YC-1 inhibits HIF-1 expression in prostate cancer cells: contribution of Akt/NF-κB signaling to HIF-1α accumulation during hypoxia


Hypoxia-inducible factor 1 (HIF-1), a transcription factor that is critical for tumor adaptation to microenvironmental stimuli, represents an attractive chemotherapeutic target. YC-1 is a novel antitumor agent that inhibits HIF-1 through previously unexplained mechanisms. In the present study, YC-1 was found to prevent HIF-1α and HIF-1β accumulation in response to hypoxia or mitogen treatment in PC-3 prostate cancer cells. Neither HIF-1α protein half-life nor mRNA level was affected by YC-1. However, YC-1 was found to suppress the PI3K/Akt/mTOR/4E-BP pathway, which serves to regulate HIF-1α expression at the translational step. We demonstrated that YC-1 also inhibited hypoxia-induced activation of nuclear factor (NF)-κB, a downstream target of Akt. Two modulators of the Akt/NF-κB pathway, caffeic acid phenethyl ester and evodiamine, were observed to decrease HIF-1α expression. Additionally, overexpression of NF-κB partly reversed the ability of wortmannin to inhibit HIF-1α-dependent transcriptional activity, suggesting that NF-κB contributes to Akt-mediated HIF-1α accumulation during hypoxia. Overall, we identify a potential molecular mechanism whereby YC-1 serves to reduce HIF-1 expression.


Accurate measurement of oxygen levels shows that hypoxia is a common feature of almost all types of solid tumors, including brain, colon, breast, cervical, prostate, and head and neck (Hockel and Vaupel, 2001). The curability of chemotherapy and surgery is compromised by hypoxia because it tends to select for tumors of greater malignant phenotype, increases mutation rates and increases expression of genes associated with angiogenesis (Brown and Wilson, 2004). Factors that regulate hypoxic events are therefore proposed to represent useful targets for anticancer therapy.

As first identified as a transcription factor that mediates hypoxia-inducible activity of the erythropoietin 3′ enhancer (Semenza and Wang, 1992), hypoxia-inducible factor 1 (HIF-1) has been regarded as the key mediator of cellular responses to reduced oxygen availability. HIF-1 exists as a heterodimeric complex consisting of HIF-1α and HIF-1β. In normoxia, HIF-1α is rapidly ubiquinated by the vonHippel–Lindau E3 ligase complex (pVHL) and subjected to proteasomal degradation (Jaakkola et al., 2001). Under hypoxic conditions, however, HIF-1α is not degraded and accumulates to form transcriptionally active complexes with HIF-1β.

In addition to serving as a response to hypoxic stress, increased expression of HIF target genes may provide a biological advantage to cells with an increased ‘metabolic load’, such as those undergoing proliferation, differentiation, development or inflammation (Bilton and Booker, 2003). Thus, a wide range of growth-promoting stimuli and cytokines also induce modest HIF-1 accumulation. In keeping with the known signal transduction pathways mediating responses to these factors, the PI3K-Akt signaling cascade is strongly implicated in HIF-1α regulation. The downstream effector termed the mammalian target of rapamycin (mTOR) is recognized to regulate the translational process through increased phosphorylation of 4E-BP and p70S6K. The resultant activation of eIF4E and p70S6K leads to increased translation of HIF-1α mRNA (Laughner et al., 2001; Treins et al., 2002).

YC-1, 3-(5′-hydroxy methyl-2′-furyl)-1-benzylindazole was first discovered by our laboratory where it was observed to possess antiplatelet activity (Ko et al., 1994). More recently, YC-1 was reported to decrease hypoxia-induced HIF-1α accumulation and expression of downstream HIF-1 target genes, including those for vascular endothelial growth factor (VEGF) and erythropoietin, in vitro (Chun et al., 2001) and to inhibit angiogenesis and tumor growth in animals (Yeo et al., 2003; Pan et al., 2005). Although YC-1 has become a valuable research tool for studies of HIF-1α (Moeller et al., 2004; Funasaka et al., 2005), the mechanism by which YC-1 exerts its antitumor effects has not been established. The growth of prostate cancer PC-3 cells is suppressed by YC-1 in vivo (Huang et al., 2005b). In the present study, therefore, PC-3 cells were employed to explore the mechanism through which YC-1 inhibits accumulation of HIF-1α.


