Simultaneous E-cadherin and PLEKHA7 expression negatively affects E-cadherin/EGFR mediated ovarian cancer cell growth

Abstract

Background

The disruption of E-cadherin-mediated adhesion is considered an important driver of tumor progression. Nevertheless, numerous studies have demonstrated that E-cadherin promotes growth- or invasion-related signaling, contrary to the prevailing notion. During tumor progression, epithelial ovarian cancer (EOC) maintains E-cadherin expression and can positively affect EOC cell growth by contributing to PI3K/AKT activation. In polarized epithelia PLEKHA7, a regulator of the zonula adherens integrity, impinges E-cadherin functionality, but its role in EOCs has been never studied.

Methods

Ex-vivo EOC cells and cell lines were used to study E-cadherin contribution to growth and EGFR activation. The expression of the proteins involved was assessed by real time RT-PCR, immunohistochemistry and western blotting. Cells growth and drug susceptibility was monitored in different 3-dimensional (3D) systems. Recombinant lentivirus-mediated gene expression, western blotting, immunoprecipitation and confocal microscopy were applied to investigate the biological impact of PLEKHA7 on E-cadherin behaviour. The clinical impact of PLEKHA7 was determined in publicly available datasets.

Results

We show that E-cadherin expression contributes to growth of EOC cells and forms a complex with EGFR thus positively affecting ligand-dependent EGFR/CDK5 signaling. Accordingly, 3D cultures of E-cadherin-expressing EOC cells are sensitive to the CDK5 inhibitor roscovitine combined with cisplatin. We determined that PLEKHA7 overexpression reduces the formation of E-cadherin-EGFR complex, EGFR activation and cell tumorigenicity. Clinically, PLEKHA7 mRNA is statistically decreased in high grade EOCs respect to low malignant potential and low grade EOCs and correlates with better EOC patient outcome.

Conclusions

These data represent a significant step towards untangling the role of E-cadherin in EOCs by assessing its positive effects on EGFR/CDK5 signaling and its contribution to cell growth. Hence, the inhibition of this signaling using a CDK5 inhibitor exerts a synergistic effect with cisplatin prompting on the design of new therapeutic strategies to inhibit growth of EOC cells. We assessed for the first time in EOC cells that PLEKHA7 induces changes in the asset of E-cadherin-containing cell-cell contacts thus inhibiting E-cadherin/EGFR crosstalk and leading to a less aggressive tumor phenotype. Accordingly, PLEKHA7 levels are lower in high grade EOC patient tumors and EOC patients with better outcomes display higher PLEKHA7 levels.

https://ift.tt/2KNqAwp

Levels of Resistance to Pyrethroid among Distinct kdr Alleles in Aedes aegypti Laboratory Lines and Frequency of kdr Alleles in 27 Natural Populations from Rio de Janeiro, Brazil

