CDK5 Upregulated by ELF3 Transcription Promotes IL-1ß-induced Inflammation and Extracellular Matrix Degradation in Human Chondrocytes.

Osteoarthritis (OA) is a common chronic disease with age-associated increase in both incidence and prevalence. The cyclin-dependent kinase 5 (CDK5), which is a member of the CDK family, is involved in many chronic diseases. This study was performed to explore the functional role of CDK5 in OA and to discuss the detailed molecular mechanisms. The expressions of CDK5 and ELF3 before or after transfection were detected with reverse transcription-quantitative PCR (RT-qPCR) and western blot. 5-ethynyl-2'-deoxyuridine (Edu) and terminal deoxynucleoitidyl transferase-mediated nick-end labeling (TUNEL) assays were used to detect the proliferation and apoptosis of C28/I2 cells. The levels of inflammatory cytokines were estimated using enzyme-linked immunosorbent assay (ELISA) while the expressions of proteins implicated in extracellular matrix (ECM) degradation- and apoptosis were detected using western blot. Additionally, the activity of CDK5 promoters and its binding with ELF3 were detected using luciferase activity assay and chromatin immunoprecipitation (CHIP) assay. In the present study, it was discovered that the mRNA and protein expressions of CDK5 were significantly increased in IL-1ß-induced C28/I2 cells. After depleting CDK5 expression, the apoptosis, inflammation and ECM in C28/I2 cells with IL-1ß induction were suppressed. It was also found that ELF3 expression was increased in IL-1ß-induced C28/I2 cells and acted as a transcription factor binding to the CDK5 promoter to regulate its transcriptional expression. The further experiments evidenced that ELF3 overexpression partially reversed the inhibitory effects of CDK5 deficiency on IL-1ß-induced apoptosis, inflammation and ECM in C28/I2 cells. Collectively, CDK5 that upregulated by ELF3 transcription could promote the development of OA.

Systemic immune inflammatory index is an independent predictor for the requirement of decompressive craniectomy in large artery occlusion acute ischemic stroke patients after mechanical thrombectomy.

Background and purpose: Following mechanical thrombectomy (MT), patients with large artery occlusive acute ischemic stroke (LAO-AIS) often have cerebral herniation due to its complications, resulting in poor prognosis. Decompressive craniectomy (DC) can markedly improve patient prognosis. This study aimed to verify the predictive value of clinical parameters such as the systemic immune-inflammatory index (SII) for DC in patients with LAO-AIS after MT. Methods: Clinical data of a total of 173 patients with LAO-AIS treated with MT between January 2020 and January 2022 were retrospectively analyzed. Patients receiving DC were grouped into an experimental group or a control group (no DC). The patients were randomly divided into the training set (n = 126, 75%) and validation set (n = 43, 25%). Multivariate logistic regression was used to construct a nomogram predictive model. Results: The SII value in the experimental group (median: 2851.1×109/L) was significantly higher than that in the control group (median: 1898.6 × 109/L) (P = 0.019). Receiver operating characteristic (ROC) analyses showed that the best cutoff value of the SII was 2505.7 × 109/L with a sensitivity of 55%, a specificity of 75.8%, and an area under the curve (AUC) of 0.649. Multivariate logistic regression indicated that the SII was an independent predictor for performing DC in patients with LAO-AIS after MT (OR = 3.579, 95% CI = 1.360-9.422, P = 0.01). The AUC was 0.728 in the training set and 0.583 in the validation set. The average error of the calibration curve was 0.032 in the training set and 0.023 in the validation set. The average error was relatively small and consistent in the training set and validation set. The C-index of the nomogram was 0.804 suggesting good accuracy. Conclusions: The SII at admission is an independent predictor for the requirement of DC in patients with LAO-AIS after MT. The SII-based nomogram helps doctors make decisions on whether DC is needed timely and rationally, and thereby may improve the prognosis of these patients.

Paired study of 172 cases of nasopharyngeal carcinoma with or without dermatomyositis.

