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.

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

期刊论文
发表日期：

2025年3月7日

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Jian Peng,
Ju Lu,
Guo-hua Li,
Meng-meng Ma,
Yi-ping Mou,
Qi-cong Zhu

摘要

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.