Thapsigargin induces apoptosis in adrenocortical carcinoma by activating endoplasmic reticulum stress and the JNK signaling pathway: an in vitro and in vivo study

Abstract

Objective: Thapsigargin (TG) is a natural product that exists in most parts of the plant Thapsia garganica L. and possesses potential anticancer activities against variety tumor cell lines. TG induces endoplasmic reticulum (ER) stress and apoptosis by inhibiting cancer growth. However, the antineoplastic effect of TG in human adrenocortical carcinoma (ACC) cells is still unknown.

Methods: In this study, two human ACC cell lines including SW-13 and NCI-H295R were employed to explore the potential role of TG in ACC. A mouse xenograft model of SW-13 cells was established to verify the role of TG in vivo. The cell viability was tested using Cell Counting Kit-8 and Transwell assays. Flow cytometry and Hoechst 33,258 staining were employed to analyze cell apoptosis. RT-qPCR and Western blot (WB) were performed to explore the underlying mechanism of TG-induced apoptosis in ACC cells.

Results: The results indicated that TG dose-dependently inhibited proliferation, migration and invasion in human ACC cells. TG significantly increased the mitochondrial rate of apoptosis and ER stress activity in ACC cells and suppressed ACC xenograft growth in vivo. In addition, the expression of Jun N-terminal kinase (JNK) signaling-related genes and proteins was upregulated by the treatment with TG.

Conclusion: Our findings suggest that TG inhibits the viability of ACC cells by inducing apoptosis through the activation of JNK signaling. Thus, TG is expected to be a potential candidate for the treatment of ACC.

Keywords: Jun N-terminal kinase signaling; adrenocortical carcinoma; apoptosis; endoplasmic reticulum ER stress; thapsigargin.

Figure 1

TG inhibited viability of ACC cell lines. (A and B) Crystal violet staining of SW-13 and NCI-H295R (×10, scale bar =400 μm). (C and D) SW-13 and NCI-H295R cells were incubated with TG at different concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 µM) for 0, 6, 12, 24, 36 and 48 h. Each experiment was performed independently in triplicate, and the data is represented as means ± SD.

Figure 2

TG suppresses the migration and invasion ability of ACC cells. The migration (A) and invasion (B) of ACC cells after treatment with or without TG (×10, scale bar =400 μm). (C and D) The number of migrated and invaded cells were quantified, and the data is represented as means ± SD (n=6). *p<0.05 vs the control group.

Figure 3

TG induced apoptosis of ACC cells. SW-13 (A) and NCI-H295R (B) cells were treated with TG at different concentrations (0, 0.5, 1, 2, 4, 8, 10 and 16 µM) for 48 h. Flow cytometry was used to analyze TG-induced apoptosis of ACC cells. (C) Early and late apoptotic events in SW-13 cells and NCI-H295R cells in the present or absent of different doses of TG were detected by flow cytometry. The top left quadrant indicates non-apoptotic cells; the top right quadrant represents late apoptosis events; the bottom right quadrant represents early apoptosis cells; and the bottom left quadrant represents living cells. Each experiment was performed in triplicate, and the data represent the means ± SD. *p<0.05 vs the control group.

Figure 4

TG induced apoptosis of ACC cells by activating ERS and JNK signaling pathways. Apoptosis of cells that induced by TG was stained with Hoechst 33,258 (A and B). The gene expression levels of JNK, ATF6, PERK, LC3B, HSAP and Bcl-2 were detected by qRT-PCR (C and D). The data is represented asmeans ± SD (n=3). *p<0.05 vs the control group.

Figure 5

Expression levels of the JNK signaling-related proteins, JNK and p-JNK, after TG treatment in SW-13 (A and B) and NCI H295R (C and D) cells. The data is represented as means ± SD (n=3). *p<0.05 vs the control group (TG 0 µM group).

Figure 6

TG induces cell apoptosis by activating JNK signaling. SW-13 was treated in the present or absent of TG or JNK inhibitor SP600125 (µM) for 48 h. The expression of JNK and p-JNK was detected by WB (A - C). Flow cytometry was used to analyze the apoptosis rate in ACC (D and E). Each experiment was performed in triplicate and the data is represented as means ± SD. *p<0.05 vs the control group.

Figure 7

TG inhibited ACC tumor growth in nude mice. Each mouse was injected subcutaneously with SW-13 cells (2×10⁶ in 200 µL of medium) under their left shoulder. When the subcutaneous tumors were approximately 0.2×0.2 cm in size, the mice were randomized divided into two groups and received intraperitoneal injections of vehicle alone (0.5% methylcellulose) or TG (1.0 mg/kg) three times a week. Body weights and tumor volumes were recorded every three days. After the experiment, the mice were anesthetized, and the tumor tissues were recieved from the mice and weighed. Tumor volumes (A), original tumors (B and C), body weights (D), tumor weights (E), and summarized data (F) are shown. The data is represented the as means ± SD (n=5). *P<0.05 vs corresponding control.

Figure 8

Expression of the JNK signaling-related proteins JNK, p-JNK, ERK, p-ERK, MAPK, p-MAPK, PERK, p-PERK, IRE1 and GRP78 in tumers in nude mice that treated with or without TG were analyzed by WB (A and B). The data is represented as means ± SD (n=5). *p<0.05 vs the control group.