Measure Cell Growth Research Articles

MicroRNA-367 Inhibits Breast Cancer and Promotes Apoptosis by Targeting AT-Rich Interactive Domain-Containing Protein 1B

Introduction: Breast cancer (BC) developed in the glandular epithelial tissue of breast. microRNA (miR)-367 is an important player in cancer progression, but has never been studied in BC. This experiment tries to probe the mechanism of miR-367 in BC treatment with downstream target gene. Materials and Methods: Human BC cell lines and healthy breast epithelium cells were applied in this study. After the transfection of miR-367 inhibitor or mimic into BC cells, functional assays were conducted to measure cell growth. Afterwards, flow cytometry was employed in apoptosis verification. Then, target relation between miR-367 and ARID1B was certified. Furthermore, ARID1B level was also measured. Results: miR-367 was underexpressed in human BC cells (p < 0.05). Besides, overexpressed miR-367 inhibited BC cell proliferation and encouraged apoptosis, while underexpressed miR-367 led to an opposite outcome (p < 0.05). This experiment then implied that miR-367 dramatically suppressed the activity of cell transfected with ARID1B-wild type. miR-367 overexpression quenched ARID1B level in BC cells; while silencing miR-367 upregulated ARID1B expression (p < 0.05). Conclusion: Our experiment discovered that miR-367 quenched BC cell growth and promoted apoptosis by targeting ARID1B. This investigation may provide novel insights in BC treatment.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

Using mathematical modeling to estimate time-independent cancer chemotherapy efficacy parameters.

One of the primary cancer treatment modalities is chemotherapy. Unfortunately, traditional anti-cancer drugs are often not selective and cause damage to healthy cells, leading to serious side effects for patients. For this reason more targeted therapeutics and drug delivery methods are being developed. The effectiveness of new treatments is initially determined via in vitro cell viability assays, which determine the of the drug. However, these assays are known to result in estimates of that depend on the measurement time, possibly resulting in over- or under-estimation of the . Here, we test the possibility of using cell growth curves and fitting of mathematical models to determine the as well as the maximum efficacy of a drug ( ). We measured cell growth of MCF-7 and HeLa cells in the presence of different concentrations of doxorubicin and fit the data with a logistic growth model that incorporates the effect of the drug. This method leads to measurement time-independent estimates of and , but we find that is not identifiable. Further refinement of this methodology is needed to produce uniquely identifiable parameter estimates.

MicroRNA-519d-3p antagonizes osteosarcoma resistance against cisplatin by targeting PD-L1.

Accumulating evidence indicates that a ligand of programmed cell death receptor-1 (PD-L1) participates in the progression and recurrence of multiple malignancies, including osteosarcoma. Nevertheless, the role of PD-L1 in chemoresistance development is not fully understood. In the current study, we aim to clarify the interaction of miR-519d-3p and PD-L1 in the development of cisplatin resistance. Immunohistochemistry, quantitative reverse-transcription polymerase reaction, and Western blot were used to evaluate PD-L1 expression. MTT and transwell migration assays were used to measure cell growth and motility, respectively. ENCORI, miRCode, and miRDB databases were recruited to predict candidate miRNAs targeting PD-L1. The binding sequences of miR-519d-3p and PD-L1 3'untranslated region were identified by dual-luciferase reporter and RNA immunoprecipitation assays. Flow cytometric analysis was conducted to measure the cycle distribution and cell apoptosis. Metastatic mouse models were generated with cisplatin-resistant sublines by intravenous injection. We found that PD-L1 expression was positively correlated to cisplatin resistance and metastasis, whereas miR-519d-3p expression was reduced in cisplatin-resistant specimens and was negatively correlated to cisplatin resistance and metastasis of osteosarcoma. We demonstrated that miR-519d-3p overexpression reversed cisplatin resistance, induced G1/S phase arrest and apoptosis. In addition, we proved that miR-519d-3p inhibited lung metastasis by establishing cisplatin-resistant MG63 metastatic xenograft models. The present findings suggest that miR-519d-3p/PD-L1 axis is a novel signaling pathway contributing to cisplatin resistance. Our study provides new clues for curing refractory osteosarcoma beyond immune checkpoint inhibitors.

