Heme Oxygenase-1 Promotes Survival of Renal Cancer Cellsthrough Modulation of Apoptosis- andAutophagy-regulating Molecules*□S

Received for publication, June 18, 2012, and in revised form, July 18, 2012 Published, JBC Papers in Press, July 26, 2012, DOI 10.1074/jbc.M112.393140

Pallavi Banerjee‡§, Aninda Basu‡§, Barbara Wegiel§¶, Leo E. Otterbein§¶, Kenji Mizumura§�, Martin Gasser**,Ana Maria Waaga-Gasser**, Augustine M. Choi§�, and Soumitro Pal‡§1

From the ‡Division of Nephrology, Children’s Hospital, Boston, Massachusetts, the §Harvard Medical School, Boston,Massachusetts 02115, the ¶Department of Surgery, Transplant Institute, Beth Israel Deaconess Medical Center, Boston,Massachusetts, the �Brigham and Women’s Hospital, Boston, Massachusetts, and the **Department of Surgery I, MolecularOncology and Immunology, University of Wurzburg, 97080 Wurzburg, Germany

Background: The cytoprotective enzyme HO-1 promotes tumor growth.Results: HO-1 down-regulated apoptosis- and autophagy-regulating proteins, and attenuated Rapamycin- and Sorafenib-induced apoptosis and autophagy in renal cancer cells.Conclusion: HO-1 protects cancer cells from chemotherapeutic drug-induced death by down-regulating apoptosis andautophagy.Significance: Inhibition of HO-1 augments efficiency of chemotherapeutic agents to kill cancer cells by promoting bothapoptosis and autophagy.

The cytoprotective enzymehemeoxygenase-1 (HO-1) is oftenoverexpressed in different types of cancers and promotes cancerprogression. We have recently shown that the Ras-Raf-ERKpathway inducesHO-1 to promote survival of renal cancer cells.Here, we examined the possible mechanisms underlying HO-1-mediated cell survival. Considering the growing evidence aboutthe significance of apoptosis and autophagy in cancer, we triedto investigate how HO-1 controls these events to regulate sur-vival of cancer cells. Rapamycin (RAPA) and sorafenib, twocommonly used drugs for renal cancer treatment, were found toinduce HO-1 expression in renal cancer cells Caki-1 and 786-O;and the apoptotic effect of these drugs was markedly enhancedupon HO-1 knockdown. Overexpression of HO-1 protected thecells from RAPA- and sorafenib-induced apoptosis and alsoaverted drug-mediated inhibition of cell proliferation. HO-1induced the expression of anti-apoptotic Bcl-xL and decreasedthe expression of autophagic proteins Beclin-1 and LC3B-II;while knockdown of HO-1 down-regulated Bcl-xL and mark-edly increased LC3B-II. Moreover, HO-1 promoted the associ-ation of Beclin-1 with Bcl-xL and Rubicon, a novel negative reg-ulator of autophagy. Drug-induced dissociation of Beclin-1from Rubicon and the induction of autophagy were also inhib-ited by HO-1. Together, our data signify that HO-1 is up-regu-lated in renal cancer cells as a survival strategy against chemo-therapeutic drugs and promotes growth of tumor cells byinhibiting both apoptosis and autophagy. Thus, application ofchemotherapeutic drugs along with HO-1 inhibitor may elevatetherapeutic efficiency by reducing the cytoprotective effects of

HO-1 and by simultaneous induction of both apoptosis andautophagy.

Hemeoxygenase-1 (HO-1)2 is a stress-inducible intracellularenzyme that degrades heme into carbon monoxide (CO), bili-verdin, and ferrous iron (1). HO-1 classically functions tomain-tain cellular homeostasis under stress conditions. This cytopro-tective effect may be attributed to the removal of a potentinflammatory agent heme, and to the functions of biologicallyactive products generated during HO-1-mediated heme degra-dation. The by-products of heme degradation play a crucial rolein the adaptive response of cells to oxidative and cellularstresses by reducing inflammation and apoptosis, and inducingcell proliferation and angiogenesis (1–3). Inhibition of apopto-sis seems to be one of the major mechanisms underlying thecytoprotective function of HO-1. HO-1 confers protectionfrom ischemia-reperfusion injury, cisplatin nephrotoxicity andendotoxic shock by attenuating apoptotic signals. Thus, HO-1is a critical player in the survival machinery of cell and plays therole of savior in many human diseases (4, 5).Despite its cytoprotective properties, recent evidences sug-

gest a role for HO-1 in promoting cancer (6–9). HO-1 is over-expressed in different types of cancers and is further induced byradiation and chemotherapy (6, 10, 11). HO-1 endorses survivalof various cancers, including hepatoma,melanoma, thyroid andlung carcinoma, while inhibition of HO-1 reduces viability ofcolon carcinoma, acute myeloid leukemia and induces apopto-sis of renal cancer cells (12, 13). The protective function ofHO-1 thus turns out to be a double-edged sword, as cancer cellsutilize the anti-apoptotic effects of HO-1 as a shield from che-

* This work was supported, in whole or in part, by National Institutes of HealthGrant R01 CA131145 (to S. P.) and a Fellowship Grant from American Soci-ety of Transplantation (to P. B.).

