Pulsed electric field induces exocytosis and overexpression of MAGE antigens in melanoma.

Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

MuSK Myasthenia Gravis-Potential Pathomechanisms and Treatment Directed against Specific Targets.

Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies target structures within the neuromuscular junction, affecting neuromuscular transmission. Muscle-specific tyrosine kinase receptor-associated MG (MuSK-MG) is a rare, often more severe, subtype of the disease with different pathogenesis and specific clinical features. It is characterized by a more severe clinical course, more frequent complications, and often inadequate response to treatment. Here, we review the current state of knowledge about potential pathomechanisms of the MuSK-MG and their therapeutic implications as well as ongoing research in this field, with reference to key points of immune-mediated processes involved in the background of myasthenia gravis.

Application of Luteolin in Neoplasms and Nonneoplastic Diseases.

Researchers are amazed at the multitude of biological effects of 3',4',5,7-tetrahydroxyflavone, more commonly known as luteolin, as it simultaneously has antioxidant and pro-oxidant, as well as antimicrobial, anti-inflammatory, and cancer-preventive, properties. The anticancer properties of luteolin constitute a mosaic of pathways due to which this flavonoid influences cancer cells. Not only is it able to induce apoptosis and inhibit cancer cell proliferation, but it also suppresses angiogenesis and metastasis. Moreover, luteolin succeeds in cancer cell sensitization to therapeutically induced cytotoxicity. Nevertheless, apart from its promising role in chemoprevention, luteolin exhibits numerous potential utilizations in patients with conditions other than neoplasms, which include inflammatory skin diseases, diabetes mellitus, and COVID-19. This review aims to present the multidimensionality of the luteolin's impact on both neoplastic and nonneoplastic diseases. When it comes to neoplasms, we intend to describe the complexity of the molecular mechanisms that underlay luteolin's anticancer effectiveness, as well as to prove the usefulness of integrating this flavonoid in cancer therapy via the analysis of recent research on breast, colon, and lung cancer. Regarding nonneoplastic diseases, this review aims to emphasize the importance of researching the potential of luteolin in areas such as diabetology, virology, and dermatology as it summarizes the most important discoveries in those fields regarding its application.

Complementary Gene Therapy after Revascularization with the Saphenous Vein in Diabetic Foot Syndrome.

Diabetic foot syndrome (DFS) is one of the most serious macroangiopathic complications of diabetes. The primary treatment option is revascularization, but complementary therapies are still being sought. The study group consisted of 18 patients diagnosed with ischemic ulcerative and necrotic lesions in DFS. Patients underwent revascularization procedures and, due to unsatisfactory healing of the lesions, were randomly allocated to two groups: a group in which bicistronic VEGF165/HGF plasmid was administered and a control group in which saline placebo was administered. Before gene therapy administration and after 7, 30, 90, and 180 days, color duplex ultrasonography (CDU) was performed, the ankle-brachial index (ABI) and transcutaneous oxygen pressure (TcPO2) were measured, and DFS changes were described and documented photographically. In the gene therapy group, four out of eight patients (50%) healed their DFS lesions before 12 weeks. During this time, the ABI increased by an average of 0.25 and TcPO2 by 30.4 mmHg. In the control group, healing of the lesions by week 12 occurred in six out of nine patients (66.67%), and the ABI increased by an average of 0.14 and TcPO2 by 27.1 mmHg. One major amputation occurred in each group. Gene therapy may be an attractive option for complementary treatment in DFS.

Effects of Nanosecond Pulsed Electric Field on Immune Checkpoint Receptors in Melanoma Cells.

