Biomedical subjects

Michihiko Kuwano

Publications and source records attributed to Michihiko Kuwano.

At least 55 records · Page 3Linked to original sources

PubMed2003

The pleiotropic functions of the Y-box-binding protein, YB-1.

The Y-box-binding protein (YB-1) represents the most evolutionary conserved nucleic-acid-binding protein currently known. YB-1 is a member of the cold-shock domain (CSD) protein superfamily. It performs a wide variety of cellular functions, including transcriptional regulation, translational regulation, DNA repair, drug resistance and stress responses to extracellular signals. As a result, YB-1 expression is closely associated with cell proliferation. In this review, we will begin by briefly describing the characteristics of YB-1 and will then summarize the pleiotropic functions brought about via DNA-RNA transaction and protein-protein interactions. In addition, we will discuss the diverse range of potential physiological and pathological functions of YB-1.

Animals↗

PubMed2003

Nuclear expression of Y-box-binding protein-1 correlates with P-glycoprotein and topoisomerase II alpha expression, and with poor prognosis in synovial sarcoma.

Nuclear expression of the Y-box-binding protein (YB-1) has been reported to correlate with the expression of P-glycoprotein in breast cancer and osteosarcoma. Overexpression of the ATP-binding cassette (ABC) superfamily, such as P-glycoprotein/multi-drug resistance (MDR) 1 and MDR-associated protein (MRP) 1, 2 and 3, has been reported in various malignant neoplasms. Fifty-four surgically resected synovial sarcomas were examined immunohistochemically for nuclear expression of YB-1 and intrinsic expression of P-glycoprotein, MRP1, MRP2, and topoisomerase II alpha, and the findings were compared with clinicopathological parameters, proliferative activities as evaluated by MIB-1 labelling index (LI), and the patients' prognoses. In addition, MDR1, MRP1, MRP2, and MRP3 mRNA levels were assessed using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method in 22 concordant frozen specimens from these cases and the findings were compared with six control skeletal muscle tissues. Independent prognostic factors were investigated using the Cox proportional hazards regression model. Nuclear expression of YB-1 protein correlated with P-glycoprotein expression (p = 0.0126). Moreover, cases with nuclear expression of YB-1 correlated with poor survival (p = 0.0495) and showed a high topoisomerase II alpha labelling index (topo II alpha LI) (p = 0.0056) and a high MIB-1 LI (p = 0.01). Multivariate Cox analysis showed that only the nuclear expression of YB-1 (p = 0.0136) and high American Joint Committee on Cancer (AJCC) stage (ie stage III or IV) (p < 0.0001) were independent factors for poor prognosis, while the expression of the YB-1 responsive gene products examined was not. These results indicate that the nuclear expression of YB-1 protein is associated with P-glycoprotein expression and proliferative activity as shown by the topo II alpha LI and the MIB-1 LI, and that expression of this protein is an important independent prognostic factor in synovial sarcoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Kinetic analysis of P-glycoprotein-mediated transport by using normal human placental brush-border membrane vesicles.

PURPOSE: P-Glycoprotein (Pgp) plays an important role in drug disposition and excretion in various tissues such as the brain, intestine, and kidney. Moreover, we have demonstrated that Pgp is expressed on the brush-border membranes of trophoblast cells in the placenta and restricts drug transfer from the maternal circulation to the fetus. However, the transport kinetics of physiologically expressed Pgp has scarcely been investigated. METHODS: In this study, we assessed the functional kinetics of transport mediated by Pgp that is physiologically expressed in normal tissue by using human placental brush-border membrane vesicles (BBMVs). Digoxin and vinblastine were used as typical substrates of Pgp. RESULTS: The uptakes of [3H]digoxin and [3H]vinblastine into BBMVs were significantly increased in the presence of an ATP-regenerating system. The ATP-dependent uptakes of [3H]digoxin and [3H]vinblastine into BBMVs exhibited saturable kinetics. The Michaelis constants (Kt values) were 2.65 +/- 1.80 microM and 21.9 +/- 3.37 microM, respectively. In the presence of a Pgp inhibitor such as verapamil, cyclosporine A, or progesterone, the ATP-dependent uptakes of [3H]digoxin and [3H]vinblastine into BBMVs were significantly reduced. Anti-Pgp monoclonal antibody C219 completely inhibited the uptake of [3H]digoxin. CONCLUSIONS: The transport kinetics of [3H]digoxin and [3H]vinblastine by physiologically expressed Pgp were successfully evaluated by using BBMVs prepared from normal human placenta. The present method enabled us to evaluate the function of physiologically expressed Pgp and is superior to the use of cultured transfectants in terms of the yield of vesicles. The present method may also be applicable to investigating the influence of various factors such as the genotype of the MDR1 gene or various pathophysiologic states of neonates on the function of Pgp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Y-box binding protein expression in thyroid neoplasms: its linkage with anaplastic transformation.

