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Despite significant advancements in osteosarcoma research, the overall survival of canine and human osteosarcoma patients has remained essentially static over the past 2 decades. Post-operative limb-spare infection has been associated with improved survival in both species, yet a mechanism for improved survival has not been clearly established. Given that the majority of canine osteosarcoma patients experiencing post-operative infections were treated with fluoroquinolone antibiotics, we hypothesized that fluoroquinolone antibiotics might directly inhibit the survival and proliferation of canine osteosarcoma cells. Ciprofloxacin or enrofloxacin were found to inhibit p21(WAF1) expression resulting in decreased proliferation and increased S-G(2)/M accumulation. Furthermore, fluoroquinolone exposure induced apoptosis of canine osteosarcoma cells as demonstrated by cleavage of caspase-3 and PARP, and activation of caspase-3/7. These results support further studies examining the potential impact of quinolones on survival and proliferation of osteosarcoma.

Chordoma, a rare neoplasm derived from intraosseous notochordal remnants, is unresponsive to conventional chemotherapy and radiotherapy. Sonic Hedgehog (Shh) is a crucial fetal notochord–secreted morphogen that directs notochordal development. The aim of this study was to determine the functional roles and therapeutic potential of Shh-Gli1 signaling in chordomas. Tissue samples and clinical profiles were collected from 42 patients with chordoma. The chordoma cell lines U-CH1 and MUG-Chor1 were used for functional experiments. Shh-Gli1 signaling pathway genetic alterations were screened, and the functions of the identified novel variants were analyzed using in silico analyses, real-time quantitative PCR, and minigene assays. Ligand-dependent Shh-Gli1 signaling activation was assessed using single- and dual-label immunostaining, western blot analysis, and a Shh-responsive Gli-luciferase reporter assay. The small-molecule inhibitor vismodegib was used to target Shh-Gli1 signaling in vitro and in vivo. Overall, 44 genetic alterations were identified, including four novel variants (c.67_69dupCTG in SMO, c.-6_-4dupGGC and c.3306 + 83_3306 + 84insG in PTCH1, and c.183-67_183-66delinsA in SUFU). Shh, PTCH1, SMO, SUFU, and Gli1 were extensively expressed in chordomas, and higher Gli1 expression correlated with poorer prognosis. A luciferase reporter assay and dual-label immunostaining indicated the occurrence of juxtacrine ligand-dependent Shh-Gli1 signaling activation. Vismodegib significantly inhibited cell proliferation and induced apoptosis and G1/S cell cycle arrest. In vivo investigation demonstrated that vismodegib effectively inhibited chordoma xenograft growth. This current preclinical evidence elucidates the therapeutic potential of Shh-Gli1 signaling pathway targeting for chordoma treatment. Vismodegib may be a promising targeted agent, and further clinical trials are warranted.

Eicosapentaenoic acid (EPA) is an unsaturated fatty acid with various bioactivities, including antitumor effects. We previously reported a synergistic antitumor effect of cisplatin (CDDP) and EPA. Here, we examined the underlying mechanism. The human oesophageal cancer cell line TE-1 was treated with the combination of EPA and CDDP. Nuclear translocation of NF-κB, a transcription factor involved in cytokine production, was detected by immunohistochemistry. IL-6 levels were measured by ELISA. Apoptosis and cell cycle distribution were evaluated by flow cytometry. Nuclear translocation of NF-κB in TE-1 cells was synergistically decreased by CDDP and EPA. IL-6 production was increased following treatment with CDDP, but treatment with EPA decreased IL-6 levels. Apoptosis was synergistically induced by CDDP and EPA. A G /M cell cycle arrest was observed with the combination of CDDP and 150 μM EPA, and S phase arrest with the combination of CDDP and 100 μM EPA. The combination of CDDP and EPA synergistically suppresses NF-κB nuclear translocation and increases apoptosis by inducing cell cycle arrest at the S or G /M phase.

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.