Scan4CFU: Low-cost, open-source bacterial colony tracking over large areas and extended incubation times

A hallmark of bacterial populations cultured in vitro is their homogeneity of growth, where the majority of cells display identical growth rate, cell size and content. Recent insights, however, have revealed that even cells growing in exponential growth phase can be heterogeneous with respect to variables typically used to measure cell growth. Bacterial heterogeneity has important implications for how bacteria respond to environmental stresses, such as antibiotics. The phenomenon of antimicrobial persistence, for example, has been linked to a small subpopulation of cells that have entered into a state of dormancy where antibiotics are no longer effective. While methods have been developed for identifying individual non-growing cells in bacterial cultures, there has been less attention paid to how these cells may influence growth in colonies on a solid surface. In response, we have developed a low-cost, open-source platform to perform automated image capture and image analysis of bacterial colony growth on multiple nutrient agar plates simultaneously. The descriptions of the hardware and software are included, along with details about the temperature-controlled growth chamber, high-resolution scanner, and graphical interface to extract and plot the colony lag time and growth kinetics. Experiments were conducted using a wild type strain of Escherichia coli K12 to demonstrate the feasibility and operation of our setup. By automated tracking of bacterial growth kinetics in colonies, the system holds the potential to reveal new insights into understanding the impact of microbial heterogeneity on antibiotic resistance and persistence.

MicroRNA-1297 suppressed the Akt/GSK3β signaling pathway and stimulated neural apoptosis in an in vivo sevoflurane exposure model.

ObjectiveCommon inhalation anesthetics used for clinical anesthesia (such as sevoflurane) may induce nerve cell apoptosis during central nervous system development. Furthermore, anesthetics can produce cognitive impairments, such as learning and memory impairments, that continue into adulthood. However, the precise mechanism remains largely undefined. We aimed to determine the function of microRNA-1297 (miR-1297) in sevoflurane-induced neurotoxicity.MethodsReverse transcription-polymerase chain reaction assays were used to analyze miR-1297 expression in sevoflurane-exposed mice. MTT and lactate dehydrogenase (LDH) assays were used to measure cell growth, and neuronal apoptosis was analyzed using flow cytometry. Western blot analyses were used to measure PTEN, PI3K, Akt, and GSK3β protein expression.ResultsIn sevoflurane-exposed mice, miR-1297 expression was up-regulated compared with the control group. MiR-1297 up-regulation led to neuronal apoptosis, inhibition of cell proliferation, and increased LDH activity in the in vitro model of sevoflurane exposure. MiR-1297 up-regulation also suppressed the Akt/GSK3β signaling pathway and induced PTEN protein expression in the in vitro model. PTEN inhibition (VO-Ohpic trihydrate) reduced PTEN protein expression and decreased the effects of miR-1297 down-regulation on neuronal apoptosis in the in vitro model.ConclusionCollectively, the results indicated that miR-1297 stimulates sevoflurane-induced neurotoxicity via the Akt/GSK3β signaling pathway by regulating PTEN expression.

Downregulation of CDC20 suppressed cell proliferation, induced apoptosis, triggered cell cycle arrest in osteosarcoma cells, and enhanced chemosensitivity to cisplatin.

Osteosarcoma (OS) is a common malignant bone tumor that occurs in adolescents or children under the age of 20, which is extremely difficult to cure and has a high recurrence rate. Recent studies showed that cell division cycle 20 (CDC20) overexpression is associated with poor prognosis in patients with osteosarcoma. However, the function of CDC20 in osteosarcoma has not been investigated clearly. In this study, we aim to explore the role of CDC20 in two independent human OS cell lines' biological phenotype and chemotherapy sensitivity. We applied multiple approaches to measure cell growth, cell cycle, and apoptosis with or without deregulation or overexpression of CDC20. We found that the downregulation of CDC20 by siRNA or apcin suppressed cell proliferation, induced apoptosis, and triggered cell cycle arrest. Consistently, overexpression of CDC20 in normal cells promoted cell growth, inhibited apoptosis. What's more, the additional treatment with siCDC20 or Apcin achieved better anticancer effects than that of cisplatin alone. Furthermore, Bim and p21 were upregulated in OS cells following Apcin treatment. Altogether, the results of the present study demonstrated that targeting CDC20 could be useful for the treatment of OS, and might be a promising solution for the treatment of the OS with cisplatin insensitivity.

Triangulation of methods using insect cell lines to investigate insecticidal mode-of-action.

