• Biomolecules & Therapeutics
• /
• v.22 no.4
• /
• pp.355-362
• /
• 2014

We have developed a fully automated high throughput drug screening (HTDS) system based on the microfluidic cell culture array to perform combinational chemotherapy. This system has 64 individually addressable cell culture chambers where the sequential combinatorial concentrations of two different drugs can be generated by two microfluidic diffusive mixers. Each diffusive mixer has two integrated micropumps connected to the media and the drug reservoirs respectively for generating the desired combination without the need for any extra equipment to perfuse the solution such as syringe pumps. The cell array is periodically exposed to the drug combination with the programmed LabVIEW system during a couple of days without extra handling after seeding the cells into the microfluidic device and also, this device does not require the continuous generation of solutions compared to the previous systems. Therefore, the total amount of drug being consumed per experiment is less than a few hundred micro liters in each reservoir. The utility of this system is demonstrated through investigating the viability of the prostate cancer PC3 cell line with the combinational treatments of curcumin and tumor necrosis factor-alpha related apoptosis inducing ligand (TRAIL). Our results suggest that the system can be used for screening and optimizing drug combination with a small amount of reagent for combinatorial chemotherapy against cancer cells.

• Journal of Life Science
• /
• v.26 no.7
• /
• pp.847-854
• /
• 2016

Cordycepin, a derivative of the nucleoside adenosine, is one of the active components extracted from fungi of genus Cordyceps, and has been shown to have many pharmacological activities. In this study, we investigated the effects of cordycepin on proliferation and apoptosis of human gastric cancer AGS cells, and its possible mechanism of action. Treatment of cordycepin resulted in significant decrease in cell viability of AGS cells in a concentration-dependent manner. A concentration-dependent apoptotic cell death was also measured by agarose gel electrophoresis and flow cytometery analysis. Molecular mechanistic studies of apoptosis unraveled cordycepin treatment resulted in an enhanced expression of tumor necrosis factor-related apoptosis-inducing ligand, death receptor 5 and Fas ligand. Furthermore, up-regulation of pro-apoptotic Bax, and down-regulation of anti-apoptotic Bcl-2 and Bcl-xL expression were also observed in cordycepin-treated AGS cells. These were followed by activation of caspases (caspase-9, -8 and -3), subsequently leading to poly (ADP-ribose) polymerase cleavage. Taken together, these findings indicate that cordycepin induces apoptosis in AGS cells through regulation of multiple apoptotic pathways, including death receptor and mitochondria. Although further mechanical studies are needed, our results revealed that cordycepin can be regarded as a new effective and chemopreventive compound for human gastric cancer treatment.

• The Korean Journal of Physiology and Pharmacology
• /
• v.25 no.4
• /
• pp.273-280
• /
• 2021

Lung cancer despite advancement in the medical field continues to be a major threat to human lives and accounts for a high proportion of fatalities caused by cancers globally. The current study investigated vanillin oxime, a derivative of vanillin, against lung cancer cells for development of treatment and explored the mechanism. Cell viability changes by vanillin oxime were measured using MTT assay. Vanillin oxime-mediated apoptosis was detected in A549 and NCI-H2170 cells at 48 h of exposure by flow cytometry. The CEBP homologous protein (CHOP) and death receptor 5 (DR5) levels were analysed by RT-PCR and protein levels by Western blotting. Vanillin oxime in concentration-dependent way suppressed A549 and NCI-H2170 cell viabilities. On exposure to 12.5 and 15 μM concentrations of vanillin oxime elevated Bax, caspase-3, and -9 levels in A549 and NCI-H2170 cells were observed. Vanillin oxime exposure suppressed levels of Bcl-2, survivin, Bcl-xL, cFLIP, and IAPs proteins in A549 and NCI-H2170 cells. It stimulated significant elevation in DR4 and DR5 levels in A549 and NCI-H2170 cells. In A549 and NCI-H2170 cells vanillin oxime exposure caused significant (p < 0.05) enhancement in CHOP and DR5 mRNA expression. Vanillin oxime exposure of A549 and NCI-H2170 cells led to significant (p < 0.05) enhancement in levels of phosphorylated extracellular-signal-regulated kinase and c-Jun N-terminal kinase. Thus, vanillin oxime inhibits pulmonary cell proliferation via induction of apoptosis through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated pathway. Therefore, vanillin oxime may be studied further to develop a treatment for lung cancer.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.11
• /
• pp.5339-5343
• /
• 2012

