An evaluation of the clinical efficacy of the application of 28mm cryoballoon for linear ablation of left atrial apex combined with enlarged pulmonary vein vestibule ablation for persistent atrial fibrillation.

OBJECTIVE: to retrospectively investigate the efficacy and safety of the application of 28 mm cryoballoon for pulmonary vein electrical isolation (PVI) combined with top left atrial linear ablation and pulmonary vein vestibular expansion ablation for persistent atrial fibrillation. METHODS: From July 2016 to December 2020, 413 patients diagnosed with persistent atrial fibrillation were evaluated, including 230 (55.7%) in the PVI group (PVI only) and 183 (44.3%) in the PVIPLUS group (PVI plus ablation of the left atrial apex and pulmonary vein vestibule). The safety and efficacy of the two groups were retrospectively analyzed. RESULTS: The AF/AT/AFL-free survival rates at 6, 12, 18, 24 and 30 months after procedure was 86.6%, 72.6%, 70.0%, 61.1% and 56.3% in the PVI group and 94.5%, 87.0%, 84.1%, 75.0% and 67.9% in the PVIPLUS group, respectively. At 30 months after procedure, the AF/AT/AFL-free survival rate was significantly higher in the PVIPLUS group than in the PVI group (P = 0.036; HR:0.63; 95% CI:0.42 to 0.95). CONCLUSION: The application of 28-mm cryoballoon for pulmonary vein electrical isolation combined with linear ablation of the left atrial apex and expanded ablation of the pulmonary vein vestibule improves the outcome of persistent atrial fibrillation.

Efficacy of extended antrum ablation based on substrate mapping plus pulmonary vein isolation in the treatment of atrial fibrillation.

INTRODUCTION AND OBJECTIVES: Pulmonary vein isolation (PVI) technique has become the cornerstone of atrial fibrillation (AF) catheter ablation. The objective of this study was to assess the efficacy and safety of extended antrum ablation based on electrophysiological substrate mapping plus PVI in AF patients who underwent cryoballoon ablation. METHODS: In this observational study, a total of 121 paroxysmal AF patients and 80 persistent AF patients who did not achieve the procedure endpoint after cryoballoon ablation received extra extended antrum ablation (EAA) based on electrophysiological substrate mapping via radiofrequency ablation (EAA group). As a control group (PVI group), among paroxysmal AF and persistent AF patients, we conducted a propensity score-matched cohort, in whom only PVI was completed. RESULTS: The average follow-up time was 15.27±7.34 months. Compared with PVI group, paroxysmal AF patients in the EAA group had a significantly higher rate of AF-free survival (90.1% vs. 80.2%, p=0.027) and AF, atrial flutter, or atrial tachycardia (AFLAT) -free rate survival (89.3% vs. 79.3%, p=0.031). Persistent AF patients in the EAA group also had a significantly higher rate of AF-free survival (90.0% vs. 75.0%, p=0.016) and AFLAT-free survival (88.8% vs. 75.0%, p=0.029) than PVI group. Complication rates did not significantly differ between both groups, in either paroxysmal AF or persistent AF patients. CONCLUSION: Our findings demonstrate that extra extended antrum ablation based on electrophysiological substrate mapping is effective and safe. Moreover, the strategy can improve the outcome of AF cryoablation.

Predictive and prognostic impact of the different features of tumor budding in stage II colorectal cancer.

Tumor budding is a significant independent prognostic factor in colorectal cancer. Routine reporting of tumor budding is now advocated for in the colorectal cancer standard approach recommended by the International Tumor Budding Consensus Conference guidelines. However, the current tumor budding assessment system only emphasizes tumor budding quantity and ignores other features. Therefore, this study aimed to further determine the prognostic value of tumor budding based on a more comprehensive feature analysis. To this end, we conducted a retrospective pathology review of the different characteristics of tumor budding (that is quantity, structure, cell atypia, location, stromal reaction, and immunohistochemical phenotype) in 224 specimens of stage II colorectal cancer at our institution between 2009 and 2015. The mean age of the patients was 60.3±9.2 years (range, 39-84 years). Among various features of tumor budding, single-cell budding, anaplasia-like cell atypia, myxoid stroma, high tumor budding quantity, and loss of CDX2 expression were independent predictors of recurrence and mortality in patients with stage II colorectal cancer. Based on these results, we suggest that in addition to tumor-budding quantity, other tumor budding features play important biological roles in the development of colorectal cancer. Our findings provide prognostic information that could help with guiding clinical management and oncology care models for patients with stage II colorectal cancer.

