| PART 1:THE ROLE AND MECHANISM OF MPPA-PDT THERAPY RESISTANCE INDUCED BY HIPPO/YAP SIGNALING PATHWAYObjective1.To study the relationship between YAP gene expression and protein expression in patients with osteosarcoma and clinical prognosis.2.To explore the possible mechanism of osteosarcoma cell resistance to MPPa-PDT treatment.3.To explore the role and mechanism of the Hippo/YAP signaling pathway in osteosarcoma cell resistance to MPPa-PDT treatment.Methods1.Tumor samples and corresponding adjacent tissues of 5osteosarcoma patients without chemotherapy were collected by biopsy from the First Affiliated Hospital of Chongqing Medical University for immunohistochemical staining（IHC）and western blotting（WB）test.2.The association between YAP gene expression and the clinical prognosis was analyzed using a public oncology dataset containing 127osteosarcoma samples.Analysis of microarray data using R2platform.YAP m RNA and protein levels were assessed in different bone tumor cell lines using data from the cancer cell line encyclopedia.The basic data obtained in CCLE was analyzed using Graph Pad Prism 8.3.Osteosarcoma cell lines HOS,MG63,U2OS,and SAOS2 were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in DMEM containing 10%FBS and penicillin/streptomycin in a5%CO2humidified incubator at 37°C.HOS and MG63 cells were treated with MPPa in a certain concentration range for 20 h,while shielded from light,and then washed twice with PBS.The medium was then added to the appropriate wells and the cells were exposed to red light（630nm,40m W/cm2）for 120s in a continuous output mode.4.Expression vector OE-YAP was obtained from Hanbio Biotechnology for a lentiviral vector containing YAP1.Short hairpin RNA（sh RNA）sequence-specific YAP1 from Hanbio Biotechnology.q PCR and Western blotting were used to assess changes in target gene expression in HOS and MG63 osteosarcoma cells infected with prepared lentivirus.5.CCK-8 was performed at 12 h post-MPPa-PDT treatment to detect the IC50 of HOS and MG63 in photosensitizer MPPa and the cell viability of HOS and MG63 cells at 12 h post-MPPa-PDT treatment.Hoechst staining was used to detect the apoptotic morphology of osteosarcoma cells at 12 h post-MPPa-PDT treatment.Immunofluorescence（IF）was used to detect YAP nuclear translocation at 12 h post-MPPa-PDT treatment for HOS and MG63 cells.Western blotting was used to detect the apoptotic marker and the key molecules of the Hippo signal pathway in osteosarcoma cells HOS and MG63 at 12 h post-MPPa-PDT.Flow cytometry（FC）assay was used to detect the apoptosis rate of osteosarcoma cells HOS and MG63at 12 h post-MPPa-PDT.Results1.The overall survival time of patients with osteosarcoma with a low expression level of YAP was significantly longer than that of patients with a high expression level of YAP.The expression level of YAP in osteosarcoma tumor tissue was also significantly higher than that in normal muscle tissue.2.HOS cell lines showed higher levels of YAP expression compared to other bone tumor cell lines.We then compared these findings with YAP levels in osteosarcoma cell lines such as HOS,MG63,U2OS,and SAOS2and obtained similar confirmation:HOS cells showed the highest levels of YAP expression.Finally,Western blotting,H&E staining,and IHC staining were performed to confirm that the expression level of YAP in tumor tissue samples from patients with osteosarcoma was higher than that in adjacent tissues.3.Next,the effects of MPPa-PDT treatment on HOS and MG63 cell viability were evaluated in vitro by the CCK-8 assay.After 24 h of treatment,it was found that MPPa-PDT induced a dose-dependent decrease in osteosarcoma cell viability,and the 50%inhibition concentration（IC50）of MPPa in HOS and MG63 cells was 0.15μM and 0.45μM,respectively.Then the MPPa-PDT treated cells were stained with Hoechst and the morphological changes of apoptosis were evaluated by fluorescence microscope.At 12 h post-MPPa-PDT treatment,we observed a significant increase in chromatin density,nuclear shrinkage,condensation,and nuclear rupture,all of which were consistent with apoptotic cell morphology.Conversely,reduced apoptosis was observed in the control group or in cells that received MPPa alone or light alone.Western blotting further showed that cleaved caspase-3,cleaved caspase-9,and