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The heightened energetic demand increases lactate dehydrogenase (LDH) activity, the corresponding oncometabolite lactate, expression of heat shock proteins (HSPs) and thereby promotes therapy resistance in many malignant tumor cell types. Therefore, we assessed the coregulation of LDH and the heat shock response with respect to radiation resistance in different tumor cells (B16F10 murine melanoma and LS174T human colorectal adenocarcinoma). The inhibition of LDH activity by oxamate or GNE-140, glucose deprivation and LDHA/B double knockout (LDH−/−) in B16F10 and LS174T cells significantly diminish tumor growth, ROS production and the cytosolic expression of different HSPs, including Hsp90, Hsp70 and Hsp27 concomitant with a reduction of heat shock factor 1 (HSF1)/pHSF1. An altered lipid metabolism mediated by a LDHA/B double knockout results in a decreased presence of the Hsp70-anchoring glycosphingolipid Gb3 on the cell surface of tumor cells, which, in turn, reduces the membrane Hsp70 density and increases the extracellular Hsp70 levels. Vice versa, elevated extracellular lactate/pyruvate concentrations increase the membrane Hsp70 expression in wildtype tumor cells. Functionally, an inhibition of LDH causes a generalized reduction of cytosolic and membrane-bound HSPs in tumor cells and significantly increases the radiosensitivity, which is associated with a G2/M arrest. We demonstrate that targeting of the lactate/pyruvate metabolism breaks the radioresistance by impairing the stress response.

The major stress-inducible protein Hsp70 (HSPA1A) is overexpressed in the cytosol of many highly aggressive tumor cells including glioblastoma multiforme and presented on their plasma membrane. Depending on its intracellular or membrane localization, Hsp70 either promotes tumor growth or serves as a target for natural killer (NK) cells. The kinetics of the membrane Hsp70 (mHsp70) density on human glioma cells (U87) was studied after different irradiation doses to define the optimal therapeutic window for Hsp70-targeting NK cells. To maintain the cells in the exponential growth phase during a cultivation period of 7 days, different initial cell counts were seeded. Although cytosolic Hsp70 levels remained unchanged on days 4 and 7 after a sublethal irradiation with 2, 4 and 6 Gy, a dose of 2 Gy resulted in an upregulated mHsp70 density in U87 cells which peaked on day 4 and started to decline on day 7. Higher radiation doses (4 Gy, 6 Gy) resulted in an earlier and more rapid onset of the mHsp70 expression on days 2 and 1, respectively, followed by a decline on day 5. Membrane Hsp70 levels were higher on cells in G2/M than in G1, however, an irradiation-induced cell cycle arrest on days 4 and 7 was not associated with an increase in the mHsp70 density. Extracellular Hsp70 concentrations in the supernatant of irradiated cells were significantly higher than sham (0 Gy) irradiated cells on days 4 and 7, but not on day 1. Functionally, elevated mHsp70 densities were associated with a significantly better lysis by Hsp70-targeting NK cells. In summary, the kinetics of changes in the mHsp70 density upon irradiation on tumor cells is time- and dose-dependent.