SESN2/sestrin 2 induction-mediated autophagy and inhibitory effect of isorhapontigenin (ISO) on human bladder cancers

Abstract

Isorhapontigenin (ISO) is a new derivative of stilbene isolated from the Chinese herb Gnetum cleistostachyum. Our recent studies have revealed that ISO treatment at doses ranging from 20 to 80 μM triggers apoptosis in multiple human cancer cell lines. In the present study, we evaluated the potential effect of ISO on autophagy induction. We found that ISO treatment at sublethal doses induced autophagy effectively in human bladder cancer cells, which contributed to the inhibition of anchorage-independent growth of cancer cells. In addition, our studies revealed that ISO-mediated autophagy induction occurred in a SESN2 (sestrin 2)-dependent and BECN1 (Beclin 1, autophagy related)-independent manner. Furthermore, we identified that ISO treatment induced SESN2 expression via a MAPK8/JNK1 (mitogen-activated protein kinase 8)/JUN-dependent mechanism, in which ISO triggered MAPK8-dependent JUN activation and facilitated the binding of JUN to a consensus AP-1 binding site in the SESN2 promoter region, thereby led to a significant transcriptional induction of SESN2. Importantly, we found that SESN2 expression was dramatically downregulated or even lost in human bladder cancer tissues as compared to their paired adjacent normal tissues. Collectively, our results demonstrate that ISO treatment induces autophagy and inhibits bladder cancer growth through MAPK8-JUN-dependent transcriptional induction of SESN2, which provides a novel mechanistic insight into understanding the inhibitory effect of ISO on bladder cancers and suggests that ISO might act as a promising preventive and/or therapeutic drug against human bladder cancer.

Keywords: MAPK8; autophagy; bladder cancer; isorhapontigenin; sestrin 2.

Figure 1.

ISO induced autophagy in human cancer cells. (A) Human bladder cancer UMUC3 and T24T cells and human cervical cancer HeLa cells, were treated with ISO at the indicated doses for 24 h. The cells were extracted and cell lysates were subjected to western blotting assay by using the indicated antibodies. (B) The GFP-LC3 construct was stably transfected into UMUC3 cells, and the transfectants were treated with various doses of ISO for 24 h. LC3 puncta formation was observed and images were captured using fluorescence microscopy. (C) Percentage of cells with GFP-LC3 puncta (left) and number of puncta per positive cell (right) were calculated and presented as described in Materials and Methods. The symbol (*) indicates a significant increase from the vehicle control (p < 0.05). (D) UMUC3 cells were treated with ISO (10 μM), together with or without 5 nM BAF for 24 h and the protein levels of LC3 were assessed by western blotting. (E) UMUC3 cells were transfected with the GFP-LC3 construct and the transfectants were treated with ISO (10 μM), with or without 5 nM BAF for 24 h. The representative images of GFP-LC3 puncta were captured using a confocal fluorescence microscope. (F) Number of puncta per GFP-LC3-positive cell was calculated and presented as described in Materials and Methods. The symbol (*) indicates a significant increase from the ISO treatment group (p < 0.05). (G) Representative images of colonies of UMUC3 treated with 10 μM ISO, 1 nM BAF or both in soft-agar assay were captured as described in Materials and Methods. (H) Colonies shown in (G) were counted under a microscope with more than 32 cells of each colony. The results are presented as colonies per 10⁴ cells, and the bars show mean ± SD from 3 independent experiments. The symbol (*) indicates a significant increase from the ISO treatment group (p < 0.05).

Figure 2.

SESN2 was required for autophagic induction and anchorage-independent growth inhibition by ISO in human bladder cancer UMUC3 cells. (A) UMUC3 cells were treated with various concentrations of ISO for 24 h. The cells were extracted and cell lysates were subjected to western blotting to detect the expression of BECN1, SESN2 and LC3. (B and D) shRNA BECN1 (B) and shRNA SESN2 (D) were stably transfected into UMUC3 cells, and the stable transfectants were identified. (C and E) The indicated stable transfectants were subjected to ISO treatment for 24 h for determination of LC3 in ISO-induced autophagy in UMUC3 cells. (F) Representative images of colonies of UMUC3 (shSESN2) and UMUC3 (Nonsense) cells in soft-agar assay in the presence or absence of various concentrations of ISO were captured using a microscope. (G) Colonies shown in (F) were counted under a microscope with more than 32 cells of each colony. The results are presented as colonies per 10⁴ cells, and the bars shows mean ± SD from 3 independent experiments. The symbol (*) indicates a significant decrease from vehicle control (p < 0.05).

Figure 3.