The induction of apoptosis in target cells is a key mechanism for most anti-tumor therapies. Bufalin is a cardiotonic steroid that has the potential to induce differentiation and apoptosis of tumor cells. Research on bufalin has so far mainly involved leukemia, prostate cancer, gastric cancer and liver cancer, and has been confined to in vitro studies. The bufadienolides bufalin and cinobufagin have been shown to induce apoptosis in a wide spectrum of cancer cell. The present article reviews the anticancer effects of bufalin. It induces apoptosis of lung cancer cells via the PI3K/Akt pathway and also suppressed the proliferation of human non-small cell lung cancer A549 cell line in a time and dose dependent manner. Bufalin, bufotalin and gamabufotalin, key bufadienolides, significantly sensitize human breast cancer cells with differing ER-alpha status to apoptosis induction by the TNF-related apoptosis-inducing ligand (TRAIL). In addition, bufadienolides induce prostate cancer cell apoptosis more significantly than that in breast epithelial cell lines. Similar effects have been observed with hepatocellular carcinoma (HCC) but the detailed molecular mechanisms of inducing apoptosis in this case are still unclear. Bufalin exerts profound effects on leukemia therapy in vitro. Results of multiple studies indicate that bufalin has marked anti-tumor activities through its ability to induce apoptosis. Large-scale randomized, double-blind, placebo or positive drug parallel controlled studies are now required to confirm the efficacy and apoptosis-inducing potential of bufalin in various cancers in the cliniucal setting.

• Journal of Life Science
• /
• v.23 no.3
• /
• pp.462-469
• /
• 2013

Withaferin A is a steroidal lactone purified from the Indian medicinal plant Withania somnifera. It exhibits a wide variety of activities, including anti-tumor, anti-inflammation, and immunomodulation properties. In this review, we focused on the anti-cancer effects of withaferin A. Withaferin A inhibits cell proliferation, metastasis, invasion, and angiogenesis in cancer cells. Furthermore, it sensitized irradiation, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-, and doxorubicin-mediated apoptosis. The results showed that multiple mechanisms were involved in withaferin A-mediated anti-cancer effects. First, withaferin A increased intracellular reactive oxygen species (ROS) production and induced ER stress- and mitochondria-mediated apoptosis. Second, withaferin A inhibited the signaling pathways (Jak/STAT, Akt, Notch, and c-Met), which are important in cell survival, proliferation, and metastasis. Third, it induced apoptosis and inhibited cancer cell migration through the up-regulation of prostate apoptosis protein-4 (Par-4). Finally, withaferin A up-regulated pro-apoptotic protein expression levels through the inhibition of proteasome activity. Our findings suggested that withaferin A is a potential, potent therapeutic agent.

• Korean Journal of Microbiology
• /
• v.52 no.1
• /
• pp.10-17
• /
• 2016

Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.

• Korean Journal of Microbiology
• /
• v.54 no.4
• /
• pp.330-342
• /
• 2018

The Streptavidin and Biotin system has been studied most extensively as the high affinity non-covalent binding of Biotin to STR ($K_D=10^{-14}M$) and four Biotin binding sites in tetrameric Streptavidin makes this system useful for the production of multivalent antibody. For the application of this system, we cloned Streptavidin amplified from Streptomyces avidinii chromosome by PCR and fused to gene of hAY4 single-chain Fv antibody specific to death receptor 4 (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand. The hAY4 single-chain Fv antibody fused to Streptavidin expressed in Escherichia coli showed 43 kDa monomer in heated SDS-PAGE. However, this fusion protein shown in both non-heated SDS-PAGE and Size-exclusion chromatography exhibited 172 kDa as a tetramer suggesting that natural tetramerization of Streptavidin by non-covalent association induced hAY4 single-chain Fv tetramerization. This fusion protein retained a Biotin binding activity similar to natural Streptavidin as shown in Ouchterlony assay and ELISA. Death receptor 4 antigen binding activity of purified hAY4 single-chain Fv fused to Streptavidin was also confirmed by ELISA and Westernblot. In addition, surface plasmon resonance analysis showed 60-fold higher antigen binding affinity of the hAY4-STR than monomeric hAY4 ScFv due to tetramerization. In summary, hAY4 single-chain Fv fused to Streptavidin fusion protein was successfully expressed and purified as a soluble tetramer in E. coli and showed both Biotin and DR4 antigen binding activity suggesting possible production of bifunctional and tetrameric ScFv antibody.