Memory/Active T-Cell Activation Is Associated with Immunotherapeutic Response in Fumarate Hydratase-Deficient Renal Cell Carcinoma.

PURPOSE: Fumarate hydratase-deficient renal cell carcinoma (FH-deficient RCC) is a rare and lethal subtype of kidney cancer. However, the optimal treatments and molecular correlates of benefits for FH-deficient RCC are currently lacking. EXPERIMENTAL DESIGN: A total of 91 patients with FH-deficient RCC from 15 medical centers between 2009 and 2022 were enrolled in this study. Genomic and bulk RNA-sequencing (RNA-seq) were performed on 88 and 45 untreated FH-deficient RCCs, respectively. Single-cell RNA-seq was performed to identify biomarkers for treatment response. Main outcomes included disease-free survival (DFS) for localized patients, objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) for patients with metastasis. RESULTS: In the localized setting, we found that a cell-cycle progression signature enabled to predict disease progression. In the metastatic setting, first-line immune checkpoint inhibitor plus tyrosine kinase inhibitor (ICI+TKI) combination therapy showed satisfactory safety and was associated with a higher ORR (43.2% vs. 5.6%), apparently superior PFS (median PFS, 17.3 vs. 9.6 months, P = 0.016) and OS (median OS, not reached vs. 25.7 months, P = 0.005) over TKI monotherapy. Bulk and single-cell RNA-seq data revealed an enrichment of memory and effect T cells in responders to ICI plus TKI combination therapy. Furthermore, we identified a signature of memory and effect T cells that was associated with the effectiveness of ICI plus TKI combination therapy. CONCLUSIONS: ICI plus TKI combination therapy may represent a promising treatment option for metastatic FH-deficient RCC. A memory/active T-cell-derived signature is associated with the efficacy of ICI+TKI but necessitates further validation.

Acceleration or retardation by a magnetic field of the anodic processes of iron in molybdate-bearing chloride solutions.

The modulation by a horizontal magnetic field of the anodic processes of iron in molybdate-bearing chloride solutions is determined. The magnetic field can accelerate or retard the anodic reaction depending on the rate-controlling steps at specified electrode potentials. The anodic current density arising from uniform dissolution from open or semi-open pits is increased by the magnetic field. The current density originating from occluded pits can be decreased by the magnetic field, where autocatalysis has a dominant effect on the pitting rate. The effect of the magnetic field on the pitting corrosion is a combination of the influence on electrochemical reactions at the interfaces of the pits and the disturbance of the autocatalysis process inside the pit enclave through the magnetohydrodynamic (MHD) effect. Micro-MHD effects for specific locations and macro-MHD effects for pitting systems are recommended to illustrate the magnetic effect on localized corrosion phenomena at various combinations of potentials and solution compositions.

Paeoniflorin alleviates AngII-induced cardiac hypertrophy in H9c2 cells by regulating oxidative stress and Nrf2 signaling pathway.

Cardiac hypertrophy is frequently associated with ventricular dysfunction and heart failure. Paeoniflorin, has been widely used to treat cardiovascular dysfunction-related diseases. However, the underlying mechanism has been unclear. Here, we investigated the potential inhibitory effects and mechanism of paeoniflorin on oxidative stress of cardiac hypertrophy induced by angiotensin II (AngII) in vitro. Using MTS assay, qRT-PCR, WGA staining assay, and western blot, different dosages (50-400 µM) of paeoniflorin were utilized to examine the antihypertrophy effects on H9c2 cells. Western blot examination revealed the presence of apoptosis-related proteins Bax, Bcl2, and Cytc, antioxidative stress-related proteins Nrf2, HO-1, SOD, and CAT, and mitophagy-related proteins PINK1 and Parkin. qRT-PCR was used to detect the mRNA expression of Bax, Bcl2, Nrf2, and HO-1. TUNEL, caspase3/9 enzyme viability, and MDA, T-AOC, and superoxide levels were all evaluated using commercial kits.The fluorescent probes DCFH-DA and JC-1 were employed to measure cellular ROS and MMP levels. Nrf2 siRNA was utilized to investigate Nrf2's role in paeoniflorin-treated cardiac hypertrophy. Paeoniflorin dramatically reduced cell section area (CSA) and hypertrophic marker (ANP, BNP) expression while inhibiting oxidative stress by modulating ROS and MDA, CAT, SOD, and T-AOC levels. Furthermore, in AngII-induced cardiomyocyte hypertrophy, paeoniflorin restores H9c2 apoptosis by restoring Bax, Bcl-2 Cyt-C, Caspase 3, and Caspase 9 levels. Paeoniflorin also restored Nrf2/HO-1 and PINK1/Parkin expression, and its anti-AngII activities were mediated by Nrf2, which was regulated by Nrf2 knockdown. In conclusion, Our data confirm that paeoniflorin alleviates cardiac hypertrophy through modulating oxidative stress and Nrf2 signaling pathway in vitro.

