Cisplatin-resistant Osteosarcoma Cells Research Articles

Groenlandicine enhances cisplatin sensitivity in cisplatin-resistant osteosarcoma cells through the BAX/Bcl-2/Caspase-9/Caspase-3 pathway

Groenlandicine is a protoberberine alkaloid isolated from Coptidis Rhizoma, a widely used traditional Chinese medicine known for its various biological activities. This study aims to validate groenlandicine’s effect on both cisplatin-sensitive and cisplatin-resistant osteosarcoma (OS) cells, along with exploring its potential molecular mechanism.The ligand-based virtual screening (LBVS) method and molecular docking were employed to screen drugs. CCK-8 and FCM were used to measure the effect of groenlandicine on the OS cells transfected by lentivirus with over-expression or low-expression of TOP1. Cell scratch assay, CCK-8, FCM, and the EdU assay were utilized to evaluate the effect of groenlandicine on cisplatin-resistant cells. WB, immunofluorescence, and PCR were conducted to measure the levels of TOP1, Bcl-2, BAX, Caspase-9, and Caspase-3. Additionally, a subcutaneous tumor model was established in nude mice to verify the efficacy of groenlandicine.Groenlandicine reduced the migration and proliferation while promoting apoptosis in OS cells, effectively damaging them. Meanwhile, groenlandicine exhibited weak cytotoxicity in 293T cells. Combination with cisplatin enhanced tumor-killing activity, markedly activating BAX, cleaved-Caspase-3, and cleaved-Caspase-9, while inhibiting the Bcl2 pathway in cisplatin-resistant OS cells. Moreover, the level of TOP1, elevated in cisplatin-resistant OS cells, was down-regulated by groenlandicine both in vitro and in vivo. Animal experiments confirmed that groenlandicine combined with cisplatin suppressed OS growth with lower nephrotoxicity.Groenlandicine induces apoptosis and enhances the sensitivity of drug-resistant OS cells to cisplatin via the BAX/Bcl-2/Caspase-9/Caspase-3 pathway. Groenlandicine inhibits OS cells growth by down-regulating TOP1 level.Therefore, groenlandicine holds promise as a potential agent for reversing cisplatin resistance in OS treatment.

Circular RNA circ-CHI3L1.2 modulates cisplatin resistance of osteosarcoma cells via the miR-340-5p/LPAATβ axis.

Resistance to chemotherapy drugs is a major factor affecting the surgical outcome and prognosis of osteosarcoma patients. Circular RNAs (circRNAs) play an important role in tumor resistance to chemotherapy. In the present study, we aimed to investigate the role and mechanism of circRNA circ-chitinase 3-like 1.2 (CHI3L1.2) in resistance to cisplatin chemotherapy in osteosarcoma. We found that circ-CHI3L1.2 levels were higher in cisplatin-resistant cells than in their parent cells. circ-CHI3L1.2 knockdown decreased the half-maximal inhibitory concentration (IC50) of cisplatin and the expression levels of P-glycoprotein (P-gp), multidrug-resistance protein 1 (MRP1), and glutathione-S-transferase Pi1 (GSTP1), and promoted apoptosis of cisplatin-resistant osteosarcoma cells. In addition, circ-CHI3L1.2 knockdown induced mesenchymal to epithelial transition (MET) and suppressed cell migration and invasion. The competitive endogenous RNA (ceRNA) mechanism indicated that circ-CHI3L1.2 targets the micro-RNA (miR)-340-5p-lysophosphatidic acid acyltransferase β (LPAATβ) axis, and inhibition of miR-340-5p alleviates the effect of circ-CHI3L1.2 knockdown. In conclusion, circ-CHI3L1.2 levels were increased in cisplatin-resistant osteosarcoma cells and circ-CHI3L1.2 knockdown sensitized cisplatin-resistant osteosarcoma cells to cisplatin through the miR-340-5p-LPAATβ axis.

Poncirin downregulates ATP-binding cassette transporters to enhance cisplatin sensitivity in cisplatin-resistant osteosarcoma cells.

