Archives of Biochemistry and Biophysics 545 (2014) 74–82

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Archives of Biochemistry and Biophysics

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Minocycline inhibits angiogenesis in vitro through the translationalsuppression of HIF-1a

0003-9861/$ - see front matter � 2014 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.abb.2013.12.023

⇑ Corresponding author. Address: Department of Microbiology, Keimyung Uni-versity School of Medicine, 1095 Dalgubeol-daero, Dalseo-gu, Daegu 704-701,Republic of Korea. Fax: +82 53 580 3788.

E-mail address: wonki@dsmc.or.kr (W.-K. Baek).

Hui-Jung Jung, Incheol Seo, Bijay Kumar Jha, Seong-Il Suh, Min-Ho Suh, Won-Ki Baek ⇑Department of Microbiology, Keimyung University School of Medicine, Daegu, Republic of Korea

a r t i c l e i n f o

Article history:Received 17 September 2013and in revised form 9 December 2013Available online 8 January 2014

Keywords:HIF-1VEGFMinocyclinemTOR

a b s t r a c t

Minocycline was recently found to be effective against cancer. However, the precise molecular mecha-nisms of minocycline in cancer are poorly understood. Hypoxia-inducible factor-1 (HIF-1, a heterodimer-ic transcription factor composed of HIF-1a and b) activates the transcription of genes that are involved inangiogenesis in cancer. In this study, we found that minocycline significantly inhibits HIF-1a proteinexpression and suppresses HIF-1 transcriptional activity. The tube formation assay showed that minocy-cline has anti-angiogenic activity and suppresses hypoxia-induced vascular endothelial growth factor(VEGF) expression. The metabolic labeling assay showed that minocycline reduces HIF-1a protein trans-lation and global protein synthesis. In addition, minocycline suppresses mTOR signaling and increases thephosphorylation of eIF2a, which is known to be related to the translational regulation of HIF-1a expres-sion. These findings collectively indicate that minocycline is a potential inhibitor of HIF-1a and providenew insight into the discovery of drugs for cancer treatment.

� 2014 Elsevier Inc. All rights reserved.

Introduction subunit. The heterodimeric HIF-1 rapidly translocates to the nu-

Minocycline is a second-generation tetracycline antibiotic withpotential therapeutic efficacy for the treatment of infectious dis-eases. In addition to its established antibiotic property, minocyclinehas shown beneficial therapeutic effects for the treatment ofinflammation and cancer [1–4]. It was recently shown that minocy-cline exhibits a variety of anticancer activities, such as anti-angio-genic effects [2] and anti-invasive effects, through the inhibitionof matrix metalloproteinase (MMPS) [5], the inhibition of mito-chondrial protein synthesis [6,7], and the induction of apoptosis[8]. Despite these known roles of minocycline, the exact mecha-nisms underlying its antitumor activity have not yet been clarified.

HIF-1 is a dimeric transcription factor composed of the HIF-1aand HIF-1b subunits. HIF-1 is a key player in the maintenance ofcellular homeostasis under hypoxia conditions through its regula-tion of the expression of many genes involved in a crucial aspect ofcancer biology [9–11]. Under normoxic conditions, prolyl hydrox-ylase mediates the hydroxylation of the proline residues of HIF-1a at two positions, which allows HIF-1a to interact with theVon Hippel–Lindau E3 ligase complex and thus be targeted for deg-radation by the ubiquitin–proteosome system [12]. Under hypoxicconditions, HIF-1a is not degraded; in contrast, it is stabilized,accumulated, and binds with the ubiquitously expressed HIF-1b

cleus, where it binds to the hypoxia-responsive element andupregulates several genes responsible for cellular survival underhypoxic conditions. One of the HIF-1-responsive genes is VEGF,which promotes angiogenesis in a tumor [13].

An increased level of HIF-1a has been found in many cancersand is associated with metastasis, poor response to treatment,and patient mortality [14–16]. HIF-1 is involved in glucose and en-ergy metabolism during intermittent tumor hypoxia and activatesthe glycolytic pathway [17,18]. These findings suggest that HIF-1plays a crucial role in cancer metabolism and indicates that theinhibition of HIF-1 may augment the response of various types ofcancer to treatment.

