Increased Serotonin Signaling Contributes to the Warburg Effect in Pancreatic Tumor Cells Under Metabolic Stress and Promotes Growth of Pancreatic Tumors in Mice

Jiang, Shu-Heng,Li, Jun,Dong, Fang-Yuan,Yang, Jian-Yu,Liu, De-Jun,Yang, Xiao-Mei,Wang, Ya-Hui,Yang, Min-Wei,Fu, Xue-Liang,Zhang, Xiao-Xin,Li, Qing,Pang, Xiu-Feng,Huo, Yan-Miao,Li, Jiao,Zhang, Jun-Feng Elsevier 2017 Gastroenterology Vol.153 No.1

<P><B>Background & Aims</B></P> <P>Desmoplasia and poor vascularity cause severe metabolic stress in pancreatic ductal adenocarcinomas (PDACs). Serotonin (5-HT) is a neuromodulator with neurotransmitter and neuroendocrine functions that contributes to tumorigenesis. We investigated the role of 5-HT signaling in the growth of pancreatic tumors.</P> <P><B>Methods</B></P> <P>We measured the levels of proteins that regulate 5-HT synthesis, packaging, and degradation in pancreata from Kras<SUP>G12D/+</SUP>/Trp53<SUP>R172H/+</SUP>/Pdx1-Cre (KPC) mice, which develop pancreatic tumors, as well as in PDAC cell lines and a tissue microarray containing 81 human PDAC samples. We also analyzed expression levels of proteins involved in 5-HT synthesis and degradation by immunohistochemical analysis of a tissue microarray containing 311 PDAC specimens, and associated expression levels with patient survival times. 5-HT level in 14 matched PDAC tumor and non-tumor tissues were analyzed by ELISA. PDAC cell lines were incubated with 5-HT and cell survival and apoptosis were measured. We analyzed expression of the 5-HT receptor HTR2B in PDAC cells and effects of receptor agonists and antagonists, as well as HTR2B knockdown with small hairpin RNAs. We determined the effects of 5-HT stimulation on gene expression profiles of BxPC-3 cells. Regulation of glycolysis by 5-HT signaling via HTR2B was assessed by immunofluorescence and immunoprecipitation analyses, as well as by determination of the extracellular acid ratio, glucose consumption, and lactate production. Primary PDACs, with or without exposure to SB204741 (a selective antagonist of HTR2B), were grown as xenograft tumors in mice, and SB204741 was administered to tumor-bearing KPC mice; tumor growth and metabolism were measured by imaging analyses.</P> <P><B>Results</B></P> <P>In immunohistochemical analysis of a tissue microarray of PDAC specimens, increased levels of TPH1 and decreased level of MAOA, which regulate 5-HT synthesis and degradation, correlated with stage and size of PDACs and shorter patient survival time. We found levels of 5-HT to be increased in human PDAC tissues compared with non-tumor pancreatic tissues, and PDAC cell lines compared with non-transformed pancreatic cells. Incubation of PDAC cell lines with 5-HT increased proliferation and prevented apoptosis. Agonists of HTR2B, but not other 5-HT receptors, promoted proliferation and prevented apoptosis of PDAC cells. Knockdown of HTR2B in PDAC cells, or incubation of cells with HTR2B inhibitors, reduced their growth as xenograft tumors in mice. We observed a correlation between 5-HT and glycolytic flux in PDAC cells; levels of metabolic enzymes involved in glycolysis, the phosphate pentose pathway, and hexosamine biosynthesis pathway increased significantly in PDAC cells following 5-HT stimulation. 5-HT stimulation led to formation of the HTR2B–LYN–p85 complex, which increased PI3K–Akt–mTOR signaling and the Warburg effect by increasing protein levels of MYC and HIF1A. Administration of SB204741 to KPC mice slowed growth and metabolism of established pancreatic tumors and prolonged survival of the mice.</P> <P><B>Conclusions</B></P> <P>Human PDACs have increased levels of 5-HT, and PDAC cells increase expression of its receptor, HTR2B. These increases allow for tumor glycolysis under metabolic stress and promote growth of pancreatic tumors and PDAC xenograft tumors in mice.</P>

