Comparison of clinical outcomes of osimertinib and first-generation EGFR-tyrosine kinase inhibitors (TKIs) in TKI-untreated EGFR-mutated non-small-cell lung cancer with leptomeningeal metastases.

BACKGROUND: Leptomeningeal metastases (LM) are devastating complications of epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC). Although osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (TKI), has better penetration into the central nervous system than first-generation EGFR-TKIs, data on the distinct activity of EGFR-TKIs in untreated advanced EGFR-mutated NSCLC with LM are lacking. PATIENTS AND METHODS: We retrospectively reviewed patients treated with EGFR-TKIs for TKI-untreated common EGFR-mutated NSCLC with LM between July 2002 and July 2021 at the National Cancer Center Hospital. The patients were divided into two groups: patients treated with osimertinib (Osi group) and those treated with gefitinib or erlotinib [first-generation (1G)-TKI group]. RESULTS: Of the 967 patients, 71 were eligible, including 29 in the Osi group and 42 in the 1G-TKI group. The median progression-free survival (PFS) and overall survival (OS) in the Osi group were better than those in the 1G-TKI group (PFS: 16.9 months versus 8.6 months, P = 0.007, and OS: 26.6 months versus 20.0 months, P = 0.158). The LM-overall response rate (ORR) and LM-PFS were significantly better in the Osi group than in the 1G-TKI group (LM-ORR: 62.5% versus 25.7%, P = 0.007; LM-PFS: 23.4 months versus 12.1 months, P = 0.021). In the subgroup analysis of EGFR mutation status, LM-PFS for patients with exon 19 deletion was significantly longer in the Osi group than in the 1G-TKI group (32.7 months versus 13.4 months, P = 0.013), whereas those with L858R mutation in exon 21 did not differ between the two groups. In the multivariate analysis, osimertinib and exon 19 deletion were significant factors for better LM-PFS and OS. CONCLUSION: Osimertinib can be more effective for untreated common EGFR-mutated NSCLC patients with LM, especially those with exon 19 deletion, compared to first-generation TKIs.

NF-kB decoy oligodeoxynucleotide preserves disc height in a rabbit anular-puncture model and reduces pain induction in a rat xenograft-radiculopathy model.

While it is known that the degenerated intervertebral disc (IVD) is one of the primary reasons for low-back pain and subsequent need for medical care, there are currently no established effective methods for direct treatment. Nuclear factor-κB (NF-κB) is a transcription factor that regulates various genes' expression, among which are inflammatory cytokines, in many tissues including the IVD. NF-κB decoy is an oligodeoxynucleotide containing the NF-κB binding site that entraps NF-κB subunits, resulting in suppression of NF-κB activity. In the present preclinical study, NF-κB decoy was injected into degenerated IVDs using the rabbit anular-puncture model. In terms of distribution, NF-κB decoy persisted in the IVDs up to at least 4 weeks after injection. The remaining amount of NF-κB decoy indicated that it fit a double-exponential-decay equation. Investigation of puncture-caused degeneration of IVDs showed that NF-κB decoy injection recovered, dose-dependently, the reduced disc height that was associated with reparative cell cloning and morphological changes, as assessed through histology. Gene expression, by quantitative real-time polymerase chain reaction (qRT-PCR), showed that NF-κB decoy attenuated inflammatory gene expression, such as that of interleukin-1 and tumor necrosis factor-α, in rabbit degenerated IVDs. NF-κB decoy also reduced the pain response as seen using the "pain sensor" nude rat xenograft-radiculopathy model. This is the first report demonstrating that NF-κB decoy suppresses the inflammatory response in degenerated IVDs and restores IVD disc height loss. Therefore, the intradiscal injection of NF-κB decoy may have the potential as an effective therapeutic strategy for discogenic pain associated with degenerated IVDs.

Dexamethasone Prolongs Cardiac Allograft Survival in a Murine Model Through Myeloid-derived Suppressor Cells.