Checkpoint molecules such as PD-1, LAG-3, and TIM-3 are currently under extensive investigation for their roles in the attenuation of the immune response in cancer. Various methods have been applied to overcome the challenges in this field. This study investigated the effects of nanosecond pulsed electric field (nsPEF) treatment on the expression of immune checkpoint molecules in A375 and C32 melanoma cells. The researchers found that the nsPEF treatment was able to enhance membrane permeabilization and morphological changes in the cell membrane without being cytotoxic. We found that the effects of nsPEFs on melanoma included (1) the transport of vesicles from the inside to the outside of the cells, (2) cell contraction, and (3) the migration of lipids from inside the cells to their peripheries. The treatment increased the expression of PD-1 checkpoint receptors. Furthermore, we also observed potential co-localization or clustering of MHC class II and PD-1 molecules on the cell surface and the secretion of cytokines such as TNF-α and IL-6. These findings suggest that nsPEF treatment could be a viable approach to enhance the delivery of therapeutic agents to cancer cells and to modulate the tumor microenvironment to promote an antitumor immune response. Further studies are needed to explore the mechanisms underlying these effects and their impacts on the antitumor immune response, and to investigate the potential of nsPEF treatment in combination with immune checkpoint inhibitors to improve clinical outcomes for cancer patients.

The mRNA expression of genes encoding selected antioxidant enzymes and thioredoxin, and the concentrations of their protein products in gingival crevicular fluid and saliva during periodontitis.

BACKGROUND: The activity of antioxidant enzymes in periodontitis is reduced, but results vary between studies and are subject to bias. In turn, the expression of genes encoding antioxidant factors has not been examined yet. OBJECTIVES: This is the first study to evaluate the expression of genes encoding superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1) and thioredoxin 1 (TXN1) in the saliva and gingival tissue of patients with periodontitis. The activity of the antioxidant enzyme protein products in the unstimulated and stimulated saliva and the gingival crevicular fluid (GCF) of patients with periodontitis was also investigated. MATERIAL AND METHODS: The prospective study involved 65 patients with periodontitis, who were divided into groups depending on the disease stage, and a control group of 31 ageand gender-matched healthy patients. RESULTS: We demonstrated that the expression of genes encoding GPX1 and TXN1 in saliva was significantly higher, and the expression of genes encoding SOD1, GPX1 and TXN1 in the gingival tissue was significantly lower in periodontitis patients as compared to the control group. We noted a lower activity of GPX1 in unstimulated saliva, of SOD1 in stimulated saliva and of both antioxidant enzymes in GCF in patients with periodontitis. CONCLUSIONS: The GPX1 transcriptome and its activity in the salivary and GCF proteome appear to be dependent on the oxidative stress related to the destructive inflammatory changes in periodontitis.

Ether Derivatives of Naringenin and Their Oximes as Factors Modulating Bacterial Adhesion.

Because of the close connection between adhesion and many vital cellular functions, the search for new compounds modulating the adhesion of bacteria belonging to the intestinal microbiota is a great challenge and a clinical need. Based on our previous studies, we discovered that O-lkyl naringenin derivatives and their oximes exhibit antimicrobial activity against antibiotic-resistant pathogens. The current study was aimed at determining the modulatory effect of these compounds on the adhesion of selected representatives of the intestinal microbiota: Escherichia coli, a commensal representative of the intestinal microbiota, and Enterococcus faecalis, a bacterium that naturally colonizes the intestines but has disease-promoting potential. To better reflect the variety of real-life scenarios, we performed these studies using two different intestinal cell lines: the physiologically functioning ("healthy") 3T3-L1 cell line and the disease-mimicking, cancerous HT-29 line. The study was performed in vitro under static and microfluidic conditions generated by the Bioflux system. We detected the modulatory effect of the tested O-alkyl naringenin derivatives on bacterial adhesion, which was dependent on the cell line studied and was more significant for E. coli than for E. faecalis. In addition, it was noticed that this activity was affected by the concentration of the tested compound and its structure (length of the carbon chain). In summary, O-alkyl naringenin derivatives and their oximes possess a promising modulatory effect on the adhesion of selected representatives of the intestinal microbiota.

Antiproliferative Activity and Impact on Human Gut Microbiota of New O-Alkyl Derivatives of Naringenin and Their Oximes.

