**5. Additional pharmacological activities of YC-1**

In addition to the effects in antiplatelet aggregation and antithrombosis, YC-1 has been demonstrated to provide several beneficial effects including cardiovascular protections; antitumor, neuroprotective, and anti-inflammatory effects; as well as optical protections.

#### **5.1 Cardiovascular protections**

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

indazole (YC-1) was selected for further investigation. The physiological property of YC-1 in stimulation of sGC was demonstrated by Ko and colleagues [49]. Potential regulatory mechanisms of YC-1 on cardiovascular protections were summarized in **Figure 1**. Ko et al. showed that YC-1 is an antithrombotic agent. It inhibits platelet aggregation by increasing platelet cGMP levels in an NO-independent manner. YC-1 action was noted to exert its antiplatelet effect through the activation of NO-independent sGC/cGMP pathway [50]. Nearly, all the newer generations of sGC stimulator except acryl-acrylamide family have been derived based on YC-1 as the parent compound [51]. YC-1 and its successors all require the presence of a reduced haem moiety within sGC to stimulate sGC, but they also act in synergy with NO by binding NO or iron-free precursor of haem to structurally resemble the NO-haem complex and stabilize sGC in its active configuration [52–54]. Stasch et al. also reported that YC-1 and its derivate, BAY 41-2272, bind to regulatory sites (cys 238 and cyst 243 regions) in the α1-subunit of sGC, resulting in conformational change and subsequent activation of recombinant sGC by NO-independent but haem-dependent mechanism [55]. Mulsch et al. also noted that the combined effect of nitrovasodilators and YC-1 in cultured VSMCs and isolated rabbit aortic rings reflected the direct synergistic action of YC-1 and NO on the sGC [56]. Wohlfart et al. reported that YC-1 can stimulate synthesis and release NO in endothelial cells independent of raising the cGMP content in a calcium-dependent manner [57]. In addition, YC-1 inhibits the cGMP-specific phosphodiesterase type 5 (PDE-5)

*Schematic overview of regulatory mechanisms of YC-1 on cardiovascular protections. cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; ECs, endothelial cells; FAK, focal adhesion kinase; GMP, guanosine monophosphate; GTP, guanosine triphosphate; HO-1, heme oxygenase-1; Hsp70, heat shock protein 70; iNOS, inducible nitric oxide synthase; MMP2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; oxLDL, oxidized low-density lipoprotein; PDE5, phosphodiesterase 5; PKG, protein kinase G; sGC, soluble guanylyl cyclase; TGF-β, transforming growth factor-beta; VASP,* 

*vasodilator-stimulated phosphoprotein; VSMC, vascular smooth muscle cells.*

**76**

**Figure 1.**

YC-1 inhibits VSMC proliferation, similar to specific guanylate cyclase inhibitors, suggesting that the antiproliferative effect of YC-1 is mediated by cGMP [62]. A similar conclusion has also been drawn by other investigators [63, 64]. As shown in **Figure 1**, NO-/cGMP-dependent processes have been suggested to modulate VSMC phenotype and the arterial response to endovascular injury [65, 66]. It has been reported that YC-1 can upregulate expression of iNOS and inducible heme oxygenase-1 (HO-1) at the transcriptional and translational level as well as stimulate sGC and cGMP production in the balloon-injured artery [63]. These results support the proposal that YC-1 can be developed as a potent new therapeutic agent for reducing restenosis *via* endogenous carbon monoxide (CO)- and/ or NO-mediated cGMP-dependent processes. Wu et al. found that two important modulators, transforming growth factor (TGF)-β1 and focal adhesion kinase (FAK), responsible for VSMC proliferation and migration were reduced in content in the cultured VSMC treated with YC-1. The effect of YC-1 on preventing balloon injury-induced vascular stenosis has also been demonstrated in a rat carotid angioplasty model [64]. Liu et al. also found that YC-1 can inhibit neointima formation in balloon-injured rat carotid through suppressing the expression and actions of matrix metalloproteinase (MMP)-2 and MMP-9 [67]. YC-1 can also prevent oxLDL-mediated apoptosis by inducing heat shock protein 70 (Hsp70) expression in VSMCs suggesting its cytoprotective effect in vascular diseases [68]. Similarly, Hsp70 overexpression has also been involved in protective effect of YC-1 on heat stroke [69]. In vivo evidence shows that YC-1 and zaprinast, an inhibitor of cGMPselective PDE, inhibit injury-induced vascular remodeling through anti-mitogenic and pro-apoptotic actions in a rat carotid artery balloon injury model [70]. Moreover, YC-1 has also been found to induce cell cycle arrest of HUVEC through upregulation of p21 and p27 protein *via* inhibition of the cyclin/cyclin-dependent kinase (CDK) system. This finding suggests that YC-1-induced antiproliferation effect in HUVEC is *via* a cGMP-independent manner [71]. Besides, the prevention effects of YC-1 on the development of hypoxia-induced pulmonary arterial hypertension (PAH), right ventricular hypertrophy (RVH), and pulmonary vascular remodeling has been clearly mentioned in animal model [72].

