J Korean Med Sci.  2004 Aug;19(4):560-566. 10.3346/jkms.2004.19.4.560.

Norcantharidin Induces Human Melanoma A375-S2 Cell Apoptosis through Mitochondrial and Caspase Pathways

• An WW
• Wang MW
• Tashiro SI
• Onodera S
• Ikejima T

• KMID: 2157686
• DOI: http://doi.org/10.3346/jkms.2004.19.4.560

Abstract

Norcantharidin (NCTD) is the demethylated form of cantharidin, which is the active substance of mylabris. To examine the pathway of NCTD-induced A375-S2 cell death, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide (MTT) assay, photomicroscopical observation, DNA agarose gel electrophoresis, caspase activity assay and Western blot analysis were carried out. A375-S2 cells treated with NCTD exhibited several typical characteristics of apoptosis. The inhibitory effect of NCTD on human melanoma, A375-S2 cells, was partially reversed by the inhibitors of pan-caspase, caspase-3 and caspase-9. The activities of caspase-3 and -9 were significantly increased after treatment with NCTD at different time. The expression of inhibitor of caspase-activated DNase was decreased in a time-dependent manner, simultaneously, the ratio of Bcl-2/Bax or Bcl-xL/Bax was decreased and the expression ratio of proteins could be reversed by caspase-3 inhibitor. The expression of cytochrome c in cytosol was increased after NCTD treatment and caspase- 3 inhibitor had no significant effect on the up-regulation of cytochrom c. These results suggest that NCTD induced A375-S2 cell apoptosis and the activation of caspase and mitochondrial pathway were involved in the process of NCTD-induced A375-S2 cell apoptosis.

Keyword

Cantharidin; Norcantharidin; Cell Line; Tumor; A375-S2 Cells; Apoptosis; Caspase; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Cytochromes c

MeSH Terms

Animals
Apoptosis/*physiology
Bicyclo Compounds, Heterocyclic/chemistry/metabolism/*pharmacology
Caspases/antagonists & inhibitors/*metabolism
Cell Line, Tumor/*drug effects
Cell Shape
DNA Fragmentation
Enzyme Activation
Humans
Mitochondria/*metabolism
Molecular Structure
Proto-Oncogene Proteins c-bcl-2/metabolism
Signal Transduction/*physiology

Figure

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  Fig. 1 Structures of cantharidin (CA) and norcantharidin (NCTD).

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  Fig. 2 Cytotoxic effects of NCTD on A375-S2 cell. Cells were treated with various doses of NCTD for 24 hr (A) or 60 µM NCTD for various time periods (B). The relative viability of cells was determined by MTT assay. Results are represented as mean±SD, n=3.

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  Fig. 3 Cellular morphology of NCTD-treated A375-S2 cells. Cells were cultured without NCTD (control) or with 60 µM NCTD for 24 hr. Morphological change were observed by fluorescent microscopy (×200). Arrows indicate condensed nuclei.

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  Fig. 4 NCTD-induced DNA fragmentation in A375-S2 cells. The cells were cultured in the presence of NCTD 60 µM for 0, 6, 12, 24 and 36 hr. Genomic DNA was extracted and analyzed via electrophoresis on 2% agarose gels. Lane M: DNA molecular markers.

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  Fig. 5 Ratio of apoptosis and necrosis in A375-S2 cells. Cells were treated with 0, 15, 30, 60,120, 240 µM NCTD for 24 hr. The ratios of LDH released from floating dead cells and the culture medium were used to distinguish the proportion of apoptotic and necrotic cells. Results are represented as mean±SD, n=5.

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  Fig. 6 Effect of caspase inhibitors on NCTD-induced A375-S2 cells apoptosis. The cells were cultured in the presence or absence of caspase inhibitors. Two hours prior to the addition of 60 µM NCTD, pan-caspase inhibitor (z-VAD-fmk, 40 µM), caspase-3 inhibitor (z-DEVD-fmk, 20 µM), caspase-8 inhibitor (z-IETD-fmk, 20 µM), caspase-9 inhibitor (Ac-LEHD-CHO, 20 µM) were added, then further incubated for 24 hr. A: NCTD-treated group; B: NCTD- and z-VAD-fmk-treated group; C: NCTD- and z-DEVD-fmk-treated group; D: NCTD- and z-IETD-fmk-treated group; E: NCTD- and Ac-LEHD-CHO-treated group; F: z-VAD-fmk-treated group; G: z-DEVD-fmk-treated group; H: z-IETD-fmk-treated group; I: z-LEHD-fmk-treated group. Results are represented as mean±SD, n=3. *p<0.05, **p<0.01 vs. group A.

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  Fig. 7 Effects of NCTD on the activation of caspase-3, -8, -9 in A375-S2 cells. Cells were treated with NCTD 60 µM for 0, 8, 18 and 28 hr. Caspase-3, caspase-8 and caspase-9 activities was performed using Caspase Apoptosis Detection Kit according to the manufacturer's instruction. Results are represented as mean±SD, n=3. *p<0.05, **p<0.01 vs. 0 hr.

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  Fig. 8 Effects of NCTD on ICAD expression in the absence or presence of caspase-3 inhibitor (z-DEVD-fmk). A375-S2 cells were treated with 60 µM NCTD only for 0, 12, 24 and 36 hr. Two hours prior to the addition of NCTD, caspase-3 inhibitor (z-DEVD-fmk, 20 µM) were added, then further incubated for 36 hr. Cell lysates were separated by 12% SDS-PAGE, and ICAD was detected by Western blot analysis.

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  Fig. 9 Effects of NCTD on Bcl-2, Bcl-xL and Bax expression in the absence or presence of caspase-3 inhibitor (z-DEVD-fmk). A375-S2 cells were treated with 60 µM NCTD only for 0, 12, 24 and 36 hr. Two hours prior to the addition of NCTD, caspase-3 inhibitor (z-DEVD-fmk, 20 µM) were added, then further incubated for 36 hr. Cell lysates were separated by 12% SDS-PAGE, and Bcl-2, Bcl-xL and Bax proteins were detected by Western blot analysis.

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