ハイポキシアを標的とする新規癌治療薬の開発

近年、腫瘍の微小環境が、癌の治療成果のみならず、癌細胞の増殖・分化、転移などを制御する重要な要因と考えられている。中でも低酸素微小環境(ハイポキシア)は癌の基本的な環境であり、これまで放射線や化学療法に対して抵抗性を示し、完治を妨げる要因として問題視されてきた。低酸素誘導因子(HIF-1α)の発見に端を発し、ハイポキシアの分子機構に関する研究が急速に進展し、細胞の低酸素ストレス応答に関わる様々な分子が明らかになってきた。その結果、新たな創薬ターゲット探索の場として、ハイポキシア研究に期待が集まっている。本総説では、低酸素微小環境を標的とする癌治療法の進歩を含む、ハイポキシア研究の最近の成果を紹介し、筆者の行った多機能性低酸素細胞放射線増感剤の開発、低酸素微小環境を標的とするhypoxic cytotoxinの分子設計、及びハイポキシア指向性ハイブリッド型ボロンキャリアの分子設計における研究成果を概説する。この研究で我々の開発した、2-nitroimidazole系放射線増感剤やheterocycle-N-oxide誘導体に、血管新生阻害作用や転移抑制効果があることを見いだした。これらの作用はHIF-1αを中心とするハイポキシア応答シグナルの阻害を介して起こるものと考えられた。この様な効果は、従来の化学療法剤にみられるDNA障害性のcytotoxic作用とは異なるcytostaticな効果である。このような薬剤は、放射線によるcytotoxic作用を補い、予後の改善とQOLの向上をもたらし、ひいては癌治療効果を増強するものと期待される。The tumor microenvironment is now recognized as a major factor that influences not only the response to conventional anti-cancer therapies, but also helps define the potential for malignant progression and metastasis. In particular, hypoxia is now considered a fundamentally important characteristic of the tumor microenvironment. Furthermore, discovery of the hypoxia inducible factor 1α (HIF-1α) has led to a rapidly increasing understanding of the molecular mechanisms involved in tumor hypoxia. This in turn has led to the current extensive interest in the signal molecules related to tumor hypoxia as potential molecular targets for cancer therapeutics. In this paper we give an overview of recent advances in hypoxia research, including cancer treatments that target tumor hypoxia. Progress in the development of hypoxia-targeting drugs are discussed, including anti-angiogenic hypoxic cell radiosensitizers, hypoxic cytotoxins and hypoxia targeting boron carriers. We have found that certain 2-nitroimidazole radiosensitizers and heterocycle-N-oxide hypoxic cytotoxins we developed have antiangiogenic activity and antimetastatic activity. We propose that these activities are based on the inhibition of signal transduction mediated by HIF-1α. The anti-tumor activities of preventing hypoxia response are considered to be cytostatic effects, in contrast to cytotoxic DNA damaging effects. The combination of these cytostatic effects that are related to radiosensitization with the cytotoxic effects of radiation should improve the prognosis and QOL of patients receiving radiation and lead to an overall response to treatment

Iron accumulation causes impaired myogenesis correlated with MAPK signaling pathway inhibition by oxidative stress

Skeletal muscle atrophy is caused by disruption in the homeostatic balance of muscle degeneration and regeneration under various pathophysiological conditions. We have previously reported that iron accumulation induces skeletal muscle atrophy via a ubiquitin ligase-dependent pathway. However, the potential effect of iron accumulation on muscle regeneration remains unclear. To examine the effect of iron accumulation on myogenesis, we used a mouse model with cardiotoxin (CTX)-induced muscle regeneration in vivo and C2C12 mice myoblast cells in vitro. In mice with iron overload, the skeletal muscles exhibited increased oxidative stress and decreased expression of satellite cell markers. Following CTX-induced muscle injury, these mice also displayed delayed muscle regeneration with a decrease in the size of regenerating myofibers, reduced expression of myoblast differentiation markers, and decreased phosphorylation of mitogen-activated protein kinase signaling pathways. In vitro, iron overload also suppressed the differentiation of C2C12 myoblast cells, but the suppression could be reversed by superoxide scavenging using tempol. Excess iron inhibits myogenesis via oxidative stress, leading to an imbalance in skeletal muscle homeostasis