Poncirin, a flavanone glycoside with bitter taste extracted from dried immature fruit of Poncirus trifoliate, exhibits multiple biological activities including anti-tumor activity. Our study aimed to determine the effect and potential mechanism of poncirin on cisplatin resistance in osteosarcoma (OS) cells. CCK-8, flow cytometry analysis, and caspase-3/7 activity assays were used to evaluate cisplatin sensitivity. The expression changes of multidrug resistance 1 (MDR1), multidrug resistance-associated protein (MRP1), breast cancer resistance protein (BCRP), and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway-related proteins were detected by RT-qPCR or western blot analyses. Results showed that poncirin exposure enhanced cisplatin sensitivity, promoted apoptosis, and increased caspase-3/7 activity in cisplatin-resistant OS cells. Poncirin decreased the expression levels of MDR1, MRP1, and BCRP, and inhibited the PI3K/Akt signaling in OS cells. Rescue experiments suggested that activation of the PI3K/Akt signaling by 740Y-P abolished poncirin-induced expression reduction of MDR1, MRP1, and BCRP, and attenuated the facilitative effects of poncirin on cisplatin sensitivity and apoptosis in cisplatin-resistant OS cells. In summary, poncirin suppressed cisplatin resistance in cisplatin-resistant OS cells by downregulating the expression of MDR1, MRP1, and BCRP through inhibiting the PI3K/Akt pathway.

WITHDRAWN: LncRNA KCNQ1OT1 promotes cisplatin resistance of osteosarcoma cancer cells through the miR-335–5p/β-catenin axis

Abstract Long noncoding RNA (lncRNA) KCNQ1OT1 (KCNQ1 opposite strand/antisense transcript 1) has been shown to participate in the tumorigenesis of several types of cancers. However, little is known about the role of KCNQ1OT1 in the chemoresistance of osteosarcoma (OS). In this study, we concentrated on the function and mechanism of KCNQ1OT1 in cisplatin resistance of OS cells. We demonstrated that KCNQ1OT1 levels were upregulated in cisplatin-resistant OS tissues and cell lines. Moreover, knockdown of KCNQ1OT1 inhibited cell proliferation, migration, and invasion and decreased cisplatin resistance in osteosarcoma 143B cells. Additionally, bioinformatics analysis and dual-luciferase reporter assays showed that miR-335-5p directly targeted KCNQ1OT1 and the 3′-UTR of β-catenin mRNA, indicating that KCNQ1OT1 regulated the expression of β-catenin via endogenous sponging of miR-335-5p. Overall, KCNQ1OT1 modulated cisplatin resistance in OS through the miR-335-5p/β-catenin axis, indicating that KCNQ1OT1 aggravates OS progression and chemoresistance to cisplatin through induction of β-catenin expression by adsorbing miR-335-5p in cisplatin-resistant OS cells, providing a theoretical basis for the treatment of chemoresistant OS.

CircUBAP2 promotes SEMA6D expression to enhance the cisplatin resistance in osteosarcoma through sponging miR-506-3p by activating Wnt/\u03b2-catenin signaling pathway

The occurrence of chemo-resistance is an essential reason for the high morbidity of osteosarcoma (OS) patients. Circular RNAs (circRNAs) have been involved in the regulation of chemo-resistance in cancers. Semaphorins 6D (SEMA6D) is abnormally expressed in many cancers. However, the roles of circUBAP2 and SEMA6D in the chemo-resistance of OS are still unclear. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of circUBAP2, SEMA6D and microRNA-506-3p (miR-506-3p). The cisplatin resistance and proliferation of cells were evaluated by 3-(4, 5-dimethyl-2 thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay. Western blot analysis was performed to measure the protein levels of Wnt/β-catenin signaling pathway biomarkers and SEMA6D. Also, the apoptosis, migration and invasion of cells were assessed by Flow cytometry and Transwell assays, respectively. Besides, Dual-luciferase reporter assay was used to verify the interaction between miR-506-3p and circUBAP2 or SEMA6D. We found that the expression levels of circUBAP2 and SEMA6D were increased in cisplatin-resistant OS tissues and cells. Knockdown of circUBAP2 inhibited the cisplatin resistance, silenced Wnt/β-catenin signaling pathway, hindered cell proliferation, migration and invasion, and promoted apoptosis in cisplatin-resistant OS cells, all of which could be reversed by overexpression of SEMA6D. MiR-506-3p could be sponged by circUBAP2 and could target SEMA6D. The suppression of miR-506-3p overexpression on the progression of OS cisplatin resistance could be reversed by SEMA6D overexpression, while miR-506-3p inhibitor also could invert the inhibitory effect of circUBAP2 silencing on the progression of OS cisplatin resistance. In conclusion, CircUBAP2 and SEMA6D played active roles in the progression of OS cisplatin resistance through miR-506-3p, which might provide some new ideas for studying the countermeasures of OS resistance.