BACKGROUND: Recently, myeloid-derived suppressor cells (MDSCs) have attracted considerable attention because of their cancer-promoting and immunosuppressive effects. The glucocorticoid dexamethasone (Dex) is an important immunosuppressive agent used to treat autoimmune diseases and organ transplant rejection. However, the mechanism by which it modulates the immune system is not completely understood. MATERIAL AND METHODS: In this study, we investigated the mechanisms by which Dex modulated the immune response in mice given an allogeneic cardiac transplant. RESULTS: Dex injection significantly prolonged heart graft survival compared with phosphate-buffered saline-injected controls. Dex treatment increased the number of splenic MDSCs. Moreover, Gr-1high/CD11b+ MDSCs and CD3+/CD4+/Foxp3+ regulatory T cells (Tregs) were significantly increased in the Dex group compared with controls. Administration of anti-Gr-1 antibody (Ab) to the Dex group significantly shortened mouse heart graft survival. In addition, anti-Gr-1 Ab treatment significantly reduced Tregs in the Dex + anti-Gr-1 co-treatment group compared with the Dex group. These observations suggest that Dex treatment increased both MDSCs and Tregs, and that MDSCs regulated the incidence of Tregs in this immunosuppressive pathway. CONCLUSION: An important role of Dex in the prevention of the rejection of cardiac grafts in mice is to expand MDSCs and Tregs.

Living Donor Kidney Transplantation After Brachytherapy for Prostate Cancer: Case Report.

INTRODUCTION: There is no obvious criterion about kidney transplantation for patients with pretransplant malignancy. Minimum tumor-free waiting periods differ according to type of cancer, staging, site of occurrence, response to therapy, and risk of cancer recurrence. We report a case of living donor kidney transplantation (LDKT) in a patient after brachytherapy for prostate cancer. CASE REPORT: The patient was a 65-year-old man with chronic kidney disease due to chronic glomerular nephritis. He received hemodialysis 3 times a week. His prostate-specific antigen level (PSA) was high (6.57 ng/mL), and he was diagnosed with prostate cancer (T1cN0M0, Gleason Score 3 + 4 = 7, 3/10) by needle biopsy in urology. He was treated with maximum androgen blockade (MAB) therapy and brachytherapy in May 2014. He underwent LDKT from a spousal donor at our department in December 2015, because urologists concluded that the prostate cancer was completely cured. Immunosuppression consisted of induction with basiliximab and maintenance with tacrolimus, mizoribine, and steroids. The postoperative course was uneventful. He discharged at postoperative day 29 with a serum creatinine level of 1.30 mg/dL. Three months after LDKT, his PSA level was 0.477 ng/mL, and there was no evidence of prostate cancer recurrence. CONCLUSION: This is the first case of LDKT for patients with prostate cancer after brachytherapy in combination with MAB. There is no recurrence of prostate cancer so far; however, careful follow-up including PSA is necessary and important.

Influences of Pre-formed Donor-Specific Anti-Human Leukocyte Antigen Antibodies in Living-Donor Renal Transplantation: Results With Graft Immunocomplex Capture Fluorescence Analysis.

BACKGROUND: Advances in immunosuppressants enable organ transplantation for sensitized patients. However, influences of pre-formed donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) have not been fully understood in renal transplantation (RT). On the other hand, immunocomplex capture fluorescence analysis (ICFA) is a reliable method to detect donor-specific anti-HLA antibodies and HLA antigen complexes. Graft ICFA can detect DSA in an allograft (g-DSA). METHODS: To elucidate the consequences of pre-formed DSA, 198 patients who underwent living-donor RT were enrolled for this study (observation period: 57.8 ± 34.9 months); 187 patients in the DSA- group (excluding ABO-incompatible cases) and 11 patients in the DSA+ group. Before RT, all DSA+ patients had undergone rituximab administration and plasmapheresis. For a graft ICFA, the biopsy specimen (1 × 105 cells) was dissolved, and HLA antigens were captured by anti-HLA beads. Finally, DSA-HLA complexes were detected by means of PE-conjugated anti-human IgG antibodies and analyzed by use of a Luminex system. A ratio (sample/blank beads, mean of fluorescence intensity) was calculated: ≥1.0 was determined as positive g-DSA. RESULTS: There were no significant differences in 5-year graft survival (87.9%/100% in the DSA-/DSA+ groups, respectively). In terms of antibody-mediated rejection (AMR), within 1 month after RT, pathologically determined AMR occurred 3.2% and 63.4% in the DSA- and DSA+ groups, respectively (P < .0001). However, interestingly, more than half of them (57.1%) indicated only subclinical AMR, that is, no fluctuation of S-Cr. As representative of 2 cases of subclinical AMR, g-DSA deposition could be confirmed (1.15 ± 0.04) at 1 hour after reperfusion by graft ICFA. Furthermore, g-DSA shifted to 2.20 ± 0.98 at 3 weeks after transplantation, along with a decline in s-DSA mean of fluorescence intensity (1718-506.5). CONCLUSIONS: Although pathologically determined AMR occurred more frequently in pre-formed DSA+ recipients, it can be argued that a successful de-sensitization protocol inhibits further production of DSA and graft destruction.

