Cytotechnology最新文献

Chinese hamster ovary (CHO) cell lines, derived as subclones from the original CHO cell line, are widely used hosts for current biopharmaceutical productions. Recently, a highly proliferative host cell line, CHO-MK, was established from the Chinese hamster ovary tissue. In this study, we assessed the fundamental culture characteristics and capabilities of CHO-MK cells for monoclonal antibody (mAb) production using specified chemically defined media. To achieve this, we established fed-batch cultures of model CHO-MK cells in shake flasks and ambr15 and 2 L bioreactors under various conditions. The mAb-producing CHO-MK cell line A produced 12.6 g/L of antibody within 7 days in the fed-batch culture using a 2 L bioreactor, with a seeding density of 1 × 10⁶ cells/mL. This performance corresponded to a space-time yield of 1.80 g/L/day, representing a productivity level that could be challengingly attained in fed-batch cultures using conventional CHO cells. In addition, when we subjected six different mAb-producing CHO-MK cell lines to fed-batch culture in the ambr15 bioreactor for 7 days, the antibody production ranged between 5.1 and 10.8 g/L, confirming that combining CHO-MK cells and specified media leads to enhanced versatility. These discoveries underscore that CHO-MK cells combined with specified media might represent a next-generation production platform, which could potentially respond to an increasing demand for antibody drugs, reducing production costs, and shortening antibody drug development times. This study is expected to serve as a benchmark for future production process development using CHO-MK cells.

Lysine-specific histone demethylase 1A (KDM1A) has emerged as an attractive therapeutic target for treating various cancers, owing to its observed overexpression. However, its function in epithelial ovarian cancer (EOC) remains uncertain. The current study sought to investigate the function of KDM1A on malignant phenotypes of EOC cells as well as the underlying mechanism. Colony formation assay, cell counting kit-8, wound healing, Transwell assays, and TUNEL assays were performed to investigate the effects of KDM1A, Zinc finger protein 64 (ZFP64), and centromere protein L (CENPL) in vitro, while subcutaneous tumor formation models were established in nude mice to evaluate their roles in vivo. KDM1A, ZFP64, and CENPL were overexpressed in EOC tissues and cells. Knockdown of KDM1A, ZFP64, or CENPL inhibited the biological behavior of EOC cells. In addition, chromatin immunoprecipitation showed that KDM1A stimulated ZFP64 expression by removing the H3K9me2 mark from its promoter. Restoration of ZFP64 promoted EOC cell malignant phenotype in the presence of KDM1A knockdown. ZFP64 activated CENPL transcription. Reactivation of CENPL promoted the growth of EOC cells in vivo inhibited by knockdown of ZFP64. Collectively, KDM1A promoted EOC cell proliferation, migration, and invasion, and reduced apoptosis by activating the ZFP64/CENPL axis, which triggered EOC progression.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00671-w.

Pancreatic adenocarcinoma (PAAD) is one of the malignant tumors with poor prognosis. This study aims to inquiry the effects of Chromobox homologue 3 (CBX3) on PAAD progression. Pan-cancer analysis of CBX3 and its correlation with PAAD progression were investigated by informatics analysis. The role of CBX3 in PAAD was explored in vitro and in vivo. Cell viability, proliferation, migration and invasion were inspected by CCK-8 assay, EdU staining, scratch test and transwell assay, respectively. The morphology of tumors was observed by hematoxylin-eosin staining. Immunohistochemistry (Ki67) was performed to inspect the proliferation of tumor tissue. The protein levels were measured by western blot. Moreover, the downstream genes of CBX3 were screened, and the effects of target gene on PAAD was investigated in vitro. CBX3 was overexpressed in multi cancers, and high CBX3 expression indicated poor prognosis in PAAD. Through the in vitro assays, knockdown of CBX3 suppressed the viability, migration and invasion of PAAD cells, and restrained tumor growth in vivo. Subsequently, kinesin family member 20A (KIF20A) was screened as the downstream gene of CBX3, which was up-regulated in PAAD and related to low overall survival. Mechanistically, we discovered that CBX3 could regulate KIF20A expression. Knockdown of CBX3 promoted the oncogenic effects of KIF20A silencing on PAAD cells, and attenuated the pro-oncogenic effects of KIF20A overexpression on PPAD. Collectively, silencing CBX3 suppressed PAAD progression through regulating KIF20A expression, providing an underlying target for PAAD treatment.