Naringenin is a 5,7,4'-trihydroxyflavanone naturally occurring mainly in citrus fruits, characterized by a wide spectrum of biological activity. Chemical modifications based on alkylation and oximation in most cases increase its bioactivity. The aim of our research was to evaluate the antiproliferative activity and influence on selected representatives of the human gut microbiota of new synthesized O-alkyl derivatives (A1-A10) and their oximes (B1-B10), which contain hexyl, heptyl, octyl, nonyl and undecyl chains attached to the C-7 or to both the C-7 and C-4' positions in naringenin. To the best of our knowledge, compounds A3, A4, A6, A8-A10 and B3-B10 have not been described in the scientific literature previously. The anticancer activity was tested on human colon cancer cell line HT-29 and mouse embryo fibroblasts 3T3-L1 using the sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. We also determined the impacts of all compounds on the growth of Gram-positive and Gram-negative bacterial strains, such as Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. The antimicrobial activity was expressed in terms of minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) values. For 7,4'-di-O-hexylnaringenin (A2), 7-O-undecylnaringenin (A9) and their oximes (B2, B9), which were safe for microbiota (MIC > 512 µg/mL) and almost all characterized by high cytotoxicity against the HT-29 cell line (A2: IC50 > 100 µg/mL; A9: IC50 = 17.85 ± 0.65 µg/mL; B2: IC50 = 49.76 ± 1.63 µg/mL; B9: IC50 = 11.42 ± 1.17 µg/mL), apoptosis assays were performed to elucidate their mechanisms of action. Based on our results, new compound B9 induced an apoptotic process via caspase 3/7 activation, which proved its potential as an anticancer agent.

miR-31-5p as a Potential Circulating Biomarker and Tracer of Clinical Improvement for Chronic Inflammatory Demyelinating Polyneuropathy.

Background: MicroRNAs are endogenous, small noncoding RNA molecules that play a pivotal role in the regulation of gene expression. MicroRNAs are involved in many biological processes such as proliferation, cell differentiation, neovascularization, and apoptosis. Studies on microRNA expression may contribute to a better understanding of the pathomechanism of chronic inflammatory demyelinating polyneuropathy (CIDP) and consequently enable the development of new therapeutic measures using antisense miRNAs (antagomirs). In this study, we evaluated the level of miR-31-5p in the serum of patients with CIDP and its correlation with the miR-31-5p level and clinical presentation and electrophysiological and biochemical parameters. Methods: The study group consisted of 48 patients, mean age 61.60 ± 11.76, who fulfilled the diagnostic criteria of a typical variant of CIDP. The expression of miR-31-5p in patient serum probes was investigated by droplet digital PCR. The results were correlated with neurophysiological findings and the patient's clinical and biochemical parameters. Results: The mean copy number of miRNA-31 in 100 µl serum was 1288.64 ± 2001.02 in the CIDP group of patients, while in the control group, it was 3743.09 ± 4026.90. There was a significant positive correlation (0.426) between IgIV treatment duration and miR-31-5p expression. Patients without IgIV treatment showed significantly lower levels of miR-31 compared to the treated group (259.44 ± 304.02 vs. 1559.48 ± 2168.45; p = 0.002). The group of patients with body weight > 80 kg showed statistically significantly lower levels of miRNA-31-5p than the patients with lower body weight (934.37 ± 1739.66 vs. 1784.62 ± 2271.62, respectively; p = 0.014). Similarly, the patients with elevated cerebrospinal fluid (CSF) protein levels had significantly higher miRNA-31-5p expression than those with normal protein levels (1393.93 ± 1932.27 vs. 987.38 ± 2364.10, respectively; p = 0.044). Conclusion: The results may support the hypothesis that miR-31-5p is strongly involved in the autoimmune process in CIDP. The positive correlation between higher miR-31-5p levels and duration of IVIg treatment may be an additional factor explaining the efficacy of prolonged IVIg therapy in CIDP.

