Anti-Angiogenic and anti-Metastatic effects of Ardisia crispa (Thunb.) A.DC. Root Hexane extract and its compound, 2-Methoxy-6-Undecyl-1,4- Benzoquinone in Colorectal cancer cell lines via Kras-related signaling pathways

Ardisia crispa is a plant that is locally known as “mata ayam” or “mata itik” and has been traditionally used to treat various ailments. Earlier investigations on the hexane extract of Ardisia crispa roots, namely ACRH, and its compound, 2- methoxy-6-undecyl-1,4-benzoquinone, denoted as BQ have been reported to exert promising anti-inflammatory and anti-angiogenic activities. Hence, this study aims to further investigate the potential of ACRH and BQ in impeding angiogenesis and metastasis in human colorectal cancer cell lines. For this, two types of CRC cell lines were used, namely HCT116 cell, which is of the primary tumor, and LoVo cell, which is of the metastatic type. The anti-proliferative effects of ACRH and BQ on HCT116 and LoVo cells were first determined via cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ACRH and BQ exhibited an IC50 of 1.93±0.08 µg/mL and 2.05±0.1 µg/mL in HCT116 cells and 1.95±0.03 µg/mL and 2.8±0.2 µg/mL in LoVo cells, respectively. Subsequent experiments were conducted with HCT 116 and LoVo cells treated with ACRH and BQ at concentrations of 2.0 µg/mL, 0.2 µg/mL, and 0.02 µg/mL, respectively and compared to sunitinib 2.0 µg/mL as the positive control. Apoptotic activity was measured using Annexin V-Propidium iodide (PI) flow cytometric assay. The results notably co-related with the cell viability assay, in which both ACRH and BQ significantly (p<0.05) reduced the percentage of healthy HCT116 and LoVo cells in a concentration dependent manner. Additionally, ACRH and BQ also significantly induced early and late apoptosis in both cells. Furthermore, ACRH and BQ, at all tested concentrations (2.0, 0.2, and 0.02 µg/mL) significantly (p<0.05) suppressed migration, invasion, and adhesion activities in HCT116 and LoVo cells, in comparison to the negative control. The suppressive effects of ACRH at high concentration (2.0 µg/mL) was observed to be more potent than sunitinib 2.0 µg/mL. Meanwhile, the inhibition of urokinase plasminogen activator (uPA) protein was only significant in ACRH treated LoVo cells only, but not in BQ in both HCT116 and LoVo cells. In contrast, zymogram assay revealed significant inhibition (p<0.05) of active matrix metalloproteinase (MMP)-2 in both HCT116 and LoVo cells treated with ACRH and BQ at all concentrations. The results suggest that ACRH and BQ could prevent extracellular matrix (ECM) degradation and thus preventing CRC cells invasion and metastasis, mediated by attenuation of these invasion-related enzymes. Moreover, ACRH and BQ, at different concentrations (0.02 µg/mL, 0.2 µg/mL, and 2 µg/mL), successfully and significantly (p<0.05) inhibited the protein expressions of vascular endothelial growth factor (VEGF)-A, VEGF-C, Kirsten rat sarcoma viral oncogene homolog (KRAS), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), extracellular regulated-signal kinase (ERK), phosphatidylinositol-3 kinase (PI3K) and protein kinase B (AKT), respectively in both HCT116 and LoVo cells. In silico study was further conducted on BQ to gain a more comprehensive understanding on its behaviour as a small molecule ligand, and its mechanism of action against the aforementioned proteins at a molecular level. Interaction of these targeted proteins with BQ exhibited good binding affinities and were postulated to be contributed by the formation of hydrogen bonds, hydrophobic interactions as well as π-interactions between the proteins and its ligand. Based on the accumulated findings in this study, it can be concluded that ACRH and BQ are able to mitigate the progression of CRC by impeding angiogenesis and metastasis, specifically by inhibition of KRAS/BRAF/ERK and KRAS/PI3K/AKT signalling pathways. Future investigation should further establish the anti-angiogenic and anti-metastatic effects of ACRH and BQ at the in vivo level. In addition, the molecular dynamics, and molecular mechanics of BQ should be further assessed to ensure its stability upon binding to these aforementioned targeted proteins.

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