In vitro assessment of the role of DpC in the treatment of head and neck squamous cell carcinoma

Abstract

The present study aimed to investigate the antitumor efficacy of di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) and di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) on head and neck squamous cell carcinoma (HNSCC) cells. The proliferation and apoptosis of HNSCC cells treated with the iron chelators DpC and Dp44mT were detected. The mechanism of DpC-induced apoptosis on HNSCC cells was investigated. The human HNSCC cell lines FaDu, Cal-27 and SCC-9 were cultured in vitro and exposed to gradient concentrations of DpC and Dp44mT. A Cell Counting Kit-8 assay was used to detect the viability of FaDu, Cal-27, SCC-9 cells. Double staining with annexin V and propidium iodide was performed for the detection of the proportion of apoptotic FaDu, Cal-27 and SCC-9 cells following treatment. The nuclear damage to Cal-27 cells that were treated with DpC was detected by Hoechst staining. Finally, western blot analysis was used to detect the expression of proteins associated with the DNA damage pathway in Cal-27 cells that were treated with DpC. The CCK-8 assay showed that treatment with DpC and Dp44mT was able to markedly inhibit the viability of FaDu, Cal-27 and SCC-9 cells in a concentration-dependent manner. In comparison to Dp44mT, treatment with DpC exhibited a more effective inhibitory effect on the viability of HNSCC cells. The proportion of apoptotic cells detected by flow cytometry increased in a dose-dependent manner in all cell lines following DpC and Dp44mT treatment, with the proportion of apoptotic HNSCC cells induced by DpC treatment being significantly higher compared with Dp44mT (P<0.05). The results of Hoechst staining revealed that the nuclei of Cal-27 cells exhibited morphological changes in response to DpC treatment, including karyopyknosis and nuclear fragmentation. The expression of DNA damage-associated proteins, including phosphorylated (p)-serine-protein kinase ATM, p-serine/threonine-protein kinase Chk1 (p-Chk-1), p-serine/threonine-protein kinase ATR (p-ATR), p-Chk-2, poly (ADP-ribose) polymerase, p-histone H2AX, breast cancer type 1 susceptibility protein, p-tumor protein P53, increased with increasing concentration of DpC in Cal-27 cells. Treatment with DpC and Dp44mT markedly inhibited cell viability and increased the apoptotic rates in human HNSCC cells in a concentration-dependent manner. DpC exhibited a stronger antitumor effect compared with Dp44mT, potentially inducing the apoptosis of HNSCC cells via the upregulation of DNA damage repair-associated proteins.

Keywords: DNA damage; apoptosis; di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone; di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone; head and neck squamous cell carcinoma; proliferation.

Figure 1.

Dp44mT and DpC inhibit the viability of Cal-27, FaDu and SCC-9 cells. The Cal-27, SCC-9 and FaDu HNSCC cell lines were treated with a range of concentrations of DpC (0, 1, 5, 10 and 50 µM) and Dp44mT (0, 1, 5, 10 and 50 µM) for 24 h, and the viability of these cell lines were subsequently assessed. The IC₅₀ values at 24 h were assessed, and it was indicated the anti-proliferative effect of DpC was stronger compared with Dp44mT in HNSCC cells. *P<0.001 vs. Dp44mT. Dp44mT, di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone; DpC, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone; HNSCC, head and neck squamous cell carcinoma; IC₅₀, half-maximal inhibitory concentration.

Figure 2.

Dp44mT and DpC promote the apoptosis of Cal-27, FaDu and SCC-9 cells. DpC (0, 1, 5, 10 and 50 µM) and Dp44mT (0, 1, 5, 10 and 50 µM) were used to treat Cal-27, SCC-9 and FaDu HNSCC cells for 24 h. (A) The apoptotic rate of the Cal-27 cells when treated with DpC (0, 1, 5, 10 and 50 µM) and Dp44mT (0, 1, 5, 10 and 50 µM). (B) The apoptotic rate of the SCC-9 cells when treated with DpC (0, 1, 5, 10 and 50 µM) and Dp44mT (0, 1, 5, 10 and 50 µM). (C) The apoptotic rate of the FaDu cells when treated with DpC (0, 1, 5, 10 and 50 µM) and Dp44mT (0, 1, 5, 10 and 50 µM). The apoptotic rate of the cells increased with the increase of the concentration of DpC and Dp44mT, indicating that DpC and Dp44mT are able to promote the apoptosis of HNSCC cells. The effect of DpC on the apoptosis of HNSCC cells was stronger compared with Dp44mT. *P<0.001. Dp44mT, di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone; DpC, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone; HNSCC, head and neck squamous cell carcinoma.

Figure 3.

DpC induces nuclear damage of Cal-27. The change in nuclear morphology by DpC on the Cal-27 cell line was detected by Hoechst staining. The experimental results showed that upon treatment with a range of concentrations of DpC (0, 2.5, 5 and 7.5 µM). (A) The nuclear staining of the Cal-27 cells when treated with 0 µM DpC. (B) The nuclear staining of the Cal-27 cells when treated with 2.5 µM DpC. (C) The nuclear staining of the Cal-27 cells when treated with 5 µM DpC. (D) The nuclear staining of the Cal-27 cells when treated with 7.5 µM DpC; the proportion of nuclei with bright blue fluorescence decreased significantly. DpC, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (magnification, ×40).

Figure 4.

Changes in the expression of proteins in the DNA-damage-associated pathway following DpC treatment in Cal-27 cells. (A) Western blot analysis was performed to detect the expression of the indicated proteins, and GAPDH was used as a loading control. Cal-27 cells treated with a range of concentrations of DpC (0, 2.5, 5 and 7.5 µM). (B) Statistical view of western blot analysis for p-ATM and p-Chk-1. (C) Statistical views of western blot analysis for p-ATR and p-Chk-2. (D) Statistical views of western blot analysis for PARP and p-histone H2A.X. (E) Statistical views of western blot analysis for BRCA1 and p-P53. As the concentration of DpC increased, the expression of (B) p-ATM, (C) p-ATR, (D) PARP, (E) BRCA1 and p-P53 also increased. *P<0.05; **P<0.01. DpC, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone; p-, phosphorylated; PARP, poly (ADP-ribose) polymerase; BRCA1, breast cancer type 1 susceptibility protein; P53, tumor protein P53.