Ultraconserved region-containing Transformer 2ß4 controls senescence of colon cancer cells.

Ultraconserved regions (UCRs) are >200 bp genomic segments with perfect human-to-rodent sequence identity. Transcribed UCRs constitute a new category of noncoding RNAs whose functions remain poorly understood. The human transformer 2ß (TRA2B) gene contains a 419-bp UCR spanning the 276-bp exon 2 and its neighboring introns. TRA2B exon 2 has premature stop codons, whereas an exon 2-containing splice variant (TRA2ß4) was expressed preferentially in the nuclei of human colon cancer cells. TRA2ß4 knockdown p53-independently stimulated CDKN1A transcription and increased p21, resulting in the appearance of senescent cells. Biotin pull-down and RNA immunoprecipitation assays revealed that TRA2ß4 interacted with Sp1 through a Sp1-binding sequence (485-GGGG-488) in a stem-loop structure of exon 2. Mutation of this sequence (485-AAGG-488) disrupted the stem-loop structure, blocked the interaction with Sp1 and increased CDKN1A transcription. Overexpression of TRA2ß4 significantly decreased CDKN1A mRNA levels and accelerated cell growth, but the introduction of the mutation in the Sp1-binding sequence completely canceled these effects. Taken together, TRA2ß4 may sequester Sp1 from occupying promoters of target genes including CDKN1A, promoting cell growth by interrupting the senescence-related gene expression program. This novel function of TRA2ß4 may uncover an oncogenic function of transcribed UCRs.

Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1ß, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1γ pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor.

Transformer 2ß and miR-204 regulate apoptosis through competitive binding to 3' UTR of BCL2 mRNA.

RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2ß (Tra2ß) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2ß antibody and microarray analysis identified a subset of Tra2ß-associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including Bcl-2 (B-cell CLL/lymphoma 2). Tra2ß knockdown in HCT116 cells decreased Bcl-2 expression and induced apoptosis. Tra2ß knockdown accelerated the decay of BCL2α mRNA that encodes Bcl-2 and full-length 3' UTR, while it did not affect the stability of BCL2ß mRNA having a short, alternatively spliced 3' UTR different from BCL2α 3' UTR. RIP assays with anti-Tra2ß and anti-Argonaute 2 antibodies, respectively, showed that Tra2ß bound to BCL2α 3' UTR, and that Tra2ß knockdown facilitated association of miR-204 with BCL2α 3' UTR. The consensus sequence (GAA) for Tra2ß-binding lies within the miR-204-binding site of BCL2 3' UTR. Mutation of the consensus sequence canceled the binding of Tra2ß to BCL2 3' UTR without disrupting miR-204-binding to BCL2 3' UTR. Transfection of an anti-miR-204 or introduction of three-point mutations into the miR-204-binding site increased BCL2 mRNA and Bcl-2 protein levels. Inversely, transfection of precursor miR-204 reduced their levels. Experiments with Tra2ß-silenced or overexpressed cells revealed that Tra2ß antagonized the effects of miR-204 and upregulated Bcl-2 expression. Furthermore, TRA2ß mRNA expression was significantly upregulated in 22 colon cancer tissues compared with paired normal tissues and positively correlated with BCL2 mRNA expression. Tra2ß knockdown in human lung adenocarcinoma cells (A549) increased their sensitivity to anticancer drugs. Taken together, our findings suggest that Tra2ß regulates apoptosis by modulating Bcl-2 expression through its competition with miR-204. This novel function may have a crucial role in tumor growth.

How long should we follow the post-transplantation patient after graft loss? A case report of renal cancer in the grafted kidney that occurred 16 years after graft loss.

