Heme Oxygenase: Drug Discovery

HO-1 has emerged as a promising therapeutic target in a broad spectrum of human diseases, such as cardiovascular, inflammatory, and neurodegenerative disorders as well as cancer ²¹. HO-1 conveys protection by mediating the production of iron-sequestering ferritin ⁴⁵², and anti-oxidants such as biliverdin and bilirubin ⁴⁵³, as well as by facilitating anti-inflammatory and anti-apoptotic effects of CO ⁴⁵⁴.

 

The heme-degrading capacity of HOs, and in particular that of HO-1, has evolved as a potent approach in blocking oxidative stress, insufficient immune responses, and pertinent disorders. Several in vitro and in vivo models of inflammation and acute lung injury established HO-1 and its product CO as main triggers of the acute inflammatory response ^{206, 455, 456}. In a model of complement-dependent inflammation, inhibition of HO-1 by tin protoporphyrin IX (SnPPIX) augmented the inflammatory response whereas HO-1 up-regulation led to a striking inhibition of the inflammation⁴⁵⁶. Of note, intra-tracheal adenoviral HMOX1 gene transfer as a model gene therapeutic approach for acute lung injury induced by inhaled pathogen diminished neutrophilic inflammation of the lung and increased production of anti-inflammatory IL-10 in macrophages after lipopolysaccharide (LPS) exposure ⁴⁵⁵. Several investigations confirmed the anti-inflammatory potential of HO-1/CO by down-regulating the production of pro-inflammatory cytokines (e.g., TNF, IL-1β, IL-6) and up-regulating the levels of anti-inflammatory cytokines (e.g., IL-10) ^{206, 359}. These effects of CO might be caused by the interference of CO with AP-1 activity via the JNK signaling pathway ³⁵⁹. A similar anti-inflammatory response can be seen in hsf1^-/- mice where the genetic deletion of HSF-1, the key transcriptional regulator of Hsp70, and/or the suppression of the Hsp70 expression attenuated the cytoprotective and immunomodulatory effects of CO in vitro and in vivo ³⁴². The HO-1 product biliverdin has also been identified to exert anti-inflammatory protection against LPS-induced acute lung injury in rats by modulating pro- and anti-inflammatory cytokine profiles ⁴⁵⁷. Anti-inflammatory protection could be achieved by transfection of mouse macrophages with HO-1 or pretreatment with heme as well, leading to the down-regulation of LPS-induced pro-inflammatory mediators such as HMGB-1 (high-mobility group box 1) ⁴⁵⁸. Induction of HO-1 also improved survival of mice in an LPS- and cecal ligation and puncture-induced sepsis model in vivo ⁴⁵⁸. The potentially safeguarding action of HO-1/CO has been estimated in a clinically-relevant model of sterile inflammation caused by mechanical ventilation which gives rise to ventilator-induced lung injury in rodents ^459-461. Also in higher mammals, anti-inflammatory actions of CO became apparent by reducing the levels of LPS-induced pro-inflammatory cytokines (IL-1β, TNF) and up-regulating anti-inflammatory IL-10 ^{462, 463}. However, the therapeutic efficacy of CO in this model rather depends on high doses that resulted in ameliorated levels of carboxyhemoglobin (CO-Hb). This study was about the first to elucidate the therapeutic ratio of CO in non-human primates in a model of acute inflammation ⁴⁶².

 

Current investigations imply a putative protective role of HO-1/CO in the inflammasome system, cytosolic protein complexes promoting the cleavage of caspase-1 and culminating in the maturation and release of pro-inflammatory cytokines such as IL-1β and IL-18. Amongst them, the NLRP-3-dependent inflammasome has been implicated in the pathogenesis of numerous acute or chronic inflammatory diseases ^{464, 465}. Jung et al. convincingly demonstrated in an NLRP-3 activation model that CO is able to block caspase-1 activation and secretion of IL-1β and IL-18 in bone marrow-derived macrophages in response to LPS and ATP ³⁴⁹. Exposure of peritoneal macrophages to the CO donor compound CORM-2 down-regulated caspase-1 activation as well as IL-1β maturation and secretion under ER stress-induced inflammatory conditions ⁴⁶⁶. CORMs (carbon monoxide releasing compounds) have been found not only to impair systemic inflammation but also to attenuate pro-adhesive vascular cell properties, as well as to prolong survival and to reduce liver injury during sepsis and inflammatory lesions ^467-469. Additionally, CORMs exert protective effects in sepsis by up-regulating bacterial clearance ⁴⁷⁰, and, on the other hand, by direct anti-bacterial activities against certain microbes including E. coli ⁴⁷¹, Pseudomonas aeruginosa ⁴⁷², Helicobacter pylori ⁴⁷³, and Salmonella typhimurium ⁴⁷⁴. It is of note that addition of CORM-3 to the preservation solution in a cardiac transplantation model resulted in a significant improvement in systolic and diastolic function as well as coronary flow ⁴⁷⁵.