Mortality and Readmission Rates After Heart Failure: A Systematic Review and Meta-Analysis.

OBJECTIVE: The current work aimed to examine the rates of and risk factors for mortality and readmission after heart failure (HF). SETTING: A systematic search was carried out in PubMed, the Cochrane Library, and EMBASE to identify eligible reports. The random-effects model was utilized to evaluate the pooled results. PARTICIPANTS: A total of 27 studies with 515,238 participants were finally meta-analysed. The HF patients had an average age of 76.3 years, with 51% of the sample being male, in the pooled analysis. PRIMARY AND SECONDARY OUTCOME MEASURES: The outcome measures were 30-day and 1-year readmission rates, mortality, and risk factors for readmission and mortality. RESULTS: The effect sizes for readmission and mortality were estimated as the mean and 95% confidence interval (CI). The estimated 30-day and 1-year all-cause readmission rates were 0.19 (95% CI 0.14-0.23) and 0.53 (95% CI 0.46-0.59), respectively, while the all-cause mortality rates were 0.14 (95% CI 0.10-0.18) and 0.29 (95% CI 0.25-0.33), respectively. Comorbidities were highly prevalent in individuals with HF. CONCLUSION: Heart failure hospitalization is followed by high readmission and mortality rates.

IL33 activates CD8+T and NK cells through MyD88 pathway to suppress the lung cancer cell growth in mice.

OBJECTIVE: In this study, we observed the effects of IL-33 on tumor immune response in lung cancer-bearing mice using wild type and MyD88-/- mice respectively. METHODS: Wild C57BL/6 (C57BL/6WT), MyD88 knockout C57BL/6 mice (C57BL/6 MyD88-/-) and Lewis cells were used in this study. Cell proliferation, cytokine release and cytotoxicity were detected. RESULTS: IL-33 could significantly up-regulate specific cellular immunity, inhibit tumor growth and improve survival time in wild type mice group, and it had dose dependent effect. However, IL-33 had no effect on cell immunity and tumor growth in MyD88-/- mice group. Compared with MyD88-/- mice, IL-33 could significantly increase the ratio of CD8+T cells to neutrophils in wild type mice, while the percentage of tumor infiltrating CD11b+ cells, Mo-MDSC, F4/80+ macrophages and mDC cells decreased significantly in wild type mice group. IL-33 could upregulate the expression of CD107a and IFN-γ in CD8+T cells and NK cells of wild type mice, while IL-33 could not upregulate them in MyD88-/- mice. IL-33 could upregulate the expression of CD40, CD80, CD86 and CD205 in DC cells in wild type mice, induce T cells to differentiate into Th1 cells and enhance tumor cell immunity. CONCLUSIONS: IL-33 could promote differentiation and maturation of DC cells through MyD88 pathway, up-regulate the tumor immunity of CD8+T cells and NK cells, and inhibit the proliferation of lung cancer cells.

One-Step Reconstruction with a Novel Suspended, Modular, and 3D-Printed Total Sacral Implant Resection of Sacral Giant Cell Tumor with Preservation of Bilateral S1-3 Nerve Roots via a Posterior-Only Approach.

