Piperine Induces Apoptosis and Cell Cycle Arrest via Multiple Oxidative Stress Mechanisms and Regulation of PI3K/Akt and MAPK Signaling in Colorectal Cancer Cells

Abstract

Piperine, a phytochemical alkaloid, exhibits notable anticancer properties in several cancer cell types. In this study, we investigated the mechanisms by which piperine induces cell death and apoptosis in colorectal cancer (CRC) cells, focusing on oxidative stress and key signaling pathways. Using MTT assay, flow cytometry, gene overexpression, and Western blot analysis, we observed that piperine significantly reduced cell viability, triggered G1 phase cell cycle arrest, and promoted apoptosis in DLD-1 cells. In addition, piperine effectively suppressed cell viability and induced apoptosis in other CRC cell lines, including SW480, HT-29, and Caco-2 cells. These effects were associated with increased intracellular reactive oxygen species (ROS) generation, mediated by the regulation of mitochondrial complex III, NADPH oxidase, and xanthine oxidase. Additionally, piperine modulated signaling pathways by inhibiting phosphoinositide 3-kinase (PI3K)/Akt, activating p38 and p-extracellular signal-regulated kinase (ERK). Pretreatment with antimycin A, apocynin, allopurinol, and PD98059, and the overexpression of p-Akt significantly recovered cell viability and reduced apoptosis, confirming the involvement of these pathways. This study is the first to demonstrate piperine induces apoptosis in CRC cells through a multifaceted oxidative stress mechanism and by critically modulating PI3K/Akt and ERK signaling pathways.

Keywords: MAPK; PI3K/Akt; apoptosis; cell cycle arrest; colorectal cancer; oxidative stress; piperine.

Figure 1

Piperine inhibits cell viability and colony formation in DLD-1 cells. (A) Cell viability of DLD-1 cells incubated for 48 h with different concentrations of piperine assessed via MTT assays. (B) Colony formation in DLD-1 cells incubated with piperine (0, 62.5, 125, and 250 μM) for 48 h (microscope magnification: 200×). Significant differences in the 0 μM-treated group are presented as p < 0.05 (*) and p < 0.001 (***).

Figure 2

Piperine induces cell cycle G1 arrest in DLD-1 cells. (A) Cell cycle of DLD-1 cells cultured for 48 h with different concentrations of piperine assessed via PI staining and flow cytometry. (B) Expression of cell cycle-regulated proteins, cyclin E, and p27 in DLD-1 cells incubated with piperine (0, 62.5, 125, and 250 μM) for 48 h. The numbers below each band represent the relative densitometric values normalized to the untreated control (set as 1.00), and were calculated to illustrate the fold changes in protein expression.

Figure 3

Piperine induces apoptosis in DLD-1 cells. (A) Percentages of apoptosis in DLD-1 cells incubated for 48 h with piperine (0, 62.5, 125, and 250 μM) assessed via TUNEL assay and flow cytometry. Boxes indicate TUNEL-positive cells, and the percentages shown within the boxes represent the proportion of TUNEL-positive cells. (B) Expression of apoptosis marker proteins, cleaved PARP, and Bax in DLD-1 cells incubated with piperine (0, 62.5, 125, and 250 μM) for 48 h. The numbers below each band represent the relative densitometric values normalized to the untreated control (set as 1.00), and were calculated to illustrate the fold changes in protein expression.

Figure 4

Piperine (PIP) induces ROS in DLD-1 cells. (A) Intracellular ROS levels in DLD-1 cells incubated with 250 μM of piperine for 1, 3, 6, and 24 h were assessed using DCFH-DA staining and flow cytometry. (B) Cells were pre-treated with various ROS-related inhibitors for 1 h followed by 250 μM piperine incubation for 1 h. Intracellular ROS levels were then analyzed using DCFH-DA staining and flow cytometry. (C) ROS levels in DLD-1 cells treated with each ROS-related inhibitor alone (without piperine) to assess their individual effects. ROS generation was evaluated by DCFH-DA staining and flow cytometry. Significant differences compared to the untreated group are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001. Significant differences compared to the piperine-treated group are indicated as # p < 0.05, ## p < 0.01, and ### p < 0.001.

Figure 5

Piperine (PIP)-mediated ROS induces cell death and apoptosis in DLD-1 cells. (A) DLD-1 cells were pre-treated with various ROS-related inhibitors for 1 h, followed by 250 μM of piperine treatment for 48 h. Cell viability was assessed by MTT assay. Significant differences compared to the piperine-treated group are indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001. (B) Cleaved PARP expression was analyzed by Western blot under the same treatment conditions as (A). (C) The cell viability of DLD-1 cells treated with each ROS-related inhibitor alone (without piperine) was evaluated using MTT assay to determine the individual cytotoxicity of each compound. Significant differences compared to the untreated group are indicated as * p < 0.05, and *** p < 0.001. The numbers below each band represent the relative densitometric values normalized to the untreated control (set as 1.00), and were calculated to illustrate the fold changes in protein expression.

Figure 6

Piperine inhibits p-Akt and regulates MAPKs in DLD-1 cells. Expression of (A) p-Akt and Akt and (B) MAPK proteins in DLD-1 cells incubated with piperine (0, 62.5, 125, and 250 μM) for 48 h. GAPDH is shown as the same loading control for p-ERK and p-JNK because these proteins were run on the same gel and membrane. The numbers below each band represent the relative densitometric values normalized to the untreated control (set as 1.00), and were calculated to illustrate the fold changes in protein expression.

Figure 7

Piperine induces cell death and apoptosis through the Akt, p38, and ERK signaling pathways. (A) DLD-1 cells were transfected with empty plasmid or pAkt-overexpressed plasmid and then treated with 250 μM of piperine for 48 h. After treatment, cell viability was assessed via MTT assay. Significant differences in the untreated and piperine-treated empty plasmid groups are presented as p < 0.001 (***) and p < 0.001 (###), respectively. (B) Pretreatment of DLD-1 cells with 50 μM of SP600125 or 50 μM of PD98059 for 1 h, then incubation with 250 μM of piperine for 48 h. Cell viability was analyzed via MTT assay. Significant differences in the piperine-treated group are presented as p < 0.01 (**). (C) p-Akt expression was detected in the empty plasmid-, or p-Akt-overexpressed plasmid-transfected DLD-1 cells. Cells were incubated with 250 μM of piperine for 48 h. Cleaved PARP was measured via Western blot. (D) Pretreatment of DLD-1 cells with 50 μM of SP600125 or 50 μM of PD98059 for 1 h, then incubation with 250 μM of piperine for 48 h. Cleaved PARP was measured via Western blot. The numbers below each band represent the relative densitometric values normalized to the untreated control (set as 1.00), and were calculated to illustrate the fold changes in protein expression.

Figure 8

Piperine reduces cell viability and induces apoptosis in SW480, HT-29, and Caco-2 CRC cells. (A) Cell viability was assessed by MTT assay following treatment of SW480, HT-29, and Caco-2 cells with increasing concentrations of piperine (62.5–250 μM) for 48 h. Data are presented as mean ± SD from three independent experiments. *** p < 0.001 versus control. (B) Cleaved PARP protein expression was evaluated by Western blot under the same treatment conditions as (A) to confirm apoptosis induction. GAPDH served as the loading control. The numbers below each band represent the relative densitometric values normalized to the untreated control (set as 1.00), and were calculated to illustrate the fold changes in protein expression.

Figure 9

Proposed model of piperine-induced apoptosis in colorectal DLD-1 cancer cells.