• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4555-4561
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• 2014

Background: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Materials and Methods: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and $IC_{50}$ values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting. Results: The $IC_{50}$ for imatinib on K562 was 362nM compared to 3,952nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down-regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562. Conclusions: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.6
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• pp.1012-1022
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• 2020

Objective: Caseins and fatty acids of milk are synthesized and secreted by the epithelial cells of the mammary gland. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, has been shown to promote mammary development. This study was conducted to determine the effect of ATRA on casein synthesis and fatty acid composition in MAC-T cells. Methods: MAC-T cells were allowed to differentiate for 4 d, treated with ATRA (0, 1.0, 1.5, and 2.0 μM), and incubated for 3 d. We analyzed the fatty acid composition, the mRNA expression of casein and fatty acid synthesis-related genes, and the phosphorylation of casein synthesis-related proteins of MAC-T cells by gas chromatography, quantitative polymerase chain reaction, and western blotting, respectively. Results: In MAC-T cells, ATRA increased the mRNA levels of α_S1-casein and β-casein, janus kinase 2 (JAK2) and E74-like factor 5 of the signal transducer and activator of transcription 5 β (STAT5-β) pathway, ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 of the mammalian target of rapamycin (mTOR) pathway, inhibited the mRNA expression of phosphoinositide 3-kinase and eukaryotic initiation factor 4E of the mTOR pathway, and promoted the phosphorylation of STAT5-β and S6K1 proteins. Additionally, ATRA increased the de novo synthesis of fatty acids, reduced the content of long-chain fatty acids, the ratio of monounsaturated fatty acids to saturated fatty acids (SFA), the ratio of polyunsaturated fatty acids (PUFA) to SFA, and the ratio of ω-6 to ω-3 PUFA. The mRNA levels of acetyl-CoA carboxylase 1, fatty acid synthase, lipoprotein lipase, stearoyl-CoA desaturase, peroxisome proliferator-activated receptor gamma, and sterol regulatory element-binding protein 1 (SREBP1) were enhanced by ATRA. Conclusion: ATRA promotes the synthesis of casein by regulating JAK2/STAT5 pathway and downstream mTOR signaling pathway, and it improves the fatty acid composition of MAC-T cells by regulating SREBP1-related genes.

• Korean Journal of Food Science and Technology
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• v.49 no.2
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• pp.192-202
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• 2017

In this study, factors involved in lipid and energy metabolism following treatment with ethanolic extract of the Polygonatum sibiricum rhizome (ID1216) were evaluated in high-fat diet-induced obese mice. ID1216-treated mice showed a significant reduction in weight gain compared to non-treated mice. ID1216 treatment increased the protein levels of AMP-dependent protein kinase, sirtuin1, peroxisome proliferator-activated receptor ${\gamma}$ coactivator 1-${\alpha}$ ($PGC1{\alpha}$), peroxisome proliferator-activated receptor ${\alpha}$ ($PPAR{\alpha}$) and uncoupling proteins in the adipose tissue, liver and muscle compared to vehicle treatment. Analysis of downstream signals of the sirtuin1 $PGC1{\alpha}$-$PPAR{\alpha}$ pathway showed that ID1216 regulates the expression of ${\beta}$-oxidation related genes such as acyl-CoA oxidase, carnitine palmitoyltransferase1, acyl-CoA dehydrogenase and adipocyte protein 2. In addition, ID1216 increased the expression of adipose triglyceride lipase. These results suggest that ID1216 has anti-obesity effects by regulating the genes involved thermogenesis, ${\beta}$-oxidation and lipolysis in a diet-induced obesity model.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.301-310
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• 2010

Bacillus subtilis strains produce a broad spectrum of bioactive peptides. The lipopeptide surfactin belongs to one well-known class, which includes amphiphilic membrane-active biosurfactants and peptide antibiotics. Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Bs-M49, obtained by means of low-energy ion implantation in wild-type Bs-916, produced significantly lower levels of surfactin, and had no obvious effects against R. solani. Occasionally, we found strain Bs-M49 decreased spore formation and the development of competence. Blast comparison of the sequences from Bs-916 and M49 indicate that there is no difference in the srfA operon promoter PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild-type Bs-916 and mutant M49 strains were significantly different. When we integrated the comA ORF into the chromosome of M49 at the amyE locus, M49 restored hemolytic activity and antifungal activity. Then, HPLC analyses results also showed the comA-complemented strain had a similar ability to produce surf actin with wild-type strain Bs-916. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis. ComA protein 3D structure prediction and motif search prediction indicated that ComA has two obvious motifs common to response regulator proteins, which are the N-terminal response regulator receiver motif and the C-terminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural prediction results implicate that three mutated residues in the ComA protein may play an important role in the formation of a salt-bridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.20-27
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• 1994

