We have previously shown that Kevetrin, a small molecule currently under development, has potent antitumor activity in human multi-drug resistant carcinoma xenograft models while being well tolerated. To investigate the mechanism of action for its potent antitumor activity, we assessed Kevetrin's effect on apoptosis, cell cycle progression, including underlying molecular mechanisms. Here we report that Kevetrin strongly induced cell cycle arrest and apoptosis in a human lung adenocarcinoma cell line (A549) using the TUNEL assay and FACS analysis; similar results were observed using a human breast carcinoma cell line (MDA-MB-231). Treatment of A549 cells with 400 µM of Kevetrin for 48 hours resulted in G2/M phase cell cycle arrest that was associated with a marked decline in levels of G2/M regulatory proteins, including CDK1 and cdc25B, and increased expression of Wee1. Further, Kevetrin-mediated growth inhibition of A549 correlated with apoptosis induction that was characterized by cleavage of procaspase-3 and poly (ADP-ribose) polymerase (PARP). The p53 tumor suppressor is a well characterized transcription factor controlling cell growth and apoptosis during times of cellular stress. The tumor suppressor p53 inhibits tumor growth primarily through its ability to induce apoptosis. In cancer development, the resistance of cells to apoptosis is one of the crucial steps. The reactivation of p53 in tumor cells should trigger massive apoptosis and eliminate the tumor. Interestingly, Kevetrin treatment caused enhanced activated p53 levels in A549 cells. Activation of p53 can lead to cell cycle arrest and apoptosis. Western blotting revealed a concentration dependent increase in phosphorylation of p53 at serine 15. The phosphorylation of p53 at serine 15 leads to reduced interaction between p53 and its negative regulator, the oncoprotein MDM2. We also found that Kevetrin increased expression of p53 target genes such as p21 (Waf1). The tumor suppressor protein p21 (Waf1) acts as inhibitor of cell cycle progression. Finally, we also found that p53 induced apoptosis by inducing the expression of PUMA. Our study shows that Kevetrin activates p53 functions in tumor cells. Thus Kevetrin is a strong candidate as an anticancer drug that targets p53. Based on our studies we plan to initiate a Phase I clinical trial in 2011. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4470. doi:10.1158/1538-7445.AM2011-4470
We have previously shown that Kevetrin, a novel molecule currently under development, was effective in reducing tumor growth in human multi-drug resistant lung cancer xenograft models while being well tolerated. In this study, we expanded our tests to include other tumor types: colon and breast. Three of the models have been shown to be multi-drug resistant: HCT-15 colon carcinoma have an overexpression of p-glycoprotein that acts as a membrane efflux pump (Iwahashi 1991, Mickley 1989), MDA-MB-435s carcinoma cells have highly active c-Abl and Arg kinases and overexpress Heat Shock Protein 27 which inhibits apoptosis thereby contributing to drug resistance (2006 Srinivasan, 2008 Shi), and A549 lung carcinoma have the K-ras mutation and overexpress STAT3 and Nrf2; mutations associated with resistance to standard chemotherapy (Akudela 2004, Mahaffey 2009, Homma 2009, Kim 2009). To demonstrate a dose response effect, in the multi-drug resistant A549 lung tumor model, nude mice bearing established tumors were treated with either 50, 100, or 200 mg/kg Kevetrin intraperitoneally (IP) every other day for 3 doses or 200 mg/kg every other day for 3 doses followed by a second cycle 10 days later. Kevetrin delayed median tumor growth in a dose dependent manner where 50, 100, and 200 mg/kg produced tumor growth delays (TGDs) of 0%, 6%, and 21%, respectively, relative to controls. When given in 2 cycles at 200 mg/kg, a significantly greater TGD of 35% was achieved. In the HCT-15 multi-drug resistant colon tumor model, nude mice bearing established tumors were treated with either 200 mg/kg Kevetrin IP every other day for 3 doses or 22 mg/kg paclitaxel intravenously (IV) daily for 4 doses. Paclitaxel had no anti-tumor activity, whereas Kevetrin produced a significant