□S This article contains supplemental Figs. S1–S3.1 To whom correspondence should be addressed: Division of Nephrology,

Children’s Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-919-2989; Fax: 617-730-0130; E-mail: soumitro.pal@childrens.harvard.edu.

2 The abbreviations used are: HO-1, heme oxygenase-1; CoPP, cobalt proto-porphyrin; CORM, CO-releasing molecule; siRNA, small interfering RNA; PI,propidium iodide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 38, pp. 32113–32123, September 14, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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motherapeutic drugs (6, 10, 14). However, there are few reportsabout anti-proliferative role of HO-1 in prostate and breastcancer (15, 16). Revelation of its effects on tumor growth andmetastasis has undoubtedly increased the significance of HO-1in the field of cancer biology. Many studies have targeted inhi-bition of this enzyme as an approach to sensitize cancer cells totherapies (14, 17).While most of the studies have tried to investigate the anti-

apoptotic role of HO-1 in promoting cancer cell survival, it is ofinterest to also explore the regulation of autophagy by theenzyme. Recent studies show that along with apoptosis, type IIcell death or autophagy plays a vital role in ascertaining the fateof cancer cells (18–20). Autophagy is a “self-eating” processresponsible for intracellular degradation of damaged and dys-functional organelles and protein aggregates. It is a non-selec-tive process where cytoplasm and organelles are encapsulatedwithin double-membrane vesicles called autophagosomes, andare eventually degraded as the autophagosomes fuse with lyso-somes (18). Protein degradation is thought to be a protein turn-over procedure. Thus, by ensuring a constant recycling ofessential nutrients and cellular components, the intracellulardegradation process autophagy serves a cytoprotective role,especially under nutrient-deficient conditions. Autophagy isinduced in tumor cells during starvation and hypoxia and canprotect the cells from apoptosis under such metabolic and oxi-dative stress conditions (21, 22). Regardless of its proposed pro-tective functions, the role of autophagy in cancer is a matter ofdebate.In contrast to studies that suggest its pro-tumorigenic func-

tion, many observations indicate a tumor-suppressing role forautophagy. An anti-cancer role for autophagy first came underconsideration when Beclin-1, a core component of theautophagosome nucleation complex was identified as a tumorsuppressor gene (23). Beclin-1 is monoallelically deleted inhuman breast, ovarian and prostate cancers, and Beclin-1�/�

mice have been found to develop spontaneous tumors (23–26).The anti-cancer function of autophagy is also supported byfacts that the tumor suppressor genes p53 and PTEN induceautophagy (27, 28), while pro-survival Bcl family members,Bcl-2 and Bcl-xL inhibit autophagy by directly associating withBeclin-1 (29). Controversy regarding the role of autophagy incancer is further augmented by conflicting reports about theeffect of autophagy on chemotherapeutic treatment. Severalchemotherapeutic drugs, including sorafenib, rapamycin(RAPA) and histone deacetylase inhibitors have been reportedto induce autophagy (18, 30, 31). While some groups suggestthat autophagy augments toxicity of the drugs to facilitate apo-ptosis of cancer cells, others propose that autophagy is inducedin cancer cells as a survival strategy against these drugs. Con-sidering the contradictory evidence regarding the role ofautophagy in cancer, it is believed that autophagy suppressesthe growth and progression of early tumors, while it supportsthe survival of established tumors and promotes cancer at laterstages (18, 32).We have recently demonstrated that HO-1 is induced in

human renal cancer cells via the activation of Ras-Raf-ERKpathway; and overexpression of HO-1 plays a pivotal role inpromoting cell survival (13). In the present study, we investi-

gated the mechanism(s) underlying HO-1-mediated cell sur-vival. We observe that HO-1 protects renal cancer cells fromapoptosis and autophagy induced by chemotherapeutic drugsRAPA and sorafenib; and HO-1-mediated cancer cell survivalinvolves modulation of regulatory proteins (particularly, Bcl-xL, Beclin-1, LC3B-II and Rubicon) for both apoptosis andautophagy.

