XPA, XPC, and XPD Modulate Sensitivity in Gastric Cisplatin Resistance Cancer Cells

Abstract

Cisplatin is an election drug widely used in clinic for the treatment of advanced gastric cancer. However, the heterogeneity of the gastric tumors and its resistance to the drugs, make in some cases the response very low and the prognosis unpredictable. In this manuscript we aim to find the molecular processes involved in cisplatin-induced apoptosis in two gastric cancer cell lines with different sensitivity to the treatment: AGS and MKN45. The apoptosis induction is higher in MKN45 than in AGS cells in response to CDDP. The intrinsic apoptotic pathway study revealed that MKN45 cells undergo degradation of Mcl-1 together with an increase of Bid and Bad levels, which results in sensitivity to CDDP. In addition, DNA repair NER pathway is impair in MKN45 cells due to low levels of XPC and the absence of translocation of XPA and XPD to the nucleus after stimuli. Altogether, these results suggest that NER and Bcl-2 protein family proteins are potential targets to improve the response to cisplatin treatment.

Keywords: Bcl-2 family; NER repair; apoptosis; cisplatin; gastric cancer.

FIGURE 1

AGS and MKN45 show different sensitivity to CDDP. (A) Cell survival percentage of AGS and MKN45 cells after 72 h of CDDP treatment. Cells were treated with increasing concentrations of CDDP (0–20 μg/ml). The percentage of viable cells was quantified by the crystal violet method. Data represent the mean values obtained in three experiments performed in quadruplicate. (B) Cell cycle profile of AGS and MKN45 cells after 24 h of CDDP treatment (10 μg/ml). DNA content was assessed by flow cytometry, and cell cycle distribution was analyzed using Cell Quest Pro software. The graph shows the percentage of each cell cycle, which is given as the mean ± standard deviation of three experiments. G2/M, cells in G2 or Mitosis; S, cells in phase of synthesis of DNA; G1, cells in G1. (C) Cleavage of PARP-1 and Caspase-3 activation were detected by western blot (WB) in cells harvested at the indicated times after CDDP treatment (10 μg/ml). α-Tubulin was used as loading control. WB images are representative of the results obtained in three different experiments made in the same conditions.

FIGURE 2

Intrinsic apoptosis pathway by CDDP is mediated by Mcl-1 in MKN45 cells. (A) Bcl-2 protein family members were detected by western blot in AGS and MKN45 cells treated with CDDP (10 μg/ml) at the indicated periods of time. Mcl-1, Bid, and Bad were detected by using specific antibodies. (B) Cells were treated as in panel (A). ERK, JNK, p38 (phosphorylated and total forms), and DUSP1 expression were detected by using specific antibodies. (C) AGS and MKN45 cells were pretreated during 30 min with 10 μM JNK Inhibitor II or p38 inhibitor (SB203580) before CDDP treatment. Mcl-1 was detected by western blot at the indicated times after treatment. α-Tubulin was used as loading control. WB images are representative of the results obtained in three different experiments made in the same conditions.

FIGURE 3

DNA repair is impaired in MKN45 cells. (A) Graph represents nuclear fluorescent intensity of DNA adducts formed after treating AGS and MKN45 with CDDP (10 μg/ml). Control cells (white bars) were untreated, or treated CDDP (black bars) for 3 h. DNA adducts were detected by using anti-(Pt-DNA) antibody. (B) Subcellular distribution of CDDP in GC cells after 3 h of 10 μg/ml treatment. White bars represent the CDDP content in nucleus and black bars in cytoplasm. Data are expressed as the mean values of the metal (nmol cisplatin) content in the nucleus or cytoplasm per million cells obtained in three different experiments. (C) AGS and MKN45 cells were treated as in panel (A), and they were allowed to recover in normal media for 1 h before harvesting. γ-H2AX foci were detected by Immunofluorescence, using DAPI to stain nuclear DNA. Graphs represent the percentage of nucleus within less than 15, between 15 and 50 and more than 50 γ-H2AX/nuclei for each condition. (D) Graphs represent the mean tail moment (TM) measured in at least 50 cells per duplicated in each condition after CDDP treatment (10 μg/ml). Tables present the percentage of residual damage (RD) after the repair period. ^∗∗∗p < 0.001.

FIGURE 4

CDDP selectively increased NER proteins in GC cells. (A) AGS and MKN45 cells were treated with 10 μg/ml CDDP and harvested at the indicated times. RT-qPCR was used to quantify the mRNA levels of XPA, XPC, XPF, XPG, and ERCC1 by using specific primers (see section “Materials and Methods”). The graphs represent the relative levels of each gene using ΔΔCT referred to the levels on a control no tumorigenic cell line HACAT, and using GAPDH as endogenous control. (B) XPC, XPD, XPA, and ERCC1 protein levels were detected by western blot in AGS and MKN45 cells after CDDP (10 μg/ml) treatment at the indicated times. α-Tubulin was used as a loading control. (C) XPA levels in AGS and MKN45 cells after 6 h of CDDP treatment (10 μg/ml) in the presence of caffeine (80 mM) and transfected with ATR^WT or ATR^DN plasmids. Arrow in XPA band indicates the non-processed protein. (D) Nuclear and cytoplasmic localization of XPC, XPD, and XPA proteins were detected by western blot in AGS and MKN45 cells after 6 h of CDDP treatment (10 μg/ml) and cellular fractioning (C, Cytoplasm; N, Nuclei). α-Tubulin was used as cytoplasmic protein loading control and lamin B1 was used as nuclear protein control. The experiments were repeated three times with similar results.

FIGURE 5

Hypothetical model to sensitize GC. DNA damage caused by CDDP leads to activation of NER, XPC is already in the nucleus and proteins XPA and XPD translocate to the nucleus to repair the lesion. Resistant cells repair properly the DNA and survive, however, if repair is not efficient, the mitochondrial apoptosis pathway is induced by Mcl-1 degradation and Bid and Bad induction. Option 1 (red circle) proposes the inhibition of XP proteins, and option 2 (green circle) proposes the use of BH₃ only mimetics.