Non-treated Cells Research Articles

Integration of Ethanol and the Immune Modulator Curcumin for Immunoablation of Hepatocellular Carcinoma

PurposeTo investigate immuno-ethanol ablation using an ethanol and immune adjuvant formulation as a potent immunoablation approach that can achieve an enhanced anticancer effect in the treatment of hepatocellular carcinoma (HCC). Materials and MethodsEthanol concentration– and exposure time–dependent cellular responses were investigated. Curcumin was combined with ethanol as an immunoablation agent. Cellular uptake of curcumin, cancer cell killing, and inflammatory markers of ethanol-curcumin treatment were characterized. To evaluate the potential in vivo anticancer immunity of ethanol-curcumin treatment, each right and left lobe of rat liver was concurrently inoculated with N1S1 HCC cells and a mixture of treated N1S1 cells (ethanol only or ethanol-curcumin) in Sprague Dawley rats (each group: 5 rats; control: nontreated N1S1 cells). Tumor growth and immune response were characterized with 7T magnetic resonance (MR) imaging, flow cytometry analysis, and immunohistology. ResultsAn optimized ethanol-curcumin (10% ethanol and 0.5% weight/volume (w/v) curcumin solution) treatment contributed to an enhanced cellular uptake of curcumin, increased cancer cell killing, and decreased inflammatory reaction. Ethanol-curcumin–treated N1S1 cell implantation in the rat liver demonstrated N1S1 HCC tumor rejection. The secondary tumor growth by nontreated N1S1 cell inoculation was significantly suppressed at the same time. Activated anticancer immunity was evidenced by significantly increased CD8+ T cell infiltration (3.5-fold) and CD8+–to–regulatory T cell ratio (4.5-fold) in the experimental group compared with those in the control group. ConclusionsEnhanced anticancer effect of immuno-ethanol ablation could be achieved with ethanol-curcumin agent. The results underscore the importance of optimized immunoablation therapeutic procedures for enhanced therapeutic outcomes.

Exosome-encapsulated lncRNA HOTAIRM1 contributes to PM2.5-aggravated COPD airway remodeling by enhancing myofibroblast differentiation.

Emphysema, myofibroblast accumulation and airway remodeling can occur in the lungs due to exposure to atmospheric pollution, especially fine particulate matter (PM2.5), leading to chronic obstructive pulmonary disease (COPD). Specifically, bronchial epithelium-fibroblast communication participates in airway remodeling, which results in COPD. An increasing number of studies are now being conducted on the role of exosome-mediated cell-cell communication in disease pathogenesis. Here, we investigated whether exosomes generated from bronchial epithelial cells could deliver information to normal stromal fibroblasts and provoke cellular responses, resulting in airway obstruction in COPD. We studied the mechanism of exosome-mediated intercellular communication between human bronchial epithelial (HBE) cells and primary lung fibroblasts (pLFs). We found that PM2.5-induced HBE-derived exosomes promoted myofibroblast differentiation in pLFs. Then, the exosomal lncRNA expression profiles derived from PM2.5-treated HBE cells and nontreated HBE cells were investigated using an Agilent Human LncRNA Array. Combining coculture assays and direct exosome treatment, we found that HBE cell-derived exosomal HOTAIRM1 facilitated the myofibroblast differentiation of pLFs. Surprisingly, we discovered that exosomal HOTAIRM1 enhanced pLF proliferation to secrete excessive collagen secretion, leading to airway obstruction by stimulating the TGF-β/SMAD3 signaling pathway. Significantly, PM2.5 reduced FEV1/FVC and FEV1 and increased the level of serum exosomal HOTAIRM1 in healthy people; moreover, serum exosomal HOTAIRM1 was associated with PM2.5-related reductions in FEV1/FVC and FVC. These findings show that PM2.5 triggers alterations in exosome components and clarify that one of the paracrine mediators of myofibroblast differentiation is bronchial epithelial cell-derived HOTAIRM1, which has the potential to be an effective prevention and therapeutic target for PM2.5-induced COPD.