Recent studies have demonstrated that Y-box binding protein (YB-1) regulates the transcription of genes linked to carcinoma progression. In this study, we investigated the expression of this protein in thyroid neoplasms to elucidate its significance. The expression of YB-1 was immunohistochemically investigated using the monoclonal antibody for various thyroid neoplasms. Normal follicles did not overexpress YB-1, and only moderate overexpression of YB-1 was observed in some follicular tumors and papillary carcinoma, especially those of a larger size. In contrast, 92.9% of anaplastic carcinoma strongly overexpressed YB-1. YB-1 immunoreactivity was seen in both cytoplasms and cell nuclei, but the former was more predominant. These findings suggest that YB-1 plays a role in regulating the transcription as well as translation of genes contributing to the anaplastic transformation of thyroid carcinoma.

Adenocarcinoma, Follicular↗

PubMed2003

The basic and clinical implications of ABC transporters, Y-box-binding protein-1 (YB-1) and angiogenesis-related factors in human malignancies.

In our laboratories, we have been studying molecular targets which might be advantageous for novel cancer therapeutics. In this review, we focus on how ATP-binding cassette (ABC) transporter superfamily genes, Y-box-binding protein-1 (YB-1), and tumor angiogenesis-associated factors could contribute to the development of novel strategies for molecular cancer therapeutics. ABC transporters such as P-glycoprotein/MDR1 and several MRP family proteins function to protect cells from xenobiotics, drugs and poisons, suggesting that ABC transporters are a double-edged sword. In this regard, P-glycoprotein/MDR1 is a representative ABC transporter which plays a critical role in the efflux of a wide range of drugs. We have reported that gene amplification, gene rearrangements, transcription factor YB-1 and CpG methylation on the promoter are involved in MDR1 gene overexpression in cultured cancer cells. Among them, two mechanisms appear to be relevant to the up-regulation of MDR1 gene in human malignancies. We first reported that MDR1 gene promoter is activated in response to environmental stimuli, and is modulated by methylation/demethylation of CpG sites on the MDR1 promoter. We also demonstrated that YB-1 modulates not only transcription of various genes associated with cell growth, drug resistance and DNA synthesis, but also translation, mRNA stabilization and DNA repair/self-defense processes. Angiogenesis is also involved in tumor growth, invasion and metastasis of various malignancies, and so angiogenesis-related molecules also offer novel molecular targets for anticancer therapeutics.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Cellular distribution of NDRG1 protein in the rat kidney and brain during normal postnatal development.

N-myc downregulated gene 1 (NDRG1) is a 43-kD protein whose mRNA is induced by DNA damage, hypoxia, or prolonged elevation of intracellular calcium. Although NDRG1 is also upregulated during cell differentiation, there are few studies on NDRG1 expression during postnatal development. Here we investigated the expression and cellular distribution of NDRG1 protein in rat kidney and brain during postnatal development. Immunohistochemical analysis revealed that the cellular localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between postnatal days 10 and 20. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period. These alterations in the cellular distribution of NDRG1 were associated with shifts in the molecular assembly on Western blots. Under non-reduced conditions, the main NDRG1 band was found only around 215 kD in both kidney and brain during the early postnatal stage. After postnatal day 10, the immunoreactive bands shifted to 43 kD in the kidney and 129 kD in the brain. These changes in the cellular distribution and state of assembly may correlate with the functional maturation of both organs.

