Forced expression of heat-shock protein 70 increases the secretion of Hsp70 and provides protection against tumour growth

Abstract

Although heat-shock protein 70 (Hsp70) has been considered an intracellular protein, we report that Hsp70 is secreted under normal cell culture conditions by human prostate cell lines, LAPC-4, PC-3, CWR-22, RWPE-1 and -2, LNCaP, and TRAMP (transgenic adenocarcinoma mouse prostate)-C2. We found that the secretion can be enhanced by transfection with cDNA encoding for Hsp70. To verify that the Hsp70 detected in the supernatant was not secondary to cell leakage, C2 cells were cotransfected with cytoplasmic Renilla luciferase as a reporter. High levels of activities were noted in the cell extracts, while no enzyme activities were detected in the supernatants. To verify that forced oversecretion of Hsp70 could protect against tumour growth, mice were injected with C2 cells transfected with an Hsp70 DNA construct and challenged with live tumour cells. Mice injected with cells transfected with the Hsp70 DNA construct demonstrated a significantly decreased rate of tumour growth compared to those injected with empty vector. In addition, a difference in survival rate as defined by a surrogate end point was noted between the two groups. In a second experiment, we developed a cell line that stably overexpressed Hsp70. Mice injected with these cells also demonstrated a significant decrease in tumour growth and significantly increased survival.

Figure 1

(A) Western analysis for Hsp70 in supernatants of pcDNA3.1+Hsp70 and Renilla luciferase or mock-transfected TRAMP-C2 cells. Spent media were collected at 24, 48, and 72 h and concentrated as in Materials and methods. (B) Western analysis for Hsp70 in cell extracts of Hsp70, Renilla luciferase, and mock-transfected TRAMP-C2 cells. Ponseau S was used for normalisation. (C) Percent of Hsp70 in supernatants (Sup) and cell extracts (CE) of Hsp70 and mock-transfected TRAMP-C2 cells as determined by densitometry. (D). Comparison of luciferase activity in cell extracts (C) vs supernatants (S) in C2 cells transfected with pcDNA3.1+Hsp70 and Renilla luciferase at various time points. Spent media and whole-cell protein extracts were prepared as above. (E) Percentage of renilla protein was determined by luminescence.

Figure 2

Western analysis for Hsp70 in supernatants (S) and cell extracts (C) of various prostate cell lines. Whole-cell protein extracts and spent media were collected at 48 h after being plated and subjected to Western analysis. Proteins were quantified with Bradford assay and equivalent amounts of proteins were loaded onto SDS–PAGE gel.

Figure 3

Western analysis for Hsp70, PSA and β-tubulin in supernatant and cell extract of LNCaP cells. Whole-cell protein extracts and spent media were prepared 16 h after treatment with various concentrations of BFA (μg ml⁻¹). Protein concentrations were quantified and equal amounts of proteins were loaded onto a single gel.

Figure 4

TRAMP-C2 tumour growth rate in C57BL/6 male mice after injecting with transiently transfected TRAMP-C2 cells with Hsp70 or empty vector. Tumours were measured every other day. P-values of <0.05 were considered statistically significant.

Figure 5

Percent survival of mice with transiently transfected Hsp70 TRAMP-C2 cells or empty vector.

Figure 6

TRAMP-C2 tumour growth rate in C57BL/6 male mice after injecting with stably transfected Hsp70 TRAMP-C2 cells. Tumours were measured every other day. P-values of <0.05 were considered statistically significant. Inlet: Western analysis of Hsp70 in pcDNA3.1+murine Hsp70 (Hsp70)- and pcDNA3.1 (E)-transfected stable clones. Cell extracts (C) and supernatants (S).

Figure 7

Percent survival of mice with stably transfected Hsp70 TRAMP-C2 cells or empty vector.