The Triple Crown: NO, CO, and H2S in cancer cell biology

Abstract

Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S) are three endogenously produced gases with important functions in the vasculature, immune defense, and inflammation. It is increasingly apparent that, far from working in isolation, these three exert many effects by modulating each other's activity. Each gas is produced by three enzymes, which have some tissue specificities and can also be non-enzymatically produced by redox reactions of various substrates. Both NO and CO share similar properties, such as activating soluble guanylate cyclase (sGC) to increase cyclic guanosine monophosphate (cGMP) levels. At the same time, H₂S both inhibits phosphodiesterase 5A (PDE5A), an enzyme that metabolizes sGC and exerts redox regulation on sGC. The role of NO, CO, and H₂S in the setting of cancer has been quite perplexing, as there is evidence for both tumor-promoting and pro-inflammatory effects and anti-tumor and anti-inflammatory activities. Each gasotransmitter has been found to have dual effects on different aspects of cancer biology, including cancer cell proliferation and apoptosis, invasion and metastasis, angiogenesis, and immunomodulation. These seemingly contradictory actions may relate to each gas having a dual effect dependent on its local flux. In this review, we discuss the major roles of NO, CO, and H₂S in the context of cancer, with an effort to highlight the dual nature of each gas in different events occurring during cancer progression.

Keywords: CBS; CSE; Cancer; Carbon monoxide; Cell signaling; Crosstalk; HO-1; Hydrogen sulfide; Nitric oxide; Reactive oxygen species; iNOS.

Figure 1.. NO, H₂S, CO biosynthesis and crosstalk.

There is an extensive level of crosstalk between the gasotransmitters. NO is enzymatically produced from L-arginine by nNOS, eNOS, and iNOS, mediates many effects through the sGC-cGMP pathway, and this signaling is terminated by the degradation of cGMP by PDE5A. Superoxide anions can reduce NO bioavailability by causing NOS uncoupling and by reacting with NO to form peroxynitrite. H₂S is enzymatically produced from L-cysteine by CBS, CSE, and 3-MST in conjunction with CAT. CO is enzymatically produced from the heme breakdown by the enzymes HO-1, HO-2, and HO-3, which additionally produce biliverdin and Fe²⁺. NO and H₂S crosstalk: NO upregulates CSE expression while it inhibits the activity of CBS. H₂S can upregulate eNOS expression and activity through intracellular Ca²⁺ release, Akt-mediated activating phosphorylation of eNOS, and eNOS S-sulfhydration, which inhibits the negative feedback regulation of NO on eNOS activity and maintains the active dimerized state. H₂S has a dual effect on iNOS activity, and is capable of both increasing and decreasing its activity through similar dual effects on activating or inhibiting the iNOS transcription factor NF-κB. H₂S reduces oxidative stress which increases NO bioavailability, and is a scavenger of peroxynitrite. H₂S augments downstream NO signaling by inhibiting PDE5A activity to allow increased cGMP accumulation, and exerting redox regulation rendering sGC more responsive to NO. NO and H₂S may also react with each other to form new chemical species such as nitrosothiols, nitroxyl, and others. NO and CO crosstalk: CO may have a dual effect on eNOS activity, both inhibiting it and augmenting it, the latter through Ca²⁺ release, Akt-mediated activating phosphorylation of eNOS, and protection of the enzyme from downregulation in inflammatory conditions. CO has dual effects on iNOS activity as well by either increasing or decreasing NF-κB transcription factor activity; additionally, CO has been found to activate PPAR-γ to downregulate iNOS as well. NO and peroxynitrite have both been seen to upregulate HO-1 expression. CO and H₂S crosstalk: CO may inhibit CBS activity but upregulate CSE expression, while H₂S may inhibit CO-1, although more investigation into these relationships is necessary.

Figure 2.. NO effects in the context of cancer.

