oxadiazoles and Osteosarcoma

Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies.

Topics: Adenosine Triphosphate; Cell Proliferation; Cell Respiration; Cellular Senescence; Electron Transport Complex I; Gene Expression Regulation, Developmental; Humans; Induced Pluripotent Stem Cells; Mitogen-Activated Protein Kinase Kinases; Mutation; Osteoblasts; Osteogenesis; Osteosarcoma; Oxadiazoles; Oxidative Phosphorylation; Piperidines; RecQ Helicases; RNA, Long Noncoding; Rothmund-Thomson Syndrome

Osteosarcoma (OS) is the predominant form of primary bone malignancy in children and adolescents. Although the combination of chemotherapy and modified surgical therapy leads to marked improvements in the survival rate, the therapeutic outcomes remain unsatisfactory. Therefore, the identification of novel drugs with higher efficacy and fewer side‑effects is urgently required. Proteasome inhibitors have been approved by the Food and Drug Administration (FDA) for the treatment of certain cancers, although none of them are directed against OS. Non‑covalent proteasome inhibitors, such as PI‑1840, are superior to covalent ones in numerous respects in view of their chemical structure; however, to date, no studies have been published on the effects of non‑covalent proteasome inhibitors on OS cells. In the present study, the antineoplastic effects of PI‑1840 were systematically evaluated in the OS cell lines, MG‑63 and U2‑OS. Cell viability and morphological changes were assessed by Cell Counting Kit‑8 (CCK‑8) and live/dead assays. The cell cycle was analyzed using flow cytometry (FCM) and western blot analysis (assessing the levels of the proteins p21, p27, and the tyrosine kinase, WEE1). The extent of cell apoptosis and autophagy were assessed by FCM, western blot analysis [of the apoptosis‑associated proteins, microtubule‑associated protein 1 light chain 3 α (LC3) and Beclin1], and mRFP‑GFP‑LC3 adenovirus transfection assay. Transwell and wound healing assays, and western blot analysis of the matrix metalloproteinases (MMPs)2 and 9 were performed to preliminarily evaluate the migration and invasion capability of the cells. In the present study, our results revealed that PI‑1840 inhibited the proliferation of OS cells and induced apoptosis, partly due to attenuation of the nuclear factor‑κB (NF‑κB) pathway. In addition, PI‑1840‑induced autophagy was detected, and inhibiting the autophagy of the OS cells led to an increase in the survival rate of the U2‑OS cells rather than of the MG‑63 cells. Furthermore, PI‑1840 attenuated the migration and invasion capabilities of the OS cells. In conclusion, the present study revealed PI‑1840 to be a promising drug for the treatment of OS.

Topics: Acetamides; Antineoplastic Agents; Apoptosis; Autophagy; Bone Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Osteosarcoma; Oxadiazoles; Proteasome Inhibitors

Nitrobenzoxadiazole derivatives (NBDs), including NBDHEX and the recently developed MC3181, are promising anticancer agents able to target glutathione transferase and inhibit both its catalytic activity and ability to sequester TNF-receptor associated factor 2 (TRAF2) and c-Jun N-terminal kinase (JNK). NBDs have been shown to impair the growth and survival of a broad-spectrum of tumor types, in vitro and in vivo. Herein, we evaluated the effects of the new compound MC3181 on U-2OS osteosarcoma cells and investigated the impact of both NBDHEX and MC3181 on autophagy.. Cell viability was evaluated by sulforhodamine B assay. The dissociation of the TRAF2-GSTP1-1 complex was detected by proximity ligation assay, while the phospho-activation of JNK was assessed by western blotting. The effects of NBDs on autophagy were evaluated by GFP-LC3 puncta formation, western blotting for LC3-II and p62, and LC3 turnover assay in the presence of bafilomycin A1. The role of JNK in the reduction of autophagic flux caused by NBDs was investigated using JNK1 shRNA-transfected cells. Fluorogenic caspase activity assay and flow cytometric analysis of DNA content were used to determine the cytotoxic effects of NBDs on JNK1-silenced cells.. Similar to NBDHEX, MC3181 reduced viability and activated TRAF2/JNK signaling in U-2OS cells. Moreover, NBDs induced the accumulation of autophagic vesicles and LC3-II while reducing both basal and nutritional stress-induced autophagic flux. Furthermore, increased levels of both LC3-II and the autophagy selective substrate p62 were observed in different tumor cell lines treated with NBDs, the concurrent increase of these markers being consistent with an impairment of autophagosome clearance. Autophagy inhibition by NBDs required JNK activity: NBDs caused autophagy inhibition and caspase-3 activation in JNK-positive U-2OS, but no autophagic flux inhibition or caspase-3 activation in JNK-silenced cells.. Our demonstration that NBDs can act as late-phase autophagy inhibitors opens new opportunities to fully exploit their therapeutic potential. This may not rely solely on their effectiveness in inducing cell cycle arrest and apoptosis, but also on their ability to weaken the capacity of tumor cells to endure stress conditions via autophagy. In addition, this study provides evidence that JNK can participate in impairing autophagy.

