Abstract
Since the first article on YC-1 was published in 1994, it has been popularly used as a pharmacological tool to activate soluble guanylate cyclase and to increase cyclic GMP levels in cultured cells or isolated tissues. In terms of the pharmacological actions of YC-1, previous studies tend to be limited to its inhibition of platelet aggregation and vascular contraction. However, recent studies have demonstrated that YC-1 has versatile pharmacological effects other than the anti-platelet and vasodilatory effects. In particular, two recent reports suggest that YC-1 could be developed as a new class of anticancer agent for rapidly growing solid tumors, because it inhibits hypoxia-inducible factor 1 (HIF-1) activity, and has been reported to halt tumor growth in vivo. We here review the cyclic GMP-dependent and independent pharmacological actions of YC-1, and its anti-HIF-1, anticancer effect.

Keywords: YC-1, soluble guanylate cyclase, cyclic GMP, hypoxia-inducible factor, anticancer effect

1. Introduction
YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole] is a chemical compound that was first introduced by Teng and colleagues [1]. They found that in isolated platelets, YC-1 inhibits platelet aggregation, ATP release, phosphoinositide breakdown, and the elevation of intracellular free calcium. They also suggested that these effects of YC-1 are caused by the activation of platelet soluble gualylate cyclase (sCG) and the elevation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) levels. Moreover, they demonstrated that YC-1 prevents intravascular thrombus formation in mice by inhibiting platelet aggregation. Similarly, YC-1 activated sCG in vascular smooth muscle cells and relaxed arterial muscle strips [2,3]. Subsequent biochemical studies that were performed with expressed sGC showed that the stimulatory effect of YC-1 on sGC was enhanced by the physiologic sGC activators nitric oxide (NO) and carbon monoxide (CO) [4-7]. Biologically, combined treatment with YC-1 and natural sGC activators synergistically inhibited platelet aggregation and arterial muscle contraction. Thus, YC-1 is viewed as a useful research tool for investigating sGC- and cGMP-mediated cellular processes. It could also be a lead compound for the development of therapeutic agents designed to prevent intravascular thrombus formation.

2. cGMP-dependent pharmacological actions of YC-1
For the past 10 years, YC-1 has been used as a direct activator of sGC, and in particular to determine whether the cGMP pathway is involved. Although NO can be used to activate sGC, it is inconvenient to apply it biological systems in some circumstances. When NO gas is applied to biological systems, specially designed equipment is needed to regulate the gas tension. It not, chemical substances are needed to spontaneously release NO. In this case, it is also difficult to maintain a constant level of NO release. In contrast, the use of YC-1 is more convenient, because it can be treated in in-vitro and in-vivo systems at precise concentrations. This is the main reason why YC-1 has become popular.

2.1. Mechanism of sGC activation
sGC is a heterodimer composed of two different subunits, an alpha subunit of 72 kDa, and a beta subunit of 65 kDa [8]. The alpha and beta subunits share a conserved domain at their C-termini, and this domain is responsible for the catalytic action, i.e., the conversion of GTP to cGMP. The N-terminal regulatory domains of both subunits, which contain one mole of prosthetic heme, are essential for the stimulation of the enzyme by NO and CO. The heme moiety, which is attached to histidine 105 of the beta subunit, is the target for the NO/CO-dependent regulation of sGC [9,10]. Binding of NO and CO to ferrous heme activates sGC, whereas the oxidation of heme iron to ferric attenuate the response to NO or CO. The NO-stimulated sGC/cGMP signal is conveyed to activate several effector molecules: cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cGMP-gated ion channels [8]. Moreover, this signaling cascade plays important roles in the cardiovascular and nervous systems, where it controls smooth muscle relaxation, modulates synaptic transmission, and inhibits platelet aggregation [11,12].

In addition to NO and CO, YC-1 is also recognized as an activator of sGC. However, YC-1 does not release NO or directly target the heme moiety of sGC like NO, as no changes were observed in the heme spectra of purified sGC after adding YC-1 [6]. In the presence of a small amount of NO, sGC activity is synergistically enhanced by the addition of YC-1. YC-1 also modulates the catalytic activity of sGC by increasing the maximum rate of cGMP formation and the affinity to GTP [13]. YC-1 also sensitizes the binding of the heme moiety to NO, because the NO concentration required to activate sGC was found to be reduced by YC-1. Moreover, YC-1 appears to stabilize the NO-heme binding in sGC by reducing the rate of NO dissociation [14,15]. Therefore, YC-1 is regarded as a NO-independent allosteric regulator of sGC.

2.2. Pharmacological actions related with increased cGMP
The direct activation of sGC is regarded as the main mechanism by which YC-1 mimics many of the known functions of NO. However, YC-1 also increases intracellular cGMP levels using an additional mechanism. For example, it has been reported that YC-1 can inhibit cGMP-hydrolyzing phosphodiesterases (PDEs), and Galle et al. [16] demonstrated that YC-1 directly inhibited five PDE isoenzymes (PDE1-5) of the rabbit aorta in the same concentration range as it induced vascular relaxation. Therefore, the non-selective inhibition of PDE activity and the activation of sGC contribute to the elevation of cGMP by YC-1, which results in the inhibition of platelet aggregation and in the relaxation of vascular smooth muscles.

