In Vitro Effect of Focused Ultrasound or Thermal Stress on HSP70 Expression and Cell Viability in Three Tumor Cell Lines

Rationale And Objectives: In this study, we compared the effect of focused ultrasound with the effect of thermal stress on the induction of a heat inducible promoter in an in vitro model using three tumor cell lines (M21, SCCVII, and NIH3T3).

Materials And Methods: We used a reporter construct that was generated using the stress-inducible promoter from the gene encoding a murine 70-kilodalton heat shock protein (Hsp70A.1) and a luciferase (luc) reporter plasmid. High-intensity focused ultrasound (HIFU) was applied in two different modes. In the first mode, an increasing voltage at constant pulse duration and in the second mode a constant voltage at increasing pulse duration was applied. HIFU or thermal stress was delivered over a range of temperatures (36-52 degrees C) for 5 minutes, and resulting luciferase activity was measured in live cells using a cooled charge-coupled device camera as a measure of reporter gene transcription. Luciferase activity was measured at set time intervals for a total of 108 hours post-stress.

Results: Both methods induced the hsp70 promoter; however, the luciferase activity under the influence of HIFU, independent of the applied mode, and thermal stress differs despite the fact that the temperature was the same. In the M21 tumor cell line, the maximum luciferase activity after focused ultrasound application was 4818 +/- 1521% at a temperature of 48 degrees C and after thermal stress 4468.2 +/- 1890.2% at a temperature of 52 degrees C with a viability of 72.3 +/- 5.2% and 85 +/- 3.4%, respectively. In the SCC tumor cell line, the maximum luciferase activity after focused ultrasound application was 6743.0 +/- 3281.4% and after only thermal stress exposure was 3910.6 +/- 2189.0% at a temperature of 44 degrees C and 50 degrees C, respectively. At the highest luciferase activity, the portion of vital cells was 72.5 +/- 8.4% and 72.5 +/- 5.9% respectively. In the NIH3T3 tumor cell line the highest luciferase activity of 428510.6 +/- 26526.8% was seen at a temperature of 42 degrees C applying focused ultrasound. Under thermal stress it was 29221.3 +/- 7205.0% at a temperature of 50 degrees C. At the highest luciferase activity, the viability analysis showed 75.3 +/- 9.2% and 72.3 +/- 7.9% viable cells, respectively.

Conclusions: Focused ultrasound induces hsp70 expression like thermal stress alone; however, HIFU is capable of inducing expression at lower temperatures than heat stress alone, indicating that nonthermal effects also play a role on the induction of hsp70.