Experimental study on the suppressive effect of B3GNT3 on the apoptosis of lung adenocarcinoma cells and its application in early screening for lung adenocarcinoma.

Background: Lung adenocarcinoma, as a common histological type in lung cancer, the overall survival is very low, and the prognosis is poor because it is difficult to find and easily recurs. Therefore, this study aimed to explore the role of the secreted protein beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) in the development of lung adenocarcinoma and to evaluate its potential significance for early clinical biomarker screening. Methods: The mRNA expression profiles of patients with lung adenocarcinoma and normal controls were analyzed via The Cancer Genome Atlas (TCGA) database. Serum samples of clinical lung cancer patients and healthy people were obtained, and the differences in B3GNT3 expression in different stages of lung adenocarcinoma and in healthy tissues were compared. Kaplan-Meier (K-M) curves were drawn to clarify the influence of high and low expression of B3GNT3 on the prognosis of patients. Peripheral blood samples from patients with lung adenocarcinoma and healthy people were obtained clinically, and receiver operating characteristic (ROC) curves were drawn to clarify the sensitivity and specificity of B3GNT3 expression for the diagnosis of lung adenocarcinoma. Lung adenocarcinoma cells were cultured in vitro, the expression of B3GNT3 was knocked down by lentivirus infection. The expression of the apoptosis-associated genes was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results: The secreted protein B3GNT3 is significantly differentially expressed in the serum of patients with lung adenocarcinoma versus normal controls. Subgroup analysis according to lung adenocarcinoma clinical stage showed that the higher the clinical stage of lung adenocarcinoma was, the higher the B3GNT3 expression. Enzyme-linked immunosorbent assay (ELISA) revealed that B3GNT3 expression was significantly increased in the serum of patients with lung adenocarcinoma and significantly decreased after surgery. By inhibiting programmed cell death-ligand 1 (PD-L1), the level of apoptosis was significantly increased and the proliferative capacity was significantly inhibited. In contrast, the level of apoptosis was significantly increased and the proliferation ability was significantly inhibited after simultaneous overexpression of B3GNT3 and inhibition of PD-L1. Conclusions: High expression of the secreted protein B3GNT3 in lung adenocarcinoma is closely related to prognosis and can serve as a potential biological marker for the early screening of lung adenocarcinoma.

Validation of a three-dimensional printed dry lab pancreaticojejunostomy model in surgical assessment: a cross-sectional study.

OBJECTIVES: Until now, there have been few tools to evaluate whether a surgeon was technically ready to perform a safe pancreaticojejunostomy (PJ). In the current study, we aimed to evaluate whether a three-dimensional model could mimic a real surgical situation and distinguish between surgeons of different levels of experiences. DESIGN: A three-dimensional PJ dry laboratory model was printed. Eight experienced pancreatic surgeons were tasked to evaluate the appearance and tactile sensation of the model. Proficiency was scored based on 15 surgeons with various levels of pancreatic experience performing a PJ on the three-dimensional model. Additionally, the time of manipulation and NASA Task Load Index (NASA-TLX) scores were recorded for each operation. SETTING: Our study was conducted in multimedical centre in China. RESULTS: Compared with real surgical situations, this model had similar appearance (3.96±0.55 out of five points) and tactile sensation (3.85±0.46 out of five points) according to the expert evaluation. Additionally, the chief surgeon group scored the best in proficiency (based on NASA-TLX scores and operative time), and there were statistical differences for performances among surgeons of various levels (p<0.05). CONCLUSION: The three-dimensional PJ model could mimic a real surgical situation and can distinguish between surgeons of different levels of experiences.

ARID1A deficient undifferentiated spindle cell and rhabdoid sarcoma of the prostate: report of a unique case with emphasis on diagnostic implications.

