Hyperthermia enhances the sensitivity of pancreatic cancer SW1990 cells to gemcitabine through ROS/JNK signaling

Abstract

Pancreatic cancer (PC) is a highly aggressive type of cancer. Gemcitabine (GEM) is a standard chemotherapeutic treatment of advanced PC; however, it requires improvement, and more effective therapeutic methods must be further explored. In the present study, hyperthermia combined with GEM was used on the PC cell line SW1990. The results revealed that mild hyperthermia (at 42°C) effectively increased the inhibitory effect of GEM on cell viability, as determined using an MTT assay, and increased the effect of GEM-induced apoptosis, as determined using an Annexin V-fluorescein isothiocyanate/propidium iodide assay, in PC SW1990 cells. Additionally, it resulted in increased S-phase arrest, downregulated the expression of the anti-apoptosis protein B-cell lymphoma 2 and upregulated the expression of the pro-apoptosis protein Bcl-2-associated X protein, cleaved caspase-3 and cleaved caspase-9, as determined using a reverse transcription-quantitative polymerase chain reaction and western blot analysis. Furthermore, it was revealed that hyperthermia resulted in the rapid generation of reactive oxygen species (ROS) and substantial activation of c-Jun-N-terminal kinase (JNK). The introduction of ROS and JNK inhibitors suppressed hyperthermia-induced apoptosis in GEM-treated cells, suggesting that hyperthermia increased GEM cytotoxicity in PC SW1990 cells by inducing apoptosis via the ROS/JNK signaling pathway.

Keywords: apoptosis; gemcitabine; hyperthermia; pancreatic cancer; reactive oxygen species/c-Jun-N-terminal kinase signaling; viability.

Figure 1.

Hyperthermia enhances the inhibitory effect of GEM on viability in SW1990 cells. (A) SW1990 cells were treated with 0, 1, 5, 10 and 20 µM GEM for 12, 24, 48 and 72 h. Cell viability was assessed using an MTT assay. (B) Cells were treated with hyperthermia at 42°C for different durations (30, 60 or 90 min), and then were treated using 5 µM GEM for 24 h. Cell viability was assessed using an MTT assay. *P<0.05 and **P<0.01 with comparisons shown by lines. Ctrl, untreated group; GEM, treated with gemcitabine alone; H30 min, treated with hyperthermia alone for 30 min; H30 min/GEM, treated with hyperthermia for 30 min in addition to gemcitabine; H60 min, treated with hyperthermia alone for 60 min; H60 min/GEM, treated with hyperthermia for 60 min in addition to gemcitabine; H90 min, treated with hyperthermia alone for 90 min; H90 min/GEM, treated with hyperthermia for 90 min in addition to gemcitabine.

Figure 2.

Hyperthermia increases GEM-induced cell apoptosis. (A) SW1990 cells were treated with hyperthermia at 42°C for 90 min, and then were treated with 5 µM GEM for 24 h. Cell apoptosis was assessed using an Annexin V-fluorescein isothiocyanate/propidium iodide assay. (B) Morphological changes of SW1990 cells were observed under a transmission electron microscope (magnification, ×6,000), including the following groups: (a) Normal cells, (b) cells treated with hyperthermia alone, (c) cells treated with GEM alone and (d) cells treated with hyperthermia combined with GEM. (C) Cell cycle distributions of PC cells were detected using flow cytometry. (D) Protein expression of the apoptosis-associated proteins, B-cell lymphoma 2, Bcl-2-associated X protein, cleaved caspase-3 and cleaved caspase-9 were evaluated using western blot analysis using respective antibodies. β-actin was detected as internal reference. *P<0.05 and **P<0.01 with comparisons shown by lines. NS, no significance; Ctrl, untreated group; H, treated with hyperthermia alone; GEM, treated with gemcitabine alone; H+GEM, treated with hyperthermia combined with gemcitabine.

Figure 3.

Involvement of survivin and ROS/JNK signaling in hyperthermia enhancing GEM-induced cell apoptosis. (A) Western blot analysis of survivin, JNK and p-JNK. β-actin was detected as internal reference. (B) Cells were treated with or without 42°C hyperthermia for 90 min, followed by treatment with 5 µM GEM for 24 h, and then ROS production was analyzed using 2′,7′-dichlorodihydrofluorescein diacetate staining. (C) SW1990 cells were treated with or without hyperthermia for 90 min at 42°C in the presence or absence of the JNK inhibitor Sp600125 (10 µM), and then were treated with 5 µM GEM for 24 h. Apoptosis was measured using an Annexin V-FITC/PI assay. (D) SW1990 cells were treated with or without hyperthermia for 90 min at 42°C in the presence or absence of the ROS inhibitor NAC (5 mM), and then were treated with 5 µM GEM for 24 h. Apoptosis was measured using an Annexin V-FITC/PI assay. **P<0.01 with comparisons shown by lines. Ctrl, untreated normal cells; H, treated with hyperthermia alone; GEM, treated with gemcitabine alone; NAC, treated with N-acetyl-L-cysteine alone; H/GEM, treated with hyperthermia in addition to gemcitabine; H/GEM/NAC, treated with hyperthermia in the presence of N-acetyl-L-cysteine in addition to gemcitabine; Sp, treated with Sp600125 alone H/GEM/Sp, treated with hyperthermia in the presence of Sp600125 plus GEM; NS, no significance; ROS, reactive oxygen species; JNK, c-Jun-N-terminal kinase; p-, phosphorylated; FITC/PI, fluorescein isothiocyanatae/propidium iodide.