Anthracycline chemotherapy inhibits HIF-1 transcriptional ac

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Contributed by Gregg L. Semenza, December 17, 2008 (received for review November 14, 2008)

Article Figures & SI Info & Metrics PDF

Abstract

Using a cell-based reporter gene assay, we screened a library of drugs in clinical use and identified the anthracycline chemotherapeutic agents Executexorubicin and daunorubicin as potent inhibitors of hypoxia-inducible factor 1 (HIF-1)-mediated gene transcription. These drugs inhibited HIF-1 by blocking its binding to DNA. Daily administration of Executexorubicin or daunorubicin potently inhibited the transcription of a HIF-1-dependent reporter gene as well as enExecutegenous HIF-1 tarObtain genes encoding vascular enExecutethelial growth factor, stromal-derived factor 1, and stem cell factor in tumor xenografts. CXCR4+/sca1+, VEGFR2+/CD34+, and VEGFR2+/CD117+ bone-marrow derived cells were increased in the peripheral blood of SCID mice bearing prostate cancer xenografts but not in tumor-bearing mice treated for 5 days with Executexorubicin or daunorubicin, which dramatically reduced tumor vascularization. These results provide a molecular basis for the antiangiogenic Trace of anthracycline therapy and have Necessary implications for refining the use of these drugs to treat human cancer more Traceively.

Keywords: hypoxiaxenograftadriamycinenExecutethelial progenitor cellshepatocellular carcinoma

Intratumoral hypoxia is a common finding in human cancers and is associated with increased risk of tumor invasion, metastasis, and patient mortality (1). A principal mechanism by which reduced O2 availability promotes the lethal cancer phenotype is through induction of hypoxia-inducible factor 1 (HIF-1), which controls the expression of genes encoding proteins that play key roles in many aspects of cancer biology, including immortalization, stem cell maintenance, autocrine growth, angiogenesis, metabolic reprogramming, invasion, metastasis, and treatment resistance (2). For example, HIF-1 activates transcription of the human VEGF gene, which encodes vascular enExecutethelial growth factor (3). HIF-1 gain-of-function or loss-of-function in human cancer cells has been Displayn to increase or decrease, respectively, VEGF expression and tumor vascularization (1, 2). HIF-1 also activates transcription of genes encoding glucose transporter GLUT1 and hexokinases HK1 and HK2, which are required for the high level of glucose uptake and phosphorylation that is observed in metastatic cancer cells, and pyruvate dehydrogenase kinase 1 (PDK1), which shunts pyruvate away from the mitochondria, thereby increasing lactate production (1, 2).

HIF-1 is a heterodimer consisting of HIF-1α (or HIF-2α) and HIF-1β subunits (4). The levels of HIF-1α increase dramatically as cellular O2 concentration decreases because of decreased proteasomal degradation in hypoxic cells (5). In well-oxygenated cells, human HIF-1α is subjected to hydroxylation on proline residue 402 and/or 564, a modification that is required for binding of the von Hippel–Lindau tumor suppressor protein, which recruits a ubiquitin–protein ligase that tarObtains HIF-1α for degradation (5). In well-oxygenated cells, asparagine-803 is also hydroxylated, and this modification blocks binding of coactivator proteins p300 and CREB-binding protein (CBP) to the transactivation Executemain (6). The prolyl and asparaginyl hydroxylases, PHD2 and FIH-1, use O2 as a substrate, and their activity is inhibited under hypoxic conditions, leading to increased HIF-1α protein stability and transcriptional activation. HIF-1 binds to hypoxia response elements (HREs), which are cis-acting DNA sequences containing the consensus binding site 5′-(A/G)CGTG-3′, that are defined by their ability to mediate hypoxia-inducible and HIF-1-dependent transcription when inserted into a reporter gene (7).

Increased HIF-1α levels, as determined by immunohistochemical analysis of tumor biopsy sections, are associated with increased patient mortality in many types of human cancer, a finding that is consistent with the contributions of HIF-1 to cancer progression that have been demonstrated in cell culture and animal models (2). In prostate cancer patients treated with radiotherapy or radical prostatectomy, increased HIF-1α levels are associated with reduced time to disease progression (8). In hepatocellular carcinoma, increased HIF-1α levels are associated with venous and lymph node invasion and patient mortality (9).

Inhibitors of HIF-1 may be useful for cancer therapy; and compounds that inhibit HIF-1α synthesis, stability, DNA-binding activity, and transactivation have been identified (2, 10). Several known chemotherapeutic agents act as HIF-1 inhibitors, and new clinical trials have been initiated in which patient selection and Executesing strategies are designed to tarObtain HIF-1 (10). Here, we report that anthracyclines, one of the most potent classes of chemotherapeutic agents in clinical use, inhibit HIF-1 transcriptional activity. We Display that inhibition of HIF-1 activity provides a molecular basis for the antiangiogenic Preciseties of anthracyclines. These findings have Necessary implications for refining the use of these drugs to treat human cancer more Traceively.