Recently, several HIF-1 inhibitors have been identified and val-idated as antitumor agents [11,19]. Minocycline has been reportedto have anti-angiogenic effects on a variety of cancer cells [20,21].In an effort to determine the anticancer mechanism induced byminocycline treatment, we aimed to determine whether minocy-cline can inhibit VEGF expression and angiogenesis through thesuppression of HIF-1. The results found in this study demonstratethat minocycline inhibits HIF-1a expression through the suppres-sion of protein translation.

Materials and methods

Cell culture, reagents, and antibodies

U87MG and DU145 cells were cultured in Dulbecco’s modifiedEagle’s medium (DMEM) containing 10% fetal bovine serum,

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penicillin, and streptomycin. Minocycline, cycloheximide (CHX),and trichloroacetic acid were purchased from Sigma. N-(meth-oxyoxoacetyl)-glycine methyl ester (DMOG), an inhibitor of prolylhydroxylase, was obtained from Cayman Chemical. Anti-HIF1aantibody was purchased from BD Bioscience. Anti-HIF1b andanti-GADD153/CHOP antibodies were purchased from Santa CruzBiotechnology. Anti-phospho-mTOR, anti-phospho-p70S6K,anti-phospho-RPS6, anti-phospho-4EBP1, anti-4EBP1, and anti-phospho-elF-2a antibodies were purchased from Cell SignalingTechnology. The anti-b-actin antibody was purchased from Sigma.

Immunoblotting

The immunoblotting assays were performed as described previ-ously [22]. The blotted membranes were blocked with 5% skimmedmilk for 1 h and incubated with primary (1:1000) and secondary(1:5000) antibodies. The membranes were developed using achemiluminescent reagent (Amersham Bioscience) and subse-quently exposed to X-ray film or analyzed with the LAS 3000(Fujifilm Co.) image analyzer using the MultiGauge software.

RT-PCR

The total RNA was extracted from cultured cells using the Trizolreagent (Invitrogen). The RT-PCR reactions were completed asdescribed previously [23].The primers used in the RT-PCR reactionswere the following: (forward primer) 50-CTCAAAGTCGGACAGCCTCA-30 and (reverse primer) 50-CCCTGCAGTAGGTTTCTGCT-30 forHIF-1a, (forward primer) 50-TTTCTGCTGTCTTGGGTGCATTGG-30

and (reverse primer) 50-TCTGCATGGTGATGTTGGACTCCT-30 forVEGF, (forward primer) 50-CGCTTCATCATCGGTGTGTA-30 and (re-verse primer) 50-TGCCGACTCTCTTCCTTCAT-30 for Glut-1, (forwardprimer) 50-CCCGGGATGCAGGAGGAGA-30 and (reverse primer) 50-CGTGCGCTTCGGGTGTTTA-30 for BNIP3, (forward primer) 50-CAGAACCAGCAGAGGTCACA-30 and (reverse primer) 50-TCACCATTCGGTCAATCAGA-30 for GADD153/CHOP, and (forward primer) 50-CGTCTTCACCACCATGGAGA-30 and (reverse primer) 50-CGGCCATCACGCCACAGTTT-30 for GAPDH. The PCR products were separated and visualizedon an agarose gel stained with ethidium bromide under UVtransillumination.

Transient transfection and luciferase assay

The pGL2-TK-HRE plasmid, in which firefly luciferase expres-sion is under the control of the hypoxia response element (HRE),was a kind gift from Dr. Giovanni Melillo (National Cancer Insti-tute, Frederick, MD, USA). U87MG and DU145 cells were seededat an initial density of 5 � 104 cells per well in a 24-well plateone day before transfection. The cells were transiently co-transfec-ted with the pGL2-TK-HRE plasmid and the pRL-TK plasmid (Pro-mega), which is a renilla luciferase expression plasmid, usingLipofectamine 2000 (Invitrogen). The cells were incubated for20 h after transfection and then with minocycline for 6 h. The rel-ative luciferase activity was evaluated based on the ratio of fireflyto renilla luciferase activity.

