Silencing of Human CutC Gene (hCutC) Induces Apoptosis in HepG2 Cells

• Published in: Biological trace element research 2016-07, Vol.172 (1), p.120-126
• Main Authors: Kunjunni, Remesh, Sandeep Sathianathan, Madhuri Behari, Parthaprasad Chattopadhyay, Vivekanandhan Subbiah
• Format: Article
• Language: English
• Subjects: Apoptosis; Apoptosis - drug effects; Apoptosis - genetics; atomic absorption spectrometry; Biochemistry; Biomedical and Life Sciences; Biotechnology; Cation Transport Proteins - deficiency; Cation Transport Proteins - genetics; cell physiology; cell viability; Copper; Copper - metabolism; Copper - pharmacology; Copper Transport Proteins; DNA fragmentation; Dose-Response Relationship, Drug; gene expression regulation; Gene Silencing; genes; Hep G2 Cells; Homeostasis; human cell lines; Humans; Life Sciences; membrane potential; Morphology; Nutrition; Oncology; Physiology; Polymerase chain reaction; proteins; quantitative polymerase chain reaction; small interfering RNA; Spectrometry; Structure-Activity Relationship; transmission electron microscopy
• ISSN: 0163-4984; 1559-0720
• DOI: 10.1007/s12011-015-0577-z

Copper is an essential microelement required for maintaining normal cell physiology. Copper transporter CutC is one of the six members of Cut family proteins, involved in prokaryotic copper homeostasis. Human homolog of CutC (hCutC) is an intracellular copper-binding protein with unknown physiological function. In the present study using HepG2 cells, we report the effects of hCutC knockdown on copper sensitivity and morphology of cells that ultimately leads to apoptosis. We silenced hCutC using specific small interfering RNA (siRNA), and its downregulation was confirmed by quantitative real-time PCR. Though there was no significant variation in total cellular copper as estimated by inductively coupled plasma-atomic emission spectrometry (ICP-AES), knockdown of hCutC caused an increase in sensitivity of HepG2 cells to copper loads when compared to control cells (studied by MTT-based cell viability assay). Morphological analysis by transmission electron microscopy (TEM) indicated onset of apoptosis in hCutC-silenced cells which was exacerbated upon copper treatment. Mitochondrial transmembrane potential (ΔΨm) assay and DNA fragmentation assay further ensured apoptosis occurring in cells upon hCutC silencing. The present study reveals copper induced damage in cells upon hCutC silencing and provides evidence for the role of hCutC protein in intracellular copper homeostasis.