EFFECT OF VITAMIN C ON APOPTOSIS OF NUCLEUS PULPOSUS CELLS INDUCED BY TUMOR NECROSIS FACTOR α AND SERUM DEPRIVATION_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

DAILibing ¹ ,  LIUZhihe ¹ ,  LIANGWeiguo ² , YAOYicun ² , XUJiake ³ , YEDongping ² , ZOULongqiang ² , SHENYan ¹

1. Guangzhou Institute of Traumatic Surgery, Medical College, Jinan University, Guangzhou Guangdong, 510220, P. R. China;
2. Department of Orthopaedics, Guangzhou Red Cross Hospital, Medical College, Jinan University, Guangzhou Guangdong, 510220, P. R. China;
3. School of Pathology and Laboratory Medicine, the University of Western Australia;

Corresponding author：

LIUZhihe, Email: zliu0731@163.com; LIANGWeiguo, Email: liangweiguo1011@126.com

Keywords：

Vitamin C; Nucleus pulposus cells; Tumor necrosis factor α; Serum deprivation; Apoptosis; Intervertebral disc degeneration

DOI：

10.7507/1002-1892.20150105

Video：

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Objective To explore the effect of Vitamin C (Vit C) on the apoptosis of human nucleus pulposus (NP) cells induced by tumor necrosis factor α (TNF-α) and serum deprivation. Methods The NP cells were isolated from patients undergoing spine corrective operation by collagenase trypsin. The experiment was divided into 3 groups:Vit C group (group A), TNF-α group (group B), and serum deprivation group (group C). Group A was reassigned to A1 subgroup (basic medium), A2 subgroup (100 μg/mL Vit C), and A3 subgroup (200 μg/mL Vit C). Group B was reassigned to B0 subgroup (control group), B1 subgroup (100 ng/mL TNF-α), B2 subgroup (100 μg/mL Vit C+100 ng/mL TNF-α), and B3 subgroup (200 μg/mL Vit C+100 ng/mL TNF-α). Group C was reassigned to C0 subgroup (Control group), C1 subgroup (2% FBS), C2 subgroup (2%FBS+100 μg/mL Vit C), and C3 subgroup (2% FBS+200 μg/mL Vit C). After C1 subgroup (2% FBS), C2 subgroup (2%FBS+100 μg/mL Vit C), and C3 subgroup (2% FBS+200 μg/mL Vit C). After application of 100 μg/mL or 200 μg/mL Vit C for 24 hours, NP cells were stimulated by TNF-α and serum deprivation, then the apoptosis rate of NP cells was detected by a flow cytometry, and the gene expressions of the extracellular matrix of NP cells (collagen type Ⅰ, collagen type Ⅱ, aggrecan, and Sox9) and apoptosis related genes (p53, FAS, and Caspase 3) were detected by real-time fluoroscent quantitative PCR. Results Group A:Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 of NP cells in A2 and A3 subgroups when compared with A1 subgroup (P<0.05), but there was no significant difference between A2 subgroup and A3 subgroup (P>0.05); Vit C could promote the expressions of the extracellular matrix (collagen type Ⅰ, collagen type Ⅱ, aggrecan, and Sox9) of NP cells in a concentration dependent manner (P<0.05). Group B:TNF-α significantly increased the apoptosis rate and the gene expressions of p53, FAS, and Caspase 3 in B1 subgroup when compared with B0 subgroup (P<0.05); however, Vit C significantly increased the apoptosis rate and the gene expressions in B2 subgroup, and significantly decreased them in B3 subgroup when compared with B1 subgroup (P<0.05). Group C:2% FBS significantly increased the apoptosis rate of NP cells and significantly reduced the gene expressions of p53, FAS, and Caspase 3 in C1 subgroup when compared with C0 subgroup (P<0.05); Vit C could significantly reduce the apoptosis rate and gene expressions of p53, FAS, and Caspase 3 in C3 subgroup, but it could significantly increase them in C2 subgroup when compared with C1 subgroup (P<0.05). Conclusion Vit C can promote the synthesis and secretion of extracellular matrix of NP cells. 200 μg/mL Vit C may delay the apoptosis induced by TNF-α and serum deprivation, indicating the potential therapeutic effect of Vit C on intervertebral disc degeneration.

