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Background Osteosarcoma (OS) is the most prevalent primary bone malignancy affecting adolescents, yet the emergence of chemoradiotherapeutic resistance has limited efforts to cure affected patients to date. Pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT) is a recently developed, minimally invasive treatment for OS that is similarly constrained by such therapeutic resistance. This study sought to explore the mechanistic basis for RhoA-activated YAP1 (YAP)-mediated resistance in OS. Methods The relationship between YAP expression levels and patient prognosis was analyzed, and YAP levels in OS cell lines were quantified. Immunofluorescent staining was used to assess YAP nuclear translocation. OS cell lines (HOS and MG63) in which RhoA and YAP were knocked down or overexpressed were generated using lentiviral vectors. CCK-8 assays were used to examine OS cell viability, while the apoptotic death of these cells was monitored via Hoechst staining, Western blotting, and flow cytometry. Tumor-bearing nude mice were additionally used to assess the relationship between lentivirus-mediated alterations in RhoA expression and MPPa-PDT treatment outcomes. TUNEL and immunohistochemical staining approaches were leveraged to assess apoptotic cell death in tissue samples. Results OS patients exhibited higher levels of YAP expression, and these were correlated with a poor prognosis. MPPa-PDT induced apoptosis in OS cells, and such MPPa-PDT-induced apoptosis was enhanced following YAP knockdown whereas it was suppressed by YAP overexpression. RhoA and YAP expression levels were positively correlated in OS patients, and both active and total RhoA protein levels rose in OS cells following MPPa-PDT treatment. When RhoA was knocked down, levels of unphosphorylated YAP and downstream target genes were significantly reduced, while RhoA/ROCK2/LIMK2 pathway phosphorylation was suppressed, whereas RhoA overexpression resulted in the opposite phenotype. MPPa-PDT treatment was linked to an increase in HMGCR protein levels, and the inhibition of RhoA or HMGCR was sufficient to suppress RhoA activity and to decrease the protein levels of YAP and its downstream targets. Mevalonate administration partially reversed these reductions in the expression of YAP and YAP target genes. RhoA knockdown significantly enhanced the apoptotic death of OS cells in vitro and in vivo following MPPa-PDT treatment, whereas RhoA overexpression had the opposite effect. Conclusions These results suggest that the mevalonate pathway activates RhoA, which in turn activates YAP and promotes OS cell resistance to MPPa-PDT therapy. Targeting the RhoA/ROCK2/LIMK2/YAP pathway can significantly improve the efficacy of MPPa-PDT treatment for OS.

The present study was performed to establish and characterize new human osteosarcoma cell lines resistant to pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT). MPPa-PDT-resistant cells are isolated from the human osteosarcoma MG63 and HOS cell lines and two resistant populations were finally acquired, including MG63/PDT and HOS/PDT. Cell Counting Kit-8 (CCK-8) assay was used to determine the MPPa-PDT, cisplatin (CDDP) resistance and proliferation of MG63, MG63/PDT, HOS and HOS/PDT cells. The intracellular ROS were analyzed using DCFH-DA staining. The colony formation, invasion and migration of parental and resistant cells were compared. FCM was employed to examine the cell cycle distribution, the apoptosis rate and the proportion of CD133+ cells. The fluorescence intensity of intracellular MPPa was observed by fluorescence microscopy and quantified using microplate reader. The protein levels were assessed by western blotting (WB). Compared with two parental cells, MG63/PDT and HOS/PDT were 1.67- and 1.61-fold resistant to MPPa-PDT, respectively, and also exhibited the resistance to CDDP. FCM assays confirmed that both MG63/PDT and HOS/PDT cells treated with MPPa-PDT displayed a significantly lower apoptosis rate in comparison with their corresponding parental cells. The expression of apoptosis-related proteins (i.e. cleaved-caspase 3 and cleaved-PARP), intracellular ROS and the antioxidant proteins (HO-1 and SOD1) in MG63/PDT and HOS/PDT cells was also lower than that in parental cells. Both MG63/PDT and HOS/PDT cells exhibited changes in proliferation, photosensitizer absorption, colony formation, invasion, migration and the cell cycle distribution as compared to MG63 and HOS cells, respectively. Compared to MG63 and HOS cells, both resistant cell lines had a higher expression of CD133, survivin, Bcl-xL, Bcl-2, MRP1, MDR1 and ABCG2, but a lower expression of Bax. The present study successfully established two resistant human osteosarcoma cell lines which are valuable to explore the resistance-related mechanisms and the approaches to overcome resistance.