YC-1 reduces accumulation of HIF-1α and HIF-1β in PC-3 cells challenged by hypoxia

YC-1 has been shown to suppress the hypoxic response by reducing HIF-1α levels. In PC-3 cells, YC-1 was found to reduce hypoxia-dependent accumulation of HIF-1α in nuclear fractions in a concentration-dependent manner after 8 h of treatment (Figure 1a). Interestingly, consistent with previous findings, HIF-1β was upregulated by hypoxia in PC-3 cells (Zhong et al., 2001) and decreased after YC-1 treatment. In experiments performed to address the kinetics of YC-1-dependent inhibition of HIF-1α accumulation, the earliest effects of YC-1 were detected at 2 h (Figure 1b).

Figure 1

YC-1 reduces HIF-1α and HIF-1β before cell viability significantly decreases. (a) PC-3 cells were pretreated with YC-1 before culture for 8 h under normoxic (N) or hypoxic conditions. Nuclear extracts were subjected to immunoblot. (b) PC-3 cells were treated at the indicated times with 60 μ M YC-1 before performing Western blot. The HIF-1α protein band (HIF-1α) and the nonspecific protein band (NS) are indicated. (c) PC-3 cells transiently transfected with HRE-Luc were treated for 24 h with YC-1 under hypoxic condition. Data have shown represented luciferase expression relative to the vehicle control. (d) PC-3 cells transfected with EGFP-N1 were treated for 24 h with YC-1(30, 60 and 100 μ M) and then harvested for the detection of EGFP using FACScan flow cytometric analysis. The percent of EGFP-positive cells was taken to indicate transfection efficiency. (e) PC-3 cells were treated with YC-1 under hypoxic conditions for 8 or 24 h. Cell number was determined by the sulforhodamine B assay and was calculated as the percentage of vehicle treatment.

The expression of HIF-1 regulated gene has been shown to decrease after YC-1 treatment in vitro and in vivo (Yeo et al., 2003). PC-3 cells transiently transfected with a luciferase gene under the control of the hypoxia response element (HRE) were therefore employed to evaluate the consequences of YC-1 treatment to HIF-1α transcriptional activity. As shown in Figure 1c, luciferase activity was concentration-dependently inhibited by YC-1 after 24 h treatment in hypoxia. Transfection efficiency, as monitored by transfection of an enhanced green fluorescent protein (EGFP)-expressing plasmid was approximately 60% (Figure 1d). As hypoxia or YC-1 treatments affected constitutive promoter-driven EGFP expression to minimal degrees, it is suggested that the HRE-driven luciferase reporter effect did not result from global modulation of the transcriptional and translational processes.

To ascertain whether inhibition by YC-1 of HIF-1 expression in hypoxic PC-3 cells correlated with decreased survival, parallel measurements of cell viability was conducted (Figure 1e). PC-3 cells became apoptotic after 24 h of treatment with YC-1 at 100 μ M, but viability was not significantly altered at 8 h of treatment at any YC-1 concentration tested. It is therefore suggested that the observed inhibition of HIF-1α accumulation by YC-1, which is observed at relatively short treatment times (8 h), is not attributable to cell death.

Inhibition of HIF-1α accumulation by YC-1 in PC-3 cells is independent of proteasomal degradation

HIF-1α is degraded mainly through the proteasomal pathway. To test the possibility that YC-1 inhibited HIF-1α accumulation by promoting degradation of the protein, PC-3 cells were pretreated with the proteasomal inhibitor Z-Leu-Leu-Leu-al (MG132) before addition of YC-1 and hypoxic challenge. As expected, pretreatment with MG132 resulted in accumulation of HIF-1α. However, the inhibition by YC-1 of HIF-1α accumulation in response to hypoxia was not blocked by MG132 (Figure 2a). HIF-1α was also immunoprecipated to examine for extent of ubiquitination and the amount of ubiquinated HIF-1α in YC-1-treated preparations was identical to that of non-treated preparations. Additionally, YC-1 did not significantly increase pVHL (Figure 2b).

Figure 2

YC-1 has no effect on degradation of HIF-1α or expression of HIF-1α mRNA. (a) PC-3 cells were treated for 4 h under hypoxic conditions with proteasome inhibitor 10 μ M MG132 and 60 μ M YC-1 before immunoblotting. (b) Hypoxic PC-3 cells were treated without (C) or with 60 μ M YC-1 (Y) in the presence of MG132 for 4 h. Lysates were subjected to immunoprecipitation with anti-HIF-1α antibody, followed by immunoblotting with anti-ubiquitin or anti-HIF-1α antibodies. For VHL immunoblotting, PC-3 cells were pretreated with YC-1 and culture for 8 h. (c) Cells exposed to 100 μ M CoCl2 overnight were pretreated without (C) or with 60 μ M YC-1 (Y) for 1 h. CHX 30 μg/ml was then added and the incubation continued for the indicated times. Nuclear extracts were then prepared for immunoblotting. The lower panel indicates quantification of the HIF-1α signal by densitometry, and values are expressed as percentage expression relative to that of non-YC-1-treated cells at 0.25 h. (d) RT–PCR analysis for HIF-1α mRNA was performed for PC-3 cells pretreated with YC-1 and cultured for 8 h.