Overcoming cisplatin resistance in osteosarcoma through the miR-199a-modulated inhibition of HIF-1α.

Dysregulation of miRNAs has been shown to contribute to multiple tumorigenic processes, as well as to correlate with tumour progression and prognosis. miR-199a has been shown to be dysregulated in multiple tumour types. However, the association between miR-199a and the chemoresistance features of osteosarcoma are not well understood, the target genes for miR-199a and the regulatory mechanisms are also unknown. In the present study, we demonstrated that miR-199a is expressed at low levels in osteosarcoma cells and patient samples. By the selection and establishment of cisplatin resistant osteosarcoma cell line, we observed a correlation between miR-199a and cisplatin resistance in osteosarcoma cells: resistant cells exhibit attenuated miR-199a expressions and exogenous overexpression of miR-199a sensitizes osteosarcoma cells to cisplatin. Moreover, we identified HIF-1α as a direct target for miR-199a. Intriguingly, cisplatin resistant osteosarcoma cells display significantly elevated HIF-1α expression under hypoxia. We report here overexpression of miR-199a resensitizes cisplatin resistant cells to cisplatin through inhibition of HIF-1α in vitro and in vivo. Finally, by analysing the clinical osteosarcoma patient samples, we demonstrate a reverse correlation between miR-199a and HIF-1α mRNAs. Our study will provide mechanisms for the miRNA-mediated anticancer therapy and miR-199a may be considered a promising therapeutic agent for osteosarcoma patients who fail to respond to conventional chemotherapy.

Long non-coding RNA ROR regulated ABCB1 to induce cisplatin resistance in osteosarcoma by sponging miR-153-3p.

Osteosarcoma (OS) is a common cancer among adolescences worldwide. Cisplatin is widely used to treat cancer, but many patients acquire chemoresistance over time. LncRNA regulator of reprogramming (ROR) has been reported to be associated with many malignancies, including OS. However, the function of ROR in cisplatin resistance and the biological mechanism of ROR remain unclear in OS. The levels of ROR, miR-153-3p, and ABCB1 in cisplatin-resistant OS tissues and cells were detected by qRT-PCR and/or Western blot assay. The interactions of miR-153-3p, ROR, and ABCB1 were predicted by miRcode Tools and StarBase v2.0 online database, respectively, and validated by Dual-Luciferase reporter assay. The cell viability in different concentrations of DDP, cell proliferative capacity, migrated cells, and invaded cells were detected by MTT assay and transwell assay, respectively. The levels of ROR and ABCB1 were both drastically increased, and miR-153-3p was apparently decreased in relapsed OS tissues, MG63/DDP, and U2OS/DDP cells. MiRcode Tools and StarBase v2.0 predicted that miR-153-3p had complementary sequences with ROR and ABCB1 3'UTR, and following Dual-Luciferase reporter assay validated that miR-153-3p was a direct target of ROR and ABCB1. Moreover, ABCB1 overexpression alleviated the inhibitory effects on cell viability in different concentrations of DDP, cell proliferative capacity, migrated cells, and invaded cells in MG63/DDP and U2OS/DDP cells transfected with sh-ROR. ROR regulated ABCB1 expression in MG63/DDP and U2OS/DDP cells by sponging miR-153-3p. We found that ROR or ABCB1 knockdown can increase the cisplatin sensitivity of MG63/DDP and U2OS/DDP cells. ROR contributed to cisplatin resistance in OS via miR-153-3p/ABCB1 axis, unraveling a new regulatory network of chemoresistance in cisplatin-resistant OS cells and may provide a therapeutic target for OS patients.

