Ceramide Regulates Anti-Tumor Mechanisms of Erianin in Androgen-Sensitive and Castration-Resistant Prostate Cancers

Abstract

Prostate cancer is the second most prevalent malignancy worldwide. In the early stages, the development of prostate cancer is dependent on androgens. Over time with androgen deprivation therapy, 20% of prostate cancers progress to a castration-resistant form. Novel treatments for prostate cancers are still urgently needed. Erianin is a plant-derived bibenzyl compound. We report herein that erianin exhibits anti-tumor effects in androgen-sensitive and castration-resistant prostate cancer cells through different mechanisms. Erianin induces endoplasmic reticulum stress-associated apoptosis in androgen-sensitive prostate cancer cells. It also triggers pro-survival autophagic responses, as inhibition of autophagy predisposes to apoptosis. In contrast, erianin fails to induce apoptosis in castration-resistant prostate cancer cells. Instead, it results in cell cycle arrest at the M phase. Mechanistically, C16 ceramide dictates differential responses of androgen-sensitive and castration-resistant prostate cancer cells to erianin. Erianin elevates C16 ceramide level in androgen-sensitive but not castration-resistant prostate cancer cells. Overexpression of ceramide synthase 5 that specifically produces C16 ceramide enables erianin to induce apoptosis in castration-resistant prostate cancer cells. Our study provides both experimental evidence and mechanistic data showing that erianin is a potential treatment option for prostate cancers.

Keywords: apoptosis; autophagy; cell cycle arrest; endoplasmic reticulum stress; sphingolipid.

Figure 1

Erianin suppresses colony formation but not migration in both LNCaP and PC3 cells. (A, B) Cells were treated with erianin at indicated concentrations for 10 days. Images are representative of three independent experiments (A) and the number of colonies was quantified (B). (C, D) Wound healing assay was performed to assess cell migration. Cells were treated with erianin at indicated concentrations for up to 48 h. Images are representative of three independent experiments (C) and the wound closure area were quantified (D). Data are expressed as mean ± SD. n=3. ***p < 0.001, versus untreated control.

Figure 2

Erianin induces ER stress-associated apoptosis in LNCaP cells. (A, B) LNCaP cell viability was determined by MTS colorimetric assay. LNCaP cells were treated with erianin at indicated concentrations for 1, 2 and 3 days (A). LNCaP cells were treated with erianin at indicated concentrations in the presence or absence of pan-caspase inhibitor z-VAD at 50 µM for 24 h (B). Data are shown as a percentage of untreated control at day 0. (C) LNCaP cells were treated with erianin at 100 nM for 24 h and apoptosis was analyzed by flow cytometry with propidium iodide (PI) and annexin V-FITC (A-V) co-staining. A-V single positive population represents early apoptotic cells, while A-V and PI double-positive population refers to late apoptotic/dead cells. (D–F) LNCap cells were treated with erianin at indicated concentrations for 24 h (D) or at 50 nM for indicated times (E, F). Apoptosis (D), ER stress (E) and Bcl-2 family members (F) were examined by Western blotting. FL-, full length; c-, cleaved; casp3, caspase 3. Data are expressed as mean ± SD. n=3. **p < 0.01; ***p < 0.001, versus untreated control.

Figure 3

Erianin induces pro-survival autophagy in LNCaP cells. (A) LNCaP cells were treated with erianin at indicated concentrations for 24 h in the presence or absence of 100 nM bafilomycin A1 (BafA1, an autophagy inhibitor). Levels of p62, LC3, cleaved caspase-3 and cleaved PARP were examined by Western blotting. FL-, full length; c-, cleaved; casp3, caspase 3. (B) LNCaP cells were transfected with GFP-tagged LC3 for 48 h. Then cells were co-treated with 100 nM BafA1 and 50 nM erianin for 24 h. Confocal images were captured. Bar = 10 µm. (C) Cells were treated with erianin at indicated concentrations in the presence or absence of 100 nM bafilomycin A1 for 24 h. Cell viability was determined by MTS colorimetric assay. Data are expressed as mean ± SD. n=4. *p < 0.05, -BafA1 versus +BafA1.