EXPERIMENTAL PROCEDURES

Reagents—Cobalt protoporphyrin (CoPP)was obtained fromFrontier Scientific. CO-releasing molecule (CORM) tricarbon-yldichlororuthenium (II) dimmer (CORM-2) was obtainedfromSigma-Aldrich. RAPAand sorafenibwere purchased fromLC Laboratories. The gene-specific small interfering RNA(siRNA) for HO-1 along with its control was purchased fromQiagen. The transfection of siRNA was performed using Lipo-fectamine 2000 (Invitrogen).Cell Lines—The human renal cancer cell lines (786–0 and

Caki-1) were obtained from American Type Culture Collec-tion. 786-0 cells were grown in RPMI 1640, and Caki-1 cellswere grown in McCoy’s medium supplemented with 10% fetalbovine serum (GIBCO).Tissue Samples—Tissue samples of human renal cell cancer

(RCC) were obtained from surgical specimens of patients whounderwent surgery at the University Hospital (Wurzburg, Ger-many). The protocol to obtain tissue samples was approved bythe review board of the hospital. Tumor tissues were graded(stages I through IV) according to Robson staging system.Tumor cells were isolated from RCC tissues by Cytospinpreparations.Plasmid—The pCMV-HO-1 cDNA plasmid was used in

transfection studies to overexpress human HO-1.Transfection Assays—786–0 or Caki-1 (2.5 � 105 cells) were

transfected with the human HO-1 overexpression plasmidusing Effectene Transfection Reagent (Qiagen), according tothe manufacturer’s protocol. The total amount of transfectedplasmidDNAwas normalized using a control empty expressionvector. Transfection efficiency was determined by co-transfec-tion of the �-galactosidase gene under control of cytomegalo-virus immediate early promoter and by measurement of �-ga-lactosidase activity using standard assay system (Promega).Immunoprecipitation Assays—Immunoprecipitations were

performed with 0.5 mg of total protein at antibody excess usinganti-Bcl-xL (Cell Signaling Technology) or anti-Rubicon(Abcam). Immunocomplexes were captured with proteinA-Sepharose beads (GE Healthcare), and bead-bound proteinswere subjected to Western blot analysis using anti-Beclin-1(Santa Cruz Biotechnology), anti-Bcl-xL or anti-Rubicon.Western Blot Analysis—Protein samples were run on SDS-

polyacrylamide gel and transferred to a polyvinylidene difluo-ride membrane (Millipore Corp.). The membranes wereincubated with anti-HO-1 (R&D Systems), anti-�-Actin (Sig-ma-Aldrich), anti-Bcl-xL, anti-Bcl-2 (Cell Signaling Technol-ogy), anti-Beclin-1, anti-LC3B (Sigma-Aldrich), or anti-Rubi-con; and subsequently incubated with peroxidase-linkedsecondary antibody. The reactive bands were detected by usingchemiluminescent substrate (Pierce).

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Cell Proliferation Assay—Cell proliferation was measured byMTT Cell Proliferation Assay (ATCC) following the manufac-turer’s protocol. Cells were plated in 96-well plates. Followingtreatment, 10 �l of MTT reagent was added to each well. Oncepurple crystals of formazan became clearly visible undermicro-scope, 100 �l of Detergent Reagent was added and the cellswere incubated at dark for 4 h. Absorbance was measured at570 nm and corrected against blanks, which consisted of cul-turemediumprocessed in the sameway as above in the absenceof cells. The reading at 570 nm is directly proportional to cellproliferation (number of viable cells).Apoptosis Assay—Cellular apoptosis was measured by

Annexin-V and propidium iodide (PI) staining using APCAnnexin-VApoptosis Detection Kit (eBioscience) according tothe manufacturer’s protocol. Following staining, the cells wereanalyzed by flow cytometry on a FACSCalibur.Autophagy Assay—Cellular autophagy was monitored using

Cyto-ID Autophagy Detection Kit (Enzo Life Sciences) follow-ing manufacturer’s protocol. The 488 nm excitable Cyto-IDGreen Autophagy Detection Reagent supplied in the kitbecomes brightly fluorescent in vesicles produced duringautophagy and thus serves as a convenient tool to detectautophagy at cellular level. Following treatment, the cells weretrypsinized, washed in phosphate-buffered saline (PBS) andresuspended in 2000� dilution of the Detection Reagent. After30 min of incubation at 37 °C, the cells were washed and ana-lyzed by flow cytometry.Immunohistochemistry—Immunohistochemistry was per-

formed on either frozen sections of RCC or tumor cells isolatedfrom RCC tissues by Cytospin preparations. Briefly, acetone-fixed sections/cells were first stained with anti-Rubicon(Abcam), and then incubated with a species-specific Cy3-con-jugated secondary antibody (Dianova). In a second step, thesections/cells were stainedwith FITC-conjugated anti-Beclin-1

(Bioss). Specimens were washed thoroughly in between incu-bations and counterstained with DAPI (4�,6-diamidino-2-phe-nylindole dihydrochloride) (Sigma-Aldrich). The sections weremounted with polyvinyl alcohol mounting medium withDABCO (Sigma-Aldrich) and visualized under fluorescencemicroscope. Co-localization of Rubicon and Beclin-1 was eval-uated by merged images.Statistical Analysis—Statistical significance was determined

by Student’s t test. Differences with p � 0.05 were consideredstatistically significant.