실험적 중증근무력증(EAMG) 실험동물 및 중증근무력증 환자에서의 CD5 양성 B-림프구 발현에 관한 비교연구

이광우,김지수,마사카수모토뮤라 대한신경과학회 1998 대한신경과학회지 Vol.16 No.3

miR-458b-5p regulates ovarian granulosa cells proliferation through Wnt/β‐catenin signaling pathway by targeting catenin beta-1

Wang Wenwen,Teng Jun,Han Xu,Zhang Shen,Zhang Qin,Tang Hui 아세아·태평양축산학회 2021 Animal Bioscience Vol.34 No.6

Objective: Ovarian follicular development, which dependent on the proliferation and differentiation of granulosa cells (GCs), is a complex biological process in which miRNA plays an important role. Our previous study showed that miR-458b-5p is associated with ovarian follicular development in chicken. The detailed function and molecular mechanism of miR-458b-5p in GCs is unclear. Methods: The luciferase reporter assay was used to verify the targeting relationship between miR-458b-5p and catenin beta-1 (CTNNB1), which is an important transcriptional regulatory factor of the Wnt/β-catenin pathway. The cell counting kit-8 (CCK-8) assay, flow cytometry with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) labeling were applied to explore the effect of miR-458b-5p on proliferation, cell cycle and apoptosis of chicken GCs. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels. Results: We demonstrated that the expression of miR-458b-5p and CTNNB1 showed the opposite relationship in GCs and theca cells of hierarchical follicles. The luciferase reporter assay confirmed that CTNNB1 is the direct target of miR-458b-5p. Using CCK-8 assay and flow cytometry with PI and Annexin V-FITC labeling, we observed that transfection with the miR-458b-5p mimics significantly reduced proliferation and has no effects on apoptosis of chicken GCs. In addition, miR-458b-5p decreased the mRNA and protein expression of CD44 molecule and matrix metallopeptidase 7, which are the downstream effectors of CTNNB1 in Wnt/β-Catenin pathway and play functional roles in cell proliferation. Conclusion: Taken together, the data indicate that miR-458b-5p regulates ovarian GCs proliferation through Wnt/β-catenin signaling pathway by targeting CTNNB1, suggesting that miR-458b-5p and its target gene CTNNB1 may potentially play a role in chicken ovarian follicular development. Objective: Ovarian follicular development, which dependent on the proliferation and differentiation of granulosa cells (GCs), is a complex biological process in which miRNA plays an important role. Our previous study showed that miR-458b-5p is associated with ovarian follicular development in chicken. The detailed function and molecular mechanism of miR-458b-5p in GCs is unclear.Methods: The luciferase reporter assay was used to verify the targeting relationship between miR-458b-5p and catenin beta-1 (<i><i>CTNNB1</i></i>), which is an important transcriptional regulatory factor of the Wnt/β-catenin pathway. The cell counting kit-8 (CCK-8) assay, flow cytometry with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) labeling were applied to explore the effect of miR-458b-5p on proliferation, cell cycle and apoptosis of chicken GCs. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels.Results: We demonstrated that the expression of miR-458b-5p and <i>CTNNB1</i> showed the opposite relationship in GCs and theca cells of hierarchical follicles. The luciferase reporter assay confirmed that <i>CTNNB1</i> is the direct target of miR-458b-5p. Using CCK-8 assay and flow cytometry with PI and Annexin V-FITC labeling, we observed that transfection with the miR-458b-5p mimics significantly reduced proliferation and has no effects on apoptosis of chicken GCs. In addition, miR-458b-5p decreased the mRNA and protein expression of CD44 molecule and matrix metallopeptidase 7, which are the downstream effectors of <i>CTNNB1</i> in Wnt/β-Catenin pathway and play functional roles in cell proliferation.Conclusion: Taken together, the data indicate that miR-458b-5p regulates ovarian GCs proliferation through Wnt/β-catenin signaling pathway by targeting <i>CTNNB1</i>, suggesting that miR-458b-5p and its target gene <i>CTNNB1</i> may potentially play a role in chicken ovarian follicular development.

