Combination Therapy of Chloroquine and C2-Ceramide Enhances Cytotoxicity in Lung Cancer H460 and H1299 Cells.

Non-small cell lung cancer (NSCLC) is a type of malignant cancer, and 85% of metastatic NSCLC patients have a poor prognosis. C2-ceramide induces G2/M phase arrest and cytotoxicity in NSCLC cells. In this study, the autophagy-inducing effect of C2-ceramide was demonstrated, and cotreatment with the autophagy inhibitor chloroquine (CQ) was investigated in NSCLC H460 and H1299 cells. The results suggested that C2-ceramide exhibited dose-dependent anticancer effects in H460 and H1299 cells and autophagy induction. Zebrafish-based acridine orange staining confirmed the combined effects in vivo. Importantly, the combination of a sublethal dose of C2-ceramide and CQ resulted in additive cytotoxicity and autophagy in both cell lines. Alterations of related signaling factors, including Src and SIRT1 inhibition and activation of the autophagic regulators LAMP2 and LC3-I/II, contributed to the autophagy-dependent apoptosis. We found that C2-ceramide continuously initiated autophagy; however, CQ inhibited autophagosome maturation and degradation during autophagy progression. Accumulated and non-degraded autophagosomes increased NSCLC cell stress, eventually leading to cell death. This study sheds light on improvements to NSCLC chemotherapy to reduce the chemotherapy dose and NSCLC patient burden.

A novel role for ß2-microglobulin: a precursor of antibacterial chemokine in respiratory epithelial cells.

We analyzed a panel of cationic molecules secreted in the culture medium of human respiratory epithelial cells (REC) upon activation by IL-1ß and different pathogen-associated molecular patterns. A 9 kDa fragment derived from ß2-microglobulin (B2M) was identified and named shed 9 kDa B2M (sB2M-9). The primary structure of sB2M-9 was revealed to increase its pI value that potentially could play an important role in innate defense. sB2M-9 exhibits antibacterial activity against Gram positive Staphylococcus aureus (SA) but not against Gram negative Klebsiella pneumonia (KP). Upon its binding to SA, sB2M-9 induces clumps, a phenomenon not observed with B2M. Migration of THP-1 monocytes exposed to SA clumps was significantly greater than that to SA without clumps. sB2M-9 binds to SA, more likely as a chemokine, to facilitate THP-1 migration. As a whole, we demonstrated that REC release a novel chemokine with antibacterial activity that is shed from B2M to facilitate THP-1 migration.

Vitamin D-binding protein is required for the protective effects of vitamin D in renal fibroblasts and is phosphorylated in diabetic rats.

Serum vitamin D is bound to vitamin D-binding protein (DBP). We studied the roles of DBP in streptozotocin-diabetic rats and high glucose (HG)-cultured cells. In diabetic rat sera, there was one upregulated (with a lower isoelectric point [pI], phosphorylated at S268, S270, S464 and T269) and one downregulated (with a higher pI, phosphorylated at S454 and S457) DBP. DBP levels with lower pI were increased in diabetic rat kidney and liver. HG (30 mM) increased DBP protein expression in NRK-49F cells and Clone-9 hepatocytes. HG decreased pI of DBP in Clone-9 hepatocytes. Moreover, DBP short hairpin ribonucleic acid attenuated 1,25-(OH)2D3-induced attenuation of HG-induced renin (but not collagen IV and fibronectin) protein expression in NRK-49F cells. Thus, DBP level is increased whereas DBP is phosphorylated in diabetic rat serum. HG increased DBP protein expression in renal fibroblasts and hepatocytes. Moreover, DBP is required for vitamin D-induced attenuation of HG-induced renin in NRK-49F cells.

Phthalic acid chemical probes synthesized for protein-protein interaction analysis.

Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO2) particles and phthalate chemical probes were manufactured from phthalic acid and APTES-SiO2. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis.

Interaction between TGF-ß and ACE2-Ang-(1-7)-Mas pathway in high glucose-cultured NRK-52E cells.

Transforming growth factor-ß (TGF-ß) is pivotal in diabetic nephropathy (DN). Angiotensin converting enzyme-2 (ACE2) converts angiotensin II (Ang II) to angiotensin 1-7 (Ang-(1-7)), which binds to Mas. Proximal tubular ACE2 is decreased in DN. ACE2 deficiency exacerbates whereas ACE2 overexpression attenuates DN. Thus, we investigated the mechanism of high glucose-decreased ACE2 in terms of the interaction between TGF-ß and ACE2-Ang-(1-7)-Mas in NRK-52E cells. We found that high glucose increased TGF-ß1. SB431542 attenuated high glucose-inhibited ACE2 and Mas and Ang-(1-7) conversion from Ang II while attenuating high glucose-induced fibronectin. TGF-ß1 also decreased ACE2 and Mas and Ang-(1-7) conversion from Ang II. A779 attenuated Ang-(1-7)-decreased TGF-ß1 and Ang-(1-7)-activated JAK2-STAT3. Moreover, A779, LY294002 and AG490 attenuated Ang-(1-7)-inhibited TGF-ß1. The combination of Ang-(1-7) and Mas attenuated TGF-ß1 (but not high glucose)-induced fibronectin. Thus, high glucose decreases ACE2 via TGF-ßR in NRK-52E cells. Additionally, there is a negative feedback function between TGF-ß and ACE2, and the combined inhibition of TGF-ß and activation of the ACE2-Ang-(1-7)-Mas may be useful for treating diabetic renal fibrosis.

