Silencing of long non-coding RNA CCHE1 inhibits the ovarian cancer SKOV3 cell invasion and migration and inactivates the p38-MAPK signaling pathway

Ovarian cancer (OC) is a major cause of cancer-related deaths among gynaecological malignancies. Emerging studiessuggest that the long non-coding RNA (lncRNA) may be the potential biomarker for the diagnosis and prognosis of the cancer.The current study was carried out to investigate the role of lncRNA CCHE1 silencing in OC cell invasion and migration.Expression of lncRNA CCHE1 in normal ovarian cell Hose and OC cell lines HO 8910, A2780 and SKOV3 was detected.LncRNA were transfected with siRNA, and then the proliferation of cells was detected by using MTT assay. Cell invasionand migration was measured by using Transwell assay and scratch test, respectively. The protein levels of E-cadherin,N-cadherin, ERK, p38-MAPK and the phosphorylation of ERK and p38-MAPK in cells after siRNA transfection weredetected by using Western blot analysis. Consequently, lncRNA CCHE1 expression was highly expressed in OC cell lines,especially in SKOV3 cells. siRNA1, siRNA2 and siRNA3 all decreased. lncRNA CCHE1 expression in SKOV3 cells andsiRNA2 showed the best silencing efficacy. Silencing of lncRNA CCHE1 decreased proliferation, invasion and migration,and reduced the protein levels of N-cadherin, ERK, p38-MAPK and the phosphorylation of ERK and p38-MAPK, whilereducing the protein level of E-cadherin in SKOV3 cells. Collectively, our study proved that the silencing of lncRNACCHE1 could inhibit SKOV3 cell invasion and migration via inactivating the p38-MAPK signaling pathway.     

17‐beta‐estradiol analog inhibits cell proliferation by induction of apoptosis in breast cell lines

Microtubules are important targets when studying potential anticancer agents since disturbance of these microtubule dynamics results in cell cycle arrest and cell death. 2‐Methoxyestradiol is a naturally occurring metabolite that exerts antiproliferative activity and induces apoptosis. Due to limited biological accessibly and rapid metabolic degradation, several analogs were synthesized. This study investigated the antiproliferative influence of an 2‐methoxyestradiol analog, (8R, 13S, 14S, 17S)‐2‐Ethyl‐13‐methyl‐7, 8, 9, 11, 12,13, 14, 15, 16, 17‐decahydro‐6H‐cyclopenta[a]phenanthrane‐3, 17‐diyl bis(sulfamate) (EMBS) on cell proliferation, morphology and apoptosis induction in a estrogen receptor‐positive breast adenocarcinoma cells line (MCF‐7), estrogen receptor‐negative highly metastatic breast cell line (MDA‐MB‐231) and a non‐tumorigenic breast epithelial cell line (MCF‐12A). Spectrophotometry results indicated that EMBS exerted differential antiproliferative activity in the three cell lines. Cell growth of the breast adenocarcinoma and highly metastatic breast cell line reached a plateau effect at 0.4 μM after 24 h of exposure. Light microscopy and polarization‐optical transmitted light differential interference contrast demonstrated compromised cell density, cells blocked in metaphase and the presence of apoptotic characteristics after EMBS exposure for 24 h in all three cell lines. Transmission electron microscopy and scanning electron microscopy revealed hallmarks of apoptosis namely the presence of apoptotic bodies, shrunken cells and cell debris in EMBS‐exposed cells. This investigation demonstrated that EMBS does exert antimitotic activity and induces apoptosis contributing to elucidating the signal transduction of EMBS in tumorigenic and non‐tumorigenic breast cell lines. Findings warrant in‐depth analysis of specific targets in vitro and subsequent in vivo investigation for anticancer therapy. Microsc. Res. Tech. 77:236–242, 2014. © 2014 Wiley Periodicals, Inc.     

