Resurfacing receptor binding domain of Colicin N to enhance its cytotoxic effect on human lung cancer cells.

Colicin N (ColN) is a bacteriocin secreted by Escherichia coli (E. coli) to kill other Gram-negative bacteria by forcefully generating ion channels in the inner membrane. In addition to its bactericidal activity, ColN have been reported to selectively induce apoptosis in human lung cancer cells via the suppression of integrin modulated survival pathway. However, ColN showed mild toxicity against human lung cancer cells which could be improved for further applications. The protein resurfacing strategy was chosen to engineer ColN by extensive mutagenesis at solvent--exposed residues on ColN. The highly accessible Asp and Glu on wild-type ColN (ColNWT) were replaced by Lys to create polycationic ColN (ColN+12). Previous studies have shown that increase of positive charges on proteins leads to the enhancement of mammalian cell penetration as well as increased interaction with negatively charged surface of cancer cells. Those solvent--exposed residues of ColN were identified by Rosetta and AvNAPSA (Average number of Neighboring Atoms Per Side-chain Atom) approaches. The findings revealed that the structural features and stability of ColN+12 determined by circular dichroism were similar to ColNWT. Furthermore, the toxicity of ColN+12 was cancer -selective. Human lung cancer cells, H460 and H23, were sensitive to ColN but human dermal papilla cells were not. ColN+12 also showed more potent toxicity than ColNWT in cancer cells. This confirmed that polycationic resurfacing method has enabled us to improve the anticancer activity of ColN towards human lung cancer cells.

Stemness-Suppressive Effect of Bibenzyl from Dendrobium ellipsophyllum in Human Lung Cancer Stem-Like Cells.

Cancer stem-like cells (CSCs) are key mediators driving tumor initiation, metastasis, therapeutic failure, and subsequent cancer relapse. Thus, targeting CSCs has recently emerged as a potential strategy to improve chemotherapy. In this study, the anticancer activity and stemness-regulating capacity of 4,5,4'-trihydroxy-3,3'-dimethoxybibenzyl (TDB), a bibenzyl extracted from Dendrobium ellipsophyllum, are revealed in CSCs of various human lung cancer cells. Culture with TDB (5-10 µM) strongly abolished tumor-initiating cells in lung cancer H460, H23, and A549 cells in both anchorage-dependent and anchorage-independent colony formation assays. Through the 3D single-spheroid formation model, attenuation of self-renewal capacity was observed in CSC-enriched populations treated with 1-10 µM TDB for 7 days. Flow cytometry analysis confirmed the attenuation of %cell overexpressing CD133, a CSC biomarker, in TDB-treated lung cancer spheroids. TDB at 5-10 µM remarkably suppressed regulatory signals of p-Akt/Akt, p-GSK3ß/GSK3ß, and ß-catenin corresponding to the downregulated mRNA level of stemness transcription factors including Nanog, Oct4, and Sox2. Moreover, the antiapoptosis Bcl-2 and Mcl-1 proteins, which are downstream molecules of Akt signaling, were evidently decreased in CSC-enriched spheroids after culture with TDB (1-10 µM) for 24 h. Interestingly, the diminution of Akt expression by specific siAkt effectively reversed suppressive activity of TDB targeting on the CSC phenotype in human lung cancer cells. These findings provide promising evidence of the inhibitory effect of TDB against lung CSCs via suppression of Akt/GSK3ß/ß-catenin cascade and related proteins, which would facilitate the development of this bibenzyl natural compound as a novel CSC-targeted therapeutic approach for lung cancer treatment.

Development of a human antibody fragment directed against the alpha folate receptor as a promising molecule for targeted application.

Alpha folate receptor (FRα) is currently under investigation as a target for the treatment of patients with non-small-cell lung cancer (NSCLC), since it is highly expressed in tumor cells but is largely absent in normal tissue. In this study, a novel human variable domain of a heavy-chain (VH) antibody fragment specific to FRα was enriched and selected by phage bio-planning. The positive phage clone (3A102 VH) specifically bound to FRα and also cross-reacted with FRß, as tested by ELISA. Clone 3A102 VH was then successfully expressed as a soluble protein in an E. coli shuffle strain. The obtained soluble 3A102 VH demonstrated a high affinity for FRα with affinity constants (Kaff) values around 7.77 ± 0.25 × 107 M-1, with specific binding against both FRα expressing NSCLC cells and NSCLC patient-derived primary cancer cells, as tested by cell ELISA. In addition, soluble 3A102 VH showed the potential desired property of a targeting molecule by being internalized into FRα-expressing cells, as observed by confocal microscopy. This study inspires the use of phage display to develop human VH antibody (Ab) fragments that might be well suited for drug targeted therapy of NSCLC and other FRα-positive cancer cells.

