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Aberrant DNA damage response pathways may predict the outcome of platinum chemotherapy in ovarian cancer.

Stefanou DT, Bamias A, Episkopou H, Kyrtopoulos SA, Likka M, Kalampokas T, Photiou S, Gavalas N, Sfikakis PP, Dimopoulos MA, Souliotis VL - PLoS ONE (2015)

Bottom Line: Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells.Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05).In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05).

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece; Department of Clinical Therapeutics, Athens University Medical School, 11528 Athens, Greece.

ABSTRACT
Ovarian carcinoma (OC) is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR) pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/C30) to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs) from OC patients, sensitive (n = 7) or resistant (n = 4) to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9) were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05). Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03). Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05). We conclude that perturbations of DNA repair pathways as measured in PBMCs from OC patients correlate with the drug sensitivity of these cells and reflect the individualized response to platinum-based chemotherapy.

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Measurement of DNA damage in ovarian carcinoma cell lines using alkaline comet assay.A2780/C30 cells (representative of the two OC cell lines) were treated with (A) cisplatin (0–150μg/ml) for 3h or (B) carboplatin (0–300μg/ml) for 24h, and analyzed at the end of the treatment using comet assay. The error bars represent standard deviation. In (C) typical comet assay images of A2780/C30 cells non-treated (NT), treated with 50μg/ml cisplatin (cis-50), 100μg/ml cisplatin (cis-100), 150μg/ml carboplatin (carbo-150) or 300μg/ml carboplatin (carbo-300). All assays were performed in triplicate.
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pone.0117654.g002: Measurement of DNA damage in ovarian carcinoma cell lines using alkaline comet assay.A2780/C30 cells (representative of the two OC cell lines) were treated with (A) cisplatin (0–150μg/ml) for 3h or (B) carboplatin (0–300μg/ml) for 24h, and analyzed at the end of the treatment using comet assay. The error bars represent standard deviation. In (C) typical comet assay images of A2780/C30 cells non-treated (NT), treated with 50μg/ml cisplatin (cis-50), 100μg/ml cisplatin (cis-100), 150μg/ml carboplatin (carbo-150) or 300μg/ml carboplatin (carbo-300). All assays were performed in triplicate.

Mentions: The levels of platinum-induced DNA damage in both cell lines were also evaluated using alkaline comet assay. This version of the comet assay detects DNA migration caused by strand breaks, alkaline labile sites, and transient repair sites [30]. Following treatment of cells with 0–150μg/ml cisplatin for 3h (Fig. 2A, C) or 0–300μg/ml carboplatin for 24h (Fig. 2B, C), a linear dose-dependent increase of DNA damage levels was obtained in both cell lines, indicating that the method has the sensitivity and accuracy to detect and quantify platinum-induced DNA damage.


Aberrant DNA damage response pathways may predict the outcome of platinum chemotherapy in ovarian cancer.

Stefanou DT, Bamias A, Episkopou H, Kyrtopoulos SA, Likka M, Kalampokas T, Photiou S, Gavalas N, Sfikakis PP, Dimopoulos MA, Souliotis VL - PLoS ONE (2015)

Measurement of DNA damage in ovarian carcinoma cell lines using alkaline comet assay.A2780/C30 cells (representative of the two OC cell lines) were treated with (A) cisplatin (0–150μg/ml) for 3h or (B) carboplatin (0–300μg/ml) for 24h, and analyzed at the end of the treatment using comet assay. The error bars represent standard deviation. In (C) typical comet assay images of A2780/C30 cells non-treated (NT), treated with 50μg/ml cisplatin (cis-50), 100μg/ml cisplatin (cis-100), 150μg/ml carboplatin (carbo-150) or 300μg/ml carboplatin (carbo-300). All assays were performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320060&req=5

pone.0117654.g002: Measurement of DNA damage in ovarian carcinoma cell lines using alkaline comet assay.A2780/C30 cells (representative of the two OC cell lines) were treated with (A) cisplatin (0–150μg/ml) for 3h or (B) carboplatin (0–300μg/ml) for 24h, and analyzed at the end of the treatment using comet assay. The error bars represent standard deviation. In (C) typical comet assay images of A2780/C30 cells non-treated (NT), treated with 50μg/ml cisplatin (cis-50), 100μg/ml cisplatin (cis-100), 150μg/ml carboplatin (carbo-150) or 300μg/ml carboplatin (carbo-300). All assays were performed in triplicate.
Mentions: The levels of platinum-induced DNA damage in both cell lines were also evaluated using alkaline comet assay. This version of the comet assay detects DNA migration caused by strand breaks, alkaline labile sites, and transient repair sites [30]. Following treatment of cells with 0–150μg/ml cisplatin for 3h (Fig. 2A, C) or 0–300μg/ml carboplatin for 24h (Fig. 2B, C), a linear dose-dependent increase of DNA damage levels was obtained in both cell lines, indicating that the method has the sensitivity and accuracy to detect and quantify platinum-induced DNA damage.

Bottom Line: Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells.Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05).In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05).

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 11635 Athens, Greece; Department of Clinical Therapeutics, Athens University Medical School, 11528 Athens, Greece.

ABSTRACT
Ovarian carcinoma (OC) is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR) pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/C30) to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs) from OC patients, sensitive (n = 7) or resistant (n = 4) to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9) were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05). Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03). Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05). We conclude that perturbations of DNA repair pathways as measured in PBMCs from OC patients correlate with the drug sensitivity of these cells and reflect the individualized response to platinum-based chemotherapy.

Show MeSH
Related in: MedlinePlus