Animals↗

PubMed2003

cis-hinokiresinol, a norlignan from Anemarrhena asphodeloides, inhibits angiogenic response in vitro and in vivo.

cis-Hinokiresinol (CHR) is a norlignan constituent from Anemarrhena asphodeloides BUNGE (Liliaceae), which shows hyaluronidase inhibitory activity. In the present studies, we have demonstrated that CHR selectively inhibited endothelial cell proliferation compared with cancer cells, and especially basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. Furthermore, endothelial cell migration and tube formation, two important steps in the angiogenic process, were also inhibited by CHR. Moreover, CHR reduced the vessel growth induced by VEGF in the mouse corneal neovascularization model. These results suggest that CHR may prove useful for the development of a novel angiogenesis inhibitor.

Anemarrhena↗

PubMed2003

1,3-dimethylisoguaninium, an antiangiogenic purine analog from the sponge Amphimedon paraviridis.

An antiangiogenic purine analog, 1,3-dimethylisoguaninium (1), was isolated from the ethanol (EtOH) extract of the Okinawan sponge Amphimedon paraviridis. The structure was elucidated on the basis of its spectral properties and X-ray crystallographic analysis. Compound 1 exhibited specific inhibition of the basic fibroblast growth factor (bFGF)-induced proliferation of bovine aorta endothelial cells (BAECs). Moreover, compound 1 reduced the tube formation of BAECs in a time-dependent manner.

Angiogenesis Inhibitors↗

PubMed2003

Genetic polymorphism at the 5' regulatory region of multidrug resistance 1 (MDR1) and its association with interindividual variation of expression level in the colon.

The multidrug resistance 1 (MDR1) is a key molecule in determining not only the resistance of cancer cells to anticancer agents but also the disposition of a variety of drugs in intestinal and other tissues. However, the mechanism underlying interindividual variations in levels of MDR1 activity and expression in various tissues remains unclear. We analyzed the nucleotide sequence polymorphisms in the 5' upstream regulatory region of the gene spanning 4 kb from the transcriptional start site of MDR1 and tried to identify any associations between polymorphisms and MDR1 expression. Within that region, we identified eight single nucleotide polymorphisms (SNPs) in the region in the Japanese population. Of the SNPs identified, -2410T>C, -1910T>C, and 692T>C were in perfect linkage disequilibrium. In normal colorectal mucosa, diplotypes at the region showed more significant association with the expression level of MDR1 mRNA than each SNP did. In an in vitro reporter assay, transcription activity of the minor-type construct carrying haplotypes 2 and 3 was significantly lower than that of the major-type construct carrying haplotype 1. We next identified two DNA binding proteins: one protein bound to the nucleotide sequence carrying -692T but not to that carrying -692C and another bound to the nucleotide sequence carrying -2352G but three times weaker than that carrying -2352A. This suggested the significance of SNP at -692 and -2352 of MDR1 in variable expression in the colon interindividually. This is the first report connecting SNPs and interindividual variety of MDR1 expression rationally.

Alleles↗

PubMed2002

Costunolide, a sesquiterpene lactone from Saussurea lappa, inhibits the VEGFR KDR/Flk-1 signaling pathway.

Costunolide (CT), a sesquiterpene lactone constituent isolated from Saussurea lappa (Compositae), exerted an antiangiogenic effect. CT selectively inhibited the endothelial cell proliferation induced by vascular endothelial growth factor (VEGF). Further, CT was also found to inhibit the VEGF-induced chemotaxis of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. From these results, we hypothesized that CT might inhibit angiogenesis by blocking the angiogenic factor signaling pathway. VEGF interacts with its cognate receptors, KDR/Flk-1 and Flt-1, and exerts its angiogenic effect. CT inhibited the autophosphorylation of KDR/Flk-1 without affecting that of Flt-1. Moreover, administration of CT reduced VEGF-induced neovascularization in a mouse corneal micropocket assay. These results suggest that CT may prove useful for the development of a novel angiogenesis inhibitor.

3T3 Cells↗

PubMed2002

Increased nuclear localization of transcription factor YB-1 in acquired cisplatin-resistant ovarian cancer.

