[Dynamic changes of ROS, MDA and SOD during arsenic-induced neoplastic transformation in human keratinocytes]

Abstract

Objective: To investigate the level of ROS, MDA and SOD in different stages of arsenic-induced neoplastic transformation in human keratinocytes.

Methods: HaCaT human immortalized keratinocytes were continuously exposed to 1.0 μmol/L arsenite for 35 passages. The secretion of active MMP-9, the proliferation rate and doubling time nd colony formation assay in soft agar colony were used to identify the malignant phenotype of the arsenite-exposed HaCaT cells. Then flow cytometry was used to detect the levels of ROS, and the level of MDA and SOD were tested by biochemical method at different passages of arsenite exposure in HaCaT cells.

Results: A marked increase in the secretion of active MMP-9 in the arsenic-treated (1.0 μmol/L NaAsO2) cells was observed in comparison to the passage-matched untreated control (0.0 μmol/L NaAsO2) cells at 28 and 35 passages. And compared with 0.0 μmol/L NaAsO2 group, the proliferation rate and doubling time in 1.0 μmol/L NaAsO2 group was much faster at 21 ((64.37 ± 15.92) h) and 28 ((64.04 ± 12.84) h) passages with a significant statistical difference at passage 35 ((54.00 ± 2.35 ) h) (P < 0.05). Furthermore, the long-term arsenite-treated cells formed significantly higher colonies (107 ± 11 in passage 35) in soft agar than control cells (P < 0.05). No obvious regularity changes of ROS and MDA levels were found before 14 passages of arsenite exposure, except for passage 1. Surprisingly, after 14 passages, with the increased passages of exposure to arsenite, both the ROS and MDA levels decreased gradually, the ROS level at passage 35 was significant lower compared to passage 0 (P < 0.05). Conversely, after passage 21, the activity of SOD was obviously enhanced and reached the highest level at passage 35.

Conclusion: Long-term exposure to low concentrations of inorganic arsenic-induced malignant transformation of HaCaT cells is accompanied by intracellular imbalance between oxidative-antioxidant, which increased expression of SOD and low levels of ROS found in the later-stage of arsenite-induced transformation.