Ascomylactam A was reported for the first time as a new 13-membered-ring macrocyclic alkaloid in 2019 from the mangrove endophytic fungus Didymella sp. CYSK-4 from the South China Sea. The aim of our study was to delineate the effects of ascomylactam A (AsA) on lung cancer cells and explore the antitumor molecular mechanisms underlying of AsA. In vitro, AsA markedly inhibited the cell proliferation with half-maximal inhibitory concentration (IC50) values from 4 to 8 μM on six lung cancer cell lines, respectively. In vivo, AsA suppressed the tumor growth of A549, NCI-H460 and NCI-H1975 xenografts significantly in mice. Furthermore, by analyses of the soft agar colony formation, 5-ethynyl-20-deoxyuridine (EdU) assay, reactive oxygen species (ROS) imaging, flow cytometry and Western blotting, AsA demonstrated the ability to induce cell cycle arrest in G1 and G1/S phases by increasing ROS generation and decreasing of Akt activity. Conversely, ROS inhibitors and overexpression of Akt could decrease cell growth inhibition and cell cycle arrest induced by AsA. Therefore, we believe that AsA blocks the cell cycle via an ROS-dependent Akt/Cyclin D1/Rb signaling pathway, which consequently leads to the observed antitumor effect both in vitro and in vivo. Our results suggest a novel leading compound for antitumor drug development.

Aflatoxins (AFs) are potent hepatotropic mycotoxins—AFB1 being the best-characterized—yet their ability to induce intrahepatic cholangiocarcinoma (iCCA) remains underexplored. Male Wistar rats received vehicle (controls; n = 5) or an AFB1-dominant AF mixture (AFB1 39.46 μg/mL; AFB2 1.13 μg/mL; AFG1 17.44 μg/mL; AFG2 0.59 μg/mL— n = 10) by daily gavage for 90 days, at a dose equivalent to 400 μg AFB1 per kg of diet. After 12 months, twelve iCCA tumors were resected and analyzed by histology (H&E) and tissue-microarray-based immunohistochemistry (Cytokeratin-19, Hep Par-1, p53, Cyclin D1, Rb, β-catenin, and PCNA). Lesions predominantly showed glandular/tubular architecture consistent with iCCA and were cytokeratin-19-positive and Hep Par-1-negative. Cell proliferation was high (PCNA ≈ 69%). p53 displayed nuclear accumulation in 83% of tumors. G1/S control was perturbed, with cyclin D1 overexpression (67%), and Rb was positive in 58% of iCCA. Aberrant Wnt activation was rare (nuclear β-catenin in 8%). Subchronic exposure to an AFB1-dominant AF mixture in rats was associated with iCCA characterized by high proliferative activity, p53 accumulation, and disruption of G1/S checkpoint components. These findings broaden the oncogenic spectrum of AFs and warrant genomic confirmation of AF mutational signatures.

Background/Aims: Reversion-inducing cysteine-rich protein with kazal motifs (RECK) is a novel tumor suppressor gene that is critical for regulating tumor cell invasion and metastasis. The expression of RECK is dramatically down-regulated in human cancers. Harmine, a tricyclic compound from Peganum harmala, has been shown to have potential anti-cancer activity. Methods: Cell proliferation assay (CCK-8 cell viability assay), cell cycle analysis (detection by flow cytometry), apoptosis staining assay (TUNEL staining), cell migration assay and invasion assay (transwell assay) were carried out to investigate the Harmine’s efficacy on non-small cell lung cancer (NSCLC) cells in vitro. A549-luciferase cell orthotropic transplantation xenograft mouse model was used to determine the effect of Harmine treatment on NSCLC in vivo. Western blotting analysis of cell growth and metastasis related signal pathways was conducted to investigate the molecular mechanism of Harmine’s inhibitory effect on NSCLC. Results: Harmine treatment effectively inhibited cell proliferation and induced the G1/S cell cycle arrest of NSCLC cells. Further study proved that Harmine treatment led to apoptosis induction. Furthermore, treatment with NSCLC cells with Hamine resulted in decreased cell migration and cell invasion in vitro. More importantly, Harmine treatment significantly suppressed the NSCLC tumor growth and metastasis in mouse xenograft model in vivo. Mechanistically, in Harmine-treated NSCLC cells, RECK expression and its downstream signaling cascade were dramatically activated. As a consequence, the expression level of MMP-9 and E-cadherin were significantly decreased. Conclusion: These findings identify Harmine as a promising activator of RECK signaling for metastatic NSCLC treatment.