Discovery of anticancer compound possessing potential to bind γ-secretase catalytic subunit and inhibit notch promoter activity

Gamma secretase (GS) is an important therapeutic target in anticancer drug discovery. Increased GS activity activates notch signaling pathway which is associated with cancer stemness and drug resistance in cancer cells. A total of 69,075 natural and their derivative compounds were screened to identify the lead compound on the basis of in silico GS catalytic domain binding potential and in vitro selective anticancer efficacy. STOCK1N-23234 showed higher dock score (−11.82) compared to DAPT (−9.2) in molecular docking experiment and formed hydrogen bond with the key amino acid (Asp385) involve in catalysis process. Molecular dynamics (MD) simulation parameters (RMSD, RMSF, Rg, SASA and hydrogen bond formation) revealed that the STOTCK1N-23234 formed structurally and energetically stable complex with the GS catalytic domain with lower binding energy (−22.79 kcal/mol) compared to DAPT (−16.22 kcal/mol). STOCK1N-23234 showed better toxicity (up to 60%) against colon and breast cancer cells (HCT-116 and MDA-MB-453) at 1–70 µM concentration. Interestingly, STOCK1N-23234 did not showed cytotoxicity against human normal breast cells (MCF-10A). STOCK1N-23234 treatment significantly decreased sphere formation, notch promoter activity, and transcription of notch target genes (Hes-1 and Hey-1) in HCT-116 cells derived colonosphere. Confocal microscopy revealed that STOTCK1N-23234 treatment at test concentration induced apoptosis related morphological changes, reduced mitochondria membrane potential and increased reactive oxygen species production in HCT-116 cells compared to non-treated cells. In conclusion, STOCK1N-23234 is a novel lead natural anticancer compound which requires in depth validation in cancer preclinical models. Communicated by Ramaswamy H. Sarma

Cell Death Induced by the Combination of Ephedra sinica Extract and Radiation in HNSCC is Positively Related to BAX and p-MLKL Expression.

Numerous studies have proven the efficacy and safety of natural products, and are widely used as attractive cancer treatments. The investigation of effective natural products for improving cancer treatment is a promising strategy. Combination treatment with radiosensitizers and radiotherapy (RT) is considered necessary for therapeutic improvement in head and neck squamous cell carcinoma(HNSCC). This study aims to investigate whether Ephedra sinica (ES) extract could induce selective cell death in cancer cells and serve as a radiosensitizer for HNSCC. HNSCC cells were pretreated with ES extract before radiation, and the radiosensitizing activity was assessed using a colony formation assay. Radiation-induced cell death was evaluated using an annexinV-FITC assay. Western blotting was performed to confirm cell death-related gene expression, including apoptosis and necrosis markers. ES extract significantly inhibited HNSCC cell viability (FaDu and SNU1076), while having minimal effect on normal HaCaT cells. When HNSCC cells were irradiated with 2, 4, or 8 Gy and cultured with ES extract (25 μg/mL), they exhibited increased radiation sensitivity compared to non-treated cells. The combination of ES extract and radiation resulted in increased cell death compared to non-treated, ES-treated, or irradiated cells. The apoptosis marker BAX and necrosis marker p-MLKL expression levels were also elevated following the combination treatment. ES extract demonstrated significant cytotoxic potential in HNSCC cells without affecting normal cells. It enhanced the radiosensitivity of HNSCC cells by upregulating BAX and p-MLKL expression, leading to increased cell death. These results suggest ES extract exhibits a potential radiosensitizing capacity in HNSCC.

Inorganic Nanoparticles Change Cancer-Cell-Derived Extracellular Vesicle Secretion Levels and Cargo Composition, Resulting in Secondary Biological Effects.

Over the past decades, the medical exploitation of nanotechnology has been largely increasing and finding its way into translational research and clinical applications. Despite their biomedical potential, uncertainties persist regarding the intricate role that nanomaterials may play on altering physiology in healthy and diseased tissues. Extracellular vesicles (EVs) are recognized as an important pathway for intercellular communication and known to be mediators of cellular stress. EVs are currently explored for targeted delivery of therapeutic agents, including nanoformulations, to treat and diagnose cancer or other diseases. Here, we aimed to investigate whether nanomaterials could have a possible impact on EV functionality, their safety, and whether EVs can play a role in nanomaterial toxicity profiles. To evaluate this, the impact of inorganic nanomaterial administration on EVs derived from murine melanoma and human breast cancer cells was tested. Cells were incubated with subtoxic concentrations of 4 different biomedically relevant inorganic nanoparticles (NPs): gold, silver, silicon dioxide, or iron oxide. The results displayed a clear NP and cell-type-dependent effect on increasing or decreasing EV secretion. Furthermore, the expression pattern of several EV-derived miRNAs was significantly changed upon NP exposure, compared to nontreated cells. Detailed pathway analysis and additional studies confirmed that EVs obtained from NP-exposed cells could influence immunological responses and cellular physiology. Together, these data reveal that NPs can have wide-ranging effects which can result in toxicity concerns or enhanced therapeutic potential as a secondary enhanced effect mediated and enhanced by EVs.

