A novel 3',5'-diprenylated chalcone induces concurrent apoptosis and GSDME-dependent pyroptosis through activating PKCδ/JNK signal in prostate cancer

Abstract

Although androgen deprivation therapy may initially be effective in prostate cancer, the disease can gradually progress to castration-resistant prostate cancer, at which point chemotherapy becomes the major clinical strategy. In this study, we demonstrated the anti-cancer potential of a novel 3',5'-diprenylated chalcone (C10), which selectively inhibited the proliferation of PC3 cells in vitro and in vivo. C10 treatment elevated the proportion of PC3 cells in sub-G1 phase and induced programmed cell death. Interestingly, C10 elicited concurrent Caspase-dependent apoptotic and gasdermin E-dependent pyroptotic events. RNA-Seq and bioinformatics analyses revealed a strong correlation between protein kinase C delta (PKCδ) and mitogen-activated protein kinase pathway activation in prostate cancer. PKCδ silencing in PC3 cells suppressed the activation of the JNK pathway and the expression of its downstream genes, including Bax, interleukin-6 and interleukin-1β, which are involved in apoptotic and pyroptotic processes. Moreover, in PC3 cell xenograft tumor tissues, C10 treatment inhibited tumor growth and upregulated PKCδ. These findings suggest that C10 treatment induces the PKCδ/JNK pathway, thereby activating Caspase-3 and inducing the cleavage of PARP and gasdermin E to execute apoptosis and cell-lytic pyroptosis in prostate cancer cells.

Keywords: 3’,5’-diprenylated chalcone; PKCδ; apoptosis; crosstalk; pyroptosis.

Figure 1

Effects of compound 10 on PCa cell viability and proliferation. (A) PC3, DU145 and RWPE-1 cells were treated with various concentrations of C10 for 24, 48 or 72 h, and cell viability was analyzed with an MTT assay. (B) Chemical structure of flavagline-like compound 10. (C) The IC₅₀ values (μM) of the indicated cell lines were measured at three time points (24, 48 and 72 h) and summarized in a table. (D, E) C10 significantly inhibited the proliferation of PC3 and DU145 cells. A clonogenic assay was performed, and the number of colonies formed was analyzed. Per condition, three independent experiments were performed. Data are shown as the mean ± SD, *P < 0.05, **P < 0.01 vs. the control group.

Figure 2

RNA-Seq analysis of overall transcriptomic changes in C10-treated PC3 cells. (A) The differentially expressed genes were redacted and visualized as a volcano plot. The red dots represent significantly differentially expressed genes, while the blue dots indicate non-significantly differentially expressed genes. (B) Heatmap of genes upregulated or downregulated by C10 treatment in PC3 cells. (C) KEGG enrichment analysis of signaling pathways altered by C10 treatment. The color and size of the dots indicate the significance of the false discovery rate and the number of differentially expressed genes in the pathway, respectively. The top 20 significantly enriched signaling pathways were profiled. (D) The dysregulated genes involved in the cell cycle, apoptosis and pyroptosis were screened from all the raw data, compiled into a new list and shown as a heatmap.

Figure 3

C10 induced sub-G1 phase arrest of PC3 cells in culture. (A, B) PC3 cells were treated with C10 (0, 4, 6, 8, 10 or 12 μM) for 24 h and stained with PI so that the DNA content could be analyzed by flow cytometry. C10 increased the proportion of PC3 cells in sub-G1 phase of the cell cycle. (C–F) Western blots of PC3 cells treated for 24 h with C10. The blots were probed with antibodies against CDK2, CDK6, c-Myc, Cyclin D1, Cyclin D2, PCNA, P21^cip1 and P27^kip1. All data shown are representative of three independent experiments. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. the control group.

Figure 4

C10 induced apoptosis in PC3 cells. (A, B) PC3 cells were treated with C10 (0, 4, 6, 8, 10 or 12 μM) for 24 h, stained with annexin-V-FITC and PI, and then analyzed by flow cytometry. C10 dose-dependently increased the percentage of annexin-V-FITC-positive apoptotic cells. (C, D) Western blot showing the expression of PARP, cleaved PARP, Caspase-3, cleaved Caspase-3, Caspase-8, cleaved Caspase-8, Caspase-9, cleaved Caspase-9, Bcl-2, Bax, cytochrome C and Survivin in PC3 cells treated with C10 for 24 h. (E, F) The phosphorylation levels of core factors in the MAPK signaling pathway (P38/MAPK and ERK1/2) were detected at different time points. β-actin was used as a loading control. Relative expression was determined based on the band intensity compared with that of the loading control. All data shown are representative of three independent experiments. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. the control group.

