• Biomolecules & Therapeutics
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• 제26권3호
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• pp.313-321
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• 2018

Anti-cancer drug resistance is a major problem in colorectal cancer (CRC) research. Although several studies have revealed the mechanism of cancer drug resistance, molecular targets for chemotherapeutic combinations remain elusive. To address this issue, we focused on the expression of cellular prion protein ($PrP^C$) in 5-FU-resistant CRC cells. In 5-FU-resistant CRC cells, $PrP^C$ expression is significantly increased, compared with that in normal CRC cells. In the presence of 5-FU, $PrP^C$ increased CRC cell survival and proliferation by maintaining the activation of the PI3K-Akt signaling pathway and the expression of cell cycle-associated proteins, including cyclin E, CDK2, cyclin D1, and CDK4. In addition, $PrP^C$ inhibited the activation of the stress-associated proteins p38, JNK, and p53. Moreover, after treatment of 5-FU-resistant CRC cells with 5-FU, silencing of $PrP^C$ triggered apoptosis via the activation of caspase-3. These results indicate that $PrP^C$ plays a key role in CRC drug resistance. The novel strategy of combining chemotherapy with $PrP^C$ targeting may yield efficacious treatments of colorectal cancer.

• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.484-490
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• 2005

Human ribosomal protein S3 (rpS3), which has a DNA repair endonuclease activity, is a multifunctional protein. This protein is involved in DNA repair, translation, and apoptosis. In particular, rpS3 has a lyase activity, which cleaves the phosphodiester bond of damaged sites such as cyclobutane pyrimidine dimers and AP sites. Here, using deletion analysis, we identified that the repair endonuclease domain resides in the C-terminal region (165-243 aa) of rpS3. We also found that ectopic expression of GST-rpS3 in bacterial strain BL21 promoted the resistance of these cells to ultraviolet (UV) radiation and hydrogen peroxide ($H_{2}O_{2}$) treatment. The repair domain of rpS3 was sufficient to exhibit the resistance to UV irradiation and recover cell growth and viability, showing that the repair activity of rpS3 is responsible for the resistance to UV irradiation. Our study suggests that rpS3 is able to process DNA damage in bacteria via its repair domain, showing the resistance to genotoxic stress. This implies that rpS3-like activity could be operative in bacteria.

• Journal of Yeungnam Medical Science
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• 제14권1호
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• pp.67-76
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• 1997

항암제에 대한 내성은 내인성 또는 획득한 내성 모두가 암의 치료에 장애가 된다. P-당단백질을 encode하고있는 mdr1 유전자의 발현이 항암제에 대해 내성을 가지고 있는 암세포에서 많이 관찰되고 있으며, 최근에는 시험관적으로 항암제에 대한 내성이 유도된 암세포주들에서 mdr1 유전자가 발현되지 않는 암세포들이 보고되고 있다. 다제내성에 관계하는 또 하나의 유전자인 MRP 발현정도를 L1210세포와 내성인 L1210변이주들에서 조사하였으며, c-myc과 c-fos 유전자의 발현변화를 관찰하였다. RT-PCR을 시행하여 L1210, L1210AdR, L1210VcR에서 MRP 유전자발현을 확인하였으며, Northern hybridization한 결과 L1210세포에 비하여 L1210AdR은 유전자 발현이 40% 정도 감소하였으며, L12l0Cis는 90% 정도의 유전자 발현감소가 관찰되었다. c-myc과 c-fos유전자의 Northern hybridization한 결과 L1210에 비하여 L1210AdR은 발현감소가 나타났으나, L1210VcR과 L1210Cis의 경우는 오히려 발현증가가 관찰되었다.

• Animal cells and systems
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• 제7권3호
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• pp.213-219
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• 2003

The response of plants to pathogens and wounding is dependent upon very sensitive perception mechanisms. Although genetic approaches have revealed a variety of resistance genes that activate common defense responses, defense-related proteins are not well characterized in plants. Therefore, we used a proteomic approach to determine which defense-related proteins are induced by salicylic acid (SA) and wounding in Chinese cabbage. We found that SA and wounding induce pathogenesis-related protein 1a (PR1a) at both protein and mRNA levels using proteomics and Northern blot analysis, respectively. This indicates that our proteomic approach is useful for identifying defense-related proteins. We also identified several other proteins that are induced by SA or wounding. Among the seven SA-induced proteins identified, four may be defense-related, including defense-related protein, phospholipase D (PLD), resistance protein RPS2 homolog, and L-ascorbate peroxidase. Out of the six wounding-induced proteins identified, three may be defense-related: heat shock cognate protein 70 (HSC70), polygalacturonase, and peroxidase P7. The precise functions of these proteins in plant defense responses await further study. However, identification of the defense-related proteins described in this study should allow us to better understand the mechanisms and signal transduction pathways involved in defense responses in Chinese cabbage.