Transfection of Unmodified MicroRNA Using Monolayer-Coated Au Nanoparticles as Gene-Delivery Vehicles.

This article describes a monolayer-coated gold nanoparticle-based transfection system for the delivery of microRNA (miRNA) into human osteosarcoma (HOS) cells. Two distinct ammonium-terminated adsorbates were used in this study, which provided a platform for ionic bonding of the miRNA onto gold nanoparticles (AuNPs). The custom-designed monolayer-coated gold nanoparticles were characterized by dynamic light scattering, gel mobility shift assay, transmission electron microscopy, ultraviolet-visible spectrometry, zeta potential, and X-ray photoelectron spectroscopy. The miRNA-loaded gold nanoparticles were transfected, and the level of intracellular miRNA delivered and taken up by cells was measured by Taqman qPCR. The overall analysis indicated a successful delivery of miRNA into the HOS cells at an ∼11,000-fold increase compared to nontreated cells.

10255-BOT-5 EXOSOMAL MIRNAS RELEASED FROM GLIOBLASTOMA CELLS AFTER BNCT TARGET CELL PROLIFERATION AND SURVIVAL SIGNALS

Abstract Boron neutron capture therapy (BNCT) is a tumor -selective particle radiation therapy. In BNCT, p-boronophenyl alanine, which is actively taken up by tumor cells through LAT1 transporter, a member of the system L family of heterodimeric, sodium-independent, amino acid transporters, is administrated to patients, and thermal or epithermal neutron is irradiated to tumor tissue of the patients. Nuclear capture and fission reactions between boron-10 and thermal neutron occurred only in tumor cells produce high- linear energy transfer (LET) alpha particles and recoiling lithium-7 (7Li). These high LET particles have very short pathlength (5-9 μmeter), and kill boron-10 containing tumor cells sparing adjacent normal tissue cells. BNCT was applied to malignant glioma, the most devastating tumor and has prolonged the survival of malignant glioma patients. In this study, we comprehensively investigated the micro RNAs (miRNAs) in exosomes derived from BNCT-treated glioblastoma U87 MG cells using microarray. Comparing with non-treated cells, more than 300 up-regulated miRNAs or 100 down-regulated miRNAs were detected in BNCT-treated cells. From the results of the prediction analysis of the target genes which interact with the miRNAs, the pathway analysis was performed. We found significantly associated pathways, such as Focal adhesion-PI3K-Akt-mTOR, Ras, MAPK, etc. miRNAs in exosomes released from BNCT-treated glioblastoma cells may suppress such essential pathways for cell proliferation and survival.

Evaluation of the effects of simulated hypoxia by CoCl2 on radioresistance and change of hypoxia-inducible factors in human glioblastoma U87 tumor cell line

PurposeGlioblastoma (GBM) is considered the most common and lethal type of brain tumor with a poor prognosis. GBM treatment has challenges due to its aggressive nature, which often causes treatment failure and recurrence. Hypoxia is one of the characteristics of glioblastoma tumors that contribute to radioresistance and malignant phenotypes of GBM. In this study, we aimed to determine the effects of hypoxia on the radiosensitivity of U87 GBM cells by the hypoxia-mimicking model. MethodsFollowing the treatment of cells with different concentrations of CoCl2, an MTT assay was used to evaluate the cytotoxicity of CoCl2. To understand the effects of Ionizing radiation on CoCl2-treated groups, cells were exposed to irradiation after pretreating with 100 μM CoCl2, and a clonogenic survival assay was performed to determine the radiosensitivity of U87 cells. Also, the intracellular Reactive oxygen level was measured by 2′,7′–dichlorofluorescein diacetate (DCFDA) probe staining. Additionally, the expression of hypoxia-associated genes, including HIF-1α, HIF-2α, and their target genes (GLUT-1), was monitored by reverse transcription polymerase chain reaction (RT-PCR). ResultsOur study revealed that the cell viability of CoCl2-treated cells was decreased in a concentration-dependent manner. Also, CoCl2 did not cause any cytotoxicity on U87 cells at a concentration of 100 μM after treatment for 24 h. Colony formation assay showed that CoCl2 pretreatment induced radioresistance of tumor cells compared to non-treated cells. Also, CoCl2 can protect cells against irradiation by the clearance of ROS. Moreover, Real-time results showed that the mRNA expression of HIF-1α and GLUT-1 were significantly upregulated following hypoxia induction and/or irradiation condition. However, the level of HIF-2α mRNA did not change significantly in hypoxia or irradiation alone conditions, but it increased significantly only in hypoxia + irradiation conditions. ConclusionTaken together, our results indicated that simulating hypoxia by CoCl2 can effectively increase hypoxia-associated genes, specially HIF-1α and GLUT-1, but did not affect HIF-2α gene expression. Also, it can increase the clearance of ROS, respectively, and it leads to inducing radioresistance of U87 cells.