YC-1 has another effect on vascular smooth muscle cells, i.e., it prevents neointima formation in luminal arterial walls injured by balloon dilation. Arterial restenosis and neointima formation is a major complication of balloon angioplasty. Neointima formation is mainly attributed to the proliferation of vascular smooth muscle cells, which is augmented by platelet aggregation, during which platelets stimulate the proliferation of smooth muscle cells by releasing growth factors, such as thrombin and platelet-derived growth factor (PDGF). Tulis and colleagues [17,18] reported that YC-1 attenuates neointima formation in balloon-injured arteries either by inhibiting smooth muscle cell proliferation or by inactivating platelets. Moreover, these effects of YC-1 were accompanied by increased cGMP levels in vascular smooth muscle cells.

cGMP is also a major intracellular mediator responsible for corpus cavernosal smooth muscle relaxation, which in turn causes penile erection. Therefore, YC-1 can be expected to aid penile erection if it increases cGMP levels in the cavernosal smooth muscle cells. Recently two studies [19,20] demonstrated that the intraperitoneal or intracavernosal injection of YC-1 evokes erectile response in male rats. YC-1 also increased cGMP levels in cultured rabbit corpus cavernosal cells and relaxed the cavernosal strips pre-contracted with phenylephrine [20]. These results support a novel clinical use of YC-1, in the treatment of male erectile dysfunction. In addition to these effects of YC-1 on smooth muscle cells, YC-1 can affect many biological functions related to the activation of cGMP-dependent protein kinases (PKGs) and of cGMP-regulated ion channels. Indeed, YC-1 up-regulated cyclooxygenase (COX) 2 expression by stimulating PKGs [21], lowered intraocular pressure with elevated levels of nitric oxide metabolites [22], and hyper-polarized vascular smooth muscle cells by opening a large-conductance, Ca²⁺-sensitive K⁺ channel (BK_Ca), which is known to be regulated by cGMP [23]. The sGC/cGMP-dependent actions and possible clinical indications for YC-1 are summarized in Fig. 1.

Fig. 1. Mechanisms, pharmacologic actions, and possible clinical indications of YC-1.
i., inhibition of; v.s.m., vascular smooth muscle; ?, uncertain

3. cGMP-independent pharmacological actions of YC-1
A growing body of evidence suggests that YC-1 has a variety of pharmacological actions that are independent of its cGMP elevating effect. Previously, we found a novel effect of YC-1 on the hypoxic adaptation of cancer cells [24]. YC-1 inhibited the hypoxic induction of hypoxia-inducible factor-1alpha (HIF-1alpha), which in turn blocked the expression of erythropoietin (EPO) and vascular endothelial growth factor (VEGF). Details regarding this effect of YC-1 are introduced in the next section. It has also been reported that YC-1 modulates the function of phagocytic immune cells. Wang and colleagues [25] demonstrated that YC-1 reduces Ca²⁺ entry in neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine; an effect probably attributable to the inhibition of tyrosine kinase. YC-1 also inhibited superoxide anion production in stimulated neutrophils and reduced the expressions of membrane-associated ADP-ribosylation factor and Rho A [26]. In a cultured rat macrophage cell-line, YC-1 augmented TNFa mRNA and protein expression stimulated by lipopolysaccharide and interferon-g [27]. In addition, YC-1 prevented NO-induced, cGMP-independent cytotoxicity in neuronal axons [28]. The sGC/cGMP-independent actions and possible clinical indications for YC-1 are summarized in Fig. 1

4. Anti-HIF, anticancer effect of YC-1

4.1. HIF-1 and Tumor promotion
To survive under oxygen-deficient conditions, organisms and cells have developed numerous adaptive mechanisms. At the cellular level, adaptation involves activated glycolysis, increased glucose uptake, and the expression of cell survival- or death-related proteins [29]. The regulation of most proteins required for hypoxic adaptation occurs at the gene level, and involves transcriptional induction via the binding of a transcription factor, hypoxia-inducible factor-1 (HIF-1), to a conserved sequence, 5-ACGTG-3, in the hypoxia response element (HRE) of regulated genes [30,31]. To date, about 40 hypoxia-inducible genes have been found to be directly regulated by HIF-1. HIF-1 is a heterodimer composed of two basic-helix-loop-helix (bHLH) proteins of the PAS family, HIF-1alpha, and aryl hydrocarbon nuclear receptor translocator (ARNT) [32]. Of these, HIF-1alpha is the key protein, as it determines the presence of HIF-1 and transactivates the hypoxia-inducible genes.

