Time and dose dependence of pluronic bioactivity in hyperthermia-induced tumor cell death

Abstract

Pluronic block copolymers have been shown to sensitize cancer cells resulting in an increased activity of antineoplastic agents. In the current study we examined a new application of Pluronic bioactivity in potentiating hyperthermia-induced cancer cell injury. DHD/K12/TRb rat adenocarcinoma cells were exposed to low-grade hyperthermia at 43 degrees C with or without Pluronic P85 or Pluronic L61. A range of Pluronic doses, pre-exposure and heat exposure durations were investigated, and the test conditions were optimized. Treatment efficacy was assessed by measurement of intracellular ATP and mitochondrial dehydrogenase activity. Both P85 and L61 in synergy with heat reduced cell viability appreciably compared to either heat or Pluronic alone. Under optimal conditions, P85 (10 mg/ml, 240 mins) combined with 15 mins heat reduced intracellular ATP to 60.1 +/- 3.5% of control, while heat alone and P85 without heat caused a negligible decrease in ATP of 1.2% and 3.8%, respectively. Similarly, cells receiving 120 mins pre-exposure of L61 (0.3 mg/ml) showed reduction in intracellular ATP to 14.1 +/- 2.1% of control. Again, heat or L61 pre-exposure alone caused a minor decrease in levels of intracellular ATP (1.5% and 4.4%, respectively). Comparable results were observed when viability was assessed by mitochondrial enzyme activity. Survival studies confirmed that the loss of viability translates to a long-term reduction in proliferative activity, particularly for L61 treated cells. Based on these results, we conclude that Pluronic is effective in improving hyperthermic cancer treatment in vitro by potentiating heat-induced cytotoxicity in a concentration and time dependent manner.

Figure 1

Effect of P85 concentration (0, 0.3, 1, 10, 30, 50 and 70 mg/ml), pre-exposure time (0–360 mins) and hyperthermia on intracellular ATP. A: ATP changes in response to Pluronic P85 in the absence of heat; and B: ATP changes in response to Pluronic P85 and hyperthermia (43°C). Arrows indicate cell response at optimal P85 dose and pre-exposure time.

Figure 2

Effect of L61 dose (0, 0.005, 0.01, 0.05, 0.1, 0.3 and 1 mg/ml), pre-exposure time (0–360 mins) and hyperthermia on intracellular ATP. A: ATP changes in response to Pluronic L61 in the absence of heat; and B: ATP changes in response to Pluronic L61 and hyperthermia (43°C). Arrows indicate cell response at optimal L61 dose and pre-exposure time.

Figure 3

Effects of Pluronic pre-exposure time on intracellular ATP at the optimal P85 (10 mg/ml) and L61 (0.3 mg/ml) concentrations. *P < 0.05.

Figure 4

Levels of mitochondrial dehydrogenase immediately (day 0) and 1, 2 and 3 days after treatment. A: P85 effect on cell survival with and without heat; B: L61 effect on cell survival with and without heat under optimal concentrations ([P85]:10 mg/ml; [L61]: 0.3 mg/ml) and pre-exposure time (P85: 240 mins; L61: 120 mins). * P < 0.05.

Figure 5

Cellular morphology of untreated cells and cells treated with heat alone (15 mins at 43°C) immediately (A, B) and 3 days (C, D) after treatment (May Grünwald/Giemsa stain ×400). A color version of the figure is available in the online journal.

Figure 6

Cellular morphology of cells pre-treated with Pluronic P85 (240 mins) or L61 (120 mins) alone or combined with heat (15 mins at 43°C) immediately (A–D) and 3 days (E–H) after treatment (May Grünwald/Giemsa stain, ×400). A color version of the figure is available in the online journal.