#### **5.2 Antitumor effects**

A growing body of evidence indicates that hypoxia-inducible factor-1 (HIF-1) contributes to tumor progression and metastasis. YC-1 inhibits HIF-1-mediated

hypoxic responses [73–76]. YC-1 enhanced radiation sensitivity by inhibiting HIF-1α expression [77]. Lau et al. also found that YC-1 suppressed both synthesis and stability of HIF-1α, *via* regulation of murine double minute (Mdm2) protein [78]. In hypoxic gastric carcinoma cell and xenograft models, low-dose YC-1 combined with glucose and insulin can effectively inhibit anaerobic glycolysis and induce hypoxia-dependent apoptosis by suppressing HIF-1α expression [79].

YC-1 also enhanced chemosensitivity of hepatocellular carcinoma cells to cisplatin through a Stat3-dependent manner [80]. Similarly, YC-1 also enhanced camptothecin toxicity by activating the caspase-8, the Bid pathway, and the mitochondria-mediated apoptotic pathway in ovarian carcinoma cell lines [81]. Additionally, it has also been found that YC-1 can suppress constitutive NF-κB activation and induce apoptosis in human prostate cancer cells [82]. YC-1 inhibited VEGF- and basic fibroblast growth factor (bFGF)-mediated ERK1/ERK2 mitogenactivated protein kinase (MAPK), AKT, and protein kinase Cα (PKCα) pathways in vitro and angiogenesis in in vivo models [83]. YC-1 arrested the cell cycle in G0/ G1 in human hepatocellular carcinoma cells by upregulating p21CIP1/WAP1 and p27KIP1 expression [84]. YC-1 arrested the cell cycle at S-phase and induced apoptosis by activating checkpoint kinases in several cancer cells [85]. Similarly, YC-1 can also increase p21 protein and decrease cyclins and CDKs to induce G0/G1 phase arrest as well as activate caspases and disrupt the mitochondrial membrane potential to trigger mitochondria-dependent apoptosis in cisplatin-resistant human oral cancer CAR cells [86]. Additionally, apoptotic mechanism of YC-1 may also be mediated by activating JNK phosphorylation and upregulating FasL and Fas receptor clustering to activate caspase-3 and caspase-8 and then trigger mitochondria-mediated and caspase-dependent pathways in renal carcinoma cells [87]. YC-1 has been shown to downregulate several invasion-related signaling proteins, such as β-catenin, caveolin, Src, and epidermal growth factor receptor (EGFR), as well as multiple growth-related proteins, including 5'-AMP-activated protein kinase α (AMPKα), phospho-acetyl-CoA carboxylase (p-ACC), human epidermal growth factor receptor 2 (HER-2), and mammalian target of rapamycin (mTOR) in nasopharyngeal carcinoma [88]. Other anti-invasion mechanisms of YC-1 have also been identified in nasopharyngeal carcinoma (NPC) cells by reverse phase protein array [88]. Activation of beta-catenin signaling has also been evidenced to involve in inhibiting the proliferation and metastasis of hepatocellular carcinoma using combination therapy with local radiofrequency ablation and YC-1 [89]. Moreover, the previous study indicated that YC-1 has a potential effect to improve drug resistance by inhibiting multidrug-resistant protein resulting in decrease of P-glycoprotein (Pgp) efflux, whose effect is modulated by the NO-cGMP-PKG-ERK signaling pathway [90]. These observations revealed together that YC-1 exerts inhibitory effects in key signaling pathways essential for maintaining cancer or endothelial cell viability and may be developed as an antitumor agent on a broad spectrum of cancer types by facilitating apoptosis and suppressing tumor angiogenesis.

