Hispidulin Suppresses Cell Growth And Metastasis By Targeting PIM1 Through JAK2/STAT3 Signaling In Colorectal Cancer

Purpose: The current study was designed to evaluate the proviral insertion site in Moloney murine leukemia virus(PIM1)as a potential therapeutic target in colorectal cancer(CRC)and to investigate the anti-cancer activities and mechanism of hispidulin in CRC.Methods: Clinical tissue sample experiment: The cancer tissues and paracancerous tissues were collected from 63 CRC patients who underwent surgery from June 2014 to February 2016.The expression of PIM1,Ki-67 and Cleaved caspase-3 in tissues was detected by IHC.PIM1 expression in CRC tissues and paracancerous tissues was compared.PIM1 expression in tissues from patients with lymph node metastasis and without lymph node metastasis was compared.The correlation between PIM1 expression with Ki-67 expression and Cleaved caspase-3 expression were analyzed,respectively.The correlation between PIM1 expression and clinicopathological features of CRC patients was also analyzed.In vitro experiments: HT29 and SW480 cells were treated with the indicated concentration(0,10 and 20 ?M)of hispidulin for 24 and 48 h and CCK-8 assay was used to detect cell viability.Flow cytometry following Anexxin V/PI double staining was used to detect apoptotic cell death.Transwell with Matrigel was used to detect cell invasion.Western blotting was used to detect the protein expression of related marker molecules and q RT-PCR was used to detect the expression of PIM1 m RNA in cells.Following treatment with different concentrations of hispidulin,ROS levels in HT29 and SW480 cells were detected using ROS detection kit.CRC cells were transfected with PIM1 si RNA or PIM1 vector for PIM1 knockdown and overexpression,respectively.JAK2-STAT3 pathway activator(IL-6)and ROS scavenger(NAC)were used to modulate the proposed signaling pathways.In vivo experiment: Xenograft model and pulmonary metastasis model were established to evaluate the effects of hispidulin on tumor growth and lung metastasis,respectively.Cell apoptosis in tumor tissues was detected by TUNEL assay.The expression of related proteins in tumor tissues was detected by immunohistochemistry.The pathological changes of lung tissue sections were detected by HE staining.The data were analyzed using SPSS 17.0 software.Results: Clinical tissue sample experiment: Immunohistochemistry results showed thatthe expression level of PIM1 in CRC tissues was significantly higher than that in paracancerous tissues,and PIM1 expression increased along with advanced TNM stage in CRC patients.In addition,PIM1 expression level was significantly higher in patients with lymph node metastasis than in patients with non-lymph node metastasis.High expression of PIM1 was positively associated with Ki-67(cell proliferation marker molecule)expression and was negatively correlated with the expression of Cleaved caspase-3(apoptotic marker molecule).Meanwhile,PIM1 expression in tumor tissues is signaificantly associated with local invasion and lymph node metastasis(p<0.01).In vitro experiments: Knockdown of PIM1 with PIM1 si RNA(knockdown efficiency is 70%)significantly inhibited CRC cell growth and invasion,and induced apoptosis.Western blot experiments also showed that PIM1 knockdown led to increased cleavage of caspase-3 and PARP,as while as down-regulation of MMP-2 and MMP-9.These combined results showed that PIM1 acts as an oncogene in CRC.CCK-8 results showed that hispidulin could reduce CRC cell viability in a concentration and time-dependent manner.In addition,hispidulin significantly induced apoptosis and inhibited invasion in CRC cells.Hispidulin could dose-dependently repress the expression level of both PIM1 m RNA and protein.Transfection of CRC cells with PIM1 si RNA and PIM1 overexpression plasmid followed by treatment with hispidulin showed that the anti-CRC effects of hispidulin could be enhanced by PIM1 si RNA and reduced by PIM1 overexpression,indicating that hispidulin exerted an anti-tumor effect through modulating PIM1.In addition,hispidulin can significantly inhibit p-JAK2 and p-STAT3 expression.Compared with 20 ?M hispidulin group,IL-6(50 ng/m L)pretreatment significantly attenuated the inhibition of 20 ?M hispidulin on PIM1 protein expression in HT29 and SW480 cells(p<0.01).Moreover,hispidulin treatment can elevate the intracellular ROS level in a dose-dependent manner.NAC(2 m M)pretreatment can significantly dampen the inhibitory effect of hispidulin on p-JAK2,p-STAT3 and PIM1 expression.Compared with the 20 ?M hispidulin treatment group,20 ?M hispidulin+IL-6(50 ng/m L)group and 20 ?M hispidulin+NAC(2 m M)group can effectively dampen the effects of hispidulin on cell viability,invasion,apoptosis and related protein expression in HT29 and SW480 cells(p<0.01).Our results indicated that hispidulin repressed the expression of PIM1 by suppressing JAK2/STAT3 signaling via generating ROS,thereby exerted anti-tumor effect in CRC.In vivo experiment: The results of in vivo experiments also showed thathispidulin could significantly inhibit tumor growth,reduce tumor volume and tumor weight,increase the number of TUNEL positive cells in tumor tissues,and down-regulate the expression of PIM1,p-JAK2 and p-STAT3 in tissues(p<0.01).Compared with the control group,40 mg/kg/day hispidulin can effectively reduce the number of metastatic nodules in the lungs of mice and did not cause pathological changes in lung tissues(p<0.01).Conclusions and Significance: PIM1 can be considered as a potential therapeutical target in CRC.Hispidulin suppresses cell growth and metastasis by targeting PIM1 through JAK2/STAT3 signaling in colorectal cancer.Our study provides an experimental basis for the application of hispidulin in the clinical treatment of CRC.