Inhibition of heat shock protein 90 sensitizes melanoma cells to thermosensitive ferromagnetic particle-mediated hyperthermia with low Curie temperature

Abstract

Heat shock protein (Hsp) 90 is a key regulator of a variety of oncogene products and cell-signaling molecules, and the therapeutic benefit of its inhibition in combination with radiation or chemotherapy has been investigated. In addition, hyperthermia has been used for many years to treat various malignant tumors. We previously described a system in which hyperthermia was induced using thermosensitive ferromagnetic particles (FMP) with a Curie temperature (Tc = 43 degrees C) low enough to mediate automatic temperature control, and demonstrated its antitumor effect in a mouse melanoma model. In the present study, we examined the antitumor effects of combining a Hsp90 inhibitor (geldanamycin; GA) with FMP-mediated hyperthermia. In cultured B16 melanoma cells, GA exerted an antitumor effect by increasing the cells' susceptibility to hyperthermia and reducing expression of Akt. In an in vivo study, melanoma cells were subcutaneously injected into the backs of C57BL/6 mice. FMP were then injected into the resultant tumors, and the mice were divided into four groups: group I, no treatment (control); group II, one hyperthermia treatment; group III, GA alone; and group IV, GA with hyperthermia. When exposed to a magnetic field, the temperature of tissues containing FMP increased and stabilized at the Tc. In group IV, complete regression of tumors was observed in five of nine mice (56%), whereas no tumor regression was seen in groups I-III. Our findings suggest that inhibition of Hsp90 with hyperthermia increases its antitumor effect. Thus, the combination of FMP-mediated, self-regulating hyperthermia with Hsp90 inhibition has important implications for the treatment of cancer.

Figure 1

Geldanamycin (GA) sensitizes cultured melanoma cells to hyperthermia (HT). Cells were incubated with GA (0.2 µM) for 16 h at 37°C, exposed to 43°C or 37°C for 30 min, and then incubated for an additional 8 h at 37°C. The cell viabilities were normalized to that of untreated control cells. Symbols depict mean ± SD of five measurements. *P < 0.05 versus control.

Figure 2

(a) Western blotting analysis of the individual and combined effects of hyperthermia (HT) and geldanamycin (GA) on Akt expression in cultured melanoma cells. (b) Akt levels were normalized to the control level obtained with untreated cells. Measurements were carried out five times in each. *P < 0.05 versus control.

Figure 3

System for induction of ferromagnetic particle (FMP)‐mediated hyperthermia. FMP were injected into the tumor and exposed to a magnetic field (600 A, 188 kHz) created using a horizontal coil in an induction heating system. In some experiments tumor and rectal temperatures were measured continuously during exposure to the magnetic field using a ceramic thermocouple and recorded on a computer system.

Figure 4

Hyperthermia induced using ferromagnetic particles (FMP) in vivo. FMP were injected directly into subcutaneous B16 melanoma tumors in mice, after which the mice were exposed to a magnetic field for 30 min. Tumor () and rectal () temperatures were measured using a ceramic thermocouple. Symbols represent mean ± SD of five mice.

Figure 5

Time courses of tumor growth in groups I (; control, n = 10), II (; one hyperthermia treatment, n = 10), III (; geldanamycin treatment alone, n = 10), and IV (; geldanamycin + hyperthermia, n = 9). Symbols depict mean ± SD. *P < 0.05 versus group I (control).

Figure 6

The TUNEL method was used to identify apoptotic melanoma cells, which stained red in groups (a) I, (b) II, (c) III, and (d) IV 20 days after ferromagnetic particle injection. The incidence of apoptosis among the target tumors was significantly greater in (d) group IV than in (a) group I. Magnification, ×400.

Figure 7

Survival rates among tumor‐bearing mice observed for a period of 40 days after injection of ferromagnetic particles (FMP): , group I (n = 10); , group II (n = 10); , group III (n = 10); and , group IV (n = 9). Survival in group IV was significantly prolonged compared to the other groups (P < 0.05 versus all other groups).