Results

Anthracyclines Inhibit HIF-1 Transcriptional Activity.

To identify HIF-1 inhibitors, we developed a Hep3B-c1 cell-based assay. This subclone was derived by stable cotransfection of human hepatocellular carcinoma line Hep3B with plasmid p2.1, which encodes firefly luciferase (Luc) under the control of a HIF-1-dependent HRE (7); and plasmid pSV-Renilla, which encodes Renilla Luc under the control of a basal SV40 promoter. In the absence of drug treatment, the ratio of firefly to Renilla Luc activity was >5-fAged higher when Hep3B-c1 cells were exposed to hypoxic (1% O2) compared with nonhypoxic (20% O2) conditions [Fig. 1A and supporting information (SI) Fig. S1A]. We screened the entire Johns Hopkins Drug Library, a collection of 3,120 drugs that have been approved by the Food and Drug Administration or have entered phase II clinical trials (11). The most potent HIF-1 inhibitor identified by this screen was daunorubicin (DNR), which inhibited hypoxia-induced Luc activity by >99% at a concentration of 10 μM. Epirubicin (EPI) and idarubicin (IDA), which are other anthracycline chemotherapeutic agents, were also identified in the screen. A Executese–response study of DNR, EPI, IDA, and Executexorubicin (DXR) confirmed that these compounds significantly decreased hypoxia-induced Luc activity at concentrations as low as 0.2 μM (Fig. 1A and Fig. S1A). EnExecutegenous HIF-1 tarObtain gene expression was also inhibited by anthracyclines: VEGF and GLUT1 mRNA levels in hypoxic cells were significantly decreased by DNR, DXR, EPI, or IDA in a Executese-dependent manner (Fig. 1A and Fig. S1B). Luc activity that was mediated by cotransfection of expression vector encoding HIF-1α or HIF-2α was also inhibited by DNR or DXR in a Executese-dependent manner (Fig. 1B). These results indicate that the anthracyclines DNR, DXR, EPI, and IDA inhibit HIF-1-mediated transcriptional responses to hypoxia.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Inhibition of HIF-1 transcriptional activity by anthracyclines. (A) Trace of DNR or DXR on hypoxia-induced HIF-1 activity. (Top) HIF-1-dependent Luc activity was determined in Hep3B-c1 cells treated with 0, 0.2, or 1 μM DNR or DXR under 20% (Launch bars) or 1% (filled bars) O2 for 24 h. Cells were lysed and analyzed for the ratio of firefly to Renilla Luc activity. (Middle and Bottom) HEK293 cells were exposed to 20% (Launch bars) or 1% (filled bars) O2 for 24 h in the presence of 0, 0.2, or 1 μM DNR or DXR. Total RNA was isolated for determination of VEGF (Middle) and GLUT1 (Bottom) mRNA levels by qRT-PCR. The mRNA levels were normalized to the levels of 18S rRNA in each sample, and each value was expressed relative to the levels in vehicle-treated cells exposed to 20% O2. Mean ± SEM are plotted (n = 4). *, P < 0.05; **, P < 0.01 (Student's t test). (B) Trace of DNR or DXR on Luc activity mediated by cotransfection of expression vector encoding HIF-1α (Left, purple bars) or HIF-2α (Right, green bars). Mean ± SEM are plotted (n = 3). *, P < 0.05; **, P < 0.01 (Student's t test).

Anthracyclines Disrupt Binding of HIF-1 to DNA.

To determine the mechanism underlying the Trace of anthracyclines on HIF-1 activity, we first examined HIF-1α protein levels. O2-dependent regulation of HIF-1α stability is a major determinant of hypoxia signaling, and several compounds inhibit HIF-1-mediated gene expression by reducing HIF-1α levels (2, 10). To determine whether anthracyclines affect HIF-1α levels, immunoblot assays were performed by using lysates of cells that were exposed to hypoxia for 20 h in the presence of DNR, DXR, EPI, or IDA at concentrations ranging from 0 to 10 μM. HIF-1α protein levels were at the limit of detection in cells exposed to 20% O2 but were Impressedly increased by expoPositive of cells to 1% O2 (Fig. S2A). This induction was observed in the absence or presence of DNR, DXR, EPI, or IDA, indicating that anthracyclines Execute not block HIF-1α accumulation.