CONCLUSION: The prognosis and late adverse effects of radiotherapy (RT) in the patients with nasopharyngeal carcinoma (NPC) with or without dermatomyositis (DM) were similar, although the NPC patients with DM had higher Epstein-Barr virus (EBV) VCA-IgA titers and more severe acute side effects. Gender, TNM stage, and chemotherapy were independent prognostic factors of overall survival for NPC with DM. Glucocorticoid treatment did not affect the survival of NPC patients with DM. OBJECTIVES: We evaluated the clinical characteristics, prognosis, and differences in the toxicity of RT in patients with NPC with or without DM. METHODS: A paired study of 172 NPC cases with DM (DM group) or without DM (control group) from Sun Yat-sen University Cancer Center was conducted. RESULTS: The DM group had higher EBV VCA-IgA titers than the control group (p = 0.017) and more acute adverse effects of RT (p < 0.001). No significant differences in the overall survival or late adverse effects were found between the two groups. Gender, TNM stage, and chemotherapy were independent prognostic factors for the overall survival in the DM group. No significant differences in the overall survival were found between the patients in the DM group who were taking glucocorticoids and those who were not.

Feasibility and efficacy of chemoradiotherapy for elderly patients with locoregionally advanced nasopharyngeal carcinoma: results from a matched cohort analysis.

BACKGROUND: To clarify the feasibility and efficacy of chemoradiotherapy (CRT) in elderly (age≥65 years) patients with locoregionally advanced nasopharyngeal carcinoma (NPC). METHODS: From January 2000 to December 2006, 101 newly diagnosed elderly non-metastatic NPC patients (age≥65 years) who received cisplatin 3-weekly or weekly concurrent CRT with/without sequential chemotherapy were recruited. Each patient from the CRT group was matched to another patient treated with radiotherapy (RT) alone based on age, gender, pathological type, performance status, overall stage, stage method, Adult Comorbidity Evaluation-27 (ACE-27) score and RT technique, from the same institute and time period. We also recruited 101 young patients (age<65 years) as the referent group, which had been matched to the CRT group based on patient characteristics and treatment parameters. Treatment tolerability and toxicity were clarified, and treatment outcomes were calculated and compared among groups. RESULTS: CRT was feasible in elderly NPC patients, while a concurrent regimen of weekly cisplatin was more tolerable. Grade≥3 acute toxicity in CRT group was similar with referent group, although it was significantly higher than the RT alone group (65.3% vs. 43.6%, P=0.002). Furthermore, patients with ACE-27 score≥2 in the CRT group had significantly higher severe acute toxicity and dose reduction. Survival was poorer in elderly patients than the referent group. Compared to RT alone, CRT significantly improved the 5-year overall survival (OS: 54.6% vs. 39.3%, P=0.009), cancer-specific survival (CSS: 56.6% vs. 42.7%, P=0.022), disease-free survival (DFS: 51.6% vs. 30.2%, P=0.028) and locoregional relapse-free survival (LRRFS: 78.4% vs. 52.2%, P=0.003), but not distant metastasis-free survival (DMFS: 69.6% vs. 63.6%, P=0.669). However, CRT did not significantly improve 5-year OS (43.6% vs. 27.3%, P=0.893) or CSS (43.6% vs. 34.1%, P=0.971) in elderly NPC patients with ACE-27 score≥2. CONCLUSIONS: CRT is feasible and effective in elderly patients with locoregionally advanced NPC without severe comorbidities. CRT should be used under serious consideration and be further tested in elderly patients with severe comorbidities. As such, it is essential to perform a comprehensive evaluation of pretreatment comorbidity status for all elderly NPC patients.

Elevated serum endostatin levels are associated with poor survival in patients with advanced-stage nasopharyngeal carcinoma.

