Objective:

2

To investigate the anticancer activity of curzerene in colorectal cancer (CRC)

in vitro

and

in vivo

models.

Methods:

2

HT29 and HCT8 cells were treated with different concentrations of curzere

ne(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:

2

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 cells from curzerene-induced 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 ac

tivation 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:

2

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.