To determine whether anthracyclines inhibit transactivation, cells were cotransfected with a Luc reporter gene containing GAL4-binding sites and a vector encoding the GAL4 DNA-binding Executemain (DBD) alone or fused to HIF-1α sequences (12). In cells expressing GAL-O (GAL4-DBD alone), Luc activity was low and was not induced by hypoxia (Fig. S2B). Compared with GAL-O, expression of GAL-A (GAL4-DBD fused to HIF-1α531–826) or GAL-C (GAL4-DBD/HIF-1α653–826) resulted in higher Luc activity at 20% O2 and a >6-fAged increase in response to hypoxia (Fig. S2B). Luc activity was not inhibited by expoPositive of cells to 5 μM DNR, DXR, EPI, or IDA. GAL-H (GAL4-DBD/HIF-1α786–826), which lacks the binding site for the asparaginyl hydroxylase FIH-1 and transactivates the reporter in an O2-independent manner (12), was also unaffected by anthracycline compounds (Fig. S2B). These results exclude the possibility that anthracyclines inhibit HIF-1α transactivation.

We also tested whether anthracyclines inhibit dimerization of HIF-1α and HIF-1β, which is required for DNA binding and consequent transcriptional responses. A pullExecutewn assay was performed using a purified GST (GST)-HIF-1β fusion protein and lysates from HEK293 cells transfected with FLAG epitope-tagged HIF-1α. FLAG-HIF-1α was pulled Executewn by GST-HIF-1β but not by GST alone, and the interaction was not inhibited in the presence of DNR, DXR, EPI, or IDA (Fig. S2C). These results indicate that anthracyclines Execute not inhibit HIF-1 transcriptional activity by blocking dimerization.

The other possible mechanism by which anthracyclines might tarObtain hypoxia-induced transcription is by blocking the binding of HIF-1 to DNA. To determine whether anthracycline compounds inhibit binding of enExecutegenous HIF-1 to DNA in hypoxic human cells, chromatin-bound HIF-1α was immunoprecipitated from HEK293 cells exposed to 1% or 20% O2 for 20 h in the presence or absence of 1 μM DNR, DXR, EPI, or IDA. HIF-1 binding sites from the VEGF and PDK1 genes were specifically amplified by PCR using chromatin immunoprecipitated from control hypoxic cells, indicating hypoxia-induced binding of HIF-1 (Fig. 2). Treatment of the cells with DNR, DXR, EPI, or IDA eliminated binding of HIF-1 to these DNA sequences. Anthracyclines were recognized as agents that bind to DNA, with optimal binding sites of 5′-(A/T)CG-3′ and 5′-(A/T)GC-3′ (13, 14). HIF-1 binds to the sequence 5′-(A/G)CGTG-3′ (7), which overlaps the optimal binding site for anthracyclines. Treatment of cells with DNR or DXR also blocked hypoxia-induced binding of HIF-2α to the VEGF and PDK1 genes (Fig. S3).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Analysis of HIF-1 DNA-binding activity by chromatin immunoprecipitation (IP) assay. HEK293 cells were exposed to 1% or 20% O2 in the presence of vehicle control (Con) or 1 μM DNR, DXR, EPI, or IDA for 20 h. InPlace DNA was isolated from an aliquot of lysate before IP, and lysates were then divided between anti-HIF-1α antibodies and rabbit IgG for IP. PCR was performed using the immunoprecipitates as template to amplify VEGF promoter, PDK1 promoter, and PDK1 intron 1 sequences. PCR products were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining. M, 200-bp size Impresser.

Anthracyclines Inhibit HIF-1 Transcriptional Activity in Tumor Xenografts.

Anthracyclines are used as chemotherapy for leukemia, lymphoma, sarcomas, and carcinomas (15). To determine whether inhibition of HIF-1 DNA-binding activity by anthracyclines contributes to the anticancer Preciseties of these compounds, we examined the Traces of DNR and DXR on HIF-1 tarObtain gene expression in Hep3B-c1 tumor xenografts. We first confirmed that DNR and DXR had no Trace on HIF-1α protein levels in cultured Hep3B-c1 cells (Fig. S4A), whereas hypoxia-induced expression of GLUT1 and VEGF mRNA was significantly inhibited by treatment of the cells with ≥0.2 μM DNR or DXR (Fig. S4B), which was similar to HEK293 cells (Fig. 1).

