Effects of protein-secretion inhibitors on Hsp60 secretion by tumor cells.

<p><b>A</b>) Hsp60 and Hsp70 detected by Western blotting in: (<b>a</b>) immunoprecipitates from conditioned media from untreated (Unt) and inhibitor-treated H292 tumor cells; and (<b>b</b>) whole-cell lysates from H292 cells. The inhibitors are listed on top of the respective lanes. Histograms to the right represent the levels of the Hsps in immunoprecipitates determined in three separate experiments as mean percentages +/− SD of arbitrary units (AU) obtained with NIH image J 1.40 analysis software. * and ** significantly different from untreated control, p<0.005 and p<0.001, respectively. The two inhibitors (listed below the bars) significantly decreased secretion of Hsp60 and Hsp70. Also, the data from whole-cell lysates show that the protein-secretion inhibitors had no detectable effect on Hsp levels inside the cells. <b>B</b>) Hsp60 levels secreted by the H292 tumor cells before and after exposure for 1 hour, followed by a 4 hours recovery period, to protein-secretion inhibitors measured by ELISA in: (<b>a</b>) conditioned media; and (<b>b</b>) exosomes. Histograms represent Hsp60 levels expressed as pg of protein normalized for mL normalised for 10⁶ cells. Data represent mean +/− SD of three different experiments in duplicate. * Significantly different from untreated control, p<0.005. The results, which are in agreement with those obtained by Western blotting, show that the inhibitors tested significantly reduced secretion of Hsp60 by the H292 tumor cells.</p

Golgi involvement in Hsp60 secretion from tumor cells. A.

<p>TEM<b>-</b>Immunogold shows Hsp60 (black dots) in tumor cells, including in the Golgi (framed by a dashed rectangle). The arrows show the plasma membrane and arrowheads indicate Hsp60 in it. Bar: 100 nm. <b>B.</b> Extracellular levels of Hsp60 decrease after Brefeldin A (BFA) treatment of the tumor cells, as measured in immunoprecipitates from the conditioned medium (left upper panel). In contrast, Hsp70 levels are not influenced by BFA treatment (left lower panel). Right hand panel: histograms showing densitometric measurements of the Western blots to the left. UT, untreated; A.U.: arbitrary units; asterisk indicates p<0.005. <b>C.</b> ELISA results demonstrate a reduction of Hsp60 levels in the conditioned culture medium from tumor cells after BFA treatment. UT: untreated. <b>D.</b> ELISA results show a reduction of Hsp60 levels in exosomes after BFA treatment of the tumor cells. UT: untreated. Asterisks in C and D, p<0.05. Error bars represent SD.</p

Hsp60 is integrated in the membrane of exosomes from tumor cells. A–C.

<p>Illustrative data on the exosome preparations utilized in this work. <b>A</b>, Transmission electron microscopy demonstrates that the dimension of isolated vesicles is equal to, or smaller than 100 nm, which is consistent with exosomes. Bar: 100 nm. <b>B</b> and <b>C</b>, acetylcholinesterase (AChEase) and ATPse enzymatic activities, respectively, typical of isolated exosomal vesicles, compared to control (conditioned culture medium). In B, the solid line represents data from exosomes, and the broken line represents results from conditioned culture medium. Vertical axis, AChEase activity in arbitrary units (AU), reflecting 412 nm absorbance; horizontal axis, time of reaction in minutes. In C, 1, marker (positive control); 2, conditioned culture medium; 3, exosomes. <b>D.</b> Treatment with sodium carbonate alone (lane 3, asterisk), or in association with proteinase K buffer (lane 4), does not remove Hsp60 from the exosomes, whereas treatment with Proteinase K does (lane 2). Lane 1, untreated exosomes (positive control).</p