Metabolic labeling, immunoprecipitation, and TCA precipitation

U87MG and DU145 cells were seeded in 60-mm culture dishes atan initial density of 4 � 105 cells. After 18 h, the cells were treatedwith minocycline for 24 h. The cells were preincubated with methi-onine/cysteine-free DMEM media for 3 h before their completeincubation. DMOG at a final concentration of 5 mM was then added,and the cells were incubated for 30 min. The cells were labeled withthe addition of 35S-methionine/cysteine (GE Healthcare Life Science)at a final concentration of 100 lCi/ml in the culture medium and

incubated for 2 h. The labeling was terminated by aspirating the cul-ture media, and the cells were harvested after washing once withPBS. Equal concentrations of the extracted proteins were immuno-precipitated with the anti-HIF-1a antibody. The immunoprecipi-tates were washed and resolved on SDS–PAGE. The gel was driedand exposed to X-ray film. To examine the general protein synthesis,U87MG and DU145 cells were treated with the indicated concentra-tion of minocycline and labeled with 35S-methionine/cysteine for1 h. After the cells were washed with PBS, the cell lysates were pre-cipitated with 10% trichloroacetic acid (TCA) for 30 min at 4 �C. Theradioactivity that was incorporated into the TCA-precipitable mate-rial in the cell lysates was measured using a liquid scintillationanalyzer (Packard Instrument Co). The total cell lysates extractedfrom the radiolabeled cells were electrophoresed throughSDS–PAGE. The gel was dried and exposed to X-ray film.

Quantitation of VEGF production

U87MG and DU145 cells were plated at a density of 2 � 105

cells per well in 6-well culture dishes for 24 h. The media fromthe cell culture plate was collected and centrifuged at 800 rpmand 4 �C for 4 min to remove all cell debris. The supernatant wascollected, and the VEGF levels in the supernatant were measuredusing the Quantikine human VEGF ELISA kit (R&D Systems) accord-ing to the manufacturer’s instruction.

In vitro tube formation assay

U87MG and DU145 cells were seeded in 6-well culture plates at adensity of 2 � 105 cells/well. After overnight incubation, the cellswere washed with PBS and cultured in fresh DMEM medium con-taining 2% FBS. The cells were treated with minocycline for 24 h un-der hypoxic conditions, and the culture medium supernatant wascollected as conditioned media. The tube formation assay was per-formed in culture plates coated with Matrigel (BD Bioscience).Matrigel (250 ll/well) was used to coat 24-well plates on ice andallowed to polymerize at 37 �C for 30 min. After polymerization, atotal volume of 250 ll of HUVECs (5 � 104/well) in diluted condi-tioned media (1:1 with EBM-2 medium from Lonza) was seededon the gel. The cells were then incubated at 37 �C for 15 h undernormoxic and hypoxic conditions. The cells were observed under amicroscope to determine any morphological changes, and photo-graphs were taken at 40�magnification to score the tube formation.

Statistical analysis

The data are presented as the mean ± SD from three indepen-dent experiments. The statistical analysis was performed usingStudent’s t test at a significance level of P < 0.05.

Results

Minocycline inhibits HIF-1a protein expression

To assess whether minocycline has an effect on the expression ofHIF-1a, U87MG and DU145 cells were treated with minocyclineunder normoxic and hypoxic conditions. Minocycline inhibitedHIF-1a protein expression in a dose-dependent manner under bothnormoxic and hypoxic conditions (Fig. 1A). To determine whetherthis decrease was due to a low level of transcriptional expression,we investigated the HIF-1a mRNA expression after minocyclinetreatment. The HIF-1a mRNA expression was not decreased byminocycline, which suggests that minocycline suppresses HIF-1aprotein expression through post-transcriptional regulation ratherthrough the inhibition of HIF-1a transcription (Fig. 1B). To

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determine the transcriptional activity of HIF-1, three downstreamtarget genes (VEGF, Glut-1, and BNIP3) were measured under theassumption that the expression of these downstream genes wouldbe decreased if the expression of HIF-1a is suppressed. As expected,minocycline decreased the mRNA expression level of all threedownstream target genes under hypoxic conditions (Fig. 1B). Wealso examined whether the decrease in HIF-1a protein expressionresults in a reduction in the HIF-1 transcriptional activity using aHIF-1 reporter assay. Both U87MG and DU145 cells were tran-siently transfected with a construct containing the Luciferasereporter gene under the control of the hypoxia response element(HRE) and then treated with minocycline. Minocycline treatmentsuppressed the basal and hypoxia-induced HRE promoter activitysignificantly (Fig. 1C). These results suggest that minocycline playsan inhibitory role on HIF-1-mediated gene transcription.