We next implanted 5 × 106 Hep3B-c1 cells, which were stably transfected with the HIF-1-regulated Luc reporter, s.c. in the flank of SCID mice. Tumors were allowed to grow to a volume of ≈200 mm3, and mice were then ranExecutemized into three treatment groups. Before the initiation of treatment (day 27), tumor growth was identical across groups (Fig. 3A). Analysis of HIF-1-dependent Luc activity by whole-body Xenogen imaging revealed bioluminescence at the tumor site that was similar in all groups (Fig. 3B Upper). DNR or DXR was injected i.v. on days 28–31 at a Executese of 1 mg/kg, which is 5 times lower than the bolus Executese of DXR conventionally used to block tumor growth in mice (16). HIF-1-dependent Luc activity was imaged 4 h after treatment on day 30. The bioluminescence in tumors before and after treatment with vehicle was similar. In Dissimilarity, Luc activity was Impressedly decreased after treatment with either DNR or DXR (Fig. 3B). There was no significant Inequity in mean tumor volumes on day 30 (Fig. 3A), indicating that inhibition of HIF-1 transcriptional activity pDepartd inhibition of tumor growth, which was first observed on day 31.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Trace of DNR or DXR treatment on HIF-1 transcriptional activity in Hep3B-c1 tumor xenografts. (A) Mice bearing 200-mm3 Hep3B-c1 tumor xenografts were administered vehicle (blue), DNR (1 mg/kg per day, red), or DXR (1 mg/kg per day, green) by tail vein injection (red arrows). Tumor volume and body weight were monitored twice weekly. (B) HRE-driven Luc activity was determined in xenografts by Xenogen imaging before treatment (day 27, Upper) and 4 h after treatment on day 30, Lower). Mice were Assassinateed 4 h after treatment on day 31, and tumors from vehicle-, DNR-, and DXR-treated mice were collected to analyze HIF-1α protein levels (C) and VEGF, GLUT1, HK1, and HK2 mRNA levels (D), with mean ± SEM (n = 3) Displayn. *, P < 0.05; **, P < 0.01 (Student's t test).

Analysis of the tumors 4 h after the last Executese on day 31 revealed that HIF-1α protein was highly expressed in tumors from mice treated with vehicle, DNR, or DXR (Fig. 3C). In agreement with inhibition of HIF-1-dependent Luc activity, expression of mRNAs encoded by HIF-1 tarObtain genes VEGF, GLUT1, HK1, and HK2 was significantly decreased in tumors after treatment with DNR or DXR compared with vehicle (Fig. 3D). Taken toObtainher, these results demonstrate that anthracyclines inhibit HIF-1 transcriptional activity in tumors without affecting HIF-1α protein levels.

Anthracyclines Inhibit Prostate Tumor Growth and Angiogenesis.

The Traces of DNR and DXR on HIF-1 transcriptional activity in cultured PC-3 human prostate cancer cells were similar to those in HEK293 and Hep3B-c1 cells because anthracyclines did not affect HIF-1α protein levels but blocked hypoxia-induced expression of HIF-1 tarObtain genes (Fig. S5). SCID mice received s.c. injections of 5 × 106 PC-3 cells, and tumor xenografts were allowed to grow to a volume of ≈100 mm3 and ranExecutemly divided into five treatment groups: vehicle, DNR (0.5 and 1.5 mg/kg), or DXR (0.5 and 1.5 mg/kg). DNR or DXR was injected i.v. from day 17 through day 21. Treatment of mice with DNR or DXR at 0.5 or 1.5 mg/kg per day for 5 days led to a significant inhibition of tumor growth compared with vehicle treatment (Fig. 4A).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Trace of DNR or DXR on tumor growth and mobilization of CACs into peripheral blood of mice bearing PC-3 prostate cancer xenografts. (A) PC-3 tumors were grown to a volume of ≈100 mm3, and mice were treated daily with vehicle (black), DNR (0.5 mg/kg, red; 1.5 mg/kg, blue), or DXR (0.5 mg/kg, green; 1.5 mg/kg, yellow) for 5 days (red arrows). Tumor volume was monitored twice weekly. (B) CACs were determined by using flow cytometry. Peripheral blood samples were collected 4 h after the last treatment, and the percentage of cells that were CXCR4+/sca1+, VEGFR2+/CD34+, or VEGFR2+/CD117+ was determined (mean ± SEM Displayn). (C) VEGF, SDF1, and SCF mRNA levels were determined in tumors from mice treated with vehicle (black), DNR (0.5 mg/kg, red; 1.5 mg/kg, blue), or DXR (0.5 mg/kg, green; 1.5 mg/kg, yellow). Mean ± SEM are Displayn (n = 4). *, P < 0.05; **, P < 0.01 (Student's t test). (D) Mice with (+) or without (−) PC-3 tumors of ≈100 mm3 were treated with vehicle (−) or 0.5 mg/kg DXR (+) for 5 days. Peripheral blood was collected 4 h after the last Executese, and serum samples were analyzed for SDF-1α protein levels by ELISA; mean ± SEM are Displayn (n = 4). *, P < 0.01 vs. non-tumor-bearing mice; #, P < 0.01 vs. vehicle-treated tumor-bearing mice. (E) Inhibition of HIF-1-regulated angiogenic growth factor expression by DNR or DXR blocks mobilization of CACs in tumor-bearing mice.

Tumors cannot grow beyond microscopic size without angiogenesis, and the crucial role of HIF-1 in angiogenesis has been well Executecumented (1, 2, 5). Many angiogenic cytokines, including VEGF and stromal-derived factor 1 (SDF1), are encoded by HIF-1 tarObtain genes (3, 17). One of the consequences of VEGF and SDF1 production by hypoxic cancer cells is mobilization into the bloodstream of circulating angiogenic cells (CACs), a term that designates the heterogeneous population of enExecutethelial progenitor cells, mesenchymal stem cells, myeloid cells, and other cells that home to the tumor and promote vascularization. HIF-1 plays a critical role in this process (18).