Chrysophanol inhibits the progression of gastric cancer by activating nod-like receptor protein-3

Aim: Gastric cancer (GC) is one of the most common malignant tumors. Chrysophanol has been reported topossess antitumor effects on a variety of cancers; however, its role in GC remains unclear. This study aimed to investigatethe effects of chrysophanol on the proliferation, pyroptosis, migration, and invasion of GC cells. Methods: Human GCcell lines MKN 28 and AGS cells were treated with different concentrations of chrysophanol, then cell proliferation,migration, invasion and pyroptosis were determined by CCK-8, colony-forming assay, wound healing assay, Transwellassay, and flow cytometry. Cell migration and invasion were reassessed in these transfected cells following thetransfection of nod-like receptor protein-3 (NLRP3) siRNA in MKN 28 and AGS cells. To examine the downstreamsignaling pathway of the NLRP3 signaling pathway, NLRP3, caspase-1, gasdermin-D, interleukin (IL)-1β, and IL-18were detected by quantitative real-time-polymerase chain reaction or western blotting. Results: Chrysophanolinhibited the proliferation of GC cells, caused pyroptosis, inhibited cell migration and invasion, and increased theexpression of NLRP3 inflammasomes in GC cells. Knockdown of NLRP3 inhibited the effects of chrysophanol onproliferation, pyroptosis, migration, and invasion of GC cells. Chrysophanol plays an anticancer role by enhancingNLRP3. Conclusions: Chrysophanol exerts anti-neoplastic effects in vitro in GC cells by modulating NLRP3, thushighlighting its therapeutic potential in GC.     

Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins

This study shows a strong association between cell attachment to substratum and activation of β1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells. We propose a mechanistic-driven approach to sensitize the cells to camptothecins. ZR-75-1 anchoragedependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 µM) or CPT-11 (40 µM) for 48 h. Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells. Because p125 focal adhesion kinase (FAK) is a transducer in the β1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins. Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11.     

Berberine inhibits the proliferation of pancreatic cancer cells by targeting pancreatic cancer stem cells through regulating EMT signaling pathway

Pancreatic ductal adenocarcinoma (PDAC) is universally acknowledged as the cancer with the highest mortalityrate. Berberine has high medicinal value and has been used as an anti-cancer agent. Hence the purpose of this study was toinvestigate the anti-cancer effect of berberine in PDAC. Berberine was shown to have a selective anti-cancer effect onPDAC by MTT assay in vitro. Pancreatic cancer stem cells (PCSCs), regulated by epithelial–mesenchymal transition(EMT), could promote the proliferation of PDAC cells. However, berberine suppressed the proliferation and stemnessof PCSCs through immunofluorescence staining, stem cell sphere assays and so forth in vitro. In vivo, berberinereduced tumor size and decreased the expression levels of Ki67, a marker of cellular proliferation, in orthotopicpancreatic tumors. In addition, berberine inhibited the EMT signaling pathway by RT-PCR and Western blottingmethods both in vitro and in vivo. Our study indicates that berberine inhibits the proliferation of PDAC cells both invivo and in vitro. The mechanism of the anti-cancer effect of berberine likely involves the inhibition of EMT.Therefore, berberine may be a novel antineoplastic drug with clinical efficacy in PDAC.     

Long non-coding RNA MIR22HG inhibits the adipogenesis of human bone marrow mesenchymal stem cells with the involvement of Wnt/β-catenin pathway

Osteoporosis is a frequently occurring bone remodeling disorder worldwide with one characteristic being decreasing bone mineral density and a predisposition to bone fracture, which diminishes patients’ quality of life. Several studies showed that imbalance between the osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) took part in the development of osteoporosis. In previous study, we found MIR22HG regulated the osteogenesis of human BMSCs positively. In this study, we found that MIR22HG was decreased during the adipogenesis of human BMSCs and exerted negative effects on adipogenesis with the involvement of Wnt/β-catenin signaling pathway both in vitro and in vivo. Nitazoxanide could inhibit Wnt signaling and relieve MIR22HG’s suppression on adipogenesis. These findings indicated that MIR22HG had great potential in clinical application for osteoporosis treatment and prevention.     