Minocycline reduces the migration of endothelial cells and inhibitsangiogenesis

HIF-1 is an important transcription factor that mediates angi-ogenesis in hypoxic tumors through the upregulation of VEGF

Fig. 1. Minocycline reduces HIF-1a protein expression. (A) U87MG and DU145 cells weconditions for 24 h. The whole cell lysate was resolved by SDS–PAGE and immunoblottetreated with different concentrations of minocycline under normoxic and hypoxic conditmeasured by RT-PCR. (C) U87MG and DU145 cells were transiently cotransfected with pand minocycline under normoxic and hypoxic conditions for 6 h. The ratio of firefly to renthe values were normalized to those of the control. � Indicates a significant differenceIndicates a significant difference compared with the vehicle-treated group under hypox

expression [24]. Because minocycline treatment inhibited theexpression of the HIF-1a protein, we investigated the effect ofminocycline on the expression of VEGF and angiogenesis. Treat-ment with minocycline reduced the hypoxia-induced VEGFproduction in a dose-dependent manner (Fig. 2A). These dataindicate that minocycline may inhibit VEGF expression throughits effect on HIF-1a. Furthermore, we attempted to confirm theanti-angiogenic property of minocycline through an in vitro tubeformation assay. The conditioned media collected from U87MGand DU145 cells was added to HUVECs, and the cells were incu-bated for 15 h at 37 �C, as described in Materials and Methods.Our data revealed that the cells exposed to conditioned mediacollected from cells exposed to hypoxia exhibited enhanced cap-illary tube formation compared with the cells exposed to condi-tioned media collected from cells not exposed to hypoxia.However, the cells treated with conditioned media collected fromcells exposed to hypoxia in the presence of minocycline exhibitedsignificantly reduced capillary tube formation (Fig. 2B). Takentogether, our data suggests that minocycline has potent anti-angiogenic activity.

re treated with various concentrations of minocycline under normoxic and hypoxicd with antibodies specific for HIF-1a and HIF-1b. (B) U87MG and DU145 cells wereions. The total RNA was extracted, and the mRNA levels of the indicated genes wereGL2-TK-HRE and pRL-TK plasmids. Both transfected cells were treated with vehicleilla luciferase activity was identified using the Dual-Glo luciferase assay system, andcompared with the vehicle-treated group under normoxic conditions (p < 0.05). #ic conditions (p < 0.05).

Fig. 2. Minocycline has anti-angiogenic activity. (A) Minocycline reduces VEGF production. U87MG and DU145 cells were treated with minocycline for 24 h under hypoxicconditions. The level of VEGF protein in the culture medium was analyzed by ELISA. � Indicates a significant difference compared with the vehicle-treated group undernormoxic conditions (p < 0.05). # Indicates a significant difference compared with the vehicle-treated group under hypoxic conditions (p < 0.05). (B) Minocycline inhibits tubeformation in HUVECs. U87MG and DU145 cells were treated with minocycline for 24 h. The conditioned media was collected and applied to HUVECs cultured in Matrigel-coated plates as described in Materials and Methods. Representative photomicrographs (40X) of three independent experiments are shown (upper). A representativehistogram of the quantified branched tubes is shown (lower). � Indicates a significant difference compared with the group treated with conditioned media collected fromcancer cells maintained under normoxic conditions (p < 0.05). # Indicates a significant difference compared with the group treated with conditioned media collected fromvehicle-treated cancer cells under hypoxic conditions (p < 0.05).

