Cellular and Biochemical Alterations in L6 Myoblast Cells Induced by 6-Aminonicotinamide

  • Published : 2007.06.30

Abstract

The effects of antimetabolite 6-AN (6-amino-nicotinamide) on viability and morphology of L6 myoblast cells have been investigated. 6-AN ($100{\mu}M$) induced a time-dependent decrease in cell viability with respect to the untreated control cells. Following 6-AN administration the viability rate started to decline sharply, reaching about 23% of the untreated control cells at 48 h. Inverted phase-contrast microscopy revealed that 6-AN caused characteristic morphological changes such as irregularly elongated and stellate shape of cells, round-shaped nucleus, cytoplasmic vacuolization, irregular cell arrangements and formation of large spaces among cell clusters. The concentrations of ATP and $NAD^{+}$ in the 6-AN treated cells were significantly lower (p < 0.01) than those of the untreated control cells. In contrast, the concentration of AMP was significantly increased by the 6-AN treatment. Activities of catalase, superoxide dismutase and glutathione peroxidase in 6-AN treated cells were significantly higher (p < 0.01) than those of the untreated control cells. The activities of glyceraldehyde-3-phosphate dehydrogenase in 6-AN treated cells were significantly lower (p < 0.01) than those of the untreated control cells. The results suggest that 6-AN caused marked reduction of cell viability and alterations of some important metabolites and enzymes.

