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

Timothy Budden,Nikola A Bowden,Andre Van Der Westhuizen

doi:10.1186/s12885-018-4010-9

Timothy Budden, Nikola A Bowden + Show 1 more

Open Access

https://doi.org/10.1186/s12885-018-4010-9

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Abstract

BackgroundMelanoma 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.MethodsTo 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.ResultsTreatment 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.ConclusionsDemethylation 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.

Highlights

Melanoma has two key features, an over-representation of Ultraviolet Radiation (UV)-induced mutations and resistance to DNA damaging chemotherapy agents
We have found that the three global genome repair (GGR) components Xeroderma Pigmentosum complementation group C (XPC), DNA damage binding protein 1 (DDB1) and DNA damage binding protein 2 (DDB2) do not respond to UV treatment in melanoma cell lines, resulting in reduced repair of UV-induced DNA damage [18, 19]
Decitabine can demethylate melanoma and increase XPC expression As an initial test to determine global methylation levels in melanoma, cell lines MM200, Sk-mel-28, Me4405 and Mel-RM were treated with the demethylating agent decitabine

Summary

Introduction

Melanoma has two key features, an over-representation of UV-induced mutations and resistance to DNA damaging chemotherapy agents. There are two features of melanoma that suggest a defect in DNA repair, an extremely high mutation load, indicative of unrepaired UV-induced DNA damage [1], and innate resistance to DNA damaging agents such as platinum based chemotherapies [2]. These can both be connected to the nucleotide excision repair (NER) pathway, The NER pathway consists of approximately 30 proteins that remove helix distorting lesions through for steps: a) damage recognition, b) unwinding of the DNA locally around damage, c) incision of damaged DNA by endonucleases, and d) DNA resynthesis and ligation [4]. The study of NER in relation to platinum chemoresistance has largely focused on ERCC1

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