Cancer Res Treat.  2004 Apr;36(2):132-139.

Synthetic CDCA Derivatives-Induced Apoptosis of Stomach Cancer Cell Line SNU-1 Cells

• Moon B
• Kim MC
• Park JS

Abstract

PURPOSE
This study was conducted to explore whether CDCA derivatives induce apoptosis in a stomach cancer cell line, and to dissect the detailed mechanism underlying apoptosis. MATERIALS AND METHODS: The human stomach cancer cell line, SNU-1, cells were treated with the synthetic CDCA derivatives, HS-1199 and HS-1200. DNA and mitochondrial stains were used to detect apoptotic cells by fluorescence imaging or flow cytometry. The caspase-3 activity was measured by Western blotting. RESULTS: Both the HS-1199 and HS-1200 induced decreased viabilities of the SNU-1 cells, in time-dependent manners. The CDCA derivatives demonstrated various apoptosis hallmarks, such as mitochondrial changes reduction of MMP, cytochrome c release, and Smac/ DIABLO translocation), activation of caspase-3 (resulting in the degradation of PARP and DFF45), DNA fragmentation and nuclear condensation. CONCLUSION: The CDCA derivatives, HS-1199 and HS- 1200, both induced apoptosis of the SNU-1 gastric cancer cells in caspase- and mitochondria-dependent fashions. Many important issues relating to their therapeutic applications remain to be elucidated.

Keyword

Chenodeoxycholic acid; Stomach neoplasm; Apoptosis; Mitochondria; Caspases

MeSH Terms

Apoptosis*
Blotting, Western
Caspase 3
Caspases
Cell Line*
Chenodeoxycholic Acid
Coloring Agents
Cytochromes c
DNA
DNA Fragmentation
Flow Cytometry
Humans
Mitochondria
Optical Imaging
Stomach Neoplasms*
Stomach*
Caspase 3
Caspases
Chenodeoxycholic Acid
Coloring Agents
Cytochromes c
DNA

Figure

• 

  Fig. 1 The viability of SNU-1 was decreased after the CDCA derivatives treatment. 50µM of both HS-1199 and HS-1200 produced significant time-dependent decreases in the cell viability (0~7 h, p<0.01), whereas CDCA did not.

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  Fig. 2 Nuclear condensation induced by CDCA derivatives was demonstrated five hours after treatment. Hoechst staining demonstrated that CDCA derivatives induced a change in the nuclear morphology. Compared to the typical round nuclei of the control or CDCA-treated cells (A&B), the cells treated with 50M of both HS-1199 and HS-1200 displayed condensed and fragmented nuclei (C&D). The percentage of dead or dying cells, as determined by nuclear morphology, was significantly increased in the CDCA derivatives-treated cells in a time-dependent manner (E; 0~7 h, p<0.01).

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  Fig. 3 CDCA derivatives produced DNA fragmentation five hours after treatment. Treatment of SNU-1 cells with 50µM of the CDCA derivatives, HS-1199 and HS-1200, resulted in DNA fragmentation, as determined by the TUNEL assay (*, p<0.01).

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  Fig. 4 CDCA derivatives produced reductions in the MMP. Loss of mitochondrial membrane potential (ΔΨm) is known to be a common event in many pathways of apoptosis induction. In this study, the potential-sensitive fluorescent probe JC-1 was employed to detect loss of ΔΨm. As depicted in figure 4, the membrane potential was rapidly reduced 5 h after treatment with 50µM CDCA derivatives. ΔΨm decreased significantly in the CDCA derivative-treated cells (*, p<0.01).

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  Fig. 5 CDCA derivatives produced cytochrome c release to cytosol. In the immunofluorescent study, the cytochrome c in the control and CDCA-treated cells were found in punctate patterns, in keeping with its normal mitochondrial location (A&B). The location of the cytochrome c in the mitochondria was confirmed by double staining of the HSP-60 in the same sample (data not shown). However, 50µM of the CDCA derivatives, HS-1199 and HS-1200, treatment led to the release of cytochrome c from the mitochondria into the cytosol (C&D). Quantification data (5 h) are also shown (E; *, p<0.01).

• 

  Fig. 6 AIF and Smac/DIABLO were released from mitochondria. In the immunofluorescent study, the AIF and Smac/DIABLO in the control and 50µM CDCA-treated cells were found in punctate patterns (A&B, E&F). The CDCA derivatives led to the release of both factors. The AIF was translocated onto the nucleus, whereas the Smac/DIABLO to the cytosol (C&D, G&H).

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  Fig. 7 Western blotting shows changes of the apoptosis-related proteins in this type of apoptosis. In the 50µM CDCA derivatives-treated cells the AIF expression increased, whereas that of the XIAP decreased. However, the CDCA derivatives showed no difference in the expression levels compared to the control or CDCA-treated cells. CDCA derivatives treatment evidently displayed activation of caspases-3, resulting in the degradations of PARP and DFF45. A 32 kDa precursor of caspase-3 was degraded, resulting in 20- and 17-kDa cleavage products. The PARP 85-kDa and DFF45 11-kDa cleavage products were shown to increase in time dependent manners by CDCA derivatives-induced apoptosis.

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  Fig. 8 The CDCA derivatives produced no specific cell cycle phase arrest. In the 50µM CDCA derivatives-treated cells, the percentage of cells showing DNA hypoploidy increased. However, arrest of specific cell cycle phase was not observed.

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