Figure 5

Combined analyses of the Taylor and STRING databases to predict the correlation between the levels of PKCδ and other core genes in pyroptotic events. (A) Plots of significant Pearson’s correlations between PKCδ levels and Bax, Survivin, Caspase-3 and Caspase-8 levels in the PCa dataset are shown. R is Pearson’s correlation coefficient, and the x and y axes denote the respective genes being analyzed. Data were obtained from the Gene Expression Omnibus. (B, C) Bioinformatics analysis of PPI and co-expression data in Homo sapiens from the STRING database, visualized using Cytoscape 3.7.1. (D, E) Western blot showing the expression of different PKC subtypes in PC3 cells treated with C10 for 24 h. (F) The mRNA levels of PKCδ, Bax, Survivin, Caspase-3 and Caspase-8 were measured by qRT-PCR in PC3 cells treated with C10 for 12 h. All data shown are representative of three independent experiments. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. the control group. (G) Diagrams of the human GSDMD and GSDME proteins. Red arrows indicate the cleavage sites of Caspases.

Figure 6

PKCδ induced PCD by activating JNK signaling in C10-treated PC3 cells. (A, B) Western blot of PC3 cells treated for the indicated times (0, 2, 4, 8, 16 or 24 h) with C10. IL-6, p-SAPK/JNK and SAPK/JNK antibodies were used. (C) Cultured PC3 cells were treated with C10 in the presence of different inhibitors (siPKCδ and the JNK-specific inhibitor Tanzisertib [CC-930]) for 24 h. The cells were then stained with Hoechst 33258 and photographed using a fluorescence microscope (magnification ×200, scale bar: 100 μm). (D, E) Cultured PC3 cells were stained with annexin-V-FITC and PI for flow cytometry analysis. (F) The mRNA levels of PKCδ, Caspase-9, IL-6, IL-8, IL-1β and Bax were measured by qRT-PCR. All data shown are representative of three independent experiments. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. the control group.

Figure 7

Inhibition of PKCδ suppressed C10-induced concurrent apoptosis and GSDME-dependent pyroptosis in PC3 cells. (A, B) Cultured PC3 cells were incubated with C10 in the presence of siPKCδ or the JNK-specific inhibitor Tanzisertib (CC-930) for 24 h to determine the link between the apoptotic and pyroptotic pathways. The protein levels of PKCδ, p-SAPK/JNK, SAPK/JNK, Survivin, Bax, PARP, cleaved PARP, Caspase-3, cleaved Caspase-3, IL-6, GSDME and GSDME-N were examined by Western blotting. (C) LDH enzyme activity was measured in the culture supernatants of PC3 cells after various treatments. (D, E) The phosphorylation levels of core factors in the MAPK signaling pathway (P38/MAPK and ERK1/2) were detected after various treatments in PC3 cells. β-actin was used as a loading control. All data shown are representative of three independent experiments. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01 vs. the control group.

Figure 8

Schematic diagram depicting the anti-PCa mechanism of C10. C10 stimulated the PKCδ/JNK/IL-6 signaling pathway and thus induced crosstalk between apoptosis and GSDME-dependent pyroptosis in PC3 cells. Red arrows: the new signal transduction pathway discovered in our study, whereby PKCδ/JNK/IL-6 lead to concurrent apoptosis and pyroptosis.

Figure 9

C10 attenuated tumor growth by inhibiting cell proliferation and inducing apoptosis and inflammation in PC3 xenograft mice. (A) PC3 cell tumor xenograft nude mice were intraperitoneally administered C10 (low-dose or high-dose) or the control treatment every two days for a total of 10 times, as indicated in the diagram. (B) The tumor sizes in the three groups were monitored and recorded at three-day intervals as soon as C10 was injected. (C) The subcutaneous tumors were weighed immediately at the end of the study. (D) The mouse weights in the three groups were recorded at three-day intervals as soon as C10 was injected. (E) Tumor spheroids generated from the control and high-dose groups were fixed, sectioned and immunohistochemically stained for PKCδ, PCNA, Bcl-2 and IL-6 expression. The levels of the indicated proteins were quantified in the control and high-dose groups (Scale bar: 50 μm). Data are shown as the mean ± SD. *P < 0.05, **P < 0.01.