OBJECTIVE: To investigate the efficacy and safety of spinopelvic reconstruction based on a novel suspended, modular, and 3D-printed total sacral implant after total piecemeal resection of a sacral giant cell tumor (SGCT) with the preservation of bilateral S1-3 nerve roots via a posterior-only approach. METHODS: Five patients who had undergone total piecemeal resection of SGCT involving upper sacral segments (S1 and S2 ) and the midline with the preservation of bilateral S1-3 nerve roots via a posterior-only approach between September 2017 and July 2018 were retrospectively reviewed. A novel suspended, modular, and 3D-printed total sacral implant had been used for reconstruction. This series included two female and three male patients, with a mean age of 42.2 years (range, 31-53 years). Surgical time, blood loss, complications, preoperative and postoperative neurological function, instrumentation failure, and local control were presented and analyzed. RESULTS: All patients underwent the operation without death or serious complications. The implant was installed on the defect, connecting the ilium and lumbar vertebrae, and fixed with a screw-rod system up to the level of L3-4 or L4-5 . The mean operative time was 502 min (range, 360-640 min) and the mean operative blood loss 4400 mL (range, 3000-7000 mL). The mean follow-up was 15 months. After the operation, pain was significantly relieved, and the patients resumed walking as early as 2 weeks later. The patients showed no neurogenic bladder dysfunction and no fecal incontinence or gait disturbance. Wound healing was poor in one patient. Patients recovered well without evidence of local recurrence. No implant failures or related clinical symptoms were detected during follow up. Satisfactory bone ingrowth and osseointegration at the bone-implant junctions was found in follow-up CT. CONCLUSION: Although technically challenging, it is feasible and safe to use a suspended, modular, and 3D-printed implant for reconstruction after total piecemeal resection with the preservation of bilateral S1-3 nerve roots in patients with SGCT. We believe that this implant can be applied to sacral reconstruction in a wide variety of diseases.

Plant-soil-foliage feedbacks on seed germination and seedling growth of the invasive plant Ageratina adenophora.

Some exotic plants become invasive because they partially release from soil-borne enemies and thus benefit from positive plant-soil feedbacks (PSFs) in the introduced range. However, reports that have focused only on PSFs may exaggerate the invader's competitiveness. Here, we conducted three experiments to characterize plant-soil-foliage feedbacks, including mature leaves (ML), leaf litter (LL), rhizosphere soil (RS) and leaves plus soil (LS), on the early growth stages of the invasive plant Ageratina adenophora. In general, the feedbacks from aboveground (ML, LL) adversely affected A. adenophora by delaying germination time, inhibiting germination rate and reducing seedling growth. The increased invasion history exacerbated the adverse effects of LL and LS feedbacks on seedling growth. These adverse effects were partially contributed by more abundant fungi (e.g. Didymella) or/and more virulent fungi (e.g. Fusarium) developed in the aboveground part of A. adenophora during the invasion. Interestingly, the aboveground adverse effects can be weakened by microbes from RSs. Our novel findings emphasize the important role of aboveground feedbacks in the evaluation of plant invasiveness, and their commonness and significance remain to be explored in other invasive systems.

Growth-promoting characteristics of potential nitrogen-fixing bacteria in the root of an invasive plant Ageratina adenophora.

Root endophytic nitrogen-fixing bacteria (reNFB) have been proposed as important contributors to the invasiveness of exotic legumes; however, the reNFB of invasive nonlegumes has received less attention. In particular, the growth-promoting effect of reNFB on invasive plants remains unknown. In this study, 131 strains of potential nitrogen-fixing bacteria were isolated and purified from the roots of the invasive plant, Ageratina adenophora, in Southwest China. Phylogenetically, these reNFB were categorized into three phyla at 97% sequence identity that included Proteobacteria (92.4%), Actinobacteria (4.6%), and Firmicutes (3.1%). The dominant isolates ranked by number were Pseudomonas (80 isolates, 61.1%), Rhizobium (12 isolates, 9.2%), and Duganella (11 isolates, 8.4%). The community composition and diversity of A. adenophora reNFB were markedly different across study regions. The capacity of these reNFB to accumulate indolyl-3-acetic acid (IAA), solubilize phosphate, and produce siderophores was determined. All 131 isolates of reNFB accumulated IAA, 67 isolates solubilized phosphate, and 108 isolates produced siderophores. Among the three dominant genera of reNFB, Pseudomonas had the highest phosphorus solubilization and siderophore production, while the accumulation of IAA in the genus Duganella was the lowest. Interestingly, the calculated reNFB Shannon diversity index of each A. adenophora individual was negatively correlated with the capacity of reNFB to produce growth-promoting products. Six randomly selected isolates from three dominant genera were further used to conduct inoculation experiments, and all isolates showed significant positive growth-promoting effects on A. adenophora seedlings. The contribution of reNFB to the root biomass was higher than that to the shoot biomass. Our results suggest that reNFB, similar to soil or nodular nitrogen-fixing bacteria, can potentially promote plant growth and may play an important role in the invasion of nonleguminous plants. More detailed studies on the correlation between reNFB and invasive plants are necessary.