The findings described above were compatible with the possibility that YC-1 reduced the half-life of HIF-1α by promoting degradation of the protein through ubiquitin- and proteasome-independent pathway. To assess more directly the effects of YC-1 on HIF-1α degradation, cycloheximide was used to suppress new protein synthesis, thus HIF-1α levels would predominantly reflect the degradation process of HIF-1α protein. Although HIF-1α accumulated to somewhat lesser degrees in YC-1-treated preparations, the rates of degradation of HIF-1α were similar in non-treated and treated preparations (Figure 2c).

Because the above findings were inconsistent with YC-1 enhancement of HIF-1α degradation, the possibility was considered that this agent inhibits HIF-1α production. HIF-1α mRNA levels of variously treated PC-3 cells were measured by reverse-transcriptase polymerase chain reaction (RT–PCR) analysis. In accord with previous observations (Chun et al., 2001; Yeo et al., 2003), HIF-1α mRNA levels were unaffected by hypoxia and treatment with YC-1 (Figure 2d). The possibility that YC-1 treatment decreases HIF-1α accumulation in hypoxic PC-3 cells via downregulation of HIF-1α mRNA translation was therefore considered.

YC-1 inhibits activation of the PI3K/Akt/mTOR pathway during hypoxia

The PI3K/Akt pathway is activated by hypoxia in certain cell types (Zundel et al., 2000; Chen et al., 2001), and interference with the Akt reduces accumulation of HIF in response to hypoxia (Zhong et al., 2000; Zundel et al., 2000; Hudson et al., 2002). The phosphorylation state of Akt in PC-3 cells, which lack phosphatase and tensin homolog (PTEN), was therefore examined following hypoxic challenge. Hypoxic treatment was observed to increase phosphorylation of Akt at serine 473, a modification associated with increased Akt activity, in a time-dependent manner (Figure 3a).

Figure 3

YC-1 inhibits activation of the PI3K/Akt/mTOR pathway during hypoxia (a) PC-3 cells were placed under normoxic (N) or hypoxic conditions for the indicated periods of time before immunoblot. (bd) Before immunoblotting, cells were pretreated with vehicle (C), YC-1, or wortmannin 1 μ M(W) followed by incubation for 8 h. (e) PC-3 cells cotransfected with HRE-Luc and Myr-Akt were treated with YC-1(30, 60 μ M) or wortmannin for 24 h under hypoxic conditions. Data represent the fold increase in luciferase expression relative to that of the empty vector (EV) control in the presence of the vehicle. The content of phospho-Akt following 8 h of hypoxia was determined by immunoblot.

It was of interest to ascertain whether YC-1 treatment promoted downregulation of the PI3K–Akt pathway in hypoxia-challenged PC-3 cells. Treatment with either YC-1 or wortmannin resulted in decreased phosphorylations of Akt, 4E-BP1 and eIF4E (Figure 3b) as well as in inhibition of HIF-1α accumulation (Figure 3c). Inhibition of PI3K was recently reported to promote degradation of HIF-1α indirectly by reducing steady-state levels of the heat-shock proteins Hsp70 and Hsp90 (Zhou et al., 2004b). However, both wortmannin and YC-1 failed to abrogate expression of Hsp70 and Hsp90 in hypoxic PC-3 cells (Figure 3d).

In hypoxic PC-3 cells YC-1 treatment suppressed activation of the Akt-regulated pathway significantly, consistent with the possibility that this agent exerted its effects through Akt. As shown in Figure 3e, transfection of constitutive active Akt partly restored the YC-1 inhibited reporter activity. These observations, which are consistent with those of others (Zhong et al., 2000, 2001; Hudson et al., 2002), highlight the involvement of the PI3K–Akt pathway in the hypoxic induction of HIF-1α in PC-3 cells. These findings also reveal that Akt is involved in YC-1-mediated HIF-1α inhibition.

