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Xeroderma Pigmentosum Group A Promotes Autophagy to Facilitate Cisplatin Resistance in Melanoma Cells through the Activation of PARP1  Rui Ge, Lin Liu,

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Presentation on theme: "Xeroderma Pigmentosum Group A Promotes Autophagy to Facilitate Cisplatin Resistance in Melanoma Cells through the Activation of PARP1  Rui Ge, Lin Liu,"— Presentation transcript:

1 Xeroderma Pigmentosum Group A Promotes Autophagy to Facilitate Cisplatin Resistance in Melanoma Cells through the Activation of PARP1  Rui Ge, Lin Liu, Wei Dai, Weigang Zhang, Yuqi Yang, Huina Wang, Qiong Shi, Sen Guo, Xiuli Yi, Gang Wang, Tianwen Gao, Qi Luan, Chunying Li  Journal of Investigative Dermatology  Volume 136, Issue 6, Pages (June 2016) DOI: /j.jid Copyright © 2016 The Authors Terms and Conditions

2 Figure 1 XPA contributes to cisplatin resistance in melanoma cell lines. (a) Total RNA was isolated from six melanoma cell lines, one melanocyte cell line (PIG1), and normal human melanocytes (MC). XPA expression was analyzed by quantitative real-time reverse transcriptase PCR. The data are expressed as the mean ± standard deviation. (b) The viability of cells was determined by Cell Counting Kit-8 assay (Dojingo Molecular Technologies, Rockville, MD), and IC50 values are presented as the mean ± standard deviation. (c) Hs294T and A2058 cells were transfected with XPA siRNA. The knockdown efficiency at 24 hours was confirmed by Western blot test. The cell lines were then treated with different doses of cisplatin for 48 hours, and the viability of cells was determined by Cell Counting Kit-8 assay. IC50 values are presented as the mean ± standard deviation. ∗∗P < Ctrl, control; IC50, half maximal inhibitory concentration; μM, μmol/L; siRNA, small interfering RNA; XPA, xeroderma pigmentosum group A. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2016 The Authors Terms and Conditions

3 Figure 2 Attenuation of XPA promotes apoptosis in melanoma cells treated with cisplatin. (a) Hs294T and A2058 cells were transfected with siRNAs against XPA or control siRNA. After a 24-hour transfection, the cell lines were treated with 20 μmol/L of cisplatin for 24 hours. The expression level of c-caspase3 and caspase3 were detected by Western blot test. Data are representative of three independently performed experiments. (b and c) Hs294T and A2058 cells were treated as described in (a), and the level of apoptosis was detected by flow cytometry assay. Bar graphs represent the mean values of flow cytometry data. Data are presented as the mean ± standard deviation. ∗P < Ctrl, control; FITC, fluorescein isothiocyanate; μM, μmol/L; siRNA, small interfering RNA; XPA, xeroderma pigmentosum group A. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2016 The Authors Terms and Conditions

4 Figure 3 XPA is required for autophagy induced by cisplatin in Hs294T and A2058 cells. (a) Western blots of LC3 and XPA in cells treated with cisplatin for different lengths of time in hours. (b) Band intensity normalized to β-actin is expressed as mean ± standard deviation. (c) Transmission electron microscopy images of autophagic vacuoles in cells treated with cisplatin. The arrows indicate the formed autophagosomes and autolysosomes. Scale bars represent 1 μm (upper row) and 500 nm (lower row). (d–f) Confocal images of LC3 puncta in XPA-knockdown cells with cisplatin treatment (d). Scale bars represent 5 μm. Statistical analyses are expressed as the mean ± standard deviation; ∗P < 0.05, ∗∗P < (e and f). (g) Western blots of LC3 in XPA-knockdown cells with cisplatin treatment for different lengths of time in hours. (h and i) The ratio of LC3-II/β-actin is reported as mean ± standard deviation. Ctrl, control; h, hours; LC, light chain; μM, μmol/L; siRNA, single interfering RNA; XPA, xeroderma pigmentosum group A. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2016 The Authors Terms and Conditions

5 Figure 4 XPA promotes cisplatin-induced autophagy through the activation of PARP1. (a) Western blots of LC3, PAR, and XPA in cells transfected with XPA siRNA for 24 hours and then treated with cisplatin. The ratio of LC3-II/β-actin is reported below as mean ± standard deviation. (b–d) Western blots of LC3 and PAR in cells transfected with siRNAs against PARP1 (b) or pretreated with DPQ (c) or olaparib (d) and then treated with cisplatin. The ratio of LC3-II/β-actin is reported below. (e) Confocal images of LC3 puncta in cells transfected with XPA siRNA for 24 hours and then treated with cisplatin. Scale bars represent 5 μm. Statistical analyses are expressed to the left as mean ± standard deviation; ∗P < 0.05, ∗∗P < Ctrl, control; DPQ, 3,4-dihydro-5-[4-(1-piperidinyl) butoxy]-1(2H)-isoquinolinone; LC, light chain; μM, μmol/L; PAR, poly(adenosine diphosphate-ribose); siRNA, single interfering RNA; XPA, xeroderma pigmentosum group A. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2016 The Authors Terms and Conditions

6 Figure 5 Inhibition of PARP1-mediated autophagy increases the sensitivity of melanoma cells to cisplatin. (a) The efficiency of ATG12 or PARP1 depletion by their respective siRNAs was verified by Western blot test. (b and c) The ability of cells transfected with control siRNA, ATG12 siRNA, or PARP1 siRNA to recover after cisplatin withdrawal was assessed with a colony formation assay. Each well shown is a representative image of at least nine similar wells (three independent experiments) (b). The columns show the number of colonies, and data are expressed as the mean ± standard deviation; ∗P < 0.05, ∗∗∗P < 0.001, one-way analysis of variance followed by Newman-Keuls test (c). siRNA, single interfering RNA. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2016 The Authors Terms and Conditions

7 Figure 6 XPA regulates both apoptosis and autophagy, contributing to the resistance of melanoma cells to cisplatin. XPA can recognize DNA lesions and recruit other NER proteins to repair damaged DNA, thus rendering melanoma cells resistant to cisplatin-induced cell death. In addition, XPA can also promote cell-protective autophagy through the activation of the PARP1-HMGB1 pathway in melanoma cells exposed to cisplatin. On the contrary, when XPA is knocked down, the DNA damage caused by cisplatin in melanoma cells leads to double strand break, causing cell death through caspase3-dependent apoptosis pathways. ADP, adenosine diphosphate; DSB, double strand break; NER, nucleotide excision repair; XPA, xeroderma pigmentosum group A. Journal of Investigative Dermatology  , DOI: ( /j.jid ) Copyright © 2016 The Authors Terms and Conditions

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