1-DEOXYNOJIRIMYCIN, A GLYCOSYLATION INHIBITOR INFLUENCES THE EXPRESSION OF EXTRACELLULAR MATRIX PROTEINS IN OSTEOSARCOMA CELLS

Malignant tumour cells secrete extracellular matrix (ECM) proteins with growth factors and cytokines to promote cell motility and adhesion for metastasis. The cell-ECM adhesion sites are comprised of integrin-bound ECM proteins, which are glycoconjugates, susceptible to the aberrant glycosylation resulting in alterations of cell-ECM adhesion dynamics. Inhibition of glycosylation could decrease the rate of invasiveness in metastatic tumour cells like osteosarcoma (OS), of which the underlying mechanism is still unknown. Hence, this study aims to determine the effect of glucosidase inhibitors towards the expression of ECM proteins in metastatic OS cell lines. OS cells (MG63) were treated with 0.5 mM of 1-deoxynojirimycin (1-DNJ). Analyses such as the glycosylation assay, ECM-cell adhesion assay, scratch assay, and Western blot analysis were performed after a 24-h treatment for each respective sample. The expression level of glycosylated proteins was the highest in non-treated MG63 cells. The cell migration rate was increased in MG63 cells treated with 1-DNJ compared to the normal osteoblast cells. The cell adhesion assay results showed an increased rate of cell adhesion towards vitronectin but decreased adhesion rate towards collagen (types I, II, and IV), fibronectin, laminin, and tenascin in 1-DNJ-treated OS cells. Western blot analysis presented a high expression level of fibronectin, vitronectin, and collagen type II in both non-treated and treated groups of MG63 cells. The inhibitory effect of 1-DNJ upregulates ECM remodelling by disrupting integrin from binding to the ECM proteins, subsequently increasing the migration rate of metastatic OS cells. Keyword(s) : Extracellular matrix protein, 1-Deoxynojirimycin, integrin glycosylation, cell adhesion rate, migration assay.

Green Tea Extract Reduced Lipopolysaccharide-Induced Inflammation in L2 Cells as Acute Respiratory Distress Syndrome Model Through Genes and Cytokine Pro-Inflammatory.

Acute Respiratory Distress Syndrome (ARDS) is a severe lung inflammatory condition that has the capacity to impair gas exchange and lead to hypoxemia. This condition is found to have been one of the most prevalent in patients of COVID-19 with a more serious condition. Green tea (Camellia sinensis L.) contains polyphenols that possess many health benefits. The purpose of this study was to assess the anti-inflammatory activities of green tea extract in Lipopolysaccharide (LPS)-induced lung cells as ARDS cells model. In this study, rat lung cells (L2) were induced by LPS to mimic the inflammation observed in ARDS and later treated with green tea extract. Pro-inflammatory cytokines such as Interleukin (IL)-12, C-Reactive Protein (CRP) as well as Tumor Necrosis Factor-α (TNF-α) were investigated using the ELISA method. Gene expression of NOD-Like Receptor Protein 3 (NLRP-3), Receptor for Advanced Glycation End-product (RAGE), Toll-like Receptor-4 (TLR-4), and Nuclear Factor-kappa B (NF-κB) were evaluated by qRTPCR. Apoptotic cells were measured using flow cytometry. The results showed that green tea extract treatment can reduce inflammation by suppressing gene expressions of NF-κB, NLRP-3, TLR-4, and RAGE, as well as pro-inflammatory cytokines such as IL-12, TNF-α, and CRP, an acute phase protein. Apoptosis levels of inflamed cells also found to be lowered when green tea extract was administered; thus, also increasing live cells compared to non-treated cells. These findings could lead to the future development of supplements from green tea to help alleviate ARDS symptoms, especially during critical moments such as the current pandemic.