This study investigated three in vitro models to assist in elucidating possible mode-of-action, which could be adopted to evaluate insecticidal activity of complex, unknown, or multi-constituent formulations. We used a combination of absorbance spectrometry, confocal scanning laser microscopy and microelectrode ion flux estimation (MIFE) to provide insight into potential target sites for insecticides. This study used two insect cell lines and evaluated three pyrethroid insecticides. We observed that the two cell lines produced distinctly different responses. Drosophila melanogaster D.mel-S2 cell line was a useful model to monitor ion flux changes, resulting from insecticides with neural toxicity; however, it was less useful to determine some metabolic pathway indicators of toxic stress. Conversely, the Spodoptera frugiperda Sf9 cell line produced acute reactive oxygen species (ROS) in response to insecticide treatments, but was not highly responsive in electrophysiological experiments. We also showed that the natural, multi-constituent botanical extract of pyrethrum elicited different Na+ , Cl- and Ca2+ ion fluxes than its synthetic, single constituent analogues, α-cypermethrin and esfenvalerate. These two methods used in combination with absorbance spectrometry measuring cell growth inhibition plus cell mortality assays shed some light on cytotoxic responses in differing model cell lines. This research highlights the importance of using multiple cell types and interdisciplinary methods to provide a better insight into mode of insecticidal action. This is especially pertinent to novel biopesticide discovery, as the underlying mechanisms for toxicity in initial screening processes are likely to be unknown.

Long Noncoding RNA LINC01485 Promotes Tumor Growth and Migration via Inhibiting EGFR Ubiquitination and Activating EGFR/Akt Signaling in Gastric Cancer.

BackgroundAlthough several long non-coding RNAs (lncRNAs) have been found to be involved in gastric cancer tumorigenesis, the more comprehensive contributions of lncRNAs to gastric cancer require further investigation. Here, we identify a cytoplasmic lncRNA, LINC01485, which promotes tumor growth and migration in gastric cancer.Materials and MethodsMicroarray and computational analysis were utilized to identify differential expression in LINC01485 and EGFR. Real-time PCR and Western blotting assays were used to confirm the expression of LINC01485 and EGFR in gastric cancer cells. Cell proliferation, wound-healing and transwell assays were performed to measure cell growth, migration and invasion. Immunoprecipitation, RNA pull-down, and RNA fluorescence in situ hybridization (RNA-FISH) assays were used to test the interaction of c-Cbl with LINC01485 and EGFR. Furthermore, tumor xenograft in nude mice was performed to test tumor growth in vivo.ResultsLINC01485 was upregulated and associated with tumor size, lymphatic metastasis and advanced pathological stage in gastric cancer. LINC01485 promoted gastric cancer cell proliferation, migration and invasion in vitro and in vivo. Furthermore, LINC01485 levels were positively correlated with EGFR expression in gastric cancer tissues and significantly increased the expression and phosphorylation (Tyr1045) of EGFR in gastric cancer cells. Mechanistically, LINC01485 competes with c-Cbl for binding to phosphorylated Tyr1045 site of EGFR, thus interfering with c-Cbl-mediated ubiquitination and subsequent degradation of EGFR.ConclusionLINC01485 promoted EGFR stabilization and activation of EGFR/Akt signaling in gastric cancer. Our findings illustrate the diversity of cytoplasmic lncRNAs in signal transduction and highlight the important roles of lncRNAs in gastric cancer.

Growth-Dependent Activation of Protein Kinases Suggests a Mechanism for Measuring Cell Growth.

In all cells, progression through the cell cycle occurs only when sufficient growth has occurred. Thus, cells must translate growth into a proportional signal that can be used to measure and transmit information about growth. Previous genetic studies in budding yeast suggested that related kinases called Gin4 and Hsl1 could function in mechanisms that measure bud growth; however, interpretation of the data was complicated by the use of gene deletions that cause complex terminal phenotypes. Here, we used the first conditional alleles of Gin4 and Hsl1 to more precisely define their functions. We show that excessive bud growth during a prolonged mitotic delay is an immediate consequence of inactivating Gin4 and Hsl1 Thus, acute loss of Gin4 and Hsl1 causes cells to behave as though they cannot detect that bud growth has occurred. We further show that Gin4 and Hsl1 undergo gradual hyperphosphorylation during bud growth that is dependent upon growth and correlated with the extent of growth. Moreover, gradual hyperphosphorylation of Gin4 during bud growth requires binding to anionic phospholipids that are delivered to the growing bud. While alternative models are possible, the data suggest that signaling lipids delivered to the growing bud generate a growth-dependent signal that could be used to measure bud growth.