Pluronics-Based Drug Delivery Systems for Flavonoids Anticancer Treatment.

This research concerns the investigation of the preparation of polymeric nanocarriers containing a flavonoid-naringenin, xanthohumol or isoxanthohumol-based on Pluronics by the thin-film formation method. The size of the formed micelles and their stability upon dilution were evaluated using Dynamic light scattering (DLS) analysis; the high values of the drug loading and the encapsulation efficiency confirmed that the proposed systems of flavonoids delivery consisting of Pluronic P123 and F127 nanomicelles could effectively distribute the drug into tumour tissues, which makes these nanocarriers ideal candidates for passive targeting of cancer cells by the enhanced permeation and retention (EPR) effect. The in vitro cytotoxicity of proposed flavonoids in the Pluronic formulations was investigated by the SRB assay with human colon cancer cells. We designed mixed polymeric micelles, which was a successful drug delivery system for the case of naringenin not being able to enhance the bioavailability and cytotoxic activity of xanthohumol and isoxanthohumol. Furthermore, it was observed that the higher amount of polymer in the formulation achieved better cytotoxic activity.

Nanoelectropulse delivery for cell membrane perturbation and oxidation in human colon adenocarcinoma cells with drug resistance.

Ultrashort electric pulses in the nanosecond range (nsPEF) can affect extra- and intracellular lipid structures and can also alternate cell functioning reversibly and irreversibly. Several of the nsPEF effects are due to the abrupt rise in intracellular free calcium levels and calcium ions influx from the outside. Calcium is one of the most important factors in cell proliferation, differentiation, and cell death (apoptosis or necrosis). Manipulating calcium levels using electroporation can have different effects on normal and malignant cells. This study aimed to examine the impact of nsPEFs, combined with 1 mM Ca2+ in human colon adenocarcinoma cell lines: sensitive- LoVo and drug resistant-LoVoDX. In this study 200 pulses of 10 ns and high voltage (12.5-50 kVcm-1) were used. Cell viability was determined by MTT and clonogenic assay. Proteasomal activity, GSH/GSSG assay, ROS production, and PALS-1 protein were evaluated as oxidative stress markers and protein damage. Cell morphology was visualized by AFM, SEM, and confocal microscopy imaging. The results revealed that nsPEF with 1 mM Ca2+ is cytotoxic, particularly for LoVoDX cells, and safe for normal cells. NsPEF provoked ROS release, altered cell polarity, and destabilized cell morphology. These results can be important for future protocols for colon adenocarcinoma using calcium nsPEF.

Two-Stage Gene Therapy (VEGF, HGF and ANG1 Plasmids) as Adjunctive Therapy in the Treatment of Critical Lower Limb Ischemia in Diabetic Foot Syndrome.

One of the most serious problems in people with diabetes is diabetic foot syndrome. Due to the peripheral location of atherosclerotic lesions in the arterial system of the lower extremities, endovascular treatment plays a dominant role. However, carrying out these procedures is not always possible and does not always bring the expected results. Gene therapy, which stimulates angiogenesis, improves not only the inflow from the proximal limb but also the blood redistribution in individual angiosomes. Due to the encouraging results of sequential treatment consisting of intramuscular injections of VEGF/HGF bicistronic plasmids followed by a month of ANG1 plasmids, we decided to use the described method for the treatment of critical ischemia of the lower limbs in the course of diabetes and, more specifically, in diabetic foot syndrome. Twenty-four patients meeting the inclusion criteria were enrolled in the study. They were randomly divided into two equal groups. The first group of patients was subjected to gene therapy, where the patients received intramuscular injections of pIRES/VEGF165/HGF plasmids and 1 month of ANG-1 plasmids. The remaining patients constituted the control group. Gene therapy was well tolerated by most patients. The wounds healed significantly better in Group 1. The minimal value of ABI increased significantly in Group 1 from 0.44 ± 0.14 (± standard deviation) to 0.47 ± 0.12 (with p = 0.028) at the end of the study. There were no significant differences in the control group. In the gene treatment group, PtcO2 increased significantly (from 28.71 ± 10.89 mmHg to 33.9 ± 6.33 mmHg with p = 0.001), while in Group 2, no statistically significant changes were found. The observed resting pain decreased significantly in both groups (Group 1 decreased from 6.80 ± 1.48 to 2.10 ± 1.10; p < 0.001; the control group decreased from 7.44 ± 1.42 to 3.78 ± 1.64 with p < 0.001). In our study, we evaluated the effectiveness of gene therapy with the growth factors described above in patients with CLI in the course of complicated DM. The therapy was shown to be effective with minimal side effects. No serious complications were observed.