Aloe-Emodin Isolated from Rheum Undulatum L. Regulates Cell Cycle Distribution and Cellular Senescence in Human Prostate Cancer LNCaP Cells

Senescence can promote hyperplastic pathologies, such as cancer. Prostate cancer is the second most common type of cancer in men. The p21-mediate cellular senescence, facilitated through the tumor suppressor p53-dependent pathway, is considered the primary mechanism for cancer treatment. Aloe-emodin, has been reported to exert anticancer effects in various types of cancers. This study aimed to investigate the bioactivity of aloe-emodin in LNCaP cells via the activation of p21-mediated cellular senescence. Aloe-emodin treatment increased the percentage of cells in the G1 phase while decreasing the percentage in the S phase. This effect was reflected in the expression levels of proteins associated with cell cycle progression, such as p21CIP, retinoblastoma protein, and cyclin-dependent kinase2/4 in LNCaP cells. However, aloe-emodin-treated LNCaP cells did not induce cell cycle arrest at G2/M checkpoint. Moreover, increased senescence-associated-galactosidase activity was observed in a dose-dependent manner following treatment with aloe-emodin. Aloe-emodin also induced DNA damage by modulating the expression of histone H2AX and lamin B1. Furthermore, aloe-emodin inhibited the proliferation of LNCaP cells, contrasting with the exponential growth observed in the nontreated cells. Importantly, this inhibition did not impact the immune system, as evidenced by the increased proliferation of splenocytes isolated from mice. These findings provide preliminary evidence of the anticancer effect of aloe-emodin in LNCaP cells, necessitating further investigations into the underlying mechanisms in vivo and human subjects.

Biocompatibility and Osteo/Odontogenic Potential of Various Bioactive Root-End Filling Materials

This in vitro study aimed to investigate if different bioactive root-end filling materials can promote osteo/odontogenic differentiation of mesenchymal stem cells (MSCs) and support their viability. MSCs from porcine tooth germs were isolated. Cells were exposed to extracts from MTA Angelus, BIOfactor MTA, Medcem MTA, Well-Root ST, and Pure Portland Cement for 7 days. Viability was determined with MTS and live/dead assay. Osteo/odontogenic differentiation was evaluated with alkaline phosphatase (ALP) activity and quantitative real-time PCR (RUNX2, DMP-1, and DSPP genes) which were compared with osteo/odontogenic-induced MSCs and non-treated cells. All the tested materials supported cell proliferation and cells maintained their viability after 7 days. Osteo/odontogenic differentiation of MSCs was promoted by the tested materials in varying levels as demonstrated by increased ALP activity and upregulation of related gene markers in comparison to the control group. Pure Portland Cement demonstrated a continuous high ALP activity on day 7, showing the highest value among all materials and significantly increased in comparison to the control group (p < 0.001). Significant RUNX2 expression and high ALP activity (p < 0.001) similar to that of osteogenically induced cells was detected for Pure Portland Cement after 7 days. Tested MTA-based cement materials are biocompatible and induce osteo/odontogenic differentiation in vitro. MTA materials performed similarly to Pure Portland Cement regarding osteo/odontogenic differentiation.

PSIII-29 A Novel Direct-Fed Microbial Supports in Vitro Intestinal Integrity Under Inflamed Conditions