Hypoxia is a common feature of rapidly growing tumors and of their metastases [33]. Since hypoxia is the universal stimulus for HIF-1alpha induction, it is no surprise that high levels of HIF-1alpha protein are observed in hypoxic tumors. In addition to hypoxia, genetic alterations in oncogenes (Her2, FRAP, Ras, and Src) and tumor suppressor genes (VHL, PTEN, and p53) also induce the expression of HIF-1alpha [34]. Tumor growth factors or cytokines also induce HIF-1alpha expression in cancer cells. These factors bind to their receptors and activate the receptor tyrosine kinases, which in turn activate the PI3K/AKT/mTOR pathway. Finally, mTOR stimulates the expression of HIF-1alpha even under normoxic conditions [35-37]. Based on the functions of the proteins up-regulated by HIF-1, HIF-1 may be expected to contribute to tumor progression and metastasis. Indeed, immunohistochemical analyses have shown that HIF-1alpha is overexpressed in human tumors [38], and HIF-1alpha levels in biopsy specimens have been associated with the vascular density in various solid tumors, such as tumors of the brain and breast [39]. More importantly, HIF-1alpha levels in tumors have been positively related to tumor aggressiveness and a poor prognosis in cancer patients [40], and several animal studies have demonstrated that HIF-1alpha enhances tumor growth and angiogenesis in xenografted tumors [41,42]. Since HIF-1alpha expression and HIF-1 activity appear central to tumor growth and progression, HIF-1 inhibition presents itself as an appropriate anticancer target.

4.2. Inhibitory effects of YC-1 on HIF-1alpha expression and tumor growth
Recently, we found that YC-1 has novel effects on HIF-1alpha expression and tumor promotion [24]. Initially, YC-1 was used as a NO mimic to examine the effect of NO signaling on hypoxic response in Hep3B hepatoma cells. YC-1 was found to diminish the hypoxic induction of both EPO and VEGF mRNA, and to suppress HRE-binding by HIF-1 and HIF-1alpha protein expression. YC-1 also inhibited non-hypoxic HIF-1alpha induction by cobalt or desferrioxamine. However, sGC inhibitors failed to block these effects of YC-1 on HIF-1alpha, and further treatment with 8-bromo-cGMP did not inhibit the hypoxic induction of HIF-1alpha. These results strongly indicate that the HIF-1-inhibitory effect of YC-1 is unlikely to be mediated by sGC/cGMP signal transduction, rather the YC-1 effect is probably achieved by a novel cellular process linked with the oxygen-sensing pathway [24]. Since HIF-1 plays a crucial role in tumor promotion and angiogenesis, YC-1, as a novel HIF-1 inhibitor, could be further developed as a novel anticancer agent targeting HIF-1 and tumor angiogenesis. Indeed, YC-1 effectively halted tumor growth in immunodeficient mice grafted with five types of human tumor cells. These tumors showed reduced HIF-1alpha expression and poor vascularization. Moreover, YC-1 suppressed the expressions of the HIF-1-regulated genes, i.e., VEGF and glycolytic enzymes, in grafted tumor tissues. In addition, the suppression of HIF-1alpha by YC-1 was associated with blocked angiogenesis, and tumor growth inhibition in YC-1-treated tumors. Moreover, YC-1 treatment did not produce serious toxicity or impair innate immunity during the treatment period [43]. Thus, we regard YC-1 as a good candidate molecule for the development of novel anti-angiogenic, anticancer agents [44,45].

4.3. Other possible indications for YC-1 as a HIF-1 inhibitor
Since HIF-1 regulates the expression of the genes essential for angiogenesis, it is clear that inhibition of HIF-1 activity could prevent the angiogenic activity of pathological tissues having hypoxic or inflamed region. Recently, new evidence has provided that inhibition of HIF-1 activity could reduce inflammation by blocking activation and infiltration of macrophages and neutrophils into affected tissues [46]. In the myocardium and the aortic wall, HIF-1alpha was expressed by mechanical wall stretch, which in turn induced VEGF [47,48]. The activation of HIF-1 in stretched muscular tissues may be associated with cardiac or vascular remodeling, such as cardiac hypertrophy or vascular wall thickening. In chronic obstructive lung disease, HIF-1 mediates the hypoxia-induced remodeling of pulmonary arterioles. This arterial thickening reduces the luminal diameter of the arterioles and increases resistance of the pulmonary circulation. In the process of pulmonary hypertension, HIF-1 may express a battery of vasoactive cytokines such as endotheline-1, angiotensin II, platelet-derived growth factor, serotonin, and thrombin, resulting in contraction and proliferation of vascular smooth muscle cells in pulmonary arterioles [49]. Besides being anticancer agent, YC-1 could be a therapeutic agent for treating various diseases associated with over-activation of HIF-1, such as angiogenesis-related diseases, immunological disorders, cardiovascular remodeling, pulmonary hypertension. These possible clinical indications for YC-1 are summarized in Fig. 1.

5. Conclusion
Initially, YC-1 was introduced as a pharmacological agent with anti-platelet and vasodilatory effects. However, few of its pharmacologic actions have been reported to date. In particular, since YC-1 inhibits both the expression and the transcriptional activity of HIF-1, and halts tumor growth in vivo, it is worth pursuing its application in cancer therapy. Although the pharmacological effects of YC-1 are generally known to depend on the activation of sGC and on increased cGMP levels, YC-1 affects cellular processes other than the sGC/cGMP signaling. Thus, YC-1 offers developmental possibilities for a range of pharmacologic agents targeted at the treatment of various disease states.

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