BACKGROUND: SWItch Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling complex functions collectively as a tumor suppressor and the inactivation of any of its constituent components is frequently associated with tumor initiation and/or progression. Most SWI/SNF deficient tumors share common rhabdoid morphology. ARID1A is the most frequently dysregulated SWI/SNF subunit in human cancer and inactivation of ARID1A is frequent across carcinomatous types while very rarely drives the tumorigenesis of sarcomas. Herein, we report a rare case of primary prostatic undifferentiated spindle cell sarcoma with focal rhabdoid morphology, harboring biallelic inactivation of ARID1A detected by next-generation sequencing with complete loss of ARID1A expression by immunohistochemistry. CASE PRESENTATION: The patient is a 58-year-old man who presented with dysuria and obstructive voiding symptoms for 3 month and was found to have a large, ill-defined, prostatic mass lesion with circumferential extension into the rectal wall on imaging studies. A needle biopsy showed a spindle cell undifferentiated sarcoma of the prostate and the patient was treated by chemotherapy of combined etoposide and cisplatin for 2 months. A subsequent imaging study showed that the tumor was significantly enlarged, and the patient underwent laparoscopically radical prostatectomy. Gross examination showed a disrupted, 10 × 7 × 5 cm, solid and cystic mass involving almost the entire prostate and sparing the seminal vesicle glands. Histologic examination showed that tumor was composed mainly of mildly atypical, oval to spindle-shaped cells, arranged in sheets and fascicles or herringbone-like patterns within a small amount of edematous to myxoid, vascularized stroma. Notably, groups of discohesive rhabdoid tumor cells with eccentric nuclei, prominent nucleoli, and abundant globular cytoplasm were observed. There were prominent mitotic figures, multifocal geographic necroses, and foci of lymphovascular invasion. Immunohistochemistry showed that the tumor cells were diffusely positive for TLE-1 and vimentin and focally positive for epithelial membrane antigen, AE1/3, Cam5.2, SATB2, and CD34 (all in less than 10% tumor cells). Next-generation sequencing showed biallelic inactivation mutation of ARID1A; the predicted inactivating effect of ARID1A deletion was confirmed by immunohistochemical staining. After the surgery, the patient received an alternative combined chemotherapy of doxorubicin and ifosfamide for 5 months. The patient died 9 months after initial presentation due to extensive abdominal metastases. CONCLUSIONS: We report an ARID1A deficient undifferentiated spindle cell and rhabdoid sarcoma of the prostate, adding to the growing spectrum of SWI/SNF driven undifferentiated sarcoma. Rhabdoid cells can be a helpful morphological clue for promoting molecular and immunohistochemical analyses for deficiency of SWI/SNF subunits, in the diagnostic workup of undifferentiated neoplasms featuring epithelioid or rhabdoid morphology.

Use of Circulating Tumor DNA for the Clinical Management of Metastatic Castration-Resistant Prostate Cancer: A Multicenter, Real-World Study.

BACKGROUND: This study aimed to describe the aberrations of DNA damage repair genes and other important driving genes in Chinese patients with metastatic castration-resistant prostate cancer (mCRPC) using circulating tumor (ctDNA) sequencing and to evaluate the associations between the clinical outcomes of multiple therapies and key genomic alterations in mCRPC, especially DNA damage repair genes. PATIENTS AND METHODS: A total of 292 Chinese patients with mCRPC enrolled from 8 centers. Multigene targeted sequencing was performed on 306 ctDNA samples and 23 matched tumor biopsies. The frequency of genomic alterations were compared with the Stand Up to Cancer-Prostate Cancer Foundation (SU2C-PCF) cohort. The Kaplan-Meier method was used to evaluate progression-free survival (PFS) following standard systemic treatments for mCRPC. Cox regression analyses were performed to determine prognostic factors associated with PFS resulting from treatments for mCRPC. RESULTS: In total, 33 of 36 (91.7%) mutations were found consistently between ctDNA and paired biopsy samples. The most common recurrent genomic alterations were found in AR (34.6%), TP53 (19.5%), CDK12 (15.4%), BRCA2 (13%), and RB1 (5.8%). The frequency of CDK12 alterations (15.4%) in our cohort was significantly higher than that in Western populations (5%-7%). AR amplification and TP53 and/or RB1 alterations were associated with resistance to abiraterone or docetaxel. Patients with a CDK12 defect showed rapid disease progression after abiraterone treatment. However, the clinical outcome after docetaxel treatment was similar between patients with and without CDK12 defects. In multivariate Cox regression analysis, a CDK12 defect was significantly associated with inferior PFS after abiraterone treatment. Patients with a BRCA2 defect showed marked response to both PARP inhibitors and platinum-based chemotherapy. CONCLUSIONS: Our study explored the genomic landscape of Chinese patients with mCRPC at different treatment stages using minimally invasive methods and evaluated the clinical implications of the driver genomic alterations on patients' response to the most widely used therapies for mCRPC. We observed a significantly higher alteration frequency of CDK12 in our cohort compared with the SU2C-PCF cohort.

Paeoniflorin and Plycyrrhetinic Acid Synergistically Alleviate MPP+/MPTP-Induced Oxidative Stress through Nrf2-Dependent Glutathione Biosynthesis Mechanisms.