SNHG16 promotes osteosarcoma progression and enhances cisplatin resistance by sponging miR-16 to upregulate ATG4B expression

BackgroundHuman osteosarcoma is the most common primary cancer of the bone. Multiple mechanisms underlying cell growth, apoptosis, bone development, and drug resistance are important in the development of osteosarcoma therapy, which remains to be fully studied. MethodsWe collected thirty-paired tumor tissues and the adjacent normal ones from osteosarcoma patients. Two osteosarcoma cell lines (SAOS2, U2OS) were used for in vitro experiments. RT-qPCR and Western blot were used for gene expression detection. We applied starBase to predict the potential binding sites. Then, the luciferase reporter assay was used to confirm the potential direct interaction. Besides, we applied CCK-8, EdU assay, and flow cytometric assays to detect cell growth and apoptosis rate. We used wound healing and transwell assays to determine cell migration and invasion abilities. Additionally, we constructed a cisplatin-resistant osteosarcoma cell line to study the potential impact of the regulatory axis on drug resistance. ResultsSmall nucleolar RNA host gene 16 (SNHG16) and autophagy-related 4B (ATG4B) were significantly upregulated in osteosarcoma tissues than the normal ones, and the higher expression level of SNHG16 predicted a poor prognosis in osteosarcoma patients. By contrast, the expression level of miR-16 was markedly lower in tumor tissues and was negatively correlated with SNHG16 (p < 0.001). SNHG16 was shown to promote cell growth, migration, and invasion, while miR-16 reversed this impact. Meanwhile, overexpression of ATG4B significantly promoted the development of osteosarcoma cells attenuated by SNHG16 knockdown or miR-16 mimics. Specifically, overexpression of ATG4B promoted cisplatin-induced autophagy and inhibited cell apoptosis rate, which enhanced the cisplatin resistance in osteosarcoma cell lines. ConclusionsOverall, our findings showed the importance of the regulatory axis of SNHG16/miR-16/ATG4B underlying osteosarcoma progression and chemoresistance to cisplatin. This research would benefit the therapy development in the treatment of osteosarcoma.

The induction of ferroptosis by impairing STAT3/Nrf2/GPx4 signaling enhances the sensitivity of osteosarcoma cells to cisplatin.

Recent studies have indicated that promoting ferroptosis is a promising approach to attenuate drug resistance of cancer cells. Hence, this study aimed to induce ferroptosis in osteosarcoma cells, thereby increasing the sensitivity to cisplatin. Osteosarcoma cells MG63 and Saos-2 were incubated with increasing doses of cisplatin to generate cisplatin-resistant strains, MG63/DDP and Saos-2/DDP. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays were performed to evaluate cell proliferation and cell death, respectively. Malondialdehyde (MDA), reactive oxygen species (ROS), and lipid oxidation in cells were measured to evaluate the degree of cell ferroptosis. MG63/DDP and Saos-2/DDP cells showed increased viability and decreased death rate compared with MG63 and Saos-2 cells, respectively, upon cisplatin treatment. Western blotting analysis indicated that protein levels of p-STAT3 (Ser727), nuclear factor erythroid 2-related factor 2 (Nrf2), and glutathione peroxidase 4 (GPx4) in drug-resistant strains increased significantly in response to cisplatin. Co-treatment with cisplatin and agonists of ferroptosis, Erastin, and RSL3, remarkably increased MDA, ROS, lipid oxidation, and sensitivity to cisplatin, in MG63/DDP and Saos-2/DDP cells. Similar results were observed by co-treatment of cells with cisplatin and a STAT3 inhibitor. The reduction of protein levels of p-STAT3 (Ser727), Nrf2, and GPx4 in MG63/DDP and Saos-2/DDP cells resulted in increased ferroptosis and sensitivity to cisplatin. These results indicate that cisplatin-resistant osteosarcoma cells inhibited ferroptosis after exposure to low doses of cisplatin. However, ferroptosis agonists and STAT3 inhibitor reactivated ferroptosis in the cells and consequently increased sensitivity to cisplatin. This study demonstrates a new approach to attenuate resistance of osteosarcoma to cisplatin in vitro .

Down-regulation of miR-377 contributes to cisplatin resistance by targeting XIAP in osteosarcoma.

