Knockdown of copper chaperone antioxidant-1 by RNA interference inhibits copper-stimulated proliferation of non-small cell lung carcinoma cells

Abstract

Copper is required for cell proliferation and tumor angiogenesis. Cellular copper metabolism is regulated by a network of copper transporters and chaperones. Antioxidant-1 (ATOX1) is a cytosolic copper chaperone important for intracellular copper transport, which plays a role in the regulation of cell proliferation by functioning as a transcription factor in cell growth signal-transduction pathways. The present study aimed to explore the role of ATOX1 in the copper-related regulation of lung cancer cell proliferation by immunohistochemical (IHC) analysis of ATOX1 expression in non-small cell lung cancer (NSCLC) tissue samples and by assessing the effects of RNA interference (RNAi)-mediated knockdown of ATOX1 on copper-stimulated proliferation of NSCLC cells. Overexpression of ATOX1 was detected in NSCLC by IHC analysis of the tissue samples from patients diagnosed with NSCLC when compared with expression of ATOX1 in non-malignant lung tissue samples. Knockdown of ATOX1 in the NSCLC cells transduced by a lentiviral vector encoding short hairpin RNA (shRNA) specific for ATOX1 was associated with reduction in copper-stimulated cell proliferation. These findings suggest that ATOX1 plays an important role in copper-stimulated proliferation of NSCLC cells and ATOX1 holds potential as a therapeutic target for lung cancer therapy targeting copper metabolism.

Figure 1

Expression of copper chaperones in NSCLC by immunohistochemical (IHC) analysis. (A) Representative IHC analysis of the expression of ATOX1, Cox17 and CCS in NSCLC tissue samples. (B) Representative IHC analysis of expression of ATOX1, Cox17, and CCS in non-malignant lung tissue samples. A high level of expression of ATOX1 and Cox17 was detected in NSCLC tissue samples, when compared with the expression of these two copper chaperones in the control non-malignant lung tissue samples. In contrast, a high level of expression of CCS was detected in the NSCLC and non-malignant lung tissue samples. Scale bar, 100 μM. ATOX1, antioxidant 1; Cox17, cytochrome c oxidase 17; CCS, copper chaperone for superoxide dismutase.

Figure 2

Expression of copper chaperones in NSCLC cells by western immunoblot assay. Expression of ATOX1 was detected in all of 6 NSCLC cell lines, at a similar expression level. Expression of Cox17 was variable, with the expression level of Cox17 in the H1355 cell lines lower than levels in the other cell lines. Expression of CCS in NSCLC cells was also variable, with its expression levels in A549 and H1703 lower than levels in the other 4 NSCLC cell lines. β-actin was used as a loading control.

Figure 3

RNAi-mediated knockdown of ATOX1 in NSCLC cells by western immunoblot assay. (A) Knockdown of ATOX1 in the SKLU-1 NSCLC cells transfected with pATOX1-shRNA plasmid vector #2 and #3 as determined by western blot assay, but not in the cells transfected with pATOX1-shRNA plasmid vector # 1, #4, or pSCR-shRNA plasmid (SCR). (B) Knockdown of ATOX1 in H1355 and A549 NSCLC cells infected with the Lenti-ATOX1-shRNA lentivirus encoding the ATOX1 shRNA sequence derived from pATOX1-shRNA plasmid vector #2, but not in the wild-type cells, and the H1355 or A549 cells infected with Lenti-SCR-shRNA virus. β-actin was used as the loading control.

Figure 4

Reduction in the copper-stimulated proliferation of NSCLC cells by RNAi-mediated knockdown of ATOX1. (A) Schematic plot showing reduction in the copper-stimulated proliferation of the H1355 NSCLC cells infected with lenti-ATOX1-shRNA virus (shATOX1-H1355) cultured in medium containing CuCl₂ dissolved in PBS buffer at a concentration of 10 μM (shATOX1-H1355 + Cu), compared with that of the control H1355 cells infected with Lenti-SCR-shRNA virus (SCR-H1355 + Cu). No significant difference was detected between the growth of the shATOX1-H1355 cells and that of the SCR-H1355 cells cultured in the medium containing no CuCl₂ (shATOX1-H1355 + PBS and SCR-H1355 + PBS). (B) Schematic plot showing reduction in the copper-stimulated proliferation of the shATOX1-A549 cells cultured in medium containing 10 μM CuCl₂ (shATOX1-A549 + Cu), compared with that of the control H1355 cells infected with the Lenti-SCR-shRNA virus (SCR-A549 + Cu). Again, no significant difference was detected between the growth of the shATOX1-A549 cells and that of the SCR-A549 cells cultured in medium containing no CuCl₂ (shATOX1-A549 + PBS and SCR-A549 + PBS). Cell number (%) is the percentage of the initially inoculated cells (0 h) at the time of the MTT assay.