Recently, combination therapy has proven to be an effective strategy for treating polygenic/multifactorial/complex disorder such as Parkinson's disease (PD). Here, we hypothesized that dual up-regulation of glutamate cysteine ligase (GCL) catalytic subunit (GCLc) and GCL modifier subunit (GCLm) via nuclear factor E2-related factor (Nrf2) contribute to the antioxidant effect of paeoniflorin (PF) synergistically with glycyrrhetinic acid (GA) (henceforth called PF/GA) in the context of MPP+/MPTP neurotoxicity. Expectedly, CompuSyn synergism/antagonism analysis showed that PF/GA exerts synergistic neuroprotection. Moreover, the antioxidant effect of PF was significantly enhanced by the combined administration of GA, although GA alone did not confer the effect. Mechanistically, PF triggered extracellular signal-regulated kinase (ERK1/2) phosphorylation, resulting in Nrf2 nuclear translocation from cytoplasmic pool via de novo synthesis in MPP+-challenged SH-SY5Y cells. Concomitantly, GA activates Akt which in turn induces nuclear accumulation of Nrf2. Especially, PF/GA up-regulated glutamate-cysteine ligase catalytic subunit (Gclc) and glutamate-cysteine ligase modifier subunit (Gclm) are formed via two separate pathways. Furthermore, these results were confirmed through pathway blockade assays using PD98059 (ERK1/2 inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), and shRNA-induced Nrf2 knockdown. Additionally, using a mouse MPTP-induced model of PD, we demonstrated that PF/GA synergistically ameliorates both motor deficits and oxidative stress in the ventral midbrain. In parallel, PF/GA also up-regulated both GCLc and GCLm expression at levels of transcription and translation. Conversely, antiparkinsonism and antioxidant effects of PF/GA were not observed in Nrf2-knockout MPTP-mice. Collectively, these results show that ERK1/2 and Akt activation contribute to the synergistic antioxidant effect of PF/GA. Hence, PF/GA regimen warrants further preclinical and possible clinical study for PD.

Dose escalation and expansion (phase Ia/Ib) study of GLS-010, a recombinant fully human antiprogrammed death-1 monoclonal antibody for advanced solid tumors or lymphoma.

BACKGROUND: GLS-010, a novel engineered fully human immunoglobin G4 monoclonal antibody, can specially block the PD-1/PD-L1/2 axis and reactivate the antitumor immunity. AIM: This phase Ia/Ib study was carried out to evaluate the safety, recommended phase II dose (R2PD), and primary antitumor effects of GLS-010 in patients with advanced, refractory lymphoma and solid tumors. METHODS: In phase Ia study, patients with refractory solid tumors and lymphoma enrolled and received GLS-010 at a dose of 1, 4, or 10 mg/kg Q2W; 240 mg Q3W or Q2W. The primary objective was to assess the dose-limiting toxicity (DLT). In phase Ib study, doses were expanded in 9 specific tumors to ensure the R2PD and explore the efficacy. Tumor mutation burden level and PD-L1 expression were also assessed with whole-exome sequencing and immunohistochemistry (SP263), respectively. RESULTS: Up to April 18, 2020, a total of 289 patients (n = 24, phase Ia; n = 265, phase Ib) were enrolled. DLT was not observed in phase Ia part. The T1/2, CLss, and Vd were similar among all dose groups and different tumors. The most common treatment-emergent adverse events (TEAEs) were anemia, leukopenia, elevated alanine aminotransaminase/asparate aminotransferase (ALT/AST), and elevated bilirubin. And hypothyroidism was the most common immune-related adverse event (irAE). The incidence of grade ≥3 TEAE was 39.8%, while grade ≥3 irAE was only 4.5%. Based on safety studies, pharmacokinetics/pharmacodynamics, and preclinical data, 240-mg Q2W was recommended as the expansion dose. The overall objective response rate was 23.6%, with 10 patients achieving complete response. Patients with a high PD-L1 expression level (31.3% Versus. 13.7%, p = 0.012) or t-issue tumor mutation burden level (31.3% Versus. 5.6%, p = 0.009) showed a significantly better response. CONCLUSION: GLS-010 showed acceptable safety profile and favorable clinical response. The dose of 240 mg Q2W was an optimal recommended dose as monotherapy.

Linc00514 promotes breast cancer metastasis and M2 polarization of tumor-associated macrophages via Jagged1-mediated notch signaling pathway.

BACKGROUND: Tumor-associated macrophages (TAMs) and tumor cells are important components of the tumor microenvironment. M2 polarization of TAMs, which is a major actor in breast cancer malignancy and metastasis, can be induced by breast cancer cells. However, the potential mechanisms of the interaction between breast cancer cells and TAMs remain unclear. METHODS: The candidate breast cancer-associated long non-coding RNAs (lncRNAs) were analyzed using the GEO database. Functional assays, including MTT assay, Transwell assay, and EdU labeling detection, were performed to investigate the oncogenic role of linc00514 in breast cancer progression. The co-culture and ELISA assays were used to assess the role of linc00514 in macrophage recruitment and M2 polarization. RNA immunoprecipitation, RNA pull-down, and luciferase reporter assays were applied to determine the mechanism of linc00514 in breast cancer metastasis. Mouse xenograft models, mouse pulmonary metastatic models, and mouse primary tumor models were used to assess the role of linc00514 in M2 macrophage polarization and breast cancer tumorigenicity. RESULTS: Linc00514 was highly expressed in clinical breast cancer tissues and breast cancer cell lines. Overexpression of linc00514 promoted the proliferation and invasion of breast cancer cells and increased xenograft tumor volumes and pulmonary metastatic nodules. Overexpression of linc00514 also increased the percentage of macrophages expressing M2 markers CD206 and CD163. Mechanistically, linc00514 promoted Jagged1 expression in a transcriptional manner by increasing the phosphorylation of a transcription factor STAT3. Subsequently, Jagged1-mediated Notch signaling pathway promoted IL-4 and IL-6 secretions in breast cancer cells and ultimately inducing M2 polarization of macrophages. CONCLUSION: Linc00514 plays an important role in regulating breast cancer tumorigenicity and M2 macrophage polarization via Jagged1-mediated Notch signaling pathway.