Citation： DAILibing, LIUZhihe, LIANGWeiguo, YAOYicun, XUJiake, YEDongping, ZOULongqiang, SHENYan. EFFECT OF VITAMIN C ON APOPTOSIS OF NUCLEUS PULPOSUS CELLS INDUCED BY TUMOR NECROSIS FACTOR α AND SERUM DEPRIVATION. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(4): 490-497. doi: 10.7507/1002-1892.20150105 Copy

1.	Zhao CQ, Jiang LS, Dai LY. Programmed cell death in intervertebral disc degeneration. Apoptosis, 2006, 11(12):2079-2088.
2.	Kokubun S, Sakurai M, Tanaka Y. Cartilaginous endplate in cervical disc herniation. Spine (Phila Pa 1976), 1996, 21(2):190-195.
3.	Esteban MA, Wang T, Qin B, et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 2010, 6(1):71-79.
4.	Shi Y, Zhao Y, Deng H. Powering reprogramming with vitamin C. Cell Stem Cell, 2010, 6(1):1-2.
5.	Rossig L, Hoffmann J, Hugel B, et al. Vitamin C inhibits endothelial cell apoptosis in congestive heart failure. Circulation, 2001, 104(18):2182-2187.
6.	Kluba T, Niemeyer T, Gaissmaier C, et al. Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc:effect of degeneration and culture system on cell phenotype. Spine (Phila Pa 1976), 2005, 30(24):2743-2748.
7.	韩成龙, 朱洪勋. 髓核细胞表型标记的研究进展. 中国修复重建外科杂志, 2011, 25(7):867-870.
8.	Murata Y, Rydevik B, Nannmark U, et al. Local application of interleukin-6 to the dorsal root ganglion induces tumor necrosis factor-alpha in the dorsal root ganglion and results in apoptosis of the dorsal root ganglion cells. Spine (Phila Pa 1976), 2011, 36(12):926-932.
9.	Wei A, Brisby H, Chung SA, et al. Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis. Spine J, 2008, 8(3):466-474.
10.	Zhang CC, Zhou JS, Hu JG, et al. Effects of IGF-1 on IL-1beta-induced apoptosis in rabbit nucleus pulposus cells in vitro. Mol Med Rep, 2013, 7(2):441-444.
11.	Kawaguchi S, Yamashita T, Katahira G, et al. Chemokine profile of herniated intervertebal discs infilltrated with monocytes and macrophages. Spine (Phila Pa 1976), 2002, 27(14):1511-1516.
12.	Valdes AM, Hassett G, Hart DJ, et al. Radiographic progression of lumbar spine disc degeneration is influenced by variation at inflammatory genes:a candidate SNP association study in the Chingford cohort. Spine (Phila Pa 1976), 2005, 30(21):2445-2451.
13.	Nam V, Christopher S, Paulo C, et al. Identification of novel TNF-α regulated extracellular matrix and cell-matrix adhesion genes in intervertebral disc cells:A microarray gene profiling of human nucleus pulposus cells. Spine Society, 2008, 8:99s-100s.
14.	Choi KM, Seo YK, Yoon HH, et al. Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation. J Biosci Bioeng, 2008, 105(6):586-594.
15.	Nandi D, Patra RC, Swarup D. Effect of cysteine, methionine, ascorbic acid and thiamine on arsenic-induced oxidative stress and biochemical alterations in rats. Toxicology, 2005, 211(1-2):26-35.
16.	Clegg HV, Zhang Y. Regulation of the p53 tumor suppressor pathway:the problems and promises of studying Mdm2's E3 ligase function. Crit Rev Eukaryot Gene Expr, 2010, 20(1):77-86.
17.	Zhang H, Gu C, Yu J, et al. Radiosensitization of glioma cells by TP53-induced glycolysis and apoptosis regulator knockdown is dependent on thioredoxin-1 nuclear translocation. Free Radic Biol Med, 2014, 69:239-248.
