Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells

Apigenin （4＇,5,7-trihydroxyflavone） is a member of the flavone subclass of flavonoids present in fruits and vegetables. The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated. Cell proliferation and viability were assessed by 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MTT） and clonogenic assays. Flow cytometry, fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy, and the role of autophagy was assessed using autophagy inhibitors. Apigenin dose- and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines. The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3, PARP cleavage and Bax/Bcl-2 ratios. The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles （AVOs） by flow cytometry. Furthermore, the results of the Western blot analysis revealed that the level of LC3-Ⅱ, the processed form of LC3-Ⅰ, was increased. Treatment with the autophagy inhibitor, 3-methyladenine （3-MA）, significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the level of PARP cleavage. Similar results were also confirmed by flow cytometry and fluorescence microscopy. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in breast cancer cells. Autophagy plays a cyto-protective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.

CUL4 E3 ligase regulates the proliferation and apoptosis of lung squamous cell carcinoma and small cell lung carcinoma

Objective:The E3 ligase,CRL4,plays diverse roles in different cellular processes,such as DNA damage,transcriptional regulation,cell cycle progression,and cell apoptosis.Our previous study showed that CUL4A and CUL4B had a strong association with tobacco smoking risk in lung squamous cell carcinoma(SCC)and small cell lung carcinoma(SCLC).This study aimed to define the potential mechanism underlying the roles of CUL4A and CUL4B in the development of SCC and SCLC.Methods:We determined the role of CUL4A and CUL4B in the cell cycle and apoptosis of SCC and SCLC,and identified the key apoptosis-related gene involved in the oncogenic activity of CUL4B by Western blot,immunohistochemical staining,flow cytometry,and enzyme inhibition experiments.Results:We found that depletion of CUL4A and CUL4B reduced the proliferation of SCC and SCLC cells.cUL4Aknockdown but not CUL4Bknockdown arrested cells in Gl phase while upregulating P21 and cU L4Bknockdown promoted cell apoptosis through upregulation o f FOXO3A.Accordingly,CUL4B decreased FO X03A expression by activating the ERK signaling pathway and mediating FOXO3A degradation via the ubiquitin-proteasome pathway.Conclusions:These results identified the function of E3 ligase CRL4 in regulating SCC and SCLC cell proliferation,which provides a potential strategy for cancer therapy by targeting FOXO3A and the E3 ligase,CRL4.

Multidisciplinary team for the diagnosis and treatment of 2cases of primary intestinal yolk sac tumor

Extragonadal primary yolk sac tumor of the intestinal tract origin is exceedingly rare. Through a multiple disciplinary team, the diagnosis and treatment of primary intestinal yolk sac tumor were further defined. We report 2 such cases with detailed histologic and immunohistochemical analysis. The two patients were a 7-year-old girl and a 29-year-old woman. Both of them preoperatively had an elevated serum alpha fetoprotein(AFP) level(≥ 1,210 ng/mL). The tumors are located in the intestine and imaging examination indicated the rectum as the primary site. Grossly the mass was grey-white and crisp texture. Microscopic examination featured reticular, microcystic, macrocystic, papillary, solid, and some glandular patterns. Immunohistochemically,tumor cells of both cases were positive for SALL4, AFP, pan-cytokeratin(AE1/AE3), and glypican-3. Simultaneously, a stain for EMA, OCT4, CD30, HCG, vimentin and CK20 were negative in all 2 neoplasms. The features of morphology,immunohistochemistry, laboratory examinations and imaging studies consist of the diagnosis of primary yolk sac tumor of the intestine.

A nomogram-based immune-serum scoring system predicts overall survival in patients with lung adenocarcinoma

Objective:The immunoscore,which is used to quantify immune infiltrates,has greater relative prognostic value than tumor,node,and metastasis(TNM)stage and might serve as a new system for classification of colorectal cancer.However,a comparable immunoscore for predicting lung adenocarcinoma(LUAD)prognosis is currently lacking.Methods:We analyzed the expression of 18 immune features by immunohistochemistry in 171 specimens.The relationship of immune marker expression and clinicopathologic factors to the overall survival(OS)was analyzed with the Kaplan-Meier method.A nomogram was developed by using the optimal features selected by least absolute shrinkage and selection operator(LASSO)regression in the training cohort(n=111)and evaluated in the validation cohort(n=60).Results:The indicators integrated in the nomogram were TNM stage,neuron-specific enolase,carcino-embryonic antigen,CD8 center of tumor(CT),CD8 invasive margin(IM),Fox P3 CT,and CD45 ROCT.The calibration curve showed prominent agreement between the observed 2-and 5-year OS and that predicted by the nomogram.To simplify the nomogram,we developed a new immune-serum scoring system(I-SSS)based on the points awarded for each factor in the nomogram.Our I-SSS was able to stratify same-stage patients into different risk subgroups.The combination of I-SSS and TNM stage had better prognostic value than the TNM stage alone.Conclusions:Our new I-SSS can accurately and individually predict LUAD prognosis and may be used to supplement prognostication based on the TNM stage.

Molecular ecological networks reveal the spatial-temporal variation of microbial communities in drinking water distribution systems

Microbial activity and regrowth in drinking water distribution systems is a major concern for water service companies.However,previous studies have focused on the microbial composition and diversity of the drinkingwater distribution systems(DWDSs),with little discussion on microbial molecular ecological networks(MENs)in different water supply networks.MEN analysis explores the potentialmicrobial interaction and the impact of environmental stress,to explain the characteristics of microbial community structures.In this study,the random matrix theory-based network analysis was employed to investigate the impact of seasonal variation including water source switching on the networks of three DWDSs that used different disinfection methods.The results showed that microbial interaction varied slightly with the seasons but was significantly influenced by different DWDSs.Proteobacteria,identified as key species,play an important role in the network.Combined UV-chlorine disinfection can effectively reduce the size and complexity of the network compared to chlorine disinfection alone,ignoring seasonal variations,which may affect microbial activity or control microbial regrowth in DWDSs.This study provides new insights for analyzing the dynamics of microbial interactions in DWDSs.

A needle tip CCEA microfluidic device based on enhanced Dean flow for cell washing

Particle/cell washing is an essential technique in biological and clinical manipulations.Herein,we propose a novel circular contraction-expansion array(CCEA)microdevice.It can be directly connected to a needle tip without connection tubes.Its small size and centrosymmetric structure are beneficial to low sample consumption,high connection stability,and a wide application range.Computational fluid dynamics(CFD)simulation results show that the CCEA structure can produce a stronger Dean flow and lead to faster particle/cell focusing than the circle structure and CEA structure with the same length.Experimentally,an optimal flow rate ratio of 1:3 and an optimal total flow rate of 120μL/min were found to ensure a stable fluid distribution.Under these conditions,rapid focusing of 10-20μm particles with high efficiencies was achieved.Compared with a normal CEA device using tubes,the particle loss rate could be reduced from 64 to 7%when washing 500μL of a rare sample.Cell suspensions with concentrations from 3×10^(5)/mL to 1×10^(3)/mL were tested.The high cell collection efficiency(>85%for three cell lines)and stable waste removal efficiency(>80%)reflected the universality of the CCEA microfluidic device.After the washing,the cell activities of H1299 cells and MCF-7 cells were calculated to be 93.8 and 97.5%,respectively.This needle-tip CCEA microfluidic device showed potential in basic medical research and clinical diagnosis.