Deoxyelephantopin from Elephantopus scaber Inhibits HCT116 Human Colorectal Carcinoma Cell Growth through Apoptosis and Cell Cycle Arrest
Deoxyelephantopin (DET), one of the major sesquiterpene lactones derived from Elephantopus scaber was reported to possess numerous pharmacological functions. This study aimed to assess the apoptosis inducing effects and cell cycle arrest by DET followed by elucidation of the mechanisms underlying cell death in HCT116 cells. The anticancer activity of DET was evaluated by a MTT assay. Morphological and biochemical changes were detected by Hoescht 33342/PI and Annexin V/PI staining. The results
... ning. The results revealed that DET and isodeoxyelephantopin (isoDET) could be isolated from the ethyl acetate fraction of E. scaber leaves via a bioassay-guided approach. DET induced significant dose-and time-dependent growth inhibition of HCT116 cells. Characteristics of apoptosis including nuclear morphological changes and externalization of phosphatidylserine were observed. DET also significantly resulted in the activation of caspase-3 and PARP cleavage. Additionally, DET induced cell cycle arrest at the S phase along with dose-dependent upregulation of p21 and phosphorylated p53 protein expression. DET dose-dependently downregulated cyclin D1, A2, B1, E2, CDK4 and CDK2 protein expression. In conclusion, our data showed that DET induced apoptosis and cell cycle arrest in HCT116 colorectal carcinoma, suggesting that DET has potential as an anticancer agent for colorectal carcinoma. Molecules 2016, 21, 385 2 of 14 is orchestrated by concerted actions of cyclin/cyclin-dependent kinase (CDK) in association with cyclin proteins. Cyclin D-CDK4/CDK6 and cyclin E-CDK2 navigate G1 transition via the restriction point, which lead to the cell cycle progression  . The cyclin E-CDK2 and cyclin A-CDK2 are essential complexes involved in the initiation and progression of the S phase  while the cyclin B-CDK1 complex regulates progression of G2 and mitosis  . These complexes in turn, are regulated by the cyclin-dependent kinase inhibitors (CDKIs), including p21 Waf1/Cip1 , a crucial regulator of cell cycle progression and apoptosis. The functional role of p21 can be initiated either by p53-dependent or -independent mechanisms [11, 12] . Elephantopus scaber Linn., commonly known as Elephant's foot, (Asteraceae) is a scabrous herb distributed on various continents, including Asia, Europe, South America and Africa. According to traditional claims, the leaves and roots are used for fever, diarrhea, dysentery, cancer and dysuria [13, 14] . E. scaber is a rich source of sesquiterpene lactones, including deoxyelephantopin (DET) and isodeoxyelephantopin (isoDET), that have been used in Ayurveda medicine in India for the treatment of cancer and leukemia  . Pharmacological studies have demonstrated the antitumor effects of on breast, nasopharyngeal, lung, cervical, Dalton lymphoma ascitis and prostate cancer      . DET induced apoptosis via intrinsic and extrinsic pathway as well as cell cycle arrest in human nasopharyngeal carcinoma epithelial CNE cells. Additionally, isoDET was shown to induce apoptosis in T47D breast carcinoma, A549 lung carcinoma and KBM-5 chronic myeloid leukemia [21, 22] . Previously, we have found the ethyl acetate fraction from E. scaber could inhibit the growth of the human colorectal cancer cell line HCT116  . In continuation of that work, here we aimed to isolate and identify the active compounds from ethyl acetate fraction of E. scaber based on its ability to inhibit HCT116 cell growth in vitro. We identified two anticancer compounds, deoxyelephantopin (DET) and isodeoxyelephantopin (isoDET). The profound cytotoxicity of DET against HCT116 cells indicates that this compound maybe valuable for colorectal cancer therapy. However, the HCT116 cell growth inhibition activities of DET, as well as its apoptosis inducing action and underlying molecular mechanisms have never been reported before. The apoptosis inducing effect of DET on HCT116 cells has been reported once previously and was only based on morphological studies  . Towards this end, the current study investigated the effect of DET in HCT116 cells on the cell cycle and apoptosis regulation. These results may help the future development of new colorectal cancer therapies. Results Isolation and Identification of the Bioactive Compounds, Deoxyelephantopin and Isodeoxyelephantopin via a Bioassay-Guided Approach Our previous study demonstrated that the ethyl acetate fraction of E. scaber exerted the most potent cytotoxic effect on HCT116 cells  . Hence, we proceeded with the isolation of its bioactive compounds via a bioassay guided approach (Figure 1) . Compounds 1 and 2 were isolated from the active fraction F2. The structures of compounds 1 and 2 were elucidated using spectroscopic analysis ( 1 H-, 13 C-NMR and HRESI-MS), and by comparison of the obtained spectral data with literature values. Compound 1 was identified as isodeoxyelephantopin (isoDET), by comparison of its 1 H-and 13 C-NMR data with the literature values of isodeoxyelephantopin (isoDET)  ( Table 1 ). The structure of compound 1 was further confirmed as isoDET by HRESI-MS, in a positive mode, which revealed a molecular ion peak at m/z 345.