단백질합성인자 eIF5B의 저 발현 효모벡터의 제조 및 특성

최상기,송진희,이준행,이병욱,성치남 한국미생물학회 2004 미생물학회지 Vol.40 No.1

eIF5B는 단백질합성의 개시 인자로서 Met-$tRNA^{Met}$을 AUG 개시코돈에 전달하고, 리보솜의 두 소단위체 결합을 유도한다. 이 인자의 기능을 연구할 목적으로 eIF5B를 코딩하는 FUN12 유전자의 5'말단 부위를 삭제하는 연구를 수행하였다. 프로모터의 대부분을 삭제한 FUN12를 함유한 pRS효모벡터를 FUN12가 삭제되어 천천히 자라는 돌연변이주 ($fun12{\Delta}g$)에 전달하였을 때 그 표현형을 부분적으로 상보하였다. 위와 같이 제조된 벡터에서 N-말단이 상실된 eIF5B 단백질이 발현되었고, 그 양이 정상 균주에서 발현되는 eIF5B 양의 약 5%에 불과하였다. 이와 같이 부분적으로 성장을 상보한 균주에서 발현된 적은 양의 단백질합성개시 인자 eIF5B는 직접적으로 그 성장을 제한하는 요소로 작용한다. 이러한 균주에서 성장의 제한인자인 eIF5B는 in vitro 에서도 역시 전체 단백질 합성의 활성을 조절하였다. eIF5B is a translation initiation factor that delivers Met-$tRNA^{Met}$ to AUG start codon and subsequently joins the small and large ribosomes. In order to study the function of eIF5B encoded by FUN12, we constructed FUN12 which lacked 5' end of its sequence. We found that this construct lacking almost all of its promoter in pRS plasmid partially complemented slow growth phenotype of fun12 deletion strain. Interestingly, this construct expressed N-terminally truncated eIF5B and its expression level was about 5% of that of wild type eIF5B. Low amount of the eIF5B expressed additionally in fun12 deletion strain played a direct role as a limiting factor for its growth. This limiting factor eIF5B in those strains also modulates activities of overall translation in vitro.

Zebrafish에서 human cytochrome b<SUB>5</SUB>의 발현

한세미(Se Mi Han),유민(Min Yoo) 한국생명과학회 2017 생명과학회지 Vol.27 No.6

본 연구에서는 zebrafish에 사람의 cyt b5 유전자를 microinjection하여 발현시키고, 그 결과를 형광으로 확인하였다. HeLa cell에서 RT-PCR을 진행한 결과 414 bp의 cyt b5 band가 증폭되었다. 염기서열 분석으로 재확인된 cyt b5 insert를 pEGFP-N3의 형광 vector에 클로닝하였고, 이렇게 준비된 pEGFP-N3-cyt b5 plasmid DNA를 1세 포기의 수정란에 microinjection하였다. cyt b5를 microinjection한 치어를 형광현미경으로 관찰한 결과 대조군 치어보다 훨씬 선명한 형광을 띠는 것이 확인되었다. 최종적으로 치어에서 RNA를 분리하여 RT-PCR하였고 전기영동과 DNA sequencing으로 fusion 단백질의 발현을 재확인하였다. Cyt b5의 발현으로 인해 zebrafish의 생존율이 다소 떨어지는 것으로 확인되었기에 독성 문제를 해결하기 위한 연구가 계속 필요할 것으로 사료된다. 이 연구는 향후 cyt b5가 결핍되었을 경우 발생할 수 있는 여러 질병들을 유전자 차원에서 치료하고, 유용 유전자 클로닝을 위한 기술 개발에 발판이 될 수 있을 것으로 기대된다. In this study, we sought to develop an effective cloning system by which human cytochrome b5 (cyt b5) is introduced and expressed in zebrafish. First, the 414 bp human cyt b5 gene was amplified from RNA extracts of HeLa cells using RT-PCR, and the amplicon was subsequently sequenced to confirm that it was intact. Next, cyt b5 was cloned into the pEGFP-N3 vector, which also encodes a fluorescent gene. One-cell stage zebrafish embryos were microinjected with the recombinant vector containing the cyt b5 gene. Fluorescence microscopy confirmed high expression of the fluorescent gene in the injected fry compared to the non-fluorescent control fry. Finally, we extracted RNA from the injected fry and performed RT-PCR to determine whether the human cyt b5 gene is expressed in the transgenic zebrafish. Sequencing analysis further confirmed that the cloned human cyt b5 gene was intact. The transgenic zebrafish model produced in this study will be a useful tool to study therapeutic approaches to cure various diseases related to the deficiency of functional human cyt b5 as well as tools for cloning useful genes in fish.

