Curzerene Induces Apoptosis in Colorectal Cancer Cells Through Inhibition of MEK/ERK Signaling Pathway

Objective: To investigate the Anticancer activity of curzerene in colorectal Cancer (CRC) in vitro and in vivo models.

Methods: HT29 and HCT8 cells were treated with different concentrations of curzerene (0, 20, 40, and 60 µg/mL) for 24 h. Cell viability was assessed using cell counting kit 8 assay, and cell proliferation was detected by colony-formation, then Apoptosis rate was assessed by flow cytometry analysis. Mitochondrial membrane potential was measured using JC-1 assay kit. Intracellular calcium levels were examined using Fluo-3AM and Mag-fluo-3AM staining. Different inhibitors of cell death, including 3-methyladenine (3-MA), cloroquine (CQ), Nec-1, and carbobenzoxy-valyl-alanylaspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK), were also utilised to validate the death mechanisms. The binding ability of curzerene to mitogen-activated extracellular signal-regulated kinase (MEK) proteins was investigated by molecular docking. In addition, the expression of key proteins such as phosphated MEK (p-MEK), phosphated extracellular regulated protein kinase (p-ERK), B-cell lymphoma-2 (Bcl-2), Bcl associated X (Bax), poly ADP-ribose polymerase (PARP) and cleaved PARP were analysed by Western blot. Finally the viable HT29 cells were injected subcutaneously into the right dorsolateral abdomen of male BALB/c nude mice for in vivo potency assessment.

Results: Curzerene inhibited proliferation and induced Apoptosis in HT29 and HCT8 cells in a time- and dose-dependent manner (all P<0.05). Subsequently, we demonstrated that the Apoptosis inhibitor Z-VAD-FMK (P<0.05) but not 3-MA, CQ or necrostatin-1 rescued curzerene-induced cell death. Compared with the control group, 60 µg/mL curzerene increased the expression of cleaved PARP by affecting intracellular calcium distribution, Reactive Oxygen Species (all P<0.01), decreasing mitochondrial membrane potential and the expressions of p-MEK, p-ERK, Bcl-2, and PARP (all P<0.05), and additionally increased the expression of cleaved PARP with a molecular binding energy of -7.1 kcal/mol. The results showed that curzerene treatment inhibited the activation of MEK/ERK signaling pathway, and pretreatment with the MEK Activator C16-PAF significantly alleviated curzerene-induced cell death (all P<0.05). The results of in vivo experiments showed that curzerene significantly inhibited the growth of subcutaneous transplantation tumours in hormonal nude mice.

Conclusion: Curzerene induces Apoptosis in CRC cells through inhibition of the MEK/ERK signaling pathway, which will hopefully be a potential chemotherapeutic agent for treating CRC patients.

Chinese medicine; apoptosis; colorectal cancer; curzerene; extracellular regulated protein kinase; mitogen-activated extracellular signal-regulated kinase.