PURPOSE: Nuclear expression of Y box-binding protein (YB-1), a member of the DNA-binding protein family, was recently reported to have a much higher concentration in cisplatin-resistant cancer cell lines than in their drug-sensitive parental counterparts, suggesting the ability to induce cisplatin resistance. Ovarian cancer has been generally treated with cisplatin-based chemotherapy and often recurs due to acquired cisplatin resistance. The aim of our study is to elucidate the association between nuclear YB-1 and cisplatin resistance in human ovarian cancer using cultured cell lines and surgical specimens. METHODS: Intracellular YB-1 localization was examined by Western blot analysis for both cisplatin sensitive and resistant human ovarian cancer cell lines. Moreover, 35 pairs of surgical specimens derived from primary and matched recurrent ovarian cancers of the same patient were evaluated for their nuclear YB-1 expression by immunohistochemical staining. RESULTS: Western blot analysis for nuclear and cytoplasmic extracts indicated that cisplatin-resistant cells showed much higher nuclear YB-1 expression than sensitive parental cells. Immunohistochemical analysis showed that ten paired cases turned from negative nuclear YB-1 in primary lesions to positive nuclear YB-1 in recurrent lesions, whereas only two paired cases showed a reverse turn from positive to negative. CONCLUSIONS: The expression of YB-1 in the nucleus seems to be associated with acquired cisplatin resistance in ovarian cancers. Nuclear YB-1 might be a useful predictive marker indicating cisplatin sensitivity and/or a target molecule to treat recurring ovarian cancers by cisplatin-based second-line chemotherapy.

Antineoplastic Agents↗

PubMed2002

Binding capacity of human YB-1 protein for RNA containing 8-oxoguanine.

8-oxoguanine (8-oxo-7,8-dihydroguanine) is generated in the cellular nucleotide pool as well as in nucleic acids, by the action of oxygen radicals produced in cells. 8-oxoguanine has the potential to pair with both cytosine and adenine, and thus, the persistence of this base in messenger RNA would cause translational errors. To prevent such an outcome, organisms should have mechanisms for preventing the misincorporation of 8-oxoguanine-containing nucleotide into RNA and for removing 8-oxoguanine-containing RNA from processes of translation. We now report that mammalian Y box-binding protein 1 (YB-1 protein) possesses the activity to bind specifically to RNA containing 8-oxoguanine. On incubation with a purified preparation of YB-1 protein, 8-oxoguanine-containing RNA forms stable complexes with the protein while normal RNA scarcely forms such a complex. Using a series of deletion mutants which produce altered forms of YB-1 protein lacking some parts of the sequence, domains of the protein necessary for RNA binding were identified. Escherichia coli cells expressing normal or truncated forms of YB-1 protein with the binding capacity acquire resistance against paraquat, a drug that induces oxidative stress in cells, whereas cells with truncated proteins lacking such an activity do not. YB-1 protein may disturb the bacterial system in recognizing oxidatively damaged RNA, thus exerting a dominant negative effect on cell growth. We propose that YB-1 protein may discriminate the oxidized RNA molecule from normal ones, thus contributing to the high fidelity of translation in cells.

CCAAT-Enhancer-Binding Proteins↗

PubMed2002

Functional characterization of mouse cation transporter mOCT2 compared with mOCT1.

We characterized the function of mouse organic cation transporter OCT2 (TC 2.A.19.1.5) in comparison with that of OCT1 (TC 2.A.19.1.1). Uptake of [(3)H]1-methyl-4-phenylpyridinium ([(3)H]MPP(+)) by Xenopus laevis oocytes injected with mOCT1 (Slc22a1) or mOCT2 (Slc22a2) cRNA was attenuated by an increase of extracellular K(+) concentration and under acidic extracellular conditions. The uptakes of [(3)H]MPP(+) via mOCT1 and mOCT2 were saturable, with similar Michaelis constants (K(t)) of 10 and 24 microM, respectively. mOCT2 also mediated the uptake of [(14)C]tetraethylammonium with a K(t) value of 36 microM, which is similar to that of mOCT1. Quinine, tetraethylammonium, cimetidine, procainamide, choline, and N(')-methylnicotinamide inhibited the uptake of [(3)H]MPP(+) via mOCT1, as well as via mOCT2, and the inhibitory potencies for mOCT1 were comparable to but slightly higher than those for mOCT2. Thus, although the transport properties of mOCT2 are similar to those of mOCT1 in respect to the membrane-potential dependency, pH-sensitivity, and affinities for MPP(+) and tetraethylammonium, several organic cations had weaker inhibitory effects on [(3)H]MPP(+) uptake by mOCT2 than by mOCT1.