NO has both cancer-combatting and cancer-promoting effects that may depend on factors such as concentration, flux, and physiological or pathological setting. NO can reduce cancer cell proliferation by inhibiting several cell survival signaling pathways, causing cell cycle arrest, and increasing cancer cell apoptosis by causing DNA damage, increasing oxidative stress, and shifting the balance of pro- and anti- apoptotic proteins in favor of mitochondrial apoptosis; NO has also been found to increase the sensitivity of cancer cells to existing chemotherapeutic and radiation treatments. On the other hand, NO may also have pro-proliferative effects in the context of cancer by enhancing several other tumor growth signaling pathways. NO can inhibit EMT through inhibition of signaling pathways implicated in this phenomenon, and can inhibit ECM remodeling by downregulating MMPs. Both of these reduce the ability of the cancer to invade and metastasize. Yet NO has also been found to induce EMT in separate studies through the activation of different pathways than those implicated in inhibiting EMT. In the context of angiogenesis, NO may either facilitate angiogenesis or inhibit it through the modulation of different signaling pathways involved in this process. NO can enhance the anticancer immune response by enhancing M1 macrophage polarization, enhancing T cell infiltration, selectively inducing Th₁ polarization, and inhibiting the immunosuppressive action of MDSCs. On the other hand, NO can suppress the host anticancer immune response by enhancing immunosuppressive MDSC accumulation, increasing M2 macrophage polarization, reducing NK cell cytotoxicity, and inhibiting cytotoxic T cell activity by reducing T cell activation and inducing apoptosis, hindering T cell infiltration, and facilitating the development of T cell tolerance. In addition to these actions, NO can play an important role in the increased infectious complications associated with cancer; this gasotransmitter has been shown to directly combat pathogens through ROS and DNA damage induction, increase mucociliary clearance, inhibit various steps of viral entry and replication, and increase the antibiotic susceptibility of bacteria.

Figure 3.. CO effects in the context of cancer.

CO has widespread effects on cancer proliferation and apoptosis, invasion and metastasis, angiogenesis, and host anticancer immunity. The gasototransmitter has both cancer-combatting and cancer-promoting effects in these arenas, which may depend on factors such as concentration, flux, and physiological or pathological setting. CO can reduce cancer cell proliferation by inhibiting several cell survival pathways, inducing cell cycle arrest, favoring mitochondrial apoptosis, and increasing cancer sensitivity to chemotherapy. On the other hand, CO has also been found to increase cancer cell proliferation by increasing the activity of different growth signaling pathways, and to increase resistance to oxidant-induced apoptosis through a mechanism involving the inhibition of K_ATP channels. CO can suppress cancer cell invasion and metastasis by inhibiting ECM remodeling through MMP downregulation, and by suppressing EMT. With regards to angiogenesis, the gasotransmitter can either increase or decrease angiogenesis by different, likely context-dependent effects on several angiogenic signaling pathways. CO can increase antitumor host immunity by inducing M1 macrophage polarization, inducing immunogenic cell death through ROS accumulation, and inducing moderate ER stress, which leads to protective autophagy and epigenetic reprogramming of T cell into a stronger antitumoral phenotype. Yet CO may also suppress host antitumor immunity by inducing M2 macrophage polarization and suppressing T cell activity. In the context of infection, CO can cause DNA damage by ROS induction, cause bactericidal inflammasome activation and phagocytosis of bacteria, inhibit biofilm formation, and inhibit viral replication.

Figure 4.. H₂S effects in the context of cancer.

H₂S also has both cancer-promoting and cancer-combatting effects resulting from its impact on cell proliferation and apoptosis, invasion and metastasis, angiogenesis, and antitumoral immunity. H₂S can inhibit tumor proliferation by inhibiting various growth signaling pathways, inducing cell cycle arrest, and increasing cancer cell apoptosis. Contrarily, H₂S can increase cancer proliferation and protect against apoptosis by activating various growth signaling pathways, maintaining DNA repair mechanisms, and suppressing ROS production. The gasotransmitter has been seen to both increase and suppress EMT induction and ECM remodeling through modulation of several different signaling pathways. In the context of angiogenesis, only a pro-angiogenic effect has been reported through increased pro-angiogenic signaling; mechanistically, NO and K_ATP channels have been found to be involved in the pro-angiogenic effect of H₂S. H₂S can boost anticancer immunity by increasing M1 macrophage polarization, enhancing T cell activation, and suppressing the immunosuppressive MDSCs; yet immunosuppressive activity of H₂S has also been described, including M2 macrophage polarization, T cell death, impaired NK cell cytotoxicity, and enhanced Treg differentiation. In the context of infection, H₂S exerts antimicrobial actions through increased oxidative stress upregulation of the antioxidant and antiviral agent GSH, increased mucociliary clearance, and inhibition of viral replication.