Enhancement of anti-TNFα monoclonal antibody production in CHO cells through the use of UCOE and DHFR elements in vector construction and the optimization of cell culture media.

Recently, there has been a high demand for anti-tumor necrosis factor-α monoclonal antibodies (mAbTNFα) in the treatment of rheumatoid arthritis and other autoimmune diseases. Thus, efficient strategies and stable high-producing cell lines need to be established to increase antibody production. In this study, we describe an efficient approach to establish a mAbTNFα high-producing clone through the optimization of expression vectors and cell culture media. The ubiquitous chromatin opening element (UCOE) and dihydrofolate reductase (DHFR)-based vectors encoding mAbTNFα were introduced into the CHO-DG44 cells using lipofection. Clones were obtained by selecting transfected cells with G418, amplifying them by treatment with methotrexate, and isolating them by limiting dilution. Different media formulated with commercial feeds and media were also screened to develop an improved medium. The antibody produced by the selected clone was purified, characterized, and compared to standard adalimumab. Using our established protocol, a cell clone obtained from stable mAbTNFα-expressing cell pools showed a 3.8-fold higher antibody titer compared to stable cell pools. Furthermore, the highest antibody yield of selected clones cultured in fed-batch mode using improved medium was 2450 ± 30 µg/mL, which was 13.2-fold higher than that of stable cell pool cultivated in batch mode using a basal medium. The purified antibody had primary chemical and biological characteristics similar to those of adalimumab. Therefore, the use of UCOE and DHFR vectors in combination with the optimization of cell culture media may help in establishing stable and high-producing CHO cell lines for therapeutic antibody production.

Bioactive chemical constituents, in vitro anti-proliferative activity and in vivo toxicity of the extract of Curcuma singularis Gagnep rhizomes.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma singularis Gagnep is a Vietnamese medicinal plant which has been commonly used as a medicinal remedy in traditional and folk medicines for improving health as well as for treating some diseases, like rheumatoid arthritis, kidney failure. However, pharmacological effects, including anti-cancer activity and the safety of this plant has not been fully investigated. AIM OF THE STUDY: This study aimed to investigate the in vitro anti-growth activity of an extract derived from Curcuma singularis rhizome extract (CSE) against cell lines as well as determine its phytochemical composition. The other goal of our study was to assess the safety of CSE in rats. MATERIALS AND METHODS: The main constituents in the extract were identified and quantitatively analyzed. The in vitro cytotoxicity of CSE was evaluated in several cancer and normal cell lines. The apoptotic activity of CSE and the expression of the apoptosis-related genes were investigated in AGS cells to clarify the underlying molecular mechanisms. The in vivo toxicity of CSE was assessed via acute and subacute oral studies on Sprague-Dawley rats, respectively according to the guidelines 425 and 407 of the Organization for Economic Cooperation and Development (OECD). The drug-related toxicity signs, mortality, body and organ weights were recoreded during the experimental period. In addition, the selected hematological and biochemical parameters, and histological alterations were determined at the end of the subacute toxicity test. RESULTS: Germacrone, ar-turmerone, and curcumol were three sesquiterpene components found in the extract. CSE showed cytotoxic effects in different cancer cells, but had minimal effects on normal cells. Apoptosis in AGS cells was caused by CSE in a concentration-dependent pattern through increase of Bax/Bcl-2 ratio, and release of cytochrome c, which leads to activation of caspase-3/-7, caspase-9, as well as cleavage of PARP. In the acute toxicity test, no signs of toxicity and no mortality were recorded in rats at both doses of 1000 and 5000 mg/kg. In the subacute toxicity study, CSE showed no drug-related adverse effects on water and food consumption, body and organ weights. CSE at a dose of 1000 mg/kg slightly increased WBC and platelet values in female rats, while it increased WBC values in male rats in all tested doses. The decrease of total cholesterol and triglyceride levels were found in female rats treated CSE at doses of 250 or 500 mg/kg. In addition, the increase of serum ALT and AST levels in rats treated at the dose of 1000 mg/kg were noted. No significant changes in histopathological structures of kidneys, spleen, heart and lungs, except liver tissue with minor modifications was found. CONCLUSIONS: Our findings indicated that CSE exhibited in vitro anti-proliferative effects on AGS cells by mainly activating the caspase-dependent mitochondrial apoptotic pathway. CSE also showed in vivo toxicity signals at the dose of 1000 mg/kg with proven minor hepatic injuries, which should be avoided the high dose for prolonged use. Curcuma singularis rhizomes may be used as a chemotherapeutic agent for the treatment of gastric cancer with in vitro anti-cancer investigation and in vivo biological safety evaluation.

Investigation of bioactive chemical constituents and anti-cancer activity of ethanol extract of Curcuma singularis Gagnep rhizomes.

Curcuma singularis Gagnep is a Vietnamese medicinal plant which has been commonly used in traditional and folk medicines for the treatment of different diseases. The goals of the present study are to investigate chemical composition and anti-proliferative activity of Curcuma singularis rhizome extract (CSE). The in vitro cytotoxicity of CSE was evaluated using WST-1 and LDH assays. The apoptosis induction was determined using nuclei DAPI staining and FACS assays. The main compounds of extract were identified and quantitatively analyzed using the validated HPLC method. The extract showed cytotoxic effects in various liver and breast cancer cells but had minimal effects on normal cells. It induced apoptosis on both Hep3B and SKBR3 cells in a dose-dependent manner. In addition, three sesquiterpene compounds, such as germacrone (3.25 ± 0.32 mg/g), ar-turmerone (1.12 ± 0.24 mg/g), and curcumol (0.31 ± 0.12 mg/g) were found as the main components of CSE. This is the first report on the in vitro cytotoxic effect of Curcuma singularis rhizomes against cancer cells.