Background. Several mutations in voltage gated sodium channel () have been identified in Aedes aegypti populations worldwide. However, only few are related to knockdown resistance to pyrethroids, most of which with variations in the 1016 and 1534 sites. In Brazil, at least two NaV alleles are known: R1, with a substitution in the 1534 (1016 Val+ + 1534 Ilekdr) and R2, with substitutions in both 1016 and sites (1016Il + 1534Cyskdr). There is also the duplication in the NaV gene, with one copy carrying the substitution Ile1011Met, although its effects on pyrethroid resistance remain to be clarified. Our goals in this study were (1) to determine the role of each kdr allele and the duplication on pyrethroid resistance and (2) to screen the frequency of the kdr alleles in 27 several natural Ae. aegypti populations from the metropolitan region of Rio de Janeiro. Methods. Pyrethroid resistance was evaluated by a knockdown time (KdT) assay, an adaptation of the WHO test tubes with paper impregnated with deltamethrin. We used laboratory-selected Ae. aegypti lineages: R1R1 and R2R2 (homozygous for the kdr R1 and R2 alleles, respectively), Dup (with duplication in the NaV gene), Rockefeller (the susceptibility reference control), and F1 hybrids among them. Genotyping of both 1016 and 1534 sites was performed in 811 Ae. aegypti sampled from 27 localities from Rio de Janeiro (17), Niterói (6) and Nova Iguaçu (4) cities, Rio de Janeiro State, Brazil, with a TaqMan real time PCR approach. Results. The laboratory lineages R1R1, R2R2, and R1R2 were the only ones that needed more than 60 minutes to knock down all the insects exposed to the pyrethroid, being the KdT R2R2 > R1R2 > R1R1, corroborating the recessive nature of the kdr mutations. Frequency of kdr alleles R1 and R2 in field-caught mosquitoes varied from 0 to 52% and 43 to 86%, respectively, evidencing high levels of "resistant genotypes" (R1R1, R1R2, and R2R2), which together summed 60 to 100% in Ae. aegypti populations from Rio de Janeiro. Conclusions. The R1 and R2 kdr alleles confer resistance to the pyrethroid deltamethrin in homozygotes and R1R2 heterozygotes, being the R2R2 most resistant genotype. The allele containing duplication in the gene, with a mutation in the 1011 site, did not confer resistance under the tested conditions. The frequencies of the "resistant genotypes" are elevated in Ae. aegypti natural populations from Rio de Janeiro.

https://ift.tt/2NCk89z

Muscle Extremely Low Frequency Magnetic Stimulation Eliminates the Effect of Fatigue on EEG-EMG Coherence during the Lateral Raise Task: A Pilot Quantitative Investigation

The aim of this study was to quantitatively investigate the effects of force load, muscle fatigue, and extremely low frequency (ELF) magnetic stimulation on electroencephalography- (EEG-) electromyography (EMG) coherence during right arm lateral raise task. Eighteen healthy male subjects were recruited. EEG and EMG signals were simultaneously recorded from each subject while three different loads (0, 1, and 3kg) were added on the forearm. ELF magnetic stimulation was applied to the subject's deltoid muscle between tasks during the resting period. Univariate ANOVA showed that all EEG-EMG coherence areas of C3, C4, CP5, and CP6 were not significantly affected by the force load (all p>0.05) and that muscle fatigue led to statistically significant reductions on the coherence area of gamma band in C3 (p=0.014) and CP5 (p=0.019). More interestingly, these statistically significant reductions disappeared with the application of muscle ELF magnetic stimulation, indicating its potential application to eliminate the effect of fatigue.

https://ift.tt/2JdWHzK

Fertility Preservation: Current and Future Perspectives for Oncologic Patients at Risk for Iatrogenic Premature Ovarian Insufficiency

Progress in recent years in the efficacy of oncologic treatment and early diagnosis of cancer has determined an increase in life expectance in cancer patients. About 10% of all cancer cases affect women younger than 45 years; therefore nowadays approximately 5-6% of the population in childbearing age consists in cancer survivors. A crucial issue is the high risk of premature ovarian insufficiency due to possible gonadotoxic effects of oncologic treatments. Considering combined chemotherapy, radiation therapy, and bone marrow transplantation, this risk can reach 92-100%, depending on the age and ovarian reserve of the patient, as well as the schedule and type of therapy. International guidelines recommend addressing all the patients diagnosed with a neoplasia treatable with potentially gonadotoxic therapies to fertility preservation. Moreover, fertility preservation also seems to reserve fascinating implications for women who want to delay childbearing for social reasons or women affected with endometriosis, who could receive unexpected opportunities. At present, the most widespread techniques to preserve fertility in adult women are embryo or oocyte cryopreservation, depending on the presence of a partner or according to legislative issues, but these procedures require time for ovarian stimulation. In prepubertal patients or when there is no possibility of delaying chemotherapy, ovarian tissue cryopreservation and subsequent transplantation represent the main strategy.

https://ift.tt/2NFllNb

Bufadienolides from Venenum Bufonis Inhibit mTOR-Mediated Cyclin D1 and Retinoblastoma Protein Leading to Arrest of Cell Cycle in Cancer Cells

