Unfolding the Apoptotic Mechanism of Antioxidant Enriched-Leaves of Tabebuia pallida in EAC Cells

Abstract

Targeting apoptosis is a promising approach to inhibit the abnormal cell proliferation of cancer progression. Existing anti-apoptotic drugs, many derived from chemical substances, have often failed to combat cancer development and progression. Therefore, identification of apoptosis-inducing anticancer agents from plant-derived sources has become a key aim in cancer research. The present study was designed to explore the regulation of apoptosis by Tabebuia pallida (T. pallida) using an Ehrlich Ascites Carcinoma (EAC) mouse model and compositional analysis by LC-ESI-MS/MS. Dried and powdered T. pallida leaves (TPL), stem bark (TPSB), root bark (TPRB) and flowers (TPF) were extracted with 80% methanol. Using cultured EAC cells and EAC-bearing mice with and without these extracts, anticancer activities were studied by assessing cytotoxicity and tumor cell growth inhibition, changes in life span of mice, and hematological and biochemical parameters. Apoptosis was analyzed by microscopy and expression of selected apoptosis-related genes (Bcl-2, Bcl-xL, NF{kappa}-B, PARP-1, p53, Bax, caspase-3 and -8) using RT-PCR. LC-ESI-MS analysis was performed to identify the major compounds from the most active extracts. In EAC mice compared with untreated controls, the TPL extract exhibited the highest cytotoxicity with significant tumor cell growth inhibition (p< 0.001), reduced ascites by body weight (p< 0.01), increased the life span (p<0.001), normalized blood parameters (RBC/WBC counts), and increased the levels of superoxide dismutase and catalase. TPL-treated EAC cells showed apoptotic characteristics of membrane blebbing, chromatin condensation and nuclear fragmentation, and caspase-3 activation, compared with untreated EAC cells. Moreover, annexin V-FITC and propidium iodide signals were greatly enhanced in response to TPL treatment, indicating apoptosis induction. Pro- and anti-apoptotic signaling after TPL treatment demonstrated up-regulated p53, Bax and PARP-1, and down-regulated NF{kappa}-B, Bcl-2 and Bcl-xL expression, suggesting that TPL shifts the balance of pro- and anti-apoptotic genes towards cell death. LC-ESI-MS data of TPL showed a mixture of glycosides, lapachol, and quercetin antioxidant and its derivatives that were significantly linked to cancer cell targets. In conclusion, the TPL extract of T. pallida possesses significant anticancer activity. The tumor suppressive mechanism is due to apoptosis induced by activation of antioxidant enzymes and caspases and mediated by a change in the balance of pro- and anti-apoptotic genes that promotes cell death. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/426226v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1112faaorg.highwire.dtl.DTLVardef@1505d1dorg.highwire.dtl.DTLVardef@1115a2aorg.highwire.dtl.DTLVardef@36a915_HPS_FORMAT_FIGEXP M_FIG C_FIG