Jorunnamycin A Suppresses Stem-Like Phenotypes and Sensitizes Cisplatin-Induced Apoptosis in Cancer Stem-Like Cell-Enriched Spheroids of Human Lung Cancer Cells.

It has been recognized that cancer stem-like cells (CSCs) in tumor tissue crucially contribute to therapeutic failure, resulting in a high mortality rate in lung cancer patients. Due to their stem-like features of self-renewal and tumor formation, CSCs can lead to drug resistance and tumor recurrence. Herein, the suppressive effect of jorunnamycin A, a bistetrahydroisoquinolinequinone isolated from Thai blue sponge Xestospongia sp., on cancer spheroid initiation and self-renewal in the CSCs of human lung cancer cells is revealed. The depletion of stemness transcription factors, including Nanog, Oct-4, and Sox2 in the lung CSC-enriched population treated with jorunnamycin A (0.5 µM), resulted from the activation of GSK-3ß and the consequent downregulation of ß-catenin. Interestingly, pretreatment with jorunnamycin A at 0.5 µM for 24 h considerably sensitized lung CSCs to cisplatin-induced apoptosis, as evidenced by upregulated p53 and decreased Bcl-2 in jorunnamycin A-pretreated CSC-enriched spheroids. Moreover, the combination treatment of jorunnamycin A (0.5 µM) and cisplatin (25 µM) also diminished CD133-overexpresssing cells presented in CSC-enriched spheroids. Thus, evidence on the regulatory functions of jorunnamycin A may facilitate the development of this marine-derived compound as a novel chemotherapy agent that targets CSCs in lung cancer treatment.

Superfine bacterial nanocellulose produced by reverse mutations in the bcsC gene during adaptive breeding of Komagataeibacter oboediens.

Nonsense mutation in the bcsC gene occurred in the ethanol-adapted strain of Komagataeibacter oboediens MSKU 3, E3 strain, resulting in the loss of the function to produce BNC. In this study, we tried to restore the BNC-producing ability of E3 strain by the following adaptive mutation through repetitive static culture, and obtained four BNC-producing revertant strains, of which the bcsC gene had InDel mutations near the frameshift mutation region in E3 strain, resulting in several amino acid alterations compared with the BcsC of MSKU 3. Each revertant produced BNCs with different productivity on the static culture. Interestingly, one of the revertants, R37-9, produced BNC with a finer structure and narrower range of fibrils width, compared to others. The genome of R37-9 strain revealed only one amino acid substitution in the bcsC gene. Thus, we concluded that N713D mutation occurred in the bcsC gene is responsible for the finer fibrils structure.

In Vitro Thermal and Ethanol Adaptations to Improve Vinegar Fermentation at High Temperature of Komagataeibacter oboediens MSKU 3.

High temperature and high ethanol concentrations obviously affect vinegar fermentation. The thermotolerant and ethanol-resistant strains are expected to become one of the technologies for effective vinegar fermentation. This study aimed to further improve thermotolerant Komagataeibacter oboediens MSKU 3 through thermal and ethanol adaptations for acetic acid fermentation. The MSKU 3 strain was independently cultured by a repetitive cultivation in gradually increasing temperature from 37 to 39 °C for thermal adaptation, while adaptation to ethanol was carried out from concentrations of 3 to 5.5% (v/v) at 37 °C. Acetic acid fermentation revealed that the thermo-adapted T4 strain could produce 2.82% acidity with 3% ethanol at 39 °C, whereas the ethanol-adapted E3 strain could produce 3.54% acidity with 5.5% ethanol at 37 °C, in contrast to the parental strain, MSKU 3, in which no fermentation occurs at either 39 °C or 5.5% ethanol. Furthermore, genome mapping analysis of T4 and E3 strains against the genome of parental strain MSKU 3 revealed several mutated genes that are associated with thermotolerance or ethanol adaptation. The occurrence of these adaptation-associated mutations during adaptive evolution was also analyzed. Therefore, adapted strains T4 and E3 revealed the potential of Komagataeibacter oboediens strain improvement to further enhance vinegar fermentation with high ethanol concentration at high temperature.