Immune Netw.  2012 Oct;12(5):189-195. 10.4110/in.2012.12.5.189.

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 14-3-3beta

• Kim JE
• Kang JS
• Lee WJ

• KMID: 2150748
• DOI: http://doi.org/10.4110/in.2012.12.5.189

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 14-3-3beta. 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.

Keyword

Vitamin C; Colon cancer; Endoplasmic raticulum; Apoptosis; Bad; 14-3-3beta

MeSH Terms

Apoptosis
Ascorbic Acid
Calcium
Cell Line
Colon
Colonic Neoplasms
Cytosol
Dissociative Disorders
Egtazic Acid
Endoplasmic Reticulum
Glycine
Humans
Incidence
Korea
Mitochondria
Vitamins
Ascorbic Acid
Calcium
Egtazic Acid
Glycine
Vitamins

Figure

• 

  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.

Reference

1. Kang JS, Cho D, Kim YI, Hahm E, Yang Y, Kim D, Hur D, Park H, Bang S, Hwang YI, Lee WJ. L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway. Cancer Immunol Immunother. 2003. 52:693–698.

2. Kang JS, Cho D, Kim YI, Hahm E, Kim YS, Jin SN, Kim HN, Kim D, Hur D, Park H, Hwang YI, Lee WJ. Sodium ascorbate (vitamin C) induces apoptosis in melanoma cells via the down-regulation of transferrin receptor dependent iron uptake. J Cell Physiol. 2005. 204:192–197.

3. Kim JE, Jin DH, Lee SD, Hong SW, Shin JS, Lee SK, Jung DJ, Kang JS, Lee WJ. Vitamin C inhibits p53-induced replicative senescence through suppression of ROS production and p38 MAPK activity. Int J Mol Med. 2008. 22:651–655.

4. Hahm E, Jin DH, Kang JS, Kim YI, Hong SW, Lee SK, Kim HN, Jung da J, Kim JE, Shin DH, Hwang YI, Kim YS, Hur DY, Yang Y, Cho D, Lee MS, Lee WJ. The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma. J Cell Biochem. 2007. 102:1002–1010.

5. Lee SK, Kang JS, Jung da J, Hur DY, Kim JE, Hahm E, Bae S, Kim HW, Kim D, Cho BJ, Cho D, Shin DH, Hwang YI, Lee WJ. Vitamin C suppresses proliferation of the human melanoma cell SK-MEL-2 through the inhibition of cyclooxygenase-2 (COX-2) expression and the modulation of insulin-like growth factor II (IGF-II) production. J Cell Physiol. 2008. 216:180–188.

6. Kim HN, Kim H, Kong JM, Bae S, Kim YS, Lee N, Cho BJ, Lee SK, Kim HR, Hwang YI, Kang JS, Lee WJ. Vitamin C down-regulates VEGF production in B16F10 murine melanoma cells via the suppression of p42/44 MAPK activation. J Cell Biochem. 2011. 112:894–901.

7. Cho D, Hahm E, Kang JS, Kim YI, Yang Y, Park JH, Kim D, Kim S, Kim YS, Hur D, Park H, Pang S, Hwang YI, Lee WJ. Vitamin C downregulates interleukin-18 production by increasing reactive oxygen intermediate and mitogen-activated protein kinase signalling in B16F10 murine melanoma cells. Melanoma Res. 2003. 13:549–554.

8. Zhang J, Dhakal IB, Zhao Z, Li L. Trends in mortality from cancers of the breast, colon, prostate, esophagus, and stomach in East Asia: role of nutrition transition. Eur J Cancer Prev. 2012. 21:480–489.

9. Ruiz de Erenchun F. The role of new agents in the treatment of colorectal cancer. Oncology. 2005. 68:162.

10. Inoue Y, Miki C, Watanabe H, Hiro J, Toiyama Y, Ojima E, Yanagi H, Kusunoki M. Schedule-dependent cytotoxicity of 5-fluorouracil and irinotecan in a colon cancer cell line. J Gastroenterol. 2006. 41:1149–1157.

11. Pallis AG, Mouzas IA. Adjuvant chemotherapy for colon cancer. Anticancer Res. 2006. 26:4809–4815.

12. Jacoby RF, Marshall DJ, Newton MA, Novakovic K, Tutsch K, Cole CE, Lubet RA, Kelloff GJ, Verma A, Moser AR, Dove WF. Chemoprevention of spontaneous intestinal adenomas in the Apc Min mouse model by the nonsteroidal anti-inflammatory drug piroxicam. Cancer Res. 1996. 56:710–714.