Long Non-Coding RNA DUXAP8 Facilitates Cell Viability, Migration, and Glycolysis in Non-Small-Cell Lung Cancer via Regulating HK2 and LDHA by Inhibition of miR-409-3p.

PurposeLong non-coding RNAs (lncRNAs) were confirmed to play important roles in human cancers. In this study, we explored the functional role of lncRNA double homeobox A pseudogene 8 (DUXAP8) in non-small-cell lung cancer (NSCLC).MethodsReal-time quantitative PCR (RT-qPCR) was used to detect DUXAP8 and microRNA-409-3p (miR-409-3p) expression. CCK-8, cell colony formation assay, and Transwell migration assay were performed to measure cell growth and migration, respectively. The expression of the relative proteins was detected by Western blot. Cell glycolysis was determined by glucose uptake, adenosine triphosphate (ATP) concentration, lactate generation, extracellular acidification rate and oxygen consumption rate assays. Bioinformatics analysis and dual-luciferase reporter assay were used to measure the interaction among DUXAP8, miR-409-3p, hexokinase 2 (HK2) and lactate dehydrogenase A (LDHA). In vivo, subcutaneous tumor formation assay was performed in the nude mice.ResultsDUXAP8 was highly expressed in NSCLC, while miR-409-3p was downregulated. High expression of DUXAP8 was positively related to the grade division and negatively associated with the 5-year survival rate of NSCLC patients. Downregulated DUXAP8 significantly suppressed cell growth, metastasis and glycolysis. Besides, DUXAP8 sponged miR-409-3p to promote HK2 and LDHA expression. DUXAP8 promoted cell viability, migration and glycolysis by regulating miR-409-3p/HK2/LDHA axis. Moreover, DUXAP8 downregulation markedly inhibited tumor growth in vivo.ConclusionOur findings demonstrated that DUXAP8 served as an oncogene in the progression of NSCLC.

FOXC2 is a prognostic biomarker and contributes to the growth and invasion of human hepatocellular carcinoma

BackgroundForkhead box C2 (FOXC2) is a crucial factor involving in various cancers. However, its functions in hepatocellular carcinoma (HCC) is unknown. Here, we explored the role of FOXC2 in the progression of HCC and its potential mechanisms.MethodsFOXC2 expression in HCC tissue and cells were detected by immunohistochemistry or western blot and real-time PCR. CCK8, wound healing and transwell assay were used to measure cell growth and invasion. Tumor formation experiment was carried out to assess the tumorigenicity of HCC cells. Regulation of FOXC2 on Ang-2 was validated by luciferase assay and complementary experiments.ResultsIncreased FOXC2 expression was found to be associated positively with more aggressive clinicopathologic features. HCC patients with higher FOXC2 expression had significantly shorter overall survival. FOXC2 expression was indentified as an independent risk factor for resectable HCC. Increased FOXC2 expression accelerated the migration and invasion of HCC cells, accompanied by enhanced Ang-2 expression. Likewise, FOXC2 knockdown yielded opposite results. Moreover, FOXC2 stimulated the activation of the Ang-2 promoter. Suppression of Ang-2 expression hindered the FOXC2-mediated EMT processs, cell migration and invasion of HCC.ConclusionsFOXC2 is a novel prognostic predictor for HCC and may facilitate the growth and invasion through Ang-2.

Electrical Control of Escherichia coli Growth Measured with Simultaneous Modulation and Imaging.

Background: The use of electricity to mediate bacterial growth is unique in providing spatial control, but requires a more detailed understanding. Methods: We use two gold wires on a glass coverslip with an overlayer of agar to image Escherichia coli cells with brightfield and fluorescence microscopy while simultaneously applying a voltage. Cells outside of the wires provide a control population to measure cell growth as a function of voltage, rather than any difference in culture conditions or growth phase. Results: An applied voltage suppresses the fraction of E. coli undergoing elongation and division with recovery to control values when the voltage is removed. Depolarization is observed over the same voltage range suggesting a membrane potential-mediated response. Conclusions: Our experiments identify and use subcytotoxic voltages to measure differences in the fraction of E. coli cells elongating and dividing as a function of applied voltage. It is hoped that this research will inform the developing field of bacterial electrophysiology.

