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In women, epithelial ovarian cancer is the leading cause of gynaecological malignancy-related deaths. Development of resistance to standard platinum and taxane based chemotherapy and recurrence of the disease necessitate development of novel drugs to halt disease progression. An established concept is to target molecular and signaling pathways that substantially contribute to development of drug resistance and disease progression. We have previously shown that, monepantel (MPL) a novel small molecule acetonitrile derivative is highly effective in suppressing growth, proliferation and colony formation of ovarian cancer cells. These effects are achieved through inhibition of the mTOR/p70S6K pathway in cancer cells. The present study was conducted to find in vivo corroboration and explore the effect of MPL om other growth stimulating putative signaling pathways. Here, female nude mice with subcutaneous OVCAR-3 xenografts were treated with 25 and 50 mg/kg doses of MPL administered (IP) three times weekly for 2 weeks. At the doses employed, MPL was modestly effective at suppressing tumor growth, but highly effective in inhibiting, mTOR, P70S6K and 4EBP1. There were also modest reductions in tumor cyclin D1 and retinoblastoma protein expression. Furthermore, it was found that MPL treatment causes down-regulation of IGF-1R, and c-MYC thus unveiling new dimensions to the growing antitumor actions of this potential anticancer drug. MPL treatment led to reduced tumor volume and weights without causing any detectable side effects. Coupled with the recent human safety data published on this molecule, expanded future trials are highly anticipated., Competing Interests: None., (AJCR Copyright © 2021.)

Hypoxia-inducible factor (HIF)-1α is the key cellular survival protein under hypoxia, and is associated with tumor progression and angiogenesis. We have recently shown the inhibitory effects of minocycline on ovarian tumor growth correlated with attenuation of vascular endothelial growth factor (VEGF) and herein report a companion laboratory study to test if these effects were the result of HIF-1α inhibition. In vitro, human ovarian carcinoma cell lines (A2780, OVCAR-3 and SKOV-3) were utilized to examine the effect of minocycline on HIF-1 and its upstream pathway components to elucidate the underlying mechanism of action of minocycline. Mice harboring OVCAR-3 xenografts were treated with minocycline to assess the in vivo efficacy of minocycline in the context of HIF-1. Minocycline negatively regulated HIF-1α protein levels in a concentration-dependent manner and induced its degradation by a mechanism that is independent of prolyl-hydroxylation. The inhibition of HIF-1α was found to be associated with up-regulation of endogenous p53, a tumor suppressor with confirmed role in HIF-1α degradation. Further studies demonstrated that the effect of minocycline was not restricted to proteasomal degradation and that it also caused down-regulation of HIF-1α translation by suppressing the AKT/mTOR/p70S6K/4E-BP1 signaling pathway. Minocycline treatment of mice bearing established ovarian tumors, led to suppression of HIF-1α accompanied by up-regulation of p53 protein levels and inactivation of AKT/mTOR/p70S6K/4E-BP1 pathway. These data reveal the therapeutic potential of minocycline in ovarian cancer as an agent that targets the pro-oncogenic factor HIF-1α through multiple mechanisms.

We have recently shown that the novel anthelmintic drug monepantel (MPL) inhibits growth, proliferation and colony formation, arrests the cell cycle and induces cleavage of PARP-1 in ovarian cancer cell lines. Here we report on the mechanism behind the anticancer properties of MPL. The cytotoxic effect of MPL on ovarian cancer cells (OVCAR-3 and A2780) was investigated employing a panel of tests used for the detection of apoptosis and autophagy. Apoptosis and autophagy were defined by caspase activity, DNA-laddering, Annexin-V and acridine orange (AO) staining. Autophagy markers such as LC3B, SQSTM1/p62 and mammalian target of rapamycin (mTOR) pathway related proteins were assessed by western blotting and ELISA techniques. MPL did not activate caspases 3 or 8, nor did it alter the percentage of Annexin V positive stained cells. Failure to cause DNA laddering and the inability of z-VAD-fmk to block the MPL antiproliferative effects led to the ruling out of apoptosis as the mechanism behind MPL-induced cell death. On the other hand, accumulation of acidic vacuoles with distinct chromatin morphology and an increase in punctuate localization of green fluorescent protein-LC3B, and MPL-induced changes in the expression of SQSTM1/p62 were all indicative of MPL-induced autophagy. Consistent with this, we found inhibition of mTOR phosphorylation leading to suppression of the mTOR/p70S6K signalling pathway. Our findings provide the first evidence to show that MPL triggers autophagy through the deactivation of mTOR/p70S6K signalling pathway.

