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. 2018 Aug;51(4):e12451.
doi: 10.1111/cpr.12451. Epub 2018 Feb 27.

Coroglaucigenin induces senescence and autophagy in colorectal cancer cells

Yong-Hao Huang  1 , Jing Lei  1 , Guo-Hui Yi  1   2 , Feng-Ying Huang  1 , Yue-Nan Li  1 , Cai-Chun Wang  1 , Yan Sun  1 , Hao-Fu Dai  3 , Guang-Hong Tan  1
Affiliations

Coroglaucigenin induces senescence and autophagy in colorectal cancer cells

Yong-Hao Huang et al. Cell Prolif. 2018 Aug.

Abstract

Objectives: Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti-cancer agent. However, the molecular mechanisms involved remain poorly understood.

Materials and methods: Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA-β-gal assay, western blotting and immunofluorescence were performed to determine CGN-induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN-induced senescence and autophagy. The anti-tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models.

Results: We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell-cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN-mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti-tumour effects in vivo.

Conclusions: Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN-mediated anti-cancer therapy.

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Conflict of interest statement

There is no competing interest.

Figures

Figure 1
Figure 1
Coroglaucigenin inhibits cell proliferation independent of apoptosis in colorectal cancer cells. (A) The structure of coroglaucigenin (CGN). (B) Cell viability was measured using MTT assays in HT‐29, SW480 and NCM460 cells treated with the indicated concentrations of CGN for 24 hours. (C) Cells were treated with the indicated concentrations of CGN for 24 hours, and the degree to which CGN inhibited cell proliferation was measured using BrdU labelling. (D and E) Cells were treated with the indicated concentrations of GCN for 24 hours, and the level of apoptosis was determined using an Annexin‐V‐FITC/PI double staining assay. (F) The level of cleavage of PARP was determined using western blotting in HT‐29 and SW480 cells treated as in D. (G) ImageJ densitometric analysis of the cleaved PARP/β‐actin ratios from immunoblots. *P  < 0.05, **P < .01, ***P < .001
Figure 2
Figure 2
Coroglaucigenin induces cell‐cycle arrest and senescence in colorectal cancer cells. (A and B) HT‐29 cells were treated with the indicated concentrations of CGN for 24 hours, and cell‐cycle distribution was determined by flow cytometry. (C and D) Cell proliferation was measured by colony formation assay in HT‐29 and SW480 cells treated with the indicated concentrations of CGN and grown for 7 d. (E and F) HT‐29 cells were treated with the indicated concentrations of CGN for 5 d and then stained for SA‐β‐gal activity 2 d after treatment. *P < .05, **P  < .01
Figure 3
Figure 3
Coroglaucigenin induces protective autophagy in colorectal cancer cells. (A) Immunoblot analysis of LC3 in HT‐29 and SW480 cells treated with the indicated concentrations of CGN for 24 hours. Protein ratios were calculated following ImageJ densitometric analysis. (B) Immunoblot analysis of LC3 in cells treated with the indicated concentrations of CGN for 24 hours in the absence or presence of 3‐methyladenine (3‐MA). (C and D) HT‐29 and SW480 cells were treated with DMSO or 4 μM of CGN for 24 hours, and the formation of endogenous LC3 puncta (arrows) was visualized under a fluorescent microscope. Scale bars, 10 μm. (E) Cell viability was measured by MTT assays in cells treated with DMSO or 4 μM of CGN for 24 hours in the presence and absence of chloroquine (CQ). (F) Cell proliferation was measured using BrdU labelling in cells treated with DMSO or 4 μM of CGN for 24 hours in the presence or absence of CQ. *P  < .05, **P  < .01
Figure 4
Figure 4
Coroglaucigenin induces senescence through the downregulation of CDK4. (A) Immunoblot analysis of CDK4 in HT‐29 and SW480 cells treated with 4 μM of CGN for 24 hours. ImageJ densitometric analysis of the CDK4/β‐actin ratios from immunoblots. (B and C) HT‐29 cells were transfected with siCDK4 or treated with 4 μM of CGN, and then stained for SA‐β‐gal activity 2 d after treatment. (D) CDK4 mRNA expression in cells treated with the indicated concentrations of CGN for 24 hours was evaluated by RT‐PCR. (E) Immunoblot analysis of CDK4 in cells treated with 4 μM of CGN for 24 hours in the presence and absence of MG132. ImageJ densitometric analysis of the CDK4/β actin ratios from immunoblots. (F) Confocal microscopy of HT‐29 cells treated with 4 μM of CGN for 24 hours in the presence and absence of MG132, then immunostained for Hsp90 (red) and CDK4 (green). Arrows indicate colocalization of green (CDK4) and red (Hsp90) signals. Scale bars, 10 μm. (G) Hsp90 interaction with CDK4 was determined by coimmunoprecipitation assays in HT‐29 cells treated with 4 μM of CGN for 24 hours in the presence and absence of MG132. *P < .05, **P < .01
Figure 5
Figure 5
Coroglaucigenin induces autophagy through disrupting the interaction of Hsp90 with Akt. (A) Immunoblot analysis of phosphorylation of Akt (Ser473) in HT‐29 and SW480 cells treated with the indicated concentrations of CGN for 24 hours. Total Akt expression served as an internal control. (B) ImageJ densitometric analysis of the p‐AKT/β‐actin ratios from immunoblots. (C) Immunoblot analysis of LC3 and p‐Akt in cells treated with 4 μM of CGN for 24 hours in the presence and absence of insulin. (D) ImageJ densitometric analysis of the p‐AKT/β‐actin and the LC3‐II/β‐actin ratios from immunoblots. (E) Cells were transfected with an empty vector or with constitutively active CA‐Akt for 48 hours, and then cells were treated with 4 μM of CGN for another 24 hours. p‐Akt and LC3 were determined by immunoblotting. (F) ImageJ densitometric analysis of the p‐AKT/β‐actin and the LC3‐II/β‐actin ratios from immunoblots. (G) SW480 cells were treated as in C. The formation of endogenous LC3 puncta (arrows) was visualized under a fluorescent microscope. Scale bars, 10 μm. (H) Hsp90 interaction with Akt was determined by coimmunoprecipitation assays in cells treated with 4 μM of CGN for 24 hours. *P < .05, **P < .01, ***P < .001
Figure 6
Figure 6
Combination therapy with coroglaucigenin and chloroquine enhances anti‐tumour activities in vivo. HT‐29 and SW480 tumour cells were injected into the right flanks of Nu/Nu mice (5 mice/group). When tumour volumes reached 50 mm3, mice were treated with indicated formulations every 2 d for 5 total treatments. (A) Images of tumour masses at day 18 in each mouse. (B) Tumour volumes at different time points. **P < .01
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