JUN activation and its binding to the AP-1 binding consensus in SESN2 promoter-mediated SESN2 transcriptional expression in human bladder cancer cells. (A) UMUC3 cells were treated with various concentrations of ISO for 12 h. RT-PCR was carried out to evaluate the expression of SESN2 mRNA. (B) The SESN2 promoter-driven luciferase reporter was transfected into UMUC3 cells. The stable transfectants were subjected to ISO treatment for 12 h to evaluate SESN2 promoter transcriptional activity. The induction fold was normalized using pRL-TK as an internal control. The results are presented as SESN2 promoter activity relative to vehicle control (relative SESN2 promoter activity). The bars show mean ± SD from 3 independent experiments. The symbol (*) indicates a significant increase from the vehicle control (p < 0.05). (C) The putative transcription factor consensus binding site in the SESN2 proximal promoter region was predicted using bioinformatics analysis. (D) The expression levels of potential transcription factors were determined following ISO treatment for 24 h using western blotting. (E) TAM67 was stably transfected into UMUC3 cells and the stable transfectants were identified by western blotting. (F and G) AP-1 luciferase reporter (F) or SESN2 promoter luciferase reporter (G) was transiently transfected into UMUC3 (TAM67) and UMUC3 (Vector) cells in combination with pRL-TK. The transfectants were treated with 10 μM ISO for 12 or 24 h and luciferase activity was determined and presented as relative AP-1 activity (F) or relative SESN2 promoter activity (G). The symbol (*) indicates a significant increase from the vehicle control (p < 0.05). (H) RT-PCR was carried out to determine the mRNA changes of SESN2 following ISO treatment for 12 h in the indicated cells. (I) ChIP was carried out using anti-JUN antibody to detect the interaction of JUN with the SESN2 promoter following ISO treatment for 12 h.

Figure 4.

JUN activation was crucial for autophagy induction and anchorage-independent growth inhibition by ISO treatment. (A and D) The stable transfectants, UMUC3 (TAM67) vs. UMUC3 (Vector) or UMUC3 (shJUN) vs. UMUC3 (Nonsense) cells, were treated with 10 μM ISO for 24 h. The cells were then extracted and the cell lyses were subjected to western blotting for determination of JUN phosphorylation, SESN2 induction and LC3-II generation as indicated. (B and E) Representative images of anchorage-independent growth of UMUC3 (TAM67) vs. UMUC3 (Vector) or UMUC3 (shJUN) vs. UMUC3 (Nonsense) in the absence or presence of various concentrations of ISO were visualized and captured using a microscope. (C and F) The colony formation was counted under a microscope with more than 32 cells of each colony, and the results presented as colonies/10⁴ cells. The bars indicate mean ± SD from 3 independent experiments. The symbol (*) shows a significant decrease from the vehicle control (p < 0.05).

Figure 5.

MAPK8 is the upstream kinase mediating for JUN activation and SESN2 expression following ISO treatment. (A) UMUC3 cells were treated with various concentrations of ISO for 24 h. The cells were extracted and cell lyses were subjected to western blotting to determine the activation of JUN and MAPK8 as well as generation of LC3-II. (B and C) shRNA MAPK8 was stably transfected into UMUC3 cells (B) and HeLa cells (C). Following treatment with ISO (10 μM) for the indicated time points, proteins were extracted and cell extracts were then subjected to western blotting for evaluation of the changes of JUN, MAPK8, SESN2 and LC3. (D) UMUC3 (shMAPK8) and UMUC3 (Nonsense) transfectants were subjected to ISO treatment and SESN2 mRNA level was detected by RT-PCR. (E) SESN2 promoter-driven luciferase reporter together with pRL-TK was transiently transfected into UMUC3 (shMAPK8) and UMUC3 (Nonsense) cells. The transfectants were treated with ISO (10 µM) for 12 h and then analyzed for luciferase activity assay. The bars show mean ± SD from 3 independent experiments. The symbol (*) indicates a significant difference between the vehicle control and ISO treatment (p < 0.05).

Figure 6.

MAPK8 is required for ISO inhibition of anchorage-independent growth in UMUC3 cells and SESN2 was downregulated in human bladder cancer tissues. (A) Representative images of colonies of UMUC3 (shMAPK8) and UMUC3 (Nonsense) cells in a soft-agar assay in the absence or presence of various concentrations of ISO were captured using a microscope. (B) The colony formation was counted under a microscope with more than 32 cells of each colony, and the results presented as colonies/10⁴ cells. The bars indicate mean ± SD from 3 independent experiments. The symbol (*) shows a significant decrease from the vehicle control (p < 0.05). (C) Clinically freshly collected human bladder cancer samples in comparison with the paired adjacent normal bladder tissue were subjected to western blotting to analyze SESN2 expression. N, normal; T, tumor. (D) The proposed model for ISO induction of SESN2 and autophagy and inhibitory effect on human bladder cancer cells.