AIMS: To evaluate the prognostic value of serum endostatin levels in patients with advanced-stage nasopharyngeal carcinoma (NPC). MATERIALS AND METHODS: Between August 2003 and March 2005, 218 patients with advanced-stage NPC were enrolled in this study, including 70 patients in the training cohort and 148 in the validation cohort. The pre-treatment serum endostatin and vascular endothelial growth factor (VEGF) levels were measured using competitive enzyme immunoassays. For the normal control, serums samples from 20 healthy individuals were also collected. RESULTS: Serum endostatin levels in the patients with advanced-stage NPC were significantly higher than those of controls, but VEGF levels were similar in the two groups. Univariate analysis revealed significant differences between the high and low endostatin level groups regarding 5 year overall survival (63.9% versus 90.5%; P = 0.003), progression-free survival (PFS) (50.2% versus 79.3%; P = 0.003) and distant metastasis-free survival (DMFS) (59.1% versus 85.3%; P = 0.01) in the training cohort. Using the same cut-off value generated from the training cohort, there were also significant unfavourable correlations between serum endostatin levels and overall survival (P = 0.001), PFS (P = 0.001) and DMFS (P = 0.002) in the second independent validation cohort. Multivariate analysis using the entire group (n = 218) revealed that the serum endostatin level was an independent unfavourable prognostic factor for overall survival (hazard ratio 4.8; 95% confidence interval 2.48-9.23; P < 0.0001), PFS (hazard ratio 3.44; 95% confidence interval 2.06-5.74; P < 0.0001) and DMFS (hazard ratio 3.65; 95% confidence interval 1.92-6.94; P < 0.0001) in patients with advanced-stage NPC. No associations were observed between the outcomes and the serum VEGF levels in patients with advanced-stage NPC. CONCLUSIONS: High endostatin levels are associated with poor survival and this knowledge may improve the risk stratification of patients with advanced-stage NPC.

Prognostic significance of the various classifications for parapharyngeal space involvement in nasopharyngeal carcinoma.

CONCLUSION: Advanced parapharyngeal space (PPS) involvement showed stronger prognostic value than PPS involvement. The classification of PPS involvement proposed by Min or Sham was the most appropriate classification for parapharyngeal extension in nasopharyngeal carcinoma (NPC). The degree of tumor extension into the PPS should be considered in future TNM staging revisions. OBJECTIVES: This study was conducted to evaluate the prognostic significance of the various classifications for PPS involvement in patients with NPC. METHODS: From January to July 2000, a total of 176 patients with pathologically diagnosed NPC were prospectively enrolled in this study. The extent of PPS involvement was examined by contrast-enhanced computed tomography (CT) scan and graded according to the four previously reported classifications (Min, Sham, Xiao, and Heng). RESULTS: The incidence of PPS involvement was 81.8%. The 5-year overall survival (OS), progression-free survival (PFS), distant metastasis-free survival (DMFS), and local relapse-free survival rates (LRFS) of the patients with and without PPS involvement were 68.1% and 90.2% (p = 0.010), 66.1% and 87.2% (p = 0.013), 76.7% and 93.6% (p = 0.032), and 84.9% and 93.1% (p = 0.220), respectively. Multivariate analysis showed that PPS involvement (yes vs no) was not an independent prognostic factor. However, graded PPS involvement was an independent factor affecting the prognosis of NPC. When the four classifications were included in a Cox model, it was shown that PPS involvement based on Min's classification was an independent factor for OS (p = 0.001). PPS involvement based on Sham's classification was an independent factor for PFS (p = 0.010) and DMFS (p = 0.009).

Estrogen receptor-beta agonist diarylpropionitrile counteracts the estrogenic activity of estrogen receptor-alpha agonist propylpyrazole-triol in the mammary gland of ovariectomized Sprague Dawley rats.

Although estrogen can bind both types of estrogen receptors, estrogen receptor-alpha (ERα) is dominant in mediating estrogenic activity in the mammary gland and uterus. Excessive estrogenic activity such as estrogen-based postmenopausal hormone replacement therapy increases the risk for breast and endometrial cancers. The adverse effect of estrogen on uterine endometrium can be opposed by progestins; however, estrogen-plus-progestin regimen imposes substantially greater risk for breast cancer than estrogen alone. In this study, we used ERα-selective agonist propylpyrazole-triol (PPT) and ERß-selective agonist diarylpropionitrile (DPN) to activate ERα and estrogen receptor-beta (ERß) separately in an ovariectomized rat model and determined whether PPT-activated ERα function in the mammary gland can be suppressed by DPN activated ERß. Ovariectomized rats were randomly divided into six groups and treated with DMSO (control), DPN, PPT, PPT/DPN, PPT/Progesterone, and PPT/Progesterone/DPN, respectively. In the mammary gland, PPT but not DPN increased cell proliferation and amphiregulin gene expression; importantly, the stimulatory effect of PPT on mammary cell proliferation and amphiregulin gene expression can be suppressed by DPN. In the uterus, the effect of PPT on uterine weight and endometrial cell proliferation was not inhibited by DPN but can be inhibited by progesterone. These data provide in vivo evidence that PPT activated ERα activity in the mammary gland can be opposed by ERß-selective agonist DPN, which may be explored for the development of better hormone replacement therapy regimen with less risk for breast cancer.