Role of hypoxia on microRNA-dependant regulation of HGFA - HGF - c-Met signalling pathway in human progenitor and mature endothelial cells.

Hepatocyte growth factor (HGF) is considered to be one of the key pro-angiogenic cytokines that stimulates endothelial cells to proliferate and migrate. The activation of the precursor form of HGF is primarily undertaken by the serine protease HGFA. Research indicates that HIF-1α hypoxia stimulates the expression of HGFA, which is synthesized by a range of cells including fibroblasts, endothelium, and macrophages. To date, little is known about the potential role of epigenetic factors in the regulation of the HGFA - HGF - c-Met signalling pathway. The literature suggests that there are several microRNAs (miRNAs, miRs) directly affecting the expression of c-Met under normoxic conditions. The main objective of the research described was to explore the effect of chemically-induced hypoxia on the expression of miRNA molecules in human progenitor and mature endothelial cells, with particulate attention paid to those miRNAs that may specifically affect the HGFA - HGF - c-Met signalling pathway. This publication sheds new light on the role of miRNAs in hypoxia, as well as identifying several miRNAs directly involved in the regulation of HGFA, HGF and c-Met expression in hypoxic conditions. The results indicate that hsa-miR-335-5p, hsa-miR-425-5p and hsa-miR-101-3p are the major miRNAs that appear to play an important role in the regulation of the HGFA - HGF - c-Met signalling pathway.

Pulsed electric fields with calcium ions stimulate oxidative alternations and lipid peroxidation in human non-small cell lung cancer.

Pulsed electric fields (PEFs) are commonly used to facilitate the delivery of various molecules, including pharmaceuticals, into living cells. However, the applied protocols still require optimization regarding the conditions of the permeabilization process, i.e., pulse waveform, voltage, duration, and the number of pulses in a burst. This study highlights the importance of electrochemical processes involved in the electropermeabilization process, known as electroporation. This research investigated the effects of electroporation on human non-small cell lung cancer cells (A549) in potassium (SKM) and HEPES-based buffers (SHM) using sub-microsecond and microsecond range pulses. The experiments were performed using 100 ns - 100 µs (0.6-15 kV/cm) bursts with 8 pulses in a sequence. It was shown that depending on the buffer composition, the susceptibility of cells to PEF varies, while calcium enhances the cytotoxic effects of PEF, if high cell membrane permeabilization is triggered. It was also determined that electroporation with calcium ions induces oxidative stress in cells, including lipid peroxidation (LPO), generation of reactive oxygen species (ROS), and neutral lipid droplets. Here, we demonstrated that calcium ions and optimized pulse parameters could potentiate PEF efficacy and oxidative alternations in lung cancer cells. Thus, the anticancer efficacy of PEF in lung cancers in combination with standard cytostatic drugs or calcium ions should be considered, but this issue still requires in-depth detailed studies with in vivo models.

The role of catechin in electroporation of pancreatic cancer cells - Effects on pore formation and multidrug resistance proteins.