YC-1 suppresses HIF-1 accumulation and translational upregulation in response to mitogens

Various mitogens, including phorbol myristic acid (PMA) (Zhong et al., 2000) and insulin (Treins et al., 2002), are reported to upregulate HIF-1α via activation of PI3K/Akt pathway. It was therefore of interest to ascertain whether PC-3 cells responded similarly to these mitogens and whether treatment with YC-1 affected the response. Both PMA and insulin increased modest HIF-1α and HIF-1β expression, and pretreatment with YC-1 inhibited the inductions (Figure 4a). Growth factor-induced activation of Akt signaling has been shown to increase de novo synthesis of HIF-1α by derepression of eIF4E and activation of p70S6K (Zhong et al., 2000; Laughner et al., 2001; Treins et al., 2002). YC-1 was found to reduce the phosphorylation of Akt as well as of components of the translational machinery including p70S6K, 4E-BP1 and eIF4E (Figure 4b). Heat-shock proteins were also inspected (Figure 4c) and showed no significant alteration after YC-1 treatment, similar with previous results in hypoxia.

Figure 4

YC-1 suppresses HIF-1 expression and translational upregulation in response to mitogens. (a and c) Before immunoblotting, PC-3 cells were pretreated for 1 h with vehicle (C) or 60 μ M YC-1 (Y) before incubation with PMA (P) or insulin (I) for 8 h. (b) Cells were treated as described above and then exposed to mitogen stimulation for 30 min. Whole-cell extracts were subjected to immunoblot.

Overall, these findings support the hypothesis that inhibition by YC-1 of HIF-1α accumulation in response to mitogens is attributable to suppression by the drug of mitogen-dependent activation of the translational process.

YC-1 suppresses hypoxia-dependent nuclear factor-κB activation through inhibition of IκBα phosphorylation

Nuclear factor-κB, a central regulator of inflammation and cancer, is required for HIF-1α induction by interleukin (IL)-1β (Jung et al., 2003) and tumor necrosis factor (TNF)-α (Zhou et al., 2003, 2004a). Selective inhibition of nuclear factor (NF)-κB also results in significant decreases in HIF-1α and EPO expression during hypoxia (Figueroa et al., 2002). Interactions between these two important transcription factors have been proposed, although the mechanisms underpinning these interactions remain to be defined.

YC-1 is reported to antagonize activation of constitutively expressed NF-κB in prostate cancer cells (Huang et al., 2005b). The effect of YC-1 on activation of NF-κB in hypoxic PC-3 cells was therefore of interest. As shown in Figure 5a, hypoxia resulted in activation of NF-κB. A decrease in NF-κB in nuclear fractions was observed, however, in the presence of YC-1. NF-κB-dependent reporter gene expression was also shown to be repressed by YC-1 in a concentration-dependent manner (Figure 5b).

Figure 5

Effect of YC-1 on NF-κB activation and IκB phosphorylation in response to hypoxia. (a and c) Before immunoblotting, PC-3 cells were pretreated without (C) or with 60 μ M YC-1 (Y) and incubated for the indicated times. (b) PC-3 cells transiently transfected with κBRE-Luc were treated for 24 h with YC-1 under hypoxic conditions, followed by measurements of reporter gene activity. Data shown represented luciferase expression relative to the vehicle control.

NF-κB activation normally proceeds through a pathway involving phosphorylation and subsequent degradation of the IκBα, resulting in the translocation of NF-κB from the cytoplasm to the nucleus. Hypoxia is also reported to promote activation of NF-κB through the phosphorylation of IκBα (Koong et al., 1994). Experiments were therefore performed to ascertain whether suppression of NF-κB activation by YC-1 in hypoxic PC-3 cells was associated with decreased phosphorylation of IκBα. As illustrated in Figure 5c, hypoxia-induced IκBα phosphorylation was suppressed by YC-1 in a time-dependent manner.

Involvement of the Akt/NF-κB signaling pathway in HIF accumulation during hypoxia

Akt can exert a positive effect on NF-κB function through phosphorylation and activation of IκB kinase (IKK). IKK induces phosphorylation, which is followed by degradation, of the NF-κB inhibitor, IκB. Based on the findings described above, the targets of YC-1 in hypoxic PC-3 cells are Akt and NF-κB. Further studies were therefore performed to explore the role of Akt/NF-κB signaling in the HIF-1α accumulation in hypoxic cells.