Autophagy is a conservative process of self degradation, in which abnormal organelles, proteins and other macromolecules are encapsulated and transferred to lysosomes for subsequent degradation. It maintains the intracellular balance, and responds to cellular conditions such as hunger or stress. To date, there are mainly three types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy. Autophagy plays a key role in regulating multiple physiological and pathological processes, such as cell metabolism, development, energy homeostasis, cell death and hunger adaptation, and so on. Increasing evidence indicates that autophagy dysfunction participates in many kinds of cancers, such as liver cancer, pancreatic cancer, prostate cancer, and so on. However, the relevant mechanisms are not yet fully understood. Baicalin is a natural flavonoid compound extracted from the traditional Chinese medicine Scutellaria baicalensis. The research has shown that after oral or intravenous administration of baicalin, it is delivered to various organs through the systemic circulation, with the highest volume in the kidneys and lungs. More and more evidence suggests that baicalin has antioxidant, anticancer, anti-inflammatory, anti-apoptotic, immunomodulatory and antiviral effects. Therefore, baicalin plays an important role in various diseases, such as cancers, lung diseases, liver diseases, cardiovascular diseases, ans so on. However, the relevant mechanisms have not yet been fully clear. Recently, increasing evidence indicates that baicalin participates in different cancer by regulating autophagy. Herein, we reviewed the current knowledge about the role and mechanism of baicalin regulation of autophagy in multiple types of cancers to lay the theoretical foundation for future related researches.

Alzheimer's disease (AD) is a progressive neurological condition that causes brain shrinkage and cell death. This study aimed to identify the role of the NORAD/miR-26b-5p axis in AD. StarBase was used to examine the binding sequences of miR-26b-5p to LncRNA NORAD or its target genes, which were verified by a double luciferase reporter assay. PC12 cells were processed with Aβ_1-42 to construct an AD model in vitro, and LncRNA NORAD and miR-26b-5p levels in PC12 cells were identified by RT-qPCR. Cell viability and apoptosis were measured using the MTT assay and flow cytometry, respectively. LDH release and oxidative stress-related indicators (MDA, SOD, and CAT) were detected using the corresponding kits, and the levels of Bcl-2 and Bax were assessed by western blotting and RT-qPCR. Aβ_1-42 distinctly decreased LncRNA NORAD and membrane metalloendopeptidase (MME) levels in PC12 cells, while miR-26b-5p was generally increased. The LncRNA NORAD can adsorb miR-26b-5p, and the target gene of miR-26b-5p is neprilysin (MME). In the Aβ_1-42 induced AD model, PC12 cell activity decreased, LDH release and apoptosis increased, oxidative stress level increased, Bax expression increased, and Bcl-2 expression decreased. LncRNA NORAD plays a protective role in AD cell models by abrogating miR-26b-5p levels. Inhibition of MME expression eliminated the protective effects of the miR-26b-5p inhibitor in AD cell models. LncRNA NORAD inhibits AD progression in vitro by modulating the miR-26b-5p-MME signaling axis. The LncRNA NORAD/miR-26b-5p is expected to be a prospective therapeutic candidate for AD.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00691-6.