Phenolic Extraction of Moringa Oleifera Leaves Induces Caspase-Dependent and Caspase-Independent Apoptosis through the Generation of Reactive Oxygen Species and the Activation of Intrinsic Mitochondrial Pathway in Human Melanoma Cells.

Moringa oleifera Lam. has long been used to treat many diseases, including diabetes, aging, inflammatory, and cancer. Many studies have revealed that the crude extract of Moringa oleifera Lam. leaves possesses anticancer property. Therefore, in this study, the extract of Moringa oleifera leaves was fractionated using different solvents to figure out the most effective fraction for anti-proliferative effect on melanoma cells. Methanol extract (MO-ME), hexane fraction (MO-HE), chloroform fraction (MO-CH), ethyl acetate fraction (MO-EA), and water-soluble fraction (MO-WA) of Moringa oleifera leaves were prepared. Total phenolic and flavonoid contents were determined. The anti-proliferative activity on melanoma cells and normal cells was investigated using WST-1 assay. The apoptotic activity was assessed by testing DNA condensation, DNA fragmentation, and phosphatidylserine (PS) externalization. The expression of apoptosis-related genes, the mitochondrial depolarization, and reactive oxygen species (ROS) were then examined to clarify the underlying molecular mechanisms. In this regard, MO-ME, MO-EA, and MO-CH inhibited the proliferation of both A375 human melanoma cells and A2058 human melanoma cells, but had little effect on WS1 normal human skin fibroblasts and primary normal human dermal fibroblasts (NHDF). Among fractions, the phenolic-rich MO-EA markedly inhibited the growth of A375 cells in a dose- and time-dependent manner. The anti-proliferation was supposed to be mediated via apoptosis, which was demonstrated by the significant increase of condensed chromatin, DNA fragmentation, and PS externalization. The apoptosis was stimulated by enhanced ROS production and reduction of mitochondrial membrane potential. MO-EA activated Bax while reducing Bcl-2 expression, leading to an increase in Bax/Bcl-2 ratio. The mechanisms of cell death involved in activation of Caspase-3/7 and Caspase-9 (Caspase-dependent pathway), activation, and translocation of apoptosis-inducing factor (AIF) into the nucleus (Caspase-independent pathway). Our study indicated that the phenolic-rich fraction exerted significant anticancer effects on melanoma cells in vitro which involved in Caspase-dependent and Caspase-independent apoptosis pathways mediated by mitochondrial ROS. These results provided a fundament for the using of phenolic-rich fraction of Moringa oleifera leaves to treat skin cancer effectively.

Mitochondria-mediated Caspase-dependent and Caspase-independent apoptosis induced by aqueous extract from Moringa oleifera leaves in human melanoma cells.

Malignant melanoma is a very aggressive and serious type of cutaneous cancer. Previous studies indicated the anti-cancer activity of aqueous extract of Moringa oleifera Lam. leaves (MOE) against a variety of cell lines. However, there has not been much research about the effect of MOE on melanoma. Therefore, this study was about to investigate the anti-proliferation mediated by apoptosis of MOE on human melanoma cell lines. Furthermore, the related molecular mechanisms of the apoptosis were also examined. An aqueous extract of Moringa oleifera leaves was prepared and the anti-proliferative activity on melanoma cells and normal cells was tested using WST-1 assay. The apoptotic hallmarks including DNA condensation and phosphatidylserine (PS) externalization were assessed. The expression of apoptosis-related genes and the depolarization of mitochondrial membrane potential were then examined to clarify the underlying molecular mechanisms. MOE inhibited cell growth of A375 cells and A2058 cells in a dose-dependent manner but had little effect on human normal fibroblasts. The cell growth inhibition was induced by apoptosis which was expressed via chromatin condensation and PS externalization. MOE decreased mitochondrial membrane potential. Additionally, MOE increased Bax/Bcl-2 ratio, activated Caspase-3/7, Caspase-9, PARP and AIF translocation, leading to apoptotic cell death. Our study indicated that MOE exerted significant anti-cancer effects on melanoma cells in vitro which involved mitochondria-mediated Caspase-dependent and Caspase-independent apoptosis pathways. These results provided a scientific approach for using Moringa oleifera leaves as an alternative therapy to treat skin cancer.

Differentiation of umbilical cord lining membrane-derived mesenchymal stem cells into endothelial-like cells.

BACKGROUND: Stem cell therapy for the treatment of vascular-related diseases through functional revascularization is one of the most important research areas in tissue engineering. The aim of this study was to investigate the in vitro differentiation of umbilical CL-MSC into endothelial lineage cells. METHODS: In this study, isolated cells were characterized for expression of MSC-specific markers and osteogenic and adipogenic differentiation. They were induced to differentiate into endothelial-like cells and then examined for expression of the endothelial-specific markers, karyotype, and functional behavior of cells. RESULTS: Isolated cells expressed MSC-specific markers and differentiated into adipocytes and osteoblasts. After endothelial differentiation, they expressed CD31, vWF, VE-cadherin, VEGFR1, and VEGFR2 at both mRNA and protein level, but their morphological changes were not apparent when compared with those of undifferentiated cells. There were no significant changes in karyotype of differentiated cells. Furthermore, angiogenesis assay and LDL uptake assay showed that differentiated cells were able to form the capillary-like structures and uptake LDL, respectively. CONCLUSION: The results indicated that umbilical CL-MSC could differentiate into functional endothelial-like cells. Also, they are suitable for basic and clinical studies to cure several vascular-related diseases.