Topics: Apoptosis; Autophagy; Caspase 3; Cell Line, Tumor; Cell Survival; Enzyme Activation; Gene Silencing; Humans; Inhibitory Concentration 50; JNK Mitogen-Activated Protein Kinases; Microtubule-Associated Proteins; Osteosarcoma; Oxadiazoles; Phagosomes; RNA-Binding Proteins; Signal Transduction; Stress, Physiological; TNF Receptor-Associated Factor 2

Wnt/β-catenin is a major regulator of stem cell self-renewal and differentiation and this signaling pathway is aberrantly activated in a several cancers, including osteosarcoma (OS). Attenuation of Wnt/β-catenin activity by tankyrase inhibitors is an appealing strategy in treatment of OS. The efficacy of the tankyrase inhibitor JW74 was evaluated in three OS cell lines (KPD, U2OS, and SaOS-2) both at the molecular and functional level. At the molecular level, JW74 induces stabilization of AXIN2, a key component of the β-catenin destruction complex, resulting in reduced levels of nuclear β-catenin. At the functional level, JW74 induces reduced cell growth in all three tested cell lines, in part due to a delay in cell cycle progression and in part due to an induction of caspase-3-mediated apoptosis. Furthermore, JW74 induces differentiation in U2OS cells, which under standard conditions are resistant to osteogenic differentiation. JW74 also enhances differentiation of OS cell lines, which do not harbor a differentiation block. Interestingly, microRNAs (miRNAs) of the let-7 family, which are known tumor suppressors and inducers of differentiation, are significantly upregulated following treatment with JW74. We demonstrate for the first time that tankyrase inhibition triggers reduced cell growth and differentiation of OS cells. This may in part be due to an induction of let-7 miRNA. The presented data open for novel therapeutic strategies in the treatment of malignant OS.

Topics: Antineoplastic Agents; Apoptosis; Axin Signaling Complex; Bone Neoplasms; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Humans; Osteogenesis; Osteosarcoma; Oxadiazoles; Tankyrases; Triazoles; Wnt Signaling Pathway

The effect of the glutathione transferase P1-1 (GSTP1-1) targeting has been investigated in both sensitive (U-2OS) and cisplatin-resistant (U-2OS/CDDP4 μg) human osteosarcoma cell lines. Despite the different enzyme's content, inhibition of GSTP1-1 by 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) causes the activation of c-Jun N-terminal kinase (JNK) and apoptosis in both cell lines. However, different time courses of JNK activation and cell responses are observed. Whereas in the U-2OS/CDDP4 μg cell line drug treatment results in an early increase of caspase activity and secondary necrosis, in the U-2OS cells it mainly causes an early cell cycle arrest followed by apoptosis. In order to elucidate the action mechanism of NBDHEX we performed a proteomic investigation by label-free nLC-MS(E). The high-throughput analysis associated with a bioinformatic tool suggested the involvement of the TNF receptor associated factor (TRAF) family in the cellular response to the drug treatment. We report experimental evidence of the interaction between GSTP1-1 and TRAF2 and we demonstrate that NBDHEX is able to dissociate the GSTP1-1 : TRAF2 complex. This restores the TRAF2 : ASK1 signaling, thereby leading to the simultaneous and prolonged activation of JNK and p38. These mitogen-activated protein kinases (MAPKs) mediate different effects: JNK is crucial for apoptosis, whereas p38 causes an increase in the p21 level and a concomitant cell cycle arrest. Our study shows that GSTP1-1 plays an important regulatory role in TRAF signaling of osteosarcoma and discloses new features of the action mechanism of NBDHEX that suggest potentially practical consequences of these findings.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Gene Expression Regulation; Glutathione S-Transferase pi; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 8; Osteosarcoma; Oxadiazoles; p38 Mitogen-Activated Protein Kinases; Proteomics; Signal Transduction; TNF Receptor-Associated Factor 2

Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most used drugs for osteosarcoma chemotherapy. By using a series of CDDP-resistant variants, which were established from the U-2OS and Saos-2 human osteosarcoma cell lines, we found that CDDP resistance was mainly associated with the increase of both the intracellular level and enzymatic activity of glutathione S-transferase P1 (GSTP1). On the basis of these findings, we evaluated the clinical effect of GSTP1 in a series of 34 high-grade osteosarcoma patients and we found that the increased expression of GSTP1 gene was associated with a significantly higher relapse rate and a worse clinical outcome. These indications prompted us to assess the in vitro effectiveness of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), a promising new anticancer agent that is a highly efficient inhibitor of GSTP1. NBDHEX was tested on a panel of 10 human osteosarcoma cell lines and 20 variants of the U-2OS or Saos-2 cell lines that were resistant to CDDP, doxorubicin, or methotrexate. NBDHEX proved to be very active on the vast majority of these cell lines, including those with higher GSTP1 levels and enzymatic activity. Drug combination studies showed that NBDHEX can be used in association with CDDP and provided useful information about the best modality of their combined administration. In conclusion, our findings show that GSTP1 has a relevant effect for both CDDP resistance and clinical outcome of high-grade osteosarcoma and that targeting GSTP1 with NBDHEX may be considered a promising new therapeutic possibility for osteosarcoma patients who fail to respond to conventional chemotherapy.

Topics: Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cisplatin; Doxorubicin; Drug Interactions; Drug Resistance, Neoplasm; Glutathione S-Transferase pi; Humans; Osteosarcoma; Oxadiazoles; Survival Rate; Tumor Cells, Cultured

Topics: Adolescent; Anti-Inflammatory Agents; Bronchitis; Child; Child, Preschool; Citrates; Female; Gastrointestinal Diseases; Growth; Humans; Infant; Infant, Newborn; Leukemia; Lung Neoplasms; Muscles; Osteosarcoma; Oxadiazoles; Pyloric Stenosis; Sarcoma; Sarcoma, Ewing