국내 감염성질환 병원체의 성상분석을 통한 표준실험실 체계 구축(Ⅱ) : 장내바이러스와 B형간염바이러스 국내 분리주의 성상분석(1)

윤재득,지영미,김기순,천두성,안정배,정윤석,신수연,이선화,이지원,조해월 국립보건연구원 2000 국립보건원보 Vol.37 No.-

고속액체크로마토그래피를 이용한 비타민 B<SUB>5</SUB> 및 B<SUB>6</SUB>의 정량 분석

김기쁨(Gi-Ppeum Kim),황영선(Young-Sun Hwang),정명근(Myoung-Gun Choung) 한국식품영양과학회 2017 한국식품영양과학회지 Vol.46 No.10

식품 함유 비타민 B<SUP>5</SUP> 및 B<SUP>6</SUP>의 최적 HPLC 분석 조건을 검토한 결과 비타민 B<SUP>5</SUP>의 경우 YMC-Pack ODS-AM(250×4.6 mm I.D.) 칼럼을 이용하고, A용매로 50 mM KH₂PO₄(pH 3.5)을, B용매는 아세토니트릴을 이동상 용매로 사용하는 A용매 95% 등용매용리 조건에서 200 nm의 파장으로 분석하는 HPLC/DAD법을 최적조건으로 확립하였다. 한편 비타민 B6의 최적분석조건은 여기파장(excitation) 290 nm, 방출파장(emission) 396 nm로 분석하는 HPLC/FLD법으로써, 칼럼은 YMC-Pack Pro RS C18(250×4.6 mm I.D.), 이 동상 용매는 A용매 20 mM CH₃CO₂Na(pH 3.6), B용매 아세토니트릴을 A용매 97% 등용매용리 조건으로 사용하였다. 비타민 B5 및 B6의 표준검량선은 R<SUP>2</SUP>값이 각각 0.9998 및 0.9999로 고도의 직선성을 나타내었고, 검출한계 및 정량한계는 비타민 B5의 경우 각각 0.4 mg/L 및 1.3 mg/L, 비타민 B6의 경우 각각 0.006 mg/L 및 0.02 mg/L로 산출되었다. Recently, many people have demanded reliable nutritional data even for minor-components. On the other hand, an analytical method for the analyses of vitamin B5 and B6 is lacking. Therefore, this study attempted to validate with accuracy and precision the analysis of vitamin B5 and B6 using a high-performance liquid chromatography (HPLC) method. The vitamin B5 and B6 contents were analyzed using an Agilent 1260 series HPLC system. YMC-Pack ODS-AM (250×4.6 mm I.D.) and YMC-Pack Pro RS C18 (250×4.6 mm I.D.) columns were used for the analyses of vitamin B5 and B6, respectively. In the case of vitamin B5, the flow rate was set to 1.0 mL/min by isocratic elution using the 50 mM KH2PO4 solution (pH 3.5)/acetonitrile (ACN) (95:5, v/v) with monitoring at 200 nm using HPLC/DAD, whereas the flow rate for vitamin B6 was set to 1.0 mL/min of flow rate by isocratic elution using a 20 mM CH₃CO₂Na solution (pH 3.6)/ACN (97:3, v/v) with monitoring by excitation at 290 nm and emission at 396 nm using HPLC/FLD. The column temperature was set to 30°C. The injection volume was 20 μL for each experiment. The specificity of the accuracy and precision for vitamin B5 and B6 were also validated by HPLC. The results showed high linearity in the calibration curve for vitamin B5 (R<SUP>2</SUP>=0.9998**), the limit of detection (LOD) and limit of quantitation (LOQ) were 0.4 mg/L and 1.3 mg/L, respectively, In contrast, for the calibration curve of vitamin B6, which showed high linearity (R<SUP>2</SUP>=0.9999**), the LOD and LOQ were 0.006 mg/L and 0.02 mg/L, respectively.