TGD of 16 days (43%) relative to controls. In a non drug-resistant colon tumor model, HT-29, Kevetrin produced a similar TGD of 18 days (49%) while 20 mg/kg 5-fluorouracil (5-FU) IP given daily for 5 doses produced a TGD of 9 days (24%). In the MDA-MB-435s drug resistant tumor model, nude mice bearing established tumors were treated as above. Paclitaxel had no significant anti-tumor activity (1% TGD), whereas Kevetrin produced a significant TGD of 20 days (65%). In a non drug-resistant breast tumor model, MDA-MB-231, Kevetrin produced a TGD of 26 days (90%), and paclitaxel produced a significant TGD of 18 days (62%). These results demonstrate that Kevetrin exerted potent anti-tumor activity against three different tumor types, e.g., lung, breast, colon, including those that have multi-drug resistant phenotypes at doses and schedules that were well-tolerated as suggested by <5% weight loss during treatments. Therefore, these studies suggest that Kevetrin has promising potential for the treatment of tumors that have become resistant to standard chemotherapy. Based on our studies we plan to initiate a Phase I clinical trial in solid tumors in 2010. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-283.
Lung cancer accounts for more than 215,000 new cases annually in the United States, and is the single largest cause of cancer death accounting for more than 130,000 deaths each year. Non-small cell lung cancer (NSCLC) accounts for 80% of all bronchogenic neoplasms with 90% of diagnosed patients dying within five years; therefore, new therapies are strongly desired. Effective therapies will come from a more detailed understanding of the molecular mechanisms underlying both the disease and the clinical response. We are developing a novel molecule, termed Kevetrin™, which was effective in reducing tumor growth in human lung cancer xenograft models while being well-tolerated. Kevetrin appeared to exert its anti-tumor activity by lowering levels of activated AKT also known as protein kinase B, of the AKT signal transduction pathway resulting in reduced tumor cell survival. Activated AKT was measured by a sandwich ELISA specific for phosphorylated AKT protein. Kevetrin was also evaluated for anti-tumor activity in vivo in the NCI-H1975 and A549 lung xenograft tumor models. Tumor-bearing nude mice were treated intravenously (IV) with either 200 mg/kg Kevetrin three times every other day or 22 mg/kg paclitaxel four times every other day. After Kevetrin was incubated with cells in vitro, phosphorylated AKT levels were reduced leading us to test for anti-tumor activity in vivo. Results with NCI-H1975 tumors showed that Kevetrin significantly delayed median tumor growth by 34 days (142%) in the first experiment and 28 days (156%) in a repeat experiment compared to controls, whereas the tumor growth delay (TGD) of paclitaxel was just 4 days (17%) and 14 days (78%), respectively. No weight loss occurred during treatment with Kevetrin. Results with A549 tumors showed that Kevetrin significantly delayed median tumor growth by 11 days (33%) in the first experiment and 30 days (111%) in a repeat experiment compared to controls, whereas the TGD of paclitaxel was just 0 days (0%) and 3 days (11%), respectively. In this study, Kevetrin resulted in <5% weight loss during treatment. These results showed that Kevetrin exerted potent anti-tumor activity against two human lung xenograft tumor models, NCI-H1975 and A549, at a dose and schedule that was well-tolerated as indicated by a small transient weight loss during treatment. NCI-H1975 have the T790M and L858R mutations in epidermal growth factor receptor (EGFR) and A549 have the K-ras mutation and overexpress STAT3 and Nrf2; these mutations are associated with resistance to standard chemotherapy. The reduction of activated AKT may be a mechanism by which Kevetrin overcame resistance and showed efficacy in these tumor models. These studies demonstrate that Kevetrin has promising potential for the treatment of lung carcinoma, particularly in cases where tumors have become resistant to standard chemotherapy. Citation Information: Cancer Res 2009;69(23 Suppl):C49.
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