18.	Halfter UM, Derbyshire ZE, Vaillancourt RR. Interferon-gamma-dependent tyrosine phosphorylation of MEKK4 via Pyk2 is regulated by annexin Ⅱ and SHP2 in keratinocytes. Biochem J, 2005, 388(Pt 1):17-28.
19.	Gerin I, Noël G, Bolsée J, et al. Identification of TP53-induced glycolysis and apoptosis regulator (TIGAR) as the phosphoglycolate-independent 2,3-bisphosphoglycerate phosphatase. Biochem J, 2014, 458(3):439-448.
20.	Wanka C, Steinbach JP, Rieger J. Tp53-induced glycolysis and apoptosis regulator (TIGAR) protects glioma cells from starvation-induced cell death by up-regulating respiration and improving cellular redox homeostasis. J Biol Chem, 2012, 287(40):33436-33446.
21.	Pan Y, Oprysko PR, Asham AM, et al. p53 cannot be induced by hypoxia alone but responds to the hypoxic microenvironment. Oncogene, 2004, 23(29):4975-4983.
22.	Sansome C, Zaika A, Marchenko ND, et al. Hypoxia death stimulus induces translocation of p53 protein to mitochondria. Detection by immunofluorescence on whole cells. FEBS Lett, 2001, 488(3):110-115.
23.	An SH, Kang JH, Kim DH, et al. Vitamin C increases the apoptosis via up-regulation p53 during cisplatin treatment in human colon cancer cells. BMB Rep, 2011, 44(3):211-216.
24.	Kim J, Lee SD, Chang B, et al. Enhanced antitumor activity of vitamin C via p53 in cancer cells. Free Radic Biol Med, 2012, 53(8):1607-1615.
25.	Petak I, Tillman DM, Houghton JA. p53 dependence of Fas induction and acute apoptosis in response to 5-fluorouracil-leucovorin in human colon carcinoma cell lines. Clin Cancer Res, 2000, 6(11):4432-4441.
26.	Lee SH, Oe T, Blair IA. Vitamin C-induced decomposition of lipid liydroperoxides to endogenous genotoxins. Science, 2001, 292(5524):2083-2086.
27.	曾锦荣, 蒋碧佳, 王绩英. 三氧化二砷联合维生素C对耐药性肺腺癌裸鼠移植瘤Caspase-3、Survivin活性的影响. 中国老年学杂志, 2011, 24(31):4827-4831.
28.	Galluzzi L, Morselli E, Kepp O, et al. Defective autophagy control by the p53 rheostat in cancer. Cell Cycle, 2010, 9(2):250-255.
29.	Maiuri MC, Galluzzi L, Morselli E, et al. Autophagy regulation by p53. Curr Opin Cell Biol, 2010, 22(2):181-185.
30.	Ascher E, Scheinman M, Hingorani A, et al. Effect of p53 gene therapy combined with CTLA4Ig selective immunosuppression on prolonged neointima formation reduction in a rat model. Ann Vasc Surg, 2000, 14(4):385-392.
31.	迟立群, 李功宋, 高长青, 等. 野生型p53基因转染和低能量激光照射防治移植静脉再狭窄的实验研究. 中华胸心血管外科杂志, 2003, 19(1):50-53.
32.	Bouvet M, Ellis LM, Nishizaki M, et al. Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human colon cancer. Cancer Res, 1998, 58(11):2288-2292.
33.	Xue Y, Li NL, Yang JY, et al. Phosphatidylinositol 3'-kinase signaling pathway is essential for Rac1-induced hypoxia-inducible factor-1(alpha) and vascular endothelial growth factor expression. Am J Physiol Heart Circ Physiol, 2011, 300(6):H2169-2176.
34.	Qin G, Kishore R, Dolan CM, et al. Cell cycle regulator E2F1 modulates angiogenesis via p53-dependent transcriptional control of VEGF. Proc Natl Acad Sci U S A, 2006, 103(29):11015-11020.

