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01.12.2018 | Research article | Ausgabe 1/2018 Open Access

BMC Cancer 1/2018

Sequential decitabine and carboplatin treatment increases the DNA repair protein XPC, increases apoptosis and decreases proliferation in melanoma

Zeitschrift:
BMC Cancer > Ausgabe 1/2018
Autoren:
Timothy Budden, Andre van der Westhuizen, Nikola A. Bowden
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12885-018-4010-9) contains supplementary material, which is available to authorized users.

Abstract

Background

Melanoma has two key features, an over-representation of UV-induced mutations and resistance to DNA damaging chemotherapy agents. Both of these features may result from dysfunction of the nucleotide excision repair pathway, in particular the DNA damage detection branch, global genome repair (GGR). The key GGR component XPC does not respond to DNA damage in melanoma, the cause of this lack of response has not been investigated. In this study, we investigated the role of methylation in reduced XPC in melanoma.

Methods

To reduce methylation and induce DNA-damage, melanoma cell lines were treated with decitabine and carboplatin, individually and sequentially. Global DNA methylation levels, XPC mRNA and protein expression and methylation of the XPC promoter were examined. Apoptosis, cell proliferation and senescence were also quantified. XPC siRNA was used to determine that the responses seen were reliant on XPC induction.

Results

Treatment with high-dose decitabine resulted in global demethylation, including the the shores of the XPC CpG island and significantly increased XPC mRNA expression. Lower, clinically relevant dose of decitabine also resulted in global demethylation including the CpG island shores and induced XPC in 50% of cell lines. Decitabine followed by DNA-damaging carboplatin treatment led to significantly higher XPC expression in 75% of melanoma cell lines tested. Combined sequential treatment also resulted in a greater apoptotic response in 75% of cell lines compared to carboplatin alone, and significantly slowed cell proliferation, with some melanoma cell lines going into senescence. Inhibiting the increased XPC using siRNA had a small but significant negative effect, indicating that XPC plays a partial role in the response to sequential decitabine and carboplatin.

Conclusions

Demethylation using decitabine increased XPC and apoptosis after sequential carboplatin. These results confirm that sequential decitabine and carboplatin requires further investigation as a combination treatment for melanoma.
Zusatzmaterial
Additional file 1: Table S1. XPC bisulfite promoter primers for PCR. (DOCX 14 kb)
12885_2018_4010_MOESM1_ESM.docx
Additional file 2: Figure S1. Representative bright-field microscopy images of senescence associated β-galactosidase staining in all four melanoma cell lines after combined decitabine and carboplatin treatment. Arrows indicate regions of positive staining, bar = 100 μm. (TIFF 42085 kb)
12885_2018_4010_MOESM2_ESM.tif
Additional file 3: Figure S2. DNA methylation pattern of the XPC CpG island in melanocytes and melanoma. Methylation levels in melanocytes (black) and each melanoma cell line at baseline (grey) was quantified by bisulfite sequencing. CpG position is shown relative to XPC transcription start site (TSS). Upstream (5′) shore = position − 2341 to − 423, CpG island = position − 364 to 568, Downstream (3′) shore = position 714 to 2596. (TIFF 603 kb)
12885_2018_4010_MOESM3_ESM.tif
Literatur
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