Akt inhibition by a PI3K specific inhibitor, LY294002 (Figure 6a), or overexpression of dominant-negative Akt (Figure 6b) significantly blunted HIF-1α and NF-κB induction in response to hypoxia. Treatment of PC-3 cells with the NF-κB inhibitor, caffeic acid phenethyl ester (CAPE), at a non-apoptotic concentration (10 μg/ml) (McEleny et al., 2004), was found to reduce HIF-1α accumulation (Figure 6c) and HIF-1-dependent transcriptional activity (Figure 6d). Consistent with previous reports (Manna and Aggarwal, 2000), suppression of Akt activity by wortmannin was associated with the attenuation of NF-κB activation (Figure 6c) and NF-κB-dependent reporter gene expression (Figure 6d). Evodiamine, an alkaloid extensively studied in our lab (Huang et al., 2005a), was recently reported to abolish NF-κB activation by inhibiting the association of IKK with Akt (Takada et al., 2005). We used it as a tool to further address whether Akt/NF-κB pathway was involved in HIF regulation during hypoxia and demonstrated that this alkaloid indeed decreased HIF-1α accumulation. It is therefore proposed that Akt-regulated NF-κB activation contributes to HIF-1α accumulation in PC-3 cells challenged by hypoxia.

Figure 6

Inhibitors of activation of the PI3K/Akt/NF-κB pathway decrease HIF-1α accumulation in response to hypoxia. (a) PC-3 cells were pretreated with LY294002 (LY) before culture for 8 h under normoxic (N) or hypoxic conditions. Nuclear extracts were subjected to immunoblot. (b) Before immunobloting, PC-3 cells transfected with empty-EV or DN-Akt were exposed to hypoxia 8 h. (c and e) PC-3 cells were treated with vehicle (C), 10 μg/ml CAPE (CA), 1 μ M wortmannin (W) or 3 μ M evodiamine (Evo) before culture for 8 h. Western blot (c) and RT–PCR analyses (e) were then performed. (d) PC-3 cells transiently transfected with HRE-Luc (or κBRE-Luc) were treated for 24 h with the pharmacological inhibitors described above under hypoxic conditions. Data are presented as the fold increase of luciferase expression relative to the vehicle control.

The NF-κB-binding site is present in the promoter region of the HIF-1α gene (Figueroa et al., 2002). Experiments were therefore performed to determine whether pharmacological inhibitor used above modulated HIF-1α expression by decreasing HIF-1α mRNA. RT–PCR analysis revealed that mRNA levels remained unchanged after drug treatment, similar with conditions of YC-1 (Figure 6e).

NF-κB contributes to Akt-mediated HIF-1α transcriptional activity during hypoxia

To investigate the possibility that NF-κB contributes to HIF-1α accumulation during hypoxia, cells transiently transfected with the IκB were examined for HIF-1-dependent transcriptional activity following hypoxic challenge. Transcriptional activity was reduced after genetic repression of NF-κB (Figure 7a). Furthermore, overexpression of NF-κB was observed to partly reverse the effects of wortmannin on HIF-1α (Figure 7b). Activation of NF-κB therefore appears to serve as a downstream signal for induction of HIF-1 expression by Akt.

Figure 7

Involvement of NF-κB in Akt-dependent induction of HIF-1α transcriptional activity. (a) PC-3 cells cotransfected with HRE-Luc and IκBαM were incubated for 24 h under normoxic or hypoxic conditions. Data shown represent the fold increase in luciferase expression relative to that of the respective normoxic control. The content of IκBα after 8 h of hypoxia was determined by immunoblot. (b) PC-3 cells cotransfected with HRE-Luc and NF-κB/p65 were treated with 1 μ M wortmannin for 24 h under hypoxic conditions. Data represent the fold increase in luciferase expression relative to the respective control. The content of p65 in nuclear extract following 8 h of hypoxia was determined by Western blotting.


Expression of HIF-1α has been found to be increased in prostate intraepithelial neoplasia and in prostate cancer as compared to expression in healthy prostate epithelium and in benign prostatic hyperplasia (Zhong et al., 2004). In addition to serving as a surrogate marker of tumor responses, HIF-1α is believed to play an active role in tumor growth. Accordingly, transplantation of tumors lacking HIF into immunodeficient mice results in reduced angiogenesis, decreased tumor growth and increased responsiveness to radiotherapy (Ryan et al., 1998). The use of antisense HIF-1α (Kung et al., 2000) also inhibits tumor growth, although it would be difficult to use clinically with current technology. As a result, the potential of HIF-1α as a target for cancer therapy lies with the development of small molecule inhibitors (Powis and Kirkpatrick, 2004). This study is the first to demonstrate that the small molecule YC-1 prevents both hypoxia-dependent and mitogen-dependent HIF-1α and HIF-1β accumulation in hormone-refractory, metastatic human prostate cancer PC-3 cells.