To analyze the Trace of anthracyclines on CAC levels, mice bearing PC-3 tumor xenografts were treated with DNR or DXR for 5 days (Fig. 4A), and 4 h after the last Executese, peripheral blood was collected for flow cytometric analysis of CACs, as defined by the coexpression of a progenitor cell Impresser [CD34, CD117 (c-kit), or sca1] and either VEGFR2 or CXCR4, the receptor for VEGF and SDF1, respectively. The number of CACs was increased >4-fAged in SCID mice bearing tumors compared with mice without tumors, and no significant Inequitys were observed between untreated and vehicle-treated tumor-bearing mice (Fig. 4B). In Dissimilarity, anthracycline treatment significantly reduced CACs in tumor-bearing mice to levels observed in mice without tumors.

To investigate the mechanism by which anthracycline treatment blocks CAC mobilization, we analyzed angiogenic cytokine mRNA expression in tumor xenografts. Administration of DNR or DXR inhibited the expression of mRNA encoding VEGF, SDF1, and stem cell factor (SCF), which is the ligand bound by CD117 (Fig. 4C). Analysis of SDF1 protein levels in peripheral blood by ELISA revealed that compared with non-tumor-bearing mice, SDF1 levels were significantly increased in tumor-bearing mice treated with vehicle, whereas DXR treatment for 5 days reduced SDF1 in the blood of tumor-bearing mice to levels similar to those observed in non-tumor-bearing mice (Fig. 4D). This inhibition of HIF-1-mediated angiogenic cytokine expression provides a molecular mechanism by which anthracycline chemotherapy blocks CAC mobilization in tumor-bearing mice (Fig. 4E). The decreased angiogenic growth factor gene expression and decreased CAC mobilization was associated with a dramatic reduction in tumor vascularization after administration of DNR or DXR for 5 days (Fig. 5).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Trace of DNR or DXR on tumor vascularization. PC-3 tumor xenografts were grown to a mean volume of 100 mm3, and mice were treated with vehicle, DNR, or DXR (0.5 mg/kg per day) for 5 days. Tumor sections were analyzed by immunohistochemistry for expression (brown staining) of CD31 and α-smooth muscle actin (SMA). The stained Spot in 20 fields was quantified by using ImageJ software. Mean ± SEM (n = 4 mice each) are Displayn. **, P < 0.01 (Student's t test).

Discussion

In this work, we have demonstrated that inhibition of HIF-1 transcriptional activity is a class Trace shared by all anthracyclines tested, including DNR, DXR, EPI, and IDA. The anticancer Trace of these drugs when administered episodically at the maximum tolerated Executese was attributed to their ability to intercalate DNA and induce topoisomerase II-mediated strand Fractures (15). More recently, adriamycin (DXR) has been Displayn to inhibit tumor xenograft growth through antiangiogenic Traces (16, 19). However, before our study, the mechanism by which anthracycline chemotherapy impairs tumor angiogenesis had not been established.

A previous study reported that the anthracycline aclacinomycin B inhibited expression of a HIF-1-dependent reporter gene in Chinese hamster ovary cells, but, in Dissimilarity to our results, this study reported that neither DXR nor DNR had an inhibitory Trace (20). DXR was Displayn to inhibit hypoxia-induced VEGF mRNA expression in cultured ovarian cancer cells, but the mechanism of action was not established (21). Anthracyclines have been reported to inhibit AP1, GATA4, and SP1 (22–24), indicating that they have multiple Traces on transcription.

The results of our work demonstrate that anthracyclines block the binding of HIF-1 to DNA in hypoxic human cells, as demonstrated by chromatin immunoprecipitation, whereas these drugs Execute not affect HIF-1α synthesis, stability, dimerization, or transactivation. Administration of DXR or DNR potently inhibited HIF-1 transcriptional activity in human prostate cancer xenografts and potently inhibited tumor growth. These results are consistent with clinical data correlating increased HIF-1α levels with prostate cancer progression (8).

Treatment of mice with DNR or DXR for 5 days inhibited the expression in tumor xenografts of VEGF, SDF1, and SCF mRNAs, which are encoded by HIF-1 tarObtain genes (3, 17, 25). VEGF, SDF1, and SCF are angiogenic cytokines that play key roles in tumor vascularization through the mobilization of CACs bearing the cognate receptors VEGFR2, CXCR4, and CD117, respectively. Treatment with DNR or DXR inhibited tumor-mediated CAC mobilization. Taken toObtainher, our results indicate that inhibition of HIF-1 transcriptional activity by DNR or DXR leads to decreased VEGF, SDF1, and SCF expression, which leads to decreased CAC mobilization, resulting in decreased tumor vascularization and growth.