Figure 4

Erianin fails to induce apoptosis in PC3 cells. (A) PC3 cell viability was determined by MTS colorimetric assay in cells treated with erianin at indicated concentrations for 1, 2 and 3 days. Data are shown as a percentage of untreated control at day 0. n=4. (B) Cells were treated with erianin at 100 nM for 24 h and apoptosis was analyzed by flow cytometry with propidium iodide (PI) and annexin V-FITC (A-V) co-staining. A-V single positive population represents early apoptotic cells, while A-V and PI double-positive population refers to late apoptotic/dead cells. n=3. (C, D) Cells were treated with erianin at indicated concentrations for 24 h (C) or at 100 nM for indicated times (D). Apoptosis and ER stress were examined by Western blotting. FL-, full length; c-, cleaved; casp3, caspase 3. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001, versus untreated control.

Figure 5

Erianin induces cell cycle arrest at the M phase in PC3 cells. (A, B) PC3 cells were treated with erianin at indicated concentrations for 24 h. (A) Cell cycle phase was determined by flow cytometry with propidium iodide (PI) staining. G₀/G₁, S and G₂/M phase populations were estimated using Flowjo software and are indicated in purple, yellow and green, respectively. (B) Percentages of cells at each phase was quantified by Flowjo software. Data are expressed as mean ± SD. n=5. *p < 0.05; ***p < 0.001, versus untreated control. (C) PC3 cells were treated with erianin at 50 nM for indicated times. Cyclin b1 and phosphorylated CDK1 substrates were examined by Western blotting. (D, E) Cells were treated with S-trityl-L-cysteine (STLC) at 10 µM for 16 h to synchronize cells at the prometaphase. Then STLC was withdrawn and cell cycle progression to metaphase, telophase and interphase were monitored in the presence ot absence of erianin at 100 nM for 1 h. Confocal images were captured. Bar = 10 µm (D). Percentages of cells at each cell cycle phase were quantified (E).

Figure 6

C16 ceramide determines PCa cell response to erianin. (A, B) LNCaP and PC3 cells were treated with erianin at indicated concentrations for 24 h. Lipids were extracted and analyzed using targeted lipidomics. Lipid heatmap shows the changes in levels of ceramides (Cer), sphingosine (Sph) and sphingosine 1-phosphate (S1P). The color key indicates the fold change on a log2 scale (A). Absolute levels of C16:0, C24:0 and C24:1 ceramides (B). Data are expressed as mean ± SD. n=4. ***, p<0.001, versus untreated LNCaP cells. (C) PC3 cells were transfected with empty vector (EV), ceramide synthase 5 (CerS5) and CerS6 for 48 h, prior to the treatment with erianin at 100 nM for an additional 24 h. CHOP and cleaved caspase-3 were examined by Western blotting.

Figure 7

Model depicting discrepant anti-tumor effects of erianin in androgen-sensitive and castration-resistant prostate cancer (PCa) cells, in which intracellular level of ceramide plays a critical role. Left panel: Androgen-sensitive PCa cells and ceramide synthase 5 (CerS5)-overexpressed castration-resistant PCa cells exhibit a high level of intracellular ceramide. Under this condition, erianin induces both adaptive and pro-death unfolded protein responses (UPRs) at the endoplasmic reticulum (ER), leading to apoptosis. Right panel: Castration-resistant PCa cells exhibit a low level of intracellular ceramide. Under this condition, erianin can only induce adaptive UPRs but not pro-death UPRs or apoptosis. Alternatively, erianin induces cell cycle arrest at the M phase. The diagram was created with BioRender.com. The 3D conformer of erianin was adapted from PubChem, https://pubchem.ncbi.nlm.nih.gov/compound/Erianin#section=3D-Conformer, PubChem CID 356759.