 

Several studies have revealed the pivotal role of heme-derived CO in the regulation of the immune system ⁴⁷⁶ and particularly the critical implication of HO-1 in modulating immune responses in tumor tissues ^477-479. CO resulting from a targeted HO-1 up-regulation exerts immunomodulatory effects by blocking CD8⁺ DC maturation and modulating cytokine production ⁴⁸⁰, suppressing proliferation and activation of CD4⁺ CD25^– effector T (T_eff) cells ²³², inducing CD4⁺ CD25⁺ regulatory T (T_reg) cells ⁴⁸¹, as well as by permitting maturation of myeloid cells ⁴⁷⁶. HO-1 induction by hemin has also been found as being linked to the blockage of allergic airway inflammation via up-regulation of CD4⁺ CD25⁺ T_reg cells ⁴⁸². In human T_reg cells, the expression of HO-1 was reported to associate positively with the presence of the T_reg-specific marker FoxP3 ⁴⁸³. HO-1-derived CO is supposed to trigger the anti-proliferative effects on CD4⁺ T cells via suppression of the production of T cell growth-inducing IL-2 ⁴⁸⁴. However, more recent investigations imply that the function of CD4⁺ CD25⁺ T_reg cells does not rely on the presence of HO-1 in these cells ⁴⁸⁵. In this regard, over-expression of HO-1 was noted to render T_reg cells resistant to CD95/Fas-mediated apoptosis ⁴⁸⁶. In human malignant gliomas, over-expression of HO-1 is associated with increased tumor grade and immune suppression obviously mediated by T_reg cells ⁴⁸⁷. Dey and collaborators convincingly demonstrated that HO-1 expression ameliorates survival of T_reg cells in glioma-bearing mice and that HO-1 inhibition by tin protoporphyrin IX (SnPPIX) reduces the number of T_reg cells, culminating in a survival advantage ⁴⁸⁸. Blancou and Anegon hypothesize that the up-regulation of HO-1 in DCs favors the emergence of CD4⁺ CD25⁺ T_reg cells and inhibits the proliferation of T cells in the tumor stromal compartment ⁴⁸⁹. It is of note that HO-1 down-regulation in tumor cells increases tumor immunogenicity by up-regulating the number of cytotoxic CD8⁺ tumor-infiltrating lymphocytes and down-regulating the number of T_reg cells, resulting in a marked delay in cancer progression ⁴⁹⁰. Pharmacological inhibition of HO-1 in different cervical cancer cell lines has been shown to increase the expression of TNF and IFN-γ in co-cultured NK cells and to restore the expression of NK activation markers including the cell surface receptors NKG2D, NKp30, and NKp46 ⁴⁹¹. NK cells play an essential role in tumor recognition as they mediate early-immune responses to cancer cells ⁴⁹².

 

There is evidence to demonstrate that the induction of HO-1 or application of exogenous CO blocks LPS-induced maturation of DCs and inhibits pro-inflammatory and allogeneic immune responses while preserving IL-10 production ⁴⁷⁸. In a transgenic mouse model of autoimmune diabetes, CO-treated DCs loaded with pancreatic beta-cell peptides were shown to suppress the accumulation and pathogenic activity of autoreactive CD8⁺ T cells in the pancreas ⁴⁹³.

 

The modulation of HO-1 expression by genetic over-expression or counter-regulation represents a promising tool in gene therapy applications. For this purpose, adenoviral and retroviral-based vectors have been developed and evaluated for HO-1 gene transfer in vivo. Adenoviral over-expression of HO-1 protected against myocardial injury in a mouse cardiac ischemia/reperfusion (I/R) injury model ⁴⁹⁴ and led to blockage of lung cell injury in response to hyperoxia in vivo after intra-tracheal administration ⁴⁹⁵. HO-1 gene transfer has also been reported to diminish hypoxia/reoxygenation-induced stasis in mice with sickle cell disease (SCD), an archetypal example of hemolysis, by inducing anti-inflammatory responses ⁴⁴⁴. Controversially, retroviral transduction of HMOX1 into severe combined immune deficient (SCID) mice has been found to increase the development of pancreatic tumors via the stimulation of angiogenesis ⁴⁹⁶. Inhibition of HO-1 by tin mesoporphyrin (SnMP) was able to transitively prolongate tumor growth in a dose-dependent manner ⁴⁹⁶. Similarly, zinc protoporphyrin IX (ZnPPIX), which suppresses the angiogenesis of pancreatic and lung cancer, blocked the metastatic potential of gastric cancer by suppressing pro-angiogenic VEGF ⁴⁹⁷.