13. Liou JY, Ghelani D, Yeh S, Wu KK. Nonsteroidal anti-inflammatory drugs induce colorectal cancer cell apoptosis by suppressing 14-3-3epsilon. Cancer Res. 2007. 67:3185–3191.

14. McDonnell MA, Wang D, Khan SM, Vander Heiden MG, Kelekar A. Caspase-9 is activated in a cytochrome c-independent manner early during TNFalpha-induced apoptosis in murine cells. Cell Death Differ. 2003. 10:1005–1015.

15. Katoh I, Tomimori Y, Ikawa Y, Kurata S. Dimerization and processing of procaspase-9 by redox stress in mitochondria. J Biol Chem. 2004. 279:15515–15523.

16. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 1995. 3:673–682.

17. Wang S, El-Deiry WS. TRAIL and apoptosis induction by TNF-family death receptors. Oncogene. 2003. 22:8628–8633.

18. von Haefen C, Wieder T, Gillissen B, Stärck L, Graupner V, Dörken B, Daniel PT. Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells. Oncogene. 2002. 21:4009–4019.

19. Torkin R, Lavoie JF, Kaplan DR, Yeger H. Induction of caspase-dependent, p53-mediated apoptosis by apigenin in human neuroblastoma. Mol Cancer Ther. 2005. 4:1–11.

20. Boya P, Cohen I, Zamzami N, Vieira HL, Kroemer G. Endoplasmic reticulum stress-induced cell death requires mitochondrial membrane permeabilization. Cell Death Differ. 2002. 9:465–467.

21. Li J, Lee B, Lee AS. Endoplasmic reticulum stress-induced apoptosis: multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem. 2006. 281:7260–7270.

22. Zhang D, Armstrong JS. Bax and the mitochondrial permeability transition cooperate in the release of cytochrome c during endoplasmic reticulum-stress-induced apoptosis. Cell Death Differ. 2007. 14:703–715.

23. Hung JH, Su IJ, Lei HY, Wang HC, Lin WC, Chang WT, Huang W, Chang WC, Chang YS, Chen CC, Lai MD. Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-kappaB and pp38 mitogen-activated protein kinase. J Biol Chem. 2004. 279:46384–46392.

24. Szegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006. 7:880–885.

25. Breckenridge DG, Germain M, Mathai JP, Nguyen M, Shore GC. Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene. 2003. 22:8608–8618.

26. Shou Y, Li L, Prabhakaran K, Borowitz JL, Isom GE. Calcineurin-mediated Bad translocation regulates cyanide-induced neuronal apoptosis. Biochem J. 2004. 379:805–813.

27. Cardoso SM, Oliveira CR. The role of calcineurin in amyloid-beta-peptides-mediated cell death. Brain Res. 2005. 1050:1–7.

28. Rapp UR, Fischer A, Rennefahrt UE, Hekman M, Albert S. BAD association with membranes is regulated by Raf kinases and association with 14-3-3 proteins. Adv Enzyme Regul. 2007. 47:281–285.

29. Claerhout S, Decraene D, Van Laethem A, Van Kelst S, Agostinis P, Garmyn M. AKT delays the early-activated apoptotic pathway in UVB-irradiated keratinocytes via BAD translocation. J Invest Dermatol. 2007. 127:429–438.

30. Levine M, Dhariwal KR, Welch RW, Wang Y, Park JB. Determination of optimal vitamin C requirements in humans. Am J Clin Nutr. 1995. 62:6 Suppl. 1347S–1356S.

31. Wolucka BA, Communi D. Mycobacterium tuberculosis possesses a functional enzyme for the synthesis of vitamin C, L-gulono-1,4-lactone dehydrogenase. FEBS J. 2006. 273:4435–4445.

32. Härtel C, Strunk T, Bucsky P, Schultz C. Effects of vitamin C on intracytoplasmic cytokine production in human whole blood monocytes and lymphocytes. Cytokine. 2004. 27:101–106.

33. Hartel C, Puzik A, Gopel W, Temming P, Bucsky P, Schultz C. Immunomodulatory effect of vitamin C on intracytoplasmic cytokine production in neonatal cord blood cells. Neonatology. 2007. 91:54–60.

34. Lin SY, Lai WW, Chou CC, Kuo HM, Li TM, Chung JG, Yang JH. Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells. Melanoma Res. 2006. 16:509–519.

35. Boyer J, McLean EG, Aroori S, Wilson P, McCulla A, Carey PD, Longley DB, Johnston PG. Characterization of p53 wild-type and null isogenic colorectal cancer cell lines resistant to 5-fluorouracil, oxaliplatin, and irinotecan. Clin Cancer Res. 2004. 10:2158–2167.