Expression of epidermal growth factor receptor and human epidermal growth factor receptor 2 in urothelial bladder carcinoma in an Egyptian cohort: Clinical implication and prognostic significance.

BACKGROUND: Bladder cancer (BC) has a particular importance in Egyptian patients due to aggressive behavior and absence of prognostic markers. OBJECTIVE: To evaluate the expression of gene and protein expression of HER2 and epidermal growth factor (EGFR) in Egyptian patients with BC and ultimately to investigate their clinical implication and prognostic significance. MATERIAL AND METHODS: The study was carried out on 46 patients with urothelial bladder BC. Tissue were obtained from transurethral resection (N = 22) and radical cystectomy (N = 24) specimens. The original hematoxylin and eosin slides were re-evaluated and the formalin fixed, paraffin-embedded (FFPE) tissues which had sufficient tumor tissue (>75%) and minimal or absent tumor necrosis were selected for immunohistochemistry (IHC) and RNA extraction. Furthermore, five control biopsies were obtained from patients with cystitis. Follow-up data were retrieved from the medical records which included the treatment regimen, disease recurrence and/or progression, and survival. RESULTS: EGFR and HER2 protein were overexpressed in 35% and 46% of patients respectively. EGFR was correlated with the tumor size, grade and pathological stage, with a similar trend for HER2. The recurrence rate was higher in patients with expression of any of the markers. Gene expression was significantly higher (10.6-folds) for EGFR and (21-folds) for HER2 in patients with BC in comparison to control patients. Survival analysis showed lower median disease-free survival in association with HER2 protein overexpression. CONCLUSIONS: Our data highlighted the prognostic significance of EGFR and HER in BC and proposed their possible use as predictive markers and potential therapeutic targets.

Xeno-free workflow exhibits comparable efficiency and quality of keratinocytes isolated from human skin biopsies.

INTRODUCTION: Regenerative solutions of the skin represent a hope for burn victims with extensive skin loss and chronic wound patients. The development of xeno-free workflow is crucial for clinical application in compliance with the directives of the European Medicines Agency. This study aimed at evaluating the outcome of the xeno-free isolation workflow of keratinocytes from human skin biopsy. METHODS: Skin biopsies were obtained from volunteers. The epidermis was digested with TrypLE™ Select, which was deactivated by dilution or with trypsin, deactivated by media with fetal bovine serum. Freshly isolated cells were compared for total cell number, viability, activity of caspase 3, gene expression and the presence of the keratinocyte surface markers cytokeratin 14. The cells were cultured in xeno-free conditions for one week and characterized regarding the number and viability as well as the metalloproteinase secretion. RESULTS: The number of obtained cells was similar in both workflows. The cell viability was less in the TrypLE group, with slight reduction of the cell surface marker cytokeratin 14. Caspase 3 activity was comparable as well as the gene expression of the apoptotic markers BAX, BCL2 and SLUG, as well as the keratinocyte markers cytokeratin 14, stratifin and filaggrin. Upon culture, the number of keratinocytes, their viability and secretion of matrix metalloproteinases 1 and 10 were equal in both groups. CONCLUSION: This study reports the possibility of isolating functioning and viable keratinocytes through a xeno-free workflow for clinical application.

Human serum albumin as a clinically accepted cell carrier solution for skin regenerative application.

The rules governing Medicinal Products in the European Union necessitates the production of cell-based therapy in good manufacturing practice facilities. The produced cells may need several hours in transportation to reach the application sites. In this study, we investigated four candidate solutions for transporting human keratinocytes. The solutions are (1) normal saline, (2) saline with 2.5% human serum albumin (Saline + HSA), (3) chemically defined, xeno-free keratinocyte media and (4) keratinocyte media with pituitary bovine extract (PBE-media). One million keratinocytes from three donors were suspended in each solution and kept at 4 °C for up to 24 h. Cells kept in Saline + HSA showed higher viability after 1, 3 and 24 h. Then, equal number of viable cells were seeded on collagenous matrix and cultured for 48 h. The adhesion and colonization were higher in the cells kept in PBE-media, while the keratinocyte surface marker, cytokeratin 14, was present in all studied groups. These results confirmed the suitability of Saline + HSA as a cell transportation solution for clinical use, which will be the choice for the planned clinical trial. Keratinocyte PBE-media can be an alternative for cells transported for research purpose, if the same media type is going to be used in the following experiments.

