Cancer Letters

Cancer Letters

Volume 334, Issue 1, 28 June 2013, Pages 69-78
Cancer Letters

Original article
Differential effects of grape seed extract against human colorectal cancer cell lines: The intricate role of death receptors and mitochondria

https://doi.org/10.1016/j.canlet.2012.12.015Get rights and content

Abstract

Failure of anti-cancer therapy in colorectal cancer (CRC) cells involves resistance to death mechanisms. We investigated grape seed extract (GSE) ability to target CRC cells and delineated the mechanisms involved in GSE-induced CRC cell death. GSE selectively induced apoptotic death in human CRC cells; efficacy increased as the metastatic potential of the cancer cells increased. Oxidative stress, loss of mitochondrial membrane potential, modulation of pro- and anti-apoptotic proteins, and involvement of both caspase-dependent/independent apoptotic pathways contributed to GSE-induced CRC cell death. GSE intervention may serve as a multi-targeted CRC therapeutic capable of inducing selective cancer cell death.

Introduction

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States [1]. The incidence of CRC is also increasing world-wide, with an over-all lifetime risk of 1 in 19, due to adoption of western lifestyle habits; high fat diet, alcohol consumption, and sedentary lifestyle [2]. Compliance with screening recommendations is low; as such 60% of new CRC cases are diagnosed at a stage where they have already progressed to advanced disease [2]. Conventional therapies for CRC, e.g. chemotherapy, radiation, and surgery, result in severe toxicity and associated therapy resistance [3], [4]. Such limitations have resulted in a shift towards additional strategies involving the use of natural dietary/non-dietary products, which target multiple pathways in cancer cells and are associated with limited or no toxicity [5], [6].

In this regard, studies have indicated that high consumption of fruits and vegetables or their bio-active components can decrease CRC incidence by ∼40% [6], [7]. Grape seed extract (GSE) is one such widely consumed dietary supplement (with 95% standardized procyanidins) that has been shown to possess anti-cancer, anti-inflammatory, anti-oxidant, anti-bacterial, anti-viral effects [5], [8], [9], [10]. Pre-clinical studies have established strong GSE efficacy against prostate, colon, lung, breast, skin, and other cancers [5], [11], [12], [13], [14], [15], [16], [17], [18], [19]. Clinical studies with GSE have indicated that its active components are bioavailable and well tolerated [20], [21], [22]. However, till date, the specificity of GSE to target CRC cells and the associated-mechanisms involved in GSE-induced CRC cell death are not well studied. Therefore, in the present study, we investigated the molecular mechanisms involved in GSE-induced CRC cell death at various stages of the malignancy. Though it has been previously shown by a number of research groups, including ours, that GSE has the ability to induce CRC cell death in both in vitro and in vivo CRC models [5], [13], [14], [15], [18], [19], [23], [24], [25], one limitation of all the in vitro studies investigating GSE efficacy in CRC was that the in vitro experimental design failed to take into consideration the different stages of this deadly malignancy as well as normal colon epithelial cells. Our present study is unique in that aspect, as we chose multiple CRC cell lines, which differ in their metastatic potential; this strategy helped in elucidating the differential effects of GSE against human CRC cell lines.

Section snippets

Reagents

Standardized preparation of GSE was a gift from Kikkoman Corp. (Nado City, Japan). The composition of the GSE preparation is listed as: 89.3% procyanidins, 6.6% monomeric flavonols, 2.24% moisture content, 1.06% of protein, and 0.8% of ash. Dimethyl Sulfoxide (DMSO) and N-acetyl cysteine (NAC) were from Sigma Chemical Co. (St. Louis, MO); Trypan blue 0.4% was from Invitrogen (Carlsblad, CA). Primary antibodies used were anti-cleaved caspase-9, anti-cleaved caspase-8, anti-cleaved caspase-3,

GSE treatment causes growth inhibition and induces death in human CRC cells

First, we evaluated the efficacy of GSE against a panel of human CRC cell lines, based on phenotypic and genetic variations, so as to cover different clinical stages of CRC, viz., SW480 (stage II CRC with mutant p53), SW620 (stage III CRC with mutant p53), HCT116 (stage IV CRC with wt p53) cells. Trypan blue exclusion assays were performed to differentiate between live and dead cells. Results indicated that GSE at 25, 50 and 100 μg/mL doses resulted in a dose-dependent growth inhibition of SW480

Discussion

Mechanisms that contribute to multi-drug tumor cell resistance are known to involve mutations that result in uncontrolled cellular proliferation and apoptosis resistance; therefore, strategies that focus on inhibiting these processes, at any stage of malignancy, would be of clinical significance [39]. The process of apoptosis is highly complex involving a cascade of molecular events; the two main pathways involved in this process are: extrinsic or death receptor pathway and the intrinsic or

Acknowledgements

This work was supported by NIH R01 AT003623 and NCI R01 CA112304.

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