Clausenidin induces caspase-dependent apoptosis in colon cancer

Abstract

Background: Clausena excavata Burm.f. is a shrub traditionally used to treat cancer patients in Asia. The main bioactive chemical components of the plant are alkaloids and coumarins. In this study, we isolated clausenidin from the roots of C. excavata to determine its apoptotic effect on the colon cancer (HT-29) cell line.

Method: We examined the effect of clausenidin on cell viability, ROS generation, DNA fragmentation, mitochondrial membrane potential in HT-29 cells. Ultrastructural analysis was conducted for morphological evidence of apoptosis in the treated HT-29 cells. In addition, we also evaluated the effect of clausenidin treatment on the expression of caspase 3 and 9 genes and proteins in HT-29 cells.

Result: Clausenidin induced a G0/G1 cell cycle arrest in HT-29 cells with significant (p < 0.05) dose-dependent increase in apoptotic cell population. The DNA fragmentation assay also showed apoptotic features in the clausenidin-treated HT-29 cells. Clausenidin treatment had caused significant (p < 0.05) increases in the expression of caspase 9 protein and gene in HT-29 cells and mitochondrial ROS and mitochondrial membrane depolarization. The results suggest the involvement of the mitochondria in the caspase-dependent apoptosis in clausenidin-treated colon cancer cells.

Conclusion: Clausenidin induces a caspase-dependent apoptosis in colon cancers through the stimulation of the mitochondria. The study demonstrates the potential of clausenidin for use in the treatment of colon cancers.

Keywords: Apoptosis; Caspase 9; Clausenidin; Colon cancer; MMP; bax; bcl 2.

Fig. 1

Structure of Clausenidin

Fig. 2

Cytotoxicity of HT-29 cells treated with clausenidin and doxorubicin. The IC₅₀ of clausenidin and doxorubicin is 13.80 ± 2.89 and 6.20 ± 0.14 μg/mL, respectively

Fig. 3

Acridine orange and propidium iodide-stained clausenidin-treated HT-29 cells. b, c, d are treatments with 5, 15, and 30 μg/mL clausenidin, respectively and (a) is the untreated control. VC - Viable cells, AP - apoptosis, MB - membrane blebbing, SN - necrosis. Analyses in triplicates. *Significant difference between means at p < 0.05

Fig. 4

Ultrastructural assessment of clausenidin-treated HT-29cells. b, c, d are treatments (13.8 μg/mL) for 24 h, 48 h, and 72 h respectively and (a) is the untreated control. VA - vacuole; LD - lipid droplet; FN - fragmented nucleus; CC - chromatin condensation; CN - convolution of nuclear outline. Analyses in triplicates

Fig. 5

Reactive oxygen species production by clausenidin-treated HT-29 cells. Analyses in triplicates. *Significant difference between means at p < 0.05

Fig. 6

DNA fragmentation in clausenidin-treated HT-29 cells. A and B are treatments (13.8 μg/mL) for 12 h and 24 h respectively, D is positive control supplied with the kit while C is the untreated control. M is the molecular marker. Analysis in triplicates

Fig. 7

Cell cycle of clausenidin-treated HT-29 cells. b, c, d are DNA contents of cell treated for 24, 48, 72 h, respectively and (a) is untreated control cells. e represents % cell count at cell cycle phases. Analyses in triplicates. (*Significant difference between means at p < 0.05)

Fig. 8

Annexin V assay of clausenidin-treated HT-29 cells. b, c, d represent viable and dead cells after treatment for 24, 48, 72 h, respectively and (a) is untreated control cells. e represents % cell count. Analyses in triplicates. (*Significant difference between means at p < 0.05)

Fig. 9

Mitochondrial membrane potential assay of clausenidin-treated HT-29cells. b, c, d represent treatment with 5, 15, and 30 μg/mL clausenidin respectively and (a) is the untreated control. Red fluorescence: cells with intact mitochondria, green fluorescence: cells with depolarized mitochondrial membrane. e represents proportion of live and apoptotic cells. Analyses in triplicates. (*Significant difference between means at p < 0.05)

Fig. 10

Caspase activities in clausenidin-treated HT-29 cells. Analyses in triplicates. (*Significant difference between means at p < 0.05)

Fig. 11

Caspase, Cyt c, Apaf 1, Bax and Bcl2 gene expressions in clausenidin-treated HT-29 cells. Analyses in triplicates. (*Significant difference between means at p < 0.05)