Honokiol Suppresses Proliferation And Induces Apoptosis Via Regulation Of The MiR-21/PTEN/PI3K/AKT Signaling Pathway In Human Osteosarcoma Cells

Honokiol(HNK)is a small biphenolic compound,which exerts anti-neoplastic effects in various types of cancer.However,the mechanism underlying the anti-tumor effects of HNK in osteosarcoma(OS)cells is not yet fully understood.Emerging evidence has indicated that micro RNAs(miRNAs/miRs)serve key roles in numerous pathological processes,including cancer.It has previously been reported that Chinese medicinal herbs harbor anticancer properties via modulating mi RNA expression.Therefore,the present study aimed to determine whether HNK could suppress OS cell growth by regulating mi-RNA expression.The MTT assay and flow cytometric analysis were used to evaluate the cell proliferation and apoptosis in human OS cells after treatment with HNK,respectively.The results demonstrated that HNK inhibits proliferation and induces apoptosis of human OS cells in a dose-dependent manner.Furthermore,HNK-induced apoptosis was characterized by up-regulation of proapoptotic proteins,including cleaved-caspase-3,cleaved-poly(ADP-ribose)polymerase and B-cell lymphoma 2(Bcl-2)-associated X protein,and down-regulation of the anti-apoptotic protein Bcl-2.RT-qPCR verified that HNK was able to induce aberrant expression of miRNAs in human OS cells,and miR-21 was one of the mi RNAs that was most significantly down-regulated.To further investigate miR-21 function,the present study validated that HNK reduces miR-21 levels in a dose-dependent manner.In addition,restoration of miR-21 expression abrogated the suppressive effects of HNK on OS cells.Luciferase assay and Western Blot analysis identified that miR-21 inhibits the expression of phosphatase and tensin homolog(PTEN)by directly targeting its 3'-UTR.Notably,HNK was able to suppress the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway;however,it was reactivated by miR-21 overexpression.Taken together,these data indicated that HNK may inhibit proliferation and induce apoptosis of human OS cells by modulating the miR-21/PTEN/PI3K/AKT signaling pathway.Therefore,miR-21 may be considered a potential therapeutic target for the treatment of osteosarcoma with HNK.Osteosarcoma(OS)is the most frequent primary malignant bone tumor,which is commonly diagnosed in children and young adolescents,with a male predominance(1).OS is highly aggressive and primarily metastasizes to the lung(2).Surgical tumor resection and multi-agent chemotherapy are the main current therapeutic strategies used to treat OS.It has previously been reported that chemotherapy may increase the 5-year survival rate for localized disease by>50%compared with surgery alone.Conversely,patients diagnosed with metastases exhibit a poor prognosis,with a 5-year survival rate of 20-30%following surgical resection and/or radiotherapy(3,4).Furthermore,currently approved agents exhibit severe side effects(3,4);therefore,the development of a novel agent with increased efficiency and reduced toxicity in OS treatment is required.Honokiol(HNK)is a biphenolic compound extracted from the magnolia tree,which has been used to treat anxiety,thrombotic stroke and gastrointestinal symptoms in traditional Chinese and Japanese medicine(5).HNK has long been known to exert antimicrobial(6),anti-inflammatory(7)and anti-angiogenic(8,9)effects.Increasing evidence has revealed that HNK exerts anti-neoplastic functions in various types of cancer,including anti-osarcoma(8),colorectal carcinoma(10),breast cancer(11)and gastric cancer(12).Furthermore,HNK may trigger apoptotic pathways that result in mitochondrial dysfunction(13),influence retinoblastoma function and E2F transcription factor 1 transcriptional activity(14),and suppress the phosphoinositide 3-kinase(PI3K)/mammalian target of rapamycin(mTOR)pathway(15).However,the molecular mechanism underlying the anticancer effects of HNK on OS cells remains to be elucidated.MicroRNAs(miRNAs/mills)are a class of small(19-24 nucleotide)noncoding RNAs that mediate post-transcriptional regulation of target genes by suppressing translation or promoting RNA degradation.MiRNAs have crucial functions in various biological and pathological processes,including cellular proliferation,differentiation,apoptosis and carcinogenesis(16).In recent years,it has been demonstrated that some natural products are able to control tumor-suppressive and oncogenic miRNAs,including curcumin(diferuloylmethane),which inhibits hepatocellular cancer cell proliferation via modulating miRNA expression(17).Furthermore,previous studies have reported that Chinese medicinal herbs exert antitumor effects by modulating miRNA expression(18,19).Zhang et al demonstrated that HNK suppresses bladder tumor growth by inhibiting the enhancer of zeste homolog 2/miR-143 axis(20).Avtanski et al also revealed that HNK rescued leptin-induced tumor progression by suppressing the Wntl-metastasis associated 1-?-catenin signaling pathway in a miR-34a-dependentmanner(11).Therefore,it may be hypothesized that HNK inhibits proliferation and induces apoptosis,via the modulation of miRNA expression,in human OS cells.The present study investigated the effects of HNK on OS tumor growth inhibition and explored the underlying molecular mechanisms.The results indicated that HNK may inhibit growth and promote apoptosis of human OS cells in a dose-dependent manner.Furthermore,the results verified that HNK induces aberrant expression of miRNAs in human OS cells,and miR-21 suppresses phosphatase and tensin homolog(PTEN)by directly targeting its 3'-untrans-lated region(3'-UTR).Notably,the results indicated that HNK blocks the PI3K/protein kinase B(AKT)signaling pathway by inhibiting miR-21 expression in human OS cells.Collectively,these results suggested that the molecular mechanism by which HNK induces apoptosis was modulated by the miR-21/PTEN/PI3K/AKT axis in human OS cells.PART I THE EFFECT OF HONOKIOL ON PROLIFERATION AND APOPTOSIS OF OSTEOSARCOMA CELLSObjectiveTo investigate the effect of honokiol on proliferation and apoptosis of osteosarcoma cells.Methods1.To investigate the anti-proliferative effects of HNK on OS cells,Saos-2 and MG-63 cells were treated with various concentrations of HNK for 24h,and the MTT assay was used to investigate the anti-proliferative effects of HNK on OS cells.2.Apoptosis analysis.To examine HNK-induced apoptosis of OS cells,the cells were analyzed by Annexin V-PI staining following treatment with HNK was used to detect cell apoptosis.3.To further explore the apoptotic mechanism,the intracellular apoptotic signaling pathway was investigated in OS cells following treatment with various concentrations of HNK.Western Blot analysis was used to detect the expression of apoptosis-related proteins in osteosarcoma cells following HNK treatment.Results1.HNK inhibits growth of human OS cells.The results of MTT assay indicated that treatment with 1-100?M HNK reduced cell viability of Saos-2 and MG-63 cells in a dose-dependent manner.The half maximal inhibitory concentration(IC50)values of HNK were 37.85?M in Saos-2 and 38.24?M in MG-63 cells.Similar IC50 values of HNK were detected in human Saos-2 and MG-63 OS cells.2.HNK induces apoptosis of human OS cells.The results of Annexin V-PI staining demonstrated that the proportion of apoptotic cells was markedly increased following HNK treatment compared with in the control group(P<0.01).Furthermore,following treatment with 10 or 40?M HNK,the number of apoptotic cells increased in a dose-dependent manner.With regards to apoptotic induction,Saos-2 and MG-63 had similar results.3.The results revealed that the protein expression levels of cleaved-caspase-3,cleaved-PARP and Bax were significantly up-regulated,and Bcl-2 was significantly down-regulated following HNK treatment.Furthermore,HNK regulated these protein expression levels in a dose-dependent manner in human OS cells.These data suggested that HNK may induce apoptosis of human OS cells by activating the intracellular apoptotic signaling pathway.Conclusion1.HNK has obvious inhibitory effect on proliferation of human osteosarcoma cells in vitro.2.HNK induces apoptosis of human OS cells.3.The results of Western Blot suggested that HNK may induce apoptosis of human osteosarcoma cells by affecting the signal transduction pathway between cells.PART II HNK INDUCES ABERRANT EXPRESSION OF MIRNAS IN HUMAN OS CELLSObjectiveThe present study aimed to determine whether HNK induces aberrant expression of miRNAs in human OS cells.Methods1.To determine whether HNK also induces aberrant expression of miRNAs in human OS cells,six miRNAs(miR-188-5p,miR-202 and miR-623 were the most significantly up-regulated;miR-21,miR-532-5p and miR-628-3p were the most significantly down-regulated)were selected based on the microarray data,and were verified by RT-qPCR.2.To investigate the function of miR-21 in human OS cells.OS cells were treated with 10-100?M HNK for 24h and the expression levels of miR-21 were determined by RT-qPCR.3.RT-qPCR was used to confirm that miR-21 expression was specifically up-regulated/knocked down following transfection with mimics/inhibitor.Subsequently,OS cell proliferation and apoptosis were assessed by MTT assay and flow cytometric analysis,respectively.Results1.To determine whether HNK induces aberrant expression of miRNAs in human OS cells,six miRNAs(miR-188-5p,miR-202 and miR-623 were the most significantly up-regulated;miR-21,miR-532-5p and miR-628-3p were the most significantly down-regulated)were selected based on the microarray data,and were verified by RT-qPCR.The results indicated that miR-202 and miR-623 were markedly up-regulated,and miR-21 was significantly down-regulated in human Saos-2 OS cells following HNK treatment(P<0.01),whereas miR-188-5p,miR-532-5p and miR-628-3p were not significantly different compared with in the vehicle group.2.To investigated the function of miR-21 in human OS cells.OS cells were treated with 10-100?M HNK for 24h and the expression levels of miR-21 were determined by RT-qPCR.The results demonstrated that HNK reduced miR-21 levels in a dose-dependent manner in human OS cells.3.Overexpression of miR-21 rescues the suppressive effects of HNK on OS cells.The proportion of apoptotic cells was significantly decreased in the HNK + miR-21 group compared with in the HNK group in Saos-2 and MG-63 cells(P<0.01).These data indicated that HNK may exert suppressive effects on human OS cells via down-regulating miR-21.ConclusionHNK reduced miR-21 levels in a dose-dependent manner in human OS cells.Overexpression of miR-21 rescues the suppressive effects of HNK on OS cells.HNK may exert antitumor effects via modulating miR-21 expression in human OS cells.PART ? THE MOLECULAR MECHANISM OF HNK IN HUMAN OS CELLS MEDIATED BY MIR-21ObjectiveTo explore the molecular mechanism of HNK in human OS cells mediated by miR-21.Methods1.The potential binding site between PTEN and miR-21 was identified using Target Scan.2.In the present study,luciferase-reporter plasmids containing wt or mut type 3'-UTR segments of PTEN were constructed.The reporters were cotransfected along-side miR-21 mimics/inhibitor or NC into Saos-2 cells,after which luciferase activity was measured.3.To further confirm that PTEN levels are modulated by miR-21,the Saos-2 human OS cell line was transfected with miR-21 mimic/inhibitor or NC,and the protein expression levels of PTEN were determined using Western Blot analysis.4.To further verify whether PTEN expression was modulated by HNK,Saos-2 cells were treated with 10-100?M HNK for 24h and PTEN expression was measured by Western Blotting.5.To determine whether HNK-mediated miR-21 modulation regulates the PI3K/AKT signaling pathway in OS cells.Saos-2 and MG-63 cells were transfected with or without miR-21 mimics following treatment with or without HNK,and Western Blot analysis was used to determine the expression levels of p-AKT,p-mTOR and p-p70S6K,which are major components of the PI3K/AKT signaling pathway.Results1.miR-21 inhibits PTEN by directly targeting PTEN 3'-UTR.2.The results demonstrated that HNK enhanced PTEN expression in a dose-dependent manner in human OS cells.3.The results demonstrated that miR-21 mimic significantly suppressed luciferase activity compared with mimic NC;however,the miR-21 inhibitor markedly enhanced luciferase activity compared with inhibitor NC in the presence of wt 3'-UTR(P<0.01).In addition,miR-21 did not affect luciferase activity of the reporter vector containing mut PTEN 3'-UTR.4.To further confirm that PTEN levels are modulated by miR-21,the human OS cell line was transfected with miR-21 mimic/inhibitor or NC,and the protein expression levels of PTEN were determined using Western Blot analysis.To further verify whether PTEN expression was modulated by HNK,OS cells were treated with 10-100?M HNK for 24h and PTEN expression was measured by Western Blotting.The results indicated that miR-21 inhibited PTEN expression in OS cells compared with the NC.5.The expression levels of AKT,mTOR and p70S6K were significantly down-regulated following HNK treatment compared with mock vehicle-treated cells or miR-21 transfection in Saos-2 and MG-63 cells;however,the expression levels of these proteins were significantly up-regulated in HNK-treated OS cells post-transfection with miR-21 mimics compared with transfection without miR-21 mimics(P<0.01).ConclusionHNK suppresses the PI3K/AKT signaling pathway by inhibiting miR-21 expression in human OS cells.