A new modified dura mater implant: characteristics in recipient dogs.

Duraplasty is critical to the maintenance of anatomical integrity and the protection of brain tissue. Allotransplantation of cadaveric dura mater was abandoned after it was found to transmit Creutzfeldt-Jakob disease (CJD). In this study, the usefulness of a xenogeneic dura mater for dural reconstruction was tested. Twelve dogs were randomly assigned to 4 groups. To simulate the condition of patients with brain surface injury, an area of approximately 2 cm x 1.5 cm of the dura mater was removed to create a defect. Xenogeneic dura mater derived from porcine pericardium was trimmed to the shape and size of the defect and sutured to the endogenous dura mater. Muscles at the apex of the skull and scalp were also sutured. Three dogs were euthanized at 3, 6, 9, and 12 months after implantation and the xenogeneic dura mater and surrounding endogenous tissue were examined macroscopically and microscopically. Three months after implantation, the graft site had begun to heal. Macroscopically, at 6, 9, and 12 months after implantation, the graft had healed completely with the surrounding tissue. No boundary between the graft and surrounding tissue was distinguishable, and the two could not be separated. The graft was smoothly epithelialized and nonadherent to the brain surface. Microscopically, the inner surface of the implant was covered with epithelial cells, and internal capillaries, subepithelial fibrous tissue deposition, and fibroblast proliferation were observed. The xenogeneic dura mater progressively degraded over time. No cysts and no neutrophilic or lymphocytic inflammatory cell response developed between the implant and the recipient brain parenchyma. The modified xenogeneic dura mater is sufficiently biocompatible to allow epithelialization of its inner surface without adherence to brain tissue. No abnormalities develop in recipients, and the xenograft is gradually biodegraded and replaced by endogenous tissue identical to the endogenous dura mater.

Involvement of RHO GTPases and ERK in synuclein-gamma enhanced cancer cell motility.

Synuclein-gamma is aberrantly expressed in more than 70% of stage III/IV breast and ovarian carcinomas. Ectopic overexpression of synuclein-gamma enhanced MDA-MB-435 cell migration in vitro and metastasis in a nude mouse model. However, the mechanism of how synuclein-gamma promotes cell motility is not clear. In our previous studies, we showed that synuclein-gamma overexpression activates ERK. In the present study, we overexpressed synuclein-gamma in several breast and ovarian cancer cell lines and evaluated the effect of synuclein-gamma on the activity of small G-protein RHO family members. We found that at least one of the RHO/RAC/CDC42 GTPases showed a higher level of the GTP-bound active form. Consistent with their role in regulating the intracellular motile machinery, inhibition of the RHO/RAC/CDC42 by C. difficile Toxin B blocked cell migration in both parental cells and synuclein-gamma overexpressing cells. The ERK inhibitor U0126 also blocked the cell migration in both parental cells and synuclein-gamma overexpressing cells. Collectively, our data indicate that synuclein-gamma might be involved in late stage breast and ovarian cancer metastasis by enhancing cell motility through activation of the RHO family small-GTPases and ERK.

Ribosomal S6 kinase as a mediator of keratinocyte growth factor-induced activation of Akt in epithelial cells.