1-Methyl-4-phenylpyridinium↗

PubMed2002

The association of death-associated protein kinase hypermethylation with early recurrence in superficial bladder cancers.

Mechanisms for bladder carcinogenesis and the development of recurrentbladder cancer remain unclear. Aberrant methylation of the 5' CpG island is thought to play an important role in the inactivation of the tumor suppressor genes in cancer. To study whether specific or bulk hypermethylation predicts intrabladder recurrence, we determined the frequency of aberrant promoter hypermethylation of seven genes, hMLH1, O(6)-methylguanine-DNA-methyltransferase (MGMT), p16, Von Hippel-Lindau (VHL), death-associated protein kinase (DAP-kinase), glutathione S-transferase P1 (GST-P1) and E-cadherin in 55 superficial bladder cancers and 5 normal urothelial epithelia by methylation-specific PCR (MSP). These patients of superficial bladder cancer had been followed prospectively by cystoscopy. Simultaneous hypermethylation of three genes or more among the seven genes was observed in 2 (7%) of 30 patients in the nonrecurrence group and 7 (28%) of 25 patients in the recurrence group. There was a significant concordance between the number of methylated genes and the development of recurrence (P = 0.012). In particular, the recurrence rate for 24 months was 88% for hypermethylation of DAP-kinase and 28% for nonmethylation of DAP-kinase. Hypermethylation of DAP-kinase is, therefore, a strong indicator of the superficial bladder cancer associated with a high recurrence rate (P < 0.001; hazards ratio, 7.01). Our results suggest that hypermethylation of DAP-kinase might be a useful prognostic marker for disease recurrence in superficial bladder cancers.

Adult↗

PubMed2002

p73 Interacts with c-Myc to regulate Y-box-binding protein-1 expression.

YB-1 is a member of the cold shock domain family of proteins that is important for signaling DNA damage and cell proliferation. YB-1 is induced by DNA damage and can also recognize cisplatin-modified DNA. In this study we observed a 6-fold increase in the steady-state level of YB-1 mRNA in response to cisplatin exposure in cells of the human cancer cell line KB. We present evidence from cotransfection experiments for a critical role of c-Myc and p73 in the transactivation of the YB-1 promoter. p73 transactivated the YB-1 promoter in experiments with Saos-2 cells, which express c-Myc, but not with HO15.19 cells, which lack c-Myc. In turn, c-Myc transactivated an intact YB-1 promoter but not a YB-1 promoter with a mutant E-box, indicating that the E-box is necessary for the response of the promoter to cisplatin. We also found that p73 interacts with c-Myc in vitro and in vivo. Using deletion mutants we showed that the DNA-binding domain of p73 and the C-terminal region of c-Myc are required for the interaction. Furthermore, p73 stimulated the interaction of Max with c-Myc and promoted binding of the c-Myc-Max complex to its target DNA. Our data suggest that p73 stimulates the transcription of the YB-1 promoter by enhancing recruitment of the c-Myc-Max complex to the E-box.

Animals↗

PubMed2002

Analysis of methotrexate and folate transport by multidrug resistance protein 4 (ABCC4): MRP4 is a component of the methotrexate efflux system.