• Genomics & Informatics
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• 제21권1호
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• pp.5.1-5.13
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• 2023

Neisseria gonorrhoeae is a Gram-negative aerobic diplococcus bacterium that primarily causes sexually transmitted infections through direct human sexual contact. It is a major public health threat due to its impact on reproductive health, the widespread presence of antimicrobial resistance, and the lack of a vaccine. In this study, we used a bioinformatics approach and performed subtractive genomic methods to identify potential drug targets against the core proteome of N. gonorrhoeae (12 strains). In total, 12,300 protein sequences were retrieved, and paralogous proteins were removed using CD-HIT. The remaining sequences were analyzed for non-homology against the human proteome and gut microbiota, and screened for broad-spectrum analysis, druggability, and anti-target analysis. The proteins were also characterized for unique interactions between the host and pathogen through metabolic pathway analysis. Based on the subtractive genomic approach and subcellular localization, we identified one cytoplasmic protein, 2Fe-2S iron-sulfur cluster binding domain-containing protein (NGFG RS03485), as a potential drug target. This protein could be further exploited for drug development to create new medications and therapeutic agents for the treatment of N. gonorrhoeae infections.

• Asian Pacific Journal of Cancer Prevention
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• 제13권11호
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• pp.5619-5625
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• 2012

Gastric carcinoma is a leading cause of cancer death in the world and multi-drug resistance (MDR) is an essential aspect of gastric carcinoma chemotherapy failure. Recent studies have shown that integrin-linked kinase (ILK) is involved in metastasis of human tumors, expression silencing of ILK inhibiting the metastasis of several types of cultured human cancer cells. However, the role and potential mechanism of ILK to reverse the multi-drug resistance in human gastric carcinoma is not fully clear. In this report, we focused on roles of expression silencing of ILK in multi-drug resistance reversal of human gastric carcinoma SGC7901/DDP cells, including increased drug sensitivity to cisplatin, cell apoptosis rates, and intracellular accumulation of Rhodamine-123, and decreased mRNA and protein expression of multi-drug resistance gene (MDR1), multi-drug resistance-associated protein (MRP1), excision repair cross-complementing gene 1 (ERCC1), glutathione S-transferase -${\pi}$ (GST-${\pi}$) and RhoE, and transcriptional activation of AP-1 and NF-${\kappa}B$ in ILK silenced SGC7901/DDP cells. We also found that there was a decreased level of p-Akt and p-ERK. The results indicated that ILK might be used as a potential therapeutic strategy to combat multi-drug resistance through blocking PI3K-Akt and MAPK-ERK pathways in human gastric carcinoma.

• 생명과학회지
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• 제17권2호통권82호
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• pp.235-240
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• 2007

본 연구는 Affymetrix Arabidopsis DNA chip을 이용하여 비 병원성 인자인 AvrRpt2 단백질에 의해서 특이적으로 전사 과정이 조절되는 애기장대 유전자들을 분리하고 병 저항성 방어체계와 관련한 이들 유전자들의 기능 분석을 시도하였다. 그 중에서 먼저 식물 호르몬인 ethylene의 신호 조절에 관여하는 ERFs (ethylene-responsive element binding factors) 전사조절 유전자 family 중에서 Bla subfamily 그룹으로 알려져 있는 AtERF11 유전자의 병 저항성 관련 기능을 규명하였다. 저항성 유전자 RPS2가 없는 경우에는 비 병원성 인자인 AvrRpt2 단백질은 기주 식물체내의 기초 병저항성을 감소시키고 병원성 세균의 증식을 향상시켜서 병증을 증대시키는 effector로 작용한다는 기존의 연구결과와 유사하게, 저항성 유전자 RPS2가 없는 조건에서 AtERF11 유전자의 발현이 AvrRpt2 단백질의 작용에 의해서 특이적으로 감소되는 것을 확인하였다. 이러한 결과를 바탕으로 AtERF11 유전자는 식물체의 병 저항성 방어기작에 있어서 positive regulator로서 작용하기 때문에 effector로 작용하는 AvrRpt2 단백질에 의해서 조절되는 것으로 추측하였다. 본 가설을 증명하기 위해 AtERF11의 발현을 증폭시킨 애기장대 형질전화체를 제작하고 P. syringae pv. tomato DC 3000에 대한 병저항성을 실험하였다. AtERF11 유전자가 대량 발현하는 형질전화 된 애기장대에서는 야생종에 비해 대략 100배 이상 세균의 증식이 억제되는 강력한 병저항성을 가진다는 것을 검증하였다.

• Asian Pacific Journal of Cancer Prevention
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• 제17권2호
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• pp.581-590
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• 2016

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

• The Plant Pathology Journal
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• 제37권6호
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• pp.555-565
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• 2021

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

• Journal of Microbiology and Biotechnology
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• 제32권11호
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• pp.1471-1478
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• 2022

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (Kan^R) should be dependent on FucT2 solubility. Kan^R was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.