An inducible ${\beta}$-lactamase gene (bla) was identified and isolated from the chromosomal DNA of multiple drug resistant strains of Staphylococcus aureus. Determined base sequence of bla and of its flanking region was compared with those of bla genes identified on the staphylococcal plasmids pPC1, pI258, pI1071, and pUB101. Base sequence of 843 base-long structural gene of our bla was same as that of pPCl-, pI258-, and pS1-bla. However, HindIII recognition site Which is found in most of the bla genes at 140 base upstream from the structural gene was moved to the site of 370 base upstream from the structural gene. And one of the two direct repeat sequence found in downstream flanking region of pI1071-bla was deleted in our bla. Amino acid sequence homology analysis of the ORF located around HindIII recognition site reveals that this 80 amino acids-long polypeptide is C-terminus of transposase of Tn4001.

• Journal of fish pathology
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• v.27 no.1
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• pp.1-9
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• 2014

The olive flounder, Paralichthys olivaceus is susceptible to viral hemorrhagic septicaemia virus (VHSV) at $15^{\circ}C$ but no mortality at $20^{\circ}C$ even though the virus can grow well in vitro at $20^{\circ}C$. Thus, we designed an experiment to know immune response of olive flounder against VHSV when the host reared at $15^{\circ}C$ or $20^{\circ}C$. cDNA microarray analysis was performed to compare the gene expression patterns of the kidney cells between the host reared at $15^{\circ}C$ or $20^{\circ}C$. The expression of MHC class I, IL-8, myeloperoxidae and endonuclease G-like having function for the antigen presentation and chemokine-factor were up-regulted both the $15^{\circ}C$ and $20^{\circ}C$ during VHSV infection. MHC class II gene existing on antigen-presenting cells and B cell lymphocytes, immunoglobulin (Ig) genes and phagocytosis related genes were down-regulated at $15^{\circ}C$ but highly expressed at $20^{\circ}C$. It can be thought that innate immune related antigen presentation by MHC class I and phagocytosis reaction against VHSV are efficiently occur both the temperature but macrophage or B cell related antigen presentation via MHC class II fails to induce downstream immune reactions (adaptive immunity) to make antibody, and it can be one of the reason that causes high mortality only at $15^{\circ}C$.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.12
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• pp.6155-6157
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• 2012

Background: Mutations in the MAPK (Mitogen Activated Protein Kinase) signaling pathway - EGFR/Ras/RAF/MEK have been associated with the development of several carcinomas. ERK2, a downstream target of the MAPK pathway and a founding member of the MAPK family is activated by cellular signals emanating at the cell membrane. Activated ERK2 translocates into the nucleus to transactivate genes that promote cell proliferation. MKP - a dual specific phosphatase - interacts with activated ERK2 via the common docking (CD) domain of the later to inactivate (dephosphorylate) and effectively terminate further cell proliferation. A constitutively active form of ERK2 carrying a single point mutation - E322K in its CD domain, was earlier reported by our laboratory. In the present study, we investigated the prevalence of this CD domain E322K mutation in 88 well differentiated OSCC tissue samples. Materials and Method: Genomic DNA specimens isolated from 88 oral squamous cell carcinoma tissue samples were amplified with primers flanking the CD domain of the ERK2 gene. Subsequently, PCR amplicons were gel purified and subjected to direct sequencing to screen for mutations. Results: Direct sequencing of eighty eight OSCC samples identified an E322K CD domain mutation in only one (1.1%) OSCC sample. Conclusions: Our result indicates that mutation in the CD domain of ERK2 is rare in OSCC patients, which suggests the role of genetic alterations in other mitogenic genes in the development of carcinoma in the rest of the patients. Nevertheless, the finding is clinically significant, as the relatively rare prevalence of the E322K mutation in OSCC suggests that ERK2, being a common end point signal in the multi-hierarchical mitogen activated signaling pathway may be explored as a viable drug target in the treatment of OSCC.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6261-6265
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• 2013

