Chloroquine Promotes the Anticancer Effect of TACE in a Rabbit VX2 Liver Tumor Model

Background: To investigate the efficacy of TACE combined with CQ, an autophagic inhibitor, in a rabbit VX2 liver tumor model.Methods: Tumor size was measured. And tumor growth rate was calculated to examine the effect of the combined treatment. Apoptosis was detected by TUNEL assay. Meanwhile, autophagic activity was detected by immunohistochemistry and Western blotting to investigate the mechanism underlying. Liver function was also examined to assess feasibility and safety of the combined therapy.Results: Tumors in the control grew more than 4 times bigger after 14 days, while that in the group of TACE alone just showed mild growth. But a slight shrinkage was shown after the treatment of CQ+TACE. Growth ratio of TACE alone was 96.45% ± 28.958% while that of CQ+TACE was -28.73% ± 12.265%. Compared with TACE alone, necrosis in CQ+TACE showed no significant difference, however, the apoptosis was much higher. There were only 14.8±3.11% apoptotic cells in TACE, but 33±4.18% in CQ+TACE, which suggests the increased apoptosis in CQ+TACE contributed to the decrease of tumor volume. In terms of autophagic activity, the result is negative when we immunostained sections of the control with LC3 antibody, but positive in TACE alone and CQ+TACE. And the result of Western blot showed that there was just a low level of LC3Ⅱexpressed in the control and CQ alone, but higher in TACE, and much higher in CQ+TACE because CQ inhibited its degradation in autophagy. Compared with control, p62 decreased in TACE, but the decrease was partially reversed in CQ+TACE. In addition, toxicity of CQ+TACE was assessed not higher than TACE alone, which supports the safety of CQ+TACE.Conclusion: CQ+TACE works better than TACE alone in rabbit VX2 liver tumor model because CQ inhibits autophagy induced by TACE. The inhibited autophagy loses its resistance to apoptosis that apoptosis increased, which contributes to the inhibition of tumor growth. This study indicates CQ may be a promising adjuvant to promote the effect of TACE.     

Activation of tumor necrosis factor-alpha-converting enzyme-mediated ectodomain shedding by nitric oxide

Ectodomain shedding of cell surface proteins is an important process in a wide variety of physiological and developmental events. Recently, tumor necrosis factor-alpha-converting enzyme (TACE) has been found to play an essential role in the shedding of several critical surface proteins, which is evidenced by multiple developmental defects exhibited by TACE knockout mice. However, little is known about the physiological activation of TACE. Here, we show that nitric oxide (NO) activates TACE-mediated ectodomain shedding. Using an in vitro model of TACE activation, we show that NO activates TACE by nitrosation of the inhibitory motif of the TACE prodomain. Thus, NO production activates the release of cytokines, cytokine receptors, and adhesion molecules, and NO may be involved in other ectodomain shedding processes.     

Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain

Tumor necrosis factor-alpha-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC(50) of 70 nm. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC(50) greater that 2 microm). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys(184) residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE.     

Reduction in Non-Protein Respiratory Quotient Is Related to Overall Survival after Hepatocellular Carcinoma Treatment

Background

Transcatheter arterial chemoembolization (TACE) is an effective treatment for hepatocellular carcinoma (HCC) that can occasionally lead to the shortening of life expectancy. We aimed to make a new and more accurate prognostic model taking into account the course of disease after TACE.

Methodology/Principal Findings

We performed a prospective cohort study involving 100 HCC patients who underwent TACE at Kobe University Hospital. Indirect calorimetry and blood biochemical examinations were performed before and 7 days after TACE. Time-dependent and time-fixed factors associated with 1-year mortality after TACE were assessed by multivariate analyses. A predictive model of 1-year mortality was established by the combination of odds ratios of these factors. Multivariate analyses showed that the ratio of non-protein respiratory quotient (npRQ) (7 days after/before TACE) and Cancer of Liver Italian Program (CLIP) score were independent factors of 1-year mortality after TACE (p = 0.014 and 0.013, respectively). Patient-specific 1-year mortality risk scores can be calculated by summarizing the individual risk scores and looking up the patient-specific risk on the graph.

Conclusions

The short-term reduction of npRQ was a time-dependent prognostic factor associated with overall survival in HCC patients undergoing TACE. CLIP score was a time-fixed prognostic factor associated with overall survival. Using the prediction model, which consists of the combination of time-dependent (npRQ ratio) and time-fixed (CLIP score) prognostic factors, 1-year mortality risk after TACE would be better estimated by taking into account changes during the course of disease.     