#### **5.3 Neuroprotective and anti-inflammatory effects**

The use of NO donors (e.g., NONOate) results in excessive NO production which may cause NO-induced axonal damage by inhibiting mitochondrial respiration, independent of cGMP [91]. YC-1 has been shown to protect white matter axons from NO toxicity. This axonoprotective action of YC-1 was unrelated to its activity on sGC but through a novel action on voltage-dependent Na<sup>+</sup> channels in the rat isolated optic nerve [92]. Lu et al. showed YC-1 inhibition of lipopolysaccharide (LPS)-induced iNOS and cyclooxygenase-2 (COX-2) expression as well as NF-κB activation, implying that YC-1 can be developed as an anti-inflammatory

**79**

*Therapeutic Applications and Mechanisms of YC-1: A Soluble Guanylate Cyclase Stimulator*

neuroprotective agent [93]. Chien et al. reported that YC-1 promoted learning behavior in Morris water maze and avoidance tests and YC-1 pretreatment reduced scopolamine-induced learning deficit. Thereby, the NO/cGMP/PKG pathway may be involved in the learning enhancement-based experiments with intracerebroventricular injection of L-NAME and PKG inhibitors [94]. Similarly, YC-1 can also improve age-related learning and memory dysfunction [95]. Furthermore, YC-1 may inhibit HIF-1α accumulation and VEGF production to protect blood-brain barrier against ischemia-/reperfusion-induced injury [96]. In addition, beneficial effect of YC-1 in ameliorating combined allergic rhinitis and asthma syndrome (CARAS) was demonstrated through reducing expressions of HIF-1α, NF-kB, and

Therapeutic application of YC-1 on sepsis has been mentioned. After administration with YC-1, several LPS-stimulated modulations, such as NF-κB activation, iNOS expression, NO overproduction, and cytokine release, were markedly inhibited, thus improving survival rate of endotoxemic mice [98]. YC-1 has also been shown to inhibit HIF-1α-induced iNOS and VEGF expressions in various tissue models. Studies showed that YC-1 inhibited optical neovascularization in the pathological stages [99, 100]. Song noted that YC-1 could prevent laser-induced choroidal neovascularization by suppressing photocoagulation-mediated HIF-1 expression [99]. The pathological retinal neovascularization could also be inhibited by YC-1 through decreasing ischemia-induced expression of HIF-1 and its downstream angiogenic mediators (e.g., VEGF) in the ischemic retina. The physiological revascularization of the retinal vascular plexuses was enhanced by YC-1 *via* inhibiting iNOS expression at mRNA and protein levels [100]. Besides, it also has been reported that YC-1 alleviated macular edema in the animal model of laser-induced experimental central retinal vein occlusion by reducing several inflammatory or angiogenesis-related

peroxisome proliferator-activated receptor α (PPARα) [97].

factors, such as interleukin-6 (IL-6), IL-8, VEGF, and HIF-1 [101].

