Apoptotic effects and chemical investigation of active extracts of Curcuma mangga rhizomes / Hong Gin Wah

Plants have a long history of use in the treatment of many ailments including cancer. The choices of plant for drug discovery based on ethnopharmacological data rather than random approach hold greater promise of finding a good candidate for investigation. Curcuma mangga known “temu mangga” in Malay has been selected for investigation use as a natural remedy for various diseases in Malaysia including cancer. Crude and fractionated extracts of C. mangga rhizomes were initially investigated for their growth inhibitory effects on four human cancer cell lines, namely colorectal adenocarcinoma cell (HT-29), colorectal carcinoma cell (HCT-116), cervical carcinoma cell (CaSki) and lung carcinoma cell (A549), and a normal human cell (non-cancer human colorectal cell line, CCD-18Co) using sulforhodamine B (SRB) colorimetric assay. Dry rhizome powder of C. mangga was soaked in dichloromethane for three days and the crude dichloromethane extract (CMD) obtained was washed with n-hexane to give the hexane-soluble extract (CMDH). The hexane-insoluble residue was dissolved completely in methanol to give the fractionated methanolic extract (CMDM). All three extracts (CMD, CMDH and CMDM) generally showed good cytotoxicity effects against HT-29, HCT-116, A549 and CaSki with IC50 value ranging from 14.3 to 21.0 μg/mL, 15.2 to 18.3 μg/mL, 14.8 to 20.0 μg/mL and 18.7 to 21.2 μg/mL respectively. All extracts exhibited lower toxicity towards CCD-18Co (IC50 value ranging from 50.3 to 55.0 μg/mL) compared with chemotherapy drug (doxorubicin), with an IC50 value of 0.11 μg/mL. Both CMDH and CMDM were subjected to chemical investigations resulted isolation and identification of five (5) components, namely longpene A, zerumin A, coronadiene, (E)-labda-8(17),12-diene-15,16-dial and calcaratarin A. Other isolated compounds could not be identified. The isolated pure compounds showed weak cytotoxicity effects against the selected cancer cell lines (IC50 values ranging from 19.1 to 34.7 μg/mL for HT-29; 17.7 to 38.6 μg/mL for HCT-116; 18.6 to 38.3 μg/mL for A549; 21.8 μg/mL to 30.2 μg/mL for CaSki). The pure compounds were less effective in preventing the proliferation of cancer cells compared with extracts. Synergism between the components maybe responsible for the observed activity. CMD was selected for further molecular investigation of its anticancer effect on HT-29 cell line for it’s good cytotoxic effects against HT-29. Typical apoptotic morphological features like membrane blebbing, formation of apoptotic bodies, cell shrinkage and condensation of chromatin were observed on treated HT-29. The CMD induced cell arrest in G2/M phase of the cell cycle after 24 hours. Externalization of phosphotidylserine from the plasma membrane was observed in a concentration- and time-dependent manner. DNA fragmentation was detected through the Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. In the Western blot analysis, the expression levels of the pro-apoptotic proteins (Bax, caspase 3, -9 and 8) were up-regulated while the anti-apoptotic proteins (Bcl-2, cIAP-2, XIAP) were down-regulated. The expression levels of cleaved PARP-1 were up-regulated. This indicated that apoptosis might have occurred through the intrinsic and extrinsic pathways. As a conclusion, the crude dichloromethane extract of C. mangga rhizomes has the potential to be further developed as an anticancer agent against HT-29.