The clinical evidence, complications and the pathogenesis of COVID-19 are not clearly understood. In COVID-19 patients, cellular immune response biomarkers and oxidative stress parameters have been used as gravity markers. Indeed, oxidative stress has been proposed to play an essential role in the genesis of COVID-19. In the present research, we investigated lipid peroxidation, protein oxidation, superoxide dismutase activity and the production of auto-antibodies against superoxide dismutase, in the blood of Tunisian patients with corona virus. To evaluate lipid peroxidation, plasma malondialdehyde and conjugated dienes, have been determined in 69 corona virus patients and 30 controls. To determine protein oxidation the thiol level was measured. Plasma superoxide dismutase activity has been measured in 30 corona virus patients and 30 controls on one hand. Utilizing a standard enzyme-linked immunosorbent assay, the level of immunoglobulin G (IgG), and M (IgM) directed against superoxide dismutase was evaluated. To investigate the implication of auto-antibody production in COVID-19 patients in the generation of oxidative stress, a correlation study between auto-antibodies production and oxidative stress parameters was performed. High levels of both malondialdehyde and conjugated dienes were found in the plasma of patients (p < 0.001, respectively). Protein oxidation was confirmed by the high level of thiol (p < 0.001). Superoxide dismutase activity was not significantly lower in COVID-19 patients (p > 0.05). The level of immunoglobulin G (IgG), and M (IgM) directed against superoxide dismutase is significantly higher in COVID-19 patients than in control group (p < 0.001 respectively). Statistical analyses have demonstrated a positive correlation between superoxide dismutase activity and IgM and IgG isotypes antibodies level against superoxide dismutase (p < 0.001). A strong positive correlation was observed between IgG and malondialdehyde level in all cases (r = 0.368; p ≤ 0.01). In addition, a significant positive correlation was noted between IgM and malondialdehyde (r = 0.290; p = 0.024). Similarly, two significant positive relationship was found between IgG / conjugated dienes (r = 0.356; p = 0.005) and between IgM / conjugated dienes (r = 0.285; p = 0.027).

The choice of media and feeds significantly influences the performance of Chinese Hamster Ovary (CHO) mammalian cell cultures in producing desired biologics like monoclonal antibodies (mAb). Sub-optimal nutrient feed/media composition can severely impact cell proliferation and the quality of the final mAb product. For instance, proper protein glycosylation, crucial for mAb stability, safety, and efficacy, heavily relies on cell culture conditions. Currently, starter CHO culture media and daily supplemental feeds used in industrial manufacturing consist of proprietary composition of nutrients critical for mAb production. Standardized optimal media/feed combinations necessary for different cell lines are often lacking, necessitating individualized optimization for each cell line and mAb product. Here, we focused on a CHO-K1 cell line engineered to produce a Trastuzumab biosimilar and evaluated the effects of fourteen commercially relevant basal media and seven feeds on cell culture parameters such as viable cell density, viability, nutrient consumption, metabolite production, mAb titer, and mAb N-glycosylation. Our findings demonstrate clearly that the compositions of the basal medium and feed play a pivotal role in enhancing cell growth and mAb production. This work offers valuable insights into strategies for optimizing feed/media composition for glycosylated monoclonal antibody production using CHO cells.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00690-7.

Cervical cancer (CC) represents one of the important cancers affecting global female population worldwide. We sought to elucidate the roles and mechanisms of KIAA1429 in the malignant properties of CC cells and the epithelial-mesenchymal transition (EMT) process. KIAA1429 was predicted to be abnormally expressed in CC and correlate with shortened survival of CC patients by GEPIA2 and GSCA databases. High expression of KIAA1429 in human CC cell lines (SiHa, HT-3) was validated by RT-qPCR and Western blot assays. A series of small interfering (si)RNAs including si-KIAA1429-1, si-KIAA1429-2, si-YTHDF2, si-BTG2, and si-negative control (NC) were utilized to interfere the expression levels of KIAA1429, YTHDF2, and BTG2, respectively. Consequently, KIAA1429 silencing attenuated the proliferation, migratory, and invasive functions of CC cells and repressed EMT while promoting CC cell apoptosis. Mechanistically, KIAA1429 could affect N6-methyladenosine (m6A) modification to attenuate the stability of BTG2 mRNA and down-regulate its expression. Additionally, loss of BTG2 partly counteracted the effects of si-KIAA1429 on repressing the malignant activities of CC cells. The aforementioned results collectively demonstrated that KIAA1429-mediated m6A modification of BTG2 and contributed to malignant progression of CC in vitro.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-024-00694-3.

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.

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.