 

Retroviral expression of miRNAs has been probed to study the targeted down-regulation of HO-1. Zhang et al. observed that HO-1 silencing using lentiviral miRNA constructs led to increased pulmonary apoptosis, decreased autophagy, and enhanced susceptibility to oxidant stress in multiple lung cell types ⁴⁹⁸. As stated by Abraham and colleagues, successful HO gene transfer aims at achieving functional HO activity in multiple ways ⁴⁹⁹. Initially, the HMOX1 gene must be supplied in a secure vector, e.g., adenoviral, retroviral or leptosome-based vectors, that are currently being used in clinical trials. Secondly, HO supply must be cell or organ-specific as achieved in rabbit ocular tissues, rat liver, kidney and vasculature, SHR kidney, and endothelial cells ⁴⁹⁹. Nonetheless, the safety and efficacy of retroviral vectors for human application represents a major concern ^{16, 499}.

 

HO-1 has generally been considered as being a cytoprotective mediator, crucially involved in cancer progression by augmenting cell proliferation, metastasis, and angiogenesis as well as by conferring resistance to phytodynamic therapy and radio- and chemotherapy ²⁰². Compared to healthy tissues, the expression of HO-1 is up-regulated in different tumor types including lymphosarcoma, adenocarcinoma, hepatoma, glioblastoma, melanoma, prostate cancers, Kaposi sarcoma, squamous carcinoma, pancreatic cancer, renal cell carcinoma as well as in brain tumors and hematological malignancies (for a review see ^{202, 363-365}). Anti-cancer approaches can further increase the expression of HO-1 and thus attenuate the efficiency of the therapy ^{25, 366, 367}. In this regard, over-expression of HO-1 has been shown to protect numerous cancer cell types from the cytostatic actions of cisplatin ^{500, 501}. Pharmacological inhibition or silencing of HO-1 by RNA interference has been noted to enhance chemosensitivity towards cisplatin in vivo and in vitro ^{502, 503}, obviously via down-regulation of MMP-9 and VEGF as well as apoptosis induction ^{500, 501}. Accordingly, knockdown or inhibition of HO-1 was reported to block tumor growth in human pancreatic cancer and non-muscle-invasive bladder cancer resistant to chemotherapy ^{504, 505}.

 

As stated before, enhanced expression of HO-1 has been associated with an unfavorable prognosis in patients with astrocytoma ³⁷⁵, glioma ³⁷⁵, and neuroblastoma ³⁷⁶. In glioma cells, inhibition of HO-1 or Nrf-2 knockdown significantly potentiated the cytostatic effects of arsenic trioxide (ATO) thereby rendering administration of ATO together with HO-1 inhibition or silencing a promising approach in the treatment of glioma ⁵⁰⁶. Furfaro and colleagues convincingly demonstrated that the Nrf-2-dependent HO-1 induction suppresses neuroblastoma cell death after bortezomib treatment or glutathione depletion, while HO-1 inhibition by ZnPPIX or silencing restores cell sensitivity towards the cytostatic drug ^507-509.

 

HO-1 is also up-regulated in various hematological malignancies including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), and multiple myeloma where it functions as an important survival factor (for a review see Li Volti et al., 2017) ³⁶³. In a mouse model of human AML, HO-1 silencing has been found by Lin et al. to prolong the survival rate of human xenografts.  Further investigations by the same group described the ability of HO-1 to block apoptosis in AML cells via the induction of the JNK signaling pathway ⁵¹⁰. Down-regulation of HO-1 could be identified as being sufficient to sensitize AML and CML cells towards first-line chemotherapeutic agents such as cytarabine, daunorubicin, and imatinib ^511-513. Retroviral expression of miRNAs has been probed to study the targeted down-regulation of HO-1. Zhang et al. observed that HO-1 silencing using lentiviral miRNA constructs led to increased pulmonary apoptosis, decreased autophagy, and enhanced susceptibility to oxidant stress in multiple lung cell types ⁴⁹⁸.

 

The definite role of HO-1 in carcinogenesis is controversial and multifaceted. In contrast to its well documented pro-tumorigenic actions, HO-1 has also been found to exert anti-tumorigenic effects, e.g., in breast ²⁴, prostate ²³, and pancreatic cancer ²⁵ as well as in NSCLC ²⁶. Studies by Zhou and collaborators recently exhibited that over-expression of HO-1 in hepatocellular carcinoma (HCC) cells impedes HCC progression by down-regulating the levels of miR-30d and miR-107 ⁵¹⁴. Accordingly, up-regulation of HO-1 has been noted to suppress tumor growth by down-regulating miR-378 and MMPs in a murine xenograft model of human lung mucoepidermoid carcinoma ³⁷⁹. From these data one might imagine that the development of specific siRNA targeting HMOX1 or experimental approaches aiming at triggering the expression of miRNA species involved in regulating HMOX1 expression might be considered as being a novel therapeutic approach in certain diseases. Also, the HO-1 byproducts biliverdin and bilirubin as well as down-regulation of ferritin appear to have therapeutic applicability in disease (for a review see ^{16, 515, 516}). The application of HO-1 inducers or inhibitors as treatment modalities for human diseases will be discussed in the section Inhibitors and Inducers. Collectively, these data validate HO-1 as a prospective novel and key therapeutic target for future treatments of a conceivably wide spectrum of disorders.