SNU-16 위암 세포주에서 p-coumaric acid의 세포성장 억제 효과

Anti-proliferative Properties of p-Coumaric Acid in SNU-16 Gastric Cancer Cells

Abstract

p-Coumaric acid (p-CA)는 항산화 및 항염 활성을 가진 식물계에서 가장 풍부한 식물화학물질이다. 그러나 위암세주포에서 p-CA의 항암 활성과 전사체 발현에 대한 연구는 아직까지 수행된 바 없다. 본 연구에서는 SNU-16 위암세포에서 p-CA에 의한 세포 증식 억제 및 전사체 프로파일에 미치는 영향을 조사하였다. p-CA는 세포사멸 단백질 발현을 조절하여 SNU-16 세포에서의 세포사멸을 유도하였다. RNA-seq 분석을 사용하여 p-CA처리에 의해 SNU-16 세포에서 차별적으로 발현된 유전자(DEGs)를 동정하였다. DEGs들의 gene ontology (GO) 술어로 유전자 산물을 검색한 결과, 주로 염증반응, 세포사멸 과정, 세포주기 및 면역 반응에 관여하는 생물학적 과정에 관여하는 것으로 나타났다. 또한, KEGG 경로분석 결과, p-CA는 주로 PI3K-Akt 와 암 신호전달 경로에 변화를 유발하였다. 본 연구결과는 p-CA가 세포증식과 암 신호 전달 경로에 관여하는 유전자 발현을 조절함으로써 위암 예방 효과를 나타낼 수 있음을 시사한다.

The ubiquitous plant metabolite p-coumaric acid (p-CA) has antioxidant and anti-inflammatory properties, but its anti-cancer activity has not been established in gastric cancer cell lines. In this study, we investigated the effects of p-CA on the proliferation and transcriptome profile of SNU16 gastric cancer cells. Treatment with p-CA induced apoptosis of the SNU-16 cells by regulating the expression of pro-apoptotic and anti-apoptotic proteins, such as Bcl-2, poly (ADP-ribose) polymerase (PARP), Bax, procaspase-3, and cleaved-caspase-3. The genes differentially expressed in response to p-CA treatment of the SNU-16 cells were identified by RNA sequencing analysis. Genes regulated by p-CA were involved mainly in the inflammatory response, apoptotic processes, cell cycle, and immune response. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the phosphatidylinositol-3-kinase-Akt and cancer signaling pathways were altered by p-CA. Protein-protein interaction (PPI) network analysis also revealed that p-CA treatment was correlated with differential expression of genes associated with the inflammatory response and cancer. Collectively, these results suggest that p-CA has potential utility in gastric cancer prevention.

Fig. 1. Effects of p-CA on cell proliferation and apoptotic protein expression in SNU-16 cells. Cells were incubated with different concentrations of p-CA for 72 hr in SNU-16 cells (A) and Hs-68 cells (B). Cell viability was determined using the MTT assay. Data are means ± standard deviation (SD) of three independent experiments. *p<0.05, ** p<0.01, and ***p<0.001 compared to the untreated group. (C) Cells were treated with different concentrations of p-CA for 48 hr. The expression of Bcl-2, Bax, procaspase-3, cleaved capase-3, and PARP were analyzed by Western blotting.

Fig. 2. Gene ontology (GO) analysis of differentially expressed genes (DEGs) regulated by p-CA in SNU-16 cells. DEGs were classified as biological process (BP), cellular component (CC), and molecular function (MF).

Fig. 3. Protein–protein interaction networks among DEGs regulated by p-CA in SNU-16 cells. The protein-protein interaction network was constructed using the STRING online tool with a confidence score >0.4. The nodes represent proteins, edges represent interactions between proteins, and the colors of the nodes represent the log2 fold change in expression level.

Fig. 4. Quantitative determination of differentially expressed genes by real-time PCR. Data are means ± standard deviation (SD) of three independent experiments. *p<0.05, **p<0.01, and ***p<0.001 compared to the untreated group.

Table 1. The primer sequences of the genes used in Real-time PCR analysis

Table 2. The pathway analysis of genes differentially regulated by p-CA in SNU-16 gastric cancer cells (p<0.05)