Enrichment of soil rare bacteria in root by an invasive plant Ageratina adenophora.

The assembly of the root-associated microbiome provides mutual benefits for the host plant and bacteria in soils. It is interesting how invasive plants interact with the local soil microbial community and establish the soil bacterial community in the endosphere of these plants in the short term. In this study, we compared the bacterial community in the rhizosphere with that in the root endosphere of an invasive plant, Ageratina adenophora, using high-throughput sequencing. The results showed that the roots of A. adenophora selectively accumulated the genera Clostridium and Enterobacter, which are rarely distributed in the rhizosphere. This selective accumulation caused a switch in the bacterial composition at the phylum level from Bacteroidetes predominant in the rhizosphere to Proteobacteria dominant in the root endosphere of A. adenophora. Our data indicated the potential existence of a highly conserved signal recognition in which hosts, either invasive or native, enrich the endosphere bacteria, such as Clostridium, Enterobacter, etc., from the rhizosphere. Moreover, the accumulated bacteria were physiologically and genetically different at the strain level and displayed distinct roles in growth between invasive and native plants. The assembly of the bacterial community in the roots may be an advantageous strategy for A. adenophora in competition with native plants.

Systematic review with meta-analysis: Chromoendoscopy versus white light endoscopy in detection of dysplasia in patients with inflammatory bowel disease.

OBJECTIVE: To compare chromoendoscopy (CE) and white light endoscopy (WLE) for dysplasia surveillance in patients with inflammatory bowel disease (IBD). METHODS: We conducted a meta-analysis of 6 randomized controlled trials (RCTs) and 5 prospective studies and compared the CE and WLE groups. A fixed-effect model was used unless the heterogeneity was high. RESULTS: CE detected more patients with dysplasia (relative risk [RR] 2.05, 95% confidence interval [CI]: 1.62-2.61) and more dysplastic lesions (RR 2.04, 95% CI: 1.40-2.98) than WLE. When magnification was added to CE, the detection rate of patients with dysplasia and dysplastic lesions improved. CE also detected more patients with dysplasia than high-definition WLE (RR 1.60, 95% CI: 1.11-2.29). Compared with WLE, the incremental yield of CE for detecting patients with dysplasia was 9%. CE was superior to WLE in detecting non-polypoid dysplastic lesions (RR 1.38, 95% CI:1.02-1.88). CE had no advantage for detecting polypoid dysplastic lesions compared with WLE. CONCLUSIONS: CE is superior to WLE in the surveillance of dysplasia in IBD patients. It is thus necessary to use CE in the surveillance of dysplasia in patients with IBD in clinical practice. Adding magnification improves the detection rate.

Tissue-Specific and Geographical Variation in Endophytic Fungi of Ageratina adenophora and Fungal Associations With the Environment.

To understand the distribution of the cultivable fungal community in plant tissues and the associations of these fungi with their surrounding environments during the geographical expansion of an invasive plant, Ageratina adenophora, we isolated the cultivable fungi from 72 plant tissues, 12 soils, and 12 air samples collected from six areas in Yunnan Province, China. A total of 4066 isolates were investigated, including 1641 endophytic fungi, 233 withered leaf fungi, 1255 fungi from air, and 937 fungi from soil. These fungi were divided into 458 and 201 operational taxonomic units (OTUs) with unique and 97% ITS gene sequence identity, respectively. Phylogenetic analysis showed that the fungi belonged to four phyla, including Ascomycota (94.20%), Basidiomycota (2.71%), Mortierellomycota (3.03%), and Mucoromycota (0.07%). The dominant genera of cultivable endophytic fungi were Colletotrichum (34.61%), Diaporthe (17.24%), Allophoma (8.03%), and Fusarium (4.44%). Colletotrichum and Diaporthe were primarily isolated from mature leaves, Allophoma from stems, and Fusarium from roots, indicating that the enrichment of endophytic fungi is tissue-specific and fungi rarely grew systemically within A. adenophora. In the surrounding environment, Alternaria (21.46%), Allophoma (19.31%), Xylaria (18.45%), and Didymella (18.03%) were dominant in the withered leaves, Cladosporium (22.86%), Trichoderma (14.27%), and Epicoccum (9.83%) were dominant in the canopy air, and Trichoderma (27.27%) and Mortierella (20.46%) were dominant in the rhizosphere soils. Further analysis revealed that the cultivable endophytic fungi changed across geographic areas and showed a certain degree of variation in different tissues of A. adenophora. The cultivable fungi in mature and withered leaves fluctuated more than those in roots and stems. We also found that some cultivable endophytic fungi might undergo tissue-to-tissue migration and that the stem could be a transport tissue by which airborne fungi infect roots. Finally, we provided evidence that the fungal community within A. adenophora was partially shared with the contiguous environment. The data suggested a frequent interaction between fungi associated with A. adenophora and those in surrounding environments, reflecting a compromise driven by both functional requirements for plant growth and local environmental conditions.

