);6.DCFH-DA detection kit was used to measure the intracellular ROS levels and JC-1 mitochondrial membrane potential detection kit was used to measure membrane potential alteration in mock and RKI-1447 treated cell,respectively;7.Confocal microscope was applied to visualize the morphology of mitochondria;8.siRNA interfere was used to knockdown endogenous CHOP mRNA transcripts,and confirm the role of CHOP in RKI-1447 caused CRC cell growth arrest;simultaneously,Crisper Cas9 was used to knockout endogenous ROCK1 and figure out the essential of ROCK1 in modulating ER stress and mitochondrial homeostasis;9.Xenograft nude mice assay was performed to confirm the anti-tumor property of RKI-1447 in vivo;western blot was used to analyze the indicated proteins as OPA1,OMA1,p-eIF2? and CHOP expression change in subcutaneous tumor;10.All statistical analyses were carried out using SPSS version 16.0 statistical software package(SPSS Inc.,Chicago,IL,USA)and presented with Graph Pad Prism version 5.0 software(Graph Pad Software,Inc.,La Jolla,CA,USA).Student's t-test was employed to compare the difference of means from two different treatment groups.All p-values are two-sided,and p<0.05 was considered as a statistically significant difference.Error bars represent the standard deviation of the data from three separate experiments.Results:1.MTT assay data showed RKI-1447 delayed cell growth in HCT-8?HCT-116?SW480 and DLD1 cells;Colony formation data indicated RKI-1447 could significantly suppress colony formation activity and induce cell apoptosis;Western blot data showed RKI-1447 could significantly increase cleavage of PARP and caspase-3 which further confirmed the activity of RKI-1447 in causing cell apoptosis;We also found RKI-1447 could suppress CRC cell growth in vivo;2.The seahorse data showed RKI-1447 could dramatically suppress both overall cellular OCR and ECAR;Simultaneously,indexes as basal respiration,maximal respiration and ATP associated oxygen consumption rate,basal aerobic glycolysis,maximal glycolysis and glycolytic capacity were all reduced upon RKI-1447 treatment;3.RKI-1447 promoted ROS generation while significantly caused loss of mitochondrial membrane potential in HCT-8 and HCT-116 cells;4.The data showed more fragmented mitochondria were found by confocal laser scanning microscope in RKI-1447 treated cells;5.Western blot showed the cleavage of OPA1 and OMA1 was significantly increased in response to RKI-1447 treatment;Simultaneously,we found ER stress indicators as p-eIF2? and CHOP were significantly increased in RKI-1447 treated cells;We also found cleavage of OPA1 and OMA1 were notably increased;simultaneously,p-eIF2? and CHOP were both increased in RKI-1447 treated tumor sections.6.The data showed depletion of CHOP could well recover RKI-1447 caused cell apoptosis,but not excessive ROS generation and loss of mitochondrial membrane potential;however,Seahorse analysis data showed down-regulation of CHOP could not be well rescued RKI-1447 caused reduction of OCR and ECAR;7.The data showed ROCK1 knockout could significantly suppress CRC tumor cell growth,promote ROS generation as well as loss of mitochondrial membrane potential.We found alteration of ROCK1 could promote cleavage of OPA1 and OMA1 as well as activating eIF2? and CHOP.Conclusion:1.RKI-1447 significantly suppresses CRC cell growth in vitro and in vivo,and induces PARP-Caspase-3 dependent cell apoptosis;2.In current study,we demonstrated RKI-1447 suppresses CRC cell growth via repressing mitochondrial respiration and aerobic glycolysis;3.We suggest that RKI-1447 induces depolarization of mitochondria and interruption of mitochondrial electric transport chain,subsequently initiates CHOP mediated ER stress,which finally contributes to RKI-1447 induced CRC cell apoptosis.Validation of this molecular mechanism would provide novel biomarker for CRC diagnosis and therapy. |
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