MiR‑21 inhibits autophagy and promotes malignant development in the bladder cancer T24cell line.

MicroRNA‑21 (miR‑21) is reported to exhibit cancer‑promoting activity in various types of cancer. It has been previously demonstrated that miR‑21 is overexpressed in bladder tumor tissue compared with normal mucosa. However, the functional mechanism of miR‑21 in bladder cancer remains largely unknown. Thus, the current study aimed to determine the roles of miR‑21 in autophagy and the malignant development of bladder cancer in T24cells. Upregulation or downregulation of miR‑21 was achieved following the transfection of miR‑21 mimic or miR‑21 inhibitor. An MTT assay was additionally performed to measure cell growth. Wound healing and transwell invasion assays were used to detect cell migration and invasion. The apoptotic potential and cell cycle were examined via flow cytometry and reverse transcription‑quantitative PCR was performed to evaluate the expression of phosphatase and tensin homolog (PTEN), beclin1, microtubule‑associated proteinl light chain3B (LC3‑II), cyclinD1, caspase‑3, E‑cadherin, matrix metallopeptidase‑9 (MMP‑9) and vimentin. The results revealed that the proliferation, migration and invasion of T24cells was greatly increased in the miR‑21 mimic group, while apoptosis was greatly inhibited. Additionally, T24cells treated with miR‑21 mimic exhibited G1‑phase arrest. In the miR‑21 mimic group, the expression of PTEN, beclin1, LC3‑II, caspase‑3 and E‑cadherin were decreased, while the expression of cyclinD1, MMP‑9 and vimentin were increased. Opposite effects were observed in the miR‑21 inhibitor group. The data of the current study may indicate that miR‑21 overexpression inhibited autophagy and promoted the proliferation, migration, invasion and epithelial to mesenchymal transition of bladder cancer T24cells. The results may further elucidate the molecular mechanism of miR‑21 in the development of bladder cancer.

<p>Regulations of miR-183-5p and Snail-Mediated Shikonin-Reduced Epithelial-Mesenchymal Transition in Cervical Cancer Cells</p>

BackgroundShikonin, the main ingredient of Lithospermum erythrorhizon, has been reported to have antitumor effects via multiple targets and signaling pathways. However, the detailed mechanism underlying the effects in cervical cancer still remained unknown.MethodsMTT, wound-healing, transwell assays and flow cytometry experiments were used to measure cell growth, migration, invasion, and cell cycle analysis. Western blot was used to examine protein levels of Snail, Vimentin and E-cadherin. The expression level of miR-183-5p was measured via qRT-PCR. The E-cadherin promoter activity was detected via Secrete-PairTM Dual Luminescence Assay Kit. The transient transfection experiments were used for silencing of E-cadherin and overexpression of Snail genes. Tumor xenograft and bioluminescent imaging experiments were carried out to confirm the in vitro findings.ResultsWe showed that shikonin inhibited cell viability, migration and invasion, and induced cell cycle arrest in a dose-dependent manner in cervical cancer Hela and C33a cells. Mechanistically, we found that shikonin increased miR-183-5p expression and inhibited expression of transcription factor Snail protein. The mimics of miR-183-5p reduced, while the inhibitors of miR-183-5p reversed shikonin-inhibited Snail protein expression. In addition, shikonin decreased Vimentin, increased E-cadherin protein expressions and E-cadherin promoter activity, the latter was reversed in cells transfected with exogenous Snail overexpression vectors. Moreover, silencing of E-cadherin significantly abolished shikonin-inhibited cervical cancer cell growth. Similar findings were also observed in vivo using one xenograft mouse model.ConclusionOur results show that shikonin inhibits EMT through inhibition of Snail and stimulation of miR-183-5p expressions, which resulted in induction of E-cadherin expression. Thus, blockade of EMT could be a novel mechanism underlying the anti-cervical cancer effects of shikonin.

Measuring Cell Growth and Junction Development in Epithelial Cells Using Electric Cell-Substrate Impedance Sensing (ECIS).