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Minocycline reduces HIF-1a protein translation

Because minocycline treatment did not reduce HIF-1a mRNAexpression, we examined the effect of minocycline on the post-transcriptional regulation of HIF-1a. First, the effect of minocyclineon HIF-1a protein stability was examined using cycloheximide(CHX), which is an inhibitor of de novo protein synthesis. The cellswere treated with CHX alone or in combination with minocyclinefor various times, and the HIF-1a protein levels were determinedby Western blot. Treatment with CHX reduced the HIF-1a proteinlevels, and the half-life of the HIF-1a protein was not significantly

different between the untreated and the minocycline-treated cells(Fig. 3A). This result suggests that minocycline does not affect HIF-1a protein stability. We then examined the effect of minocyclineon HIF-1a protein synthesis. The accumulation rate of HIF-1a pro-tein was determined using the prolyl hydroxylase inhibitor DMOGto block HIF-1a protein degradation and thereby cause HIF-1a pro-tein accumulation. As expected, DMOG led to a rapid accumulationof HIF-1a protein in U87MG and DU145 cancer cells after 90 min oftreatment (Fig. 3B). However, the addition of minocycline withDMOG led to a significant reduction in the HIF-1a protein accumu-lation. This finding suggests that minocycline may have an

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inhibitory effect on HIF-1a protein translation. This inhibitory ef-fect of minocycline on protein translation was further examinedthrough metabolic labeling and immunoprecipitation using anHIF-1a antibody (Fig. 3C). The cells were labeled with 35S methio-nine/cysteine in the presence of DMOG to block the degradation ofnewly synthesized HIF-1a protein. Treatment with minocycline re-duced the accumulation of newly synthesized HIF-1a protein in adose-dependent manner. Taken together with the results fromthe CHX experiment, these findings suggest that minocycline re-duces HIF-1a protein expression through the suppression of HIF-1a protein synthesis rather than an increase in HIF-1a proteindegradation.

Fig. 3. Minocycline inhibits HIF-1a protein translation without affecting protein stabilityand cycloheximide (50 lM) was added for the indicated time period. The cell lysates wermembranes were immunoblotted with mouse monoclonal HIF-1a antibody. The lowerprotein levels were normalized to that of b-actin in the respective cell line. The HIF-1a prwere pretreated with minocycline for 24 h, and 5 mM DMOG was added for the indicaWestern blot analysis using a specific HIF-1a antibody. The lower panel shows the quantrespective cell line. The HIF-1a protein level of untreated cells was arbitrarily set to 10DU145 cells were pretreated with the indicated concentration of minocycline for 24 hsimultaneously exposed to both DMOG (5 mM) and 35S-methionine/cysteine for 30 mantibody.

Minocycline inhibits protein translation

Because minocycline inhibits HIF-1a protein translation, weexamined whether the inhibitory effect was associated with themodulation of global protein translation. First, we measured the ef-fects of minocycline on general protein synthesis using a TCA pre-cipitation assay, as described in Materials and Methods.Minocycline decreased the amount of radioactively labeled proteinprecipitates (Fig. 4A). We then examined the global protein synthe-sis using the lysates obtained from the metabolic-labeled cells bySDS–PAGE, as described in Materials and Methods. Minocyclinetreatment presented results similar to those obtained with theTCA precipitation assay (Fig. 4B). These findings suggest that the

. (A) U87MG and DU145 cells were pretreated with minocycline or vehicle for 24 h,e assayed by Western blot using an equal amount of protein from each sample. Thepanel shows a quantitative representation of three independent experiments; the

otein level in untreated cells was arbitrarily set to 100%. (B) U87MG and DU145 cellsted times. An equal amount of protein extract from each sample was subjected toitative representation of the HIF-1a protein level normalized to that of b-actin in the0%. (C) Metabolic labeling assay and immunoprecipitation of HIF-1a. U87MG and, and 35S-methionine/cysteine was added to the medium for 2 h. The cells were

in. The cell lysates were immunoprecipitated with a specific HIF-1a monoclonal

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minocycline-induced suppression of HIF-1a protein translation isassociated with the inhibition of global protein translation.

It has been reported that mTOR acts as an upstream activator ofHIF-1 in cancer cells and forms part of an important signaling

Fig. 4. Minocycline inhibits global protein synthesis. (A) TCA precipitation assay. U87MGfor 24 h, and 35S-methionine/cysteine was added to the medium for 2 h. The cell lysate wausing a liquid scintillation analyzer. The bar graph shows the quantitative representationlines. � Indicates a significant difference compared with untreated cells (p < 0.05). (Bminocycline for 24 h, and 35S-methionine/cysteine was added to the medium for 1 h. Thradioactivity was detected by autoradiography. The representative bar diagram shows thsignaling and induces phosphorylation of eIF2a. U87MG and DU145 cells were treated wconditions. The expression of the indicated proteins was analyzed by Western blot.

