Investigation of the combined cytotoxicity induced by sodium butyrate and a flavonoid quercetin treatment on MCF-7 breast cancer cells.

Quercetin (QUE) belonging to the flavonoid class is a common phytochemical present in the daily diet of some individuals. Quercetin is an important source of free radical scavengers. This property makes this flavonoid a reliable antioxidant with the following properties: anti-inflammatory, anti-diabetic, antimicrobial and anti-carcinogenic. Sodium butyrate (NaBu) acts as a histone deacetylase inhibitor (HDACi) and is known to regulate apoptosis in cancer cells. Combining natural flavonoids such as QUE with different substances may synergistically enhance their anti-carcinogenic capacity. Thus, the aim of this study was to examine the combined treatment effects of QUE and NaBu in hormone-sensitive breast cancer cells in vitro. MCF-7 breast cancer cells were treated with QUE alone, NaBu alone, as well as QUE and NaBu combined to determine the following: cell proliferation, levels of protein annexin A5 (ANXA5) and reactive oxygen species (ROS), mRNA protein expression, as well as cell and nuclear morphology. Data demonstrated that either QUE or NaBu alone inhibited cell proliferation, and reduced levels protein ANXA5, ROS and mRNA protein expression, The combination of QUE and NaBu produced a significant synergistic inhibitory effect compared to treatment groups of QUE or NaBu alone. In conclusion, our findings showed that the combination treatment of QUE and NaBu may constitute a promising therapeutic approach to breast cancer treatment but this needs further molecular and in vivo investigations.

Long term culture promotes changes to growth, gene expression, and metabolism in CHO cells that are independent of production stability.

Phenotypic stability of Chinese hamster ovary (CHO) cells over long term culture (LTC) presents one of the most pressing challenges in the development of therapeutic protein manufacturing processess. However, our current understanding of the consequences of LTC on recombinant (r-) CHO cell lines is still limited, particularly as clonally-derived cell lines present distinct production stability phenotypes. This study evaluated changes of culture performance, global gene expression, and cell metabolism of two clonally-derived CHO cell lines with a stable or unstable phenotype during the LTC (early [EP] vs. late [LP] culture passages). Our findings indicated that LTC altered the behavior of CHO cells in culture, in terms of growth, overall gene expression, and cell metabolism. Regardless whether cells were categorized as stable or unstable in terms of r-protein production, CHO cells at LP presented an earlier decline in cell viability and loss of any observable stationary phase. These changes were parallelled by the upregulation of genes involved in cell proliferation and survival pathways (i.e., MAPK/ERK, PI3K-Akt). Stable and unstable CHO cell lines both showed increased consumption of glucose and amino acids at LP, with a parallel accumulation of greater amounts of lactate and TCA cycle intermediates. In terms of production stability, we found that decreased r-protein production in the unstable cell line directly correlated to the loss in r-gene copy number and r-mRNA expression. Our data revealed that LTC produced ubiquitious effects on CHO cell phenotypes, changes that were rooted in alterations in cell transcriptome and metabolome. Overall, we found that CHO cells adapted their cellular function to proliferation and survival during the LTC, some of these changes may well have limited effects on overall yield or specific productivity of the desired r-product, but they may be critical toward the capacity of cells to handle r-proteins with specific molecular features.

In vitro chemo-protective effect of Eisenia foetida coelomic fluid against histone deacetylase inhibitor-induced oxidative toxicity in breast cancer cells.

Developing new drugs from natural products is important for therapeutic effects to minimise tissue toxicity of drugs used in cancer treatment. Eisenia foetida is a worm with a double transport system consisting of coelomic fluid (ECF) that can be used as alternative medicine. It is important to eliminate or reduce the high cytotoxicity of sodium butyrate (NaBu), a chemotherapeutic agent used in breast cancer treatment, for both neoplastic and normal cells. We aimed to evaluate the effect of ECF on the oxidative toxicity induced by NaBu in MCF-7 cells, changes in ROS production levels and expression of cell death and ROS-related genes. It was determined that the expression levels of Bax and Bcl-2 genes remained unchanged, while the amount of ROS decreased significantly in MCF-7 cells exposed to NaBu with ECF. Thus, ECF may be a potential therapeutic molecule with fewer side effects in cancer treatment in the future.

Evaluation of the antioxidative and genotoxic effects of sodium butyrate on breast cancer cells.

Oncogenic stimulation shows a rise in reactive oxygen species (ROS), and ROS can eventually induce carcinogenesis by causing DNA damage. In this context, this study aims to evaluate some biochemical and genotoxic changes in the control of cell death caused by NaBu (Sodium butyrate). treatment in breast cancer cells. NaBu's impact on cell proliferation was determined via WST-1 assay. The lipid peroxidation (MDA), reduced glutathione (GSH), Nitric Oxide (NO), hydrogen peroxide (H2O2), and superoxide dismutase (SOD) enzyme levels were determined biochemically. NaBu-induced genotoxic damage was estimated via single-cell gel electrophoresis (SCGE). NaBu reduced cell viability and potentially induced GSH, but decreased SOD enzyme activity and the level of MDA and NO decreased also H2O2 decreased at different times and NaBu concentrations. Higher NaBu concentrations amplified DNA damage in MCF-7 cells compared to the control group. NaBu shows anticancer and genotoxic effects, especially through antioxidant enzymes, one of the oxidative stress parameters in breast cancer. However, the anticancer and genotoxic effects of NaBu is changed in the oxidative stress parameters with time and treatment concentration of NaBu in MCF-7 cells. Furthermore, his oxidative stress-dependent effect changes need to be clarified by further evaluation with molecular and more biochemical parameters.