Catechin is a bioflavonoid known for its anti-cancer properties. In the present study, we combined theoretical and experimental approaches to reveal the potential of catechin application in the electroporation (EP) or electrochemotherapy (ECT) of pancreatic cancer cells. The molecular dynamics simulations were implemented to examine the interactions of catechin with a model of a membrane, its influence on the membrane's thickness, and the impact of the catechin-membrane interaction on the pore formation. The data were confronted with experimental measurement of the threshold electric field required for permeabilization of pancreatic cancer cells to a fluorescent dye YO-PRO-1. Further, we examined the influence of catechin on cell viability following electroporation with cisplatin or calcium ions. Finally, we investigated the catechin impact on four proteins associated with multidrug resistance: P-glycoprotein, MRP1, BCRP, and LRP. We demonstrated that catechin may boost the effects of electroporation through various mechanisms: i) increasing the cell permeability prior to electroporation ii) increasing the electroporation threshold iii) sensitization of cells to chemotherapeutic compounds. We showed that catechin incubation influences mRNA levels and mitigates the immunoreactivity of Pgp, MRP1, BCRP, and LRP but these changes did not translate to the efficacy of electrochemotherapy.

RCCS Bioreactor-Based Modeled Microgravity Affects Gastric Cancer Cells and Improves the Chemotherapeutic Effect.

(1) Background: The main purpose of the study was to determine whether altered gravity might alter cell viability, improve drug delivery and modulate the expression of drug resistance-related genes. (2) Methods: This study investigated the intracellular mechanisms activated by microgravity in human resistant and sensitive gastric cancer cells (EPG85-257 RDB) and (EPG85-257 P). We used a rotary cell culture system (RCCS) developed by NASA to expose cells to altered gravity. The antitumor potential of microgravity was simulated by the RCCS bioreactor, and its effectiveness was evaluated in sensitive cell lines compared to chemotherapy-resistant cells concerning drug-sensitive cancer cells. Microgravity with chemotherapy was estimated by the viability assay, cytoskeleton imaging, MDR (multidrug resistance) gene expression analysis, MTCO-1 (mitochondrially encoded cytochrome C oxidase I), and 8-OHdG immunocytochemical analysis. (3) Results: We found that altered gravity combined with doxorubicin was cytotoxic to cancer cells. Cells following simulated microgravity revealed decreased expression of genes related to drug resistance and increased DNA/RNA damage marker expression. Cytoskeleton evaluation demonstrated significant reorganization of F-actin fibers after exposure to changed gravity conditions. (4) Conclusions: Intracellular alterations caused by simulated microgravity can increase gastric cancer cells' sensitivity to chemotherapy. We have obtained satisfactory results showing the correlation between altered gravity and MDR phenomena which seems promising in future therapeutic applications.

Anticancer Efficacy of 6-Gingerol with Paclitaxel against Wild Type of Human Breast Adenocarcinoma.

Breast cancer is one of the most common malignant neoplasms, and despite the dynamic development of anticancer therapies, 5-year survival in the metastatic stage is still less than 30%. 6-Gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3-decanone) is a substance contained in ginger, which exhibits anti-cancer properties. Paclitaxel is a cytostatic substance used to treat breast cancer, but its therapeutically effective dose has many adverse effects. The aim of the presented study was to assess the anticancer effect of 6-gingerol and the possibility of increasing the effectiveness of Paclitaxel in the death induction of wild type human breast cancer cells. MCF-7/WT cells were treated with drugs-6-gingerol and paclitaxel at selected concentrations. The mitochondrial activity assay, caspase 7 activity assay, ATP assay, microscopy studies, and RT-PCR assays were performed to evaluate the antitumor activity and mechanism of action of both compounds, alone and in combination. After 72 h of incubation, the mitochondrial activity showed that the combination of 5 nM Paclitaxel with 10 µM 6-Gingerol led to the same decrease in viability as the use of 20 nM Paclitaxel alone; 10 µM 6-Gingerol led to an enhancement of caspase 7 activity, with the highest activity observed after 24 h of incubation. A real-time PCR study showed that 6-Gingerol induces the simultaneous transcription of Bax with TP53 genes in large excess to BCL-2. In contrast, 5 nM Paclitaxel induces TP53 transcription in excess of BCL-2 and Bax. Our results suggest that 6-Gingerol may act as a cell death-inducing agent in cancer cells and, in combination with paclitaxel, and increase the effectiveness of conventional chemotherapy.