Electric Cell-substrate Impedance Sensing (ECIS) is an automated method that can be used to quantify processes such as cell attachment, growth, migration and barrier functions (i.e., the properties of tight junctions). The method provides simultaneous information on cell number and tight junction function by detecting electric parameters of cells grown on electrodes. Samples are probed with small alternating current (AC) over a range of frequencies, and changes in capacitance and impedance are measured over time. Capacitance reflects the degree of electrode coverage by cells, that correlates with cell number, and can be used to assess cell proliferation or migration. Impedance values inform about barrier function. Obtaining real-time simultaneous information on these parameters is unique to this system and is of great value for addressing fundamental questions such as the role of tight junction proteins in cell growth and migration. This protocol describes the use of ECIS to follow cell growth and tight junction-dependent barrier generation in tubular epithelial cells. We used this method to explore how depleting claudin-2, a tight junction protein affects tubular cell growth and barrier function. During the process, cells are transfected with control or claudin-2-specific siRNA, and 24h later plated on electrodes. ECIS automatically collects information on cell growth and barrier as the monolayer develops. The data are initially analyzed using the ECIS software and exported into a graph software for further processing.

Interaction Of c-Jun And HOTAIR- Increased Expression Of p21 Converge In Polyphyllin I-Inhibited Growth Of Human Lung Cancer Cells.

BackgroundLung cancer is a leading cause of cancer-related death worldwide. Previously we demonstrated that polyphyllin I (PPI), a bioactive component extracted from Paris polyphylla, inhibited the growth of non-small cell lung cancer (NSCLC) cells through the SAPK/JNK-mediated suppressing p65, DNMT1 and EZH2 expressions. However, the molecular mechanism underlying anti-lung cancer effect by PPI still remain elusive.PurposeIn this current study, we further explored the molecular mechanism underlying the anti-lung cancer effect of PPI.MethodsMTT, Cell-LightTM EdU DNA cell proliferation and colony formation assays were used to measure cell growth. Western blot were used to examine protein levels of c-Jun and p21. The expression level of long non-codingth RNA HOX transcript antisense RNA (HOTAIR) was measured by qRT-PCR. The p21 promoter activity was measured by Dual-Luciferase Reporter Assay System. The transient transfection experiments were used to silence and overexpression of c-Jun, p21 and HOTAIR. Tumor xenograft and bioluminescent imaging experiments were carried out to confirm the in vitro findings.Results We showed that PPI suppressed growth of NSCLC cells. Mechanistically, we observed that PPI reduced expression of HOTAIR, while increased transcription factor c-Jun protein levels. Additionally, PPI also induced protein expression and promoter activity of p21, a cyclin-dependent kinase inhibitor. While exogenously expressed HOTAIR showed no effect on c-Jun levels, silencing of c-Jun significantly reversed the PPI-inhibited HOTAIR expression. Moreover, excessive expressed c-Jun further enhanced PPI-inhibited HOTAIR expression and PPI-induced p21 protein levels. Intriguingly, overexpression of HOTAIR and silencing of c-Jun overcame the PPI-induced p21 protein and promoter activity. Finally, silencing of p21 neutralized the PPI-inhibited cell proliferation. Similar results were also found in one xenograft mouse model.Conclusion Our results demonstrate that PPI inhibits growth of NSCLC cells through regulation of HOTAIR and c-Jun expressions, which lead to induction of p21 gene. The interactions among HOTAIR, c-Jun and p21 regulatory axis converge in the overall anti-lung cancer effect of PPI. This study unveils an additional new mechanism for the anti-lung cancer role of PPI.

LRP6 regulates Rab7‐mediated autophagy through the Wnt/β‐catenin pathway to modulate trophoblast cell migration and invasion

