Search

There are numerous reports suggesting that fibroblast activating protein-α (FAP-α) plays an important role in invasion of various tumor types. We studied the expression pattern of FAP-α in esophageal squamous cell carcinoma (ESCC) patients who had not been treated primarily and those who had received neoadjuvant radiochemotherapy. Our goal was to establish whether readily available tissue specimens fixed in formalin and stored in paraffin blocks for years might still be a source of FAP-α RNA for PCR analysis. The study included 20 patients divided into two groups, 10 patients in each group. We evaluated the expression of FAP-α by PCR techniques in fresh frozen and in paraffin-embedded tissues, and compared it to the expression in non-cancer tissues. To detect the protein expression level of FAP-α in paraffin-embedded tissues we used chromogenic immunohistochemical (IHC) staining. Data were analyzed by t-test or the nonparametric Wilcoxon matched pair test using Statistica 12.5 software. We observed an increased level of the FAP-alpha gene and protein expression in cancer tissues when compared with their corresponding normal tissues. However, statistically significant differences were found only in the group of patients untreated before surgery. RNA extracted from paraffin-embedded tissue sections had very low quality, especially in the context of degradation. FAP-α remains a highly altered participant of a complex microenvironment in esophageal squamous cell carcinoma, and its role in cell signaling requires further study. In this paper, we conclude that the use of a regular RT-PCR method for diagnostic purposes, which we have presented in an earlier paper, can be as good as qRT-PCR. Also, immunohistochemistry proved to be very useful and the only reliable method that can be used on formalin-fixed, paraffin-embedded tissues stored long term.

Abstract Resistance of cancer cells to anticancer drugs remains a major challenge in modern medicine. Understanding the mechanisms behind the development of chemoresistance is key to developing appropriate therapies to counteract it. Nowadays, with advances in technology, we are paying more and more attention to the role of the tumor microenvironment (TME) and intercellular interactions in this process. We also know that important elements of the TME are not only the tumor cells themselves but also other cell types, such as mesenchymal stem cells, cancer-associated fibroblasts, stromal cells, and macrophages. TME elements can communicate with each other indirectly (via cytokines, chemokines, growth factors, and extracellular vesicles [EVs]) and directly (via gap junctions, ligand–receptor pairs, cell adhesion, and tunnel nanotubes). This communication appears to be critical for the development of chemoresistance. EVs seem to be particularly interesting structures in this regard. Within these structures, lipids, proteins, and nucleic acids can be transported, acting as signaling molecules that interact with numerous biochemical pathways, thereby contributing to chemoresistance. Moreover, drug efflux pumps, which are responsible for removing drugs from cancer cells, can also be transported via EVs.

Abstract 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.

Background: We performed gene therapy for critical limb ischemia in thromboangiitis obliterans (TAO) by the intramuscular administration of plasmids of the vascular endothelial growth factor gene (VEGF 165) with or without bone marrow-derived stem cells. Methods: The 21 patients were randomly assigned to three groups: A—with dual therapy, cells and plasmid; B—plasmid only; and C—control group, where patients received intramuscular injections of saline. Serum VEGF levels, the ankle–brachial index (ABI), transcutaneous oxygen pressure (TcPO2), and the rest pain measured by the visual analog scale (VAS) were determined sequentially before treatment, and then 1 and 3 months after treatment. Results: In the treatment groups, serum VEGF levels increased by 4 weeks and returned to baseline values after 3 months. ABI after 12 weeks increased by an average of 0.18 in group A, and 0.09 in group B and group C. TcPO2 increased by an average of 17.3 mmHg in group A, 14.1 mmHg in group B, and 10.7 mmHg in group C. The largest pain decrease was observed in group A and averaged 5.43 less pain intensity. Conclusions: Gene therapy using the VEGF plasmid along with or without bone marrow-derived mononuclear cells administered intramuscularly into an ischemic limb in TAO is a safe and effective therapy.

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.

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.

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.

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.

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.

(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.

(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 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.

A series of 18 aminochalcone derivatives were obtained in yields of 21.5−88.6% by applying the classical Claisen-Schmidt reaction. Compounds 4−9, 14 and 16−18 with 4-ethyl, 4-carboxy-, 4-benzyloxy- and 4-benzyloxy-3-methoxy groups were novel, not previously described in the scientific literature. To determine the biological properties of the synthesized compounds, anticancer and antimicrobial activity assays were performed. Antiproliferative potential was evaluated on four different human colon cancer cell lines—HT-29, LS180, LoVo and LoVo/DX —using the SRB assay and compared with green monkey kidney fibroblasts COS7. Anticancer activity was described as the IC50 value. The best results were observed for 2′-aminochalcone (1), 3′-aminochalcone (2) and 4′-aminochalcone (3) (IC50 = 1.43−1.98 µg·mL−1) against the HT-29 cell line and for amino-nitrochalcones 10−12 (IC50 = 2.77−3.42 µg·mL−1) against the LoVo and LoVo/DX cell lines. Moreover, the antimicrobial activity of all derivatives was evaluated on two strains of bacteria: Escherichia coli ATCC10536 and Staphylococcus aureus DSM799, the yeast strain Candida albicans DSM1386 and three strains of fungi: Alternaria alternata CBS1526, Fusarium linii KB-F1 and Aspergillus niger DSM1957. In the case of E. coli ATCC10536 almost all derivatives hindered the bacterial growth (∆OD = 0). Furthermore, the best results were observed in the presence of 4′-aminochalcone (3), that completely limited the growth of all tested strains at the concentration range of 0.25−0.5 mg·mL−1. The strongest bacteriostatic activity was exhibited by novel 3′-amino-4-benzyloxychalcone (14), that prevented the growth of E. coli ATCC10536 with MIC = 0.0625 mg·mL−1.

In our investigation, we concentrated on naringenin (NG)—a widely studied flavanone that occurs in citrus fruits. As a result of a reaction with a range of alkyl iodides, 7 novel O-alkyl derivatives of naringenin (7a⁻11a, 13a, 17a) were obtained. Another chemical modification led to 9 oximes of O-alkyl naringenin derivatives (7b⁻13b, 16b⁻17b) that were never described before. The obtained compounds were evaluated for their potential antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The results were reported as the standard minimal inhibitory concentration (MIC) values and compared with naringenin and its known O-alkyl derivatives. Compounds 4a, 10a, 12a, 14a, 4b, 10b, 11b, and 14b were described with MIC of 25 µg/mL or lower. The strongest bacteriostatic activity was observed for 7-O-butylnaringenin (12a) against S. aureus (MIC = 6.25 µg/mL). Moreover, the antitumor effect of flavonoids was examined on human colon cancer cell line HT-29. Twenty-six compounds were characterized as possessing an antiproliferative activity stronger than that of naringenin. The replacement of the carbonyl group with an oxime moiety significantly increased the anticancer properties. The IC50 values below 5 µg/mL were demonstrated for four oxime derivatives (8b, 11b, 13b and 16b).