Synergistic Inhibition of Colon Cancer Cell Proliferation via p53, Bax, and Bcl-2 Modulation by Curcumin and Plumbagin Combination

Abstract

Cancer is a major contributor to global morbidity and mortality. Among the different forms of cancer, colorectal cancer (CRC) is the third most frequently diagnosed cancer in men and the second most common cancer type in women globally. We aimed to explore the possible synergistic anticancer potential of curcumin (Cur) and plumbagin (PL) in the human colon cancer cell line (HCT-116). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)/cytotoxicity assay revealed IC₅₀ values of 7.7 and 7.5 μM for Cur and PL, respectively, as a separate entity. However, the combined treatment of Cur + PL significantly enhanced the cancer cell growth inhibitory potential compared with solitary treatments with an IC₅₀ value of 6.8 μM. The combined treatment also led to the induction of apoptosis by 41%, cell cycle arrest at the G2/M phase, while Bax and p53 genes were found to be upregulated and the Bcl-2 gene was downregulated compared to the untreated/solvent control. Furthermore, combined treatment elevated reactive oxygen species (ROS) production by 59% and resulted a decline in the mitochondrial membrane potential (MMP) compared to the control. Catalase and superoxide dismutase (SOD) activities were significantly reduced, leading to enhanced lipid peroxidation (LPO) and compromised membrane integrity, which were also confirmed by 4',6-diamidino-2-phenylindole (DAPI) + propoidium iodide (PI) staining were also noted. Our in vitro data were further supported by molecular docking, which showed a higher binding energy of the proteins (Bax, Bcl-2, and p53) with Cur + PL. Overall, our findings highlight the potent synergistic effects of the Cur and PL combination, which can be exploited as a combination therapy for CRC.

Figure 1

Percentages of cell viability (A: PCS-201-013-Cur treatment B: PCS-201-013-PL treatment, C: HCT-116-Cur treatment, D: HCT-116-PL treatment, E: HCT-116 Cur + PL treatment, and F: PCS-201-013-Cur+PL treatment, where a = control, b = treated with IC₅₀ concentration of Cur, c = treated with IC₅₀ concentration of PL, and d = treated with IC₂₅ concentration of Cur+PL, and e = treated with IC₅₀ concentration of Cur+PL).

Figure 2

Morphological alteration in HCT-116 cells after 24 h of treatment (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells).

Figure 3

Scratch wound healing assay in HCT-116 cell lines at 0 and 24 h (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells). E: Percentage migration. Left 0 h; Right, 24 h.

Figure 4

Mitochondrial membrane potential in HCT-116 cells (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells). E: Percentage change in MMP in response to individual compounds and their combination.

Figure 5

Reactive oxygen species (ROS) production in HCT-116 cells (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells). E: Percentage change in ROS in response to individual compound and their combination.

Figure 6

DAPI staining of HCT-116 cells (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells).

Figure 7

Catalase activity assay of HCT-116 cells (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells).

Figure 8

SOD activity assay of HCT-116 cells (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells).

Figure 9

Lipid peroxidation in HCT-116 cells (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells).

Figure 10

Cell cycle analysis by flow cytometry (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells).

Figure 11

Induction of apoptosis by flow cytometry (A: Untreated control cells, B: Cur-treated cells, C: PL-treated cells, and D: Cur + PL-treated cells). (E, F): Change in the shapes of control and combined treated cells, respectively. (Note: In the quadrant, R5 is total viable cells, R4 is early apoptotic cells, above R4 is late apoptotic cells, and above R5 is necrotic cells. In addition, Annexin V-positive/PI-negative (early apoptosis), Annexin V-positive/PI-positive (late apoptosis), and Annexin V-negative/PI-negative (live cells).

Figure 12

Molecular docking of (A) curcumin, (B) plumbagin, and (C) Cur + PL to Bax protein.

Figure 13

Molecular docking of (A) curcumin, (B) plumbagin, and (C) Cur + PL to the Bcl-2 protein.

Figure 14

Molecular docking of (A) curcumin, (B) plumbagin, and (C) Cur + PL to p53 protein.