Background: Cisplatin-based chemotherapy is the standard regimen for patients with newly diagnosed advanced head and neck squamous cell carcinoma (HNSCC). However, chemo-resistance to cisplatin is frequent, unpredictable, and contributes to treatment failure in over 90% of HNSCC patients with metastasis. Cisplatin acts mainly through the induction of various types of DNA damage. The mechanisms of cisplatin resistance include reduced uptake, increased DNA damage repair and decreased apoptosis. DNA repair capacity is the most promising predictor of cisplatin sensitivity, but, to date, no assay has been able to accurately predict the probability of a given tumor to respond to platinum-based chemotherapy. Recently, we developed a novel and highly sensitive primer-anchored DNA damage detection assay (PADDA) that accurately quantifies the in vivo levels of DNA damage. Here, we used PADDA to quantify DNA damage induced by cisplatin and assessed PADDA's ability to predict tumor cell response to cisplatin.
Aim: (1) To document PADDA's sensitivity to quantify in vivo oxidative DNA damage induced by free radicals, such as those induced by cisplatin treatment. (2) To determine PADDA's ability to quantify DNA damage and repair after cisplatin treatment, and therefore predict cisplatin resistance in cancer cells.
Methods: Induced oxidative DNA damage was quantified in human squamous cell carcinoma (SCC) cells exposed to diverse doses of H2O2 and allowed to recover for 0, 3 and 24 h. To determine the in vivo levels of cisplatin induced DNA damage, SCC cell lines were exposed to escalating doses of cisplatin and allowed to recover for up to 48 h. Overall DNA damage was measured using PADDA on a high-throughput setting. DNA double strand breaks were detected by immunofluorescence analysis of γ-H2AX. Cell viability and cell growth were determined by MTT assay. Data were analyzed by Student's t test and exact non-parametric tests.
Results: PADDA detected a dose-dependent increase in oxidative DNA damage induced by H2O2 and documented an inverse correlation between damage repair and H2O2 dose. Cisplatin treatment induced DNA damage detectable by PADDA in all doses and SCC cell lines. A significant decrease in cell viability and cell growth was observed in both cisplatin sensitive and resistance cell lines up to 48 h. However, a linear dose-dependent increase in in vivo DNA damage was observed only in cisplatin sensitive cells.
Conclusion: We show for the first time that PADDA is highly sensitive to quantify cisplatin-induced DNA damage and subsequent repair. These types of data are essential to determine whether resistance to cisplatin is due to increased DNA damage removal or damage tolerance and will facilitate the development of individualized strategies targeting drug resistance. Of high clinical relevance, PADDA detects a dose-dependent increase in DNA damage only in cisplatin sensitive cells, suggesting that PADDA can be used to predict patient response to cisplatin therapy.
Citation Format: Vengatesh Ganapathy, Ilangovan Ramachandran, Lurdes V.F. Queimado. A novel assay to predict cisplatin resistance in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2831. doi:10.1158/1538-7445.AM2014-2831