Down-regulation of argininosuccinate synthetase is associated with cisplatin resistance in hepatocellular carcinoma cell lines: implications for PEGylated arginine deiminase combination therapy - PubMed (original) (raw)

Down-regulation of argininosuccinate synthetase is associated with cisplatin resistance in hepatocellular carcinoma cell lines: implications for PEGylated arginine deiminase combination therapy

Jennifer A McAlpine et al. BMC Cancer. 2014.

Abstract

Background: Many advanced human tumors, including hepatocellular carcinomas (HCC) are auxotrophic for arginine due to down-regulation of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in arginine synthesis. The arginine-lowering agent PEGylated arginine deiminase (ADI-PEG 20) has shown efficacy as a monotherapy in clinical trials for treating arginine-auxotrophic tumors and is currently being evaluated in combination with cisplatin in other cancer types. Epigenetic silencing via methylation of the ASS1 promoter has been previously demonstrated in other cancer types, and a reciprocal relationship between ASS1 expression and cisplatin resistance has also been observed in ovarian cancer. However, the mechanism of ASS1 down-regulation, as well as the correlation with cisplatin resistance has not been explored in HCC. The present study investigates ADI-PEG 20 and cisplatin sensitivities in relation to ASS1 expression in HCC. In addition, we show how this biomarker is regulated by cisplatin alone and in combination with ADI-PEG 20.

Methods: ASS1 protein expression in both untreated and drug treated human HCC cell lines was assessed by western blot. The correlation between ASS1 protein levels, ADI-PEG 20 sensitivity and cisplatin resistance in these cell lines was established using a luminescence-based cell viability assay. Epigenetic regulation of ASS1 was analyzed by bisulfite conversion and methylation-specific PCR.

Results: A good correlation between absence of ASS1 protein expression, ASS1 promoter methylation, sensitivity to ADI-PEG 20 and resistance to cisplatin in HCC cell lines was observed. In addition, cisplatin treatment down-regulated ASS1 protein expression in select HCC cell lines. While, at clinically relevant concentrations, the combination of ADI-PEG 20 and cisplatin restored ASS1 protein levels in most of the cell lines studied.

Conclusion: ASS1 silencing in HCC cell lines is associated with simultaneous cisplatin resistance and ADI-PEG 20 sensitivity which suggests a promising combination therapeutic strategy for the management of HCC.

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Figures

Figure 1

Figure 1

Effect of ASS1 expression on the sensitivity of HCC cell lines to ADI-PEG 20 and cisplatin treatment. (A) ASS1 protein levels from cell lysates were determined by western blot. GAPDH was run as a loading control. The signal intensities of the bands were quantified and normalized by taking the level of HepG2 cells as 1. (B and C) Sensitivity of three representative HCC cell lines to ADI-PEG 20 and cisplatin. Cells were cultured in medium containing various concentrations of (B) ADI-PEG 20 and (C) cisplatin. Duplicate samples were assessed for cell viability after 72 h using the Promega luminescence assay. Percent cell viability from either ADI-PEG 20 or cisplatin-treated cells was calculated relative to the viability in corresponding matched DMSO-treated cells, which was designated as 100%. The data are representative of three or more independent experiments. Error bars represent S.D.

Figure 2

Figure 2

Methylation status of the ASS1 promoter in HCC cancer cell lines. DNA bisulfite conversion was performed directly from the cells and MSP was subsequently carried out to determine the methylation status of the ASS1 promoter. UM denotes unmethylated and M denotes methylated. Unmethylated and methylated controls (UC and MC) were included to assess bisulfite conversion efficiency. Expected band sizes are as follows: ASS1: 188 bp; control DNA: 274 bp. Data is representative of 3-4 independent experiments. (A) Control: ovarian cell lines A2780 and A2780CR. (B) ASS1-negative/low cells: SNU398, Sk-Hep1 and Tong. (C) ASS1-positive cells: HCC36, SNU182, Malhavu and HepG2.

Figure 3

Figure 3

Cisplatin treatment down-regulates ASS1 protein in HCC cell lines. Cells were treated with indicated cisplatin concentrations in identical rows of a 96-well microplate. After 72 h, triplicate samples were assessed for cell viability by reading the luminescence and lysates were made out of the remaining identical rows. Luminescence values were used to load equal amounts of protein and ASS1 expression was assessed by western blot. ASS1 levels were normalized to GAPDH at each cisplatin concentration and expressed relative to zero drug (100%). The data are representative of three to four independent experiments. Error bars represent S.D. An unpaired _t_-test was conducted to determine the significance of the change in ASS1 protein levels after each cisplatin concentration treatment as compared to the untreated, or zero drug sample (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001). (A) HepG2 cells. (B) HCC36 cells. (C) SNU182 cells.

Figure 4

Figure 4

ASS1 expression during cisplatin and ADI-PEG 20 combination treatment in HCC cells. Cells were simultaneously treated with a cisplatin concentration of either 5 μM or 7.5 μM and increasing concentrations of ADI-PEG 20: 0.5, 1, 2, 4 and 8 nM in the same well. After 72 h, triplicate samples were assessed for cell viability by reading the luminescence and lysates were made out of the remaining identical rows. Luminescence values were used to load equal amounts of protein and ASS1 levels were assessed by western blot. ASS1 levels were normalized to GAPDH at each treatment condition and expressed relative to zero drug (100%). The data are representative of two to three independent experiments. Error bars represent S.D. An unpaired _t_-test was conducted to determine the significance of the change in ASS1 protein levels after each cisplatin and ADI-PEG 20 combination treatment as compared to the untreated, or zero drug sample (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001). (A) HepG2 cells. (B) HCC36 cells. (C) SNU182 cells.

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Pre-publication history
    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/621/prepub

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