This study aimed to assess the effects of hexavalent chromium on zebrafish (Danio rerio) embryo development. The zebrafish embryos were treated with solutions containing chromium at different concentrations (0.1, 1, 3.125, 6.25, 12.5, 50, and 100 µg/mL). The development of zebrafish embryos was estimated by the determination of survival rate, heart rate, and the measurement of larvae body length. Real time RT-PCR and Western blot were performed to assess the expression of apoptosis- and antioxidant-related genes. The results showed that the reduced survival rate of zebrafish embryos and larvae was associated with an increase in chromium concentration. The exposure of higher concentrations resulted in a decrease in body length of zebrafish larvae. In addition, a marked increase in heart rate was observed in the zebrafish larvae under chromium treatment, especially at high concentrations. The real-time RT-PCR analysis showed that the transcript expressions for cell-cycle-related genes (cdk4 and cdk6) and antioxidant-related genes (sod1 and sod2) were downregulated in the zebrafish embryos treated with chromium. Western blot analysis revealed the upregulation of Caspase 3 and Bax, while a downregulation was observed in Bcl2. These results indicated that hexavalent chromium induced changes in zebrafish embryo development by altering apoptosis- and antioxidant-related genes.

Anoectochilus roxburghii Lind. (A. roxburghii) has promising anti-oxidant, hyperglycemic, hepatoprotective, and immunomodulatory activities as well as anti-tumor effects. However, the pharmacological actions of in vitro cultured plants remain to be determined. Therefore, the objective of the study was to assess in vitro cytotoxicity and in vivo potential toxicity of an extract derived from in vitro cultivated A. roxburghii, termed as iARE. The total flavonoid content and predominant flavonoid compounds of extract were identified and quantitatively analyzed. The in vitro cytotoxicity of iARE was examined using several cancer and normal cell lines. The apoptotic activity and expression of apoptosis-associated genes were also examined in MCF7 cells to determine the underlying mechanisms related to anti-proliferative effects. In vivo potential toxicity of iARE was assessed following acute and subchronic oral administration in Sprague Dawley rats. Quercetin, kaempferol, and isorhamnetin were three flavonoid components identified in iARE. The extract exerted cytotoxic effects on various cancer cells but not normal fibroblasts. Apoptosis in MCF7 cells was induced by iARE in a concentration-dependent manner associated with increased Bax/Bcl-2 ratio and reduced mitochondrial membrane potential ΔΨm, leading to release of cytochrome c, activation of caspase-3/7 and caspase-9, and cleavage of PARP. In the acute oral toxicity study, no mortality or toxicological signs were observed in rats at 1000 or 5000 mg/kg. In a subchronic oral toxicity study, iARE at a dosage of up to 1000 mg/kg produced no mortality or treatment-related adverse effects on general behavior, food intake, body weight, relative organ weights. No apparent marked changes in the histopathology of the liver and kidney were detected. Data demonstrated that iARE induced in vitro cytotoxic effects in cancer cells are associated with lackof invivo toxicity. Thus, iARE was suggested to be considered as apotential therapeutic candidate for cancer treatment.

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.

Curcuma aromatica Salisb. (C. aromatica) is one of the traditional herbs used to treat microbial infection, skin eruption, coronary heart disease, and other diseases, including cancer. However, the inhibitory effects and underlying mechanisms of action of C. aromatica on gastric cancer cells have not yet been fully elucidated. Our study aimed to examine the possible molecular mechanisms underlying the cytotoxic effects attributed to C. aromatica rhizome standardized extract against gastric cancer cells. The components of two major active compounds in C. aromatica rhizome extract were quantitatively analyzed using a simple and validated HPLC method. Cytotoxicity was determined in different gastric cancer and non-cancer cell lines. The biological activities of the extract targeting apoptosis and cell cycle-related genes on gastric cancer AGS cells were also investigated to elucidate the mechanisms relating to the anti-proliferative effect of C. aromatica rhizomes. The two major active compounds curdione and germacrone, in the C. aromatica extract were standardized to 0.64% and 1.12% w/w, respectively. The standardized extract (CAE) exerted cytotoxic effects on various cancer cells, whereas minimal effects at equivalent doses were noted for normal cells. CAE concentration-dependently suppressed growth of gastric cancer AGS cells via induction of apoptosis. Further studies revealed that CAE treatment disrupted mitochondrial membrane potential (ΔΨm), increased Bax/Bcl-2 ratio, and cytochrome c release, resulting in activation of caspase-9/-3 and subsequent cleavage of PARP. Further, the inhibitory effects of caspase-9/-3 expression by a synthetic pan-caspase inhibitor partially protected cells against apoptosis following CAE treatment. In addition, CAE significantly promoted cell death in AGS cells via an accumulation of cells in the G0/G1 phase. This effect was associated with upregulation of the CDK inhibitor p21 and downregulation of cyclin D1, cyclin E, CDK4, and CDK2 expression. Our data indicated that CAE exerted anti-proliferative activity by activating the mitochondria-mediated caspase-dependent apoptotic pathway and arresting the p21-mediated G0/G1 cell cycle on human gastric cancer AGS cells.