Tubeimoside-1 inhibits the proliferation and metastasis by promoting miR-126-5p expression in non-small cell lung cancer cells.

Tubeimoside-1 (TBMS1) possesses broad anticancer activities, including the cytostatic and anti-angiogenesis effects in lung cancer. However, the effect of TBMS1 on the metastasis of non-small cell lung cancer (NSCLC) cells and the potential underlying mechanism remain unclear. In the present study, a cell counting kit-8 assay revealed that TBMS1 suppressed the proliferation of NCI-H1299 cells significantly, particularly following 48 h of treatment. Further studies showed that TBMS1 notably enhanced the apoptosis, and inhibited the migration and invasion of NCI-H1299 cells upon treatment for 48 h. A total of 14 NSCLC tissues and 14 normal adjacent tissues were collected, reverse transcription-quantitative polymerase chain reaction revealed decreased expression of microRNA (miR)-126-5p in NSCLC tissues compared with adjacent NSCLC tissues, which was reversed following TBMS1 administration in NCI-H1299 cells. The overexpression of miR-126-5p induced by TBMS1 was demonstrated to target and downregulate vascular endothelial growth factor (VEGF)-A. Simultaneously, the expression of VEGF-R2 was reduced notably, along with a significant declined in the phosphorylation levels of dual specificity mitogen-activated protein kinase kinase 1 and extracellular signal-regulated kinase (ERK)1/2. Overall, the aforementioned results indicated that TBMS1 inhibited the proliferation and metastasis, and promoted the apoptosis of NCI-H1299 cells, which may be mediated by overexpressing miR-126-5p, which inactivates the VEGF-A/VEGFR2/ERK signaling pathway. Therefore, TBMS1 may be a promising drug for prevention and treatment of NSCLC.

Antitumor effects of Tubeimoside-1 in NCI-H1299 cells are mediated by microRNA-126-5p-induced inactivation of VEGF-A/VEGFR-2/ERK signaling pathway.

Tubeimoside-1 (TBMS1), a triterpenoid saponin isolated from the tuber of Bolbostemma paniculatum (Maxim) Franquet, serves an universal suppressive role in multiple cancer types, including lung cancer. However, the mechanism involved in non­small cell lung cancer (NSCLC) cells by which TBMS1 elicits its antitumor effects is not yet comple-tely understood. The present study indicated that 10 µmol/l TBMS1 significantly enhanced apoptosis and notably blocked the migration and invasion of NCI­H1299 cells. These effects were reversed following transfection with miR­126­5p inhi-bitor into TBMS1­treated NCI­H1299 cells. Vascular endo-thelial growth factor-A (VEGF­A) is a target gene for miR­126­5p. Notably, results suggested that the downregulated VEGF­A and VEGFR­2 in TBMS1­treated NCI­H1299 cells were upregulated after inhibiting miR­126­5p, and overexpression of VEGF­A or VEGFR­2 could significantly reduce apoptosis and promote the migration and invasion of TBMS1­treated NCI­H1299 cells. Furthermore, TBMS1 combined with TBHQ (an ERK activator) dramatically suppressed TBMS1­induced apoptosis and stimulated TBMS1­reduced migration and invasion in NCI­H1299 cells, suggesting that TBMS1 inhibits the ERK signaling pathway and represses the growth and metastasis of NCI­H1299 cells. Further study demonstrated that either inhibiting miR­126­5p or overexpressing VEGF­A and VEGFR­2 in TBMS1­treated NCI­H1299 cells elevated the mRNA expression levels and phosphorylation levels of MEK1, as well as ERK. To conclude, TBMS1 increases miR­126­5p expression, whereas overexpressing miR­126­5p inactivates VEGF­A/VEGFR­2/ERK signaling pathway, which ultimately actuates the pro­apoptotic and anti­metastatic effects in NCI­H1299 cells. Therefore, the present findings provide a theoretical foundation for TBMS1 as a potential candidate in NSCLC treatment.

Resveratrol alleviate hypoxic pulmonary hypertension via anti-inflammation and anti-oxidant pathways in rats.