The keratinocyte growth factor receptor (KGFR) is a member of the fibroblast growth factor receptor (FGFR) superfamily. The proximal signaling molecules of FGFRs are much less characterized compared with other growth factor receptors. Using the yeast two-hybrid assay, we have identified ribosomal S6 kinase (RSK) to be a protein that associates with the cytoplasmic domain of the KGFR. The RSK family of kinases controls multiple cellular processes, and our studies for the first time show association between the KGFR and RSK. Using a lung-specific inducible transgenic system we have recently demonstrated protective effects of KGF on the lung epithelium and have demonstrated KGF-induced activation of the prosurvival Akt pathway both in vivo and in vitro. Here we show that a kinase inactive RSK mutant blocks KGF-induced Akt activation and KGF-mediated inhibition of caspase 3 activation in epithelial cells subjected to oxidative stress. It was recently shown that RSK2 recruits PDK1, the kinase responsible for both Akt and RSK activation. When viewed collectively, it appears that the association between the KGFR and RSK plays an important role in KGF-induced Akt activation and consequently in the protective effects of KGF on epithelial cells.

Response markers and the molecular mechanisms of action of Gleevec in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs), defined by the presence of constitutively activated KIT, are the most common gastrointestinal mesenchymal malignancies. This observation has been successfully exploited in clinical trials of Gleevec (also known as imatinib mesylate, STI-571) for patients with unresectable and/or metastatic GISTs. The biological mechanisms of Gleevec as well as its downstream molecular effects are generally unknown. We used a DNA microarray-based approach to identify gene expression patterns and signaling pathways that were altered in response to Gleevec in GIST cells. We identified a total of 148 genes or expressed sequence tags (of 10,367) that were differentially regulated; 7 known genes displayed a durable response after treatment. The significantly down-regulated genes were SPRY4A, FZD8, PDE2A, RTP801, FLJ20898, and ARHGEF2. The only up-regulated gene was MAFbx. On a functional level, we demonstrated that imatinib inhibited phosphorylation of KIT, AKT, and extracellular signal-regulated kinase 1/2 without affecting the total level of these proteins and that differential expression of these response genes involved activation of mitogen-activated protein kinase-dependent and -independent pathways. In an attempt to correlate these in vitro findings to clinical data, we examined GIST needle biopsy specimens taken from patients before and after Gleevec administration according to the CSTI571-B2222 Phase II trial and demonstrated that expression levels of the two gene transcripts evaluated correlated well with clinical response. This study emphasizes the potential value of an in vitro cell model to investigate GIST response to imatinib in vivo, for the purpose of identifying important genetic markers of clinical response, mechanisms of drug action, and possible therapeutic targets.

p21-activated protein kinase 4 (PAK4) interacts with the keratinocyte growth factor receptor and participates in keratinocyte growth factor-mediated inhibition of oxidant-induced cell death.

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family (also known as FGF-7), is an important protective factor for epithelial cells. The receptor for KGF (also called FGFR2-IIIb), which has intrinsic tyrosine kinase activity, is expressed specifically on epithelial cells and in the lung epithelium. Administration of KGF has been shown to protect the lung from various insults, but the mechanism of protection is not well understood. To understand the mechanism by which KGF exerts protective functions on epithelial cells, we used the yeast two-hybrid assay to identify proteins that interact with the KGF receptor (KGFR). Here we show that the cytoplasmic domain of KGFR interacts with p21-activated protein kinase (PAK) 4, which is a new member of the PAK family. The PAKs are regulated by the Rho-family GTPases Rac and Cdc42. PAK4 is the most divergent member of the PAK family of proteins and may have distinct functions. However, stimuli that regulate PAK4 activity are not known. Our data show that PAK4 can associate with the KGFR, which is dependent on KGFR tyrosine kinase activity. We show that a dominant negative mutant of PAK4 blocks KGF-mediated inhibition of caspase-3 activation in epithelial cells subjected to oxidant stress. Our data demonstrate that PAK4 is an important mediator of the anti-apoptotic effects of KGF on epithelial cells.

Gamma-synuclein promotes cancer cell survival and inhibits stress- and chemotherapy drug-induced apoptosis by modulating MAPK pathways.