Immunomodulatory MicroRNAs in cancer: targeting immune checkpoints and the tumor microenvironment.

Cancer immunotherapy represents a promising new era in cancer management due to the relatively high safety margins and selectivity, compared to the classical cancer chemotherapeutic agents. However, there is an imperative need to overcome tumor resistance in order to improve clinical outcomes and maximize the benefits of cancer immunotherapy. The interaction between the programmed cell death-1 (PD-1) receptor and its ligand PD-L1 is a vital immune checkpoint that is often adopted by cancer cells to undergo immune evasion. PD-1/PD-L1 signaling is regulated at multiple levels through the crosstalk with other immune targets or relevant signaling partners involved in the cancer progression. Among the significant epigenetic players that are implicated in modulating the immune system are microRNAs (miRNAs). A complex system of these noncoding RNAs regulates the gene expression at the post-transcriptional level and plays a significant role in the modulation of both innate and the adaptive immune systems. The expression profile of immune-modulatory miRNAs might be useful as a predictive biomarker for the response and clinical outcomes in cancer immunotherapy. Therefore, in the current review, we highlighted the role of miRNAs in cancer immune evasion through a critical discussion of their impact on key immune checkpoints as well as the role of miRNAs in cancer progression and resistance.

Paradoxical effects of the epigenetic modifiers 5-aza-deoxycytidine and suberoylanilide hydroxamic acid on adipogenesis.

Adipogenesis is an important biological process that is linked to obesity and metabolic disorders. On the other hand, fat regeneration is crucial as a restorative approach following mastectomy or severe burn injury. Furthermore, optimizing an in-vitro model of adipogenesis, which would help in understanding the possible effects and/or side effects of fat-soluble drugs and anti-obesity remedies, in addition to the developmental studies. Epigenetic is an important factor that is involved in cellular differentiation and commitment. This study aimed at investigating the effect of DNA methylation and histone deactylases inhibitors, 5-Aza-deoxycytidine (5-Aza-dC) and Suberoylanilide hydroxamic acid (SAHA), on the adipogenic differentiation process. The two modifiers were applied according to our previously published protocol, followed by three cycles of a classical, two-step adipogenesis protocol. The cells pretreated with SAHA showed enhanced expression of the many adipogenic genes, including peroxisome proliferator-activated receptor-γ as well as the accumulation of intracytoplasmic fat as shown by oil red and Nile red staining and the secretion of adipokines, such as MCP-1 and IP-10. On contrary, 5-Aza-dC inhibited all these markers. In conclusion, adding the reported step with SAHA to the differentiation protocols could have an impact on the progress of the in-vitro fat regenerative approach. The possible role of 5-Aza-dC in the inhibition of adipogenesis can be of clinical interest and will need further characterization in the future.

Interplay between Epigenetics, Expression of Estrogen Receptor- α, HER2/ERBB2 and Sensitivity of Triple Negative Breast Cancer Cells to Hormonal Therapy.

Triple negative breast cancer (TNBC) cells are resistant to hormonal/targeted therapies. This study aims to investigate epigenetic differences between TNBC and other types of breast cancer and the effect of epigenetic modulation on the response of TNBC cells to hormonal therapy. Thus, we investigated (i) the expression of different epigenetic markers, (ii) the effect of epigenetic modifying agents on the expression of ERα and HER2/ERBB2 and (iii) the effect on the response to tamoxifen in four breast cancer cell lines with different hormonal receptor status. Our results revealed a differential expression patterns of epigenetic markers in the four breast cancer cells. In TNBC cells, histone deacetylases (HDAC) 1 and 2 were less expressed, whereas HDACs 4 and 6 were overexpressed. Interestingly, treatment with epigenetic modifiers resulted in (i) a pronounced increase in the expression of ERα and HER2/ERBB2 along with (ii) an increase in the sensitivity of TNBC cells to tamoxifen. Collectively, this study indicates a different epigenetic background for TNBC cells, which represses the expression of ERα and HER2/ERBB2. Furthermore, we provide here the rationale for the use of epigenetic modifiers to enhance the response of TNBC to hormonal therapy through upregulation of ERα.

Caffeic acid phenethyl ester protects against glucocorticoid-induced osteoporosis in vivo: Impact on oxidative stress and RANKL/OPG signals.

