Vitamin C Induces Apoptosis in Human Colon Cancer Cell Line, HCT-8 Via the Modulation of Calcium Influx in Endoplasmic Reticulum and the Dissociation of Bad from 14-3-3β

Abstract

It has been reported that vitamin C plays an effective role in the treatment and prevention of cancer, but its specific mechanisms are still largely unknown. The incidence of colon cancer is now increasing in Korea. Therefore, we have examined here the effect of vitamin C on the induction of the apoptosis on colon cancer and its related mechanisms. We have found that remarkable increase of the apoptosis and the calcium influx in endoplasmic reticulum (ER) in human colon cancer cell line, HCT-8. However, vitamin C-induced apoptosis was effectively inhibited by the pre-treatment of BAPTA-AM (1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid), which is well-known as a calcium specific chelator. During the apoptosis, we found the increase of the translocation of Bad to mitochondria from cytosol, after releasing from 14-3-3β. In this process, the expression of Bax, a well-known pro-apoptotic protein, was also increased. Taken together, vitamin C induces apoptosis of colon cancer cell line, HCT-8 through the increase of 1) the calcium influx in endoplasmic reticulum (ER), 2) the translocation of Bad to mitochondria, and 3) the expression of Bax.

Keywords: 14-3-3β; Apoptosis; Bad; Colon cancer; Endoplasmic raticulum; Vitamin C.

Figure 1

Dose and time kinetic study of vitamin C for induction of apoptosis in human colon cancer cell line, HCT-8. (A) Cells (2×10⁶) were incubated in the presence of 2 and 4 mM of vitamin C for 24 hrs. (B) Cells (2×10⁶) cells were incubated in the presence of 2 mM of vitamin C and incubated for 12, 18 and 24 hrs. Then the cells were collected and the effect of vitamin C on induction of apoptosis was measured by Annexin V-FITC/7-AAD staining. The result is representative of three experiments.

Figure 2

Apoptosis of HCT-8 via the increase of intracellular calcium level by the treatment of vitamin C. (A) Cells (1×10⁶) were incubated in the abscence or presence of 2 mM of vitamin C for 0.5, 1 and 2 hrs, and then loaded with 0.5 µM of Fluo-3/AM at 37℃. Each sample was analyzed for detection of increased cytosolic calcium levels through changing in fluorescence of the dyes using the standard filters following a 488 nm excitation. (B) Cells were pre-incubated for 30 min with (B) BAPTA-AM (1.25 µM), respectably, prior to treatment with 2 mM of vitamin C for 24 hrs. And then cells were collected and the effect of vitamin C on induction of apoptosis was measured by Annexin V-FITC/7-AAD staining. Result is representative of three experiments. Data present as Mean±SD.

Figure 3

Increase of Bax expression and the dissociation of Bad expression from 14-3-3β by the treatment of vitamin C. (A and B) Cells (2×10⁶) were incubated for 3, 6, 9 and 12 hrs in the presence or absence of 2 mM of vitamin C. Then protein was extracted and subjected to immunoblotting done by using (A) anti-Bax antibodies and (B) anti-Bad, anti-14-3-3β and anti-14-3-3σ antibodies as described in materials and methods. The result is representative of more than three experiments. (C) After cells were cultured in the presence or absence of 2 mM of vitamin C for 0.5, 1, 3, and 6 hrs, protein was extracted and subjected to immunoprecipitation with a monoclonal antibody against 14-3-3β. The immunoprecipitatedprotein was used to analyze for detection of associated Bad by immunobloting. The blotwas stripped out and reprobed by anti 14-3-3β antibody as described in materials and methods. The result is representative of more than three experiments. The density of each band was measured by densitometry, and the values were expressed as the ratio Bad/14-3-3β.

Figure 4

The translocation of Bad from 14-3-3β to mitochondria by the treatment of vitamin C. (A) Cells (2×10⁶) was incubated for 3, 6 and 9 hrs in the presence or absence of 2 mM of vitamin C. Mitochondrial fraction was prepared as described in materials and methods. Then western blotting was done by using antibodies against Bad and cytochrome C. (B) The density of each band was measured by densitometry, and the values were expressed as the ratio Bad/Cytochrome C.