Resveratrol, a plant-derived polyphenolic compound and a phytoestrogen, was shown to possess multiple protective effects including anti-inflammatory response and anti-oxidative stress. Hypoxic pulmonary hypertension (HPH) is a progressive disease characterized by sustained vascular resistance and marked pulmonary vascular remodeling. The exact mechanisms of HPH are still unclear, but inflammatory response and oxidative stress was demonstrated to participate in the progression of HPH. The present study was designed to investigate the effects of resveratrol on HPH development. Sprague-Dawley rats were challenged by hypoxia exposure for 28 days to mimic hypoxic pulmonary hypertension along with treating resveratrol (40 mg/kg/day). Hemodynamic and pulmonary pathomorphology data were then obtained, and the anti-proliferation effect of resveratrol was determined by in vitro assays. The anti-inflammation and anti-oxidative effects of resveratrol were investigated in vivo and in vitro. The present study showed that resveratrol treatment alleviated right ventricular systolic pressure and pulmonary arterial remodeling induced by hypoxia. In vitro experiments showed that resveratrol notably inhibited proliferation of pulmonary arterial smooth muscle cells in an ER-independent manner. Data showed that resveratrol administration inhibited HIF-1 α expression in vivo and in vitro, suppressed inflammatory cells infiltration around the pulmonary arteries, and decreased ROS production induced by hypoxia in PAMSCs. The inflammatory cytokines' mRNA levels of tumor necrosis factor α, interleukin 6, and interleukin 1ß were all suppressed by resveratrol treatment. The in vitro assays showed that resveratrol inhibited the expression of HIF-1 α via suppressing the MAPK/ERK1 and PI3K/AKT pathways. The antioxidant axis of Nuclear factor erythroid-2 related factor 2/ Thioredoxin 1 (Nrf-2/Trx-1) was up-regulated both in lung tissues and in cultured PASMCs. In general, the current study demonstrated that resveratrol may prevent pulmonary hypertension through its anti-proliferation, anti-inflammation and antioxidant effects. Hence, the present data may offer novel targets and promising pharmacological perspective for treating hypoxic pulmonary hypertension.

12-Deoxyphorbol 13-palmitate inhibits the expression of VEGF and HIF-1α in MCF-7 cells by blocking the PI3K/Akt/mTOR signaling pathway.

Vascular endothelial growth factor (VEGF) is an essential component for angiogenesis, and hypoxia-inducible factor-1α (HIF-1α), which controls the switch of glycolytic and oxidative metabolism, activates the transcription of VEGF. 12-Deoxyphorbol 13-palmitate (DP) is a compound isolated from the roots of Euphorbia fischeriana, and has been revealed to possess anticancer activity. In the present study, we found that DP is an effective inhibitor of VEGF and HIF-1α in MCF-7 cells. DP markedly reduced cell viability as determined by MTT assay. ELISA, western blotting and RT-qPCR assays indicated that DP significantly decreased the protein and mRNA expression of VEGF and the protein expression of HIF-1α, while HIF-1α mRNA remained unchanged. In addition, the entrance of HIF-1α into the nucleus was blocked after DP treatment as detected by immunofluorescence analysis. In a further study, we proved that the effects mentioned above were associated with constitutive interference of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. DP effectively inhibited the phosphorylation of PI3K and its downstream factors p-Akt and p-mTOR, oppositely enhanced the expression of TSC1 (hamartin) and TSC2 (tuberin), which could be reversed by the co-treatment with the PI3K inhibitor wortmannin. Moreover, the addition of wortmanin further downregulated the protein levels of VEGF and HIF-1α. The results revealed that DP inhibited the expression of VEGF and HIF-1α through the PI3K/Akt/mTOR signaling pathway, confirming that DP may be a potential therapeutic candidate for breast cancer.

Predicting chemosensitivity to gemcitabine and cisplatin based on gene polymorphisms and mRNA expression in non-small-cell lung cancer cells.

AIM: We used a panel of 17 non-small-cell lung cancer cell lines to investigate whether the presence of polymorphisms in the RRM1, ERCC1, ABCB1 and MTHFR genes and alterations in their mRNA expression can affect the in vitro chemosensitivity to cisplatin and gemcitabine. MATERIALS & METHODS: Polymorphisms in these genes were evaluated by direct sequencing. mRNA expression levels were assessed by realtime PCR. In vitro chemosensitivity to cisplatin and gemcitabine was expressed as IC50 values, using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. RESULTS: There was a significant, positive correlation between RRM1 mRNA expression and IC50 values for gemcitabine (r = 0.6533, p = 0.0045), and there was a significant, negative correlation between ABCB1 mRNA expression and IC50 values for cisplatin (r = -0.5459, p = 0.0287). When examining the association between the polymorphisms and IC50, we found that only the MTHFR 1298A>C polymorphism showed a tendency to be more chemosensitive to gemcitabine (p = 0.0440). CONCLUSION: These in vitro results suggest that mRNA expression levels of the RRM1 and ABCB1 genes may be useful indicators of chemosensitivity to gemcitabine and cisplatin, respectively. The MTHFR 1298A>C polymorphism was associated with gemcitabine chemosensitivity, which require further functional analysis with co-expressed genes and should be explored in prospective clinical studies to determine its potential clinical application as a predictive biomarker. Original submitted 11 February 2014; Revision submitted 3 November 2014.

miR-29a-3p attenuates hypoxic pulmonary hypertension by inhibiting pulmonary adventitial fibroblast activation.

