Cytotechnology最新文献

The expression spectra of connexin (Cx) isoforms were investigated in three mouse melanoma cell lines: B16-F1 (F1), B16-F10 (F10), and B16-BL6 (BL6). Metastatic potential intensity was higher in the order of F1, F10, and BL6. A remarkable behavior of Cx45 was found among 20 isoforms. The expression level of Cx45 was highest in F1 and lowest in BL6. It was inductively predicted that Cx45 might be a novel suppressor of metastasis. A Cx45-overexpressing BL6 cell line (Cx45 ⁺BL6) was developed and its properties were compared with those of a wild-type cell line of BL6 (W-BL6). Compared to W-BL6, Cx45 ⁺BL6 showed reduced wound healing, Transwell® permeability, and matrix metalloproteinase 9 expression, suggesting the suppression of cellular migration and invasion. The expression of E-cadherin and integrin β1 in Cx45 ⁺BL6 was also lower than in W-BL6, suggesting reduced cell adhesion. The decrease in cell adhesion was supported by the cell washing-out assay. In contrast, no difference between W-BL6 and Cx45 ⁺BL6 was observed in cell proliferation, suggesting no effect on cell-cycle regulating factors. Finally, an in vivo assay revealed a significant decrease in the number of metastatic colonies of Cx45 ⁺BL6 (176 ± 25/lung) in comparison with those of W-BL6 (252 ± 23/lung) in a mouse model. In conclusion, Cx45 is a novel suppressor of melanoma metastasis.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-022-00563-x.

Growing consumer interest in skin whitening has led to intensive investigations of whitening methods. In this study, we evaluated the effect of sphingomyelin, a component of cell membranes, on melanin production. B16 mouse melanoma cells were treated with lauroyl-sphingomyelin (SM) or its metabolite lauroyl-ceramide (CER) and measured for cell viability, melanin content, and direct and indirect tyrosinase activity. Expression of melanin synthesis-related genes encoding tyrosinase (Tyr), tyrosinase-related proteins (Trp1 and Trp2), and microphthalmia-associated transcription factor (Mitf) were quantified by real-time PCR, and SM content in cells was measured by fluorescence high-performance liquid chromatography. SM treatment decreased melanin content in a concentration-dependent manner, without significantly altering the number of viable cells. By contrast, treatment with CER at the same concentrations did not decrease melanin content. SM inhibited the activity of intracellular tyrosinase, but not mushroom-derived tyrosinase. Gene expression levels of Tyr and Mitf were significantly reduced by treatment with SM, while those of Trp2 and Mitf were significantly reduced by CER. Fluorescence-labeled SM was converted to fluorescence-labeled CER in cells over time. In conclusion, CER was found to inhibit melanogenesis without inhibiting tyrosinase activity, suggesting that SM is more water soluble than CER, and is more effectively taken up into cells.

β-Conglycinin was conjugated with ε-polylysine (PL) by means of microbial transglutaminase (MTGase) to improve its function. The β-conglycinin-PL conjugate was purified by dialysis. Composition of the β-conglycinin-PL was β-conglycinin:PL = 1:18 (molar ratio) which was confirmed by amino acid analysis. The β-conglycinin-PL was further conjugated with dextran (Dex) by the Maillard reaction. The β-conglycinin-PL-Dex conjugate was purified by dialysis. Conjugation was confirmed by SDS-PAGE and PAS staining. Composition of the β-conglycinin-PL-Dex was β-conglycinin-PL:Dex = 1:41 (molar ratio) which was confirmed by UV spectra measurement and phenol sulfuric acid method. Solubility of β-conglycinin in the acidic range was much improved by conjugation with PL and further improved by further conjugation with Dex. Emulsifying property of β-conglycinin in acidic pH range was much improved by conjugation with PL and Dex. Immunogenicity of β-conglycinin was decreased by conjugation with PL and Dex.

