Heme oxygenase-1 promotes survival of renal cancer cells through modulation of apoptosis- and autophagy-regulating molecules

Abstract

The cytoprotective enzyme heme oxygenase-1 (HO-1) is often overexpressed in different types of cancers and promotes cancer progression. We have recently shown that the Ras-Raf-ERK pathway induces HO-1 to promote survival of renal cancer cells. Here, we examined the possible mechanisms underlying HO-1-mediated cell survival. Considering the growing evidence about the significance of apoptosis and autophagy in cancer, we tried to investigate how HO-1 controls these events to regulate survival of cancer cells. Rapamycin (RAPA) and sorafenib, two commonly used drugs for renal cancer treatment, were found to induce HO-1 expression in renal cancer cells Caki-1 and 786-O; and the apoptotic effect of these drugs was markedly enhanced upon HO-1 knockdown. Overexpression of HO-1 protected the cells from RAPA- and sorafenib-induced apoptosis and also averted drug-mediated inhibition of cell proliferation. HO-1 induced the expression of anti-apoptotic Bcl-xL and decreased the expression of autophagic proteins Beclin-1 and LC3B-II; while knockdown of HO-1 down-regulated Bcl-xL and markedly increased LC3B-II. Moreover, HO-1 promoted the association of Beclin-1 with Bcl-xL and Rubicon, a novel negative regulator of autophagy. Drug-induced dissociation of Beclin-1 from Rubicon and the induction of autophagy were also inhibited by HO-1. Together, our data signify that HO-1 is up-regulated in renal cancer cells as a survival strategy against chemotherapeutic drugs and promotes growth of tumor cells by inhibiting both apoptosis and autophagy. Thus, application of chemotherapeutic drugs along with HO-1 inhibitor may elevate therapeutic efficiency by reducing the cytoprotective effects of HO-1 and by simultaneous induction of both apoptosis and autophagy.

FIGURE 1.

RAPA and sorafenib treatment was associated with induced HO-1 expression in renal cancer cells. A and B, Caki-1 and 786-O cells were treated with different doses of RAPA, sorafenib or vehicle for 48 h. The cells were lysed, and the expression of HO-1 was analyzed by Western blot (top). β-Actin was used as loading control (bottom). The bar graphs beside Western blots illustrate the relative expression of HO-1 by densitometry, wherein the signals were standardized to the expression of the internal control β-actin. Results are representative of three independent experiments. Columns, average of relative intensity of HO-1 expression from three different blots; bars, S.E. *, p < 0.05, and **, p < 0.005 compared with vehicle-treated cells.

FIGURE 2.

Inhibition of HO-1 promotes RAPA- and sorafenib-induced apoptosis. A and B, 786-O cells were transfected with either HO-1 siRNA (50 nm) or control siRNA. After 24 h of siRNA transfection, the cells were treated with either 10 ng/ml RAPA (A) or 20 μm sorafenib (B) for 48 h; control cells were treated with vehicle alone. Apoptotic index of the cells was determined by Annexin-V (APC) and propidium iodide staining. C, knockdown of HO-1 in siRNA-transfected cells was confirmed by Western blot. Data shown are representative of three independent experiments.

FIGURE 3.

Overexpression of HO-1 inhibits RAPA- and sorafenib-induced apoptosis. A and B, 786-O cells were transfected with either HO-1 overexpression plasmid (0. 5 μg) or the empty vector. After 24 h of plasmid transfection, the cells were treated with either 10 ng/ml RAPA (A) or 20 μm sorafenib (B) for 48 h; control cells were treated with vehicle alone. Apoptotic index of the cells was determined by Annexin-V (APC) and propidium iodide staining. C, overexpression of HO-1 in plasmid-transfected cells was confirmed by Western blot. Data shown are representative of three independent experiments.

FIGURE 4.

Overexpression of HO-1 prevents RAPA- and sorafenib-mediated inhibition of cell proliferation. Caki-1 cells were transfected with either HO-1 overexpression plasmid (1.0 μg) or the empty vector. After 24 h of transfection, the cells were treated with 10 ng/ml RAPA, 20 μm sorafenib or vehicle alone for 48 h. Cell proliferation was measured by MTT assay. Data shown are representative of three independent experiments. Columns, average of triplicate readings of two different samples; bars, S.E. *, p < 0.05, **, p < 0.005.

FIGURE 5.

HO-1 modulates the expression of apoptosis- and autophagy-regulating molecules, and promotes the association of Beclin-1 and Bcl-xL. A, Caki-1 cells were treated with either different concentrations of CoPP or vehicle alone for 48 h. B, Caki-1 cells were transfected with either different concentrations of HO-1 overexpression plasmid or the empty vector for 48 h. C, Caki-1 cells were transfected with either different concentrations of HO-1 siRNA or control siRNA for 72 h. D, 786–0 cells were treated with either different concentrations of CORM-2 or vehicle alone for 24 h. A–D, cells were lysed, and expression of Bcl-xL, Bcl-2, Beclin-1, LC3B (I & II), HO-1, and β-actin was analyzed by Western blot. Data shown are representative of three independent experiments. E, Caki-1 and 786-O cells were transfected with different concentrations of HO-1 overexpression plasmid or empty vector for 24 h. Cell lysates were immunoprecipitated (IP) with anti-Bcl-xL. Immunoprecipitates were captured by protein A-Sepharose beads, boiled in SDS buffer and separated by SDS-PAGE. The expression of Beclin-1 and Bcl-xL in the immunoprecipitate, and β-actin in the cell lysate was analyzed by Western blot. Results are representative of three independent experiments.

FIGURE 6.

Beclin-1 and Rubicon are expressed and co-localized in renal cancer tissues and tumor cells. Representative photomicrographs illustrating double fluorescence labeling of both Beclin-1 (green) and Rubicon (red) in high stage and low stage human renal cell cancer tissues (A) and also tumor cells obtained from renal cancer tissues by Cytospin preparations (B). DAPI (blue) was used to visualize nuclei. Merged images are shown to indicate co-localization (yellow) of Beclin-1 and Rubicon. Scale bar, 50 μm. Results (A-B) are representative of three independent experiments.

FIGURE 7.

HO-1, RAPA, and sorafenib modulate the association of Beclin-1 with Rubicon, and HO-1 inhibits RAPA-induced autophagy of renal cancer cells. A, Caki-1 and 786-O cells were transfected with different concentrations of HO-1 overexpression plasmid or empty vector for 24 h. B, Caki-1 cells were transfected with either HO-1 overexpression plasmid (0.5 μg) or empty vector, and treated with 10 ng/ml RAPA, 20 μm sorafenib or vehicle alone for 24 h. C, Caki-1 cells were transfected with either 50 nm HO-1 siRNA or control siRNA. After 48 h of siRNA transfection the cells were treated with either 20 μm sorafenib or vehicle for another 24 h. A–C, cell lysates were immunoprecipitated (IP) with anti-Rubicon. Immunoprecipitates were captured by protein A-Sepharose beads, boiled in SDS buffer and separated by SDS-PAGE. The expression of Beclin-1 and Rubicon in the immunoprecipitate, and β-actin in the cell lysate was analyzed by Western blot. Data shown are representative of three independent experiments. D, 786–0 cells were treated with different combinations of 50 ng/ml RAPA, 10 μm (left panel) or 20 μm (right panel) CoPP, or vehicle for 72 h. The cells were stained with Cyto ID Green Autophagy Detection Reagent as described in “Experimental Procedures,” and analyzed by flow cytometry. Data shown are representative of three independent experiments.