RESULTS

HO-1 IsOverexpressed in Renal Cancer Cells Following RAPAand Sorafenib Treatment—We have recently shown that thecytoprotective enzyme HO-1 is overexpressed in human renalcancer cells and promotes cell survival (13). In addition, tumorcells may bypass the killing effects of different chemotherapeu-tic agents because of overexpression of HO-1 (6, 14). Here, weexamined if there is any change in HO-1 expression in renalcancer cells (786-0 and Caki-1) following treatments withRAPA and sorafenib, two approved drugs that are being used totreat renal cancer. The cells were treatedwith different concen-trations of either RAPA (10 and 20 ng/ml) or sorafenib (10 and20 �M); control cells were treated with vehicle alone. Westernblot analysis showed that treatments with both RAPA andsorafenib were associated with a marked increase in HO-1 pro-tein expression compared with vehicle-treated controls (Fig. 1,A and B). In accordance with current literature, it is expectedthat the overexpressed HO-1 may mediate a protective func-tion against RAPA- and sorafenib-mediated killing of renal can-cer cells.Inhibition of HO-1 Augments RAPA- and Sorafenib-induced

Apoptosis of Renal Cancer Cells—Treatments with RAPA andsorafenib can promote apoptosis of cancer cells. As our previ-

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 treatedwith 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). �-Actin was usedas loading control (bottom). The bar graphs beside Western blots illustrate the relative expression of HO-1 by densitometry, wherein the signals werestandardized to the expression of the internal control �-actin. Results are representative of three independent experiments. Columns, average of relativeintensity of HO-1 expression from three different blots; bars, S.E. *, p � 0.05, and **, p � 0.005 compared with vehicle-treated cells.

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ous experiment suggested that treatments with RAPA andsorafenib are associated with HO-1 overexpression, here wewished to evaluate if the knockdown of HO-1 could facilitateRAPA- and sorafenib-induced apoptosis of renal cancer cells.To this end, 786-0 cells were transfected with either HO-1siRNA or control siRNA. Cells were then treated with eitherRAPA or sorafenib; and control cells were treated with vehiclealone. The cells were stained with Annexin-V and propidiumiodide and analyzed by flow cytometry to check the apoptoticindex. As shown in Fig. 2A, RAPA treatment increased cellularapoptosis in control siRNA-transfected renal cancer cells com-pared with vehicle-treated controls; the percentage of apopto-tic cells (early � late apoptotic cells) increased from 3.29%(1.79� 1.5%) to 13.7% (10.5� 3.2%). However, the knockdownof HO-1 significantly increased cellular apoptosis in RAPA-treated cells; the percentage of apoptotic cells increased from13.7% (control siRNA-transfected and RAPA-treated cells) to30.44% (HO-1 siRNA-transfected and RAPA-treated cells).Similarly, as shown in Fig. 2B, sorafenib treatment increased

cellular apoptosis in control siRNA-transfected renal cancercells compared with vehicle-treated controls; the percentage ofapoptotic cells (early � late) increased from 2.73% (vehicle-treated control cells) to 13.56% (control siRNA-transfected andsorafenib-treated cells). However, the knockdown of HO-1 sig-nificantly increased cellular apoptosis in sorafenib-treated

cells; the percentage of apoptotic cells increased from 13.56%(control siRNA-transfected and sorafenib-treated cells) to36.69% (HO-1 siRNA-transfected and sorafenib-treated cells).The knockdown of HO-1 was confirmed by Western blot (Fig.2C). These observations clearly suggest that the knockdown ofHO-1 can augment RAPA- and sorafenib-induced apoptosis ofrenal cancer cells.Overexpression of HO-1 Inhibits RAPA- and Sorafenib-in-

duced Apoptosis of Renal Cancer Cells—In our earlier experi-ment, wemeasured the status of RAPA- and sorafenib-inducedapoptotic cells followingHO-1 knockdown.Here, we examinedthe effect of HO-1 overexpression on RAPA- and sorafenib-induced renal cancer cell apoptosis. We used a HO-1 plasmid(pCMV-HO-1) that overexpresses human HO-1. 786-0 cellswere transfectedwith either theHO-1 plasmid or empty vector.Cells were then treated with either RAPA or sorafenib; andcontrol cells were treated with vehicle. As shown in Fig. 3A,treatment with RAPA increased cellular apoptosis in vector-transfected renal cancer cells compared with vehicle-treatedcontrols; the percentage of apoptotic cells (early � late)increased from 1.81% (vehicle-treated control cells) to 10.95%(vector-transfected and RAPA-treated cells). However, theoverexpression of HO-1 significantly decreased cellular apo-ptosis in RAPA-treated cells; the percentage of apoptotic cellsdecreased from 10.95% (vector-transfected and RAPA-treated