Asymmetric Total Synthesis and Absolute Configuration Determination of (-)-Verrupyrroloindoline.

The first asymmetric total synthesis of (-)-verrupyrroloindoline (20% overall yield in 6 steps) is described. The short approach was enabled by Buchwald's Cu(II)-catalyzed asymmetric conjugate reduction, DMDO-triggered one-pot four-step tandem reaction, and the first amide-selective Ir-catalyzed direct reduction of ß-carboethoxy tertiary lactam. Along with the total synthesis, the absolute configuration of natural verrupyrroloindoline was determined as 7 R,10 R,11 R.

Antimicrobial and antioxidant activities of Flammulina velutipes polysaccharides and polysaccharide-iron(III) complex [corrected].

FVP is polysacchrides obtained from Flammulina velutipes. A polysacchride named FVP2 was isolated from FVP by DEAE cellulose-52 chromatography and Sephadex G-100 size-exclusion chromatography. FVP-Fe and FVP2-Fe were synthesized by neutralization of FeCl3 carbohydrate solution. The antibacterial and antifungal activities of FVP, FVP2, FVP-Fe, FVP2-Fe were investigated and their antioxidant effects on hydroxyl, 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion, 2,2'-azobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, reducing power, inhibition of malondialdehyde (MDA) were assessed in vitro. The results suggested that FVP-Fe and FVP2-Fe significantly suppressed the growth of bacteria Staphylococcus aureus, Escherichia coli, and Bacillus subtilis, and have relatively strong antioxidant activity to scavenge superoxide anion radical. In addition, FVP exhibited strong antioxidant activity to eliminate hydroxyl, DPPH, ABTS radicals, had high reducing power and inhibited the MDA production of health mice liver homogenate induced by auto-oxidation and Fe2+-H2O2 system.

Transnasal Endoscopic Biopsy Approach to Atlas Tumor with X-ray Assisted and Related Radiographic Measure.

OBJECTIVE: To develop an endoscopic transnasal approach to atlas tumors and study its practicability. METHODS: This article comprises two components: an illustrative case report and observational data on 50 volunteers. As to the case report, a 34 year old man presented with occipital pain for more than 3 months and underwent systematic investigation in Qilu Hospital of Shandong University. CT and MRI scans showed bony destruction in the craniovertebral junction (CVJ) suggestive of tumor. Via an endoscopic transnasal approach to the suspected atlas tumor through the inferior nasal meatus, a Gallini biopsy needle was used to obtain tissue for examination. The procedure was performed endoscopically with double orientation X-ray guidance and coaxial technology after establishing the shortest distance for the biopsy track and range of target tissue and was assisted by manual palpation. As to the observational data, 50 volunteers underwent atlas-related morphometric image measurement using gemstone CT equipment. Biopsy track angles, range for biopsy of the atlas and biopsy track distances were measured by a blinded operator on CT images. CASE REPORT: pathological examination of the biopsy resulted in diagnosis of a chordoma. There were no complications such as bleeding, infection or spinal cord injury. One month later, the patient underwent tumor resection and reconstruction in other hospital and the diagnosis of chordoma was confirmed by pathological examination of the resected specimen. Observational data: measurements obtained from CT scans of the 50 volunteers were as follows. Biopsy track angles: mean leaning inside angle 3.53° ± 0.39° and mean posterior slope angle 13.05° ± 1.39°. Range for atlas biopsy: transverse diameter 11.84 ± 1.24 mm and longitudinal diameter 9.67 ± 0.90 mm. Biopsy track distances: from atlas to nostril, and to anterior and posterior edges of the inferior turbinate mucosa were 94.52 ± 5.03 mm, 78.21 ± 4.63 mm, and 33.51 ± 3.13 mm, respectively. CONCLUSIONS: An endoscopic transnasal approach enables biopsy and diagnosis of tumors in the anterior arch of the atlas. Relevant measurements were obtained by assessing CT scans of 50 volunteers to assist operators to determine the effective and safe range for transnasal atlas biopsy.