Abstract Ruminants are often exposed to pathogenic and non-pathogenic challenges during daily handling procedures and management activities. These responses, in turn, may trigger the release of pro-inflammatory compounds that can have negative effects on the host. Based on this rationale, we hypothesized that 1) pro-inflammatory cytokines (TNF-α and IFN-γ) would compromise the in vitro gut barrier integrity, and 2) a novel direct-fed microbial (DFM) formulation would support the gut barrier integrity under inflammatory conditions. Therefore, our objective was to evaluate the ability of a novel DFM mixture to support barrier integrity of intestinal epithelial cell monolayers in the absence or presence of TNF-α and IFN-γ by measuring transepithelial electrical resistance (TEER). Human cancer-derived epithelial intestinal Caco-2 cell monolayers were seeded on transwells for approximately 20 days. A DFM mixture containing Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus licheniformis, and B. subtilis (BOVAMINE DEFEND Plus; Chr. Hansen A/S, Hørsholm, Denmark) at 1.0 × 108 CFU/transwell was then administered to the apical side of the cell monolayers. Then, two hours later a cocktail containing TNF-α / IFN-γ at a 10:1 ratio (100:10 ng/mL, respectively) was added to the basolateral side after which TEER was measured over a 16-h period and used to calculate the area under the curve (AUC). Following the administration of the pro-inflammatory challenge, TEER was decreased, reaching its least concentrations at 5.5 h post-challenge, whereas overall AUC decreased by 21.4% compared with non-challenged control cells. The DFM mixture by itself was able to support the integrity of the intestinal cells by stimulating TEER increase over time and increasing overall AUC vs. non-treated cells (P < 0.01). Moreover, when cells receiving the DFM were challenged with the pro-inflammatory cocktail, TEER concentrations were still above 100% and no decrease was observed over the evaluation period (16 h). Moreover, when compared with non-DFM-treated but challenged cells, AUC was greater for DFM vs. challenged control cells (P < 0.05). In conclusion, these data support our hypothesis that a DFM mixture containing L. animalis, P. freudenreichii, B. licheniformis, and B. subtilis (BOVAMINE DEFEND Plus) supports the gut barrier integrity with or without the pro-inflammatory challenge.

Protein Disulfide Isomerase Family A Member 3 Knockout Abrogate Effects of Vitamin D on Cellular Respiration and Glycolysis in Squamous Cell Carcinoma.

PDIA3 is an endoplasmic reticulum disulfide isomerase, which is involved in the folding and trafficking of newly synthesized proteins. PDIA3 was also described as an alternative receptor for the active form of vitamin D (1,25(OH)2D3). Here, we investigated an impact of PDIA3 in mitochondrial morphology and bioenergetics in squamous cell carcinoma line A431 treated with 1,25(OH)2D3. It was observed that PDIA3 deletion resulted in changes in the morphology of mitochondria including a decrease in the percentage of mitochondrial section area, maximal diameter, and perimeter. The 1,25(OH)2D3 treatment of A431∆PDIA3 cells partially reversed the effect of PDIA3 deletion increasing aforementioned parameters; meanwhile, in A431WT cells, only an increase in mitochondrial section area was observed. Moreover, PDIA3 knockout affected mitochondrial bioenergetics and modulated STAT3 signaling. Oxygen consumption rate (OCR) was significantly increased, with no visible effect of 1,25(OH)2D3 treatment in A431∆PDIA3 cells. In the case of Extracellular Acidification Rate (ECAR), an increase was observed for glycolysis and glycolytic capacity parameters in the case of non-treated A431WT cells versus A431∆PDIA3 cells. The 1,25(OH)2D3 treatment had no significant effect on glycolytic parameters. Taken together, the presented results suggest that PDIA3 is strongly involved in the regulation of mitochondrial bioenergetics in cancerous cells and modulation of its response to 1,25(OH)2D3, possibly through STAT3.

Cultured lymphocytes' mitochondrial genome integrity is not altered by cladribine.

Cladribine tablets are a treatment for multiple sclerosis with effects on lymphocytes, yet its mode of action has not been fully established. Here, we analyzed the effects of cladribine on mitochondrial DNA integrity in lymphocytes. We treated cultured human T-cell lines (CCRF-CEM and Jurkat) with varying concentrations of cladribine to mimic the slow cell depletion observed in treated patients. The CCRF-CEM was more susceptible to cladribine than Jurkat cells. In both cells, mitochondrial protein synthesis, mitochondrial DNA copy number, and mitochondrial cytochrome-c oxidase-I mRNA mutagenesis was not affected by cladribine, while caspase-3 cleavage was detected in Jurkat cells at 100 nM concentration. Cladribine treatment at concentrations up to 10 nM in CCRF-CEM and 100 nM in Jurkat cells did not induce significant increase in mitochondrial DNA mutations. Peripheral blood mononuclear cells from eight multiple sclerosis patients and four controls were cultured with or without an effective dose of cladribine (5 nM). However, we did not find any differences in mitochondrial DNA somatic mutations in lymphocyte subpopulations (CD4+, CD8+, and CD19+) between treated versus nontreated cells. The overall mutation rate was similar in patients and controls. When different lymphocyte subpopulations were compared, greater mitochondrial DNA mutation levels were detected in CD8+ (P = 0.014) and CD4+ (P = 0.038) as compared to CD19+ cells, these differences were independent of cladribine treatment. We conclude that T cells have more detectable mitochondrial DNA mutations than B cells, and cladribine has no detectable mutagenic effect on lymphocyte mitochondrial genome nor does it impair mitochondrial function in human T-cell lines.