Wang and his colleagues evidenced that YC-1 inhibited bone resorption and induced extrinsic apoptosis of osteoclasts to reduce bone loss, which implied that YC-1 has potential application for use as an antiresorptive drug in postmenopausal osteoporosis [102]. Besides, YC-1 and its derivatives also have been mentioned to improve hepatic fibrosis, which mechanisms may be caused by inhibiting liver neutrophil infiltration as well as decreasing in TNF-α signaling and macrophage

Extensive studies have been conducted to explore possible systemic actions of YC-1 in disease models in animals to demonstrate that YC-1 has versatile physiological activities to be a potent candidate drug for a number of vascular disorders. In the cardiovascular and hematological systems, it has been reported that local extravascular administration of YC-1 could prevent neointima formation in a rat carotid artery model of balloon angioplasty [63, 64, 67]. In a study of experimental thrombosis model, YC-1 conferred beneficial effect through its anti-aggregating and pro-fibrinolytic effects [105]. BAY 63-2521 (riociguat™), a NO-independent but heme-dependent sGC stimulators like TC-1, is currently in clinical development

*DOI: http://dx.doi.org/10.5772/intechopen.84572*

**5.4 Optical protections**

**5.5 Other activities**

aggregation [103, 104].

**6. Clinical significance of YC-1**

*Therapeutic Applications and Mechanisms of YC-1: A Soluble Guanylate Cyclase Stimulator DOI: http://dx.doi.org/10.5772/intechopen.84572*

neuroprotective agent [93]. Chien et al. reported that YC-1 promoted learning behavior in Morris water maze and avoidance tests and YC-1 pretreatment reduced scopolamine-induced learning deficit. Thereby, the NO/cGMP/PKG pathway may be involved in the learning enhancement-based experiments with intracerebroventricular injection of L-NAME and PKG inhibitors [94]. Similarly, YC-1 can also improve age-related learning and memory dysfunction [95]. Furthermore, YC-1 may inhibit HIF-1α accumulation and VEGF production to protect blood-brain barrier against ischemia-/reperfusion-induced injury [96]. In addition, beneficial effect of YC-1 in ameliorating combined allergic rhinitis and asthma syndrome (CARAS) was demonstrated through reducing expressions of HIF-1α, NF-kB, and peroxisome proliferator-activated receptor α (PPARα) [97].

#### **5.4 Optical protections**

*Vascular Biology - Selection of Mechanisms and Clinical Applications*

facilitating apoptosis and suppressing tumor angiogenesis.

The use of NO donors (e.g., NONOate) results in excessive NO production which may cause NO-induced axonal damage by inhibiting mitochondrial respiration, independent of cGMP [91]. YC-1 has been shown to protect white matter axons from NO toxicity. This axonoprotective action of YC-1 was unrelated to its

the rat isolated optic nerve [92]. Lu et al. showed YC-1 inhibition of lipopolysaccharide (LPS)-induced iNOS and cyclooxygenase-2 (COX-2) expression as well as NF-κB activation, implying that YC-1 can be developed as an anti-inflammatory