Since the identification of HIF-1β near decades ago, relatively few studies have been performed that focus on the regulation of this important transcription factor. In human prostate cancer cells, HIF-1α and HIF-1β may share common pathways for nuclear accumulation. Similarly to HIF-1α, expression of HIF-1β is increased by hypoxia or PMA and is altered by suppression of the PI3K pathway regardless of a high level of basal expression (Zhong et al., 2001). In the present study, hypoxia, PMA or insulin was found to increase HIF-1β whereas YC-1 was observed to antagonize the PI3K pathway and decrease HIF-1β accumulation. Significant evidence has been provided to support the idea that an interaction with HIF-1β contributes to the stability of HIF-1α. HIF-1α acquires a new conformational state upon dimerization with HIF-1β, rendering HIF-1α more resistant to proteolytic digestion (Kallio et al., 1997). YC-1 decreased HIF-1β accumulation in PC-3 cells but HIF-1α stability was not significantly changed under the experimental conditions utilized. It is conceivable that HIF-1β-mediated stabilization of HIF-1α is specific for Hsp90-dependent, but not for VHL-mediated degradation (Isaacs et al., 2004). In this case, downregulation of HIF-1β by YC-1 would play a more important role in VHL-deficient cells, such as renal cell carcinoma.

In the present study, neither HIF-1α protein half-life nor mRNA level was affected by YC-1. These observations supports the proposal that the YC-1-dependent decrease in HIF-1α accumulation is due to downregulation of HIF-1α mRNA translation. In eukaryotes, most translation is cap-dependent with regulation of eIF4E availability serving as a major control point. Under normoxic conditions, upregulation of translation through the PI3K–Akt–mTOR pathway serves to promote HIF-1α protein accumulation and expression of eIF4E is also sufficient to elevate HIF-1α protein levels (Karni et al., 2002). Our results suggested that YC-1 interfered with translation activated by PMA or insulin, both of which are reported to induce HIF-1α accumulation via PI3K/Akt. Although further studies are required to determine the relative contributions of p70S6K, 4E-BP and eIF4E to YC-1-mediated translational inhibition, YC-1 was found in the present study to inhibit phosphorylation of 4E-BP1 most significantly whether in normoxia or hypoxia.

Regulation of HIF-1α expression at the translational step during hypoxia is also essential because HIF-1α mRNA translation must circumvent the general reduction in translation rates that occurs under hypoxic conditions. Certain compounds, such as 2-methoxyestradiol (Mabjeesh et al., 2003) and topotecan (Rapisarda et al., 2004), have been reported to inhibit hypoxia-induced HIF-1α accumulation through a translation-dependent mechanism. An RNA element involved in promotion of gene expression during inhibition of cap-dependent translation is the internal ribosome entry site (IRES) and experiments have been performed to confirm its presence in the 5′-untranslated region of HIF-1α mRNA (Lang et al., 2002). Although the regulation of this IRES in responses to hypoxia remains unclear, TNF-α provokes IRES-dependent translation of HIF-1α required the functional integrity of NF-κB, PI3K, and mitogen-activated protein kinase signaling (Zhou et al., 2004a). In the present study, YC-1 was found to inhibit NF-κB and PI3K-Akt signaling during hypoxia. It is therefore attractive to speculate that inhibition of HIF-1α mRNA translation during hypoxia is linked to modulation of the IRES.

Almost every step in activation of the PI3K-cascade is reported to be involved in HIF-1α expression in tumor cells, including activation by growth factors such as epidermal growth factor and insulin, increased activation of PI3K or Akt, loss of PTEN, and downstream molecular alterations involving mTOR, MDM2, GSK3β and FOXO4 (Mottet et al., 2003; Tang and Lasky, 2003; Nieminen et al., 2005). Akt can stimulate NF-κB function by phosphorylation and activation of IKK, and activation of the Akt pathway by IL-1β (Jung et al., 2003) or hypoxia (Figueroa et al., 2002) is reported to promote activation of NF-κB and subsequent upregulation of HIF-1α expression. Zhou et al. (2003) demonstrated that activation of NF-κB induced the accumulation of both unmodified and ubiquitinated HIF-1α but not of HIF-1α mRNA, implicating translational upregulation in the induction (Zhou et al., 2004a). In the present study, the activation of NF-κB by hypoxia was found to be reduced by YC-1. To determine whether inhibition of HIF-1α accumulation is a general property of NF-κB targeting agents or is specific to YC-1, the effects of other NF-κB inhibitors that exert direct or indirect effects through Akt were examined. Treatment of hypoxic PC-3 cells with the PI3K inhibitor, wortmannin, or with the inhibitor of Akt–IKK association, evodiamine, resulted in attenuation of nuclear NF-κB accumulation and activity. Moreover, utilization of CAPE, an inhibitor of NF-κB activation, or overexpression of IκB resulted in significant blockade of HIF-1-dependent transcriptional activity. Pyrrolidinedithiocarbamate (PDTC), a putative inhibitor of NF-κB, was also examined in this study; however, relatively weak inhibitions of HIF-1α accumulation were observed (data not shown). This result is consistent with previous reports and may attribute to PDTC alter cell redox environment (Haddad et al., 2000) or serve as a proteasome inhibitor (Kim et al., 2004). Details of the mechanism whereby activation of NF-κB is linked to upregulation of HIF-1α mRNA translation remain to be elucidated. Nevertheless, the findings reported here support the proposal that the Akt/NF-κB signaling pathway participates in hypoxia-induced HIF-1α accumulation in prostate cancer cells.