pathway that regulates global protein synthesis and HIF-1a pro-tein translation [25,26]. mTOR regulates the phosphorylation ofribosomal protein S6 (RPS6) through p70S6K to increase proteinsynthesis. 4EBP1 is regulated through mTOR and undergoes

and DU145 cells were pretreated with the indicated concentration of minocyclines treated with 10% TCA for 30 min. The TCA-precipitable protein level was estimatedof the percentage value obtained in the absence of minocycline in the respective cell) U87MG and DU145 cells were pretreated with the indicated concentration ofe cells were harvested, and the cell lysates were electrophoresed by SDS–PAGE. Thee quantification of the signal densities of each lane. (C) Minocycline inhibits mTOR

ith minocycline at the indicated concentration for 24 h under normoxic and hypoxic

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phosphorylation at multiple sites. The phosphorylation of 4EBP1(Thr37/46) plays an important role in the release of eIF4E from4EBP1 [27]. Therefore, the effect of minocycline on the phosphory-lation of mTOR and its downstream targets p70S6K, RPS6, and4EBP1 was investigated. Minocycline treatment decreased thephosphorylation of mTOR, p70S6K, RPS6, and 4EBP-1 in a dose-dependent manner in parallel with the reduction in HIF-1a proteinexpression in both U87MG and DU145 cells (Fig. 4C). To confirmthe phosphorylation status of 4EBP-1, we performed a Westernblot using anti-4EBP-1 antibody in addition to a phospho-specificantibody. 4EBP-1 was detected as three distinct bands (a, b, andc): a hyperphosphorylated, slow-migrating c form (uppermost),two faster-migrating hypophosphorylated a (lowermost) and b(middle) forms. These three forms are considered a condition of4EBP-1 hypophosphorylation [28]. Minocycline treatmentincreased the levels of the hypophosphorylated forms of 4EBP-1in a dose-dependent manner (Fig. 4C). Various cellular stressescan initiate the phosphorylation of eIF2a on serine 51 and inhibitglobal protein synthesis [29]. We therefore examined the phos-phorylation status of eIF2a. Minocycline treatment induced eIF2aphosphorylation in U87MG and DU145 cells (Fig. 4C). Collectively,these results suggest that minocycline affects global protein syn-thesis through the modulation of multiple cellular signalingpathways.

Minocycline induces the endoplasmic reticulum (ER) stress

We next examined whether minocycline induces the ER stress,because minocycline increased the phosphorylation of eIF2a whichis a well-known indicator of the ER stress [30,31]. Recent reportsuggests that minocycline stimulates autophagy through ER stress,and induces caspase-dependent cell death when autophagy isblocked [32]. We investigated the mRNA and protein expressionpatterns of CHOP, a molecular indicator of ER stress in U87MGand DU145 cells. Minocycline treatment increased the expressionof mRNA and protein of CHOP as compared with that of the control(Fig. 5). Taken together with the dose-dependent increasing data ofeIF2a phosphorylation, these results indicate that minocycline in-duces ER stress.

Fig. 5. Minocycline induces ER stress in U87MG and DU145 cells. U87MG and DU145 cellThe cell lysates were assayed by Western blot using an equal amount of protein from eaand phospho-eIF2a. b-actin was used as the internal control. (B) The total RNA was extrused as a loading control. Thapsigargin (TG) at the concentration of 3 lM was used fexperiments.

Minocycline inhibits cell proliferation in U87MG and DU145 cells

To examine the effect of minocycline in U87MG and DU145cells, cell proliferation assay was performed by MTS assay after24, 48, and 72 h exposure to minocycline at various concentrations.Minocycline treatment inhibited growth in a dose-dependentmanner. The effect was similar in both of the cell lines (Fig. 6).The vehicle control did not decrease cell viability in both of the celllines. These data indicate that minocycline inhibits cell prolifera-tion in U87MG and DU145 cells.