Characterization of protein adducts formed by toxic alkaloids by nano-scale liquid chromatography with mass spectrometry.

Betel quid chewing is associated with cytotoxicity, genotoxicity and carcinogenicity in diseases such as oral cancer, liver cirrhosis, hepatocellular carcinoma and diabetes mellitus. Arecoline and arecaidine, which are the main alkaloids in the areca nut, are potential exposure biomarkers in habitual betel quid users. This study developed a method of detecting arecoline- and arecaidine-protein adducts by mass spectrometry (MS). First, bovine serum albumin was used to predict and confirm the binding sites of proteins modified by arecoline or arecaidine. Cells were then treated with arecoline to identify new protein adducts after cellular metabolic processing. Finally, human plasma was used to model long-term exposure to arecoline and arecaidine. Following isolation proteins were tryspin digested. The peptides afforded were separated and analyzed by nano-scale liquid chromatography with MS using an LTQ Orbitrap mass spectrometer. The experimental findings showed that cysteine is the predominant amino acid in protein adduct formation. The goal of this study was to establish a screening platform for identifying novel protein adducts that form covalent bonds with arecoline or arecaidine. Use of this strategy to survey new protein-toxic adducts may help to identify novel biomarkers of betel nut exposure.

Proteomic comparison of human embryonic stem cells with their differentiated fibroblasts: Identification of 206 genes targeted by hES cell-specific microRNAs.

Human embryonic stem (hES)-T3 (T3ES) cells were spontaneously differentiated into autogeneic fibroblast-like T3DF cells, as feeder cells with the capacity to support the growth of undifferentiated hES cells. The proteomes of undifferentiated T3ES cells and their differentiated T3DF fibroblasts were quantitatively compared. Several heterogeneous nuclear ribonucleoproteins and glycolytic enzymes, including l-lactate dehydrogenase A (M), were found to be abundantly and differentially expressed in T3ES cells and T3DF fibroblasts, respectively. Both miRNA and mRNA profiles from the undifferentiated T3ES cells and their differentiated T3DF fibroblasts had been previously determined. In this investigation, 206 genes were found to be targets of the four hES cell-specific miRNAs of miR-302d, miR-372, miR-200c, and/or miR-367 by using two-fold differential expression and inverse expression levels (highly negative correlations) of miRNAs to their target mRNAs. That YWHAZ (14-3-3 zeta) is a target of miR-302d and miR-372 was further confirmed by proteomic comparison between T3ES cells and their differentiated T3DF fibroblasts. According to GeneOntology analyses, almost 50% of these 206 target proteins are nuclear and are involved in gene transcription. Identifying the target mRNAs of hES cell-specific miRNAs will provide a better understanding of the complex regulatory networks in hES cells. Furthermore, these miRNA-targeted proteins play important roles in differentiation of hES cells and during embryo development.

UBE2M-mediated p27(Kip1) degradation in gemcitabine cytotoxicity.

Gemcitabine (2'-deoxy-2', 2'-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27(Kip1) protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27(Kip1) protein degradation. The induction of UBE2M and reduction of p27(Kip1) by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27(Kip1). Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27(Kip1). Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27(Kip1) expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27(Kip1) in drug resistance.

A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: comparative expression profiles of mRNAs, microRNAs and proteins among different feeders and conditioned media.

BACKGROUND: Human embryonic stem (hES) cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF) feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages. RESULTS: A feeder-free culture on Matrigel in hES medium conditioned by the autogeneic feeder cells (T3HDF) was established to maintain the undifferentiated growth of hES-T3 cells (T3/CMHDF) for 8 passages in this investigation. The gene expression profiles of mRNAs, microRNAs and proteins between the undifferentiated T3/HDF and T3/CMHDF cells were shown to be very similar, and their expression profiles were also found to be similar to those of T3/MEF and T3/CMMEF cells grown on MEF feeder and feeder-free Matrigel in MEF-conditioned medium, respectively. The undifferentiated state of T3/HDF and T3/CMHDF as well as T3/MEF and T3/CMMEF cells was evidenced by the very high expression levels of "stemness" genes and low expression levels of differentiation markers of ectoderm, mesoderm and endoderm in addition to the strong staining of OCT4 and NANOG. CONCLUSION: The T3HDF feeder and T3HDF-conditioned medium were able to support the undifferentiated growth of hES cells, and they would be useful for drug development and toxicity testing in addition to the reduced risks of xenogeneic pathogens when used for medical applications such as cell therapies.