Immunohistochemistry has been utilized to demonstrate nuclear localization of NF-κB, which indicates activation states of NF-κB, in primary prostate cancer patient tumor samples. Although the percentage of positive nuclear staining in tumor specimens varied between 15–54.7% in different studies (Lessard et al., 2003; Ross et al., 2004; Domingo-Domenech et al., 2005; Lessard et al., 2005), emerging data suggest NF-κB nuclear localization links to poor clinical outcome (Lessard et al., 2003; Ayala et al., 2004; Fradet et al., 2004). In particular, constitutive NF-κB activity correlates with progression to bone metastasis (Lessard et al., 2003). In bone metastases-derived PC-3 cells, prominent constitutive NF-κB activation was also observed and its in vivo growth was suppressed by YC-1 (Huang et al., 2005b). Therefore, in the present study, PC-3 cells were employed to explore the mechanism of YC-1 and the link between NF-κB and HIF-1α expression during hypoxia.

Although the genetic features of PC-3 cells including loss of PTEN, constitutive activation of NF-κB and p53 negativity may somehow affect mechanism study of YC-1, PC-3 cells can still serves as a good model, as aberrant expression of multiple genes is common in advanced malignancy. In the present study, multiple targets of YC-1, such as HIF-1α, Akt, and NF-κB have been explored with the goal of achieving multifocal signal modulation therapy (McCarty, 2004). Our findings reveal the mechanism, whereby YC-1 reduces HIF-1 and the existence of a mechanistic link between Akt/NF-κB and HIF-1α expression during hypoxia. These findings also provide a rationale to investigate Akt/NF-κB targeting agents, such as CAPE and evodiamine, for their ability to block tumor adaptation to hypoxia.

Materials and methods


YC-1 was obtained from Yung-Shin Pharmaceutical Industry Co. Ltd. (Taichung, Taiwan). Rosewell Park Memorial Institute (RPMI)-1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco BRL Life Technologies (Grand Island, NY, USA). Sulforhodamine B (SRB), MG132, N-ethylmaleimide, cycloheximide, wortmannin, PMA, insulin, LY294002, PDTC and CAPE were ordered from Sigma Chemical (St Louis, MO, USA). Evodiamine was purchased from Matsuura Yakugyo Co. Ltd. (Nagoya, Japan). HIF-1α and VHL antibody were from Novus Biologicals (Littleton, CO, USA). Anti-HIF-1β was purchased from Abcam (Cambridge, UK). Antibodies against nucleolin (major nuclear protein as internal control), ubiquitin, Hsp70, Hsp90 NF-κB/p65, actin and α-tubulin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-Akt (Ser473), phospho-p70S6K (Ser389), phospho-4E-BP1, phospho-eIF4E and phospho-IκBα antibodies came from Cell Signaling Technologies (Beverly, MA, USA). Reported plasmid 5 × HRE-Luc was kindly provided by Professor Giaccia AJ (Stanford University, Stanford, CA, USA). EGFP-N1 plasmid (Clontech, Mountain View, CA, USA) contains the EGFP under the control of cytomegalovirus promoter. Myr-Akt, IκBα mutant (IκBαM), DN-Akt plasmids were kindly provided by Professor C-H Lin (Taipei Medical University, Taipei, Taiwan). Reporter plasmid κBRE-Luc was a kind gift from Professor C-C Chen (National Taiwan University, Taipei, Taiwan). NF-κB/p65 plasmid was kindly provided by Professor W-W Lin (National Taiwan University, Taipei, Taiwan).