cytochrome C levels were significantly increased at 12 h post-MPPa-PDT treatment compared with other treatment groups.The level of anti-apoptotic protein Bcl-2 was significantly decreased.Annexin V/PI staining was performed and the apoptosis rate was assessed by flow cytometry.At 12 h after MPPa-PDT treatment,no significant difference in apoptosis rate was observed between the control group,MPPa,and the light control group,while apoptotic death was significantly increased in the MPPa-PDT group.4.To evaluate the ability of MPPa-PDT to induce YAP activation in HOS and MG63 cells,Western blotting analysis was performed to demonstrate that p-LATS/LATS and p-YAP/YAP protein levels decreased at 12 h post-MPPa-PDT compared with the other three treatment groups.Meanwhile,CTGF and CYR61 protein levels were elevated.Cell immunofluorescence staining confirmed that YAP was predominantly localized in the nucleus at 12 h post-MPPa-PDT treatment,whereas in the three controls,YAP was localized in the cytoplasm.These results suggest that MPPa-PDT can induce osteosarcoma cell apoptosis and YAP activation.5.To further determine the functional importance of YAP in osteosarcoma cells,we then knocked down or overexpressed this gene in HOS and MG63 cells,and confirmed the change of YAP expression by Western blotting and q PCR.YAP knockdown and overexpression also resulted in a significant decrease or increase in the levels of YAP downstream target proteins（CTGF and CYR61）,respectively.CCK-8assay was used to detect the viability of MPPa-PDT-treated osteosarcoma cells after YAP knockdown or overexpression.After 12 h,these cells were treated with PDT in combination with a wide range of photosensitizer doses.These photosensitizers showed concentration-dependent effects related to target cell viability in subsequent PDT results.At 12 h post-MPPa-PDT treatment,the 50%inhibitory concentration（IC50）of MPPa in osteosarcoma cells was 0.155μM in HOS-sh NC cells,0.107μM in HOS-sh YAP cells,and 1.766μM in HOS-OE-YAP cells.0.448μM in MG63-sh NC cells,0.069μM in MG63-sh YAP cells,and 0.982μM in MG63-OE-YAP cells.CCK-8 assay showed that YAP overexpression was sufficient to inhibit MPPa-PDT-induced apoptosis of osteosarcoma HOS and MG63cells,while YAP knockdown enhanced the ability of MPPa-PDT to drive apoptosis in these osteosarcoma cell lines.Hoechst staining and flow cytometry further confirmed the role of YAP in the apoptosis of osteosarcoma cells induced by MPPa-PDT.Similarly,Western blotting showed that YAP knockdown was associated with increased Bax,cleaved caspase-3,cleaved caspase-9,and reduced Bcl-2 protein levels induced by MPPa-PDT,whereas YAP overexpression had the opposite effect.Together,these results suggest that YAP can enhance the therapeutic resistance of osteosarcoma cells to MPPa-PDT.Conclusions1.Patients with osteosarcoma show upregulation of YAP associated with poor prognosis.2.MPPa-PDT induces osteosarcoma cell apoptosis and YAP activation.3.YAP enhances the resistance of osteosarcoma cells to MPPa-PDT treatment.PART 2: EFFECT AND MECHANISM OF MPPA-PDT ON RHOAACTIVATION AND YAPACTIVATION VIA THE MEVALONATE PATHWAYObjective1.To investigate the relationship between RHOA gene expression and clinical prognosis in patients with osteosarcoma.2.To explore the possible mechanism of the mevalonate pathway in resistance to MPPa-PDT treatment in osteosarcoma cells.3.To explore the role and mechanism of the mevalonate pathwayRho A-YAP signaling pathway in the treatment resistance of osteosarcoma to MPPa-PDT.Methods1.A common oncology dataset containing 127 samples of osteosarcoma was used to analyze the relationship between YAP and RHOA expression in osteosarcoma samples and the relationship between RHOA gene expression and clinical prognosis.Analysis of microarray data using R2 platform.2.Osteosarcoma cell lines HOS and MG63 were cultured in DMEM containing 10% FBS and penicillin/streptomycin in a 5%CO2 humidified incubator at 37°C.Cells were treated with MPPa in a certain concentration range for 20 h,while shielded from light,and then washed twice with PBS.The medium was then added to the appropriate wells and the cells were exposed to red light（630nm,40 m W /cm2）for 120 s in a continuous output mode.3.Expression vector OE-RHOA was obtained from Hanbio Biotechnology.Short