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Background: The human cancer-related nucleoside triphosphatase (HCR-NTPase) is overexpressed in several tumour tissues including neuroblastoma. HCR-NTPase is an enzyme exhibiting a slow in vitro activity in hydrolysing nucleosidetriphosphates. However, its in vivo function is still unknown. To learn more about the physiological role of HCR-NTPase, we both overexpressed and silenced it in the neuroblastoma cell line SH-SY5Y. Findings: No effect was observed when the expression of endogenously expressed HCR-NTPase in the cells was silenced by RNA interference. On the other hand, overexpression of HCR-NTPase led to cytotoxicity of the protein in SH-SY5Y cells. Even if the catalytic essential amino acid glutamate 114 was replaced by alanine (E114A-HCR-NTPase), the protein remained cytotoxic. The results could be confirmed by successfully rescuing the cells via RNA interference. Conclusion: Although expressed in several tumours, at least in SH-SY5Y, HCR-NTPase is not essential for the cells to survive. Increased levels of the protein lead to cytotoxicity due to physical intracellular interactions rather than hydrolysis of nucleosidetriphosphates by its intrinsic residual enzymatic activity.