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

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cells) to 4.98% (HO-1 plasmid-transfected and RAPA-treatedcells).Similarly (Fig. 3B), the overexpression of HO-1 significantly

decreased sorafenib-induced apoptosis of renal cancer cells; thepercentage of apoptotic cells (early� late) decreased from 8.0%(vector-transfected and sorafenib-treated cells) to 2.96% (HO-1plasmid-transfected and sorafenib-treated cells). Overexpres-sion ofHO-1 following transfectionwith theHO-1 plasmidwasconfirmed by Western blot (Fig. 3C). Together, these observa-tions suggest thatHO-1 can play amajor role in limiting cellularapoptosis of renal cancer cells following treatments with RAPAand sorafenib.Overexpression of HO-1 Prevents RAPA- and Sorafenib-me-

diated Down-regulation of Renal Cancer Cell Proliferation—Wesought to determine if overexpression of HO-1 couldmodulatethe effect of RAPA and sorafenib on renal cancer cell prolifer-ation. Caki-1 cells were first transfected with either the HO-1overexpression plasmid or empty vector, and then treated witheither RAPA or sorafenib; control cells were treated with vehi-cle. Following treatments, cells were subjected to a cell prolif-eration assay. As shown in Fig. 4 and supplemental Fig. S1,treatment with either RAPA or sorafenib decreased the prolif-eration of renal cancer cells compared with vehicle-treatedcontrols; however, following overexpression of HO-1, treat-

ments with RAPA or sorafenib could not decrease cell prolifer-ation up to similar level. We found a similar effect of HO-1 in786-0 cells; however, the effect wasmore prominent and signif-icant in Caki-1 cells. These results suggest that the overexpres-

FIGURE 3. Overexpression of HO-1 inhibits RAPA- and sorafenib-induced apoptosis. A and B, 786-O cells were transfected with either HO-1 overexpressionplasmid (0. 5 �g) or the empty vector. After 24 h of plasmid transfection, the cells were treated with either 10 ng/ml RAPA (A) or 20 �M 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, overexpressionof 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-medi-ated inhibition of cell proliferation. Caki-1 cells were transfected witheither HO-1 overexpression plasmid (1.0 �g) or the empty vector. After 24 h oftransfection, the cells were treated with 10 ng/ml RAPA, 20 �M sorafenib orvehicle alone for 48 h. Cell proliferation was measured by MTT assay. Datashown are representative of three independent experiments. Columns, aver-age of triplicate readings of two different samples; bars, S.E. *, p � 0.05, **, p �0.005.

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sion of HO-1 can prevent RAPA- and sorafenib-mediateddown-regulation of renal cancer cell proliferation.Induction of HO-1 Is Associated with Increase in the Expres-

sion of Anti-apoptotic Bcl-xL in Renal Cancer Cells—Our ear-lier experiments suggested that the overexpression of HO-1 inrenal cancer cells can significantly down-regulate cellular apo-ptosis induced by chemotherapeutic agents. It has been shownthat with increased expression of Bcl-2 gene family (Bcl-2 orBcl-xL), levels of apoptosis are minimal in renal cell cancer,which may assist in cancer progression and resistance to che-motherapeutic treatments (33). Here, we testedwhether induc-tion of HO-1 in renal cancer cells is also associated with mod-ulation of the expression of Bcl-2 family proteins. HO-1 wasoverexpressed in Caki-1 cells by either CoPP treatment (1–20�M) or transfection with the HO-1 plasmid (0.5–1.0 �g); con-trol cells were either treated with vehicle or transfected withempty vector. We observed that overexpression of HO-1 pro-moted marked induction of Bcl-xL (Fig. 5,A and B, top panels);however, there was no significant change in the expression ofBcl-2 (Fig. 5,A andB, second panel; and supplemental Fig. S2,AandB).We also confirmed that siRNA-mediated knockdown ofHO-1markedly decreased the expression of Bcl-xLwithout anysignificant change in the expression of Bcl-2 (Fig. 5C, top twopanels, and supplemental Fig. S2C).These findings prompted us to investigate if increased level

of CO, which arises from HO-1-mediated heme degradation,

can also regulate Bcl-xL. We treated 786–0 cells with the COreleasing molecule CORM-2. As illustrated in Fig. 5D (toppanel) and supplemental Fig. S2D, CORM-2 treatment indeedincreased the expression of Bcl-xL compared with vehicle-treated control. Together, our results suggest that inducedexpression of Bcl-xL can be one of the possible mechanism(s)for the anti-apoptotic effect of HO-1 in renal cancer cells.Induction of HO-1 Down-regulates the Expression of

Autophagy-regulating Molecules Beclin-1 and LC3B in RenalCancer Cells—Recent studies show that along with apoptosis,autophagy also plays a vital role in regulating the fate of cancercells (18). Interestingly, it has been shown that sorafenib canpromote the killing of cancer cells through autophagy (30).Thus, we wanted to determine if the induction of HO-1 in renalcancer cell could also modulate the expression of twoautophagy-regulating genes, Beclin-1 and LC3B. To this end,HO-1 was overexpressed in Caki-1 cells by either CoPP treat-ment or transfection with the HO-1 plasmid as describedabove. We found that the induction of HO-1 was associatedwith a marked decrease in the expression of Beclin-1 andLC3B-II (Fig. 5, A and B, third and fourth panels; and supple-mental Fig. S2, A and B). We confirmed that knockdown ofHO-1 in these cells markedly increased the expression ofLC3B-II (Fig. 5C, third panel; and supplemental Fig. S2C).We also checked the effect of CO on the expression of