Effect of plasma-activated water on the biofilm-forming ability of Salmonella enterica serovar Enteritidis and expression of the related genes

In recent years, microbial decontamination with plasma-activated water (PAW) has attracted a lot of research attention in the field of food industry. Despite several studies showing that PAW effectively inactivates planktonic bacteria, few studies have been conducted on biofilms. The present study was, therefore, designed to evaluate the effect of PAW on the biofilm formation characteristics of Salmonella Enteritidis. Comparing the expression patterns of biofilm-related genes in PAW-treated and non-treated planktonic and biofilm cells provided insight into how PAW regulates this process. The results showed that a 30-minute exposure to PAW at room temperature significantly reduced S. enteritidis planktonic cells. This exposure resulted in a decreased expression of the genes involved in the early stages of biofilm formation (csgD, agfA, fimA, lpfE, and rpoS), and an increased expression of the csrA gene in S. enteritidis planktonic cells. These results indicated the inhibitory effect of PAW on the biofilm formation process in S. enteritidis. Results of the initial attachment assay confirmed these findings, where, after 6 h, the number of PAW-treated cells attached to the stainless steel surfaces were significantly lower than non-treated ones. Furthermore, biofilm development assay revealed that the number of PAW-treated biofilm cells were significantly lower than non-treated ones after 24 h incubation at 37 °C. These findings were confirmed by measurements of the major components of biofilm i.e., extracellular DNA (eDNA), protein and carbohydrate. The amount of these components in 24-hour biofilms produced by PAW-treated S. enteritidis cells was significantly lower than that of non-treated cells. PAW's treatment on preformed 24-hour biofilms for 30 min led to a decrease in the expression of genes involved in quorum sensing and cellulose synthesis (csgD, bapA, adrA, luxS and sdiA) and an increase in the expression of the csrA gene. This treatment also reduced the number and metabolic activity of biofilm cells compared to non-treated biofilm cells. In total, the present study demonstrated that PAW has an inhibitory effect on the process of biofilm formation in S. enteritidis and hence, the food industry should pay special attention to PAW as a promising treatment to eliminate bacterial biofilms.

Nedaplatin/Peganum harmala Alkaloids Co-Loaded Electrospun, Implantable Nanofibers: A Chemopreventive Nano-Delivery System for Treating and Preventing Breast Cancer Recurrence after Tumorectomy.

Currently, the main pillars in treating breast cancer involve tumorectomy pursued by hormonal, radio, or chemotherapies. Nonetheless, these approaches exhibit severe adverse effects and might suffer from tumor recurrence. Therefore, there is a considerable demand to fabricate an innovative controlled-release nano-delivery system to be implanted after tumor surgical removal to guard against cancer recurrence. In addition, combining platinum-based drugs with phytochemicals is a promising approach to improving the anticancer activity of the chemotherapeutics against tumor cells while minimizing their systemic effects. This study designed polycaprolactone (PCL)-based electrospun nanofiber mats encapsulating nedaplatin (N) and Peganum harmala alkaloid-rich fraction (L). In addition to physicochemical characterization, including average diameters, morphological features, degradation study, thermal stability, and release kinetics study, the formulated nanofibers were assessed in terms of cytotoxicity, where they demonstrated potentiated effects and higher selectivity towards breast cancer cells. The dual-loaded nanofiber mats (N + L@PCL) demonstrated the highest antiproliferative effects against MCF-7 cells with a recorded IC50 of 3.21 µg/mL, as well as the topmost achieved selectivity index (20.45) towards cancer cells amongst all the tested agents (N, L, N@PCL, and L@PCL). This indicates that the dual-loaded nanofiber excelled at conserving the normal breast epithelial cells (MCF-10A). The combined therapy, N + L@PCL treatment, resulted in a significantly higher percent cell population in the late apoptosis and necrosis quartiles as compared to all other treatment groups (p-value of ≤0.001). Moreover, this study of cell cycle kinetics revealed potentiated effects of the dual-loaded nanofiber (N + L@PCL) at trapping more than 90% of cells in the sub-G1 phase and reducing the number of cells undergoing DNA synthesis in the S-phase by 15-fold as compared to nontreated cells; hence, causing cessation of the cell cycle and confirming the apoptosis assay results. As such, our findings suggest the potential use of the designed nanofiber mats as perfect implants to prevent tumor recurrence after tumorectomy.

