Cytotechnology最新文献

S-Sulfocysteine (SSC) is a metabolite derived from the metabolism of sulfur-containing amino acids. It has been implicated in neurotoxicity observed in children with sulfite oxidase deficiency. The aim of our study was to confirm the neurotoxic effects of SSC using a mouse hippocampal cell line (HT-22) and to investigate the role of apoptosis in these effects, especially in terms of caspase-3 activation and genotoxicity. Based on the viability graph obtained following increasing concentrations of SSC, we determined the LC50 dose of SSC to be 125 µM by probit analysis. The cytotoxic effects of SSC were not reversed by glutamate receptor blocker administration. However, SSC treatment did not induce caspase-3 activation or induce DNA damage. Our results showed that SSC has a cytotoxic effect on neurons like glutamate, but glutamate receptor blockers reversed glutamate-induced toxicity, while these blockers did not protect neurons from SSC toxicity. The absence of caspase-3 activation and DNA fragmentation, which are indicative of apoptosis, in SSC-induced cell death suggests that alternative cell death pathways, such as necrosis and oxytosis may be implicated. Further research is necessary to fully elucidate SSC-induced cell death. The aim of our study was to confirm the neurotoxic effects of SSC using a mouse hippocampal cell line (HT-22) and to investigate the role of apoptosis in these effects, especially in terms of caspase-3 activation and genotoxicity.

The chemical substance bisphenol A (BPA) is widely used in household products, and its effect on human health has frequently been the focus of research. The aim of this study was to explore the potential molecular regulatory mechanism of BPA on the proliferation and migration of lung cancer cells. In this study, the H1299 and A549 lung cancer cell lines were selected as the study objects. The cells were treated with different concentrations of BPA (0, 0.1, 1, or 10 μM), and cell viability, proliferation, and migration were evaluated by CCK-8, EdU, clonogenic, and scratch test assays. Western blotting and RT‒qPCR were used to detect the expression of related proteins and genes. Our findings indicated that BPA markedly enhanced both the proliferation and migration capacities of lung cancer cells. In BPA-treated lung cancer cells, the level of miR-141-3p was decreased, PTGER4 expression was significantly increased, and PTGER4 knockdown reduced BPA-induced lung cancer cell proliferation and migration. In addition, miR-141-3p can target and negatively regulate the expression of PTGER4 and further inhibit PI3K/AKT signaling pathway activation and MMPs expression. Moreover, PTGER4 overexpression weakened the inhibitory effect of the miR-141-3p mimic on the proliferation and migration of lung cancer cells. In conclusion, miR-141-3p can inhibit the proliferation and migration of BPA-induced lung cancer cells by downregulating PTGER4, providing a new potential target for the treatment and prevention of lung cancer.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00692-5.

Mechanical and thermal cell damage can occur due to invasive procedures related to drilling, the insertion of dental implants, and periodontal treatments. Necrotic cells release the content of their cytoplasm and membrane fragments, thereby signaling the need for repair, which includes bone resorption by osteoclasts and inflammation. Here we screened lysates from human gingival fibroblasts, HSC2 and TR146 oral squamous carcinoma cell lines, as well as murine IDG-SW3 osteocytic and RAW264.7 macrophage cell lines for their potential to modulate in vitro osteoclastogenesis in murine bone marrow cultures. We also tested the impact of necrotic lysates on modulating the expression of inflammatory cues in murine ST2 bone marrow stromal cells. We report here that independent of human or murine origin, all cell lysates significantly reduced in vitro osteoclastogenesis in bone marrow cultures, as indicated by the expression of the osteoclast marker genes cathepsin K and tartrate-resistant acid phosphatase and the respective histochemical staining in multinucleated cells. We also found that lysates from HSC2 and TR146 cells significantly pushed the expression of CCL2, CCL5, CXCL1, IL1, and IL6 in ST2 cells. These findings suggest that oral cell lysates reduce in vitro osteoclastogenesis, but only damaged oral squamous carcinoma cells can force murine stromal cells to produce an inflammatory environment.

The tumor microenvironment (TME) is important in the recurrence and metastasis of colorectal cancer (CRC). Phillygenin is an effective component of Forsythiae fructus that has long been used in cancer therapy. The mechanism by which phillygenin regulates the TME remains unknown. Methods and Results: A co-culture system of CRC cells and Jurkat T cells was used to simulate the TME in vitro. Network pharmacology and Human XL cytokine arrays were used to preliminarily evaluate the role of phillygenin in the TME. The target of phillygenin was determined using transfection of plasmid-producing overexpression of hypoxia-inducible factor 1 alpha (HIF-1α) overexpression or abrogated HIF-1α expression via short hairpin RNA plasmid. The therapeutic effect of phillygenin in vivo was assessed in a subcutaneous tumor mouse model. In vitro, phillygenin enhanced the immune response of T cells and prevented the immune escape of cancer cells via the inhibition of HIF-1α. Phillygenin upregulated interleukin (IL)-2 and downregulates IL-10 and FOXP3 in Jurkat T cells co-cultured with CRC cells. Phillygenin inhibited expressions of HIF-1α, transforming growth factor-beta, vascular endothelial growth factor, and CD31 in CRC cells cultured alone or with Jurkat T cells. Phillygenin considerably suppressed tumor growth and improved the TME in vivo. Conclusions: Phillygenin can enhance the immune response and inhibit angiogenesis in the TME in CRC by inhibiting HIF-1α.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00679-2.