BACKGROUND: Renal cancers commonly occur in the native kidneys of renal transplant recipients, whereas renal cancer in the grafted kidney has been reported occasionally. Renal cancer in the grafted kidney occurred 16 years after graft loss in this case, which would be a more rare case. CASE REPORT: A 60-year-old man who had a kidney transplant from his mother at the age of 31 years and had hemodialysis again because of chronic rejection from the age of 44 years had right lower abdominal pain. Computerized tomography (CT) showed tumor involvement in the grafted kidney. Positron-emission tomography-CT also showed hot spots in the liver, cervical vertebra, and costal bone. Needle biopsy for grafted kidney and liver tumors were done, and pathologic findings revealed renal cancer of grafted kidney and metastatic liver tumor. Graftectomy was done, and renal cancer was diagnosed as spindle cell carcinoma. Irradiation for cervical bone metastasis was done after the surgery. He complained of abdominal pain and eating disturbance 2 months after the surgery. CT showed a huge recurrence tumor and multiple tumor dissemination. Small intestine was involved and obstructed by the main tumor. He died of recurrence of renal cancer 3 months after the surgery. CONCLUSIONS: It is reported that the rate of renal cell carcinoma in the grafted kidney was 0.19%-0.5% and it occurred at a mean of 12.6 years after renal transplantation. Herein, we report a rare case of renal cancer that occurred 29 years after renal transplantation. Long-term observation should be required for recipients who had rehemodialysis.

Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells.

Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth.

Acute renal failure caused by hyperuremic acidemia in ABO-incompatible kidney transplant maintained with cyclosporine and high-dose mizoribine: a case report.

INTRODUCTION: The shortage of cadaver organs has led to expansion of living donor kidney transplantations with, 30% increase among ABO-incompatible cases in Japan and the use of marginal extended donors. Herein we have reported the outcome after an ABO-incompatible kidney transplantation from an aged living-related donor who suffered from mild diabetes mellitus and hypertension. CASE REPORT: A 48-year-old man underwent ABO-incompatible kidney transplantation from his 76-year-old father, using anti-CD20 antibody induction, followed by cyclosporine (CsA), mycophenolate mofetil (MMF), and prednisolone. After the operation, MMF was switched to high-dose mizoribine (MZ). He was discharged from the hospital on postoperative day (POD) 28 with a serum creatinine (sCr) of 1.47 mg/dL. On POD 34 when the sCr was 8.14 mg/dL, his urine examination showed uric acid crystals with serum uric acid of 24.6 mg/dL. Biopsy findings showed no evidence of acute rejection but mild tubulointerstitial injury. Hemodialysis performed twice to reduce uric acid was accompanied by hydration. CsA/MZ was switched to tacrolims/MMF; benzbromarone, to febuxostat to treat hyperuric acidemia. On POD 58, sCr reduced to 1.75 mg/dL he was discharged. On POD 416, graft function was stable with sCr of 1.70 mg/dL. CONCLUSION: Common side effect of MZ is hyperuricemia which presumably caused acute renal failure of this aged marginal donor kidney.

Gene expression profile of a newly established choriocarcinoma cell line, iC3-1, compared to existing choriocarcinoma cell lines and normal placenta.

Gestational choriocarcinoma is a malignant trophoblastic tumor that usually occurs in the uterus after pregnancy. The tumor is curable with advanced chemotherapy, but the molecular mechanism of choriocarcinoma tumorigenesis remains unclear. This is partly because the low incidence makes it difficult to obtain clinical samples for investigation and because an appropriate choriocarcinoma cell model to study the tumorigenesis has not been developed. We have established a new choriocarcinoma cell line, induced choriocarcinoma cell-1 (iC(3)-1), that possesses unique characteristics compared to other choriocarcinoma cell lines, including production of tumors that consist of the two types of cells commonly found in choriocarcinoma and mimicking of the clinical pathology. Existing trophoblast cell lines utilized in previous choriocarcinoma studies have had significantly dissimilar gene expression profiles. Therefore, it is important to choose an appropriate cell line for a particular study based on the characteristics of the cell line. In this study, to clarify the genetic characteristics of iC(3)-1 and to explore the tumorigenesis mechanism, we examined the gene profile of iC(3)-1 compared to those of existing cell lines and normal placental tissue. Bioinformatics analysis showed that several characteristic genes, IGF1R, CHFR, MUC3A, TAF7, PARK7, CDC123 and PSMD8, were significantly upregulated in iC(3)-1 compared to BeWo and JEG3 cells. Interestingly, HAS2, CD44 and S100P were significantly upregulated in iC(3)-1 compared to parental HTR8/SVneo cells and normal third trimester placenta. Choriocarcinoma samples also showed immunoreactivity to HAS2, CD44 and S100. In summary, the gene expression profile of iC(3)-1 suggests that studies using this cell line can make an important contribution to improved understanding of choriocarcinoma tumorigenesis.