channels in

activity on sGC but through a novel action on voltage-dependent Na<sup>+</sup>

**5.3 Neuroprotective and anti-inflammatory effects**

hypoxic responses [73–76]. YC-1 enhanced radiation sensitivity by inhibiting HIF-1α expression [77]. Lau et al. also found that YC-1 suppressed both synthesis and stability of HIF-1α, *via* regulation of murine double minute (Mdm2) protein [78]. In hypoxic gastric carcinoma cell and xenograft models, low-dose YC-1 combined with glucose and insulin can effectively inhibit anaerobic glycolysis and induce hypoxia-dependent apoptosis by suppressing HIF-1α expression [79]. YC-1 also enhanced chemosensitivity of hepatocellular carcinoma cells to cisplatin through a Stat3-dependent manner [80]. Similarly, YC-1 also enhanced camptothecin toxicity by activating the caspase-8, the Bid pathway, and the mitochondria-mediated apoptotic pathway in ovarian carcinoma cell lines [81]. Additionally, it has also been found that YC-1 can suppress constitutive NF-κB activation and induce apoptosis in human prostate cancer cells [82]. YC-1 inhibited VEGF- and basic fibroblast growth factor (bFGF)-mediated ERK1/ERK2 mitogenactivated protein kinase (MAPK), AKT, and protein kinase Cα (PKCα) pathways in vitro and angiogenesis in in vivo models [83]. YC-1 arrested the cell cycle in G0/ G1 in human hepatocellular carcinoma cells by upregulating p21CIP1/WAP1 and p27KIP1 expression [84]. YC-1 arrested the cell cycle at S-phase and induced apoptosis by activating checkpoint kinases in several cancer cells [85]. Similarly, YC-1 can also increase p21 protein and decrease cyclins and CDKs to induce G0/G1 phase arrest as well as activate caspases and disrupt the mitochondrial membrane potential to trigger mitochondria-dependent apoptosis in cisplatin-resistant human oral cancer CAR cells [86]. Additionally, apoptotic mechanism of YC-1 may also be mediated by activating JNK phosphorylation and upregulating FasL and Fas receptor clustering to activate caspase-3 and caspase-8 and then trigger mitochondria-mediated and caspase-dependent pathways in renal carcinoma cells [87]. YC-1 has been shown to downregulate several invasion-related signaling proteins, such as β-catenin, caveolin, Src, and epidermal growth factor receptor (EGFR), as well as multiple growth-related proteins, including 5'-AMP-activated protein kinase α (AMPKα), phospho-acetyl-CoA carboxylase (p-ACC), human epidermal growth factor receptor 2 (HER-2), and mammalian target of rapamycin (mTOR) in nasopharyngeal carcinoma [88]. Other anti-invasion mechanisms of YC-1 have also been identified in nasopharyngeal carcinoma (NPC) cells by reverse phase protein array [88]. Activation of beta-catenin signaling has also been evidenced to involve in inhibiting the proliferation and metastasis of hepatocellular carcinoma using combination therapy with local radiofrequency ablation and YC-1 [89]. Moreover, the previous study indicated that YC-1 has a potential effect to improve drug resistance by inhibiting multidrug-resistant protein resulting in decrease of P-glycoprotein (Pgp) efflux, whose effect is modulated by the NO-cGMP-PKG-ERK signaling pathway [90]. These observations revealed together that YC-1 exerts inhibitory effects in key signaling pathways essential for maintaining cancer or endothelial cell viability and may be developed as an antitumor agent on a broad spectrum of cancer types by

**78**

Therapeutic application of YC-1 on sepsis has been mentioned. After administration with YC-1, several LPS-stimulated modulations, such as NF-κB activation, iNOS expression, NO overproduction, and cytokine release, were markedly inhibited, thus improving survival rate of endotoxemic mice [98]. YC-1 has also been shown to inhibit HIF-1α-induced iNOS and VEGF expressions in various tissue models. Studies showed that YC-1 inhibited optical neovascularization in the pathological stages [99, 100]. Song noted that YC-1 could prevent laser-induced choroidal neovascularization by suppressing photocoagulation-mediated HIF-1 expression [99]. The pathological retinal neovascularization could also be inhibited by YC-1 through decreasing ischemia-induced expression of HIF-1 and its downstream angiogenic mediators (e.g., VEGF) in the ischemic retina. The physiological revascularization of the retinal vascular plexuses was enhanced by YC-1 *via* inhibiting iNOS expression at mRNA and protein levels [100]. Besides, it also has been reported that YC-1 alleviated macular edema in the animal model of laser-induced experimental central retinal vein occlusion by reducing several inflammatory or angiogenesis-related factors, such as interleukin-6 (IL-6), IL-8, VEGF, and HIF-1 [101].

#### **5.5 Other activities**

Wang and his colleagues evidenced that YC-1 inhibited bone resorption and induced extrinsic apoptosis of osteoclasts to reduce bone loss, which implied that YC-1 has potential application for use as an antiresorptive drug in postmenopausal osteoporosis [102]. Besides, YC-1 and its derivatives also have been mentioned to improve hepatic fibrosis, which mechanisms may be caused by inhibiting liver neutrophil infiltration as well as decreasing in TNF-α signaling and macrophage aggregation [103, 104].
