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°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°C and after thermal stress 4468.2 ± 1890.2% at a temperature of 52°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°C and 50°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°C applying focused ultrasound. Under thermal stress it was 29221.3 ± 7205.0% at a temperature of 50°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 .
High-intensity focused ultrasound (HIFU) has been used clinically for thermal ablation of tumors ( ) and has promising potential as a therapeutic modality and for drug and gene delivery. So far, ultrasound has been used to permeabilize cell membranes for protein transport ( ), to enhance reporter gene expression in benign and malignant cell types both in culture and in vivo ( ), and to induce apoptosis in cell culture ( ) as well as in vivo in rabbit brain ( ) and human breast cancer ( ). HIFU causes physical changes in tissues both through physical alteration, primarily thought to be indirectly caused by either the sonochemical or sonomechanical effects of acoustic cavitation on the cells ( ), and through energy deposition leading to localized heating.
The ability of ultrasound to induce localized heating could be exploited by using HIFU in conjunction with a heat inducible promoter. It has been demonstrated that the promoter for the heat-shock protein (HSP) can activate gene expression in response to hyperthermia ( ). HSP are highly conserved and found in nearly all subcellular compartments ( ), where they perform a variety of chaperoning functions, including antigen presentation ( ) and folding/unfolding processes of nascent polypeptides and proteins ( ). These functions of the HSPs may be important in the expression of thermotolerance ( ). HSPs are not only induced by stress, but they are also associated with physiologic processes such as the cell cycle, cell proliferation, and differentiation ( ). The induced synthesis of HSPs is, at the molecular level, the cellular response to a wide range of threatening stimuli such as heat shock, heavy metals, oxidative stress, fever, inflammation, ethanol, amino acid analogs, anoxia, and agents capable of perturbing protein structure ( ).
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Materials and methods
Reporter Construct
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Cell Culture and Transfection
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Application of Heat Stress
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Application of HIFU
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Table 1
Focused Ultrasound Parameter
Temp (°C) mV Pulse Duration (Milliseconds) Frequency (Hz) Intensity (W/cm 2 ) Total Energy (W/cm 2 ) P (kPa) TAP (W) Increasing voltage constant pulse duration 36 70 50 1 28.0 8,404.0 289.8 8.3 38 80 50 1 40.0 12,030.2 346.4 11.9 40 90 50 1 54.1 16,238.5 402.8 16.1 42 100 50 1 70.1 21,029.3 458.5 20.8 44 110 50 1 88.0 26,402.1 513.8 26.2 46 120 50 1 107.8 32,357.4 568.6 32.1 48 130 50 1 129.6 38,894.8 623.5 38.6 50 140 50 1 153.3 46,014.4 678.1 45.7 52 150 50 1 179.0 53,716.5 732.8 53.4 Constant voltage increasing pulse duration 36 100 20 1 28.0 8,411.0 289.8 20.8 38 100 29 1 40.0 12,107.0 346.4 20.8 40 100 39 1 54.6 16,402.8 404.7 20.8 42 100 50 1 70.1 21,029.3 458.5 20.8 44 100 63 1 88.3 26,496.8 514.6 20.8 46 100 77 1 107.8 32,385.0 568.6 20.8 48 100 92 1 128.9 38,693.8 621.8 20.8 50 100 110 1 154.2 46,264.3 680.1 20.8 52 100 128 1 179.4 53,834.8 733.6 20.8
High-intensity focused ultrasound was applied in a pulsed mode with increasing voltage and constant pulse duration and with constant voltage and increasing pulse duration achieving nearly the same intensity and the same temperatures in the conical thin-walled thermocycler tubes as measured by placing a temperature probe into the small tube.
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Imaging of Luciferase Activity
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Viability Assays and Normalization
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Statistical Analysis
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Results
Imaging of Luciferase Activity
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Table 2
Comparison of Luciferase Activity After Application of HIFU and Thermal Stress in the M21-HSP70-Luc Tumor Cell Line
M21_HSP70_Luc HIFU (Flux − Photons/Second) Increasing Voltage Constant Pulse Duration HIFU (Flux − Photons/Second) Constant Voltage Increasing Pulse Duration Thermal Stress (Flux − Photons/Second) Comparison HIFU Increase Voltage − Constant Voltage Comparison HIFU Increase Voltage − Thermal Stress Comparison HIFU Constant Voltage − Thermal Stress_n_ = 50 Mean ± SD Mean ± SD Mean ± SD_t_ -test_t_ -test_t_ -test Control 421,418 ± 173,991 434,681 ± 121,231 426,664 ± 133,896 0.431 0.831 0.561 36°C 421,906 ± 170,488 445,617 ± 151,211 414,779 ± 174,227 0.594 0.794 0.622 38°C 429,606 ± 169,231 431,114 ± 124,236 455,916 ± 178,908 0.641 0.340 0.452 40°C 551,918 ± 235,590 567,541 ± 241,210 435,181 ± 169,626 0.421 <0.001 0.003 42°C 718,143 ± 561,433 731,515 ± 512,341 446,993 ± 167,036 0.561 <0.001 0.001 44°C 1,053,806 ± 1,086,360 1,145,115 ± 913,413 458,181 ± 159,640 0.625 <0.001 <0.001 46°C 1,401,019 ± 1,651,575 1,346,131 ± 1,314,263 583,893 ± 283,335 0.551 <0.001 <0.001 48°C 2,886,344 ± 4,131,662 2,416,211 ± 5,242,421 927,062 ± 849,297 0.421 <0.001 <0.001 50°C 2,321,466 ± 2,528,159 2,145,124 ± 5,135,316 2,011,609 ± 2,900,564 0.623 0.472 0.621 52°C 1,141,216 ± 972,667 1,716,271 ± 311,523 2,779,119 ± 3,908,967 0.723 <0.001 <0.001
HIFU: high-intensity focused ultrasound; SD: standard deviation.