Esophageal cancer remains a formidable challenge in oncology, characterized by its poor prognosis and limited therapeutic options. Recent investigations have unveiled the potential of repurposing existing drugs for cancer treatment. Notably, etomidate, an anesthetic agent traditionally used for inducing general anesthesia, has emerged as a promising candidate demonstrating significant anticancer properties across various tumor types. The present study aims to investigate the effects of etomidate on esophageal carcinoma cells, with a specific focus on its ability to modulate the PI3K/AKT signaling pathway and inhibit tumor proliferation. This study employed both in vitro and in vivo methodologies to assess the effects of etomidate on esophageal cancer cells. In vitro experiments evaluated the effects of etomidate on cell proliferation, migration, invasion, and glycolytic processes. An in vivo xenograft mouse model was established to investigate the therapeutic potential of etomidate on tumor growth and assess its impact on the PI3K/AKT signaling pathway in a physiologically relevant context. Etomidate demonstrated a significant inhibitory effect on the proliferation, migration, invasion, and glycolytic capacity of esophageal cancer cells. This multifaceted suppression of tumorigenic properties was closely associated with the inhibition of the PI3K/AKT pathway, as evidenced by reduced phosphorylation levels of PI3K and AKT. In vivo studies using a murine model of esophageal cancer corroborated these findings. Etomidate administration resulted in a substantial reduction in tumor volume and mass, accompanied by increased apoptotic activity and the inhibition of the PI3K/AKT pathway within the tumor tissue. This study demonstrates etomidate's potent inhibition of esophageal cancer progression through suppression of the PI3K/AKT pathway. These promising results warrant further clinical investigation of etomidate as a potential therapeutic strategy for esophageal cancer.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00661-y.

Breast cancer is the most frequent cancer in women with a 20% mortality rate. The fate of patients suffering from breast cancer can be influenced by immune cells and tumor cells interaction in the tumor microenvironment (TME). Immune checkpoints such as Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) are regulators of the immune system and defend normal tissues from immune cell attacks but they can be expressed in breast cancer tissue and facilitate immune evasion of tumoral cells. Based on this, here we studied the role of CTLA-4 silencing by specific siRNA in MCF-7 breast cancer cell line together with Docetaxel treatment which is one of the robust chemotherapy agents to demonstrate the significance of combining chemotherapy with efficient targeted therapy in tumor regression. The MCF-7 breast cancer cell line was transfected with CTLA-4-siRNA through the electroporation method, then received an appropriate dose of Docetaxel determined by MTT assay. Flow cytometry was utilized to investigate the consequence of simultaneous CTLA-4 gene silencing and Docetaxel treatment on the apoptosis and cell cycle of MCF-7 cells. The expression levels of Bax and Bcl-2 were also investigated using quantitative real-time PCR. Compared to control groups, CTLA-4-suppressed and Docetaxel-treated cells became more susceptible to apoptosis and cell cycle arrest at the G2-M phase. The additive effect of CTLA-4 knockdown together with Docetaxel treatment significantly downregulated BCL-2 level and upregulated BAX expression. Our findings support the idea that combining chemotherapy such as Docetaxel with efficient targeted therapy against inhibitory immune checkpoints can be a promising strategy in cancer treatment.

Cytotoxins (CTXs), proteins found in cobra venom, selectively inhibit tumor cell proliferation. Herein, we selected CTX-XII because of its potent antitumor activity to investigate the effect of solution pH on its response. MTT assay results showed significantly higher inhibition rates for CTX-XII at pH 5.72 (75.79 ± 3.48%) than that at pH 7.32 (50.75 ± 3.8%). Flow cytometry demonstrated that apoptosis rates in B16F10 cells induced by CTX-XII were also higher at pH 5.72 (44.92 ± 7.94%) and 4.12 (42.87 ± 1.89%) than at pH 7.32 (23.5 ± 4.02%). Confocal laser scanning microscopy images showed that red fluorescence, representing CTX-XII concentration, was more intense around tumor cells at pH 5.72, with higher levels in the cytoplasm, than at pH 7.32. In the murine melanoma model, tumor weight in the pH 5.72 CTX-XII group (0.45 ± 0.19 g) was significantly lower than that in the pH 7.32 CTX-XII group (0.84 ± 0.42 g). These results indicate that pH has a strong influence on the antitumor activity of CTX-XII, likely due to pH-dependent ionization changes in CTX-XII that increase its affinity for and penetration into tumor cell membranes. This study provides new insights into the antitumor effects of CTXs and factors influencing their activity.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00681-8.