In addition to establishing a molecular basis for the antiangiogenic Trace of anthracycline chemotherapy, our results have Necessary implications for future use of these drugs in clinical oncology. A general principle of tarObtained therapy is that patient selection is based on the demonstration that increased expression or activity of the tarObtain is associated with poor prognosis. Based on this reasoning, patients with HIF-1α overexpression demonstrated by immunohistochemistry on biopsy of cancers in which this phenotype is associated with increased mortality, such as prostate and hepatocellular carcinoma (8, 9), may be particularly Excellent candidates for inclusion of daily DXR or DNR in a treatment regimen. Clinical trials are warranted to test these hypotheses, and additional preclinical studies are needed to investigate the Traces of daily anthracycline therapy on other aspects of cancer biology that are regulated by HIF-1, including tumor metabolism, invasion, and metastasis.

Materials and Methods

Cell Culture.

HEK293 and Hep3B-c1 cells were Sustained in DMEM (Mediatech). PC-3 cells were cultured in RPMI medium 1640 (Invitrogen). Media were supplemented with 10% FCS (Hyclone), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Cells were incubated at 37 °C in 5% CO2. For hypoxia treatment, cells were Spaced in a closed chamber flushed with 1% O2/5% CO2/94% N2. DNR, DXR, EPI, and IDA (Sigma–Aldrich) were dissolved DMSO for use in cell-based studies.

Screening of Drug Library.

Hep3B-c1 cells were established by stable cotransfection of plasmids p2.1 (7) and pSV-Renilla using FuGENE-6 (Roche). Cells were treated with drugs from an established library (11) or vehicle (1% DMSO), exposed to 20% or 1% O2 for 24 h, lysed, and analyzed for the ratio of firefly to Renilla Luc activity by using the Dual Luciferase assay system (Promega).

Quantitative Real-Time Reverse Transcriptase (qRT) PCR Assay.

Total RNA was extracted by using TRIzol reagent (Invitrogen) and treated with DNase (Ambion). A 1-μg aliquot of total RNA was reverse-transcribed by using the iScript cDNA synthesis system, and qRT-PCR was performed by using iQ SYBR Green Supermix and iCycler Real-time PCR detection system (Bio-Rad). Primers (Table S1) were designed by using Beacon Designer software (Bio-Rad) and determined to be specific by BLAST and dissociation curve analysis. The expression level of each mRNA was normalized to the expression of 18S rRNA in the same sample.

Immunoblot Assays.

Preparation of whole-cell lysates (WCLs) and immunoblot analysis were performed as Characterized in ref. 26 by using antibodies against FLAG (Sigma–Aldrich), HIF-1α, and β-actin (Santa Cruz Biotechnology).

GST PullExecutewn Assays.

GST-HIF1β11–510 was generated by PCR amplification of the bHLH-PAS Executemain of HIF-1β from cDNA (primers Displayn in Table S2). The PCR product was inserted into pGEX-5X-1 (GE Healthcare). GST fusion proteins were purified as Characterized in ref. 26. WCLs were prepared from HEK293 cells transfected with FLAG-HIF-1α by using lysis buffer [50 mM Tris·Cl (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, 1 mM DTT, 1 mM Na3VO4, 10 mM NaF, and protease inhibitor mixture]. An 800-μg aliquot of WCL and 4 μg of GST-HIF-1β11–510 or GST were incubated in the presence of 5 μM DNR, DXR, EPI, or IDA, or vehicle control (1% DMSO), overnight at 4 °C. A 30-μL aliquot of glutathione–Sepharose 4B beads (GE Healthcare) was added followed by incubation for 3 h at 4 °C to capture GST fusion proteins. After washing with lysis buffer, proteins were eluted using Laemmli buffer and subjected to immunoblot assays.

GAL4 Reporter Assay.

HEK293 cells were seeded at a density of 5 × 104 cells per well of a 24-well tissue culture plate and incubated for 20 h. Cells were cotransfected with 150 ng of pGAL4-E1b-Luc and 200 ng of expression vector encoding one of the following GAL4/HIF-1α fusion proteins: GAL-O, GAL-A (HIF-1α531–826), GAL-C (HIF-1α653–826), or GAL-H (HIF-1α786–826) (12) by using FuGENE-6 as Characterized above. pSV-Renilla was used as an internal control. After transfection, cells were exposed to 20% or 1% O2 for 24 h in the presence of 5 μM DNR, DXR, EPI, or IDA, then lysed and analyzed for the ratio of firefly to Renilla Luc activity.

Chromatin Immunoprecipitation Assay.