Human MRP4 (ABCC4, MOAT-B) is a lipophilic anion transporter that is able to confer resistance to nucleotide analogues and methotrexate (MTX). We previously investigated the implications of the ability of MRP4 to confer resistance to nucleotide analogues and determined that the pump is competent in the MgATP-energized transport of cyclic nucleotides and estradiol 17beta-D-glucuronide. Here we examine the potential role of MRP4 in conferring resistance to MTX and related processes by determining the selectivity of the transporter for MTX, MTX polyglutamates, and physiological folates. In so doing, it is shown that MRP4 is active in the transport of MTX as well as the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin). MTX, FA, and leucovorin are subject to high capacity [V(max(MTX)), 0.24 +/- 0.05 nmol/mg/min; V(max (FA)), 0.68 +/- 0.14 nmol/mg/min; V(max(leucovorin)), 1.95 +/- 0.18 nmol/mg/min], low affinity [K(m(MTX)), 0.22 +/- 0.01 mM; K(m(FA)), 0.17 +/- 0.02 mM; K(m (leucovorin)), 0.64 +/- 0.23 mM] transport by MRP4. In addition, as would be expected were MRP4 a component of the MTX efflux system, its capacity to transport this agent is abrogated by the addition of a single glutamyl residue. It is also shown that glutamylation similarly affects the ability of MRP2 to transport MTX. On the basis of these transport properties, it is concluded that the efflux system for MTX includes MRP2 and MRP4, in addition to MRP1 and MRP3, and that MRP4 represents a common efflux system for both MTX and certain nucleotide analogues.

Animals↗

PubMed2002

Suppression of thymidine phosphorylase-mediated angiogenesis and tumor growth by 2-deoxy-L-ribose.

Thymidine phosphorylase (TP), an enzyme involved in the reversible conversion of thymidine to thymine, is identical to an angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF). Both TP and one of the TP-degradation products of thymidine 2-deoxy-D-ribose (dRib) display endothelial cell chemotactic activity in vitro and angiogenic activity in vivo. Recently, we demonstrated that 2-deoxy-L-ribose (lRib) could abolish the inhibitory effect of dRib on hypoxia-induced apoptosis. This suggested that lRib may be a useful inhibitor of dRib and thereby of TP functions. Therefore, we investigated the ability of lRib to inhibit the range of biological activities of TP and dRib. lRib suppressed both dRib-induced endothelial cell migration in a chemotaxis assay and endothelial tube formation induced by dRib in a collagen gel. lRib could also suppress the biological effects of TP in vivo assays of angiogenesis and tumor growth. Thus, in a corneal assay of angiogenesis, lRib inhibited angiogenesis induced by the implantation of recombinant TP. In a dorsal air sac assay of angiogenesis, lRib inhibited angiogenesis induced by the implantation of KB cells overexpressing TP (KB/TP). In a tumor growth assay, lRib treatment considerably decreased the growth rate of KB/TP cells xenografted into nude mice and also resulted in an increase in the proportion of apoptotic cells in KB/TP tumors. These findings demonstrate that TP and dRib play an important role in angiogenesis and tumor growth, and that these effects can be inhibited by lRib. Thus, lRib is a potentially useful agent for the suppression of TP-dependent angiogenesis and tumor growth.

Animals↗

PubMed2002

ZD1839 (Iressa) induces antiangiogenic effects through inhibition of epidermal growth factor receptor tyrosine kinase.

Epidermal growth factor receptor (EGFR) tyrosine kinase is a potential target for anticancer therapy. ZD1839 (Iressa) is a selective inhibitor of EGFR tyrosine kinase. In this study, we investigated the question as to whether the antitumor effect of ZD1839 is partly attributable to antiangiogenic activity and the potential mechanisms involved. Both ZD1839 and SU5416 [a vascular endothelial growth factor (VEGF)-receptor tyrosine kinase inhibitor] inhibited the migration of human umbilical vein endothelial cell cocultivated with EGF-stimulated cancer cells. ZD1839 also inhibited EGF-induced migration and the formation of tube-like structures by human microvascular endothelial cells. Moreover, ZD1839 almost completely blocked EGF-induced neovascularization of mice cornea, and SU5416 partially blocked neovascularization. In contrast, ZD1839 did not inhibit VEGF-induced angiogenesis. However, EGF-induced up-regulation of the angiogenic factors, VEGF and IL-8, was almost completely blocked by ZD1839. The antitumor effects of ZD1839 could, therefore, be mediated in part by the inhibition of tumor angiogenesis through direct effects on microvascular endothelial cells that express EGFR and also through reduced production of proangiogenic factors by tumor cells.

Angiogenesis Inhibitors↗