1,3,5-triazines inhibit osteosarcoma and avert lung metastasis in a patient-derived orthotopic xenograft mouse model with favorable pharmacokinetics.

Objectives: Osteosarcoma is a major solid malignant tumor of bone, possessing significant burden on healthcare due to non-availability of specific anticancer agents. The current study was conducted to identify novel 1,3,5-triazine derivatives against osteosarcoma. Materials and Methods: The compounds were synthesized in a straight-forward two-step reaction and subsequently tested against PI3K and mTOR kinase and anticancer activity against osteosarcoma cells (MG-63, U2-OS, and Saos-2). The effect of the most potent compound was evaluated on apoptosis and cell phase of Saos-2 cells. The pharmacological activity was further established in the patient-derived orthotopic xenograft (PDOX) mouse model. Results: The developed compounds 8 (a-f) showed significant inhibitory activities against PI3K, mTOR, and OS cells. Among the tested series, compound 8a showed highly potent PI3K/mTOR inhibitory activity with significant anticancer activity against Saos-2 cells compared with Imatinib as standard. It also induces apoptosis and causes G2/M arrest in Saos-2 cells. Compound 8a significantly improved body weight, reduced tumor volume, and inhibited lung metastasis in athymic nude mice in a PDOX mouse model. It also showed optimal pharmacokinetic parameters in SD rats. Conclusion: In summary, 1,3,5-triazine analogs were identified as new PI3K/mTOR inhibitors against osteosarcoma.

Novel 1,3,5-triazine-nicotinohydrazide derivatives induce cell arrest and apoptosis in osteosarcoma cancer cells and inhibit osteosarcoma in a patient-derived orthotopic xenograft mouse model.

The present study deals with developing novel 1,3,5-triazine-nicotinohydrazide derivatives as potent CDK9 inhibitors in a straightforward synthetic route with potent anti-osteosarcoma activity. The most potent CDK9 inhibitor compound 5k inhibits proliferation of MG-63 cells via induction of apoptosis and G2/M cell cycle arrest. It reduces tumor progression in the patient-derived orthotopic xenograft (PDOX) mouse model with significant antioxidant and anti-inflammatory activity. In tumor tissue homogenates, it caused significant inhibition of CDK9 and inhibited the phosphorylation of RNAPII ser2 and reduced MCL-1 expression in Western blot analysis. Compound 5k also showed considerable bioavailability in SD mice. Our results demonstrated that compound 5k inhibits growth of OS in vitro and in vivo via inhibition of CDK9 which attenuated the downstream phosphorylation of RNAPII ser2 and represses expression of the anti-apoptotic protein, MCL-1 for the induction of apoptosis in OS.

Design and development of novel 1,2,3-triazole chalcone derivatives as potential anti-osteosarcoma agents via inhibition of PI3K/Akt/mTOR signalling pathway.

Osteosarcoma (OS) is an uncommon tumour that mainly affects bone in children and adolescents. The current treatment options of OS are of limited significance due to their immense side effects. In the present manuscript, we have developed a novel series of 1,2,3-triazole chalcone derivatives as potential agents against OS. The compounds were synthesized and evaluated for their PI3K and mTOR inhibitory activity using luminescent kinase assay, and Lance ultra assay, resp. The entire set of compounds showed significant to moderate inhibition of both kinases in the nanomolar range. The three most active compounds: 4e (N-(4-(3-(1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)acryloyl)phenyl)-4-nitrobenzamide), 4f (N-(4-(3-(1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)acryloyl)phenyl)-4-chlorobenzamide) and 4g (4-bromo-N-(4-(3-(1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)acryloyl)phenyl)benzamide), were evaluated for anticancer activity against human OS cancer cell line (MG-63), liver cancer cell line (HepG2), lung cancer cell line (A549) and cervical cancer (HeLa), using MTT assay. Among the tested series, compound 4e showed a better inhibitory profile than gedatolisib against PI3K and was approximately comparable to that of gedatolisib against mTOR. The most significant inhibitory activity was observed for compound 4e against all cell lines (MG-63, HepG2, A549 and HeLa), still somewhat lower to comparable to that of gedatolisib, but with the highest potency against MG-63 cells. Compound 4e was further tested for anti-cancer activity against other OS cells and showed to be equipo-tent to gedatolisib against U2OS and Saos-2 cells. Moreover, it was also found non-toxic to normal cells (BEAS-2B and MCF 10A). The effect of compound 4e was further determined on apoptosis of Saos-2 cells by Annexin-PI assay, where it significantly amplified the percentage of apoptotic cells. Novel 1,2,3-triazole chalcone derivatives are potential agents against OS.

Inhibitory effect of quercetin on titanium particle-induced endoplasmic reticulum stress (ERS)-related apoptosis and in vivoosteolysis.