Involvement of TNF-α Converting Enzyme in the Development of Psoriasis-Like Lesions in a Mouse Model

TNF-α plays a crucial role in psoriasis; therefore, TNF inhibition has become a gold standard for the treatment of psoriasis. TNF-α is processed from a membrane-bound form by TNF-α converting enzyme (TACE) to soluble form, which exerts a number of biological activities. EGF receptor (EGFR) ligands, including heparin-binding EGF-like growth factor (HB-EGF), amphiregulin and transforming growth factor (TGF)-α are also TACE substrates and are psoriasis-associated growth factors. Vascular endothelial growth factor (VEGF), one of the downstream molecules of EGFR and TNF signaling, plays a key role in angiogenesis for developing psoriasis. In the present study, to assess the possible role of TACE in the pathogenesis of psoriasis, we investigated the involvement of TACE in TPA-induced psoriasis-like lesions in K5.Stat3C mice, which represent a mouse model of psoriasis. In this mouse model, TNF-α, amphiregulin, HB-EGF and TGF-α were significantly up-regulated in the skin lesions, similar to human psoriasis. Treatment of K5.Stat3C mice with TNF-α or EGFR inhibitors attenuated the skin lesions, suggesting the roles of TACE substrates in psoriasis. Furthermore, the skin lesions of K5.Stat3C mice showed down-regulation of tissue inhibitor of metalloproteinase-3, an endogenous inhibitor of TACE, and an increase in soluble TNF-α. A TACE inhibitor abrogated EGFR ligand-dependent keratinocyte proliferation and VEGF production in vitro, suggesting that TACE was involved in both epidermal hyperplasia and angiogenesis during psoriasis development. These results strongly suggest that TACE contributes to the development of psoriatic lesions through releasing two kinds of psoriasis mediators, TNF-α and EGFR ligands. Therefore, TACE could be a potential therapeutic target for the treatment of psoriasis.     

Acetylenic TACE inhibitors. Part 3: Thiomorpholine sulfonamide hydroxamates

A series of thiomorpholine sulfonamide hydroxamate TACE inhibitors, all bearing propargylic ether P1' groups, was explored. In particular, compound 5h has excellent in vitro potency against isolated TACE enzyme and in cells, oral activity in a model of TNF-alpha production and a collagen-induced arthritis model, was selected as a clinical candidate for the treatment of RA.     

Acetylenic TACE inhibitors. Part 1. SAR of the acyclic sulfonamide hydroxamates

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors, all bearing a butynyloxy P1' group, was explored. In particular, compound 5j has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and MMP-9, and oral activity in an in vivo model of TNF-alpha production and a collagen-induced arthritis model.     

Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch

TNF-alpha-converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. For example, the TACE-mediated cleavage of the type II 75-kDa TNF receptor (TNFR2) generates a soluble TNF-binding protein that modulates TNF bioactivity. TACE is synthesized as a latent proenzyme that is retained in an inactive state via an interaction between its prodomain and catalytic domain. Although the formation of an intramolecular bond between a cysteine in the prodomain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine-switch motif is not required. Here, we hypothesized that the amino terminus of the TACE prodomain might contribute to the ability of the prodomain to maintain TACE in an inactive state independently of a cysteine-switch mechanism. We synthesized a 37-amino acid peptide corresponding to TACE amino acids 18-54 (N-TACE(18-54)) and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE(18-54) attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Furthermore, N-TACE(18-54) inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30-48, demonstrated partial inhibitory activity. In summary, we have identified a subdomain within the amino terminus of the TACE prodomain that attenuates TACE catalytic activity independently of a cysteine-switch mechanism, which provides new insight into the regulation of TACE enzymatic activity.     

Hydantoins, triazolones, and imidazolones as selective non-hydroxamate inhibitors of tumor necrosis factor-alpha converting enzyme (TACE)

We have discovered selective and potent inhibitors of TACE that replace the common hydroxamate zinc binding group with a hydantoin, triazolone, and imidazolone heterocycle. These novel heterocyclic inhibitors of a zinc metalloprotease were designed using a pharmacophore model that we previously described while developing hydantoin and pyrimidinetrione (barbiturate) inhibitors of TACE. The potency and binding orientation of these inhibitors is discussed and they are modeled into the X-ray crystal structure of TACE and compared to hydroxamate and earlier hydantoin TACE inhibitors which share the same 4-[(2-methyl-4-quinolinyl)methoxy]benzoyl P1' group.     