Keywords

References

  1. Bai J, Rodriguez AM, Melendez JA, and Cederbaum AI (1999) Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury. J Biol Chem 274: 26217-26224 https://doi.org/10.1074/jbc.274.37.26217
  2. Bielicki L, and Krieglstein J (1976) Decreased GABA and glutamate concentration in rat brain after treatment with 6- aminonicotinamide. Naunyn-Schmiedeberg's Arch Pharmacol 294: 157-160 https://doi.org/10.1007/BF00507848
  3. Eliasson MJ, Sampei K, Mandir AS, Hurn PD, Traystma RJ, Bao J, Pieper A, Wang ZQ, Dawson TM, and Snyder SH (1997) Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral-ischemia. Nat Med 3: 1089-1095 https://doi.org/10.1038/nm1097-1089
  4. Engliez SA, and Park IK (2005) 6-aminonicotinamide induces G1 arrest by elevating $p27^{kip1}$ as well as inhibiting cdk2, cyclin E and p-Rb in IMR32 neuroblastoma cell line. Integrative Bioscience 9: 191-198 https://doi.org/10.1080/17386357.2005.9647270
  5. Fong Y, Moldawer LL, Marano MA, Wei H, Barber A, Fisehman DA, and Lowry SF (1989) starvation leads to decreased levels of MRNA for proteins. J Surg Res 46: 457-461 https://doi.org/10.1016/0022-4804(89)90160-1
  6. Fritz PJ (1967) Rabbit lactate dehydrogenase isozymes. Science 156: 82-84 https://doi.org/10.1126/science.156.3771.82
  7. Griffiths IR, Kelley PA, and Grome JJ (1981) Glucose utilization in the central nervous system in the acute gliopathy due to 6- aminonicotinamide. Lab. Invest 44: 547-5542
  8. Haghighat N, and McCandless DW (1996) Effect of 6- aminonicotinamide on metabolism of astrocytes and C6- glioma cells. Metab Brain Dis 12: 29-45 https://doi.org/10.1007/BF02676352
  9. Herouart CR, Van Montagu M, and Inze D (1993) Redoxactivated expression of the cytosolic copper/zinc superoxide dismutase gene in Nicotiana. Proc Natl Acad Sci 90: 3108- 3112
  10. Hunting D, Gowans B, and Henderson JF (1985) Effects of 6- aminonicotinamide on cell growth, poly(ADP-ribose) synthesis and nucleotide metabolism. Biochem Pharmacol 34: 3999- 4003 https://doi.org/10.1016/0006-2952(85)90379-X
  11. Jung HK, and Park IK (1992) Effects of 6-aminonicotinamide on nucleotide and catecholamine. Korean J Zool 35: 23-28
  12. Kim HI, and Park IK (1995) Effects of prolonged starvation on acitivities of malic enzyme and acetylcholinesterase in tissues of quail. Int J Biochem Cell Biol 27: 1161-1167 https://doi.org/10.1016/1357-2725(95)00088-7
  13. Kim JY, and Park IK (1998) Effects of 6-aminonicotinamide on levels of soluble proteins and enzyme acitivities in various tissues of Japanese quail. Int J Biochem Cell Biol 30: 1337- 1344 https://doi.org/10.1016/S1357-2725(98)00102-2
  14. Klaidman LK, Mukherjee SK, and Adams JD Jr (2001) Oxidative changes in brain pyridine nucleotides and neuroprotection using nicotinamide. Biochim Biophys Acta 1525: 136-148 https://doi.org/10.1016/S0304-4165(00)00181-1
  15. Kohler E, Barrach HJ, and Neubert D (1970) Inhibition of NADP dependent oxidoreductases by the 6-aminonicotinamide analogue of NADP. FEBS Lett 6: 225-228 https://doi.org/10.1016/0014-5793(70)80063-1
  16. Krieglstein J, and Stock R (1975) Decreased glycolytic flux rate in the isolated perfused rat brain after pretreatment with 6- aminonicotinamide in rats. Naunyn-Schmiedeberg's Arch Pharmacol 290: 323-327 https://doi.org/10.1007/BF00510561
  17. Martin DS, Stolfi RL, Colofiore JR, and Nord LD (1996) Marked enhancement in vivo of paclitaxel's tumorregressing activity by ATP depleting modulation. Anticancer Res 7: 655-659 https://doi.org/10.1097/00001813-199608000-00006
  18. Lee YB, and Park IK (2001) Effects of neurotoxin 6-aminonicotinamide on levels of enzyme acitivities and metabolites in quail plasma. Int J Biochem Cell Biol 33: 613-620 https://doi.org/10.1016/S1357-2725(01)00036-X
  19. Leist M, Single B, Castoldi AF, Kuhnle S, and Nicotera P(1997) Intracellular ATP concentration: A switch in the decision between apoptosis and necrosis. J Expt Med 185: 1481-1486 https://doi.org/10.1084/jem.185.8.1481
  20. Mossmann T (1983) Rapid colorimetric assay for the cellular growth and survival: application to the proliferation and cytotoxicity assay. J Immunol Methods 65: 55-63 https://doi.org/10.1016/0022-1759(83)90303-4
  21. Ogata S, Takeuchi M, Fujita H, Shibata K, Okumura K, and Taguchi H (2000) Apoptosis induced by nicotinamide-related compounds and quinolinic acid in HL-60 cells. Biosci Biotechnol Biochem 64: 327-332 https://doi.org/10.1271/bbb.64.327
  22. Paglia DE, and Valentine WN (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70: 158-169
  23. Park IK, Shin S, and Marquardt RR (1991) Effects of niacin deficiency on the relative turnover rates of proteins in various tissues of quail. Int J Biochem 23: 1005-1012 https://doi.org/10.1016/0020-711X(91)90137-C
  24. Park IK, and Marquardt RR (1996) Effects of niacin deficiency on the thermal stability of NAD- and NADP-dependent dehydrogenases in liver and pectoral muscle of quail. Int J Biochem Cell Biol 28: 1169-1178 https://doi.org/10.1016/1357-2725(96)00047-7
  25. Penkowa M, Camats J, Hadberg H, Quintana A, Rojas S, Giralt M, Molinero A, Campbell IL, and Hidalgo J (2003) Astrocytetargeted expression of interleukin-6 protects the central nervous system during neurological degeneration induced by 6- aminonicotinamide. J Neurosci Res 73: 481-496 https://doi.org/10.1002/jnr.10681
  26. Schraufstatter IU, Hinshaw DB, Hyslop PA, Spragg RG, and Cochrane CG (1986) Oxidant injury cells: DNA strand-breaks activate PARP polymerase and lead to depletion of NAD. J Clin Invest 77: 1312-1320 https://doi.org/10.1172/JCI112436
  27. Shapiro DM, Dietrich LS, and Shils ME (1956) Quantitative biochemical differences between tumor and host as a basis for cancer chemotherapy. Cancer Res 17: 600-604
  28. Sternberg SS, and Philips FS (1958) 6-Aminonicotinamide and acute degenerative changes in the central nervous system. Science 127: 644-645
  29. Street JC, Mahmood U, Ballon D, Alfieri AA, and Koutcher JA (1996) $^{13}C$ and $^{31}P$ NMR investigation of effects of 6- aminonicotinamide on metabolism of RIF-1 tumor cells in vitro. J Biol Chem 271: 4113-4119 https://doi.org/10.1074/jbc.271.8.4113
  30. Sweet S, and Singh G (1995) Accumulation of human promyelocytic leukemia at two energetic cell cycle checkpoints. Cancer Res 55: 5164-5167
  31. Velick SF (1963) Glyceraldehyde-3-phosphate dehydrogenase, In: P.D. Boyer, H. Lardy, K. Myrback, Editors, The Enzymes 7243
  32. Wheeler CR, Salzman JA, Elsayed NM, Omaye ST, and Korte DW Jr (1990) Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity. Anal Biochem 184: 193-199 https://doi.org/10.1016/0003-2697(90)90668-Y
  33. Yang YC, Kim JY, and Park IK (1998) Neurotoxin 6- aminonicotinamide affects levels of soluble proteins and enzyme acitivities in various tissues of golden hamsters. Int J Biochem Cell Biol 32: 549-556 https://doi.org/10.1016/S1357-2725(99)00150-8
  34. Zhang J, Dawson VL, Dawson TM, and Snyder SH (1994) Nitric oxide activation of poly(ADP-ribose) synthetase in neurotoxicity. Science 265: 722-723 https://doi.org/10.1126/science.8047876
  35. Zeitz M, Lange K, Keller K, and Herken H (1978) Effects of 6- aminonicotinamide on growth and acetylcholinesterase activity during differentiation of neuroblastoma cells in vitro. Naunyn-Schmiedeberg's Arch Pharmacol 305: 117-121 https://doi.org/10.1007/BF00508280