autophagy-regulating genes in renal cancer cells. As shown in

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 differentconcentrations 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 siRNAor 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, andexpression of Bcl-xL, Bcl-2, Beclin-1, LC3B (I & II), HO-1, and �-actin was analyzed by Western blot. Data shown are representative of three independentexperiments. E, Caki-1 and 786-O cells were transfected with different concentrations of HO-1 overexpression plasmid or empty vector for 24 h. Cell lysateswere immunoprecipitated (IP) with anti-Bcl-xL. Immunoprecipitates were captured by protein A-Sepharose beads, boiled in SDS buffer and separated bySDS-PAGE. The expression of Beclin-1 and Bcl-xL in the immunoprecipitate, and �-actin in the cell lysate was analyzed by Western blot. Results are represent-ative of three independent experiments.

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Fig. 5D (second and third panels) and supplemental Fig. S2D,the treatment with CORM-2 markedly decreased the expres-sion of Beclin-1 and LC3B-II compared with vehicle-treatedcontrol. Together, these observations suggest that the overex-pressed HO-1 in part also promotes survival of renal cancercells through down-regulation of autophagy-regulatingproteins.Overexpression of HO-1 Promotes the Association between

Bcl-xL and Beclin-1—The Bcl-2 family members (Bcl-2 andBcl-xL) classically function as anti-autophagy proteins bydirectly associating with Beclin-1 (29, 34). In our earlier exper-iments, we observed that induction of HO-1 in renal cancercells promotes overexpression of Bcl-xL. Here, we sought toevaluate if there is any change in the status of Bcl-xL-Beclin-1complex following HO-1 overexpression. Caki-1 and 786-0cells were transfected with either the HO-1 plasmid or emptyvector. We observed that induction of HO-1 in both cell typesmarkedly increased the complex formation betweenBcl-xL andBeclin-1 compared with vector-transfected controls (Fig. 5E,top panel). As observed in our earlier experiments, the expres-

sion of Bcl-xL was also increased following HO-1 induction(second panel). These findings suggest that the overexpressionof HO-1 in renal cancer cells leads to increased associationbetween Bcl-xL and Beclin-1, which may promote cell survivalthrough decreased autophagy.There Is Co-localization of Beclin-1 and Rubicon in Human

Renal Cancer Cells, and This Association Is Increased FollowingInduction of HO-1—A novel molecule Rubicon has recentlybeen identified as a Beclin-1-interacting protein that sup-presses autophagosomematuration and inhibits autophagy (35,36).We first checked the expression of Beclin-1 and Rubicon inhuman renal cancer tissues and tumor cells (Cytospin prepara-tions). As shown in Fig. 6,A and B, we found that both proteinswere expressed and co-localized in cancer tissues and tumorcells. However, the co-localization was greater in low-stagerenal tumor cells compared with high stage tumor cells.Next, we examined if the induction of HO-1 could also mod-

ulate the association betweenBeclin-1 andRubicon. Caki-1 and786-0 cells were transfected with either the HO-1 plasmid orempty vector. We observed that overexpression of HO-1 in

FIGURE 6. Beclin-1 and Rubicon are expressed and co-localized in renal cancer tissues and tumor cells. Representative photomicrographs illustratingdouble 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 cellsobtained 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 �m. Results (A-B) are representative of three independent experiments.

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both cell types was associated with increased complex forma-tion between Beclin-1 and Rubicon compared with vector-transfected controls (Fig. 7A). Thus, our observations suggestthat induction ofHO-1 in renal cancer cellsmay down-regulateautophagy through increased association between Beclin-1 andRubicon.HO-1 Inhibits RAPA- and Sorafenib-induced Dissociation of

Beclin-1 from Rubicon—We sought to determine the effect ofRAPA and sorafenib treatment on the complex formationbetween Beclin-1 and Rubicon, and how HO-1 may modulatethe process. Caki-1 cells were transfected with either HO-1plasmid or empty vector, and treated with RAPA/sorafenib. Asshown in Fig. 7B (top panel), both RAPA (lane 3) and sorafenib(lane 5) treatment down-regulated the complex formationbetween Beclin-1 and Rubicon compared with vehicle-treatedcontrol (lane 1); however, RAPA and sorafenib-mediateddown-regulation of this complex formation (Beclin-1-Rubicon)was markedly inhibited following overexpression of HO-1(lanes 4 and 6). We observed that there was some decrease inthe expression of Rubicon followingRAPA treatment; however,there was no significant change in Rubicon expression follow-ing sorafenib treatment (Fig. 7B, second panel).We also confirmed this finding by using siRNA. Caki-1 cells