Cell culture

PC-3 cells (derived from the American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI-1640 medium supplemented with 10% FBS (v/v) and penicillin (100 U/ml)/streptomycin (100 μg/ml). Cultures were maintained in a humidified incubator containing 21% O2 and 5% CO2 in air (normoxia, N). For hypoxia exposure, cells were placed in a airtight chamber (BioSpherix, Redfield, NY, USA) flushed with a mixture of 1% O2, 5% CO2 and 94% N2 to maintain O2 concentration at 1% with Pro-Ox model 110 O2 regulator (BioSperix). Cells were subjected to hypoxic induction at a cell density of 1 × 105 cells/cm2.

Western blot analysis

Cells were seeded and allowed to attach for 24 h. The cells were starved 24 h and then pretreated for 1 h with drugs as mentioned in the figure legends in fresh media with 0.1% FBS, followed by hypoxia treatment or addition of 100 nM PMA, 200 nM insulin. After the indicated exposure time, cells were lysed in the radio immunoprecipitation buffer to obtain total cell lysate. For analysis of HIF-1α, HIF-1β, NF-κB and nucleolin, nuclear extracts were prepared as previously described (Huang et al., 2005b). Equivalent aliquots of protein were resolved in sodium dodecyl sulfate (SDS)–polyacrylamide gels and transferred to poly(vinylidene difluoride) (PVDF) membranes. All immunoblots were developed using enhanced chemiluminescence reagents (Amersham, Piscataway, NJ, USA).

Luciferase assays

PC-3 cells (5 × 104) were seeded into 24-well plates in standard growth medium. After an overnight culture, the cells were transfected with 0.72 μg plasmid DNA (0.24 μg HRE-Luc (or κBRE-Luc) and 0.48 μg empty vector (or Myr-Akt, IκBαM, p65) and 1.8 μl Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's instruction. At 8 h post-transfection, the cells were starved 16 h and then pretreated with drugs for 1 h as mentioned in the figure legends. The cells were incubated for 24 h in hypoxia chamber and lysed with passive lysis buffer (Promega, Madison, WI, USA). Luciferase activities of the cell extracts were determined using Luciferase assay system (Promega) with a liquid scintillation counter. Data showed were normalized for protein content. Transfection efficiency was measured by flow cytometric analysis of EGFP expression in 2.5 × 105 cells (seeded into six-well plates) transfected with 3.6 μg EGFP-N1 under the same treated condition mentioned aboved.

Sulforhodamine B assay

PC-3 cells were inoculated into 96-well plates (2 × 104 cells/well) in complete media. After an overnight culture, cells were starved 24 h followed by pretreatment with various concentrations of YC-1 for 1 h in 0.1% FBS culture media. Cells were incubated in hypoxia for an additional 8 or 24 h. The assay was terminated and the cell growth was measured by SRB assay described in the previous study (Chiang et al., 2005).


Immunoprecipitation was done as described previously (Chiang et al., 2005), besides N-ethylmaleimide was added to the cell lysis buffer at a final concentration of 10 mM to preserve poly-ubiquitinated protein conjugates.


For RT–PCR analysis, total cellular RNA was isolated with Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). First strand cDNA was synthesized with random primer and moloney murine leukemia virus reverse transcriptase (M-MLV RT). The primers and PCR conditions used for HIF-1α were according to Kaluzova et al. (2004). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard. PCR products were separated on 1.5% agarose gel and visualized by ethidium bromide staining. The gel was photographed using an image analyzer (Gel DOC 2000, BioRad, Hercules, CA, USA).

Statistical analysis

Each experiment was performed at least three times, and representative data are shown. Data in bar graph are given as the means±s.e.m. Means were checked for statistical difference using the t-test and P-values less than 0.05 were considered significant (*P<0.05, **P<0.01, ***P<0.001).


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We thank Yu-Chun Huang, Yi-Fan Ma, Paul Yueh-Jen Hsu and Po-Cheng Chiang for valuable discussion of experimental technique. We also thank Shu-Hui Chiang for providing us with critical reagents that made this work possible. This work was supported by a research grant from Yung-Shin Pharmaceutical Industry Co. Ltd. and NSC 94-2320-B002-038 from the National Science Council of the Republic of China, Taiwan.

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Correspondence to C-M Teng.

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Sun, HL., Liu, YN., Huang, YT. et al. YC-1 inhibits HIF-1 expression in prostate cancer cells: contribution of Akt/NF-κB signaling to HIF-1α accumulation during hypoxia. Oncogene 26, 3941–3951 (2007). https://doi.org/10.1038/sj.onc.1210169

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  • YC-1
  • HIF-1
  • Akt
  • NF-κB
  • prostate cancer

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