Chemoresistance is one of the obstacles for effective treatment of cancers, and recent evidence has shown that microRNAs play critical roles in drug resistance. In this study, we investigated the effect of miR-377 on cisplatin resistance in osteosarcoma (OS). Tumor tissues from chemoresistant and control OS patients were subjected to Real-time polymerase chain reaction (PCR) to assess miR-377 expression. The effect and mechanism of miR-377 on cisplatin resistance were assessed using Cell Counting Kit (CCK) 8, flow cytometry, Western blot, and luciferase assays in cisplatin-resistant OS cells lines. Down-regulation of miR-377 was found in chemoresistant OS tissues and cisplatin-resistant OS cell lines. Overexpression of miR-377 re-sensitizes cisplatin resistant OS cells to cisplatin-induced caspase-3 dependent apoptosis. MiR-377 directly represses X-linked inhibitor of apoptosis protein (XIAP) expression through binding to its 3' untranslated region (UTR) of mRNA. Overexpression of XIAP partially cancelled the cisplatin-sensitizing effect of miR-377. These data uncovered an essential function of miR-377/XIAP signaling axis in regulating cisplatin resistance of OS. MiR-377 may have potential therapeutic values in tackling OS chemoresistance.

MiR-133b induces chemoresistance of osteosarcoma cells to cisplatin treatment by promoting cell death, migration and invasion.

As an important chemotherapeutic agent for the treatment of osteosarcoma, the effectiveness of cisplatin is considered to be due to its unique properties, which allow it to penetrate the cell membrane and form various DNA-platinum adducts, resulting in genetic alterations or DNA damage. However, chemoresistance to cisplatin remains a major challenge for its use and chemotherapeutic effects. In the present study, an isogenic model of a cisplatin resistant osteosarcoma cell line, MG63-DDP, was generated from the original MG63 cell line. The expression level of microRNA (miR)-133b in the MG63-DDP cisplatin-resistant osteosarcoma cell line was analyzed by reverse transcription-quantitative polymerase chain reaction (PCR). Cisplatin-DNA adduct formation, cell death (carboxyfluorescein succinimidyl ester/propidium iodide staining) and clonogenic survival assays (crystal violet staining) were performed, comparing various cell types. The effect of miR-133b on migration (scratch wound assay) and invasion (Transwell assay) was also evaluated. Characterization studies have previously revealed an increased level of miR-133b in MG63-DDP cells compared with normal MG63 cells. Upregulation of miR-133b was associated with the accumulation of cisplatin-DNA adducts and an increase in cisplatin-induced cell death. Furthermore, increased miR-133b expression levels enhanced the migration and invasion of MG63 cells under cisplatin stress. Concordantly, in MG63-DDP cells the neutralization of miR-133b demonstrated opposite effects, as compared with the upregulation of miR-133b. To the best of our knowledge, the present study demonstrated for the first time that cisplatin-resistant MG63 cells exhibit an increased level of miR-133b expression. The endogenous expression level of miR-133b is sufficient for inducing cisplatin resistance, which suggests that miR-133b may be a biomarker for cisplatin resistance in osteosarcoma.

MiR-214 modulates cisplatin sensitivity of osteosarcoma cells through regulation of anaerobic glycolysis.

Osteosarcoma is the most frequent primary bone tumor originating from adolescents and young adults. Despite improvements in the chemo- or radio- therapy of osteosarcoma patients, survival rate has not increased and drug resistance becomes a major factor that limits the effectiveness. Therefore, investigation of new treatment modalities is urgently required to optimize therapeutic options. Our previous study described an oncogenic role of miR-214 through promotion of osteosarcoma cells proliferation. In this study, we report miR-214 contributes to cisplatin resistance in osteosarcoma cells. Overexpression of miR-214 decreased the cisplatin sensitivity. By establishing an osteosarcoma cisplatin resistant cell line, we find miR-214 is significantly upregulated in cisplatin resistant cells. Moreover, we show miR-214 promotes anaerobic glycolysis rates of osteosarcoma cells but suppresses mitochondrial oxidative phosphorylation. Consistently, cisplatin resistant cells exhibit upregulated glycolysis but decreased mitochondrial oxidative phosphorylation, a phenotype called "Warburg effect". Finally, we demonstrate inhibition of glycolysis by either glycolysis inhibitor or miR-214 inhibition significantly re-sensitizes cisplatin resistant osteosarcoma cells. In summary, this study illustrates a miRNA-involved chemosensitivity of osteosarcoma and will contribute to the developments of therapeutic agents for the anti-chemoresistance treatments.