MicroRNA-382 inhibits the proliferation of mouse spermatogonia by targeting <i>Kmt5a</i>

Spermatogenesis is a highly efficient and intricate process in the testis by which mature spermatozoa areproduced daily to maintain lifelong male fertility. Essential to this process are spermatogonia capable of bothproliferation and differentiation. Nevertheless, the underlying mechanisms for spermatogonial proliferation anddifferentiation remain poorly understood. MicroRNAs (miRNAs) are a category of non-coding small RNAs withregulatory functions by binding to the 3’ untranslated region (UTR) of the target mRNA. Previous studies havedemonstrated that miRNAs are capable of modulating cell proliferation, differentiation and apoptosis, but the roles ofindividual miRNAs in spermatogonial fate determination remain largely elusive. Here, by using a mousespermatogonial cell line (GC-1), we investigated the role for miRNA-382 in spermatogonial proliferation. We foundthat pre-miRNA-382 was expressed in spermatogonia. The luciferase reporter assay demonstrated Kmt5a but notTop1 as a target gene of miRNA-382. Overexpression of miRNA-382 by transfecting a miRNA mimic downregulatedKmt5a at both RNA and protein levels, and further reduced the proliferation and viability of spermatogonia.Knockdown of Kmt5a by RNA interference (RNAi) resulted in a uniform phenotype in spermatogonia. We thereforeconclude that miRNA-382 inhibits the proliferation of mouse spermatogonia by targeting Kmt5a. Our finding extendsthe knowledge about the regulatory roles of miRNAs in spermatogonia and lays the groundwork for diagnosis andtreatment of male infertility.     

Cloud‐based decision support system for the detection and classification of malignant cells in breast cancer using breast cytology images

The advancement of computer‐ and internet‐based technologies has transformed the nature of services in healthcare by using mobile devices in conjunction with cloud computing. The classical phenomenon of patient–doctor diagnostics is extended to a more robust advanced concept of E‐health, where remote online/offline treatment and diagnostics can be performed. In this article, we propose a framework which incorporates a cloud‐based decision support system for the detection and classification of malignant cells in breast cancer, while using breast cytology images. In the proposed approach, shape‐based features are used for the detection of tumor cells. Furthermore, these features are used for the classification of cells into malignant and benign categories using Naive Bayesian and Artificial Neural Network. Moreover, an important phase addressed in the proposed framework is the grading of the affected cells, which could help in grade level necessary medical procedures for patients during the diagnostic process. For demonstrating the e effectiveness of the proposed approach, experiments are performed on real data sets comprising of patients data, which has been collected from the pathology department of Lady Reading Hospital of Pakistan. Moreover, a cross‐validation technique has been performed for the evaluation of the classification accuracy, which shows performance accuracy of 98% as compared to physical methods used by a pathologist for the detection and classification of the malignant cell. Experimental results show that the proposed approach has significantly improved the detection and classification of the malignant cells in breast cytology images.     

Chaperone-mediated autophagy targeting chimeras (CMATAC) for the degradation of ERα in breast cancer

Estrogen receptor alpha (ERα/ESR1) is overexpressed in over half of all breast cancers and is considered avaluable therapeutic target in ERα positive breast cancer. Here, we designed a membrane-permeant Chaperonemediated Autophagy Targeting Chimeras (CMATAC) peptide to knockdown endogenous ERα protein throughchaperone-mediated autophagy. The peptide contains a cell membrane-penetrating peptide (TAT) that allows thepeptide to by-pass the plasma membrane, an αI peptide as a protein-binding peptide (PBD) that binds specifically toERα, and CMA-targeting peptide (CTM) that targeting chaperone-mediated autophagy. We validated that ERαtargeting peptide was able to target and degrade ERα to reduce the viability of ERα positive breast cancer cells. Takentogether, our studies provided a new method to reduce the level of intracellular ERα protein via CMATAC, and thusmay provide a new strategy for the treatment of ERα positive breast cancer.     