1. Zhao CQ, Jiang LS, Dai LY. Programmed cell death in intervertebral disc degeneration. Apoptosis, 2006, 11(12):2079-2088.
2. Kokubun S, Sakurai M, Tanaka Y. Cartilaginous endplate in cervical disc herniation. Spine (Phila Pa 1976), 1996, 21(2):190-195.
3. Esteban MA, Wang T, Qin B, et al. Vitamin C enhances the generation of mouse and human induced pluripotent stem cells. Cell Stem Cell, 2010, 6(1):71-79.
4. Shi Y, Zhao Y, Deng H. Powering reprogramming with vitamin C. Cell Stem Cell, 2010, 6(1):1-2.
5. Rossig L, Hoffmann J, Hugel B, et al. Vitamin C inhibits endothelial cell apoptosis in congestive heart failure. Circulation, 2001, 104(18):2182-2187.
6. Kluba T, Niemeyer T, Gaissmaier C, et al. Human anulus fibrosis and nucleus pulposus cells of the intervertebral disc:effect of degeneration and culture system on cell phenotype. Spine (Phila Pa 1976), 2005, 30(24):2743-2748.
7. 韩成龙, 朱洪勋. 髓核细胞表型标记的研究进展. 中国修复重建外科杂志, 2011, 25(7):867-870.
8. Murata Y, Rydevik B, Nannmark U, et al. Local application of interleukin-6 to the dorsal root ganglion induces tumor necrosis factor-alpha in the dorsal root ganglion and results in apoptosis of the dorsal root ganglion cells. Spine (Phila Pa 1976), 2011, 36(12):926-932.
9. Wei A, Brisby H, Chung SA, et al. Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis. Spine J, 2008, 8(3):466-474.
10. Zhang CC, Zhou JS, Hu JG, et al. Effects of IGF-1 on IL-1beta-induced apoptosis in rabbit nucleus pulposus cells in vitro. Mol Med Rep, 2013, 7(2):441-444.
11. Kawaguchi S, Yamashita T, Katahira G, et al. Chemokine profile of herniated intervertebal discs infilltrated with monocytes and macrophages. Spine (Phila Pa 1976), 2002, 27(14):1511-1516.
12. Valdes AM, Hassett G, Hart DJ, et al. Radiographic progression of lumbar spine disc degeneration is influenced by variation at inflammatory genes:a candidate SNP association study in the Chingford cohort. Spine (Phila Pa 1976), 2005, 30(21):2445-2451.
13. Nam V, Christopher S, Paulo C, et al. Identification of novel TNF-α regulated extracellular matrix and cell-matrix adhesion genes in intervertebral disc cells:A microarray gene profiling of human nucleus pulposus cells. Spine Society, 2008, 8:99s-100s.
14. Choi KM, Seo YK, Yoon HH, et al. Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation. J Biosci Bioeng, 2008, 105(6):586-594.
15. Nandi D, Patra RC, Swarup D. Effect of cysteine, methionine, ascorbic acid and thiamine on arsenic-induced oxidative stress and biochemical alterations in rats. Toxicology, 2005, 211(1-2):26-35.
16. Clegg HV, Zhang Y. Regulation of the p53 tumor suppressor pathway:the problems and promises of studying Mdm2's E3 ligase function. Crit Rev Eukaryot Gene Expr, 2010, 20(1):77-86.
17. Zhang H, Gu C, Yu J, et al. Radiosensitization of glioma cells by TP53-induced glycolysis and apoptosis regulator knockdown is dependent on thioredoxin-1 nuclear translocation. Free Radic Biol Med, 2014, 69:239-248.
18. Halfter UM, Derbyshire ZE, Vaillancourt RR. Interferon-gamma-dependent tyrosine phosphorylation of MEKK4 via Pyk2 is regulated by annexin Ⅱ and SHP2 in keratinocytes. Biochem J, 2005, 388(Pt 1):17-28.