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Unreasonable application of nitrogen fertilizer to cropland decreases nitrogen use efficiency of crop. A large amount of nitrogen loss to environment through runoff, leaching, ammonia volati-lization, nitrification-denitrification, etc., causes water and atmospheric pollution, poses serious environmental problems and threatens human health. The type of nitrogen fertilizer and its application rate, time, and method have significant effects on nitrogen loss. The primary reason for nitrogen loss is attributed to the supersaturated soil nitrogen concentration. Making full use of environmental nitrogen sources, reducing the application rate of chemical nitrogen fertilizers, applying deep placement fertilizing method, and applying organic fertilizers with chemical nitrogen fertilizers, are effective practices for reducing nitrogen loss and improving nitrogen use efficiency. It is suggested that deve-loping new high efficiency nitrogen fertilizers, enhancing nitrogen management, and strengthening the monitoring and use of environmental nitrogen sources are the powerful tools to decrease nitrogen application rate and increase efficiency of cropland.

The asymmetric total synthesis of (+)-N-acetyl norloline.

The asymmetric total synthesis of (+)-N-acetyl norloline, the putative biogenic precursor of all known loline alkaloids, has been achieved in 12 steps from commercially available (R)-glyceraldehyde acetonide. The synthesis relies on the Rassu/Casiraghi's vinylogous aldol reaction, an intramolecular oxa-heteroconjugate addition and a reductive amination to establish the four contiguous stereogenic centers and construct the strained oxygen-bridge under mild conditions.

The Association of Serum IL-33 and sST2 with Breast Cancer.

Breast cancer is one of the most common malignant diseases in women. The main cause of death from breast cancer is its metastases at distant sites in the body. Interleukin-33 (IL-33) is a cytokine of the IL-1 family and found overexpressed in various cancers. The aim of the present study was to explore the association of serum IL-33 and sST2 with breast cancer. Here, the serum levels of Interleukin-33 (IL-33) and sST2 were found significantly higher in breast cancer patients than in healthy volunteers. Serum levels of vascular endothelial growth factor (VEGF), metalloproteinase-11 (MMP-11), and platelet-derived growth factor-C (PDGF-C) were also greater in breast cancer patients compared to healthy volunteers. We found that serum levels of IL-33 or sST2 were positively correlated with the serum levels of VEGF, MMP-11, and PDGF-C. Moreover, breast cancer dataset downloaded from The Cancer Genome Atlas showed that patients with higher level of MMP-11 or PDGF-C expression had shorter survival time than those with lower level of these proteins. In conclusion, IL-33 and sST2 may serve as noninvasive diagnosis markers for breast cancer. IL-33 and sST2 were significantly associated with MMP-11 or PDGF-C which indicated poor prognosis of breast cancer patients.

Equol inhibits proliferation of human gastric carcinoma cells via modulating Akt pathway.

