Materials and methods: 3D-mediated inhibition on cell viability was evaluated by MTT and real-time cell proliferation was measured by xCelligence RTDP instrument. Western blotting was used to measure pro-apoptotic, anti-apoptotic proteins and JAK2-STAT3 phosphorylation. Flow cytometry was used to measure ROS production and apoptosis.
Results: Our study revealed that 3D treatment significantly reduced the viability of human CRC cells HT-29 and SW620. Furthermore, 3D treatment induced the generation of reactive oxygen species (ROS) in human CRC cells. Confirming our observation, N-acetylcysteine significantly inhibited apoptosis. This is further evidenced by the induction of p53 and Bax; release of cytochrome c; activation of caspase-9, caspase-7 and caspase-3; and cleavage of PARP in 3D-treated cells. This compound was found to have a significant effect on the inhibition of antiapoptotic proteins Bcl2 and BclxL. The results further demonstrate that 3D inhibits JAK2-STAT3 pathway by decreasing the constitutive and IL-6-induced phosphorylation of STAT3. 3D also decreases STAT3 target genes such as cyclin D1 and survivin. Furthermore, a combination study of 3D with doxorubicin (Dox) also showed more potent effects than single treatment of Dox in the inhibition of cell viability.
Conclusion: Taken together, these findings indicate that 3D induces ROS-mediated apoptosis and inhibits JAK2-STAT3 signaling in CRC.
METHODS AND RESULTS: The leaves extracts were analysed for its antiproliferative effect on breast cancer (MCF7) cells and normal epithelial breast (MCF 10A) cells using Sulforhodamine B (SRB) assay. The selective extract was evaluated for its ability to induce apoptosis using Annexin V-FITC apoptosis staining and the expression of molecular genes using qualitative reverse transcription-polymerase chain reaction (RT-PCR) against MCF7 cells. Gas chromatography-mass spectrometry (GC-MS) was used to identify the compounds from the selective extract. The findings showed that dichloromethane fraction (CV-Dcm) extract had high antiproliferative effect against MCF7 cells (IC50 = 24 µg/mL, selective index (SI) = 8.17). The percentages of apoptosis cells in CV-Dcm-treated MCF7 cells was 58.8%. The CV-Dcm extract induced downregulation of PCNA level. The apoptotic genes were also triggered in both extrinsic and intrinsic signaling pathways, affecting a 1.5-fold increase in BAX, 1.4-fold increase in cytochrome c, 1.3-fold increase in caspase-8, 1.7-fold increase in caspase-3 and 0.5-fold-decrease in BCL-2. Treated MCF7 cells also activated P53-dependent apoptotic death pathway.
CONCLUSIONS: The present work strongly suggests that high efficacy of CV-Dcm extract was attributed to its antiproliferative and apoptosis-inducing activation in MCF7 cells, most likely due to its favourable compounds.