Activation of pulmonary adventitial fibroblasts plays a key role in the pulmonary vascular remodeling in hypoxic pulmonary hypertension. Previous studies showed that miRNAs participated in the regulation of fibroblast activation. This study explored the role of miR-29 in the activation of pulmonary adventitial fibroblasts and the therapeutic potential in hypoxic pulmonary hypertension. We found that hypoxia-induced pulmonary adventitial fibroblasts activation was accompanied with a drastic decrease of miR-29a-3p expression. Knockdown of hypoxia-inducible factor-1 α or Smad3 reversed the hypoxia-induced decrease of miR-29-3p in cultured pulmonary adventitial fibroblasts. In vitro, miR-29a-3p mimic inhibited the hypoxia-induced proliferation, migration, and secretion of pulmonary adventitial fibroblasts, suppressed the hypoxia-induced expression of α-smooth muscle actin and extracellular matrix collagen in pulmonary adventitial fibroblasts; however, miR-29a-3p inhibitor mimicked the effect of hypoxia on the activation of pulmonary adventitial fibroblasts. Further studies revealed that preventative or therapeutic administration of miR-29a-3p significantly decreased pulmonary artery pressure and right ventricle hypertrophy index and ameliorated pulmonary vascular remodeling in hypoxic pulmonary hypertension rats. These findings suggest that miR-29a-3p regulates the activation and phenotype of pulmonary adventitial fibroblasts in hypoxia and has preventative and therapeutic potential in hypoxic pulmonary hypertension.

Antitumor effects of artesunate on human breast carcinoma MCF-7 cells and IGF-IR expression in nude mice xenografts.

PURPOSE: The objective of this study was to investigate the anti-tumor effects and analyze the mechanism of artesunate (ART) action on breast cancer in vivo using tumor transplanted nude mice. METHODS: The human breast tumor cell line MCF-7 was transplanted into nude mice, and the animals were treated with various doses of ART alone or in combination with cyclophosphamide (CTX) or normal saline (NS). The tumor inhibitory effects were observed and compared, and the ultrastructural morphology of the transplanted tumor cells was observed by electron microscopy. The apoptosis rates and cell cycle status were detected by flow cytometry (FCM). The expression of apoptosis-related proteins p53, Bcl-2, Bax and Caspase-3 were detected by immunohistochemistry and IGF-IR was detected by western blot. The expression correlation for these proteins was also analyzed. RESULTS: The tumor inhibition rates in the low dose ART group, high dose ART group, CTX group and combined drug therapy group were (24.39±10.20)%, (40.24±7.02)%, (57.01±5.84)% and (68.29±5.1)%, respectively. The cell cycle was arrested in phase G0/G1 after treatment with ART. The expression of Bcl-2 was significantly reduced, and the expression levels of Bax and Caspase-3 were significantly increased in the ART group compared to the negative control saline group. There was no significant difference detected in p53 expression. The Bcl-2 level was negatively related to Bax and Caspase-3. The western blotting results showed IGF-IR downregulation. CONCLUSIONS: ART inhibits the growth of MCF-7 breast tumor cell xenografts in nude mice. The anti-tumor mechanism of ART for human breast carcinoma in nude mice might be correlated with the alteration of apoptosis related protein expression, which may further induce apoptosis and inhibit cell proliferation.

Analyzing 395,793 samples shows significant association between rs999737 polymorphism and breast cancer.

Large-scale genome-wide association studies (GWAS) have been conducted and reported the association between rs999737 polymorphism at 14q24.1 (RAD51L1) and breast cancer risk. Following studies investigated rs999737 polymorphism in European and Asian populations. However, some of these studies reported weak and no significant association. Here, we reevaluated this association using large-scale samples from previous 11 studies (n=395,793; 162,261 cases and 233,532 controls) from the PubMed database. We evaluated the genetic heterogeneity among the selected studies. The pooled odds ratio (OR) is calculated by the fixed effect model. All statistical tests for heterogeneity and meta-analysis were computed using R package. We did not identify significant heterogeneity among the included studies using the allele model (P=0.1314 and I (2)=33.4 %). We observed significant association between rs999737 and breast cancer using the allele model (P=2.47E - 35, OR=0.92, 95 % confidence interval (CI) 0.91-0.93). Our analysis further supports previous findings that the rs999737 polymorphism contributes to breast cancer susceptibility. We believe that our finding will be very useful for future genetic studies in breast cancer.

Efficacy, safety, and tolerability of exenatide once weekly in patients with type 2 diabetes mellitus: an integrated analysis of the DURATION trials.