AIM: To investigate the anti-tumor effects of equol in gastric cancer cells and the underlying molecular mechanisms. METHODS: MGC-803 cells were employed for in vitro experiments in this study. Cells were treated with control (vehicle, 0.1% DMSO) or equol under specified dose titration or time courses. Cell viability was examined by MTS assay, and the levels of Ki67 were determined by qPCR and immunofluorescent assay. Changes in cell cycle distribution and apoptosis rate were detected by flow cytometry. The mRNA expression of cyclin E1 and P21(WAF1) was determined by qPCR. The protein levels of cell cycle regulators, PARP and Caspase-3 cleavage, and the phosphorylation of Akt were examined by Western blot. In addition, to characterize the role of elevated Akt activation in the anti-tumor effect exerted by equol, Ly294002, a PI3K/AKT pathway inhibitor, was used to pretreat MGC-803 cells. RESULTS: Equol (5, 10, 20, 40, or 80 µmol/L) inhibited viability of MGC-803 cells in a dose- and time-dependent manner after treatment for 24, 36, or 48 h (P < 0.05 for all). Equol also decreased the mRNA (P < 0.05 for 12 and 24 h treatment) and protein levels of Ki67. Equol treatment significantly induced G0/G1 cell cycle arrest (P < 0.05), with the percentages of G0/G1 cells of 32.23% ± 3.62%, 36.31% ± 0.24%, 45.58% ± 2.29%, and 65.10% ± 2.04% for equol (0, 10, 20, or 30 µmol/L) treatment, respectively, accompanied by a significant decrease of CDK2/4 (P < 0.05 for 24 and 48 h treatment) and Cyclin D1/Cyclin E1 (P < 0.05), and an increased level of P21(WAF1) (P < 0.05). A marked increase of apoptosis was observed, with the percentages of apoptotic cells of 5.01% ± 0.91%, 14.57% ± 0.99%, 37.40% ± 0.58%, and 38.46% ± 2.01% for equol (0, 5, 10, or 20 µmol/L) treatment, respectively, accompanied by increased levels of cleaved PARP and caspase-3. In addition, we found that equol treatment increased P-Akt (Ser473 and Thr308) at 12 and 24 h compared to vehicle-treated control; longer treatment for 48 h decreased P-Akt (Ser473 and Thr308). P-Akt at Thr450, however, was decreased by equol treatment at all time points examined (P < 0.05 for all). Moreover, Akt inhibition by Ly294002 could not prevent but led to enhanced G0/G1 arrest and apoptosis. CONCLUSION: Equol inhibits MGC-803 cells proliferation by induction of G0/G1 arrest and apoptosis. Its anti-cancer effects are likely mediated by dephosphorylation of Akt at Thr450.

Effects of melatonin combined with Cis-platinum or methotrexate on the proliferation of osteosarcoma cell line SaOS-2.

OBJECTIVE: To evaluate the effects of melatonin (Mel) combined with cis-platinum (DDP) or methotrexate (MTX) on the proliferation of osteosarcoma cell line SaOS-2, and to explore whether Mel combined with DDP or MTX could play a synergistic antitumor effect. METHODS: SaOS-2 was treated with Mel alone or Mel combined with DDP or MTX. Cell counting kit-8 assay was used to measure the cell activities. Combination index(CI) value was used to evaluate the combined effects: CI<1 indicating synergetic effect, CI=1 additive, and CI>1 antagonistic.Flow cytometry was used to analyze cell cycle distribution and cell apoptosis. RESULTS: After treated with Mel (0.5,1,2,4,5 mmol/L), DDP (6.67, 16.67, 33.33, 66.66 µmol/L) or MTX(0.1, 0.5, 1, 2, 4 mmol/L)alone,SaOS-2 cell activities decreased in a dose-dependent manner (all P<0.05). The activities of SaOS-2 cell treated with both Mel (1 mmol/L) and DDP or MTX were significantly lower than that of DDP or MTX alone (all P<0.05).CI values of cells exposed to 1 mmol/L Mel plus 6.67, 16.67, 33.33, and 66.66 µmol/L DDP were 1.18, 1.21, 1.09, and 0.84, respectively,and CI values of cells exposed to 1 mmol/L Mel plus 0.1, 0.5, 1, 2, and 4 mmol/L MTX were 0.88, 0.88 ,0.83, 0.78, and 0.81, respectively. The G1-stage cells were increased and the S-stage cells were reduced when the cells were treated with Mel (1 mmol/L) alone or combined with MTX (0.5 mmol/L) (P<0.05). The S-stage cells were increased when the cells treated with MTX (0.5 mmol/L) (P<0.05). The apoptotic cells were increased when they treated with Mel (1 mmol/L) alone or combined with DDP (16.67 µmol/L) or MTX (0.5 mmol/L) (P<0.05). When the cells were treated with Mel combined with DDP or MTX, the apoptotic cells were more than that of DDP or Mel alone(P<0.05). CONCLUSIONS: Mel can inhibit SaOS-2 cells activity,block the cell cycle at G1-stage,and induce apoptosis. Mel has an antagonistic effect with lower concentration of DDP but a synergistic effect with MTX or higher concentration of DDP.