Monepantel (MPL) is a new anthelmintic agent approved for the treatment of nematode infections in farm animals. As a nematicide, it acts through a nematode-specific nicotinic receptor subtype which explains its exceptional safety in rodents and mammals. In the present study, we evaluated its potential as an anticancer agent. In vitro treatment of epithelial ovarian cancer cells with MPL resulted in reduced cell viability, inhibition of cell proliferation and suppression of colony formation. Proliferation of human ovarian surface epithelial cells and other non-malignant cells were however minimally affected. MPL-induced inhibition was found to be independent of the acetylcholine nicotinic receptor (nAChR) indicating that, its target in cancer cells is probably different from that in nematodes. Analysis of MPL treated cells by flow cytometry revealed G1 phase cell cycle arrest. Accordingly, MPL treated cells expressed reduced levels of cyclins D1 and A whereas cyclin E2 expression was enhanced. Consistent with a G1 phase arrest, cellular levels of cyclin dependent kinases (CDKs) 2 and 4 were lower, whereas expression of CDK inhibitor p27(kip) was increased. In cells expressing the wild-type p53, MPL treatment led to increased p53 expression. In line with these results, MPL suppressed cellular thymidine incorporation thus impairing DNA synthesis and inducing cleavage of poly (ADP-ribose) polymerase (PARP-1). Combined these pre-clinical findings reveal for the first time the anticancer potential of monepantel.

Background: malignant peritoneal mesothelioma (MPM) is a rare peritoneal mesothelial neoplasm. Ki67 and BCL2 are established prognostic markers in several cancers. High Ki67 expression indicates tumour progression, whilst similar expression of BCL2 retards tumour replication. Traditionally, prognosis in MPM is gauged with a single biomarker assessed separately in a dichotomous manner. Here, we examine prognosis with dual biomarkers incorporated in a model to predict survival., Materials and Methods: Forty two MPM archival patient tumours were screened for Ki67 and BCL2 by immunohistochemistry and evaluated using standard methods. Ki67 and BCL2 expression was incorporated into a prognostic model to develop Ki67-BCL2 index. Using this index, three hazard groups were identified (high, medium and low risk). Kaplan-Meier survival analysis was performed to assess the significance of these hazard groups in the various clinicopathological categories., Results: In all clinicopathological categories, high risk group showed poor prognosis compared to low risk group (p = < 0.001). Compared to medium risk, high risk group carried poor prognosis in all tumours, females, epitheloid tumours, peritoneal cancer index (PCI) < 20, ≥ 20, age at diagnosis (AAD) < 60, and ≥ 60 years. Independent of the Ki67-BCL2 index, male, sarcomatoid, PCI ≥ 20 and AAD ≥ 60 were poor prognostic factors. High risk group was an independent poor prognostic factor in all tumours, males, females and age < 60 years. The distribution of high risk: low risk group in male and female was 3: 2 and 2: 3, respectively, indicating a gender difference. Comparing hazard ratios generated by Ki67-BCL2 index to that of either Ki67 or BCL2, as a single prognostic biomarker, there was a reduction of HR values., Conclusion: Ki67-BCL2 index seems to suggest a more sensitive method of predicting prognosis. However, the current model needs further evaluation in an independent large cohort sample.

Unlabelled: Background Malignant peritoneal mesothelioma (MPM) is a rare neoplasm of the peritoneal membrane that is causally related to asbestos exposure. Survival after treatment is poor. Current therapy involving hyperthermic intraperitoneal chemotherapy has improved survival in selective patients. In the past, several prognostic factors have been identified in MPM patients and this has prompted the development of new therapies and patient management. Since BCL2, an antiapoptotic oncoprotein, is a favourable prognostic factor in breast cancer, we investigated to determine the significance of BCL2 in MPM. Materials and Methods Forty two archival patient tumour sections embedded in paraffin blocks were sectioned and subjected to immunohistochemistry to detect BCL2. The staining intensity and abundance was classified using standard procedures and classified into two groups (0-4 = low & 5-8 = high expression). The distribution of BCL2 groups was examined in the different clinicopathological categories to determine prognosis using Kaplan-Meier survival analysis., Results: Univariate analysis revealed that in almost all clinicopathological categories, high BCL2 expression predisposed patients to a favourable prognosis. Independent of BCL2 expression, univariate analysis also showed that male gender, sarcomatoid histology, high PCI and age at diagnosis ≥ 60 years were associated poor prognosis. Multivariate analysis indicated that for all tumours, males and females, high BCL2 expression was associated with good prognosis. Further, independent of BCL2, age ≥ 60 years is an unfavourable prognostic factor., Conclusion: Expression of BCL2 may serve to distinguish prognosis within the individual clinicopathological categories. BCL2 is also an independent variable in all tumours, males and females, with high expression being associated with good prognosis.