Anthranilate derivatives as TACE inhibitors: Docking based CoMFA and CoMSIA analyses

Anthranilic acid based derivatives (ANTs) have been identified as a novel class of potent tumor necrosis factor-α converting enzyme (TACE) inhibitors. A computational strategy based on molecular docking studies, followed by CoMFA and CoMSIA analyses has been performed to elucidate the atomic details of the TACE/ANT interactions and also to identify the most important features impacting TACE inhibitory activity of ANTs. The CoMSIA model resulted to be slightly more predictive than CoMFA model, and gave conventional r² 0.991, r_cv² 0.793, q² 0.777, SEE 0.050, F-value 655.610, and r_test² 0.871. The 3D-QSAR field contributions and the structural features of the TACE binding site showed a good correlation. These studies will be useful to design new TACE inhibitors with improved potency.     

Tumor necrosis factor-alpha converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability

We previously implicated tumor necrosis factor-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming growth factor-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary keratinocytes, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal growth factor (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-2) were born with open eyes significantly more often than Tace(+/+)Egfr(wa-2)(/)(wa-2) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.     

Structure-based design of TACE selective inhibitors: manipulations in the S1'-S3' pocket

A series of beta-sulfonyl hydroxamate TACE inhibitors, bearing a butynylamino or a butynyloxy P1' group, was designed and synthesized. Of the compounds investigated, 22 has excellent potency against isolated TACE enzyme, shows good selectivity over MMP-2 and MMP-13, and oral activity in an in vivo mouse model of TNF-alpha production.     

Multiple metalloproteinases process protransforming growth factor-alpha (proTGF-alpha)

Shedding of TNF-alpha requires a single cleavage event, whereas the ectodomain of proTGF-alpha is cleaved at N-proximal (N-terminal) and membrane proximal (C-terminal) sites to release mature TGF-alpha. Tumor necrosis factor-alpha converting enzyme (TACE) was shown to have a central role in the shedding of both factors. Here we show that cleavage of the proTGF-alpha C-terminal site, required for release of mature growth factor, is less sensitive to a panel of hydroxamates than TNF-alpha processing. Recombinant TACE cleaves TNF-alpha and N-terminal TGF-alpha peptides 50-fold more efficiently than the C-terminal TGF-alpha peptide. Moreover, fractionation of rat liver epithelial cell membranes yields two populations: one contains TACE and cleaves peptides corresponding to TNF-alpha and both proTGF-alpha processing sites, while the other lacks detectable TACE and cleaves only the C-terminal proTGF-alpha processing site. Activities in both fractions are inhibited by hydroxamates and EDTA but not by cysteine, aspartate, or serine protease inhibitors. Both membrane fractions also contain ADAM 10. ADAM 10 correctly cleaves peptides and a soluble form of precursor TGF-alpha (proTGFecto) at the N-terminal site but not the C-terminal site. However, the kinetics of N-terminal peptide cleavage by ADAM 10 are 90-fold less efficient than TACE. Our findings indicate that while TACE is an efficient proTGF-alpha N-terminal convertase, a different activity, distinguishable from TACE, exists that can process proTGF-alpha at the C-terminal site. A model that accounts for these findings and the requirement for TACE in TGF-alpha shedding is proposed.     

Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis

Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). TNF-alpha plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-alpha levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-alpha and NOx- levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-alpha. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-alpha release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-alpha release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.     

Anthranilate sulfonamide hydroxamate TACE inhibitors. Part 2: SAR of the acetylenic P1' group

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups was explored. In particular, compound 4t bearing a butynyloxy P1' moiety has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and oral activity in an in vivo model of TNF-alpha production.     

