Monepantel antitumor activity is mediated through inhibition of major cell cycle and tumor growth signaling pathways

Abstract

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.

Keywords: IGF-1R; Ovarian cancer; c-MYC; mTOR; monepantel; tumor signaling.

Figure 1

Monepantel suppresses growth of OVCAR-3 xenografts in nude mice. Antitumor effects of MPL was tested in nude mice bearing rapidly growing subcutaneous OVCAR-3 xenografts. Mice were treated (IP injections; 0.1 mL) for 2 weeks with the vehicle (control group) or MPL (25 or 50 mg/kg). Vehicle or drug administration took place 3 times weekly starting on day 7 post cell inoculation and termination on day 20 of the experiment. Sterile 0.5% HPMC was used as the vehicle for MPL and also as the treatment for the control group. Calliper measurement of tumour volumes were recorded 3 times weekly. Values are mean ± SD from the measurements over the course of the experiment. (A) Tumor volumes during the 22 day course of MPL or vrhicle administration. (B) MPL treatment did not affect animal weights. (C) Tumor weights in nude mice weight in nude mice at the time of sacrifice (day 22 of study) and (D) representative photos of mice bearing OVCAR-3 tumors and being treated with either the vehicle or monepantel (25 or 50 mg/kg).

Figure 2

MPL inhibits mTOR/p70S6K signaling pathway. Immunoblot analysis of ovcar-3 tumor lysates to determine levels of mTOR, P70S6K and 4EBP1 together with their related phosphorylated proteins in tumor lysates. Blots were cropped to ensure clearer presentation of the data. Densitometry was performed to quantify changes arising from MPL treatment (25, 50 mg/kg three times weekly for two weeks). Vehicle treated values (control) are expressed as 100% and the drug treated values are presented relative to control. GAPDH was used as the housekeeping gene confirm similar protein loading and blot transfer. * < 0.05 compared to control (vehicle treated).

Figure 3

MPL affects cell cycle regulators. Treatment of mice bearing OVCAR-3 tumors with monepantel (25 or 50 mg/kg × 3 weekly) for two weeks led to down-regulation of cell cycle regulatory proteins. Western blot analysis of lysates prepared from the tumours demonstrate down-regulation of cyclins D1 and E2, CDK2 and the phosphorylated form of the retinoblastoma protein. This aligns well with the cell cycle data showing substantial arrest of the cells at the G1 phase of the cell cycle. The cell cycle is tightly controlled at specific points by CDKs, and in particular CDK2 and CDK4 which seem essential for entry in G1 and G1-S transition.

Figure 4

MPL induces necrosis and down-regulates IGF1R and TGF-β. (A) Histological images of H&E stained tumors shown on top, indicating profound drug-induced necrosis (black arrow; magnification, 10 ×). Analysis of necrosis was performed based on number of the field/section, section = 5, % of necrosis = necrosis area/Total area × 100. Western blot analysis of monepantel treated tumors show down-regulation of IGF1R-β (B) and TGFβ (C) proteins. Blots were cropped to ensure clearer presentation of the data.

Figure 5

MPL suppresses c-MYC. Overexpression of the MYC oncogene is a key feature of many human malignancies. Treatment of mice (n = 5 per treatment group) bearing SC OVCAR-3 tumours with monepantel for a period of two weeks led to significant (P < 0.05) inhibition of c-MYC. Monepantel was administered 3 times weekly as a bolus IP injection at the doses of 25 or 50 mg/kg. Dosing was initiated on day 7 post cell inoculation. The control group received 0.5% HPMC as the vehicle. Western blot analysis for detection of c-MYC protein was performed on lysates prepared from tumors excised from mice treated with the vehicle or the drug. Blots were cropped to ensure clearer presentation of data. Based on the results obtained in this investigation, a tentative mechanism has been proposed to describe the antitumor effects of monepantel in this experimental model of ovarian cancer.