ZBTB7A Enhances Osteosarcoma Chemoresistance by Transcriptionally Repressing lncRNALINC00473-IL24 Activity

Chemoresistance remains a major drawback to osteosarcoma treatment. ZBTB7A, a member of the POK transcription repressor family, was shown to play an important role in tumorigenesis. However, the effect of ZBTB7A on osteosarcoma chemoresistance is completely unknown. In this study, we found that ZBTB7A is increased in cisplatin-resistant osteosarcoma cells and that elevated ZBTB7A inhibits cisplatin-induced apoptosis by repressing LINC00473 expression. Further mechanistic studies revealed that ZBTB7A directly binds to the promoter and suppresses the transcription of LINC00473. Additionally, our data indicate that LINC00473 interacts with the transcript factor C/EBPβ, facilitating its binding to the promoter of IL24, leading to decrease chemoresistance. Thus, these findings indicate that the ZBTB7A-mediated LINC00473-C/EBPβ-IL24 pathway is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

Telomerase reverse transcriptase promotes chemoresistance by suppressing cisplatin-dependent apoptosis in osteosarcoma cells

Cisplatin is one of the most efficacious antimitotic drugs used in the treatment of a range of malignant tumors. However, treatment failures are common due to the development of chemoresistance. In addition to its telomere maintenance function, telomerase plays a pro-survival role, inducing decreased apoptosis and increased resistance against DNA damage. Elucidation of the molecular mechanisms underlying this effect is critical to improve treatment outcomes. Previously, our group showed higher telomerase reverse transcriptase(TERT) expression in cisplatin resistant osteosarcoma cells. In this study, confocal fluorescence microscopy experiments revealed that TERT translocates from the nucleus to mitochondria in cisplatin treated osteosarcoma cells. We observed decreased apoptosis rate and improved mitochondrial function in TERT-overexpressing cells following cisplatin treatment. Based on these results, we further established that TERT inhibits cisplatin-induced apoptosis independently of telomerase reverse transcriptase activity. Moreover, TERT suppressed cisplatin-induced apoptosis and improved mitochondrial function via alleviating intracellular ROS in osteosarcoma cells. Our finding that TERT shuttles from the nucleus to the mitochondrion in response to cisplatin treatment and inhibits cisplatin-induced apoptosis in osteosarcoma cells may be especially important to overcome drug resistance.

Silencing LPAATβ inhibits tumor growth of cisplatin-resistant human osteosarcoma in vivo and in vitro.

Cisplatin-resistance has become a major impediment in the medical treatment of cancers such as osteosarcoma, the most common primary malignancy of bone. Since lysophosphatidic acid acyltransferase β (LPAATβ) was reported to be critically involved in osteosarcoma, our study investigated the role of LPAATβ in human osteosarcoma with cisplatin-resistance. Expression of LPAATβ or other relevant proteins were analyzed in 40 osteosarcoma patients by immunohistochemistry analysis (IHC), and in cisplatin‑resistant sublines by real-time PCR and western blotting. Next, the synthesized siRNA was inserted into the lentivirus vector and silencing of LPAATβ expression was employed to determine the effect of LPAATβ on cisplatin-resistant osteosarcoma cell viability invitro and osteosarcoma tumor growth in vivo with cisplatin treatment. Exogenous LPAATβ mediated by heritable RNAi decreased cisplatin‑resistant sensitivity through activating the PI3K/Akt/mTOR signaling pathway. We further demonstrate that silencing LPAATβ effectively inhibited tumor growth in nude mice with xenografts of cisplatin‑resistant osteosarcoma cells. IHC assay results showed that PI3K/Akt/mTOR signaling pathway was also involved in this process. Our results suggested that LPAATβ may play an important role in osteosarcoma and silencing LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of cisplatin-resistance.

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy

ABSTRACTRecent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

Cisplatin selects for stem-like cells in osteosarcoma by activating Notch signaling.