Osteosarcoma is the most common primary malignant tumor of the bone found predominantly in children and teenagers and results in early metastasis and poor prognosis. The present study primarily focused on the impact of celastrol on apoptosis and autophagy of osteosarcoma HOS cells, as well as the related mechanisms. Following the appropriate treatment, the human osteosarcoma cell line HOS was assessed for viability, Ca2+ in cells, apoptosis and changes in cell morphology using Cell Counting Kit-8, flow cytometry, inverted phase contrast microscope, Hoechst staining and transmission electron microscopy. The expression levels of various proteins, including endoplasmic reticulum stress (ERS)-related proteins (Bip, PERK, p-PERK, IRE1α, calnexin, PDI and Erol‑Lα), apoptosis-related proteins (CHOP, cleaved caspase‑12), mitochondrial apoptosis-related proteins (Bax, Bcl-2 and cytochrome c), cleaved caspase-3, and autophagy-related proteins (LC3-Ⅰ, LC3-Ⅱ and P62) and β-actin, were assessed with western blotting. Celastrol significantly inhibited the viability of HOS cells in a dose-dependent manner and promoted the expression of ERS-related, apoptosis-related and mitochondrial apoptosis-related proteins. The ERS inhibitor tauroursodeoxycholate promoted celastrol-induced autophagy and apoptosis of HOS cells. Pretreatment with the PERK inhibitor GSK2656157 significantly promoted celastrol-induced death and attenuated HOS cell autophagy. Our results indicated that the ERS pathway and the mitochondrial pathway were involved in celastrol-induced apoptosis of HOS cells. The ERS/PERK pathway may protect HOS cells from apoptosis by celastrol and may play a complicated role in the process of autophagy.

Photodynamic therapy (PDT), which is a new method for treating tumors, has been used in the treatment of cancer. In-depth research has shown that PDT cannot completely kill tumor cells, indicating that tumor cells are resistant to PDT. Glucose regulatory protein 78 (GRP78), which is a key regulator of endoplasmic reticulum stress, has been confirmed to be related to tumor resistance and recurrence, but there are relatively few studies on the further mechanism of GRP78 in PDT. Our experiment aimed to observe the role of GRP78 in HOS human osteosarcoma cells treated with pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPα-PDT) and to explore the possible mechanism by which the silencing of GRP78 expression enhances the sensitivity of HOS osteosarcoma cells to MPPα-PDT. HOS osteosarcoma cells were transfected with siRNA-GRP78. Apoptosis and reactive oxygen species (ROS) levels were detected by Hoechst staining and flow cytometry, cell viability was detected by Cell Counting Kit-8 assay, GRP78 protein fluorescence intensity was detected by immunofluorescence, and apoptosis-related proteins, cell proliferation-related proteins, and Wnt pathway-related proteins were detected by western blot. The results showed that MPPα-PDT can induce HOS cell apoptosis and increase GRP78 expression. After successful siRNA-GRP78 transfection, HOS cell proliferation was decreased, and apoptosis-related proteins expressions was increased, Wnt/β-catenin-related proteins expressions was decreased, and ROS levels was increased. In summary, siRNA-GRP78 enhances the sensitivity of HOS cells to MPPα-PDT, the mechanism may be related to inhibiting Wnt pathway activation and increasing ROS levels.