Micro- and Nanosecond Pulses Used in Doxorubicin Electrochemotherapy in Human Breast and Colon Cancer Cells with Drug Resistance.

(1) Background: Pulsed electric field (PEF) techniques are commonly used to support the delivery of various molecules. A PEF seems a promising method for low permeability drugs or when cells demonstrate therapy resistance and the cell membrane becomes an impermeable barrier. (2) Methods: In this study, we have used doxorubicin-resistant and sensitive models of human breast cancer (MCF-7/DX, MCF-7/WT) and colon cancer cells (LoVo, LoVoDX). The study aimed to investigate the susceptibility of the cells to doxorubicin (DOX) and electric fields in the 20-900 ns pulse duration range. The viability assay was utilized to evaluate the PEF protocols' efficacy. Cell confluency and reduced glutathione were measured after PEF protocols. (3) Results: The obtained results showed that PEFs significantly supported doxorubicin delivery and cytotoxicity after 48 and 72 h. The 60 kV/cm ultrashort pulses × 20 ns × 400 had the most significant cytotoxic anticancer effect. The increase in DOX concentration provokes a decrease in cell viability, affected cell confluency, and reduced GSSH when combined with the ESOPE (European Standard Operating Procedures of Electrochemotherapy) protocol. Additionally, reactive oxygen species after PEF and PEF-DOX were detected. (4) Conclusions: Ultrashort electric pulses with low DOX content or ESOPE with higher DOX content seem the most promising in colon and breast cancer treatment.

Modifications of Plasma Membrane Organization in Cancer Cells for Targeted Therapy.

Modifications of the composition or organization of the cancer cell membrane seem to be a promising targeted therapy. This approach can significantly enhance drug uptake or intensify the response of cancer cells to chemotherapeutics. There are several methods enabling lipid bilayer modifications, e.g., pharmacological, physical, and mechanical. It is crucial to keep in mind the significance of drug resistance phenomenon, ion channel and specific receptor impact, and lipid bilayer organization in planning the cell membrane-targeted treatment. In this review, strategies based on cell membrane modulation or reorganization are presented as an alternative tool for future therapeutic protocols.

Biological Response Induced in Primary Human Gingival Fibroblasts upon Exposure to Various Types of Injectable Astringent Retraction Agents.

Traditional chemo-mechanical retraction/displacement materials can impact the gingival margin tissues. This study was undertaken to analyze biological responses induced in human gingival fibroblasts (HGFs) upon application of injectable astringent-based agents used in the cordless retraction technique. HGFs were exposed to hemostatic agents (five gels, three pastes, and one foam) based on aluminium chloride, aluminium sulphate and ferric sulphate. Changes in cell viability and proliferation were evaluated using an MTT assay and a BrdU assay. The cytoskeleton structure organization (zyxin and F-actin) was visualized by confocal laser scanning microscopy. Oxidative stress was determined using the Griess Reagent System. The RNA expression levels of antioxidant enzymes were quantified by real-time RT-PCR. The statistical significance was evaluated using Student's t-test and one-way ANOVA with post-hoc Tukey HSD test. The evaluated agents did not downregulate fibroblast viability or proliferation. No significant cytoskeleton reorganization was observed. Only one agent (Expasyl) induced oxidative stress, demonstrated by the increased level of nitrites. Incubation with the studied agents significantly increased the RNA expression of some antioxidant enzymes (SOD1, SOD3, GPX1). However, no significant influence on the expression of SOD2 and HMOX1 was detected. The injectable forms of chemical retraction agents revealed biocompatibility with HGFs, suggesting their potential clinical usefulness in gingival margin retraction.