Sulforaphane (SFN) extracted from broccoli sprout has previously been investigated for its potential properties in cancers, however, the underlying mechanisms of the anticancer activity of SFN remain not fully understood. In the present study, we investigate the effects of SFN on cell proliferation, cell cycle, cell apoptosis, and also the expression of several cell cycle and apoptosis-related genes by MTT assay, flow cytometry and western blot analysis in gastric cancer (GC) cells. The results showed that SFN could impair the colony-forming ability in BGC-823 and MGC-803 cell lines compared with the control. In addition, SFN significantly suppressed cell proliferation by arresting the cell cycle at the S phase and enhancing cell apoptosis in GC cells in a dose-dependent manner. Western blot results showed that SFN treatment significantly increased the expression levels of p53, p21 and decreased CDK2 expression, which directly regulated the S phase transition. The Bax and cleaved-caspase-3 genes involved in apoptosis executive functions were significantly increased in a dose-dependent manner in BGC-823 and MGC-803 cells. These results suggested that SFN-induced S phase cell cycle arrest and apoptosis through p53-dependent manner in GC cells, which suggested that SFN has a potential therapeutic application in the treatment and prevention of GC.

Scutellaria baicalensis extract (SbE) has been shown to exert chemopreventive effects on several types of cancer. Baicalin, a hydrophilic flavonoid found in SbE, may have opposing effects that decrease the antitumor potential of SbE against colorectal cancer. In this study, after removing baicalin, we prepared an aglycone-rich fraction (ARF) of SbE and evaluated its anti-proliferative activity and mechanisms of action. The flavonoids found in ARF, baicalin fraction (BF) and SbE were determined by high-performance liquid chromatography (HPLC). The effects of ARF, BF, SbE and representative flavonoids on the proliferation of HCT-116 and HT-29 human colorectal cancer cells were determined by an MTS assay. The cell cycle, the expression of cyclins A and B1 and cell apoptosis were assayed using flow cytometry. Apoptosis-related gene expression was visualized by quantitative real-time polymerase chain reaction (PCR), and mitochondrial membrane potential was estimated following staining with JC-1. HPLC analysis showed that ARF contained two hydrophobic flavonoids, baicalein and wogonin, and that BF contained only baicalin. SbE had little anti-proliferative effect on the colorectal cancer cells; cancer cell growth was even observed at certain concentrations. ARF exerted potent anti-proliferative effects on the cancer cells. By contrast, BF increased cancer cell growth. ARF arrested cells in the S and G2/M phases, increased the expression of cyclins A and B1, and significantly induced cell apoptosis. Multiple genes in the mitochondrial pathway are involved in ARF-induced apoptosis, and subsequent cellular functional analysis validated the involvement of this pathway. These results suggest that removing baicalin from SbE produces an ARF that significantly inhibits the growth of colorectal cancer cells, and that the mitochondrial apoptotic pathway plays a role in hydrophobic flavonoid-induced apoptosis.

The present study compared the effect of resveratrol on HCT116 and Caco-2 human colon cancer cells. Annexin V/propidium iodide staining, MTT assay and western blot analysis revealed that resveratrol induced cycle arrest in the two cell lines, which was evidenced by cell cycle analysis and changes in the expression of the cell cycle proteins cyclin-dependent kinase (CDK) 2, CDK4, cyclin D1, proliferating cell nuclear antigen and P21. Furthermore, resveratrol was found to have a strong apoptosis-inducing effect, which was evidenced through the high percentage of annexin V positive cells and high protein expression of cleaved-caspase-7, cleaved-caspase-9 and cleaved-poly(ADP-ribose) polymerase in the resveratrol-treated cancer cells. In conclusion, these results demonstrated that resveratrol had greater growth inhibitory and cell cycle arrest effects on Caco-2 cells than HCT116 cells, through caspase-dependent and cyclin-CDK pathways.