hairpin RNA（sh RNA）sequence-specific RHOA from Hanbio Biotechnology.q PCR and Western blotting were used to assess changes in target gene expression in HOS and MG63 osteosarcoma cells infected with prepared lentivirus.4.Simvastatin,an HMGCR inhibitor,and CCG-1423,a Rho A inhibitor,were treated with CCK-8 to detect the cellular viability of HOS and MG63 cells.Western blotting was used to detect the changes of ROCK2/LIMK2/Cofilin/F-actin signaling pathway after RHOA knockdown or overexpression and the changes of Rho A and HMGCR proteins at 12 h post-MPPa-PDT treatment in HOS and MG63 cells.Western blotting was used to detect Rho A,YAP,and downstream target genes of osteosarcoma treated with simvastatin or CCG-1423.Results1.To evaluate the expression of RHOA in patients with osteosarcoma,we started by analyzing the m RNA microarray data of the GEO database（GSE42352）and found that YAP was highly positively correlated with RHOA gene expression.Patients diagnosed with osteosarcoma with low RHOA expression levels had significantly longer survival time without metastasis than patients with high RHOA expression levels.2.Next,the effects of simvastatin or CCG-1423 treatment on HOS and MG63 cell viability were evaluated in vitro by CCK-8 assay.We observed a dose-dependent reduction in osteosarcoma cell viability induced by simvastatin or CCG-1423 at 24 h after treatment with simvastatin or at48 h after treatment with CCG-1423.3.To evaluate the ability of MPPa-PDT to induce RhoA activation in HOS and MG63 cells,Western blotting assay was performed next,and it was proved that total Rho A and activated Rho A protein levels increased at12 h post-MPPa-PDT treatment compared with the other three treatment groups.HMGCR protein expression was also elevated.4.To determine the functional importance of Rho A in osteosarcoma cells,we then knocked down or overexpressed this gene in HOS and MG63 cells,and confirmed the change of Rho A expression by Western blotting and q PCR.RHOA knockdown or overexpression also significantly reduced or increased the levels of activated YAP and its downstream target protein（CTGF）,respectively,and caused changes in ROCK2/LIMK2/Cofilin/F-actin signaling pathway proteins.5.To further determine the functions of mevalonic acid and RhoA in osteosarcoma cells,simvastatin or CCG-1423 were used in HOS and MG63 cells,respectively,and Western blotting was used to confirm the changes in the expressions of activated Rho A,activated YAP,and their downstream target genes.Use of simvastatin or CCG-1423 resulted in significant reductions in activated Rho A,activated YAP,and their downstream target proteins（CTGF and CYR61）,respectively,which were partially reversed by mevalonate supplementation.Conclusions1.Patients with osteosarcoma showed RHOA up-regulation associated with poor prognosis,and the expression of YAP was highly positively correlated with RHOA.2.Mevalonate pathway is involved in the resistance of osteosarcoma to MPPa-PDT treatment.3.The Rho A/ROCK2/LIMK2/Cofilin/F-actin signal axis regulates YAP activity.4.MPPa-PDT activates Rho A and YAP via the mevalonate pathway.PART 3: THE ROLE AND MECHANISM OF RHOA IN PROMOTING THE RESISTANCE OF OSTEOSARCOMA TO MPPA-PDT THERAPYObjective1.To investigate the role and mechanism of Rho A in the treatment resistance of osteosarcoma with MPPa-PDT.Methods1.Osteosarcoma cell lines HOS and MG63 were cultured in DMEM containing 10% FBS and penicillin/streptomycin in a 5%CO2 humidified incubator at 37°C.Cells were treated with MPPa in a certain concentration range for 20 h,while shielded from light,and then washed twice with PBS.The medium was then added to the appropriate wells and the cells were exposed to red light（630nm,40 m W /cm2）for 120 s in a continuous output mode.2.Expression vector OE-RhoA was obtained from Hanbio Biotechnology.Short hairpin RNA（sh RNA）sequence-specific RHOA（sh RHOA）from Hanbio Biotechnology.HOS and MG63 osteosarcoma cells were infected with prepared lentivirus to obtain stable cell lines.3.HMGCR inhibitor simvastatin,mevalonate,and Rho A inhibitor CCG-1423 were pretreated with osteosarcoma cells respectively,and Western blotting was used to detect the changes of YAP and Rho A,CTGF,and CYR61 proteins at 12 h after HOS and MG63 were treated with MPPaPDT.CCK8 detected the