Effect of Tetrodotoxin from Crude Puffer Fish (Tetraodon fluviatilis) Liver Extract on Intracellular Calcium Level and Apoptosis of HeLa Cell Culture

Natanael Untario, Titik Cinthia Dewi, M. Aris Widodo, Pudji Rahaju

Abstract


Cervical cancer is the third most commonly diagnosed cancer and fourth leading cause of women death with 8% of total death caused by cancer in women in 2008. Tetrodotoxin (TTX) is a potent neurotoxin found in inner organs puffer fish, with the specific mechanism of sodium channel blocking, and widely used for research purposes. Previous reports claimed that TTX has the capability of inhibiting the metastatic process of cancer and apoptotic effect. Studies also show that apoptosis is a process involving the increase of intracellular calcium level, yet the connection between TTX and increase of intracellular calcium level, therefore triggering apoptosis, has not been established. This is an experimental study with post test only control group design, carried out by exposing HeLa cell culture to a crude liver extract of a puffer fish species, Tetraodon fluviatilis. Crude puffer fish liver extract is administered into HeLa cell culture well in different concentrations 10-4, 10-2, and 10-1. Intracellular calcium level and apoptosis were then measured after 18 hours of incubation. Measurements of intracellular calcium level were done by using CLSM with Fura-2AM staining, and apoptosis by using flowcytometry with Annexin V/PI.  The result shows that there is a significant difference between samples both in intracellular calcium (p < 0.05) and apoptosis (p < 0,05). Both intracellular calcium and apoptosis levels are proportional to liver fish extract concentration. Pearson’s correlation test shows correlation between treatment and intracellular calcium levels (p = 0.000), between treatment and apoptosis (p = 0.002), but not between intracellular calcium and apoptosis (p = 0.05). These results suggest that TTX induces an increase in intracellular calcium level and apoptosis, but calcium pathway is not the sole cause of the apoptosis. 


Keywords


Apoptosis, HeLa cell culture, intracellular calcium, Tetraodon fluviatilis, tetrodotoxin

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References


Ferlay J, Shin HR, Bray F et al. (2010) Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. International Journal of Cancer 127: 2893–2917.

Gupta A, Mazumder UK, Kumar RS, Kumar TS (2004) Anti-tumor activity and anti-oxident role of Bauhinia racemosa against Ehrlich ascites carcinoma in Swiss albino mice. Acta Pharmacologica Sinica 25 (8): 1070–1076.

X-Plain (2011) Patient education: Chemoterapy. http://online.x-plain.com/. Accessed: March 2015.

Benzer TI (2007) Toxicity, Tetrodotoxin. http://emedicine.medscape.com/. Accessed: March 2015.

Hagen NA, Fisher KM, Lapointe B et al. (2007) An open-label, multi-dose efficacy and safety study of intramuscular tetrodotoxin in patients with severe cancer-related pain. Journal of Pain Symptom Manage 34 (2): 171-182.

Madhavan A, He L, Stuber GD et al. (2010) Micro-Opioid receptor endocytosis prevents adaptations in ventral tegmental area GABA transmission induced during naloxone-precipitated morphine withdrawal. The Journal of Neuroscience 30 (9): 3276–3286.

Zhao X, Yeh JZ, Narahashi T (2001) Post-stroke dementia. Nootropic drug modulation of neuronal nicotinic acetylcholine receptors. Annals of the New York Academy of Sciences 939: 179-186.

Brackenbury W, Djamgoz M (2007) Nerve growth factor enhances voltage-gated Na+ channel activity and Transwell migration in Mat-LyLu rat prostate cancer cell line. Journal of Cell Physiology 210 (3): 602–608.

Fouda FM (2005) Anti-tumor activity of tetrodotoxin extracted from the Masked Puffer fish Arothron diadematus. Egyptian Journal of Biology 7:1-13.

Orrenius S, Zhivotovsky B, Nicotera P (2003) Regulation of cell death: The calcium-apoptosis link. Nature Publisihing 4 (7): 552–565.

Zhou M, Shum FHK (2003) Method of extracting tetrodotoxin. https://www.google.com/patents/US6552191 Accessed: January 2015.

Asakawa M, Shida Y, Miyazawa K, Naguchi T (2012) Chromatography - The most versatile method of chemical analysis, Dr. Leonardo Calderon (Ed.), InTech. http://www.intechopen.com/ Accessed: February 2015. doi: 10.5772/48668.

Chakrabarti R, Chakrabarti R (2006) Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels. Journal of Cellular Biochemistry 99 (6): 1503–1516.

Bragadeeswaran S, Therasa D, Prabhu K, Kathiresan K (2010) Biomedical and pharmacological potential of tetrodotoxin-producing bacteria isolated from marine pufferfish Arothron hispidus (Muller, 1841). The Journal of Venomous Animals and Toxins including Tropical Diseases 16 (3): 421–431. doi: 10.1590/S1678-91992010000300008.

Abd El-Dayem SM, Fouda FM, Ali EH, Abd El Motelp BA (2012) The antitumor effects of tetrodotoxin and/or doxorubicin on Ehrlich Ascites carcinoma-bearing female mice. Toxicology and Industrial Health 29 (5): 404–417. doi: 10.1177/0748233711434955.

Rusnak M, Tóth ZE, House SB, Gainer H (2007) Depolarization and neurotransmitter regulation of vasopressin gene expression in the rat suprachiasmatic nucleus in vitro. The Journal of Neuroscience 27 (1): 141–151. doi: 10.1523/JNEUROSCI.3739-06.2007.

Ferri KF, Kroemer G (2001) Organelle-specific initiation of cell death pathways. Nature Cell Biology 3 (11): E255–E263.

Martinou JC, Desagher S, Antonsson B (2000) Cytochrome c release from mitochondria: all or nothing. Nature Cell Biology 2 (3): E41-E43.

Joza N, Susin SA, Daugas E et al. (2001) Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410 (6828): 549–554.

Verhagen AM, Ekert PG, Pakusch M et al. (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102 (1): 43–53.

Parrish J, Li L, Klotz K et al. (2001) Mitochondrial endonuclease G is important for apoptosis in C. elegans. Nature 412 (6842): 90–94. doi: 10.1038/35083608.

Hengartner MO (1998) Apoptosis: Death cycle and Swiss army knives. Nature 391: 441–442. doi:10.1038/35036

Abd El-Motelp BA, Zaazaa AM (2013) Potential antitumor effect of tetrodotoxin and/or quercetin against ehrlich carcinoma in mice. World Journal of Pharmacy and Pharmaceutical Sciences 2 (6): 6653–6667.

Tanaka T, Nangaku M, Miyata T et al. (2004) Blockade of calcium influx through L-type calcium channels attenuates mitochondrial injury and apoptosis in hypoxic renal tubular cells. Journal of the American Society of Nephrology 15 (9): 2320–2333. doi: 10.1097/01.ASN.0000138287.468

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DOI: http://dx.doi.org/10.11594/jtls.07.01.04

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