Dehydrodiisoeugenol inhibits colorectal cancer growth by endoplasmic reticulum stress-induced autophagic pathways

Abstract

Background: Dehydrodiisoeugenol (DEH), a novel lignan component extracted from nutmeg, which is the seed of Myristica fragrans Houtt, displays noticeable anti-inflammatory and anti-allergic effects in digestive system diseases. However, the mechanism of its anticancer activity in gastrointestinal cancer remains to be investigated.

Methods: In this study, the anticancer effect of DEH on human colorectal cancer and its underlying mechanism were evaluated. Assays including MTT, EdU, Plate clone formation, Soft agar, Flow cytometry, Electron microscopy, Immunofluorescence and Western blotting were used in vitro. The CDX and PDX tumor xenograft models were used in vivo.

Results: Our findings indicated that treatment with DEH arrested the cell cycle of colorectal cancer cells at the G1/S phase, leading to significant inhibition in cell growth. Moreover, DEH induced strong cellular autophagy, which could be inhibited through autophagic inhibitors, with a rction in the DEH-induced inhibition of cell growth in colorectal cancer cells. Further analysis indicated that DEH also induced endoplasmic reticulum (ER) stress and subsequently stimulated autophagy through the activation of PERK/eIF2α and IRE1α/XBP-1 s/CHOP pathways. Knockdown of PERK or IRE1α significantly decreased DEH-induced autophagy and retrieved cell viability in cells treated with DEH. Furthermore, DEH also exhibited significant anticancer activities in the CDX- and PDX-models.

Conclusions: Collectively, our studies strongly suggest that DEH might be a potential anticancer agent against colorectal cancer by activating ER stress-induced inhibition of autophagy.

Keywords: Anticancer agent; Autophagy inhibition; Colorectal cancer; Dehydrodiisoeugenol (DEH); Endoplasmic reticulum (ER) stress.

Fig. 1

DEH inhibits the growth of colorectal cancer cells in vitro. a Colorectal cells (HCT 116 and SW620) and normal human colon epithelial cell NCM460 were incubated with a series of different concentrations of DEH for 48 h. Cell viability was measured by the MTT assay. The IC₅₀ values of DEH for 48 h in the tested cells are marked in the lower-left corner. b Dose- and time-dependent effects of DEH on HCT 116 and SW620 cells. The cells were incubated DEH at different concentrations for 24, 48, and 72 h. Cell viability was measured by MTT assay. The results are represented as the means ±SD (N = 3). c Cell morphology of HCT 116 and SW620 cells after incubation with the indicated concentrations of DEH or DMSO for 48 h. Scale bar: 10 μm. The histograms represent the effect of DEH on the cell viability. d Images and quantification of -positive HCT 116 and SW620 cells after treatment with DEH for 48 h. Scale bar: 100 μm. e Colony formation and self-renewal capability were investigated by soft agar assay after incubation with DMSO, 20, or 60 μM DEH, Scale bar: 15 μm. The number of clones was counted and statistically represented as mean ± SD. The notability analysis was performed by the Unpaired Student’s t-test, and a p-value less than 0.05 was considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

DEH inhibits cell growth by arresting the cell cycle at the G1/S phase. a Cell cycles of HCT 116 and SW620 cells were investigated via flow cytometry after treatment with or without DEH for 48 h. The distribution ratio of G1, S, and G2 of panel A was determined. b Western blotting assays were performed to detect the expression of p21, CDK2, CDK4, Cyclin D1, Cyclin E1, Cyclin E2, and Tubulin in HCT 116 and SW620 cells after treatment with DEH and the densitometry of western blotting bands of panel c. The protein expression levels of p21, CDK4, Cyclin D1, and Tubulin in DEH-treated colorectal cancer cells with time gradient after treatment with 60uM DEH and the densitometry of western blotting bands of panel. All the data were analyzed using the Unpaired Student’s t-test, and p-values less than 0.05 were considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

DEH induces autophagy in colorectal cancer cells. a Immunofluorescence staining of LC3B (green) and tubulin (red) in HCT 116 and SW620 cells treated with or without 60 μM DEH for 48 h. The nuclei were counterstained with DAPI (blue). Scale bars: 15 μm. The histogram shows quantification of the percentage of cells with LC3B puncta. b Fluorescence images of GFP-MAP 1LC3B puncta in HCT 116 and SW620 cells incubated with or without 60 μM DEH for 48 h. GFP-MAP 1LC3B puncta were quantified and presented in the bar chart on the right. Scale bars: 10 μm. c Autophagic vesicles detected by TEM in HCT 116 and SW620 cells treated with or without 60 μM DEH for 48 h. Scale bar: 1 μm. N: nucleus. d Protein levels of LC3B, p62, and ATG7 were detected by western blotting after HCT 116 and SW620 were treated with the indicated concentrations of DEH for 48 h. The densitometry of western blotting is shown to the right of the pane. The statistical results are presented as mean ± SD. All the data were analyzed by using the Unpaired Student’s t-test and p-values less than 0.05 were considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