BACKGROUND AND AIM: Exenatide is a glucagon-like peptide-1 receptor agonist demonstrated to improve glycemic control with low hypoglycemia risk in patients with type 2 diabetes mellitus. The Diabetes Therapy Utilization: Researching Changes in A1C, Weight, and Other Factors Through Intervention With Exenatide Once Weekly (DURATION) program comprised 6 randomized, comparator-controlled, 24- to 30-week trials of exenatide once weekly (EQW), an extended-release formulation. This post hoc analysis pooled data from patients taking EQW across 6 trials to assess efficacy and safety in a large, varied patient population. MATERIALS AND METHODS: The intent-to-treat (ITT) population contained 1379 patients (baseline mean ± standard deviation glycated hemoglobin [HbA1c] levels of 8.4% ± 1.1%) who were treated with EQW over the course of 24 to 30 weeks. Changes from baseline in efficacy parameters for the ITT population and a completer population (1195 patients with ≥ 22 weeks of exposure) were evaluated. RESULTS: The ITT population experienced significant reductions from baseline (least-squares mean [95% CI]) in HbA1c levels (-1.4% [-1.5% to -1.4%]), fasting blood glucose levels (-36 mg/dL [-38.4 mg/dL to -33.8 mg/dL]), and body weight (-2.5 kg [-2.8 kg to -2.3 kg]) after 24 to 30 weeks of EQW treatment. Reductions in HbA1c and fasting blood glucose levels were observed across baseline HbA1c level strata; patients with higher baseline HbA1c levels experienced greater reductions. Treatment with EQW was associated with modest, significant reductions in blood pressure (systolic blood pressure, -2.8 mm Hg [-3.5 mm Hg to -2.1 mm Hg]; diastolic blood pressure, -0.8 mm Hg [-1.2 mm Hg to -0.4 mm Hg]), and fasting lipid levels (total cholesterol, -6.5 mg/dL [-8.2 mg/dL to -4.7 mg/dL]; low-density lipoprotein cholesterol, -3.9 mg/dL [5.3 mg/dL to -2.5 mg/dL]; and triglyceride [geometric least-squares mean percent change (95% CI)], -6% [-8% to -4%] levels). Similar reductions were observed in the completer population. Exenatide once weekly was generally well tolerated. Transient, mild-to-moderate gastrointestinal treatment-emergent adverse events and injection-site treatment-emergent adverse events were reported most frequently, but were seldom treatment limiting. No major hypoglycemic events were observed; minor hypoglycemic events occurred infrequently in patients not using a sulfonylurea. CONCLUSION: This post hoc analysis of > 1300 patients demonstrated that EQW was associated with significant reductions in HbA1c levels, body weight, blood pressure, and fasting lipid levels, with minimal hypoglycemia risk. Consistent with established safety profiles, EQW therapy was generally well tolerated. TRIAL REGISTRATION: www.ClinicalTrials.gov identifiers: NCT00308139, NCT00637273, NCT00641056, NCT00676338, NCT00877890, NCT01029886.

Endogenous estrogen attenuates hypoxia-induced pulmonary hypertension by inhibiting pulmonary arterial vasoconstriction and pulmonary arterial smooth muscle cells proliferation.

Exogenous estrogen was shown to exert various beneficial effects on multiple diseases including hypoxia-induced pulmonary hypertension (HPH). However, the effect of endogenous estrogen on HPH was seldom investigated. In the present study, we explored the protective effects and mechanisms of endogenous estrogen on hypoxia-induced pulmonary hypertension. Male, female, pregnant and ovariectomized rats were housed in a hypoxic condition for 21 days, and then hemodynamic together with morphologic indexes of pulmonary circulation were measured. The right ventricular systolic pressure, mean pulmonary artery pressure, right ventricular hypertrophy index, and arterial remodeling index were significantly elevated after chronic hypoxia exposure. Experimental data showed less severity in female, especially in pregnant rats. In vitro, artery rings of different sex or estrus cycle rats were obtained, and then artery rings experiments were performed to investigate pulmonary vasoconstriction by recording the maximum phase II vasoconstriction. Data showed that the vasoconstriction was milder in proestrus female than diestrus female or male groups, which could be leveled by treating U0126 (a MAPK pathway inhibitor). Pulmonary arterial smooth muscle cells isolated from different sex or estrus cycle rats were cultured in the condition of 2% oxygen for 24 hours, and cell proliferation was evaluated by the [3H]-thymidine incorporation assay. Cells from proestrus rats exhibited lower proliferation than the other groups, which could be countered by both U0126 and raloxifene (a selective estrogen receptor modulator). Serum estradiol levels were detected, and rats with higher levels showed less severity of pulmonary hypertension. Conclusively, endogenous estrogen may alleviate hypoxia-induced pulmonary hypertension by attenuating vasoconstriction through non-genomic mechanisms and inhibiting smooth muscle cells proliferation through both genomic and non-genomic mechanisms.