were transfected with either HO-1 siRNA or control siRNA,

and then treated with sorafenib or vehicle alone. As shown inFig. 7C (top panel), both knockdown of HO-1 (lane 2) andsorafenib treatment (lane 3) down-regulated the complex for-mation between Beclin-1 and Rubicon; however, there was fur-ther and marked increase in sorafenib-mediated down-regula-tion of Beclin-1-Rubicon complex formation followingknockdown of HO-1 (lane 4). We found similar results (datanot shown) in RAPA-treated cells following HO-1 knockdown.Our observations suggest that HO-1 may inhibit RAPA andsorafenib-induced autophagy in renal cancer cells through themodulation of Beclin-1 and Rubicon complex.Overexpression of HO-1 Inhibits RAPA-induced Autophagy

in Renal Cancer Cells—To confirm our earlier observationsthat HO-1 can inhibit therapeutic drug-induced autophagy inrenal cancer cells, we investigated the status of autophagy(functional read out) in RAPA-treated cells following HO-1overexpression. 786-0 cells were treated with RAPA in theabsence or presence of different doses of CoPP, an inducer ofHO-1. The cells were stained with Cyto-ID Green AutophagyDetection Reagent and analyzed by flow cytometry. As shownin Fig. 7D and supplemental Fig. S3, RAPA significantly pro-moted autophagy in the cells, while induction of HO-1 mark-edly attenuated both basal as well as RAPA-induced autophagy.Thus, our data show that HO-1 protects renal cancer cells from

FIGURE 7. HO-1, RAPA, and sorafenib modulate the association of Beclin-1 with Rubicon, and HO-1 inhibits RAPA-induced autophagy of renal cancercells. 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 weretransfected with either HO-1 overexpression plasmid (0.5 �g) or empty vector, and treated with 10 ng/ml RAPA, 20 �M 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 �M sorafenibor vehicle for another 24 h. A–C, cell lysates were immunoprecipitated (IP) with anti-Rubicon. Immunoprecipitates were captured by protein A-Sepharosebeads, boiled in SDS buffer and separated by SDS-PAGE. The expression of Beclin-1 and Rubicon in the immunoprecipitate, and �-actin in the cell lysate wasanalyzed by Western blot. Data shown are representative of three independent experiments. D, 786 – 0 cells were treated with different combinations of 50ng/ml RAPA, 10 �M (left panel) or 20 �M (right panel) CoPP, or vehicle for 72 h. The cells were stained with Cyto ID Green Autophagy Detection Reagent asdescribed in “Experimental Procedures,” and analyzed by flow cytometry. Data shown are representative of three independent experiments.

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both apoptosis and autophagy induced by chemotherapeuticdrugs.

DISCUSSION

The cytoprotective enzyme HO-1, which plays an essentialrole in maintaining cellular homeostasis under stress condi-tions, is often highly up-regulated in tumor tissues and canfacilitate tumor growth and metastasis. In this study, we showthat the overexpression of HO-1 can promote survival of renalcancer cells through regulation of both apoptosis andautophagy. HO-1 significantly attenuates RAPA- andsorafenib-induced apoptosis of cancer cells; and HO-1 overex-pression is associated with an induction of Bcl-xL and inhibi-tion of Beclin-1 and LC3B-II. In addition, HO-1 promotes theassociation of Beclin-1 with Bcl-xL and Rubicon, which is anovel negative regulator of autophagy. Finally, we show thatRAPA-induced autophagy is significantly inhibited by HO-1.Revelation of its role in cancer has given a new dimension to

studies involving HO-1. We have recently shown that activa-tion of the Ras-Raf-ERK pathway promotes overexpression ofHO-1 and survival of renal cancer cells (13). Induction of HO-1in cancer is often associated with resistance of cancer cells tochemotherapeutic drugs (6, 37, 38); and inhibition of HO-1 incombinationwith chemotherapy can be a possible and effectivetherapeutic strategy to enhance efficiency of cancer treatment(8, 10, 14). In this study, we demonstrate that knockdown ofHO-1 significantly augments RAPA- and sorafenib-inducedapoptosis of renal cancer cells. It has been reported that in addi-tion to induction of apoptosis, the knockdown ofHO-1 can alsodecrease angiogenesis, proliferation and growth of varioustumors (7, 39, 40). Importantly HO-1 inhibition has beenshown to sensitize cancer cells to cisplatin, gemcitabine andother drug treatments (11, 12, 14). Thus, the mechanisms ofHO-1-mediated cell survival need to be explored to understandhow the function of HO-1 can be modulated to increase theefficacy of cancer therapy.The Bcl2 family, comprised of both pro- (Bax, Bak, Bad) and

