Abstract 4089: Calmodulin antagonists inhibit cholangiocarcinoma tumorigenesis independent of gemcitabine by the activating of caspase 2
Authors: Jing Gu , Yuan Kaiyu , Turk Amy N. , McDonald Jay M. , Chen Yabing 
Published: July 2012

Abstract
Cholangiocarcinoma is a highly malignant tumor with limited therapeutic options. Gemcitabine (GMT) is currently used as a first line chemotherapeutical reagent for many cancers, including cholangiocarcinoma. However, it is often used in combination with other therapeutical strategies, due to high toxicity of GMT. We have previously reported that antagonists of calmodulin (CaM), including tamoxifen (TMX), trifluoperazine (TFP) and W7, induce apoptosis of cholangiocarcinoma cells and reduce cholangiocarcinoma tumorigenesis in mice. In the present studies, we determined the effect of combination therapy of TMX and GMT on cholangiocarcinoma tumorigenesis and investigated the mechanisms responsible for their efficacy.
The effect of TMX and GMT on cholangiocarcinoma cell growth and apoptosis in vitro was determined by MTS and Annexin V staining analysis. The expression/activation of apoptotic signaling molecules were assessed by Western blot analysis. A nude mouse xenograft model was utilized for in vivo cholangiocarcinoma tumorigenesis as we previously reported. TMX (15mg/kg), GMT (15mg/kg) and combination of both were administrated by intraperitoneal injection. Tumor growth was measured every 3 days. Apoptosis in xenograft tumors was determined by TUNEL and cleaved caspase 3 staining.
GMT inhibited cell growth and induced apoptosis of cholangiocarcinoma cells in a concentration-dependent manner. TMX enhanced GMT-induced apoptosis and GMT inhibition of cell growth in cholangiocarcinoma cells. GMT (15mg/kg, every third day) inhibited cholangiocarcinoma tumorigenesis in nude mice by 50%. TMX (15mg/kg, two out of three days) enhanced the inhibitory effect of GMX on tumorigenesis by 33%. The inhibition of tumor growth correlated with enhanced apoptosis in tumor tissues. To elucidate the mechanisms underlying the additive effects of TMX on GMT-induced apoptosis, we determined activation of caspases in cholangiocarcinoma cells exposed to GMT, TMX or both. Activation of caspase 9 and caspase 3, as well as cytochrome c release to the cytosol were demonstrated in cells exposed to either reagent alone. By contrast, TMX but not GMT activated caspase 2. Pharmacologic inhibition of caspase 2 activation decreased TMX-, but not GMT-, induced activation of caspase 3 and apoptosis of cholangiocarcinoma cells. Similarly, activation of caspase 2 was found in tumors from TMX-treated mice, but not GMT-treated mice. Therefore, the enhanced effect of TMX on GMT-induced cholangiocarcinoma cell death is partially mediated by activation of caspase 2. TMX and GMT both induce apoptosis and inhibit cholangiocarcinoma tumorigenesis, which may be attributed to the activation of distinct apoptosis signals by TMX and GMT. Our studies provide in vivo evidence and molecular insight to support the use of TMX and GMT in combination as an effective therapy for cholangiocarcinoma.
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 4089. doi:10.1158/1538-7445.AM2011-4089