Research article
Apigenin up-regulates transgelin and inhibits invasion and migration of colorectal cancer through decreased phosphorylation of AKT

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Abstract

Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. Apigenin is a flavonoid that possesses various clinically relevant properties such as anti-tumour, anti-platelet and anti-inflammatory activities. Our results showed that apigenin has anti-proliferation, anti-invasion and anti-migration effects in three kinds of colorectal adenocarcinoma cell lines, namely SW480, DLD-1 and LS174T. Proteomic analysis with SW480 indicated that apigenin up-regulated the expression of transgelin (TAGLN) in mitochondria to exert its anti-tumour growth and anti-metastasis effects. Real-time quantitative polymerase chain reaction (RQ-PCR) and western blot confirm the up-regulation in all the three colorectal adenocarcinoma cells. An inverse correlation was observed between TAGLN expression and CRC metastasis in tissue microarray staining. TAGLN siRNA increased the viability of SW480. Apigenin decreased the expression of MMP-9 in a dose-dependent manner. Transfection of three truncated forms of TAGLN and wild type has identified TAGLN as a repressor of MMP-9 expression. A synergetic effect was observed in overexpression of TAGLN wild type and apigenin treatment which manifested as lowered phosphorylation of AKT Ser473 and ATK Thr308. In an orthotopic CRC model, apigenin inhibited tumour growth and metastasis to liver and lung. In conclusion, our research provided direct evidence that apigenin inhibited tumour growth and metastasis both in vitro and in vivo. Apigenin up-regulated TAGLN and hence down-regulated MMP-9 expression through decreasing phosphorylation of Akt at Ser473 and in particular Thr308 to prevent cell proliferation and migration.

Introduction

Colorectal cancer (CRC) is a major cause of morbidity and mortality in the United States (US). The estimated new cases of CRC in 2012 are 143 460, and the estimated deaths are 51 690, both accounting for 9% among the total ten leading cancers of US. It affects men and women equally and is the third leading cause of death [1]. The incidence and mortality of CRC is rising rapidly in Asia [2], and it is one of the three cancers with the greatest increasing incidence in China between 1991 and 2005 [3]. The incidence and mortality rates of CRC increased by 38.56% and 15.30% from 1988 to 2002 in 10 cities and counties in China [4]. Thus, measurements for the prevention and treatment of CRC in the whole population are of long run significance in future.

Although about 50% of the tumours can be curatively treated by surgery alone, metastatic growth is one of the major factors that compromised the outcome of cancer treatment, which is particularly true for the high mortality rate of CRC [5], [6]. Approximately half of CRC patients will develop local recurrence or distant metastasis during the course of the illness [7]. Although lymph node metastasis represents the primary indicator for systemic disease spread, distant metastases formation constitutes the most important prognostic factor in colorectal cancer and accounts for the majority of deaths [5]. Therefore the prevention of CRC metastasis, especially distant metastasis, becomes more important.

Apigenin is a flavonoid that widely existed in fruits and vegetables. It structurally belongs to the flavone class and is chemically known as 4′,5,7,-trihydroxyflavone. It has been found to possess various clinically relevant properties such as anti-tumour, anti-platelet and anti-inflammatory activities [8], [9]. It has also been reported to suppress tumour metastasis in an orthotopic ovarian tumour model [10] and to inhibit hepatocyte growth factor promoted lung metastasis [11]. Apigenin significantly decreased the incidence of peritoneal metastasis of azoxymethane and bombesin induced intestinal adenocarcinomas [12]. Our previous work showed that apigenin inhibited the growth of colorectal cancer xenografts [13]. However, whether apigenin could decrease the metastasis in an orthotopic CRC model is unknown.

The process of dissemination of CRC to discontiguous vicinity or distant secondary sites comprises several necessary steps: invasion, adhesion, migration and proliferation at the secondary site [14]. In the current study, the effects of apigenin on cell proliferation, migration and invasion were addressed. As many studies focused on phosphatidylinositol 3-kinases /Akt pathway [10], [11], a two-dimensional electrophoresis-based proteomic analysis was adopted to screen new target proteins of apigenin. The relationship between phosphatidylinositol 3-kinases/Akt and the new target proteins would be discussed further.

Section snippets

Chemicals

Apigenin, dimethyl sulfoxide (DMSO), propidium iodide (PI) and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640, FBS and antibiotics were purchased from Invitrogen (Gibco, Grand Island, NY, USA).

Animals and orthotopic CRC tumour model

All procedures involving laboratory animal use were in accordance with the guidelines of the Instituted Animal Care and Use Committee of Southern Medical University. All protocols were submitted and validated by Animal Care Ethics Committee of

Apigenin inhibits cell proliferation and invasion

Apigenin significantly decreased cell viability in a time and dose-dependent manner ranging from 20 to 120 μM. Cell viabilities decreased to 52.29%, 50.62% and 45.10% in SW480, DLD-1 and LS174T cells when treated with 40 μM apigenin for 48 h compared to the control (Fig. 1A). Therefore, apigenin 40 μM was chosen in the further study. A rat intestinal epithelial cell line (IEC-6) was also included in our study. Further analysis showed that the viabilities of SW480, DLD-1 and LS174T were higher

Discussion

Apigenin is a flavonoid structurally belonging to the flavone class and is found in traditional Chinese herbs such as Chrysanthemum morifolium Ramat [18] and tree peony flowers [19], etc. It has been reported that apigenin inhibited colon carcinoma cell growth via induction of G2/M arrest [20], [21]. Three kinds of colorectal adenocarcinoma cell lines including SW480-eGFP were utilized in this study for the convenience of fluorescence imaging of cell invasion and tumour metastasis. The

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    This work was supported by the National Science Foundation of China (No. 81273621), Planned Science Technology Project of Guangdong Province, China (No. 2011B031700001) and Baiyun District Science and Technology Program (No. 2012-KZ-81).

    1

    These authors contributed to this work equally.

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