Table 3
Comparison of Luciferase Activity After Application of HIFU and Thermal Stress in the SCC-HSP70-Luc Tumor Cell Line
SCC_HSP70_Luc HIFU (Flux − Photons/Second) Increasing Voltage Constant Pulse Duration HIFU (Flux − Photons/Second) Constant Voltage Increasing Pulse Duration Thermal Stress (Flux − Photons/Second) Comparison HIFU Increase Voltage − Constant Voltage Comparison HIFU Increase Voltage − Thermal Stress Comparison HIFU Constant Voltage − Thermal stress_n_ = 50 Mean ± SD Mean ± SD Mean ± SD_t_ -test_t_ -test_t_ -test Control 424,675 ± 217,638 431,415 ± 225,632 412,925 ± 211,250 0.352 0.729 0.621 36°C 467,625 ± 185,618 441,223 ± 145,712 407,866 ± 191,376 0.645 0.046 0.031 38°C 511,938 ± 227,591 561,231 ± 256,448 393,014 ± 172,130 0.643 <0.001 <0.001 40°C 603,588 ± 408,266 653,482 ± 421,461 395,226 ± 162,483 0.534 <0.001 <0.001 42°C 1,144,500 ± 1,775,573 1,244,501 ± 1,275,321 479,101 ± 15,801 0.451 0.001 <0.001 44°C 2,331,000 ± 4,284,601 2,431,020 ± 4,414,641 555,601 ± 222,601 0.432 <0.001 <0.001 46°C 1,773,338 ± 2,056,824 1,972,321 ± 2,455,225 584,581 ± 391,753 0.342 <0.001 0.001 48°C 1,649,375 ± 1,511,220 1,949,241 ± 1,311,921 1,437,239 ± 2,417,385 0.443 0.506 0.406 50°C 928,415 ± 646,355 948,426 ± 316,325 2,476,126 ± 3,545,785 0.621 <0.001 <0.001 52°C 593,578 ± 202,190 653,271 ± 212,112 946,500 ± 719,872 0.564 <0.001 <0.001
HIFU: high-intensity focused ultrasound; SD: standard deviation.
Table 4
Comparison of Luciferase Activity After Application of HIFU and Thermal Stress in the NIH3T3-HSP70-Luc Tumor Cell Line
NIH3T3_HSP70_Luc HIFU (Flux − Photons/Second) Increasing Voltage Constant Pulse Duration HIFU (Flux − Photons/Second) Constant Voltage Increasing Pulse Duration Thermal stress (Flux − Photons/Second) Comparison HIFU Increase Voltage − Constant Voltage Comparison HIFU Increase Voltage − Thermal Stress Comparison HIFU Constant Voltage − Thermal Stress_n_ = 50 Mean ± SD Mean ± SD Mean ± SD_t_ -test_t_ -test_t_ -test Control 89,308 ± 13,244 93301 ± 52151 81308 ± 15466 0.446 0.450 0.421 36°C 340,100 ± 88,669 361103 ± 432051 89949 ± 15034 0.412 < .001 <0.001 38°C 1,788,038 ± 558,251 1928031 ± 441665 80795 ± 14381 0.643 <0.001 <0.001 40°C 24,939,438 ± 10,979,380 26137431 ± 12153151 95583 ± 19097 0.453 <0.001 <0.001 42°C 41,946,200 ± 13,729,074 44156101 ± 19351315 372036 ± 97891 0.553 0.001 <0.001 44°C 19,110,763 ± 8,288,854 21170263 ± 7166261 456849 ± 127791 0.425 <0.001 <0.001 46°C 11,117,225 ± 1,722,295 18112269 ± 1218155 773999 ± 277775 0.557 <0.001 0.001 48°C 8,713,475 ± 1,894,318 8114411 ± 1224151 1005249 ± 228737 0.467 <0.001 <0.001 50°C 6,141,975 ± 1,203,724 6521271 ± 1281517 2241386 ± 573161 0.663 <0.001 <0.001 52°C 1,435,525 ± 1,350,411 1531472 ± 1121212 2470636 ± 494259 0.361 <0.001 <0.001
HIFU: high-intensity focused ultrasound; SD: standard deviation.
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Viability
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Discussion
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