Preclinical evaluation of novel PI3K/mTOR dual inhibitor SN202 as potential anti-renal cancer agent

Abstract

ABSTRACT The PI3K/mTOR pathway is one of the most frequently aberrantly activated pathways in human malignancies, such as renal cell carcinoma (RCC), and contributes to resistance to antitumor therapies. Thus, PI3K/mTOR is an attractive target for the development of antitumor agents. In this study, we evaluated the preclinical effects of a novel inhibitor SN202. We examined Akt/mTOR activities in renal cancer cells after SN202 treatment. The preclinical effects of SN202 on tumor growth were evaluated in renal cancer cells in vitro and in murine xenografts in vivo. SN202 inhibits PI3Kα, PI3Kγ, and mTOR, the corresponding IC₅₀ values were 3.2, 3.3, and 1.2 nM, respectively. In A498, 786-0, and ACHN renal cancer cell lines, SN202 inhibits cell proliferation in a dose-dependent manner and significantly inhibits 786-0 cell growth. Western blot analysis revealed that SN202 decreases the phosphorylation of PI3K downstream signaling molecules, Akt and S6K, in 786-0 renal cancer cells. Furthermore, oral administration of SN202 results in significant inhibition in human renal carcinoma xenografts in nude mice and favourable pharmacokinetic properties in rats. These results suggest that SN202 might be a promising therapeutic agent against RCC as a dual PI3K/mTOR inhibitor.

Keywords: PI3K/mTOR inhibitor; SN202; antitumor growth; renal cell carcinoma.

Figure 1.

Chemical structure and biochemical activity of SN202. (A) The chemical structure of SN202. Inhibition rate of SN202 on kinase activity of mTOR (B), PI3Kα (C) and PI3Kγ (D).

Figure 2.

Inhibition of Akt and S6K phosphorylation by SN202. 786-0 cells were incubated for the indicated time periods with 10 µM SN202. Zero time samples were treated with DMSO. These cells were harvested and analyzed by Western blot using antibodies specific for either serine- or threonine-phosphorylated S6K and Akt. β-actin was used as a loading control. Results are expressed as the mean ± SEM of three independent experiments. One-way ANOVA for SN202 treatment versus the control group: *P < 0.05, **P< 0.01.

Figure 3.

Inhibition potential of SN202 against RCC. The inhibitory effect of SN202 on cell proliferation was determined by CCK-8 assay. 786-0 (A), ACHN (B) and A498 (C) cells were treated with varying concentrations of SN202, BKM120 and BEZ235. Data are presented as the means of three independent experiments.

Figure 4.

Inhibition of 786-0 cell cycle progression by SN202. (A) and (C) 786-0 cells were treated with 8 μM SN202 for 8, 16, and 24 h. (B) and (D) 786-0 cells were incubated in the presence of 1, 3, and 8 μM SN202 for 16 h. Cell cycle was determined by a flow cytometry using propidium iodide staining. Results are representative of three independent experiments at minimum.

Figure 5.

Inhibition of tumor growth by SN202 in 786-0 xenograft model. (A) Mean tumor volume after the start of treatment. (B) Mean body weight of mice after the start of treatment. (C) Tumors resected from nude mice on 21 d. Renal carcinoma 786-0 cells (10⁶ cells) were implanted into the left armpit of each nude mouse to develop tumors. Mice bearing tumors around 120 mm³ were orally treated with SN202 (5 mg/kg and 20 mg/kg) once per day for 21 days. Tumor volume and body weight were recorded twice per week. Data are expressed as mean ± SEM. **P< 0.01 versus the vehicle group.

Figure 6.

Mean plasma concentration-time profile of SN202 after iv or oral administration. Rats were given a single iv bolus at 1 mg/kg or a single oral administration at 2 mg/kg. At the indicated times after dosing, blood samples were obtained via tail vein of individual rats. Plasma was separated and then determined by LC/MS/MS.