Incomplete radiofrequency ablation accelerates proliferation and angiogenesis of residual lung carcinomas via HSP70/HIF-1α

Abstract

Radiofrequency ablation (RFA) therapy has been proved effective and feasible for lung cancer. However, the molecular mechanisms of local lung cancer recurrence following RFA are poorly understood. The present study aimed to evaluate the ability of HSP70/HIF-1α to affect the proliferation and angiogenesis of non-small cell lung cancers (NSCLCs) following insufficient RFA to uncover the molecular mechanisms of local recurrence. In vitro heat treatment was used to establish sublines of NCI-H1650 cells. The NCI-H1650 subline that was established by heat treatment at 54˚C had a relatively higher viability and significantly elevated heat tolerance (compared to the parental strain). After treatment with the HSP70 inhibitor VER-155008, the HIF-1α inhibitor YC-1 and PI3K/Akt inhibitor wortmannin, the viability and proliferation rate of the cells was measured. At the same time, HSP70, HIF-1α and Akt were detected by real-time PCR and western blotting. In vivo xenograft tumors were created by subcutaneously inoculating nude mice with NCI-H1650 cells. HSP70, HIF-1α and Akt were detected by western blotting, and CD34 expression was detected by immunohistochemistry before and after RFA or treatment with the VER-155008, YC-1 or wortmannin inhibitors. The heat-adapted NCI-H1650 subline established in vitro had a higher viability and proliferative activity compared to parental cells. Inhibiting HSP70/HIF-1α abolished this difference. Blocking the PI3K/Akt signaling pathway decreased HSP70/HIF-1α expression levels. In vivo, we found that incomplete RFA treatment promoted HSP70/HIF-1α and CD34 expression. Additionally, the combination of RFA and treatment targeting HSP70/HIF-1α resulted in a synergistic reduction in tumor growth compared to incomplete RFA alone. The PI3K/Akt signaling pathway is also involved in regulating HSP70/HIF-1α expression during this process. We conclude that the accelerated proliferation and angiogenesis potential of residual lung carcinomas following RFA treatment was induced by HSP70/HIF-1α, expression of which is regulated by the PI3K/Akt signaling pathway.

Figure 1

Treatment groups and schedule.

Figure 2

The viability of NCI-H1650 cells and sublines derived from NCI-H1650 cells after hyperthermia. (A) NCI-H1650 cells were cultured after 42°C heat treatment. The 24, 48 and 72 h cell viability of NCI-H1650 cells with or without 42°C heat treatment were measured using MTT assay (p<0.05, control group vs. 42°C heat treatment group). (B) Eight sublines were established following 42, 46, 50, 54, 58, 62, 66 and 70°C heat treatment for 15 min as described in Materials and methods. The 24, 48 and 72 h viability was evaluated by MTT assay after these 8 sublines had been established. par, parental NCI-H1650 cells; a–h, sublines derived from the NCI-H1650 cells. (C) The 24, 48 and 72 h viability of parental NCI-H1650 and 54°C heat-adapted NCI-H1650 cells was evaluated by MTT assay (p<0.05, parental NCI-H1650 cells vs. NCI-H1650-d cells). (D) A growth curve of parental NCI-H1650 and NCI-H1650-d cells was drawn. Data are the representative results of three independent experiments (p<0.05 from day 2–7 parental NCI-H1650 cells vs. NCI-H1650-d cells).

Figure 3

(A) Heat-adapted NCI-H1650 cells (54°C) express elevated levels of HSP70 and HIF-1α. (A) Expression of HSP70 and HIF-1α in parental NCI-H1650 and NCI-H1650-d cells treated with or without VER-155008 (50 µg) or YC-1 (20 µg) for 24 h was analyzed by western blot and semi-quantitative analyses. (B) The mRNA expression of HSP70 and HIF-1α in parental NCI-H1650 and NCI-H1650-d cells treated with or without VER-155008 (50 µg) or YC-1 (20 µg) was assayed using real-time PCR. (C) Growth curve of parental NCI-H1650 cells treated with or without VER-155008 or YC-1 (ns, parental cell group vs. VER-155008 group day 2–7; ^#p<0.05, parental cell group vs. YC-1 group day 2–7. (D) Growth curve of NCI-H1650-d cells treated with or without VER-155008 or YC-1 (^*p<0.05, NCI-H1650-d cells vs. VER-155008 day 2–7; ^#p<0.05, NCI-H1650-d cell group vs. YC-1 group day 2–7). ns, no significance.