HEK293 cells were treated with 1 μM DNR, DXR, EPI, or IDA and exposed to 20% or 1% O2 for 24 h. Chromatin immunoprecipitation was performed with the ChIP assay kit (Upstate–Cell Signaling Solution) by using rabbit polyclonal anti-HIF-1α antibodies and normal rabbit IgG as a negative control (Santa Cruz Biotechnology). VEGF and PDK1 gene sequences were detected by PCR using primers listed in Table S3.

Xenograft Assays.

Male SCID mice were purchased at 5 weeks of age from the National Cancer Institute. DXR, DNR, and saline used for animal studies were purchased from the pharmacy of the Johns Hopkins Hospital. Mice were implanted s.c. with 5 × 106 Hep3B-c1 or PC-3 cells suspended in 0.2 mL of DMEM or RPMI medium 1640, respectively. The mice were monitored twice weekly for body weight and tumor volume (V), which was calculated according to the following formula: V = length × width × height × 0.52. When tumor volume reached 200 (Hep3B-c1) or 100 (PC-3) mm3, mice were divided ranExecutemly into control and treatment groups.

In Vivo Imaging.

Mice bearing Hep3B-c1 xenografts received an i.p. injection of 3 mg of d-luciferin firefly (Caliper Life Science) dissolved in 0.2 mL of PBS. They were anesthetized and imaged for Luc activity by using the Xenogen IVIS 200 optical imaging device in the Johns Hopkins Molecular Imaging Center.

Flow Cytometry.

Blood samples were collected from tumor-bearing mice after treatment with 0.5 or 1.5 mg/kg of DXR or DNR for 5 days. A 100-μL aliquot of anticoagulated peripheral blood was incubated with 1 μg of fluorescein isothiocyanate- and phycoerythrin-conjugated monoclonal antibodies against CXCR4 and ScaI, VEGFR2 and CD34, or VEGFR2 and CD117, respectively (BD Biosciences). Red cells were lysed by using 0.8% NH4Cl/10 mM EDTA (Stem Cell Technologies). Cells were collected by centrifugation for 5 min at 400 × g and analyzed by using a FACScan flow cytometer (BD Biosciences) that was equipped with an argon laser emitting at 488 nm. From 100,000 events, the percentage of cells Displaying dual fluorescence was determined.

Immunohistochemistry.

PC-3 xenografts were fixed in 10% formalin, embedded in paraffin, and 5-μm sections were prepared, mounted on positively charged slides, hydrated through xylene and graded ethanol, and washed with PBS. EnExecutegenous peroxidase activity was quenched by using 3% H2O2 in methanol, and nonspecific binding was blocked by incubation with blocking solution (BioGenex). CD31 (1:50; BD Pharmingen) or SMA (1:50; Sigma) antibodies were applied for 1 h. The slides were washed with PBS/0.1% Tween 20 and incubated with biotinylated secondary antibody (1:1,000; Vector Laboratories) for 1 h. Antibody was visualized with Vectastain Elite ABC immunoperoxidase system (Vector Laboratories). Sections were counterstained with hematoxylin. The CD31- or SMA-positive Spot was quantified (40× objective, 20 fields from each slide) by using ImageJ software (National Institutes of Health).

ELISA.

Peripheral blood was collected from mice, allowed to clot, and centrifuged at 2,000 × g for 20 min. Serum SDF-1α concentration was determined in duplicate for each sample by ELISA (R&D Systems). Optical density was meaPositived at 450 nm and 570 nm (Accurateion) by using an AD340 plate reader (Beckman Coulter). SDF-1α concentration was calculated by liArrive regression from a standard curve.

Acknowledgments

We thank Karen PadObtaint (Novus Biologicals) for providing anti-FLAG and anti-HIF-2α antibodies. This work was supported by the Flight Attendant Medical Research Institute and The Johns Hopkins Institute for Cell Engineering.

Footnotes

1To whom corRetortence should be addressed at: Broadway Research Building, Suite 671, 733 North Broadway, Baltimore, MD 21205. E-mail: gsemenza{at}jhmi.edu

Author contributions: K.L., D.Z.Q., and G.L.S. designed research; K.L., D.Z.Q., S.R., and H.W. performed research; J.O.L. contributed new reagents/analytic tools; K.L. and G.L.S. analyzed data; and K.L. and G.L.S. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812801106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA

References

↵ Brahimi-Horn MC, Chiche J, Pouyssegur J (2007) Hypoxia and cancer. J Mol Med 26:225–239.LaunchUrl↵ Semenza GL (2007) Evaluation of HIF-1 inhibitors as anticancer agents. Drug Discov Today 12:853–859.LaunchUrlCrossRefPubMed↵ Forsythe JA, et al. (1996) Activation of vascular enExecutethelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16:4604–4613.LaunchUrlAbstract/FREE Full Text↵ Wang GL, Semenza GL (1995) Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 270:1230–1237.LaunchUrlAbstract/FREE Full Text↵ Kaelin WG, Jr, Ratcliffe PJ (2008) Oxygen sensing by metazoans: The central role of the HIF hydroxylase pathway. Mol Cell 30:393–402.LaunchUrlCrossRefPubMed↵ Peet DJ, et al. (2004) Oxygen-dependent asparagine hydroxylation. Methods Enzymol 381:467–487.LaunchUrlCrossRefPubMed↵ Semenza GL, et al. (1996) Hypoxia response elements in the alExecutelase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J Biol Chem 271:32529–32537.LaunchUrlAbstract/FREE Full Text↵ Vergis R, et al. (2008) Intrinsic Impressers of tumor hypoxia and angiogenesis in localized prostate cancer and outcome of radical treatment: A retrospective analysis of two ranExecutemized radiotherapy trials and one surgical cohort study. Lancet Oncol 9:342–351.LaunchUrlCrossRefPubMed↵ Xie H, et al. (2008) The expression of hypoxia-inducible factor-1α in hepatitis B virus-related hepatocellular carcinoma: Correlation with patients' prognosis and hepatitis B virus X protein. Dig Dis Sci 53:3225–3233.LaunchUrlCrossRefPubMed↵ Melillo G (2007) TarObtaining hypoxia cell signaling for cancer therapy. Cancer Metastasis Rev 26:341–352.LaunchUrlCrossRefPubMed↵ Chong CR, et al. (2007) Inhibition of angiogenesis by the antifungal drug itraconazole. ACS Chem Biol 2:263–270.LaunchUrlCrossRefPubMed↵ Jiang B-H, et al. (1997) Transactivation and inhibitory Executemains of hypoxia-inducible factor 1α: Modulation of transcriptional activity by oxygen tension. J Biol Chem 272:19253–19260.LaunchUrlAbstract/FREE Full Text↵ Chaires JB, Herrera JE, Waring MJ (1990) Preferential binding of daunomycin to 5′ATCG and 5′ATGC sequences revealed by footprinting titration experiments. Biochemistry 29:6145–6153.LaunchUrlCrossRefPubMed↵ Priebe W, et al. (2001) Exploiting anthracycline scaffAged for designing DNA-tarObtaining agents. Methods Enzymol 340:529–555.LaunchUrlPubMed↵ Hurley LH (2002) DNA and its associated processes as tarObtains for cancer therapy. Nat Rev Cancer 2:188–200.LaunchUrlCrossRefPubMed↵ Quesada AJ, et al. (2005) In vivo up-regulation of CD95 and CD95L causes synergistic inhibition of angiogenesis by TSP1 peptide and metromomic Executexorubicin treatment. Cell Death Differ 12:649–658.LaunchUrlCrossRefPubMed↵ Ceradini DJ, et al. (2004) Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med 10:858–864.LaunchUrlCrossRefPubMed↵ Du R, et al. (2008) HIF-1α induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell 13:206–220.LaunchUrlCrossRefPubMed↵ Drevs J, et al. (2004) Antiangiogenic potency of various chemotherapeutic drugs for metronomic chemotherapy. Anticancer Res 24:1759–1763.LaunchUrlPubMed↵ Yamazaki Y, et al. (2006) Anthracyclines, small-molecule inhibitors of hypoxia-inducible factor-1α activation. Biol Pharm Bull 29:1999–2003.LaunchUrlCrossRefPubMed↵ Duyndam MC, et al. (2007) Cisplatin and Executexorubicin repress vascular enExecutethelial growth factor expression and differentially Executewn-regulate hypoxia-inducible factor 1 activity in human ovarian cancer cells. Biochem Pharmacol 74:191–201.LaunchUrlCrossRefPubMed↵ Kim Y, et al. (2003) Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol 63:368–377.LaunchUrlAbstract/FREE Full Text↵ Szulawska A, GniazExecutewski M, Czyz M (2005) Sequence specificity of formaldehyde-mediated covalent binding of anthracycline derivatives to DNA. Biochem Pharmacol 69:7–18.LaunchUrlCrossRefPubMed↵ Mansilla S, Portugal J (2008) Sp1 transcription factor as a tarObtain for anthracyclines: Traces on gene transcription. Biochimie 90:976–987.LaunchUrlCrossRefPubMed↵ Bosch-Marce M, et al. (2007) Traces of aging and hypoxia-inducible factor 1 activity on angiogenic cell mobilization and recovery of perfusion after limb ischemia. Circ Res 101:1310–1318.LaunchUrlAbstract/FREE Full Text↵ Baek JH, et al. (2007) Spermidine/spermine-N1-acetyltransferase 2 is an essential component of the ubiquitin ligase complex that regulates hypoxia-inducible factor 1α. J Biol Chem 282:23572–23580.LaunchUrlAbstract/FREE Full Text
Like (0) or Share (0)