Mild heat shock induces autophagic growth arrest, but not apoptosis in U251-MG and U87-MG human malignant glioma cells

Although hyperthermia has been used as a treatment of malignant brain tumors, it is not yet clear what is the mechanism of the cell growth inhibition by heat shock, especially by the temperature which has clinically been applied to tumor-brain border-zone, 42-43degreesC. Therefore, we evaluated the change of U251-MG and U87-MG human malignant glioma cells after 43degreesC-heat shock comparing with that of 45degreesC. First, we observed that cell growth was transiently inhibited after 43degreesC-heat shock for 3 or 5 days, in U251-MG or U87-MG cells, respectively, which was followed by regrowth. During the period of transient growth inhibition, mild G2/M arrest was observed. However, apoptosis was observed in only 2.7% or 1.5%, of 43degreesC-heated cells, in U251-MG or U87-MG cells, respectively. Instead, transmission electron micrography showed the formation of vacuoles, degeneration of mitochondria, and autophagosomes. Moreover, in the both cell lines, flow-cytometric analysis with acridine orange revealed the induction of acidic vesicle organelles, which was blocked by 3-methyladenine (3-MA), suggesting the involvement of autophagy. Furthermore, while 3-MA did not increase the anti-tumor effect of 43degreesC-heat shock, bafilomycin A1, another autophagy inhibitor, did significantly enhance the effect in U251-MG cells. Taken together, mild heat shock (43degreesC for 2 h) causes autophagy and mild G2/M arrest, but does not induce apparent apoptosis in U251-MG and U87-MG glioma cells. Inhibition of autophagy with bafilomycin A1 may increase the anti-tumor efficacy of mild heat shock against some malignant glioma cells.