Identification of a small molecule with synthetic lethality for K-ras and protein kinase C iota - PubMed (original) (raw)

Identification of a small molecule with synthetic lethality for K-ras and protein kinase C iota

Wei Guo et al. Cancer Res. 2008.

Abstract

K-Ras mutations are frequently found in various cancers and are associated with resistance to treatment or poor prognosis. Similarly, poor outcomes have recently been observed in cancer patients with overexpression of protein kinase C iota (PKCiota), an atypical protein kinase C that is activated by oncogenic Ras protein and is required for K-Ras-induced transformation and colonic carcinogenesis in vivo. Thus far, there is no effective agent for treatment of cancers with K-Ras mutations or PKCiota overexpression. By synthetic lethality screening, we identified a small compound (designated oncrasin-1) that effectively kills various human lung cancer cells with K-Ras mutations at low or submicromolar concentrations. The cytotoxic effects correlated with apoptosis induction, as was evidenced by increase of apoptotic cells and activation of caspase-3 and caspase-8 upon the treatment of oncrasin-1 in sensitive cells. Treatment with oncrasin-1 also led to abnormal aggregation of PKCiota in the nucleus of sensitive cells but not in resistant cells. Furthermore, oncrasin-1-induced apoptosis was blocked by siRNA of K-Ras or PKCiota, suggesting that oncrasin-1 is targeted to a novel K-Ras/PKCiota pathway. The in vivo administration of oncrasin-1 suppressed the growth of K-ras mutant human lung tumor xenografts by >70% and prolonged the survival of nude mice bearing these tumors, without causing detectable toxicity. Our results indicate that oncrasin-1 or its active analogues could be a novel class of anticancer agents, which effectively kill K-Ras mutant cancer cells.

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Figures

Figure 1

Figure 1

Library screening. (A) Chemical structures of oncrasin-1. (B) Dose effect of oncrasin-1 in T29, T29Ht1, and T29Kt1 cells. The cells were treated with various concentrations (ranging from 0.1 μM to 33 μM) of oncrasin-1. Cell viability was determined 3 days after treatment by SRB assays. Control cells were treated with solvent (DMSO), and their value was set as 1. (C) Dose-Response in human lung cancer cell lines. Lung cancer cells with various oncogenic Ras gene status were treated with oncrasin-1 at various concentrations. Cell viability was determined as in B. The values shown are the means + SD of two assays done in quadruplicate. Control cells were treated with solvent (DMSO), whose value was set as 1. (D) The status of Ras gene and p53 gene mutations, and IC50 of oncrasin-1 in lung cancer cell lines tested in C. *Based on published data and the Cancer Genome Project Database (

http://www.sanger.ac.uk/genetics/CGP/

). Wt, wild type; UN, unknown; HBEC, human bronchial epithelial cells.

Figure 2

Figure 2

Apoptosis induction by oncrasin-1. (A) T29, T29Kt1, and H460 cells were treated with 10 μM (for T29 or T29Kt1) or 1 μM (for H460) oncrasin-1 and then harvested 12 hours later. Apoptosis was detected by flow cytometiric analysis. The number in each panel indicates percentage of apoptotic cells. (B) Western blot analysis. H460 cells were treated with various concentrations of oncrasin-1 as indicated or with 30 μM indole-3-carbinol (I3C), an inactive analogue, for 24 hours. Activation of caspases 3 and 8 was detected by Western blot analysis. β-actin was used as the loading control.

Figure 3

Figure 3

K-Ras knockdown inhibited oncrasin-mediated apoptosis. (A) H460 human lung cancer cells were treated with either control (luciferase [Luc]) siRNA or K-Ras siRNA and then treated with DMSO or 1 μM oncrasin-1 for 12 hours. The values represent differences of percentage of apoptotic cells between oncrasin-1 + siRNA and DMSO + siRNA groups. The values shown are the means + SD of two experiments. (B) Western blot analysis of siRNA-mediated K-Ras knockdown in H460 cells. cells treated with phosphate-buffered saline, K-Ras siRNA, or control siRNA were harvested for testing K-Ras proteins by Western blot analysis. β-Actin was used as the loading control.

Figure 4

Figure 4

Oncrasin-induced aggregation of PKCι T29, T29Kt1, and H460 cells were treated with DMSO or 10 μM (for T29 or T29Kt1) or 1 μM (for H460) oncrasin-1 for 12 hours, and then immunofluorescent staining was performed to test for intracellular localization of PKCι. Nuclear PKCι aggregated into large foci after oncrasin-1 treatment in sensitive T29Kt1 and H460 cells but not in the resistant T29 cells.

Figure 5

Figure 5

Effects of PKCι on oncrasin-1-induced cytotoxicity. (A) Susceptibility to oncrasin-1 after PKCι Knockdown. Six plasmids encoding PKCι specific shRNA were divided into two groups (PKCι i-1 and PKCι i-2) and were used in the experiment. PKCζ shRNA plasmids (a mixture of 4) were used as a control. Cell viability was determined as described in Figure 1. Western blot analysis of PKCι in T29Kt1 cells after plasmid transfection and a brief selection was also shown. (B) Knockdown of PKCι abolished abnormal nuclear PKCι aggregation. The experiment was performed as described in Figure 4.

Figure 6

Figure 6

Antitumor activity in vivo. (A) Tumor growth. Mice with subcutaneous tumors derived from H460 cells were treated with oncrasin-1 or solvent. Tumor volumes were monitored over time after the treatments. The values are the means ± SD of data from 5 mice per group. The mean tumor volume in the mice treated with oncrasin-1 differed significantly from that of the solvent-treated mice (p <.05). (B) Survival curve. The mean survival times in mice treated with solvent and oncrasin-1 were 24 and 32 days, respectively.

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