Human umbilical cord blood mesenchymal stem cells conditioned media inhibits hypoxia-induced apoptosis in H9c2 cells by activation of the survival protein Akt

This work aimed to study the beneficial role of human umbilical cord blood-derived mesenchymal stem cellconditioned medium (MSC-CM) in hypoxia-induced apoptosis in H9c2 cardiomyoblasts, in which the serine/heroinekinases (Akt) pathway would be involved. For this, CM was collected by culturing MSCs in serum-free DMEMmedium for 24 h, and paracrine factors were analyzed by protein chip. H9c2 cells were divided into the followinggroups: control group, hypoxia group, MSC-CM intervention group (CM group), MSC-CM + Akt phosphorylationinhibitor (LY294002) group (LY group). Apoptosis of the H9c2 cells was tested with chromatin dye Hoechst 33342and FITC-conjugated Annexin V apoptosis detection kit by flow cytometer after a hypoxia/serum deprivation (H/SD)for 24 h. The apoptosis-related proteins were evaluated by Western blot. MSC-CM displayed significantly elevatedlevels of growth factors, anti-inflammatory, and anti-apoptosis cytokines. On Hoechst 33342 apoptosis staining, theH9c2 cell morphology displayed a lower proportion of apoptosis in the CM group than those in the hypoxia group,while apoptosis was increased in LY group. Flow cytometer analysis revealed the apoptosis ratio in the CM group waslower than the hypoxia group (12.34 ± 2.00% vs. 21.73 ± 2.58%; p < 0.05), while the LY group was significantly higher(22.54 ± 3.89%). Active caspase-3 expression was increased in hypoxia group than control group (p < 0.05), butdecreased in CM group (p < 0.01). Umbilical cord blood-derived mesenchymal stem cell-conditioned media secretemultiple paracrine factors that are able to inhibit hypoxia-induced H9c2 cardiomyoblasts apoptosis, and in which theactivation of Akt phosphorylation is involved to achieve the protective effect.     

Observations on fretting damage transition to cracking; state of the art and preliminary observations

Interrupted fretting fatigue experiments were performed on 7075-T6 aluminum alloy and fretting damage characterized by confocal and scanning electron microscopy. Strain, induced by specimen fatigue, produces a small amplitude oscillatory motion between the fatigue specimen and the fretting pad. A fretting fatigue damage threshold exists in this material. Hundred percent fretting fatigue was defined as the average total cycles to fracture based on specimens 1–5 (both axial and normal forces were applied). Specimens had fretting damage induced at 100%, 80%, 60%, 40%, and 20% of the maximum fretting fatigue cycles to fracture. A positive correlation was not found between the depth of fretting damage and crack formation, but there appeared to be a stronger relationship between the fretting damaged surface areas, proximity of pits and crack nucleation sites.     

Arsenic trioxide inhibits the activity of SphK1 by decreasing the level of phosphatidylserine and phosphatidic acid in the human gastric cancer cell line MGC-803

Sphingosine kinase 1 (SphK1) is an important synthetase during the synthesis of sphingosine-1-phosphate (S1P)from sphingosine (Sph). Previous studies demonstrated that arsenic trioxide (As₂O₃) could reduce the level of S1P in humangastric cancer cell line MGC-803, indicating that As₂O₃ may inhibit the activity of SphK1. In this study, the effect of As₂O₃on the SphK1 activation pathway was investigated. Western blot and quantitative real-time PCR analysis were used toevaluate the changes in protein and mRNA levels. The multi-dimensional mass spectrometry-based shotgun lipidomicsmethod (MDMS-SL) was used for the quantitative detection of phosphatidylserine (PS) and phosphatidic acid (PA). Theresults revealed that As₂O₃ did not affect the protein and mRNA expression of SphK1 in the MGC-803 cells. However,As₂O₃ increased the levels of p-ERK1/2 and CIB1 in the SphK1 activation pathway and decreased the levels of PS andPA in the MGC-803 cells. The outcomes suggested that As₂O₃ may enhance the activity of SphK1 by increasing thelevels of p-ERK1/2 and CIB1 and decrease the activity of SphK1 by decreasing the levels of PS and PA. It was suggestedthat the inhibition effect is stronger and resulting in an overall decrease in the activity of SphK1.     

