The effect of antioxidants on UV-irradiated melanocytes and melanoma cells

Banjar, A 2015, The effect of antioxidants on UV-irradiated melanocytes and melanoma cells, Doctor of Philosophy (PhD), Medical Sciences, RMIT University.


Document type: Thesis
Collection: Theses

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Title The effect of antioxidants on UV-irradiated melanocytes and melanoma cells
Author(s) Banjar, A
Year 2015
Abstract The effect of ultraviolet radiation (UVR) on cell viability, melanin content, ROS levels, Tumour Necrosis Factor-α (TNF-α), TNF-α Converting Enzyme (TACE), and furin expression as well as the phosphorylation of Mitogen-activated protein kinases (MAPK) including p-38, c-Jun N-terminal kinases (JNK), extracellular-signal-regulated kinases (ERK), and B-RAF were investigated. The effect of vitamin C (1 mM) or the vitamin E analogue trolox (0.1 mM) on the above mentioned changes were examined using human lightly pigmented MM418-C1, darkly pigmented MM418-C5 melanoma cells and primary melanocytes (HEM).

Cell viability was measured 24 h post-UV-irradiation. MM418-C1 and MM418-C5 cells were more sensitive to UVB and UVAB radiation than were HEM cells. Only vitamin C conferred a protective effect to UVB-irradiated MM418-C1 cells, but not the other cells.

The effect of acute or chronic doses of UVR were examined to see if a single UV dose enhanced tyrosinase expression and increased melanin levels to a greater extent than did two smaller doses given 24 h apart. Acute and chronic doses of UVA or/and UVB did not significantly alter tyrosinase expression. Only acute doses of UVB and UVAB radiation significantly increased melanin levels in MM418-C1 cells. Antioxidant treatment had no effect on intracellular melanin levels in the irradiated cells.

Intracellular ROS (peroxide but not superoxide levels) were increased in all cells exposed to UVB and UVAB radiation. Melanin may have a negative regulatory effect post-UVR exposure, as the least pigmented cells (MM418-C1 and HEM cells) had the highest increase in peroxide levels.

TNF-α levels released from MM418-C1 cells were higher than those from MM418-C5 cells. Exposure to UVB and UVAB greatly increased the levels of TNF-α secreted from MM418-C1 cells but not MM418-C5 cells. Similarly, Interleukin-1α (IL-1α) stimulated the release of TNF-α from MM418-C1 cells but not MM418-C5 cells. Neither TACE nor furin expression, were altered post-UVR exposure in both melanoma cell lines, even if IL-1α was present. Antioxidant treatment did not affect TNF-α release, nor the expression of TACE and furin. As TNF-α levels released from UV-irradiated MM418-C1 cells were higher than that from MM418-C5 cells, it suggests that high melanin levels may negatively regulate TNF-α formation/release from these irradiated cells.

The effect of antioxidants on intracellular signalling pathways in the UV-irradiated cells was examined. Intracellular p-p38 and p-JNK levels reached a peak 30 min post-irradiation. The highest increase in p-p38 and p-JNK levels was seen in the UV-irradiated MM418-C5 cells. Antioxidant treatment did not affect the expression of these signalling intermediates in all cell lines. It seems that following exposure to UVR, high melanin levels increased p38 signalling activity but reduced JNK-1 signalling activity, which was opposite to that seen in cells containing less melanin.

In summary, vitamin C and trolox did not confer protection to both pigmented melanoma cell lines and melanocytes from UVR-induced cell death. We conclude that the use of UVR doses causing 50% cell death probably overwhelmed the ability of these cells to overcome UV-induced damage, thereby negating any potential protective effect that may have been conferred by the antioxidants.

Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Medical Sciences
Keyword(s) Antioxidants
Melanoma
Melanocytes
Ultraviolet radiation
Melaninogenesis
Cell signalling
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Created: Fri, 06 Nov 2015, 10:21:44 EST by Denise Paciocco
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