anti-apoptotic (Bcl-2, Bcl-xL) members, is a key regulator ofapoptosis (41). It is believed that along with enhanced cell pro-liferation, impaired apoptosis is a crucial factor in tumor devel-opment. Thus, overexpression of anti-apoptotic members ofthe Bcl-2 family, or loss of their pro-apoptotic relatives canresult in oncogenesis and tumor progression. Overexpressionof Bcl-xL and other anti-apoptotic Bcl-2 members has beenfound to be associated with resistance to chemotherapeuticdrugs (42, 43). Interestingly, we found that HO-1 markedlyinduces Bcl-xL in renal cancer cells, with no significant changein expression of Bcl-2. Although both Bcl-xL and Bcl-2 areknown to promote anti-apoptotic signals, it is not uncommonwhen only one of these pro-survival members takes active partin protecting the cells from apoptosis (44, 45). Considering theanti-apoptotic role of Bcl-xL and its involvement in chemore-sistance, it is possible that inhibition of apoptosis by modula-tion of Bcl-xL is one of the possible mechanisms underlyingHO-1-mediated cancer cell survival.While apoptosis has long been known to play a major regu-

latory role in tumorigenesis, autophagy is fast gaining impor-tance (18–20). Recent studies indicate that cancer cell survival

is controlled by a complex interplay of these two cell deathpathways. The precise role of autophagy in cancer has alwaysevoked controversy; there are enough evidences to support itsrole as a killer and also as a savior of cancer cells. It is reasonableto believe that autophagy serves a “quality control” task in theearly stages of cancer, by eliminating cells with defective organ-elles and proteins, thereby inhibiting oncogenesis and tumorgrowth (18, 23, 46). However, during late or advanced stages ofcancer, when rapidly dividing tumor cells are under metabolicstress due to hypoxia and nutrient deficiency, the “protein turn-over” process of autophagy comes as a savior; it supplementsthe growing needs of the cells with excess supply of oxygen andnutrients, thus supporting tumor growth and progression (21,22, 32). The fact that autophagy is promoted with chemother-apeutic drugs has also been justified by two conflicting ration-ale. While some groups believe that autophagy (primarily toxicautophagy) promotes apoptotic functions of chemotherapeuticdrugs, others suggest that it may act as a survival strategy ofcancer cells to protect themselves from toxic effects of thedrugs (18, 30, 31). Our data in this study show thatHO-1 down-regulates some key mediators (Beclin-1 and LC3B-II) of theautophagy pathway, and also inhibits drug-induced autophagyin renal cancer cells. Of several molecules participating in theautophagy pathway, one of the most critical roles is served byBeclin-1. Beclin-1 is an integral part of a class III PI-3K multi-protein complex that is essential for autophagosome nucle-ation. Binding of Beclin-1 to anti-apoptotic members of Bcl-2family abrogates autophagy-promoting functions of Beclin-1,while the dissociation of Beclin-1 from these anti-apoptoticproteins promotes autophagy (29, 34). Our findings indicatethat HO-1 promotes the association of Beclin-1 with its inhib-itor Bcl-xL, and thereby may down-regulate autophagy.Recently, Rubicon was identified as another novel Beclin-1-binding partner that negatively regulates autophagy and endo-cytosis (35, 36). Rubicon associates with UVRAG-Beclin-1complex and suppresses autophagosomematuration and endo-cytic trafficking. Interestingly, our data show a critical role ofHO-1 in inhibiting RAPA- and sorafenib-induced dissociationof Rubicon from Beclin-1. Thus, increased association of Rubi-con and Beclin-1 can act as one of the possible mechanisms forHO-1-mediated down-regulation of drug-induced autophagy.Although apoptosis and autophagy are completely different

events, convergence of these two pathways has been reported.Inducers of apoptosis can also regulate autophagy, and mem-bers of Beclin-1 and Bcl-2 family may serve as a point of cross-talk between these two events (47). It is important to note thatunder specific circumstances, instead of protecting cells fromapoptosis, autophagy can mediate cell death (48). As discussedearlier, chemotherapeutic treatments can promote autophagyto kill tumor cells (30), and it has been suggested thatautophagic cell death can occur in cells that may not die byapoptosis (47). We observe that inhibition of both apoptosisand autophagy play a key role in HO-1-mediated survival ofrenal cancer cells. In support of our findings, it has been shownthat the anti-autophagic property of Bcl-2may play a key role intumor growth and progression (49).In summary, this study elucidates an important mechanism

underlying HO-1-mediated survival of renal cancer cells. It

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explains howHO-1 can protect cells from death by suppressingboth apoptosis and autophagy induced by chemotherapeuticdrugs. Thus, our work highlights the significance of HO-1 as apossible therapeutic target for renal cancer treatment. Applica-tion of chemotherapeutic drugs along with an HO-1 inhibitormay augment the efficiency of therapy by promoting both apo-ptosis and autophagy induced by these drugs.

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Martin Gasser, Ana Maria Waaga-Gasser, Augustine M. Choi and Soumitro PalPallavi Banerjee, Aninda Basu, Barbara Wegiel, Leo E. Otterbein, Kenji Mizumura,

of Apoptosis- and Autophagy-regulating MoleculesHeme Oxygenase-1 Promotes Survival of Renal Cancer Cells through Modulation

doi: 10.1074/jbc.M112.393140 originally published online July 26, 20122012, 287:32113-32123.J. Biol. Chem. 

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