5,6-dehydrokawain from the rhizome of Alpinia mutica Roxb. induced proangiogenic tumour-derived VEGF of HT-29 colorectal cancer

Abstract Vascular endothelial growth factor (VEGF) is a glycoprotein vital to the regulation of vascular endothelial cells proliferation, migration and angiogenesis. The expression of VEGF is required for the formation of new blood vessels critical in supplying oxygen and nutrition in the course of tumorigenesis. The present study investigated the effect of 5,6-dehydrokawain isolated from the rhizomes of Alpinia mutica on VEGF expression in vitro using HT-29 cell line. The results revealed that 5,6-dehydrokawain induced the expression of proangiogenic tumour-derived VEGF of HT-29 cells, which may explain the inability of 5,6-dehydrokawain in suppressing cancer cells proliferation.


Introduction
Vascular endothelial growth factor (VEGF) has been described as mediator of angiogenesis and a critical regulator of vascular growth during embryogenesis and the formation of new blood vessels (Olsson et al. 2006). However, the abnormal growth vessels attributed to excessive angiogenesis is implicated in tumour progression. Consequently, the tumour requires these new blood vessels to deliver oxygen and nutrition by employing proangiogenic proteins such as VEGF essential in triggering tumour angiogenesis (Fu et al. 2015). 5,6-dehydrokawain is a phenolic lactone molecule ( Figure 1) found in the rhizomes (Jantan et al. 2004;Malek et al. 2011) and leaf (Sirat and Jani 2013) of Alpinia mutica, Alpinia zerumbet (Kumagai et al. 2016) and Polygonum hydropiper (Xiao et al. 2017). Whilst 56DKW have been known as potent platelet-activating factor (PAF) (Jantan et al. 2004) and thromboxane A 2 inhibitor (Teng et al. 1990), the compound have recently been reported to promote differentiation of osteoblastic MC3T3-E1 cells (Kumagai et al. 2016), activate Nrf2/ARE of PC12 cells (Tanaka et al. 2010) and induced autophagy of T24 cells .
Founded by the above information, the present study was primarily designed to investigate the effect of 56DKW on VEGF expression in vitro using HT-29 cell line. Our result revealed for the first time that 5,6-dehydrokawain induced the expression of proangiogenic tumour-derived VEGF of HT-29 cells suggesting the inability of the bioactive compound to suppress cancer cells proliferation.

Results and discussion
56DKW was obtained as pale yellow amorphous solid and identified as a phenolic lactone molecule. The bioactive compound was assigned and conformed specifically based on spectral similarities, which were consistent and found to exhibit near similar spectroscopic data to the established spectral data previously reported elsewhere (Dharmaratne et al. 2002). Previously, the ineffective cytotoxicity of 56DKW screened against different cancer cell lines to include HT-29 has been demonstrated elsewhere (Malek et al. 2011). Apparently, similar result was also observed in the present study at IC 50 values of 55.37 μg/mL (242.85 μM). S1 Figure shows a dose-response curve of 56DKW compared with 5-flourouracil with IC 50 values  In the present study, human immunoassay approach was used to quantify the expression of VEGF in HT-29 cells treated with either 56DKW or curcumin. Justifiably, curcumin was used as a positive control due to its potent VEGF receptors suppressor. Whilst curcumin inhibit the VEGF expression of HT-29 cells in a dose-dependent manner (12.5, 25, and 50 μM), 56DKW on the other hand induced the expression of VEGF in a similar fashion (12.5, 25, and 50 μM) ( Figure S2). VEGF-induced angiogenesis is often associated with thromboxane A 2 expression, which regulate angiogenesis via VEGF inhibition (Ashton and Ware 2004). Consequently, the potent inhibition of thromboxane A 2 by 56DKW might explain the expression of VEGF observed in the present study. Furthermore, previous studies have shown that autophagy is associated with angiogenesis (Du et al. 2012). Likewise, the ability of 56DKW to induced autophagy may also suggest its ability to stimulate angiogenesis.
Founded by the above information, our finding revealed that 56DKW induced the expression of proangiogenic tumour-derived VEGF of HT-29 cells in vitro, which may explain the inability of the bioactive compound to inhibit cancer cells proliferation. Further studies are, therefore, recommended for ultimate validation of the relevant studies.

Supplementary material
The experimental section, cell viability ( Figure S1), graph of VEGF expression ( Figure S2), 1 H NMR, 13 C NMR and DIMS spectral data of 56DKW, raw data from VEGF expression.