Increased plasma LIGHT levels in patients with atopic dermatitis.

LIGHT [the name of which is derived from 'homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for herpes simplex virus entry mediator (HVEM), and expressed by T lymphocytes'], is a member of the tumour necrosis factor superfamily that is involved in various inflammatory diseases. We aimed to estimate the relevance of plasma LIGHT levels as a biomarker for atopic dermatitis (AD). In order to understand the putative role of LIGHT in AD pathogenesis, we also investigate the effects of LIGHT on a monocytic cell line, human acute monocytic leukaemia cell line (THP-1). We examined plasma LIGHT levels, total serum IgE, serum value of CCL17 and peripheral blood eosinophil counts in patients with AD and healthy subjects. The effects of LIGHT on activation and apoptosis in THP-1 cells were also investigated. The plasma concentrations of LIGHT in AD patients were significantly higher than those in healthy individuals and the concentrations decreased as the symptoms were improved by treatment. The LIGHT plasma concentrations correlated with IgE levels and the Severity Scoring of AD (SCORAD) index. In addition, LIGHT stimulation increased expression of CD86 and induced production of interleukin-1ß in THP-1 cells. Apoptosis was inhibited, the Bcl-2 level increased and the caspase-3 level decreased in THP-1 cells stimulated with LIGHT, compared to unstimulated control cells. These results suggest that plasma LIGHT levels may be one of the promising biomarkers for AD.

Potential role of autoantibody in severe neutropenia of a patient with Kawasaki Syndrome.

Neutropenia associated with Kawasaki Syndrome (KS) has been rarely reported, and the detailed mechanisms responsible for this state are not yet elucidated. The aim of this study was to clarify the mechanisms of neutropenia in KS. We examined antibodies to known neutrophil antigens (HNA1a, HNA1b, HNA null, HNA2, HNA3, HNA4 and non-HLA antigen 9a) in a KS patient with neutropenia. We also performed the granulocyte immunofluorescence test (GIFT) using patient or control neutrophils incubated with the patient's serum at serial time points over the patient's clinical course. No specific antibody to known neutrophil antigens was detected. Flow cytometric analysis showed that autoantibodies bound to immature CD13-positive myeloid cells, which resulted in myeloid lineage maturation arrest in the bone marrow. GIFT showed that neutrophil-specific autoantibodies were produced by the patient, and the amount of autoantibody inversely correlated with the patient's neutrophil counts. The presence of an autoantibody to a novel antigen on immature myeloid cells or neutrophils is the likely the cause of severe neutropenia in this patient with KS.

Transfection of NF-κB decoy oligodeoxynucleotide suppresses pulmonary metastasis by murine osteosarcoma.

Nuclear factor-kappa B (NF-κB) has a pivotal role in the progression and distant metastasis of cancers, including malignant bone tumors. To inhibit NF-κB activation, a new molecular therapy using synthetic double-stranded oligodeoxynucleotide (ODN) as a 'decoy' cis element against NF-κB has been developed. To determine whether pulmonary metastasis of osteosarcoma is reduced by inhibiting the action of NF-κB, NF-κB decoy ODN was transfected into the nuclei of murine osteosarcoma cells with high pulmonary metastatic potential, the LM8 cell line, using a three-dimensional alginate spheroid culture model. An in vitro study demonstrated the successful transfection of LM8 cells cultured in alginate beads by 'naked' NF-κB decoy ODN and that the activation of NF-κB signaling was significantly suppressed. Tumor growth was not affected by transfection of NF-κB decoy ODN, however, the expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) mRNA was markedly decreased. Furthermore, the transfection of 'naked' NF-κB decoy ODN effectively suppressed pulmonary metastasis in an in vivo alginate bead transplantation model. Our results suggest that NF-κB has a central and specific role in the regulation of tumor metastasis and could be a molecular target for development of anti-metastatic treatments for osteosarcoma.

Ursolic acid and oleanolic acid, members of pentacyclic triterpenoid acids, suppress TNF-α-induced E-selectin expression by cultured umbilical vein endothelial cells.