Notch signaling regulates normal stem cells and is also thought to regulate cancer stem cells (CSCs). Recent data indicate that Notch signaling plays a role in the development and progression of osteosarcoma, however the regulation of Notch in chemo-resistant stem-like cells has not yet been fully elucidated. In this study we generated cisplatin-resistant osteosarcoma cells by treating them with sub-lethal dose of cisplatin, sufficient to induce DNA damage responses. Cisplatin-resistant osteosarcoma cells exhibited lower proliferation, enhanced spheroid formation and more mesenchymal characteristics than cisplatin-sensitive cells, were enriched for Stro-1+/CD117+ cells and showed increased expression of stem cell-related genes. A similar effect was observed in vivo, and in addition in vivo tumorigenicity was enhanced during serial transplantation. Using several publicly available datasets, we identified that Notch expression was closely associated with osteosarcoma stem cells and chemotherapy resistance. We confirmed that cisplatin-induced enrichment of osteosarcoma stem cells was mediated through Notch signaling in vitro, and immunohistochemistry showed that cleaved Notch1 (NICD1) positive cells were significantly increased in a relapsed xenograft which had received cisplatin treatment. Furthermore, pretreatment with a γ-secretase inhibitor (GSI) to prevent Notch signalling inhibited cisplatin-enriched osteosarcoma stem cell activity in vitro, including Stro-1+/CD117+ double positive cells and spheroid formation capacity. The Notch inhibitor DAPT also prevented tumor recurrence in resistant xenograft tumors. Overall, our results show that cisplatin induces the enrichment of osteosarcoma stem-like cells through Notch signaling, and targeted inactivation of Notch may be useful for the elimination of CSCs and overcoming drug resistance.

(Z)-3,4,3',5'-tetramethoxystilbene, a natural product, induces apoptosis and reduces viability of paclitaxel-and cisplatin-resistant osteosarcoma cells.

Osteosarcoma is a common bone tumor and the development of drug resistance in therapy of osteosarcoma is a general rule. The natural compounds isolated from medicinal plants represent a valuable resource for anticancer therapeutics. (Z)-3,4,3', '-tetramethoxystilbene is one of them. In this work, we investigated the potential anti-cancer activities of (Z)-3,4,3' ,'5-tetramethoxystilbene in paclitaxel- and cisplatin-resistant osteosarcoma cells. ATP assay was used to examine cell viability. Cell nuclei staining assays with Hoechst or propidium iodide (PI) were used to evaluate cell apoptosis. Xenograft tumor model was used to evaluate the in vivo anti-cancer activities of (Z )-3,4,3',5'-tetramethoxystilbene. TUNEL staining assay was used to evaluate the apoptosis of tumor cells. We found that (Z)-3,4,3',5'-tetramethoxystilbene could effectively reduce viability of both paclitaxel-and cisplatin-resistant osteosarcoma cells. Moreover, (Z)-3,4,3',5'-tetramethoxystilbene induced dramatic apoptosis in resistant cells. Importantly, (Z)-3,4,3',5'-tetramethoxystilbene significantly suppressed in vivo tumor growth of cisplatin-resistant osteosarcoma. This is the first report on anti-cancer activity of (Z)-3,4,3','5- tetramethoxystilbene in resistant osteosarcoma cells. Our studies suggest that (Z)-3,4,3',5'-tetramethoxystilbene is a promising therapeutic drug for overcoming drug resistance in osteosarcoma.

Chloroquine blocks the autophagic process in cisplatin-resistant osteosarcoma cells by regulating the expression of p62/SQSTM1

Cisplatin (DDP) is one of the most effective chemotherapeutic drugs against osteosarcoma (OS), the most common malignant bone-specific tumor. However, the acquired resistance to DDP limits its effectiveness in tumor treatment. In this study, in order to elucidate the mechanisms of drug resistance in cancer cells, we investigated cell death induced by DDP in OS cells. We evaluated the contribution of autophagy in the process of drug resistance in a panel of four OS cell lines, MG-63, U-2OS, MNNG/HOS and Saos-2. The cells were treated with DDP (0-50 µM) for 48 h and then cell vaibility was assessed using the Cell Counting kit-8 (CCK-8). Apoptosis was detected by flow cytometry and the green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) expression vector was used to visualize the formation of autophagic vesicles. Our results demonstrated that autophagy was induced by DDP in the drug-resistant cell line, Saos-2, which does not respond to DDP with apoptosis. DDP-induced autophagy protected the Saos-2 cells from apoptotic cell death. Moreover, the inhibition of autophagy by chloroquine, an inhibitor of lysosomal proteases, accelerated the DDP-induced cell death in Saos-2 cells. We also found that during DDP treatment, the protein expression level of the autophagic regulator, p62/sequestosome 1 (SQSTM1), decreased during the first hour of treatment, followed by a rapid recovery. Therefore, our data suggest a potential clinical therapy by targeting autophagy with chloroquine or monoclonal antibodies for the treatment of drug-resistant OS.