Glucocorticoid-induced osteoporosis (GIO) is one of the most common causes of secondary osteoporosis. Given that glucocorticoids are considered as a main component of the treatment protocols for a variety of inflammation and immune-mediated diseases besides its use as adjuvant to several chemotherapeutic agents, it is crucial to find ways to overcome this critical adverse effect. Caffeic acid phenethyl ester (CAPE), which is a natural compound derived from honeybee propolis displayed promising antiosteoporotic effects against mechanical bone injury in various studies. The current work aimed at investigating the potential protective effect of CAPE against GIO in vivo with emphasis on the modulation of oxidative status and receptor activator of NF-kB ligand (RANKL)/osteoprotegrin (OPG) signaling. The results showed that CAPE opposed dexamethasone (DEX)-mediated alterations in bone histology and tartarate-resistant acid phosphatase (TRAP) activity. In addition, CAPE restored oxidative balance, Runt-related transcription factor 2 (RunX2) expression and reduced caspase-3 activity in femur tissues. Co-administration of CAPE with DEX normalized RANKL/OPG ratio and Akt activation indicating a reduction in DEX-osteoclastogenesis. In conclusion, concurrent treatment of CAPE with DEX exhibited promising effects in the protection against DEX-induced osteoporosis through opposing osteoclastogenesis and protecting osteoblasts. The potent antioxidant activity of CAPE is, at least in part, involved in its anti-apoptotic effects and modulation of RunX2 and RANKL/OPG signals. The use of CAPE-enriched propolis formulas is strongly recommended for patients on chronic glucocorticoid therapy to help in the attenuation of GIO.

The effect of circadian rhythm on pharmacokinetics and metabolism of the Cdk inhibitor, roscovitine, in tumor mice model.

Roscovitine is a selective Cdk-inhibitor that is under investigation in phase II clinical trials under several conditions, including chemotherapy. Tumor growth inhibition has been previously shown to be affected by the dosing time of roscovitine in a Glasgow osteosarcoma xenograft mouse model. In the current study, we examined the effect of dose timing on the pharmacokinetics, biodistribution and metabolism of this drug in different organs in B6D2F1 mice. The drug was orally administered at resting (ZT3) or activity time of the mice (ZT19) at a dose of 300 mg/kg. Plasma and organs were removed at serial time points (10, 20 and 30 min; 1, 2, 4, 6, 8, 12 and 24 h) after the administration. Roscovitine and its carboxylic metabolite concentrations were analyzed using HPLC-UV, and pharmacokinetic parameters were calculated in different organs. We found that systemic exposure to roscovitine was 38% higher when dosing at ZT3, and elimination half-life was double compared to when dosing at ZT19. Higher organ concentrations expressed as (organ/plasma) ratio were observed when dosing at ZT3 in the kidney (180%), adipose tissue (188%), testis (132%) and lungs (112%), while the liver exposure to roscovitine was 120% higher after dosing at ZT19. The metabolic ratio was approximately 23% higher at ZT19, while the intrinsic clearance (CLint) was approximately 67% higher at ZT19, indicating faster and more efficient metabolism. These differences may be caused by circadian differences in the absorption, distribution, metabolism and excretion processes governing roscovitine disposition in the mice. In this article, we describe for the first time the chronobiodistribution of roscovitine in the mouse and the contribution of the dosing time to the variability of its metabolism. Our results may help in designing better dosing schedules of roscovitine in clinical trials.

Epigenetic modifiers influence lineage commitment of human bone marrow stromal cells: Differential effects of 5-aza-deoxycytidine and trichostatin A.

Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. However, current in vitro methods for the differentiation of human bone marrow stromal cells (HBMSCs) do not, to date, produce homogeneous cell populations of the osteogenic or chondrogenic lineages. As epigenetic modifiers are known to influence differentiation, we investigated the effects of the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) or the histone deacetylase inhibitor trichostatin A (TSA) on osteogenic and chondrogenic differentiation. Monolayer cultures of HBMSCs were treated for 3 days with the 5-aza-dC or TSA, followed by culture in the absence of modifiers. Cells were subsequently grown in pellet culture to determine matrix production. 5-aza-dC stimulated osteogenic differentiation as evidenced by enhanced alkaline phosphatase activity, increased Runx-2 expression in monolayer, and increased osteoid formation in 3D cell pellets. In pellets cultured in chondrogenic media, TSA enhanced cartilage matrix formation and chondrogenic structure. These findings indicate the potential of epigenetic modifiers, as agents, possibly in combination with other factors, to enhance the ability of HBMSCs to form functional bone or cartilage with significant therapeutic implications therein.