Inhibitory action of iron on erythropoietin via oxidative stress-HIF-2α pathway

Renal anemia is a major complication in chronic kidney disease (CKD). Iron supplementation, as well as erythropoiesis-stimulating agents, are widely used for treatment of renal anemia. However, excess iron causes oxidative stress via the Fenton reaction, and iron supplementation might damage remnant renal function including erythropoietin (EPO) production in CKD. EPO gene expression was suppressed in mice following direct iron treatment. Hypoxia-inducible factor-2 alpha (HIF-2α), a positive regulator of the EPO gene, was also diminished in the kidney of mice following iron treatment. Anemia-induced increase in EPO and HIF-2α expression was also inhibited by iron-treatment. In in vitro experiments using EPO-producing HepG2 cells, iron stimulation reduced the expression of the EPO gene, as well as HIF-2α. Moreover, iron treatment augmented oxidative stress, and iron-induced reduction of EPO and HIF-2α expression was restored by tempol, an anti-oxidant compound. HIF-2α interaction with the EPO promoter was inhibited by iron treatment, and was restored by tempol. These findings suggested that iron supplementation reduced EPO gene expression via an oxidative stress-HIF-2α-dependent signaling pathway

Dietary iron restriction alleviates renal tubulointerstitial injury induced by protein overload in mice

Increased proteinuria causes tubulointerstitial injury due to inflammation in chronic kidney disease (CKD). Iron restriction exhibits protective effects against renal dysfunction; however, its effects against protein overload-induced tubulointerstitial damage remain unclear. Here, we investigated dietary iron restriction effect on tubulointerstitial damage in mice with protein-overload tubulointerstitial injury. Renal tubulointerstitial injury in animal model was induced by intraperitoneal injection of an overdose of bovine serum albumin (BSA). We divided mice into three groups: normal saline + normal diet (ND), BSA + ND, and BSA + iron-restricted diet (IRD). BSA overload induced renal tubulointerstitial injury in the ND mice, which was ameliorated in the IRD mice. Inflammatory cytokines and extracellular matrix mRNA expression was upregulated in BSA + ND mice kidneys and was inhibited by IRD. BSA-induced increase in renal superoxide production, NADPH oxidase activity, and p22phox expression was diminished in the IRD mice. IRD suppression increased BSA-induced renal macrophage infiltration. Moreover, BSA mice exhibited nucleotide-binding oligomerisation domain-like receptor pyrin domain-containing protein (NLRP) inflammasome activation, which was inhibited by IRD. Ferrous iron increased in kidneys with BSA overload and was inhibited by IRD. Thus, iron restriction inhibited oxidative stress and inflammatory changes, contributing to the protective effect against BSA overload-induced tubulointerstitial injury

A pH-Adjustable Tissue Clearing Solution That Preserves Lipid Ultrastructures: Suitable Tissue Clearing Method for DDS Evaluation

Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethylenimine was required for a wide pH range adjustment of the tissue clearing solution.The combination of polyethylenimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained with lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Moreover, spatial distribution of liposomes in the liver was visualized. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing, while preserving lipid ultrastructures, which is suitable for drug delivery system evaluations

Chemical tools for detecting Fe ions

Owing to its distinctive electrochemical properties with interconvertible multiple oxidation states, iron plays a significant role in various physiologically important functions such as respiration, oxygen transport, energy production, and enzymatic reactions. This redox activity can also potentially produce cellular damage and death, and numerous diseases are related to iron overload resulting from the dysfunction of the iron regulatory system. In this case, “free iron” or “labile iron,” which refers to iron ion weakly bound or not bound to proteins, causes aberrant production of reactive oxygen species. With the aim of elucidating the variation of labile iron involved in pathological processes, some chemical tools that can qualitatively and/or quantitatively monitor iron have been utilized to investigate the distribution, accumulation, and flux of biological iron species. Since iron ions show unique reactivity depending on its redox state, i.e., Fe²⁺ or Fe³⁺ (or transiently higher oxidative states), methods for the separate detection of iron species with different redox states are preferred to understand its physiological and pathological roles more in detail. The scope of this review article covers from classical chromogenic to newly emerging chemical tools for the detection of Fe ions. In particular, chemical tools applicable to biological studies will be presented

Organelle-specific analysis of labile Fe(ii) during ferroptosis by using a cocktail of various colour organelle-targeted fluorescent probes

Ferroptosis is an emerging type of cell death mode that is dependent of iron. Unfortunately, the detailed analysis of the function of organelle labile Fe(II) in oxidative damage and lethality of the cells has not been demonstrated so far, mainly due to the lack of efficient methods to visualize labile Fe(II) at the targeted organelles. We have recently reported a series of Fe(II)-selective fluorescent probes, i.e. Ac-MtFluNox, Lyso-RhoNox, and ER-SiRhoNox, which can detect Fe(II) specifically in mitochondria, lysosomes, and endoplasmic reticulum (ER), respectively. These probes demonstrate the similar reaction rates and off/on contrasts with various colours and intracellular distributions, enabling simultaneous multi-colour imaging that allows the monitoring of labile Fe(II) levels at each targeted organelle. In this paper, by using a cocktail of these probes, we successfully visualised the aberrant elevation of labile Fe(II) in lysosomes and ER prior to HT1080 cell death induced by erastin, which is an inducer of ferroptosis