Figure 4

The PI3K/Akt signaling pathway is involved in regulating HSP70 and HIF-1α expression. (A) The expression levels of Akt, p-Akt, HSP70 and HIF-1α in parental NCI-H1650 cells treated with or without wortmannin (20 µM) for 24 h were analyzed by western blotting. (B) The expression levels of Akt, p-Akt, HSP70 and HIF-1α in heat-adapted cells treated with or without wortmannin (20 µM) for 24 h were analyzed by western blotting. (C) Semi-quantitative analysis of Akt, p-Akt, HSP70 and HIF-1α expression. ns, no significance.

Figure 5

HSP70/HIF-1α are involved in proliferation and angiogenesis of subcutaneously transplanted tumors following incomplete radiofrequency ablation. (A) After the subcutaneously transplanted tumor had formed, the RFA probe was inserted into the tumor tissue. (B) Growth curve of subcutaneously transplanted tumors in control, RFA, VER-155008 + RFA and YC-1 + RFA group (^*p<0.05 from day 3–7 control group vs. RFA group; ^★p<0.05 from day 3–7 RFA group vs. VER-155008 + RFA group; ^#p<0.05 from day 3–7 RFA group vs. YC-1 + RFA group). (C) In the control, RFA, VER-155008 + RFA, YC-1 + RFA, VER-155008 alone and YC-1 alone groups, HSP70 and HIF-1α expression in subcutaneously transplanted tumors was measured by western blot and semi-quantitative analyses (^*control group vs. RFA group: HSP70, p<0.05; HIF-1α, p<0.05. ^#RFA group vs. VER-155008 + RFA group: HSP70, p<0.05; HIF-1α, p<0.01. ^྿RFA group vs. YC-1 + RFA group: HSP70, ns; HIF-1α, p<0.01. ^▲Control group vs. VER-155008 group: HSP70, ns; HIF-1α, ns. ^★Control group vs. YC-1 group: HSP70, ns; HIF-1α, p<0.05). (D) At day 7 following RFA treatment, the numbers of new microvessels marked with CD34 expression in the subcutaneous tumors were detected by immunohistochemistry analysis and quantified by performing counts of 10 random fields at a magnification of ×400 (^*p<0.05 control group vs. RFA group; ^#p<0.05 RFA group vs. VER-155008 + RFA group; ^྿p<0.05 RFA group vs. YC-1 + RFA group; ^▲ns control group vs. VER-155008 group; ^★p<0.05 control group vs. YC-1 group). ns, no significance.

Figure 6

The PI3K/Akt signaling pathway regulates HSP70/HIF-1α expression and residual tumor growth following incomplete radiofrequency ablation. (A) In the control, RFA, wortmannin + RFA and wortmannin groups, HSP70 and HIF-1α expression in the subcutaneously transplanted tumors was measured by western blot and semi-quantitative analyses (^*control group vs. RFA group: HSP70, p<0.05; HIF-1α, p<0.05; ^#RFA group vs. wortmannin + RFA group: HSP70, p<0.05; HIF-1α, p<0.05; ^★control group vs. wortmannin group: HSP70, ns; HIF-1α, p<0.05). (B) Growth curve of subcutaneously transplanted tumors in the control, RFA, wortmannin + RFA and wortmannin group (^*p<0.05 from day 3–7 control group vs. RFA group; ^྿p<0.05 from day 3–7 RFA group vs. wortmannin + RFA group; ^#p<0.05 from day 3–7 control group vs. wortmannin group).