Pre-eclampsia is a common complication during pregnancy; however, the underlying mechanisms of the crosstalk between low-density lipoprotein receptor-related protein 6 (LRP6) and autophagy in trophoblast cells are still not fully explored. Messenger RNA (mRNA) and protein levels of LRP6, beclin 1, Unc-51-like autophagy activating kinase 1 (ULK1), p62, vimentin, matrix metallopeptidase-9 (MMP-9), β-catenin, c-Myc, and Rab7, as well as the ratio of LC3-II/LC3-I, were analysed by quantitative real-time polymerase chain reaction or Western blot analysis, respectively. An MTT assay was used to measure cell growth, and transwell and wound healing assays were carried out to evaluate the invasion and migration abilities of the trophoblasts used. An immunofluorescence assay was used to measure LC3. The mRFP-GFP-LC3 tandem fluorescence assay was applied to detect autophagic flow. LRP6 overexpression was achieved by constructing pcDNA3.1-LRP6 vectors. LRP6 was expressed at low levels in HTR-8/SVneo cells under hypoxia/reoxygenation (H/R) conditions. H/R inhibited the activation of autophagy. LRP6 overexpression promoted cell proliferation and activated autophagy, which led to the upregulation of beclin 1 and ULK1, as well as the ratio of LC3-II/LC3-I and the downregulation of p62. Furthermore, LRP6 overexpression elevated the migration and invasion abilities of the indicated cells and increased vimentin and MMP-9 expression levels. Furthermore, LRP6 upregulated Rab7 and activated autophagy through the Wnt/β-catenin pathway. The late autophagy inhibitor bafilomycin A1 (Baf-A1) and the Wnt/β-catenin pathway inhibitor PKF115-584 reversed the effects of LRP6 on trophoblast autophagy, migration and invasion. LRP6 promotes Rab7-mediated autophagy by activating the Wnt/β-catenin pathway, which leads to increasing migration and invasion of trophoblast cells. Our study paves a new avenue for clinical treatment, and LRP6 may serve as an essential target in pre-eclampsia.

Improvement of the Anticancer Activity of Chlorambucil and Ibuprofen via Calix[4]arene Conjugates

One of the possible ways of improving the activity and selectivity profile of anticancer agents is to design drug carrier systems employing nanomolecules. Calix[4]arene derivatives and chlorambucil and ibuprofen are important compounds that exhibit interesting anticancer properties. The objective of this article is the synthesis of new calix[4]arene-derivative conjugates of chlorambucil or ibuprofen with potential anticancer activity. Cytotoxicity assays were determined using the protein-binding dye sulforhodamine B (SRB) in microculture to measure cell growth as described [19, 20]. Conjugates of chlorambucil and resorcinarene-dendrimers were prepared in 2% DMSO and added into the culture medium immediately before use. Control cells were treated with 2% DMSO. Thus, calix[4]arene-derivative conjugates of chlorambucil or ibuprofen showed good stability of the chemical link between drug and spacer. Evaluation of the cytotoxicity of the calix[4]arene chlorambucil or ibuprofen conjugates employing a sulforhodamine B (SRB) assay in K-562 (human chronic myelogenous leukemia cells) and U-251 (human glioblastoma cells) demonstrated that the conjugate was more potent as an antiproliferative agent than free chlorambucil and ibuprofen. The conjugates did not show any activity against the COS-7 African green monkey kidney fibroblast cell line. In the paper, we report the synthesis and spectroscopic analyses of new calix[4]arene derivative conjugates of chlorambucil or ibuprofen. Cytotoxicity assays revealed that at 10 μM, the conjugates were very active against K-562 (human chronic myelogenous leukemia cells) and U- 251 (human glioblastoma cells) cancer cells' proliferation. In order to explain the molecular mechanisms involved in the anticancer activity of calix[4]arene chlorambucil or ibuprofen conjugates, our research will be continued.

Accurate theoretical modeling of cell growth by comparing with visualized data in high-pressure foam injection molding

A novel simulation model has been developed that can predict cell growth tailored to high-pressure foam injection molding (HP-FIM) processes during the entire processing period. This was done together with systematic HP-FIM experiments using a visualization technique. The mathematical model that has been developed is based on the well-known Cell Model to describe the initial bubble growth accurately. To improve the model’s robustness and accuracy for the whole processing period, we used the Simha-Somcynsky equation of state for the PS/CO2 mixture, which facilitated the accurate prediction of realistic transport and rheological properties (that is, gas diffusivity, surface tension, viscosity, and relaxation time) that are changing as a function of the gas content. All of these properties were also a function of the temperature and the pressure in order to accurately capture the behavior of the fluid flow and the transport phenomenon associated with cell growth. By inputting the initial gas concentration and the transient pressure and temperature profiles, the proposed model could predict the cell growth profile under different HP-FIM conditions. The proposed model was validated by comparison with the experimental data obtained from the visualized HP-FIM trials. Reasonable quantitative agreement was achieved between the simulated and measured cell growth throughout the HP-FIM process. Moreover, a sensitivity analysis was conducted by varying the model’s material properties. It was found that an increase in the gas’s diffusivity increased the growth rate of the cell, whereas changes in the surface tension only led to minor changes in the growth profile. Meanwhile, a longer relaxation time promoted the growth rate, whereas a higher zero-shear viscosity restrained growth.