Cultivated and wild Pleurotus ferulae ethanol extracts inhibit hepatocellular carcinoma cell growth via inducing endoplasmic reticulum stress- and mitochondria-dependent apoptosis

Abstract

Pleurotus ferulae is a kind of editable mushroom and has various biological functions such as antitumor, antioxidation and immunoregulation. Wild P. ferulae was successfully domesticated but the antitumor function and mechanisms of cultivated and wild P. ferulae need to be compared and explored. Here, we prepared cultivated and wild P. ferulae ethanol extracts (PFEE-C and PFEE-W) and compared their antitumor effect on hepatocellular carcinoma. Our data showed that PFEE-C and PFEE-W significantly inhibited the growth of H22 and HepG2 cells through induction of apoptosis. PFEE-W exhibited higher antitumor activity than PFEE-C. Both PFEE-C and PFEE-W induced endoplasmic reticulum (ER) stress characterized by the up-regulated levels of phosphorylated JNK, cleaved caspase-12 and HSP70, and mitochondrial dysfunction characterized by the reduction of mitochondrial membrane potential and the release of cytochrome c, which promoted the cleavage of caspase-3, -7, -9 and PARP. Moreover, PFEE-C and PFEE-W significantly increased ROS generation in H22 cells and suppressed H22 cell migration through reducing the levels of matrix metalloproteinase -2 and -9. Further, PFEE-C inhibited H22 tumor growth in mouse model and improved the survival of tumor mice. These results indicated that PFEE-C and PFEE-W could inhibit hepatocellular carcinoma cell growth through ER stress- and mitochondria-dependent apoptotic pathways.

Figure 1

Effects of PFEE-C and PFEE-W on the proliferation of H22 cells and splenocytes in vitro. (a) The morphological changes of H22 cells after PFEE-C and PFEE-W treatment for 24 h. (b) The viability of H22 cells after PFEE-C and PFEE-W treatment for 24, 48 and 72 h, respectively. (c) The proliferation of splenocytes after PFEE-C and PFEE-W treatment for 24, 48 and 72 h, respectively. Data are from 3 independent experiments and analyzed by ANOVA. **p < 0.01; ***p < 0.001 compared to untreated group.

Figure 2

The apoptosis of H22 cells induced by PFEE-C and PFEE-W treatment. Different concentrations of PFEE-C and PFEE-W were used to treat H22 cells for 24 h. (a) The apoptosis and necrosis of H22 cells were analyzed by flow cytometry. The individual dot plots were shown in left panels and the summary data were shown in right panels. (b) The nuclear morphology of H22 cells. The above H22 cells were stained with Hoechst 33258 and observed by inverted fluorescence microscopy. The arrows indicated the chromosomal condensation. Data are from 3 independent experiments and analyzed by ANOVA. *p < 0.05; **p < 0.01 compared to untreated group.

Figure 3

Cell cycle distribution in H22 cells upon PFEE-C and PFEE-W treatment. H22 cells were treated with different concentrations of PFEE-C and PFEE-W for 24 h. After PI staining, cell cycle distribution was analyzed by flow cytometry. Data are from 3 independent experiments and analyzed by ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 compared to untreated group.

Figure 4

The effects of PFEE-C and PFEE-W on Δψ_m and intracellular ROS generation in H22 cells. H22 cells were treated with different concentrations of PFEE-C and PFEE-W. After 48 h, cells were stained with JC-1 and the fluorescence changes were observed using inverted fluorescence microscopy (a) and analyzed by flow cytometry (b). (c) After 24 h, proteins were isolated and the levels of Bax, Bcl-2 and cytochrome (cyto) c were detected by Western blot. Cropped blots are shown and full-length blots are included in the Supplementary Information. Grayscale scanning data were obtained by Image J. The ratios of Bax/Bcl-2 and cyto c/β-actin were shown in lower panels.

Figure 5

PFEE-C and PFEE-W activated caspases in H22 cells. H22 cells were treated with different concentrations of PFEE-C and PFEE-W. After 24 h, proteins were isolated and the levels of cleaved-caspases and -PARP were detected by Western blot. Cropped blots are shown and full-length blots are included in the Supplementary Information. Grayscale scanning data were obtained by Image J. The ratios of cleaved-caspases/caspases and cleaved-PARP/PARP were shown in lower panels. Data are from 3 independent experiments and analyzed by ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 compared to untreated group.

Figure 6

PFEE-C and PFEE-W induce ER stress in H22 cells. H22 cells were treated with PFEE-C and PFEE-W. After 24 h, proteins were isolated and the levels of ER stress-related proteins were detected by Western blot. Cropped blots are shown and full-length blots are included in the Supplementary Information. Grayscale scanning data were obtained by Image J. The ratios of HSP70/β-actin, P-JNK/β-actin and cleaved-cas-12 /cas-12 were shown in lower panels. Data are from 3 independent experiments and analyzed by ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 compared to untreated group.

Figure 7

The effects of PFEE-C and PFEE-W on intracellular ROS generation in H22 cells. H22 cells were treated with different concentrations of PFEE-C and PFEE-W. (a) After 48 h, cells were stained with DCFH-DA and samples were analyzed by flow cytometry. (b) The cells were pretreated with or without 10 mM NAC for 2 h, and treated with PFEE-C and PFEE-W for 24 h, then the apoptosis of cells was analyzed by flow cytometry. Data are from 3 independent experiments and analyzed by ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 compared to untreated group.

Figure 8

PFEE-C and PFEE-W inhibited H22 cell migration in vitro. (a) After PFEE-C and PFEE-W treatment for 24 h and 48 h, H22 cell migration was observed by inverted microscope and analyzed by Image J. The width of scratches was shown in lower panels. (b) After PFEE-C and PFEE-W treatment for 24 h, proteins were isolated from H22 cells to detect the levels of MMP-2 and MMP-9 by Western blot. Cropped blots are shown and full-length blots are included in the Supplementary Information. The ratios of MMP-2/β-actin and MMP-9/β-actin were shown in lower panels. Data were analyzed by ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001 compared to untreated group.

Figure 9

The inhibition of tumor growth in vivo. Tumor mouse model was induced by injection of H22 cells. After 3 days, tumor mice (7 mice per group) were treated with or without PFEE-C. Body weight, tumor volume and survival rate were monitored at the indicated time points.