Metabolic profiling of Chinese hamster ovary cell cultures at different working volumes and agitation speeds using spin tube reactors.

Culture systems based on spin tube reactors have been consolidated in the development of manufacturing processes based on Chinese hamster ovary (CHO) cells. Despite their widespread use, there is little information about the consequences of varying operational setting parameters on the culture performance of recombinant CHO cell lines. Here, we investigated the effect of varying working volumes and agitation speeds on cell growth, protein production, and cell metabolism of two clonally derived CHO cell lines (expressing an IgG1 and a "difficult-to-express" fusion protein). Interestingly, low culture volumes increased recombinant protein production and decreased cell growth, while high culture volumes had the opposite effect. Altering agitation speeds exacerbated or moderated the differences observed due to culture volume changes. Combining low agitation rates with high culture volumes suppressed growth and recombinant protein production in CHO cells. Meanwhile, high agitation rates narrowed the differences in culture performance between low and high working volumes. These differences were also reflected in cell metabolism, where low culture volumes enhanced oxidative metabolism (linked to a productive phenotype) and high culture volume generated a metabolic profile that was predominately glycolytic (linked to a proliferative phenotype). Our findings indicate that the culture volume influence on metabolism modulates the balance between cell growth and protein production, a key feature that may be useful to adjust CHO cells toward a more productive phenotype.

Improved CHO Cell Line Stability and Recombinant Protein Expression During Long-Term Culture.

Therapeutic proteins require proper folding and posttranslational modifications to be effective and biologically active. Chinese hamster ovary (CHO) cells are by far the most frequently used host for commercial production of therapeutic proteins. However, an unpredictable decrease in protein productivity during the time required for scale up impairs process yields, time, finance, and regulatory approval for the desired product. Therefore, it is important to assess cell lines at stages throughout the period of long-term culture in terms of productivity and various molecular parameters including plasmid and mRNA copy numbers and location of the plasmid on the host cell chromosome. Here, we describe methods, which are frequently used to analyze stability of the recombinant CHO cells over long-term culture. These procedures include the following; western blotting, ELISA to evaluate protein production, real-time PCR to analyze plasmid and mRNA copy numbers, and fluorescent in situ hybridization (FISH) to assess the location of the inserted plasmid on host cell chromosomes.

Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification.

The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non-UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long-term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.

Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells.

CHO cells are the most frequently used host for commercial production of therapeutic proteins, and DHFR-mediated gene amplification is extensively applied to generate cell lines with increased protein production. However, decreased protein productivity is observed unpredictably during the time required for scale-up with consequences for yield, time, finance and regulatory approval. In this study, we have examined the interaction between Ubiquitous Chromatin Opening Elements (UCOE) and DHFR-linked amplification in relation to cell expression stability. In summary, the inclusion of UCOE elements generated cells that (1) achieved higher cell densities and exhibited increased production of recombinant mRNA per cell and protein yield, (2) allowed isolation of greater numbers of high-producing clones, (3) resulted in greater mRNA recovery per recombinant gene copy, (4) retained stable mRNA and protein expression after amplification provided Methotrexate (MTX) was present (but not in the absence of MTX when instability was observed) and (5) conferred copy number-dependent expression to linked transgene, suggesting that they are resistant to positional gene-silencing effects. It was concluded that the inclusion of UCOEs within expression constructs offers significant advantages for certainty of cell line generation (and the number of recovered clones for more detailed characterisation/optimisation) and that UCOEs are compatible with DHFR amplification protocols. The data suggested that enhanced cell line recovery by transcriptional enhancement of selection markers, such as DHFR, could be achieved.

Evaluating the interaction between UCOE and DHFR-linked amplification and stability of recombinant protein expression.

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry. In the creation of mammalian cell lines plasmid DNA carrying the gene-of-interest integrates randomly into the host cell genome, which results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long-term culture. This means that a large number of clones need to be screened in order to isolate stable, high producing cell lines making mammalian cell line development a long and laborious process. In this study an expression platform incorporating a Ubiquitous Chromatin Opening Element (UCOE; which are proposed to maintain chromatin in an open state) has been utilised for the expression of eGFP in CHO cells. Cell lines containing a UCOE vector, showed a significantly higher and more consistent eGFP expression than the non-UCOE cell lines without DHFR amplification. To further improve recombinant protein production cell lines were amplified with methotrexate (MTX). UCOE cell lines showed improved growth in MTX therefore amplification to 250 nM MTX was achieved following a one-step amplification procedure. However, non-UCOE cell lines showed higher levels of eGFP production following MTX amplification. In addition, UCOE cell lines did not improve stability during long-term culture in the absence of selective pressure. Stable eGFP production was achieved for all cell lines when MTX is present. Finally, UCOE cell lines displayed more consistent response to external stimuli than non-UCOE cell lines, suggesting that UCOE cell lines are less prone to clonal variability.