Horseradish Peroxidase-Mediated Bioprinting via Bioink Gelation by Alternately Extruded Support Material.

Horseradish peroxidase (HRP)-mediated extrusion bioprinting has a significant potential in tissue engineering and regenerative medicine. However, they often face challenges in terms of printing fidelity and structural integrity when using low-viscosity inks. To address this issue, a method that alternately extrudes bioinks and support material was developed in this study. The bioinks consisting of cells, HRP, and phenolated polymers, and the support material contained hydrogen peroxide (H2O2). The support material not only prevented the collapse of the constructs but also supplied H2O2 to facilitate the enzymatic reaction. 3D constructs with tall and complex shapes were successfully printed from a low-viscosity ink containing 10 U/mL HRP and 1.0% w/v phenolated hyaluronic acid (HA-Ph), with a support material containing 10 mM H2O2. Over 90% viability of mouse fibroblasts (10T1/2) was achieved following the printing process, along with a morphology and proliferation rate similar to that of nontreated cells. Furthermore, human hepatoblastoma (HepG2) cells showed an increased spheroid size over 14 days in the printed constructs. The 10T1/2 cells adhered and proliferated on the constructs printed from inks containing both phenolated gelatin and HA-Ph. These results demonstrate the great potential of this HRP-mediated extrusion bioprinting technique for tissue engineering applications.

Piper chaba, an Indian spice plant extract, inhibits cell cycle G1/S phase transition and induces intrinsic apoptotic pathway in luminal breast cancer cells.

Piper chaba (Piperaceae) is a medicinal spice plant that possesses several pharmacological activities. In the present study, we for the first time studied the effect of P. chaba extract on breast cancer cells. P. chaba stem methanolic (PCSM) extract produced time and dose dependent cytotoxicity in luminal breast cancer cells (MCF-7 and T47D) with a minimal toxicity in breast normal cells (MCF-10A) at 10-100 µg/mL concentration. PCSM extract exerts 16.79 and 31.21 µg/mL IC50 for T47D and MCF-7 cells, respectively, in 48 h treatment. PCSM significantly arrests the T47D cells at the G0/G1 phase by reducing the CCND1 and CDK4 expression at mRNA and protein levels. PCSM extract treatment significantly altered nuclear morphology, mitochondria membrane potential, and production of reactive oxygen species in T47D cells at IC50 concentration. Extract treatment significantly altered the Bax/Bcl-2 ratio and altered caspase 8 and 3 mRNA/protein levels in T47D cells. Confocal microscopy showed an increase in late apoptosis in PCSM extract-treated breast cancer cells at IC50 . Further, an increased caspase 9 and caspase 3/7 enzymatic activity was observed in test cells compared with nontreated cells. In conclusion, P. chaba phytocompound possesses the potential to induce cell cycle arrest and induce apoptosis in luminal breast cancer cells.

Multitargeting Prodrugs that Release Oxaliplatin, Doxorubicin and Gemcitabine are Potent Inhibitors of Tumor Growth and Effective Inducers of Immunogenic Cell Death.

A multitargeting prodrug (2) that releases gemcitabine, oxaliplatin, and doxorubicin in their active form in cancer cells is a potent cytotoxic agent with nM IC50s ; it is highly selective to cancer cells with mean selectivity indices to human (136) and murine (320) cancer cells. It effectively induces release of DAMPs (CALR, ATP & HMGB1) in CT26 cells facilitating more efficient phagocytosis by J774 macrophages than the FDA drugs or their co-administration. The viability of CT26 cells co-cultured with J774 macrophages and treated with 2 was reduced by 32 % compared to the non-treated cells, suggesting a synergistic antiproliferative effect between the chemical and immune reactions. 2 inhibited in vivo tumor growth in two murine models (LLC and CT26) better than the FDA drugs or their co-administration with significantly lower body weight loss. Mice inoculated with CT26 cells treated with 2 showed slightly better tumor free survival than doxorubicin.