Cytotoxic Activity of Epigallocatechin and Trans- Cinnamaldehyde in Gastric Cancer Cell Line

Gastric cancer is one of the main causes of cancer related death all over the worlds [1]. Treatment strategies for these patients comprisesurgery and chemotherapy. But, beneficial effects of chemotherapeutic agents are not good enough and these drugs have numerous side effects [2]. Bioactive dietary agents have displayed the capability to decrease cancer growth with noticeable immediate effects on cancer cells. Throughseparation and examination of the effective ingredients in these therapeutic agents, regarding cancer cell inhibition, a perfect mechanism of action might beassociated with animproved understanding of the profits of these natural products [3]. Recently herbal Abstract


Introduction
Gastric cancer is one of the main causes of cancer related death all over the worlds [1]. Treatment strategies for these patients comprisesurgery and chemotherapy. But, beneficial effects of chemotherapeutic agents are not good enough and these drugs have numerous side effects [2].
Bioactive dietary agents have displayed the capability to decrease cancer growth with noticeable immediate effects on cancer cells. Throughseparation and examination of the effective ingredients in these therapeutic agents, regarding cancer cell inhibition, a perfect mechanism of action might beassociated with animproved understanding of the profits of these natural products [3]. Recently herbal lines with (−)-epigallocatechin-3-gallate (EGCG), the active tea catechin. Nonetheless, the molecular basis for the suggested mechanisms and its efficacy as an anti-cancer agent in vivo are not evidentlyrecognized [5]. The exposure of human stomach cancer cells to green tea catechin extract and EGCG, lead to growth inhibition and the apoptosis induction. Accordingly Morphological changes display apoptotic body in the cells exposedtoEGCG and green tea catechin extract [6]. Other polyphenol of green tea, could affect development of numerous cancers [7]. So in this study the anti-cancer effects of traditional herbal medicines components; EGCG and TC in single and combined cases was examined in gastric AGS cell line.

Materials and Methods
AGS, a gastric adenocarcinoma cell line, was purchased from Pasteur Institute, Tehran, Iran. The cells were seeded in RPMI 1640 medium, with 10% (v/v) FBS and 0.1% penicillin streptomycin (at 37 °C in a humidified incubator containing 5% CO 2 atmosphere). ECCG and TC was obtained from sigma-Aldrich Company. Absorbance was measured by ELISA reader at the wavelength of 490 nm.
Data were presented as mean ± standard deviation of values attained by independent tests. one-way ANOVA was utilized for multiple comparisons. p-values > 0.05 were considered as statistically significant. Statistical analysis of data was carried out with SPSS (Version 16: SPSS. Link. USA).

Results
In this study we examined cell proliferation rate of AGS gastric cancer cells against various concentrations of TC and EGCG in single and double treated cases.
As shown in Figure 1, the cytotoxic effects of ECGC and TC increased in dose dependent manner. It could be indicate that the dose response curve of both agents were partially similar.
Then, we tested the lower doses than IC50 in combination of TC and EGCG on AGS cells. As shown in Figure 2, we found that in double combined cases (0.75×IC50 and 0.3×IC50) cellular viability decreased in compared to IC50 doses of each single agents. Also, there were significant decrease in cellular viability in all single and double treated cases toward untreated cells. In double combinations there were significant higher toxicity in 0.75×IC50 concentrations of both EGCG and TC in compared with double combinations at 0.2×IC50 and 0.1×IC50 doses (P<0.05). Also double combinations in 0.3×IC50 and 0.2×IC50doses exerted higher growth inhibitory effects versus double combination in 0.1×IC50 dose (P <0.05).

Discussion
There are many types of cancers such as ovarian cancer, lung cancer, and breast cancer [9][10][11][12][13]. Gastric cancer considered as one of the leading cause of cancer mortality world width. indeed, numerous gastric cancer patients are identified when the tumor is at unrespectable stage. In this cases, chemotherapeutic agents in single and combined cases remains main treatment options. Previous data show that TC show inhibitory effects on cancer cell growth [3] but in many cases of cancer , favorable effects of chemotherapeutic agents are not good enough [2]. So the use of natural compound from herbal medicine could be helpful [14]. cytotoxicity of TC and EGCG could be indicated that these two agents might be acts in cooperative manner in induction of cell death.
In this regards it has been reported that EGCG repressed gastric tumour growth through inhibiting Wnt/βcatenin signalling. Indeed, EGCG decreased gastric cancer cell proliferation [7]. Also TC promote cancer cell death and apoptosis induction [17]. It seem that these effects was increased in our studied combinatorial cases.
To the best of our knowledge we didn't find any more related studies and these combination were evaluated in this study for the first time. Further studies should be conducted to verify the possible mechanisms which engaged in cytotoxic effects of EGCG and TC in combined cases.
In this regards, Cinnamaldehyde and cinnamaldehydederived compounds are proposed as possible potential agents as anticancer drugs [15]. Also EGCG, showed the inhibitory effects on cell growth in a various cultured cells [16].
Consequently, we examined effects of EGCG and TC in AGScells (human gastric carcinoma). The proliferation of this cell line was inhibited with EGCG and TC in a dose-dependent manner. Also in combination of this two agents in lower concentrations (>IC50) cell growth of AGS cells inhibited in compared with single treatments.
In similar to our results, Horie et al confirmed the synergistic impact of epicatechin with EGCG on the apoptosis induction in gastric cancer cells.in this study, caspases-3, -8 and -9 activities were assessed in EGCG-treated cells, which confirmed the caspases are engaged in the possible mechanism of EGCG action [16].
Also other study showed the potential efficacy of Cinnamaldehyde as a new antitumor agent. The mechanisms of action include the regulation of apoptosis adhesion and invasion related genes [17].
Chiang et al in a recent study on breast cancer cell line and xenograft animal models that treated with various concentrations of visfatin combined with CA and FK866 (a visfatin inhibitor) tested the cell toxicity. In the breast cancer cell and the xenograft animal model, visfatin enhanced proliferation-related protein, nevertheless combination with CA or FK866 significantly decreased visfatin-induced carcinogenic impacts. They indicate that TC decreased visfatin-related breast cancer progression.
In describing the possible mechanism of increased Data are presented as mean± standard deviation. *Significant differences compared with untreated control cells. # Significant differences in compared to IC50 of each single agents. Epigallocatechin gallate; EGC. trans-cinnamaldehyde; TC.