DEH inhibits autophagic flux in colorectal cancer cells. a Fluorescence imaging photographs of HCT 116 and SW620 cells infected with mRFP-GFP-LC3B recombinant adenovirus. The cells were infected with adenovirus for 24 h and then incubated with DMSO, EBSS, 10 μM CQ, and 60 μM DEH for 48 h. Nuclei were stained with DAPI. Scale bar: 10 μm. The average number of autophagosomes (yellow) and autolysosomes (red) per detected cell was counted. All the data were analyzed using the Unpaired Student’s t-test, and p-values less than 0.05 were considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. b Western blot analysis of LC3B-II levels in HCT 116 and SW620 cells incubated with or without 60 μM DEH in the absence or presence of 10 mM 3-MA for 48 h. The western blot analysis of LC3B-II levels in HCT 116 and SW620 cells incubated with or without 60 μM DEH in the absence or presence of 10 μM CQ for 48 h. c Western blot analysis of LC3B-II levels in HCT 116 and SW620 cells incubated in normal medium or EBSS with or without 60 μM DEH for 6 h. Western blot analysis of LC3B-II levels in HCT 116 and SW620 cells incubated with or without 60 μM DEH in the absence or presence of 10 nM Baf A1 for 48 h

Fig. 5

DEH induces ER stress in colorectal cancer cells. a Gene set enrichment analysis of UPR genes between control and DEH-treated cells. b The thermodynamic chart of the mRNA expression level of genes related to ER stress in colorectal carcinoma cells after incubation with DEH for 48 h. c The subcellular structure of colorectal cancer cells after treatment with or without 60 μM DEH for 48 h were observed by TEM. Scale bar: 2 μm. N: nucleus. The ER is circled in red. d Western blotting assays were performed to detect the expression of BiP, Ero1-Lα, PERK, eIF2α, p-eIF2α, IRE1α, XBP-1 s, CHOP, and Tubulin in HCT 116 and SW620 cells after treatment with or without DEH. The densitometry of western blotting in the right panel. The data were presented as means ±SD. All the data were analyzed by the Unpaired Student’s t-test and p-values less than 0.05 were considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

EDH induces autophagy through PERK/eIF2α and IRE1α/XBP-1 s/CHOP pathways in colorectal cancer cells. a The MTT assay was used to evaluate the inhibition rate of colorectal carcinoma cells, which were transfected with PERK or IRE1α siRNAs, followed by incubation with the indicated concentrations of DEH for another 48 h. b The cell activity was detected by colony formation assay. Cells were transfected with PERK or IRE1α siRNAs, followed by incubation with 60 μM DEH for 10 days. The cells were stained with crystal violet staining solution. Scale bar: 200 nm. The number of clones was quantitated and presented to the right of the panel. c The western blotting assay was used to detect the expression of IRE1α, LC3B, and Tubulin. Tubulin was used as an internal control. d Cellular activity was also detected by colony formation assay. The cells were pretreated with 4U8C, followed by incubation with DEH for 10 days. The cells were stained with crystal violet staining solution. Scale bar: 200 nm. The number of clones were quantified and presented below the panel. e The expression of IRE1α, LC3B- II, and Tubulin was detected after DEH treatment with the inhibitor of IRE1α, 4U8C, or DMSO for 48 h. All the data were analyzed using the Unpaired Student’s t-test, and p-values less than 0.05 were considered to be statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

Effects of DEH on the growth of colorectal cancer in vivo. a HCT 116, SW620, and the tumor tissue from colon cancer patient were injected or transplanted into the flanks of NOD/SCID mice. The tumor-bearing mice were treated with DMSO or 40 mg/kg DEH by intraperitoneal injection when tumors were palpable. Tumor volume was measured every 2 days. Two weeks later, the mice were anesthetized and killed, and the tumors were imaged and analyzed. b Hematoxylin and eosin (H&E) staining of the indicated xenograft tumors. Scale bar: 50 μm. c Immunohistochemical (IHC) staining of the indicated xenograft tumors. Scale bar: 50 μm. d H&E staining of the heart, liver, spleen, lung, and kidney in mice treated with DMSO or 50 mg/kg DEH. Scale bar: 50 μm. e The expression of BiP, PERK, and IRE1α of xenograft tumors was detected by western blotting. f The expression of LC3B, p62, and tubulin in xenograft tumors was detected by western blotting. All the data were presented as means ±S.D. and are representative of three independent experiments. P-value < 0.05 was considered to be significant. **P < 0.01; ***P < 0.001

Fig. 8

Diagram of the predicted mechanism of DEH treatment in colorectal cancer. Dehydrodiisoeugenol (DEH), as the monomer extracted from the Nutmeg seeds, showing an excellent anti-tumor activity to colorectal cancer both in vivo and in vitro. On the one hand, DEH can inhibit the proliferation of colorectal cancer cells via arresting cell cycle in a dose- and time-dependent manner. On the other hand, DEH cause endoplasmic reticulum (ER) stress and subsequently stimulates autophagy inhibition through the PERK-eIF2α / IRE1α-XBP-1 s-CHOP signal pathways. So as to inhibit the cell growth proliferation of colorectal cancer more effectively. Taken together, our studies suggest that DEH may be a promising therapeutic agent against colorectal cancer in the future