E-selectin is an early response adhesion molecule expressed on the surface of endothelial cells during inflammatory responses. We examined the effects of two pentacyclic triterpenoid acids, ursolic acid (UA) and oleanolic acid (OA), on the expression of E-selectin by cultured human umbilical vein endothelial cells (HUVECs). Treatment of the cells with UA or OA alone did not influence expression of E-selectin. Expression of E-selectin mRNA and surface antigen by HUVECs was induced by treatment with tumor necrosis factor-α (TNF-α) in a dose- and time-dependent manner. TNF-α-induced up-regulation of E-selectin was abrogated by pre-treatment of the cells with UA or OA which decreased expression of E-selectin mRNA. The repression of E-selectin mRNA expression caused by the pentacyclic triterpenoid acids paralleled the inhibition of NF-κB activation and nuclear translocation, as evaluated by electrophoretic mobility shift assays, although the degree of repression by UA was approximately two times more effective than that of OA. The results suggest that UA and OA suppress the inflammatory cytokine-induced expression of E-selectin in endothelial cells by decreasing E-selectin transcription via inhibition of NF-κB activation. Thus, UA and OA function as anti-inflammatory agents. The differences in the inhibitory efficacy between UA and OA may be due to conformational differences in ring-E of the two pentacyclic triterpenoid acids.

Expression levels of adiponectin receptors and periodontitis.

BACKGROUND AND OBJECTIVE: We recently showed that adiponectin, an adipocyte-derived cytokine, may function as a negative regulator of the Toll-like receptor signaling pathway and of osteoclast formation in periodontal disease. In this study, we investigated whether the expression levels of adiponectin receptors (AdipoR1 and AdipoR2) are related to the presence of periodontitis. MATERIAL AND METHODS: We initially examined, using RT-PCR, the expression of the AdipoR1 and AdipoR2 genes at the mRNA level in several oral tissues of C57BL mice. Next, we investigated (using real-time PCR assays) whether inflammatory cytokines, such as tumor necrosis factor-alpha, could affect the expression levels of these genes in human gingival fibroblasts. Lastly, we compared the expression levels of these receptor proteins in gingival tissues between two healthy subjects and five patients with severe periodontal disease using western blotting analysis. RESULTS: The AdipoR1 and AdipoR2 receptors were ubiquitously expressed in the oral tissues of mice. We observed that treatment with tumor necrosis factor-alpha could significantly reduce the expression levels of both AdipoR1 and AdipoR2 genes in human gingival fibroblasts. Moreover, we found that the expression of both receptors was lower in periodontal tissues from patients with severe periodontitis than in patients with healthy periodontal tissues. CONCLUSION: These observations suggest that adiponectin may not function efficiently in sites of periodontal disease because of a decrease in the number of its receptors, and this probable dysfunction may play a role in worsening periodontitis in patients.

Complex regulation of cell-cycle inhibitors by Fbxw7 in mouse embryonic fibroblasts.

The F-box protein Fbxw7 (also known as Fbw7, SEL-10, hCdc4 or hAgo) mediates the ubiquitylation and thereby contributes to the degradation of proteins that positively regulate cell cycle. Conditional ablation of Fbxw7 in mouse embryonic fibroblasts (MEFs) induces cell-cycle arrest accompanied by abnormal accumulation of the intracellular domain of Notch1 (NICD1) and c-Myc. However, the molecular mechanisms by which the accumulation of NICD1 and c-Myc induces cell-cycle arrest have remained unclear. We have now examined the expression of cell-cycle inhibitors in Fbxw7-deficient MEFs and found that the abundance of p27(Kip1) and p57(Kip2) is paradoxically decreased. This phenomenon appears to be attributable to the accumulation of NICD1, given that it was recapitulated by overexpression of NICD1 and blocked by ablation of RBP-J. Conversely, the expression of p16(Ink4a) and p19(ARF) was increased in an NICD1-independent manner in Fbxw7-null MEFs. The increased expression of p19(ARF) was recapitulated by overexpression of c-Myc and abolished by ablation of c-Myc, suggesting that the accumulation of c-Myc is primarily responsible for that of p19(ARF). In contrast, the upregulation of p16(Ink4a) appeared to be independent of c-Myc. These results indicate that cell-cycle inhibitors undergo complex regulation by the Fbxw7-mediated proteolytic system.