Objective. Bufadienolides, the main components in Venenum Bufonis secreted from toads, have been proved to be with significant anticancer activity aside from the positive inotropic action as cardenolides. Here an underlying anticancer mechanism was further elucidated for an injection made from Venenum Bufonis containing nine bufadienolides. Methods. One solution reagent and cell cycle analyses were for determining effect of bufadienolides on cancer cells. Western blotting was used for protein expression. Results. Bufadienolides inhibit cell proliferation and arrest cells in G1 phase. Bufadienolides also inhibit the mammalian target of rapamycin (mTOR) signaling pathway, which is evidenced by the data that bufadienolides inhibit type I insulin-like growth factor- (IGF-1-) activated phosphorylation of mTOR by a concentration- and time-dependent way, as well as phosphorylation of p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). Subsequent results indicated that cyclin D1 expression and phosphorylation of retinoblastoma protein (Rb)—two characterized regulators in cell cycle of G1—are also inhibited and the process is dependent on mTOR pathway. Conclusion. Bufadienolides inhibit proliferation partially due to arresting cell cycle in G1 phase, which is mediated by inhibiting mTOR-cyclin D1/Rb signal pathway.

https://ift.tt/2L578uz

Combination of Epicatechin 3-Gallate from Euphorbia hirta and Cefepime Promotes Potential Synergistic Eradication Action against Resistant Clinical Isolate of Pseudomonas aeruginosa

Pseudomonas aeruginosa is naturally resistant to many classes of antipseudomonal antibiotics due to the species ability to easily acquire resistance. Plant-based antibacterial agent in combination with the existing antibiotic proposes an alternative treatment regimen for the eradication of resistant bacterial infections. The antibacterial effects of the isolated epicatechin 3-gallate compound from Euphorbia hirta in combination with cefepime were investigated in vitro against resistant P. aeruginosa. The fractional inhibitory concentration index of the combination was determined using checkerboard broth microdilution method. Epicatechin 3-gallate combined with cefepime had produced synergistic effect against P. aeruginosa (with average FIC index of 0.24). The MIC of epicatechin 3-gallate was effectively reduced to MIC/4, MIC/8, MIC/16, and MIC/32 in the presence of cefepime. Time-kill study of epicatechin 3-gallate combined with cefepime exhibited remarkable bactericidal activity where the eradication of P. aeruginosa occurred within 4 h of treatment. Scanning electron micrographs revealed apparent cell membrane damage and leakage of cytoplasmic contents from P. aeruginosa cells which eventually led to the cell lysis after the combination treatment of epicatechin 3-gallate and cefepime. The potential of epicatechin 3-gallate to act synergistically with cefepime against clinically resistant P. aeruginosa strain possibly will maximize the successful outcomes when choosing empirical antibiotic treatment in hospitals or health care institutions.

https://ift.tt/2L8nDCU

Exploring the Therapeutic Ability of Fenugreek against Type 2 Diabetes and Breast Cancer Employing Molecular Docking and Molecular Dynamics Simulations

Fenugreek (Trigonella foenum-graecum) is used as a spice throughout the world. It is known for its medicinal properties such as antidiabetic, anticarcinogenic, and immunological activities. The present study shows the properties and the nutritional quality of fenugreek seed extract and focuses on screening of active compounds in drug designing for type 2 diabetes and breast cancer. Quantitative analysis was used to calculate the percentages of protein, carbohydrates moisture, fatty acid, galactomannan, oil, and amino acid. Phytochemical analysis revealed the presence of flavonoids, terpenoids, phenols, proteins, saponins, and tannins in fenugreek seed extracts. Molecular docking and molecular dynamics simulation-based computational drug discovery methods were employed to address the role of fenugreek seed constituents against type 2 diabetes and breast cancer. The computational results reveal that the compound galactomannan can be ascribed as potential drug candidate against breast cancer and type 2 diabetes rendered by higher molecular dock scores, stable molecular dynamics (MD) simulations results, and lower binding energy calculations.

https://ift.tt/2L1wny2