Characterization of the effective atomic number for first row transition elements by the ratio of coherent to compton scattering intensities obtained by wavelength dispersive X-ray fluorescence

The effective atomic numbers of compounds of the first row transition elements were determined experimentally by a scattering method using wavelength dispersive X-ray fluorescence spectrometer. A calibration curve was created by using the intensity ratios of coherent to Compton scattered peaks of pure elements from atomic number 13–48. This relationship was employed to determine the effective atomic numbers of the compounds. The effective atomic numbers were also calculated by using empirical formulas from the literature. Mass attenuation coefficients were calculated using software. The experimentally measured values of the effective atomic numbers with the calculated values by empirical formulas were comparable.     

Correlative fluorescence and transmission electron microscopy: an elegant tool to study the actin cytoskeleton of whole-mount (breast) cancer cells

Elucidating the structure and dynamics of lamellipodia and filopodia in response to different stimuli is a topic of continuing interest in cancer cells as these structures may be attractive targets for therapeutic purposes. Interestingly, a close functional relationship between these actin-rich protrusions and specialized membrane domains has been recently demonstrated. The aim of this study was therefore to investigate the fine organization of these actin-rich structures and examine how they structurally may relate to detergent-resistant membrane (DRM) domains in the MTLn3 EGF/serum starvation model. For this reason, we designed a straightforward and alternative method to study cytoskeleton arrays and their associated structures by means of correlative fluorescence (/laser)- and electron microscopy (CFEM).
  CFEM on whole mounted breast cancer cells revealed that a lamellipodium is composed of an intricate filamentous actin web organized in various patterns after different treatments. Both actin dots and DRM's were resolved, and were closely interconnected with the surrounding cytoskeleton. Long actin filaments were repeatedly observed extending beyond the leading edge and their density and length varied after different treatments. Furthermore, CFEM also allowed us to demonstrate the close structural association of DRMs with the cytoskeleton in general and the filamentous/dot-like structural complexes in particular, suggesting that they are all functionally linked and consequently may regulate the cell's fingertip dynamics. Finally, electron tomographic modelling on the same CFEM samples confirmed that these extensions are clearly embedded within the cytoskeletal matrix of the lamellipodium.     

Weighted gene co-expression network analysis identifies a novel immune-related gene signature and nomogram to predict the survival and immune infiltration status of breast cancer

Breast cancer is one of the most common cancers in the world and seriously threatens the health of women worldwide. Prognostic models based on immune-related genes help to improve the prognosis prediction and clinical treatment of breast cancer patients. In the study, we used weighted gene co-expression network analysis to construct a co-expression network to screen out highly prognostic immune-related genes. Subsequently, the prognostic immune-related gene signature was successfully constructed from highly immune-related genes through COX regression and LASSO COX analysis. Survival analysis and time receiver operating characteristic curves indicate that the prognostic signature has strong predictive performance. And we developed a nomogram by combing the risk score with multiple clinical characteristics. CIBERSORT and TIMER algorithms confirmed that there are significant differences in tumor-infiltrating immune cells in different risk groups. In addition, gene set enrichment analysis shows 6 pathways that differ between high- and low-risk group. The immune-related gene signature effectively predicts the survival and immune infiltration of breast cancer patients and is expected to provide more effective immunotherapy targets for the prognosis prediction of breast cancer.