19. Gerin I, Noël G, Bolsée J, et al. Identification of TP53-induced glycolysis and apoptosis regulator (TIGAR) as the phosphoglycolate-independent 2,3-bisphosphoglycerate phosphatase. Biochem J, 2014, 458(3):439-448.
20. Wanka C, Steinbach JP, Rieger J. Tp53-induced glycolysis and apoptosis regulator (TIGAR) protects glioma cells from starvation-induced cell death by up-regulating respiration and improving cellular redox homeostasis. J Biol Chem, 2012, 287(40):33436-33446.
21. Pan Y, Oprysko PR, Asham AM, et al. p53 cannot be induced by hypoxia alone but responds to the hypoxic microenvironment. Oncogene, 2004, 23(29):4975-4983.
22. Sansome C, Zaika A, Marchenko ND, et al. Hypoxia death stimulus induces translocation of p53 protein to mitochondria. Detection by immunofluorescence on whole cells. FEBS Lett, 2001, 488(3):110-115.
23. An SH, Kang JH, Kim DH, et al. Vitamin C increases the apoptosis via up-regulation p53 during cisplatin treatment in human colon cancer cells. BMB Rep, 2011, 44(3):211-216.
24. Kim J, Lee SD, Chang B, et al. Enhanced antitumor activity of vitamin C via p53 in cancer cells. Free Radic Biol Med, 2012, 53(8):1607-1615.
25. Petak I, Tillman DM, Houghton JA. p53 dependence of Fas induction and acute apoptosis in response to 5-fluorouracil-leucovorin in human colon carcinoma cell lines. Clin Cancer Res, 2000, 6(11):4432-4441.
26. Lee SH, Oe T, Blair IA. Vitamin C-induced decomposition of lipid liydroperoxides to endogenous genotoxins. Science, 2001, 292(5524):2083-2086.
27. 曾锦荣, 蒋碧佳, 王绩英. 三氧化二砷联合维生素C对耐药性肺腺癌裸鼠移植瘤Caspase-3、Survivin活性的影响. 中国老年学杂志, 2011, 24(31):4827-4831.
28. Galluzzi L, Morselli E, Kepp O, et al. Defective autophagy control by the p53 rheostat in cancer. Cell Cycle, 2010, 9(2):250-255.
29. Maiuri MC, Galluzzi L, Morselli E, et al. Autophagy regulation by p53. Curr Opin Cell Biol, 2010, 22(2):181-185.
30. Ascher E, Scheinman M, Hingorani A, et al. Effect of p53 gene therapy combined with CTLA4Ig selective immunosuppression on prolonged neointima formation reduction in a rat model. Ann Vasc Surg, 2000, 14(4):385-392.
31. 迟立群, 李功宋, 高长青, 等. 野生型p53基因转染和低能量激光照射防治移植静脉再狭窄的实验研究. 中华胸心血管外科杂志, 2003, 19(1):50-53.
32. Bouvet M, Ellis LM, Nishizaki M, et al. Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human colon cancer. Cancer Res, 1998, 58(11):2288-2292.
33. Xue Y, Li NL, Yang JY, et al. Phosphatidylinositol 3'-kinase signaling pathway is essential for Rac1-induced hypoxia-inducible factor-1(alpha) and vascular endothelial growth factor expression. Am J Physiol Heart Circ Physiol, 2011, 300(6):H2169-2176.
34. Qin G, Kishore R, Dolan CM, et al. Cell cycle regulator E2F1 modulates angiogenesis via p53-dependent transcriptional control of VEGF. Proc Natl Acad Sci U S A, 2006, 103(29):11015-11020.

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EFFECT OF VITAMIN C ON APOPTOSIS OF NUCLEUS PULPOSUS CELLS INDUCED BY TUMOR NECROSIS FACTOR α AND SERUM DEPRIVATION

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