Piperine inhibits the proliferation of colorectal adenocarcinoma by regulating ARL3- mediated endoplasmic reticulum stress

Abstract

Colorectal adenocarcinoma (COAD) is a significant cause of cancer-related mortality worldwide, necessitating the identification of novel therapeutic targets and treatments. This research aimed to investigate the role of ARL3 in COAD progression and to explore the effects of Piperine on ARL3 expression, cell proliferation, epithelial-mesenchymal transition (EMT), and endoplasmic reticulum (ER) stress. Bioinformatics analysis of The Cancer Genome Atlas (TCGA)-COAD, GSE39582, and GSE44861 datasets assessed ARL3 expression levels. Immunohistochemical data from the Human Protein Atlas (HPA) database confirmed ARL3 overexpression in COAD. The association of ARL3 with COAD clinical parameters and prognosis was also examined. COAD cells were treated with Piperine, and in vitro assays evaluated cell proliferation, apoptosis, EMT marker expression, and ER stress (ERS) responses. ARL3 overexpression in COAD correlated with poor prognosis and varied across pathological stages. Piperine treatment inhibited COAD cell proliferation in a concentration- and time-dependent manner, as indicated by reduced Ki-67 levels and decreased colony-forming ability. Piperine induced S-phase cell cycle arrest and facilitated apoptosis in COAD cells, evidenced by changes in Bax, Bcl-2, cleaved caspase-3, and cleaved Poly (ADP-ribose) polymerase (PARP) levels. Moreover, Piperine downregulated ARL3 expression in COAD cells, thereby suppressing transforming growth factor beta (TGF-β)-induced EMT. Additionally, Piperine attenuated the ARL3-mediated ER stress response, significantly reducing binding immunoglobulin protein (BiP), inositol-requiring enzyme 1 alpha (p-IRE1α), activating transcription factor 6 (ATF6), and C/EBP homologous protein (CHOP) levels. Piperine exerts anti-cancer effects in COAD by modulating ARL3 expression, disrupting cell cycle progression, inhibiting the EMT pathway, and regulating ERS. These findings suggest that Piperine holds promise as a therapeutic agent for COAD through its targeting of ARL3.

Figure 1.

Identification of DEGs in COAD and the prognostic significance of ARL3. (A–C) DEGs screening of TCGA-COAD dataset, GSE39582 dataset, and GSE44861 dataset. Red represents upregulated DEGs, green represents downregulated DEGs, and gray represents insignificant genes; (D–F) Expression of ARL3 in tumor samples and normal samples in the TCGA-COAD dataset, GSE39582 dataset, and GSE44861 dataset. Red represents tumor samples, and green represents normal samples; (G) Immunohistochemical staining of ARL3 in COAD detected by HPA database. The left panel (ARL3 tumor) shows a representative tissue section from a COAD showing high staining intensity, and the right panel (ARL3 normal) shows a section from adjacent normal colon tissue with moderate staining intensity. Catalog number HPA036292 refers to the antibody used for staining; (H) OS prognosis of high ARL3 expression and low ARL3 expression. Red represents high expression and green represents low expression. ***P < 0.001, ****P < 0.0001. COAD: Colorectal adenocarcinoma; DEGs: Differentially expressed genes; TCGA: The Cancer Genome Atlas; OS: Overall survival prognosis; HPA: Human Protein Atlas.

Figure 2.

Piperine inhibits COAD cell proliferation by inducing cell cycle arrest. (A and B) CCK-8 detected changes in the proliferation ability of COAD cells (HCT116 and HT29) after induction with different concentrations of Piperine at different times; (C and D) WB detected changes in the expression level of Ki-67 protein in HCT116 and HT29 cells after treatment with 50 and 150 µM Piperine; (E and F) A colony formation assay was used to detect the effects of 50 µM Piperine and 150 µM Piperine on the proliferation of HCT116 and HT29 cells for 48 h, and quantitative analysis was performed; (G–J) Flow cytometry detected cell cycle changes after COAD cells are treated with 50 µM Piperine and 150 µM Piperine; (K–M) WB detection of the effects of 50 µM Piperine and 150 µM Piperine treatment on COAD cell cycle proteins. *P < 0.05, **P < 0.01, ***P < 0.001. COAD: Colorectal adenocarcinoma; CCK-8: Cell counting kit-8; WB: Western blot.

Figure 3.

Piperine induces apoptosis in COAD cells. (A–C) Flow cytometry was used to detect the effects of 50 µM Piperine and 150 µM Piperine on COAD cell apoptosis after 48 h of treatment, and quantitative analysis was performed; (D–F) WB detection of the effects of 50 µM Piperine and 150 µM Piperine on apoptotic proteins (Bax, caspase 3, cleaved caspase 3, Bcl-2, PARP, cleaved PARP) in HCT116 and HT29 cells. **P < 0.01, ***P < 0.001. COAD: Colorectal adenocarcinoma; WB: Western blot.

Figure 4.

Piperine inhibits ARL3 expression and inhibits TGF-β-induced EMT. (A–C) qRT-PCR and WB detected the expression of ARL3 after COAD cells were treated with 50 µM Piperine and 150 µM Piperine; (D–F) qRT-PCR and WB detected the expression of ARL3 in COAD cells after treatment with 10 ng/mL TGF-β and 150-µM Piperine; (G–J) Transwell was used to detect the changes in cell invasion and migration ability after COAD cells were treated with 10 ng/mL TGF-β and 150 µM Piperine for 48 h, and quantitative analysis was performed; (K–M) WB analysis of expression changes of EMT marker proteins in COAD cells treated with 10 ng/mL TGF-β and 150 µM Piperine for 48 h. *P < 0.05 or **P < 0.01 vs Control group, ^#P < 0.05 vs 10 ng/mL TGF-β group. COAD: Colorectal adenocarcinoma; qRT-PCR: Quantitative real-time polymerase chain reaction; WB: Western blot; EMT: Epithelial–mesenchymal transition.

Figure 5.

Piperine promotes overexpression of ARL3 and antagonizes ER stress. (A–C) qRT-PCR and WB detected the overexpression efficiency of ARL3 in COAD cells; (D) CCK-8 detection of COAD cell proliferation in different treatment groups. The groups included: Control, over-ARL3, over-ARL3+50 µM Piperine, and over-ARL3+150 µM Piperine; (E–G) Flow cytometry was used to detect the apoptosis rate of COAD cells in different treatment groups. The groups included: Control, over-ARL3, over-ARL3+50 µM Piperine, and over-ARL3+150 µM Piperine; (H–J) WB detection of changes in ER stress-related proteins (BIP, IRE1α, p-IRE1α, ATF6, CHOP) in COAD cells in different treatment groups. The groups included: Control, over-ARL3, over-ARL3+50 µM Piperine, and over-ARL3+150 µM Piperine. *P < 0.05 or **P < 0.01 vs control group, ^#P < 0.05 vs over-ARL3 group. COAD: Colorectal adenocarcinoma; qRT-PCR: Quantitative real-time polymerase chain reaction; WB: Western blot; ER: Endoplasmic reticulum; CCK-8: Cell counting kit-8.

Figure S1.

Comparison of characteristics of the COAD patients with high and low ARL3 expression. (A) The percentage of male and female patients in ARL3 high and low expression samples; (B) The percentage of different races in high/low gene expression samples; (C) The percentage of different pT stages in high/low gene expression samples; (D) The percentage of different pN stages in high/low gene expression samples; (E) The percentage of different pM stages in high/low gene expression samples; (F) The percentage of different pTNM stages in high/low gene expression samples.