Background: Recent studies have suggested that expression of the RAS protein activator like-1 gene (RASAL1) is decreased in gastric carcinoma tissues and cell lines, indicated a role in tumorigenesis and development of gastric cancer. Reduced expression of RASAL1 could result in aberrant increase of activity of RAS signaling pathways in cancer cells. However, the exact mechanism which induces down-regulation of the RASAL1 gene remains unclear. This study aimed to determine the methylation status and regulation of RASAL1 in gastric cancer. Materials and Methods: Using the methylation-specific polymerase chain reaction (MSP), the methylation status of CpG islands in the RASAL1 promoter in gastric cancers and paired adjacent non-cancerous tissues from 40 patients was assessed and its clinicopathological significance was analyzed. The methylation status of RASAL1 in gastric cancer lines MKN-28, SGC-790l, BGC-823, as well as in normal gastric epithelial cell line GES-l was also determined after treatment with a DNA methyltransferase inhibitor, 5-aza-2'-doexycytidine (5-Aza-CdR). RAS activity (GAS-GTP) was assessed through a pull-down method, while protein levels of ERK1/2, a downstream molecule of RAS signaling pathways, were determined by Western blotting. Results: The frequencies of RASAL1 promoter methylation in gastric cancer and paired adjacent non-cancerous tissues were 70% (28/40) and 30% (12/40) respectively (P<0.05). There were significantly correlations between RASAL1 promoter methylation with tumor differentiation, tumor size, invasive depth and lymph node metastasis in patients with gastric cancer (all P<0.05), but no correlation was found for age or gender. Promoter hypermethylation of the RASAL1 gene was detected in MKN-28, SGC-790l and BGC-823 cancer cells, but not in the normal gastric epithelial cell line GES-1. Elevated expression of the RASAL1 protein, a decreased RAS-GTP and p-ERK1/2 protein were detected in three gastric cancer cell lines after treatment with 5-Aza-CdR. Conclusions: Aberrant hypermethylation of the RASAL1 gene promoter frequently occurs in gastric cancer tissues and cells. In addition, the demethylating agent 5-Aza-CdR can reverse the hypermethylation of RASAL1 gene and up-regulate the expression of RASAL1 significantly in gastric cancer cells in vivo. Our study suggests that RASAL1 promoter methylation may have a certain relationship with the reduced RASAL1 expression in gastric cancer.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.159-159
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• 2017

Drought, a common environmental constraint, induces a range of physiological, biochemical and molecular changes in plants, and can cause severe reductions in crop yield. Consequently, understanding the molecular mechanisms of drought tolerance is an important step towards crop biotechnology. Here, we report that the rice (Oryza sativa) homeodomain-leucine zipper class IV transcription factor gene, ${\underline{R}ice}$ ${\underline{o}utermost}$ ${\underline{c}ell-specific}$ gene 10 (Roc10), enhances drought tolerance and grain yield by increasing lignin accumulation in ground tissues. Overexpression of Roc10 in rice significantly increased drought tolerance at the vegetative stages of growth and promoted both more effective photosynthesis and a reduction in water loss rate, compared with non-transgenic controls or RNAi transgenic plants. Importantly, Roc10 overexpressing plants had a higher drought tolerance at the reproductive stage of growth and a higher grain yield compared with the controls under field-drought conditions. Roc10 is mainly expressed in outer cell layers including the epidermis and the vasculature of the shoots, which coincides with areas of cell wall lignification. Roc10 overexpression elevated the expression levels of lignin biosynthetic genes in shoots, with a concomitant increase in the accumulation of lignin, while the overexpression and RNAi lines showed opposite patterns of lignin accumulation. We identified downstream target genes of Roc10 by performing RNA-seq and chromatin immunoprecipitation (ChIP)-seq analyses of shoot tissues. Roc10 was found to directly bind to the promoter of PEROXIDASEN/PEROXIDASE38, a key gene in lignin biosynthesis. Together, our findings suggest that Roc10 confers drought stress tolerance by promoting lignin biosynthesis in ground tissues.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.13
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• pp.5311-5316
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• 2014

Nuclear factor erythroid 2-related factor 2 (Nrf2) has been recognized as a transcription factor that controls mechanisms of cellular defense response by regulation of three classes of genes, including endogenous antioxidants, phase II detoxifying enzymes and transporters. Previous studies have revealed roles of Nrf2 in resistance to chemotherapeutic agents and high level expression of Nrf2 has been found in many types of cancer. At physiological concentrations, luteolin as a flavonoid compound can inhibit Nrf2 and sensitize cancer cells to chemotherapeutic agents. We reported luteolin loaded in phytosomes as an advanced nanoparticle carrier sensitized MDA-MB 231 cells to doxorubicin. In this study, we prepared nano phytosomes of luteolin to enhance the bioavailability of luteolin and improve passive targeting in breast cancer cells. Our results showed that cotreatment of cells with nano particles containing luteolin and doxorubicin resulted in the highest percentage cell death in MDA-MB 231cells (p<0.05). Furthermore, luteolin-loaded nanoparticles reduced Nrf2 gene expression at the mRNA level in cells to a greater extent than luteolin alone (p<0.05). Similarly, expression of downstream genes for Nrf2 including Ho1 and MDR1 were reduced significantly (p<0.05). Inhibition of Nrf-2 expression caused a marked increase in cancer cell death (p<0.05). Taken together, these results suggest that phytosome technology can improve the efficacy of chemotherapy by overcoming resistance and enhancing permeability of cancer cells to chemical agents and may thus be considered as a potential delivery system to improve therapeutic protocols for cancer patients.