ShRNA knock‐down of CXCR7 inhibits tumour invasion and metastasis in hepatocellular carcinoma after transcatheter arterial chemoembolization

To investigate the effects of lentiviral vector‐mediated shRNA suppressing CXCR7 on tumour invasion and metastasis in hepatocellular carcinoma (HCC) after transcatheter arterial chemoembolization (TACE). HCCLM3 cell lines were cultured and assigned into the CXCR7‐shRNA, negative control (NC) and blank groups. The qRT‐PCR and Western blotting were applied to detect the mRNA and protein expressions of CXCR7, CXCR4 and MMP‐2 in HCCLM3 cells. Cell proliferation and invasion were evaluated by MTT and Transwell assays. A Buffalo rat model of HCC was established. Fifty model rats were divided into the CXCR7‐shRNA + TACE, CXCR7‐shRNA, TACE, NC and control groups. Immunohistochemistry was performed to detect the expressions of CXCR7, MMP‐2, vascular endothelial growth factor (VEGF) and intratumoral CD31‐positive vessel count in tumour tissues of mice. Compared with the blank and NC groups, the mRNA and protein expressions of CXCR7 and MMP‐2 were decreased in the CXCR7‐shRNA group. The cell proliferation and invasion rates of the CXCR7‐shRNA group were lower than the blank and NC groups. At the 4th week after TACE, tumour weight of the CXCR7‐shRNA + TACE group increased continuously. The CXCR7‐shRNA + TACE group showed longer survival time and smaller tumour sizes than other groups. Compared with other groups, the CXCR7‐shRNA + TACE and CXCR7‐shRNA groups had less number of lung metastatic nodules and lower expressions of CXCR7, MMP‐2, VEGF and CD31‐positive vessel count. CXCR7‐shRNA inhibits tumour invasion and metastasis to improve the efficacy of TACE in HCC by reducing the expressions of CXCR7, MMP‐2 and VEGF.     

肝动脉导管化疗栓塞序贯经皮微波消融治疗原发性肝癌的临床应用

目的:探讨肝动脉导管化疗栓塞(TACE)序贯B超/CT精准引导下经皮微波消融(MWA)在原发性肝癌中的治疗应用,分析比较疗效。方法:回顾性分析2016年1月至2018年7月在上海交通大学附属第一人民医院接受治疗的96例原发性肝癌患者,42例行TACE序贯联合B超/CT精准引导下MWA治疗(联合组),另54例仅行单纯TACE治疗(TACE组)。术后1月、3月、6月、1年、2年复查增强CT/MRI、AFP、肝功能,随访2年比较两组患者肿瘤坏死、复发、进展和生存情况,评价两组疗效。结果:联合组肿瘤坏死率92.9%,TACE组肿瘤坏死率48.1%,差异有统计学意义(P0.05);联合组肿瘤复发率7.1%,TACE组肿瘤复发率24.1%,差异有统计学意义(P0.05);联合组肿瘤进展率19.1%,TACE组肿瘤进展率27.8%,差异无统计学意义(P0.05);联合组肿瘤进展时间13.2个月,TACE组肿瘤进展时间7.6个月,差异有统计学差异(P0.05);联合组1年生存率83.3%,TACE组1年生存率57.4%,差异有统计学意义(P0.05);联合组2年生存率62%,TACE组2年生存率31.5%,差异有统计学意义(P0.05);联合组中位生存时间28.9个月,TACE组中位生存时间16.9个月,差异有统计学意义(P0.05)。结论:TACE序贯MWA治疗肝癌安全有效,互补增益,是肝癌综合治疗的新模式。     

Mapping and characterization of the functional epitopes of tissue inhibitor of metalloproteinases (TIMP)-3 using TIMP-1 as the scaffold: a new frontier in TIMP engineering

Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) is responsible for the release of TNF-alpha, a potent proinflammatory cytokine associated with many chronic debilitating diseases such as rheumatoid arthritis. Among the four variants of mammalian tissue inhibitor of metalloproteinases (TIMP-1 to -4), TACE is specifically inhibited by TIMP-3. We set out to delineate the basis for this specificity by examining the solvent accessibility of every epitope on the surface of a model of the truncated N-terminal domain form of TIMP-3 (N-TIMP-3) in a hypothetical complex with the crystal structure of TACE. The epitopes suspected of interacting with TACE were systematically transplanted onto N-TIMP-1. We succeeded in transforming N-TIMP-1 into an active inhibitor for TACE (K(i)(app) 15 nM) with the incorporation of Ser4, Leu67, Arg84, and the TIMP-3 AB-loop. The combined effects of these epitopes are additive. Unexpectedly, introduction of "super-N-TIMP-3" epitopes, defined in our previous work, only impaired the affinity of N-TIMP-1 for TACE. Our mutagenesis results indicate that TIMP-3-TACE interaction is a delicate process that requires highly refined surface topography and flexibility from both parties. Most importantly, our findings confirm that the individual characteristics of TIMP could be transplanted from one variant to another.     

Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE.