Discovery of orally bioavailable inhibitors of MALT1 with in vivo activity for psoriasis.

We report the design, synthesis, and biological activity of a series of compounds that exhibit potent mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) inhibition. Structural transformation of the substructures of a starting compound gave amidomethyl derivatives and sulfonylguanidine derivatives that exhibited potent inhibition of MALT1. Compound 37 had good oral bioavailability and showed anti-psoriatic activity in an imiquimod-induced psoriasis mouse model after oral administration.

Structure-activity relationship studies of 3-substituted pyrazoles as novel allosteric inhibitors of MALT1 protease.

We report the discovery of a novel series of 1,5-bisphenylpyrazoles as potent MALT1 inhibitors. Structure-activity relationship exploration of a hit compound led to a potent MALT1 inhibitor. Compound 33 showed strong activity against MALT1 (IC50: 0.49 µM), potent cellular activity (NF-κB inhibition and inhibition of IL2 production), and high selectivity against caspase-3, -8, and -9. The results of a kinetics study suggest that compound 33 is a non-competitive inhibitor of MALT1 protein.

Toll-like receptor 4 signaling promotes the migration of human melanoma cells.

Immune cell Toll-like receptors (TLRs) recognize conserved microbial components, leading to immune and inflammatory responses. However, TLRs are also expressed in cancer cells, including melanoma cells, which express TLR2-4. TLR4 ligands have received attention as immunotherapies; therefore, we assessed the expression of TLR4 in human melanoma specimens (29 primary lesions and 28 metastatic lesions) representing different types of melanoma. A high percentage (≥ 90%) of melanoma lesions expressed TLR4, as judged by immunohistochemistry. Next, the role of TLR4 in cell proliferation and migration was assessed using the TLR4-positive (TLR4(+)) melanoma cell lines 501mel and 888mel, and TLR4-negative (TLR4(‒)) 928mel melanoma cells. Lipopolysaccharide (LPS), a TLR4 agonist, increased the proliferation of TLR4(+) melanoma cells but not of TLR4(‒) 928mel cells. The proliferation-inducing effect of LPS in 888mel cells was abolished by blockade of TLR4 signaling via treatment with short interfering RNA (siRNA) targeting TLR4 or myeloid differentiation primary response gene 88 (MyD88), a molecule downstream of TLR4. However, knockdown of TLR4 or MyD88 expression did not affect the LPS-induced proliferation of 501mel cells, suggesting that residual TLR4 signaling is sufficient to maintain cell proliferation. By contrast, LPS increased the migration of TLR4(+) melanoma cells, and this effect was substantially inhibited by TLR4 or MyD88 knockdown. Furthermore, TLR4 knockdown decreased cell migration even in the absence of LPS, suggesting the presence of an endogenous TLR4 ligand(s) in melanoma cells. TLR4 signaling may contribute to melanoma progression, and caution should be exercised when using TLR4 ligands as adjuvant therapy for cancer.

A prostacyclin analog prevents the regression of renal microvascular network by inhibiting mitochondria-dependent apoptosis in the kidney of rat progressive glomerulonephritis.

We have previously demonstrated that renoprotective effects of a prostacyclin analog, beraprost sodium, on the kidney of anti-glomerular basement membrane glomerulonephritis (GN) rats. The aim of this study is to address the renoprotection mechanism of beraprost sodium, especially in the terminal stage of GN. Beraprost sodium was orally administrated from 2 to 7 weeks after induction of GN, and renal function, morphology, protein and mRNA levels were analyzed. We found the beraprost sodium treatment suppressed the structural regression of renal microvascular network and decline of renal blood flow occurred in the kidney of GN rats. To address the mechanism of the structural maintenance, we focused on apoptosis because the increased number of apoptotic renal microvascular endothelial cells and tubular epithelial cells was observed in the kidneys of GN rats as compared with normal and beraprost sodium treated rats. Protein and mRNA analyses demonstrated that mitochondria-dependent apoptotic pathway was activated in the kidneys of GN rats, and beraprost sodium suppressed the activation by modulating the expression patterns of pro- and anti-apoptotic factors. These results suggest that inhibition of mitochondria-dependent apoptosis of renal cells in GN kidney and consequent maintenance of renal functional structures, including microvascular network might contribute to the renoprotective effect of beraprost sodium in GN.

Beraprost sodium improves survival rates in anti-glomerular basement membrane glomerulonephritis and 5/6 nephrectomized chronic kidney disease rats.

Beraprost sodium, a stable prostacyclin analog, was showed to improve survival rates in two different rat models, anti-glomerular basement membrane (GBM) glomerulonephritis (GN) and 5/6 nephrectomized (Nx) chronic kidney disease (CKD) rats. In the anti-GBM rat, beraprost sodium (0.2 and 0.6 mg/kg/day) improved survival rate (hazard ratio for beraprost sodium 0.6 mg/kg/day group, 0.10; 95% confidence interval, 0.01 to 0.68). Subsequently, in the 5/6 Nx CKD rat, beraprost sodium (0.6 mg/kg/day) improved survival rate (hazard ratio for beraprost sodium, 0.46; 95% confidence interval, 0.23 to 0.92), serum creatinine doubling time and the slope of the reciprocal of serum creatinine. In the anti-GBM GN rats, beraprost sodium suppressed the serum accumulation of representative uremic toxins such as indoxyl sulfate. Furthermore, beraprost sodium inhibited human aortic endothelial cell (HAEC) injury induced by indoxyl sulfate, indicating that beraprost sodium might have a protective effect against cardiovascular damage due to CKD. These results show that beraprost sodium can improve the survival rates in two rat models of anti-GBM GN and 5/6 Nx CKD rats by protecting endothelial cells and thereby ameliorating decreased renal function. Therefore, clinical studies are needed in patients with chronic kidney failure to determine whether beraprost sodium will become a useful medication in CKD.

Immune suppression and resistance mediated by constitutive activation of Wnt/ß-catenin signaling in human melanoma cells.

Cancer-induced immunosuppression is a major problem reducing antitumor effects of immunotherapies, but its molecular mechanism has not been well understood. We evaluated immunosuppressive roles of activated Wnt/ß-catenin pathways in human melanoma for dendritic cells (DCs) and CTLs. IL-10 expression was associated with ß-catenin accumulation in human melanoma cell lines and tissues and was induced by direct ß-catenin/TCF binding to the IL-10 promoter. Culture supernatants from ß-catenin-accumulated melanoma have activities to impair DC maturation and to induce possible regulatory DCs. Those immunosuppressive culture supernatant activities were reduced by knocking down ß-catenin in melanoma cells, partly owing to downregulation of IL-10. Murine splenic and tumor-infiltrating DCs obtained from nude mice implanted with human mutant ß-catenin-overexpressed melanoma cells had less ability to activate T cells than did DCs from mice with control melanoma cells, showing in vivo suppression of DCs by activated Wnt/ß-catenin signaling in human melanoma. This in vivo DC suppression was restored by the administration of a ß-catenin inhibitor, PKF115-584. ß-catenin-overexpressed melanoma inhibited IFN-γ production by melanoma-specific CTLs in an IL-10-independent manner and is more resistant to CTL lysis in vitro and in vivo. These results indicate that Wnt/ß-catenin pathways in human melanoma may be involved in immunosuppression and immunoresistance in both induction and effector phases of antitumor immunoresponses partly through IL-10 production, and they may be attractive targets for restoring immunocompetence in patients with Wnt/ß-catenin-activated melanoma.

Effect of RNA interference of BID and BAX mRNAs on apoptosis in granulosa cell-derived KGN cells.

In mitochondrion-dependent type II apoptosis, BH3-interacting domain death agonist (BID) and BCL-2-associated X protein (BAX) promote death ligand and receptor-mediated cell death. In porcine ovaries, the levels of BID and BAX increase in follicular granulosa cells during atresia. In the present study, to confirm the pro-apoptotic activity of BID and BAX in granulosa cells, we examined the effect of RNA interference of BID or BAX on apoptosis using a human ovarian granulosa tumor cell line, KGN. By reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, expression of BID and BAX was detected in KGN cells. Then, we suppressed BID and BAX mRNA expression in KGN cells using small interfering RNA (siRNA). When BID or BAX was suppressed, a significant decrease in the apoptotic cell rate was noted. In granulosa-derived cells, BID and BAX showed pro-apoptotic activity. These results suggest that BID and BAX act as signal-transducing factors in mitochondrion-dependent type II apoptosis.

Role of cell-death ligand-receptor system of granulosa cells in selective follicular atresia in porcine ovary.

In the mammalian ovary, more than 99% of follicles degenerate without ovulation and few oocytes ovulate and succeed to the next generation. Granulosa cell apoptosis plays a critical role in this process, follicular atresia. However, the molecular mechanisms responsible for the regulation of granulosa cell apoptosis have not been clarified. Death ligand and receptor systems are major apoptosis-inducing factors. This review describes the granulosa cell apoptosis via death ligand and receptor systems during follicular atresia in the porcine ovary.

Characteristic distribution of melanin columns in the cornified layer of acquired acral nevus: an important clue for histopathologic differentiation from early acral melanoma.

Clinical and histopathologic differentiation between early acral melanoma and acral nevus is often difficult. Dermoscopy is helpful in this differentiation. On dermoscopy, early acral melanoma shows the parallel ridge pattern showing band-like pigmentation on the ridges of the surface skin markings, whereas a representative dermoscopic pattern in acquired acral nevus is the parallel furrow pattern showing parallel linear pigmentation along the surface furrows. The parallel furrow pattern suggests that melanocytes of acral nevus preferentially proliferate in the crista profunda limitans, an epidermal rete ridge underlying the surface furrow. In the present study, however, we found that in 13 of 18 acquired acral nevi, proliferation of melanocytes were detected not only in the crista profunda limitans but also in the crista profunda intermedia (CPI), an epidermal rete ridge underlying the surface ridge. Very interestingly, Fontana-Masson staining of these acral nevi revealed that even when proliferation of melanocytes was prominent in the CPI, melanin granules in the cornified layer were observed as regular melanin columns situated under the surface furrows and were hardly detected under the surface ridges. These findings indicate that in acral nevus, melanin granules produced by melanocytes in the CPI are not transferred to the upper epidermis. Hence, we must be careful not to overdiagnose an acral melanocytic lesion in which an increased number of melanocytes are detected in the CPI. Even in such a case, if melanin granules in the cornified layer are detected as melanin columns regularly distributed under the surface furrows, the lesion is strongly suggested to be a benign acral nevus.

Bid and Bax are involved in granulosa cell apoptosis during follicular atresia in porcine ovaries.

More than 99% of follicles undergo "atresia" during follicular development and growth. Follicular atresia is predominantly regulated by granulosa cell apoptosis. However, the intracellular signaling pathway of apoptosis in granulosa cells has not been revealed. In the present study, we examined changes in the expression of BH3-interacting domain death agonist (Bid) and Bcl-2-associated X protein (Bax), which are considered to promote the cell death ligand/receptor-mediated process in mitochondrion-dependent type II apoptosis, in porcine granulosa cells during atresia. Levels of mRNA and protein of Bid and Bax were determined by the reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting techniques, respectively. Levels of Bid and Bax mRNA and protein were markedly increased in granulosa cells of early atretic follicles compared with those of healthy follicles. In situ hybridization and immunohistochemical staining revealed that mRNA and protein of Bid and Bax were present in the granulosa cells, though only traces were found in healthy follicles; however, strong staining was noted in atretic follicles. These results indicate that Bid and Bax appear to be signal transduction factors in granulosa cells during follicular atresia and appear to play proapoptotic roles and confirm that the porcine granulosa cell is a mitochondrion-dependent type II apoptotic cell.

Expression and function of apoptosis initiator FOXO3 in granulosa cells during follicular atresia in pig ovaries.

In mammalian ovaries, most follicles are lost by atresia before ovulation. It has become apparent that the apoptosis of granulosa cells induces follicular atresia. Forkhead box O3 (FOXO3), also called FKHRL1 (forkhead in rhabdomyosarcoma-like 1), is a proapoptotic molecule that belongs to the FOXO subfamily of forkhead transcription factors. Foxo3-deficient female mice were reported to be infertile because of abnormal ovarian follicular development, but the precise influences of FOXO3 on follicular atresia of mature ovary have not been determined. Therefore, we examined the expression and function of FOXO3 in porcine ovarian follicles and granulosa-derived cells. FOXO3 mRNA levels in granulosa cells of porcine ovaries increased during atresia, while FOXO3 protein was abundant in granulosa cells of early atretic follicles. By immunohistochemistry, the inner surface area of the granulosa layer in early atretic follicles was strongly stained with anti-FOXO3 antibody. The granulosa cells expressing FOXO3 coincided with apoptotic cells, indicating a role of FOXO3 as a proapoptotic factor in granulosa cells of porcine ovaries. In porcine (JC-410) and human (KGN) granulosa-derived cells, cell death was induced by transfection of FOXO3 expression vectors. Expression of the proapoptotic factors Fas ligand (FASLG) and BCL2-like 11 (BCL2L11) was upregulated by FOXO3 in KGN cells. In conclusion, FOXO3 is expressed in porcine ovarian follicles and induces apoptosis in granulosa cells, suggesting that it is a candidate for the initiator of follicular atresia.

Aberrant fatty acid-binding protein-7 gene expression in cutaneous malignant melanoma.

Fatty acid-binding protein-7 (FABP7) has been shown to be expressed in cutaneous melanoma; however, its role in tumor progression is unclear. Expression of FABP7 was assessed during melanoma progression through assessment of various clinicopathology stages of primary tumor progression and metastasis. FABP7 mRNA was highly expressed in 60 of 87 (69%) primary melanomas, compared with significant (P<0.0001) reduction in 13 of 68 (19%) metastatic melanomas. Analysis of 37 paired primary and metastatic melanomas by immunohistochemistry with anti-FABP7 Ab showed 73 and 27% positivity, respectively (P<0.001). FABP7 detection of metastatic tissues was inversely correlated with relapse-free (P<0.0001) and overall (P<0.0001) survival. To examine FABP7 expression loss in advanced melanomas, loss of heterozygosity (LOH) was assessed using microsatellite markers encompassing the FABP7 gene. LOH was identified in 10 of 20 (50%) metastatic melanomas at 6q22.31, compared with 0 of 14 primary melanomas (P=0.0017). FABP7 as a surrogate biomarker for circulating tumor cells (CTCs) in the blood was assessed by quantitative real-time (qRT)-PCR from melanoma patients' blood (n=134). Assessment of patients' blood showed that FABP7(+) CTC decreased with disease progression. FABP7 may function as a tumor progression gene and can be used as a potential diagnostic biomarker of early-stage melanoma systemic spreading in blood.

Polyclonality of BRAF mutations in acquired melanocytic nevi.

Melanocytic nevi are thought to be senescent clones of melanocytes that have acquired an oncogenic BRAF mutation. BRAF mutation is considered to be a crucial step in the initiation of melanocyte transformation. However, using immunomagnetic separation or laser-capture microdissection, we examined BRAF mutations in sets of approximately 50 single cells isolated from acquired melanocytic nevi from 13 patients and found a substantial number of nevus cells that contained wild-type BRAF mixed with nevus cells that contained BRAF(V600E). Furthermore, we simultaneously amplified BRAF exon 15 and a neighboring single nucleotide polymorphism (SNP), rs7801086, from nevus cell samples obtained from four patients who were heterozygous for this SNP. Subcloning and sequencing of the polymerase chain reaction products showed that both SNP alleles harbored the BRAF(V600E) mutation, indicating that the same BRAF(V600E) mutation originated from different cells. The polyclonality of BRAF mutations in acquired melanocytic nevi suggests that mutation of BRAF may not be an initial event in melanocyte transformation.

Cancer Cells Expressing Toll-like Receptors and the Tumor Microenvironment.

Toll-like receptors (TLRs) play a crucial role in the innate immune response and the subsequent induction of adaptive immune responses against microbial infection or tissue injury. Recent findings show that functional TLRs are expressed not only on immune cells but also on cancer cells. TLRs play an active role in carcinogenesis and tumor progression during chronic inflammation that involves the tumor microenvironment. Damage-associated molecular patterns (DAMPs) derived from injured normal epithelial cells and necrotic cancer cells appear to be present at significant levels in the tumor microenvironment, and their stimulation of specific TLRs can foster chronic inflammation. This review discusses how carcinogenesis, cancer progression, and site-specific metastasis are related to interactions between cancer cells, immune cells, and DAMPs through TLR activation in the tumor microenvironment.

mRNA expression and BRAF mutation in circulating melanoma cells isolated from peripheral blood with high molecular weight melanoma-associated antigen-specific monoclonal antibody beads.

BACKGROUND: The detection of circulating tumor cells (CTCs) in the peripheral blood of melanoma patients by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis correlates with a poor prognosis. The assessment of CTCs from blood has been difficult because of lack of a good monoclonal antibody (mAb) directed against surface cell antigens to capture melanoma cells. METHODS: Blood was collected prospectively from 57 melanoma patients (43 test and 14 test-development cases) and 5 healthy donors. High molecular weight melanoma-associated antigen (HMW-MAA)-specific mAbs bound to immunomagnetic beads were used to isolate CTCs. mRNA and/or DNA were extracted from CTCs. Testing for the expression of a melanoma-associated gene panel (MLANA, MAGEA3, and MITF) with qRT-PCR and for the presence of BRAFmt (a BRAF gene variant encoding the V600E mutant protein) verified the beads-isolated CTCs to be melanoma cells. A peptide nucleic acid-clamping PCR assay was used for BRAFmt analysis. RESULTS: Spiking of peripheral blood cells (PBCs) with melanoma cells showed that the beads-based detection assay can detect approximately 1 melanoma cell in 5 x 10(6) PBCs. qRT-PCR analysis detected MLANA, MAGEA3, and MITF expression in 19 (44%), 29 (67%), and 19 (44%) of the patients, respectively. At least one biomarker of the panel was positive in 40 (93%) of the 43 melanoma patients. BRAFmt was detected in 17 (81%) of the 21 assessed stage IV melanoma patients. CONCLUSION: The assay of bead capture coupled with the PCR has utility for assessing CTCs in melanoma patients, which can then be characterized for both genomic and transcriptome expression.