Discussion

HIF-1a is constantly upregulated in various types of cancers andpromotes tumorigenesis [10]. In addition, HIF-1a is considered anattractive therapeutic target in cancer. It has been demonstratedthat U87MG and DU145 cells have the significant expression ofHIF-1a and strong angiogenic potentials [33–35]. Thus, we se-lected these two cell lines for this study. To the best of our knowl-edge, this study provides the first demonstration that minocyclineplays an inhibitory role in HIF-1a expression through suppressionof translation levels in cancer cells. Minocycline is one of the tetra-cycline antibiotics and is active against a broad range of bacteria[36,37]. In addition to its anti-bacterial activity, various studieshave shown that minocycline exerts an anti-angiogenic effect.HIF-1 triggers the expression of the major angiogenic factor VEGF,and the suppression of HIF-1 reduces the production of VEGF andinhibits angiogenesis [38,39]. We have shown that minocyclineeffectively suppresses HIF-1a and VEGF. In addition, we found thatminocycline inhibits the accumulation of HIF-1a without affectingits degree of protein degradation and its mRNA level (Figs. 1 and3A). These data further support the novel strategy of using minocy-cline for cancer therapy. It is well known that Glut-1 and BNIP3 arethe downstream targets of HIF-1 that help tumor cells attain theirmetabolic needs under hypoxic conditions [40,41]. Consistent withthis fact, it was found that minocycline downregulates the mRNAlevels of GLUT-1 and BNIP3, which indicates that this downregula-tion is concomitant with the inhibition of HIF-1a protein transla-tion after minocycline treatment. Because HIF-1 is a major

s were incubated with different concentrations of minocycline or vehicle for 24 h. (A)ch sample. The membranes were immunoblotted with antibodies specific for CHOPacted, and the mRNA level of the CHOP gene was measured by RT-PCR. GAPDH wasor ER stress inducer. All data shown are the representative of three independent

Fig. 6. Minocycline inhibits cell proliferation. U87MG and DU145 cells were treatedwith the indicated concentrations of minocycline for 24, 48, and 72 h. The viabilityof the cells was detected by MTS assay (Promega Co.). The data are presented as themean percentage decrease ± SD of three independent experiments relative to thevehicle-treated control. � Indicates a significant difference compared with thevehicle treated cells (p < 0.05).

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transcription factor that controls VEGF gene expression, the sup-pressive activity of minocycline on HIF-1 could be an importantmechanism for its anti-angiogenesis activity. In accordance withthis, minocycline reduced the expression of VEGF (Fig. 2A). Our re-sults are also in close agreement with those of previous studiesthat found that angiogenesis is inhibited by minocycline [20,42].VEGF is the crucial regulator for endothelial cell migration [43]and tumor angiogenesis [44,45]. Our study revealed a significantcorrelation between the effects of minocycline on the productionof HIF-1a, VEGF, and angiogenesis. The decreased level of HIF-1acaused by minocycline treatment resulted in low levels of VEGFexpression and angiogenesis (Fig. 2B). This new link between min-ocycline and its effect on HIF-1a downregulation may explain whyminocycline inhibits angiogenesis. Minocycline has also been re-ported to suppress angiogenesis through the inhibition of matrixmetalloproteinases (MMP-2 and MMP-9) [42], and it is also re-ported that MMPs are target genes of HIF-1a [46]. Taken togetherwith our results, these reports suggest that the inhibitory effects ofminocycline on HIF-1a lead to the suppression of angiogenesisthrough multiple mechanisms, including the transcriptional regu-lation of VEGF and MMPs [42].

Minocycline has shown beneficial therapeutic effects for thetreatment of inflammation, however, the molecular mechanismof minocycline’s property as anti-inflammatory is unclear. Previousstudies show that granulocytes and macrophages rely on HIF-1function that critically involves in nitric oxide synthase (iNOS)upregulation and nitric oxide production for pathogen elimination[47–49]. Also HIF-1 is involved in the regulation of chemokineCCL2 in the lung, suggesting that HIF-1 plays pivotal role in the air-ways inflammation [50]. Thus, our study indicates that minocy-cline may exert anti-inflammatory property through HIF-1asuppression. Furthermore, it has been reported that minocyclinehas an immunosuppressive effects [51,52]. Our data demonstratesthat minocycline inhibits the general protein translation throughsuppression of mTOR signaling pathway. Inhibition of mTOR path-way enhances immunosuppressive effect [53]. This suggests thatminocycline may act as immunosuppressive agent through theinhibition of mTOR pathway.

The HIF-1a protein levels are modulated through their synthe-sis and degradation. In an effort to better understand the relation-ship between minocycline and the decrease in HIF-1a expression,we have shown that minocycline (even at high doses) has no effecton HIF-1a protein degradation (Fig. 3A). Additionally, minocyclinedoes not change the HIF-1a mRNA level (Fig. 1B). Minocycline wasfound to decrease HIF-1a protein synthesis, as was shown by themetabolic labeling assay, and to decrease the rate of HIF-1a accu-mulation in the presence of a prolyl hydroxylase inhibitor. Re-cently, Li et al. reported that minocycline reduced HIF-1a proteinexpression by enhancing degradation of HIF-1a protein [54]. How-ever, in our experiments, the degradation rate of HIF-1a proteinwas very similar with untreated control cells suggesting thatHIF-1a proteins suppression by minocycline was due to the sup-pression of HIF-1a proteins translation rather than the enhance-ment of the protein degradation. The data showed in severalreports support that minocycline has a potential to modulate pro-tein translation through multiple targets such as eIF2a, mTOR, andeIF4I [32,55].

Recently, Liu et al. showed that cytotoxic effects of minocyclinein glioma cancer cells are associated with minocycline-induced ERstress [32]. In agreement with this report, our experiment showedthat minocycline induces expression of CHOP and phosphorylationof eIF2a. This result, taken together with previous report, suggestsminocycline’s anticancer effects would be associated with ER stress[56]. Taken together, it is likely that the ultimate molecular targetof minocycline remains unidentified, and it seems that the ob-served molecular mechanisms occur downstream of modulationof an upstream key target.

It is well established that the mTOR pathway is activated duringtumorigenesis [57]. Recent studies have shown that the upregula-tion of mTOR leads to the production of a high amount of HIF-1athrough translational regulation factors, such as p70S6K and4EBP1 [57,58]. The suppression of HIF-1a accumulation is associ-ated with the dephosphorylation of mTOR and its effectorsp70S6K and 4EBP1 [59]. In the present study, minocycline wasfound to decrease the expression of HIF-1a protein and dephos-phorylated p70S6K and 4EBP1 under both normoxic and hypoxicconditions. These data support the hypothesis that p70S6K and4EBP1 enhance the translation of HIF-1a and suggest that theinhibitory effect of minocycline on HIF-1a is associated with thesuppression of mTOR. In addition to its inhibition of HIF-1a thatwas demonstrated in our study, minocycline has also been re-ported to affect neurite outgrowth through the mTOR signalingpathway [55]. Moreover, minocycline potentiate the nerve growthfactor (NGF)-induced neurite outgrowth with the involvement ofeukaryotic translation initiation factor eIF4I protein [55]. In ourexperiment, we found that minocycline induces the phosphoryla-tion of eIF2a. This finding is in agreement with previous studies,

82 H.-J. Jung et al. / Archives of Biochemistry and Biophysics 545 (2014) 74–82

which demonstrated the eIF2a phosphorylation leads to the sup-pression of HIF-1a [60,61]. Several studies have suggested thatthe inhibition of protein translation initiation can mediate an anti-cancer effect in various types of cancer cells [62–64]. Our findingsare important because minocycline reduces protein translation byincreasing the phosphorylation of eIF2a. The inhibition of globalprotein translation by minocycline corroborates the hypothesisthat minocycline may be a promising new candidate for cancertherapy. In addition, recent report suggests that glycylcycline anti-biotic tigecycline (a tetracycline derivative) exhibits anticanceractivity against acute myeloid leukaemia (AML) stem cells throughselective shut down of energy production by blocking mitochon-drial protein synthesis in the cancer cells [65]. In conclusion, ourdata suggest that minocycline inhibits HIF-1a expression by inhib-iting its translation rather than changing its stability. Furthermore,we also demonstrated that the inhibitory role of minocycline onprotein translation is associated with a repression of mTOR signal-ing and an increase in eIF2a phosphorylation.

Acknowledgments

We would like to thank Dr. Giovanni Melillo (National CancerInstitute, Frederick, MD, USA) for providing the pGL2-TK-HRE plas-mid. This research was supported by Basic Science Research Pro-gram through the National Research Foundation of Korea (NRF)funded by the Ministry of Education, Science and Technology(NRF-2011-0013913).

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