Synucleins are a family of highly conserved small proteins predominantly expressed in neurons. Recently we and others have found that gamma-synuclein is dramatically up-regulated in the vast majority of late-stage breast and ovarian cancers and that gamma-synuclein over-expression can enhance tumorigenicity. In the current study, we have found that gamma-synuclein is associated with two major mitogen-activated kinases (MAPKs), i.e. extracellular signal-regulated protein kinases (ERK1/2) and c-Jun N-terminal kinase 1 (JNK1), and have shown that over-expression of gamma-synuclein leads to constitutive activation of ERK1/2 and down-regulation of JNK1 in response to a host of environmental stress signals, including UV, arsenate, and heat shock. We also tested the effects of gamma-synuclein on apoptosis and activation of JNK and ERK in response to several chemotherapy drugs. We have found that gamma-synuclein-expressing cells are significantly more resistant to the chemotherapeutic drugs paclitaxel and vinblastine as compared with the parental cells. The resistance to paclitaxel can be partially obliterated when ERK activity is inhibited using a MEK1/2 inhibitor. Activation of JNK and its downstream caspase-3 by paclitaxel or vinblastine is significantly down-regulated in gamma-synuclein-expressing cells, indicating that the paclitaxel- or vinblastine-activated apoptosis pathway is blocked by gamma-synuclein. In contrast to paclitaxel and vinblastine, etoposide does not activate JNK, and gamma-synuclein over-expression has no apparent effect on this drug-induced apoptosis. Taken together, our data indicate that oncogenic activation of gamma-synuclein contributes to the development of breast and ovarian cancer by promoting tumor cell survival under adverse conditions and by providing resistance to certain chemotherapeutic drugs.

OVCA2 is downregulated and degraded during retinoid-induced apoptosis.

Retinoids, the natural and synthetic derivatives of vitamin A, have been shown to regulate the growth and differentiation of a wide variety of cell types and consequently have enormous potential as chemotherapeutic agents. We have previously identified 2 genes, termed OVCA1 and OVCA2, which are located in a small region showing a high frequency of allelic loss in breast and ovarian tumors and share a common exon. Recent studies have suggested that expression of OVCA1 may be influenced by retinoids. Therefore, we analyzed the expression of OVCA1 and OVCA2 in cells in response to treatment with all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR), or under conditions of low serum and confluence, to determine further the roles of OVCA1 and OVCA2 in cell growth, apoptosis and differentiation. We show that OVCA2 mRNA and protein are ubiquitously expressed and that they are downregulated in the lung cancer cell line Calu-6 after treatment with RA and 4HPR. In addition, we observed that OVCA2 protein is proteolytically degraded in response to RA and 4HPR treatment in a time- and dose-dependent manner in the promyelocytic leukemia cell line HL60. In contrast, expression of the candidate tumor suppressor OVCA1 was not downregulated by these treatments. Furthermore, we demonstrate that OVCA2 is evolutionarily conserved and shows regional homology with dihydrofolate reductases (DHFRs), specifically with hydrolase folds found in alpha-beta hydrolases. Our results are in contrast to a previous report and show that OVCA2, not OVCA1 mRNA and protein, is downregulated in response to RA and 4HPR.

MSX2 expression in the apical ectoderm ridge is regulated by an MSX2 and Dlx5 binding site.

The apical ectodermal ridge (AER) is a specialized ectodermal region essential for limb outgrowth. Msx2 expression patterns in limb development strongly suggest an important role for Msx2 in the AER. Our previous studies identified a 348-bp fragment of the chicken Msx2 gene with AER enhancer activity. In this study, the functions of four potential homeodomain binding TAAT sites in this enhancer were studied using transgenic mice and in vitro protein-DNA interactions. Transgenic studies indicate that the four TAAT sites are not redundant and that only the B-TAAT site is critical for AER enhancer activity. The expression patterns of Msx2 and Dlx5 genes in the AER suggest that they might be involved in the regulation of Msx2. In support of this hypothesis, we found that Msx2 and Dlx5 can bind to the B-TAAT site as well as to a fragment containing the D- and E-TAAT sites in the Msx2 AER enhancer sequences. (c)2002 Elsevier Science (USA).