cell viability of osteosarcoma cells treated with MPPa-PDT after RHOA knockdown or overexpression.4.Simvastatin,mevalonate,and CCG-1423 were used to pretreat osteosarcoma cells with knockdown or overexpression of Rho A.Apoptosis morphology was detected by Hoechst staining,apoptosis rates were valued by flow cytometry,and molecular changes of apoptosis markers of osteosarcoma cells at 12 h post-MPPa-PDT treatment were detected by Western blotting.5.Whether MPPa-PDT can induce osteosarcoma apoptosis in vivo was evaluated by subcutaneous injection of osteosarcoma cells in nude mice,and the effect of RHOA knockdown or overexpression on osteosarcoma apoptosis was verified in vivo.In addition,TUNEL was used to detect the apoptosis level of tumor tissue in nude mice,and an immunohistochemical method was used to detect the expression of Cleaved PARP and Cleaved caspase-3 protein in tumor tissue in nude mice.Results1.To further determine the functional importance of MPPa-PDT in the treatment of Rho A activation and YAP in osteosarcoma cells,we then performed Western blotting on the following treatment groups: control group,MPPa-PDT,simvastatin + MPPa-PDT,CCG-1423 + MPPa-PDT,and mevalonate + simvastatin + MPPa-PDT.Analysis showed that simvastatin and CCG-1423 significantly reduced Rho A,activated YAP,and downstream target gene levels in osteosarcoma cells after MPPa-PDT treatment,while mevalonate can partially rescue YAP activation that was inhibited by simvastatin.2.Next,a CCK-8 assay was used to evaluate the cell viability after knockdown or overexpression of RHOA on HOS and MG63.These cells were treated in combination with PDT with a wide range of photosensitizer doses.These photosensitizers showed concentration-dependent effects related to target cell viability in subsequent PDT results.At 12 h postMPPa-PDT treatment,the IC50 of MPPa in osteosarcoma cells were0.158μM in HOS-sh NC cells,0.111μM in HOS-sh RHOA cells,3.460μM in HOS-OE-RHOA cells,0.456μM in MG63-sh NC cells,0.104μM in MG63-sh RHOA cells,and 0.801μM in MG63-OE-Rho A cells respectively.To observe the effect of Rho A on MPPa-PDT treatment in osteosarcoma cells,a CCK-8 test was performed,indicating that Rho A overexpression was sufficient to reverse the apoptosis of HOS and MG63 cells,while Rho A knockdown had the opposite effect.3.Hoechst staining and flow cytometry further confirmed this result.The application of CCG-1423,a Rho A inhibitor,was sufficient to rescue the inhibition of apoptosis induced by RHOA overexpression.Western blotting also showed that RHOA overexpression could partially reverse MPPa-PDT-induced apoptosis of HOS and MG63 cells,while Rho A knockdown promoted such apoptosis.This was demonstrated by increased cleaved PARP,Bax,and Cleaved caspase-3levels,as well as reduced Bcl-2 levels.4.Compared with the shNC group,tumor growth of mice implanted with sh RHOA osteosarcoma HOS cells was significantly reduced,while tumor growth of mice implanted with OE-RHOA osteosarcoma HOS cells was significantly enhanced.The inhibition of tumor growth was strongest in the sh RHOA group treated with MPPa-PDT.In contrast,mice treated with MPPa-PDT with OE-RHOA tumors showed more robust tumor growth than mice treated with MPPa-PDT with sh NC.5.TUNEL staining was performed on tumor tissue sections to evaluate the apoptotic cell death after MPPa-PDT treatment.The results showed that the apoptosis index values of different treatment groups（sh RHOA,OE-RHOA,MPPa-PDT,sh RHOA/MPPa-PDT,and OE-RHOA /MPPaPDT）were different.We also assessed apoptosis markers including cleaved PARP and cleaved caspase-3 to demonstrate RHOA knockdown and MPPa-PDT inhibition of osteosarcoma growth.Levels of these two apoptosis marker proteins were significantly increased in the sh RHOA and MPPa-PDT groups compared with the control group.Notably,the expression levels of these apoptotic proteins were highest in combination with sh RHOA/MPPa-PDT.Conclusions1.Rho A enhances the resistance of osteosarcoma cells to MPPaPDT.2.Rho A knockdown or in combination with MPPa-PDT is sufficient to inhibit the growth of osteosarcoma in vivo,while the best results of Rho A knockdown and MPPa-PDT therapy suggest that MPPa-PDT resistance can be eliminated by Rho GTPase knockdown. |
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