Oncology/endocrine
Modification of Gene Products Involved in Resistance to Apoptosis in Metastatic Colon Cancer Cells: Roles of Fas, Apaf-1, NFκB, IAPs, Smac/DIABLO, and AIF

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Background

Colon cancer becomes resistant to apoptosis as it acquires metastatic potential. SW480 and SW620 colon cancer cells were established from the same patient at different stages of tumor progression. The stage III colorectal cancer cell line (SW620) is more resistant to apoptosis. In the present report, we investigated the apoptotic gene products that might account for colon cancer evasion of immune attack and chemoradioresistance-induced apoptosis.

Methods

SW480 and SW620 cells were used for this experiment. Type 1 apoptosis was induced by CH-11. Type 2 apoptosis was induced by cisplatin and ionizing radiation. Apoptosis was determined by caspase-3 activity and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. Gene products Fas, TRAIL, c-FLIP, Bid, BAX, Bcl-2, Bcl-xL, Apaf-1, nuclear factor-kappa B, Smac/DIABLO, apoptosis inducing factor, and the inhibitors of apoptosis were investigated by immunocytochemistry and Western blot analyses.

Results

SW620 cell lines were more resistant to both Type 1 and Type 2 apoptosis induced by CH-11, cisplatin, and ionizing radiation, respectively. Examination of the extrinsic pathway demonstrated Fas receptor to be down-regulated in SW620. Apaf-1 was decreased in SW620 cells; while other members of the mitochondrial pathway including Bax, Bid, Bcl-xL, and Bcl-2 demonstrated minimal alterations of protein levels in both cell lines. Survivin and XIAP protein levels were increased in SW620 cells, which correlated with nuclear expression of nuclear factor-kappa B in SW620 cells but not SW480. Mitochondrial-released factors including Smac/DIABLO and apoptosis inducing factor were increased in SW480 cells.

Conclusions

SW620 cells have acquired genetic defects both in the intrinsic and extrinsic pathways of apoptosis, which may explain in part the ability of colon cancer cells to escape the immune system and to become chemoradioresistant. These genes may be potential targets for chemoradiosensitization in advanced colorectal cancer.

Introduction

Since the vast majority of cytotoxic modalities exert their antitumor effects by induction of apoptosis, programmed cell death has rapidly emerged as a potential target for cancer treatment at various stages of tumor progression [1]. Immunoregulation and chemoradiosensitization are potential targets where insight in apoptotic mechanisms may lead to improvement of chemoradiotherapeutic modalities. In colon cancer, apoptosis decreases as the colonocyte progresses in the adenoma to carcinoma sequence of colon carcinogenesis and metastasis [2].

The extrinsic pathway of apoptosis is important in the elimination of unwanted cells by the immune system. Analysis of how defects in the extrinsic pathway of apoptosis permit colon cancer cells to escape the immune system may provide treatment options whereby the body’s immune system could again recognize and eliminate unwanted cells [3, 4].

The intrinsic pathway of apoptosis plays a pivotal role in the elimination of damaged cells that have undergone intracellular stress [2]. Defects in this pathway of apoptosis, therefore, may lead to chemoradioresistance. The study of apoptosis in colon cancer has been dependent on ex vivo systems that demonstrate apoptosis at a single point in time. In vitro systems have investigated only a few apoptotic mediators at a time in different colon cancer cell lines [2, 5, 6, 7]. It is difficult to determine how changes of one mediator of apoptosis will affect the fate of other proapoptotic or antiapoptotic molecules in the entire process of programmed cell death when more than one cell line is examined. Examination of a single system where defects in the various pathways of apoptosis are established would permit the study of how chemotherapeutic interventions alter the entire cascade of apoptosis.

An in vitro model of colon carcinogenesis has been validated to represent the progression of colon carcinogenesis from a primary tumor to metastatic disease [8]. The SW480 cell line was derived from a primary, Dukes’ stage B (colon adenocarcinoma) tumor from a 50-y-old Caucasian male. The SW620 cell line was cultured from a lymph node metastasis in the same patient at a later time [9, 10]. Because these cells are derived from the same patient, the gene product modifications that have occurred might be the result of changes the cells acquire as they progress into a metastatic phenotype. The aims of the present study are: (1) to demonstrate the relative sensitivity of Fas-, cisplatin-, and ionizing radiation-induced apoptosis between SW480 and SW620 cells; and (2) to investigate the specific gene modifications that might account for the observed differences in the rate of apoptosis between these two cell lines.

Section snippets

Cell Culture and Reagents

Scott and White (SW)480 and (SW)620 human colon cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA): SW480 (colon adenocarcinoma CCL-228 passage number: 96) and SW620 (colon adenocarcinoma, lymph node metastasis CCL-227 passage number: 83). These cell lines were maintained at 37°C and 5% CO2, in DMEM (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, 1% (vol/vol) L-glutamine. For every experimental condition, the cells

CH-11-Mediated Apoptosis

To confirm previous finding by Hewitt et al. [8], we incubated SW480 and SW620 cells with 40 ng/mL and 100 ng/mL CH-11. SW620 cells demonstrated poor induction to apoptosis (<5%) with CH-11. In contrast, there was a >20% apoptosis as measured by caspase-3 activity in the SW480 cells (Fig. 1).

Cisplatin-Mediated Apoptosis

We have previously demonstrated that SW620 cells are consistently more resistant to cisplatin-mediated apoptosis in a dose dependent manner by propidium iodide (PI) staining determination of DNA content [14

Discussion

The pivotal role of apoptosis in colon cancer has been previously reviewed [2]. Defects in the process of apoptosis translate into inability of the immune system to recognize unwanted cells as well as resistance of malignant cells to chemoradiotherapeutic modalities. In colon cancer, the relative roles of the mediators of apoptosis and the timing of their loss (adenoma versus metastatic cancer) are poorly defined. In the present study, we interrogated multiple mediators in machinery of

Acknowledgments

This work was supported by a VISN 17 New Investigator Award (SH) and the Hudson-Penn Surgery Funds. YMA-H holds a Ph.D. grant scholarship from Consejo Nacional de Ciencia y Tecnología, México (CONACyT).

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