Characterization of the anti-cancer effects of Gleditsia sinensis in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC) occurs at a relatively high frequency in the Asian regions including Chinese mainland. In Hong Kong, the incidence of ESCC accounts for more than 90% of esophageal cancers, making this disease of intense local importance for investigation. Current modalities of therapy for this disease offer relatively poor survival and cure rates, and thus more investigations on the novel therapeutic agents are essential for further improving the treatments of ESCC. Multiple lines of epidemiological, experimental and clinical evidences showed that the overexpression of cyclooxygenase-2 (COX-2), an inducible enzyme not expressed in most normal tissues, participates in the carcinogenesis of human cancers including ESCC through multiple mechanisms. Previous studies revealed that administration of non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 inhibitors could suppress the growth of ESCC by inhibiting COX-2 activities, but the values are limited by the accompanied side effects, including gastrointestinal complications and the increased risk of myocardial infarction. Thus novel therapeutic agents targeting on COX-2 remain to be identified. The anti-cancer effects of the anomalous fruit extract of Gleditsia sinensis (GSE) on human cancers, especially ESCC, were extensively reported before. However, the anti-cancer actions of GSE targeting on COX-2 in ESCC has not been investigated before, despite its well-known anti-inflammatory actions as described in the traditional Chinese pharmacopoeias. The present study showed that GSE could effectively inhibit the proliferation of a panel of ESCC cell lines including KYSE30, 150, 450, 510, 520 and HKESC-3, HKESC-4 and SLMT-1 in both time- and dose-dependent manner as shown in the MTS cytotoxicity assays. Multiplex RT-PCR analysis also revealed that COX-2 mRNA was frequently overexpressed in 11 out of 13 (85%) ESCC cell lines and 11 out of 13 (85%) showed no change in COX-1 mRNA expression with respect to the non-tumor esophageal epithelial cells. Western blotting analysis performed on all the ESCC cell lines with overexpressed COX-2 also indicated the overexpression of COX-2 protein but not COX-1. After GSE treatment with the respective doses, all the tested ESCC cell lines (8/8) showed the attenuated COX-2, but not COX-1, mRNA level and protein expression in multiplex RT-PCR, Western blotting and immunohistochemical analyses, and such observations were also GSE dose-dependent. GSE also showed the dose-dependent suppression of PGE2 production, which reflects the COX-2-coupled PGE2 synthesis, in 7 out of 8 (88%) ESCC cell lines using PGE2 enzyme immunoassay. Furthermore, a pilot study with the use of oral doses of GSE (0, 50 and 100 mg/kg/day; 3 mice for each group) for 31 days also demonstrated the growth inhibition on all the SLMT-1 xenograft-bearing nude mice with reduced COX-2 protein at the histological level compared with the controls. The overall results of the present study provide the first evidence that GSE can induce its anti-cancer actions on ESCC through targeting on COX-2 expression at the mRNA and protein levels in the ESCC cell lines and xenograft tumors. The findings of the present study thus provide a window for further exploration of GSE as an alterative therapeutic agent for ESCC or even other cancers.