Cystatin SN inhibits auranofin-induced cell death by autophagic induction and ROS regulation via glutathione reductase activity in colorectal cancer

Abstract

Cystatin SN (CST1) is a specific inhibitor belonging to the cystatin superfamily that controls the proteolytic activities of cysteine proteases such as cathepsins. Our previous study showed that high CST1 expression enhances tumor metastasis and invasiveness in colorectal cancer. Recently, auranofin (AF), a gold(I)-containing thioredoxin reductase 1 (TrxR1) inhibitor, has been used clinically to treat rheumatoid arthritis. AF is a proteasome-associated deubiquitinase inhibitor and can act as an anti-tumor agent. In this study, we investigated whether CST1 expression induces autophagy and tumor cell survival. We also investigated the therapeutic effects of AF as an anti-tumor agent in colorectal cancer (CRC) cells. We found that CRC cells expressing high levels of CST1 undergo increased autophagy and exhibit chemotherapeutic resistance to AF-induced cell death, while those expressing low levels of CST1 are sensitive to AF. We also observed that knockdown of CST1 in high-CST1 CRC cells using CST1-specific small interfering RNAs attenuated autophagic activation and restored AF-induced cell mortality. Conversely, the overexpression of CST1 increased autophagy and viability in cells expressing low levels of CST1. Interestingly, high expression of CST1 attenuates AF-induced cell death by inhibiting intracellular reactive oxygen species (ROS) generation, as demonstrated by the fact that the blockage of ROS production reversed AF-induced cell death in CRC cells. In addition, upregulation of CST1 expression increased cellular glutathione reductase (GR) activity, reducing the cellular redox state and inducing autophagy in AF-treated CRC cells. These results suggest that high CST1 expression may be involved in autophagic induction and protects from AF-induced cell death by inhibition of ROS generation through the regulation of GR activity.

Figure 1

Upregulated expression of CST1 mRNA and protein in colon cancer tissues and effect of auranofin (AF) treatment on cell viability. (a) qPCR analysis of CST1 expression in 21 paired non-tumor colon (Normal) and colorectal tumor (Tumor) samples. (b) Immunohistochemistry assay with NC (negative control), anti-CST1 antibody and relative fold-change analysis were performed using tissue microarray chips consisting of 9 normal colons, 2 stage I, 11 stage II, 15 stage III, and 12 stage IV colorectal adenocarcinomas. (c and d) the basal level of CST1 expression in various colon cancer cell lines by qPCR (c) and western blot analysis, including relative band intensity (d). (e) WST-1 assay in colon cancer cell lines following treatment with various doses of AF for 24 h. All data shown are the mean±S.D. of three independent experiments. *P<0.05, ^#P<0.01 versus control

Figure 2

High-CST1 colon cancer cell lines are resistant to AF-induced cell death. (a) Cell viability assay in LoVo, RKO, HT-29, and SW480 cells following treatment with 2.5 μM AF for various lengths of time. (b) Percentage of apoptotic cells as assessed by DAPI staining after treatment with 2.5 μM AF for 24 h. (c) AF-induced cell death as measured by Annexin V-PI staining. Each cell was treated with 2.5 μM AF for 16 h. (d) Cell death is dependent on CST1 expression, as analyzed by western blot using the apoptosis markers caspase-3, cleaved caspase-3, and PARP. All data shown are the mean±S.D. of three independent experiments. *P<0.05, ^#P<0.01 versus control

Figure 3

Autophagy inhibits AF-induced cell death. (a) Western blot of autophagic marker expression in LoVo, RKO, SW480, and HT-29 cells after treatment with 2.5 μM AF for various lengths of time. (b) LoVo, RKO, HT-29, and SW480 cells were transfected with GFP-LC3B. Accumulation of GFP-LC3B punctae was detected after treatment with 2.5 μM AF for 16 h. Graph shows the number of cells with GFP-LC3B punctae. (c) Confirmation of autophagy induction in LoVo, RKO, HT-29, and SW480 cells by autophagy flux experiment using western blot. Cells were treated with autophagy inhibitors 3-MA (5 mM), CQ (20 μM), and 2.5 μM AF for 24 h. (d) WST-1 cell viability assay showing effect of autophagy inhibitors on AF-induced cell death. All data shown are the mean±S.D. of three independent experiments. *P<0.05, ^#P<0.01 versus control

Figure 4

CST1 protects against AF-induced cell death through regulation of autophagy. (a) CST1 mRNA expression and WST-1 assay of HT-29 and SW480 shCST1 knockdown cell lines following treatment with 2.5 μM AF for 24 h. (b) Western blot of apoptosis markers in shCST1 knockdown HT-29 and SW480 cells. (c) Western blot of autophagy markers was in shCST1 knockdown HT-29 and SW480 cells. (d) CST1 mRNA expression and WST-1 assay of LoVo and RKO CST1-overexpressing lines following treatment with 2.5 μM AF for 24 h. (e) Western blot of apoptosis markers in CST1-overexpressing LoVo and RKO cells. (f) Western blot of autophagy markers in CST1-overexpressing LoVo and RKO cells. All data shown are the mean±S.D. of three independent experiments. *P<0.05, ^#P<0.01 versus control

Figure 5

CST1 inhibits AF-induced cell death by regulating intracellular ROS. (a) CM-H₂DCFDA was used for detection of AF-induced ROS by flow cytometry after 2 h of incubation with 2.5 μM AF. (b) Effect of ROS scavengers NAC (1 mM) and M-TEMPO (100 μM) on AF-induced cell death as measured by WST-1 assay. ROS scavengers were co-treated with 2.5 μM AF for 24 h. (c) Western blot analysis to detect AF-induced cell death after co-incubation of AF and ROS scavengers for 24 h. (d and e) Intracellular ROS levels of CST1-overexpressing LoVo and RKO cells (d) and shCST1 knockdown HT-29 and SW480 cells (e) as measured by flow cytometry after 2 h of incubation with AF. (f) TrxR1 expression and activity in LoVo, RKO, HT-29, and SW480 cells after 12 h of incubation with AF as measured by western blot and TrxR1 assay kit. All data shown are the mean±S.D. of three independent experiments. *P<0.05, ^#P<0.01 versus control

Figure 6

CST1 regulates cellular GSH to protect from AF-induced oxidative stress. (a) Western blot of GR levels in LoVo, RKO, HT-29, and SW480 cells following AF treatment for various lengths of time. (b) Cellular GSH levels in LoVo and HT-29 cells with or without AF treatment. (c) GSH levels in CST1-overexpressing LoVo and RKO cells after 12 h of incubation with AF. (d) GSH levels in shCST1 knockdown HT-29 and SW480 cells after 12 h of incubation with AF. (e) Western blot of GR and TrxR1 expression in CST1-overexpressing LoVo and RKO cells and shCST1 knockdown HT-29 and SW480 cells. (f) Western blot of catalase (Cat), SOD1, and SOD2 expression in CST1-overexpressing LoVo and RKO cells and shCST1 knockdown HT-29 and SW480 cells following AF treatment for 24 h. (g) Western blot of beclin-1 and LC3B expression in RKO and HT-29 cell lines following treatment with AF and ROS scavengers NAC (1 mM), M-TEMPO (100 μM), or DPI (2.5 μM)