Mycoplasma contamination is a significant problem in cell culture replication and maintenance. From more than 200 known species, a limited number of Mycoplasma species have been detected in cell cultures, representing new species or variants that can escape detection systems. A qPCR commercial kit was used for Mycoplasma detection in cell cultures. Furthermore, an amplified Mycoplasma species was sequenced and summited for sequence assembly, clustering, and evolutionary analysis study. Our work has identified a new and unusual variant or species of Mycoplasma that possesses a high degree of homology with species related with M. mycoides cluster. This variant is usually associated with cattle but has been detected contaminating a cell culture. Mycoplasma testing (even for unusual species) in cell cultures is essential to ensure the validity and reproducibility of research that uses cell cultures and to ensure the quality of cell line deposits in biobanks. For this reason, it is necessary to perform continuous checks for the absence of Mycoplasma in cell cultures and engage in the continuous adaptation of relevant detection systems.

β-Conglycinin was conjugated with carboxymethyl dextran (CMD) by the Maillard reaction to improve its function. The β-conglycinin-CMD conjugate was purified by dialysis. Conjugation was confirmed by SDS-PAGE with CBB and PAS staining. Composition of the β-conglycinin-CMD was β-conglycinin:CMD = 1:2.7 (molar ratio) which was confirmed by BCA method and phenol sulfuric acid method. Solubility of β-conglycinin in the range of pH 2.0-7.0 was much improved by conjugation with CMD. Emulsifying property of β-conglycinin at pH 7 and in presence of salt was improved by conjugation with CMD. Immunogenicity of β-conglycinin was reduced by conjugation with CMD. Conjugation method performed in this study was considered to be valuable in that it can be used in food processing.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cancer with a low overall survival rate, necessitating effective treatments. This study reports the anti-OTSCC effect of vorinostat and selinexor. OTSCC cell lines SCC-4 and SCC-25 were cultured to determine the effects of vorinostat and/or selinexor on cell survival, invasion, migration, and apoptosis. The transplanted tumor model of SCC-25 in nude mice was established to observe the therapeutic effects of vorinostat and/or selinexor. Western blotting was used to determine protein expressions in tumor cells. The results showed that histone deacetylase 1 (HDAC1) and exportin 1 (XPO1) were highly expressed, while nuclear maspin was expressed at a low rate in SCC-4 and SCC-25 compared to the normal tongue tissue. In vitro, both vorinostat and selinexor effectively inhibited cell viability, invasion, and migration, promoted cell apoptosis, down-regulated HDAC1, Matrix Metalloproteinase 2 (MMP2), and B cell leukemia/lymphoma 2 (Bcl-2), and up-regulated nuclear maspin and cleaved caspase 3. In vivo, both vorinostat and selinexor inhibited the growth of SCC-25-bearing tumors, down-regulated the expression of Ki67, HDAC1, MMP2, and Bcl-2, and promoted the expression of nuclear maspin and cleaved caspase 3. The combination of these two drugs exhibited synergistic effects both in vivo and in vitro. Our evidence shows that vorinostat combined with selinexor is an effective treatment for OTSCC. The mechanism may be that selinexor promotes the accumulation of maspin in the nucleus, an endogenous HDAC1 inhibitory protein to inhibit the HDAC1 activity of vorinostat and exert a synergistic anti-OTSCC effect.

In order to ensure the proper use and interpretation of results from laboratory test systems, it is important to know the characteristics of your test system. Here we compare mitochondria and the handling of reactive oxygen species (ROS) in two gill epithelial cell lines, the well-known RTgill-W1 cell line from Rainbow trout and the newly established ASG-10 cell line from Atlantic salmon. Rotenone was used to trigger ROS production. Rotenone reduced metabolic activity and induced cell death in both cell lines, with RTgill-W1 far more sensitive than ASG-10. In untreated cells, the mitochondria appear to be more fragmented in RTgill-W1 cells compared to ASG-10 cells. Furthermore, rotenone induced mitochondrial fragmentation, reduced mitochondria membrane potential (Δψm) and increased ROS generation in both cell lines. Glutathione (GSH) and catalase is important to maintain the cellular oxidative balance by eliminating hydrogen peroxide (H₂O₂). In response to rotenone, both GSH and catalase depletion were observed in the RTgill-W1 cells. In contrast, no changes were found in the GSH levels in ASG-10, while the catalase activity was increased. In summary, the two salmonid gill cell lines have different tolerance towards ROS, probably caused by differences in mitochondrial status as well as in GSH and catalase activities. This should be taken into consideration with the selection of experimental model and interpretation of results.