Wear particle-induced periprosthetic osteolysis is the main cause of aseptic loosening of orthopaedic implants. The aim of this study is to determine the protective effect of quercetin (QUE) against titanium (Ti) particle-induced ERS-related apoptosis and osteolysis. In this study, RAW264.7 cells were pretreated with different concentrations (40, 80, and 160 µmol/l) of QUE for 30 min and then treated with Ti particle (5 mg/ml) for 24 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC apoptosis detection kit, respectively. Protein and mRNA expressions of ERS-related genes were examined by western blot and real-time PCR, respectively. The release of inflammatory cytokines was detected by ELISA. Then a mouse calvarial osteolysis model was established. Histological sections of calvaria were stained with H&E or TRAP. The results showed that Ti particle reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of Ti particle were dramatically inhibited by QUE pretreatment. Interestingly, we found that QUE also significantly reduced Ti particle-induced up-regulation of the expression levels of PERK, IRE1, GRP78, CHOP, caspase-12 and caspase-3 and enhanced the down-regulation of Bcl-2. In addition, QUE decreased Ti particle-induced inflammatory cytokines release from RAW264.7 cells. Moreover, treatment with QUE markedly decreased osteoclast number. In a mouse calvarial osteolysis model, QUE inhibited Ti particle-induced osteolysis in vivo by inhibiting osteoclast formation and expressions of ERS-related genes. In conclusion, QUE can protect RAW264.7 cells from Ti particle-induced ERS-related apoptosis and suppress calvarial osteolysis in vivo.

Protective Effects of Garlic-Derived S-Allylmercaptocysteine on IL-1ß-Stimulated Chondrocytes by Regulation of MMPs/TIMP-1 Ratio and Type II Collagen Expression via Suppression of NF-κB Pathway.

BACKGROUND: Garlic-derived S-allylmercaptocysteine (SAMC) has widely been used in many disease therapies. However, the potential effects and mechanism of SAMC on IL-1ß-stimulated chondrocytes are unclear. METHODS: Chondrocytes were isolated, and 5 ng/mL of IL-1ß was added to mimic the in vitro osteoarthritis (OA) model. SAMC (20 and 60 µM) was used for the treatment in OA model. Cell viability was assessed by MTT method. Western blotting, Quantitative RT-PCR, and ELISA were performed to evaluate the mechanisms in SAMC treated OA model. RESULTS: Following 48 h of IL-1ß exposure, SAMC exhibited protection effect on IL-1ß-injured chondrocyte viability. Type II collagen was elevated with reduced degradation products, as a consequence of altered MMPs/TIMP-1 ratio after SAMC treatment in IL-1ß-treated chondrocytes. The protein and mRNA level of TNF-α in cellular supernatant and cells were downregulated in a dose-dependent manner. Besides, IκBα in cytoplasmic fraction was increased, while p65 level in nuclear fraction was decreased after SAMC treatment in OA. CONCLUSIONS: This study showed that SAMC may play a protective role in IL-1ß induced osteoarthritis (OA) model. This effect may be through inhibiting the NF-κB signaling pathway, therefore altering the MMPs/TIMP-1 ratio change which induced type II collagen destruction and decreasing inflammatory cytokine secretion such as TNF-α.

Long non-coding RNA Sox4 promotes proliferation and migration by activating Wnt/ß-catenin signaling pathway in osteosarcoma.

Osteosarcoma is a bone tumor without effective treatment in the world. Recently, long non-coding RNAs (lncRNAs) are considered as essential regulators in cancer progression. LncSox4 plays crucial roles in liver tumor-initiating cells self-renewal and tumor initiation. However, the effect of lncSox4 in osteosarcoma remains largely unclear. Quantitative real-time PCR (qRT-PCR) and Northern blot were performed to detect lncSox4 expressions in osteosarcoma. The functions of lncSox4 in osteosarcoma were determined using cell viability and migration assays. In addition, the proteins associated with lncSox4 were further evaluated by western blot. We found that lncSox4 was expressed highly in U-20S and Mg63 cells and osteosarcoma tumor tissues (all P < 0.001). LncSox4 silencing attenuated but lncSox4 overexpression promoted cell viability (all P < 0.001) and migration (P < 0.01) in the Mg63 cells. Next, we found lncSox4 regulation of osteosarcoma is involved in ß-catenin, and overexpression of lncSox4 could stable ß-catenin expression. Further, Wnt agonist CID11210285 completely abolished the decrease of Mg63 cells viability induced by lncSox4 silencing when cells cultured for 3 and 4 days (both P < 0.01), while Wnt inhibitor IWP-3 abolished the increase of Mg63 cells viability induced by overexpression of lncSox4 after treatment for 2 (P < 0.01), 3 (P < 0.001) and 4 (P < 0.01) days. Our study offers evidence for the first time that lncSox4 plays a positive role in osteosarcoma development and progression, and could act as a potential prognostic and therapy biomarker.