[논문]SNU-16 위암 세포주에서 p-coumaric acid의 세포성장 억제 효과

장미경; 고희철; 김세재

doi:10.5352/jls.2019.29.7.809

초록
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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.

Abstract ▼ AI-Helper

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 gast...

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.

주제어

표/그림 (6)

그림 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.
표 Table 1. The primer sequences of the genes used in Real-time PCR analysis
그림 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).
표 Table 2. The pathway analysis of genes differentially regulated by p-CA in SNU-16 gastric cancer cells (p<0.05)
그림 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.
그림 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.

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가설 설정

However, the effects of p-CA on cell growth and transcriptome profiles in SNU-16 gastric cancer cells have not yet been examined. In this study, we investigated the effects of p-CA on cell proliferation and transcriptome profiles in SNU-16 cells.

제안 방법

Barcodes were introduced during amplification. High-throughput sequencing was performed as paired-end 100 sequencing using HiSeq 2500 (Illumina, Inc., Foster City, CA, USA).
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, Netherlands), and RNA quantification was performed using the ND-2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For gene expression profiling, RNA sequencing (RNAseq) was performed at Ebiogen Inc.

대상 데이터

RPMI 1640 medium, fetal bovine serum (FBS), and penicillin–streptomycin (PS) were purchased from Gibco BRL (Grand Island, NY, USA). Antibodies against B-cell/lymphoma 2 (BCL2), BCL2-associated X protein (BAX), procaspase3, and poly (ADP-ribose) polymerase (PARP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against cleaved caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA).
SNU-16 human gastric cancer cells were obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI-1640 medium (Gibco BRL) supplemented with 10% FBS and 1% PS (100 units penicillin/ml and 100 pg streptomycin/ml).

데이터처리

All data are expressed as means ± standard deviation. Statistical differences between groups were examined using one-way ANOVA. Differences with p-values <0.

이론/모형

Cells in the exponential growth phase were used throughout the experiments. Cell viability was determined using the MTT assay. Briefly, cells were plated in 96-well plates at 2.
Fragments per kilobase of exon per million fragments (FPKM) was used as the method of determining the expression levels of the gene regions. Gene classification was based on searches conducted using DAVID (http://david.abcc.ncifcrf.gov/).
Differentially expressed genes (DEGs) were determined based on counts from unique and multiple alignments using coverage in Bedtools [23]. The RT (Read Count) data were processed based on the quantile normalization method using EdgeR within R (R development Core Team, 2016) using Bioconductor [6]. The alignment files were used to assemble transcripts, estimate their abundances, and detect differential expression of genes or isoforms using cufflinks.
The expression level of each gene was normalized to an endogenous control (GAPDH) and was calculated using the 2−ΔΔCt method.

성능/효과

A total of 2,877 DEGs, comprising 2,216 upregulated and 661 downregulated genes, were identified in 1,500μM p-CA-treated cells compared with untreated control cells.
The BCL2 family plays a critical role in the regulation of apoptosis, and includes the anti-apoptotic proteins BCL2 and BCL-xL, as well as the pro-apoptotic proteins BAX, BCL2 antagonist/killer (BAK), BCL2-associated agonist of cell death (BAD), BH3 interacting domain death agonist (BID), and BAX inhibitor motif (BIM) [2, 5, 7, 14]. Our results showed that p-CA-activated executioner caspase-3 and the cleavage of PARP resulted in mitochondria-mediated apoptosis, even though the mechanism of apoptosis induction by p-CA in gastric cancer SNU-16 cells remains to be determined.

후속연구

Thus, it is suggested that IC₅₀ values against SNU-16 cell growth may be achievable internally, and may exert chemopreventive properties. However, further research is needed to elucidate the underlying mechanisms of p-CA’s effects in greater detail.

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SNU-16 위암 세포주에서 p-coumaric acid의 세포성장 억제 효과
Anti-proliferative Properties of p-Coumaric Acid in SNU-16 Gastric Cancer Cells 원문보기

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SNU-16 위암 세포주에서 p-coumaric acid의 세포성장 억제 효과 Anti-proliferative Properties of p-Coumaric Acid in SNU-16 Gastric Cancer Cells 원문보기

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SNU-16 위암 세포주에서 p-coumaric acid의 세포성장 억제 효과
Anti-proliferative Properties of p-Coumaric Acid in SNU-16 Gastric Cancer Cells 원문보기

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