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Supplementary information: The online version contains supplementary material available at 10.1007/s10616-022-00560-0.

The standard treatment for non-muscle-invasive bladder cancer is intravesical Bacillus Calmette-Guérin (BCG) therapy, which is considered the only intravesical therapy that reduces the risk of progression to muscle-invasive cancer. BCG unresponsiveness, in which intravesical BCG therapy is ineffective, has become a problem. It is thus important to evaluate the effectiveness of BCG treatment for patients as soon as possible in order to identify the optimal therapy. Urine cytology is a noninvasive, easy, and cost-effective method that has been used during BCG treatment, but primarily only to determine benign or malignant status; findings concerning the efficacy of BCG treatment based on urine cytology have not been reported. We investigated the relationship between BCG exposure and nuclear an important criterion in urine cytology, i.e., nuclear chromatin patterns. We used three types of cultured cells to evaluate nuclear chromatin patterns and the cell cycle, and we used T24 cells to evaluate the phosphorylation of retinoblastoma protein (pRb) in six-times of BCG exposures. The results revealed that after the second BCG exposure, (i) nuclear chromatin is distributed predominantly at the nuclear periphery and (ii) the dephosphorylation of threonine-821/826 in pRb occurs. This is the first report of a dynamic change in the nuclear chromatin pattern induced by exposure to BCG. Molecular findings also suggested a relationship between this phenomenon and cell-cycle proteins. Although these results are preliminary, they contribute to our understanding of the cytomorphological changes that occur with BCG exposure.

Oral squamous cell carcinoma (OSCC) is an epithelial malignant tumor with great challenges of tumor metastasis and drug resistance. Nutlin-3 is a MDM2 inhibitor that can potently activate tumor suppressor gene p53. However, the exact role of Nutlin-3 in OSCC has not been identified yet. SCC-9 cells were treated with 0, 2.5, 5, 10, 20 μM Nutlin3. MDM2 and p53 protein levels were assessed using western blot analysis. Then, CCK8 assay, clone formation assay, TUNEL staining, wound healing and transwell assays were conducted to analyze the influences of Nutlin3 on the proliferation, apoptosis, migration, and invasion in SCC-9 cells. Moreover, SCC-9 cells were co-treated with 0, 0.5, 1, 2.5, 5 μM cisplatin and Nutlin3 to determine the effect of Nutlin3 on cisplatin chemosensitivity in OSCC. As expected, Nutlin-3 inhibited MDM2 but restored p53 in OSCC in a concentration-dependent manner. Meanwhile, Nutlin-3 suppressed the proliferation, clone formation, migration, invasion and epithelial-mesenchymal transition of SCC-9 cells and both boosted the apoptosis. In addition, Nutlin-3 caused a reduced cell viability and an elevated cell apoptosis rate in cisplatin-treated SCC-9 cells, indicating that Nutlin-3 enhanced cisplatin chemosensitivity in OSCC cells. Taken together, Nutlin-3 may suppress tumorigenesis and progression of OSCC and enhance chemosensitivity to cisplatin in OSCC.

The unlimited proliferation capacity of embryonic stem cells (ESCs) coupled with their capability to differentiate into several cell types makes them an attractive candidate for studying the molecular mechanisms regulating self-renewal and transition from pluripotent state. Although the roles of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family (PFKFB1-4) in cell survival, proliferation, and differentiation in tumor cells have been studied, their role in mouse ESC (mESC) biology is currently unkown. In the current study, Pfkfb isoenzyme expressions were analyzed in R1 and J1 mESCs that were cultured in the presence and absence of leukemia inhibitory factor (LIF). We report that expression of the Pfkfb3 isoenzyme was markedly increased when mESCs were promoted to differentiate upon LIF removal. We then demonstrated that Pfkfb3 silencing induced the differentiation marker Brachyury suggesting that Pfkfb3 may be required for the regulation of mesodermal differentiation of mESCs. Furthermore, we show that the increase in Pfkfb3 expression is required for the growth of early differentiated mESCs. Although these results provide important insights into the early differentiation of mESCs with regard to Pfkfb expressions, further mechanistic studies will be needed for understanding the pathways and mechanisms involved in regulation of proliferation and early differentiation of mESCs through Pfkfb3.