Primary hepatocytes (PHs) are indispensable for studying liver function, drug screening, and regenerative medicine. However, freshly isolated PHs only survive for a few hours in non-adherent suspension culture. This study proposes treatment with PEG-GRGDS, a polymer-peptide conjugate comprising polyethylene glycol (PEG) and the pentapeptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS), to sustain the viability of dispersed single PHs under non-adherent conditions. As a proof of concept, PHs treated with the PEG-GRGDS molecule were cultured in a microarray with single-cell-sized microwells. After 24 h of culture, enhanced cell survival was confirmed via esterase activity alongside activity for Cytochrome P450 1A1 (CYP1A1). Some liver-specific functionalities, including albumin secretion, were observed in the treated PHs. Additionally, it was observed that the length of the PEG-chain in the conjugates influenced the maintenance of single-cell dispersion and the levels of polymerized actin in the cells. These findings suggest that treatment with a polymer-peptide like PEG-GRGDS might provide a promising platform for the short-term culture of non-adherent single PHs.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00696-1.

High-intensity exercise can cause excessive generation of ROS and induce oxidative stress injury in the body, which is a major reason accounting for muscle damage following exercise. The previous study demonstrated that IBS008738, the activator of TZA, was able to enhance myogenesis in mouse myogenic C2C12 cells, prevent dexamethasone-induced muscle atrophy, and facilitate muscle repair in cardiotoxin-induced muscle injury. Accordingly, our study was designed to probe into the potential role of IBS008738 in muscle damage in mouse models induced by high-intensity exercise. Mice were first administrated with IBS008738, and then subjected to high-intensity eccentric exercise to induce muscle damage after 24 h. During the experiment, mouse weight change and food take were recorded. At the end of the experiment, blood samples were collected through cardiac puncture and centrifugated. Serum levels of blood urea nitrogen (BUN), creatinine, glucose, lactate dehydrogenase (LDH), creatinine kinase (CK), and C-related protein were evaluated using an autoanalyzer. After mice were sacrificed, the gastrocnemius muscles were dissected for DCFH-DA assay of ROS generation, thiobarbituric acid-reactive substances (TBARS) assay of MDA content, hematoxylin-eosin (H&E) staining of histological examination, and western blotting analysis of Akt/mTOR/S6K1 signaling expression. IBS008738 and/or exercise exert significant effects on mouse weight and food take. High-intensity exercise markedly increased ROS generation and lipid peroxidation, upregulated serum levels of CK, LDH, and C-related protein, ameliorated muscle histological damage, and reduced TAZ, phosphorylated (p)-Akt, p-mTOR, and p-S6K1 protein levels in mice. However, IBS008738 administration reversed the above changes induced by high-intensity exercise in mice. IBS008738 alleviates oxidative stress and muscle damage in mice after high-intensity exercise by activating TAZ and the Akt/mTOR/S6K1 signaling pathway.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00667-6.

Shake tubes (ST) are widely employed to assist the development of the stirred tank reactor (STR) perfusion cell culture. However, cell lysis may be frequently underrestimated and lead to culture performance discrepency between these systems, rendering the ST model ineffective in designing the STR perfusion cultures. In this study, perfusion culture performance bewteen the STR and ST was investigated under various conditions with the analysis of cell lysis. Comparable performance was observed bewteen the two systems at low perfusion rates ( $D$ ≤1.0 VVD), except that the specific productivity ( $q_P$ ) of the STR was decreased at $D$ =0.5 VVD, which was related to product degradation by cell lysis. In contrast, significant differences in cell maintenance, metabolism, and $q_P$ were found at $D$ =2.0 VVD. By the analysis of the authentic cell growth and death kinetics, it was found that cell growth arrest, potentially due to the limited availability of oxygen, led to the stable cell maintenance at VCD≈90 × 10⁶ cells/ml and altered cellular metabolism for the ST, while the continuous decline of VCD and $q_P$ in the STR were related to excessive cell death, subsequently ascribed to the harmful hydrodynamic stress conditions. We further demonstrated that cell lysis accounted for 57.62-76.29% of the total generated biomass in both the reactors and significantly impacted the estimation of process descriptors crucial for understanding the true cellular states. With cell lysis in sight, cell performance can therefore be accurately described and this knowledge can be further leveraged to expedite process development for the perfusion cell culture processes.