고속액체크로마토그래피를 이용한 비타민 B5 및 B6의 정량 분석

김기쁨,황영선,정명근 한국식품영양과학회 2017 한국식품영양과학회지 Vol.46 No.10

식품 함유 비타민 B5 및 B6의 최적 HPLC 분석 조건을 검토한 결과 비타민 B5의 경우 YMC-Pack ODS-AM(250×4.6 mm I.D.) 칼럼을 이용하고, A용매로 50 mM KH2PO4(pH 3.5)을, B용매는 아세토니트릴을 이동상 용매로 사용하는 A용매 95% 등용매용리 조건에서 200 nm의 파장으로 분석하는 HPLC/DAD법을 최적조건으로 확립하였다. 한편 비타민 B6의 최적분석조건은 여기파장(excitation) 290 nm, 방출파장(emission) 396 nm로 분석하는 HPLC/FLD법으로써, 칼럼은 YMC-Pack Pro RS C18(250×4.6 mm I.D.), 이동상 용매는 A용매 20 mM CH3CO2Na(pH 3.6), B용매 아세토니트릴을 A용매 97% 등용매용리 조건으로 사용하였다. 비타민 B5 및 B6의 표준검량선은 R2값이 각각 0.9998 및 0.9999로 고도의 직선성을 나타내었고, 검출한계 및 정량한계는 비타민 B5의 경우 각각 0.4 mg/L 및 1.3 mg/L, 비타민 B6의 경우 각각 0.006 mg/L 및 0.02 mg/L로 산출되었다. Recently, many people have demanded reliable nutritional data even for minor-components. On the other hand, an analytical method for the analyses of vitamin B5 and B6 is lacking. Therefore, this study attempted to validate with accuracy and precision the analysis of vitamin B5 and B6 using a high-performance liquid chromatography (HPLC) method. The vitamin B5 and B6 contents were analyzed using an Agilent 1260 series HPLC system. YMC-Pack ODS-AM (250×4.6 mm I.D.) and YMC-Pack Pro RS C18 (250×4.6 mm I.D.) columns were used for the analyses of vitamin B5 and B6, respectively. In the case of vitamin B5, the flow rate was set to 1.0 mL/min by isocratic elution using the 50 mM KH2PO4 solution (pH 3.5)/acetonitrile (ACN) (95:5, v/v) with monitoring at 200 nm using HPLC/DAD, whereas the flow rate for vitamin B6 was set to 1.0 mL/min of flow rate by isocratic elution using a 20 mM CH3CO2Na solution (pH 3.6)/ACN (97:3, v/v) with monitoring by excitation at 290 nm and emission at 396 nm using HPLC/FLD. The column temperature was set to 30°C. The injection volume was 20 μL for each experiment. The specificity of the accuracy and precision for vitamin B5 and B6 were also validated by HPLC. The results showed high linearity in the calibration curve for vitamin B5 (R2=0.9998**), the limit of detection (LOD) and limit of quantitation (LOQ) were 0.4 mg/L and 1.3 mg/L, respectively, In contrast, for the calibration curve of vitamin B6, which showed high linearity (R2=0.9999**), the LOD and LOQ were 0.006 mg/L and 0.02 mg/L, respectively.

Detection of RET (rearranged during transfection) variants and their downstream signal molecules in <i>RET</i> rearranged lung adenocarcinoma patients

Kim, Jeong- Oh,Shin, Jung -Young,Kim, Min Young,Son, Kyoung Hwa,Jung, Chan Kwon,Kim, Tae-Jung,Kim, Su Young,Park, Jae Kil,Sung, Sook Whan,Bae, Sang Ju,Min, Hyun Jung,Kang, Jin- Hyoung Elsevier 2018 Surgical oncology Vol.27 No.1

<P><B>Abstract</B></P> <P><B>Background</B></P> <P>We screened resected tumor tissues from patients with lung cancer for <I>EGFR</I> mutations, <I>ALK</I> rearrangements, and rearranged during transfection (<I>RET</I>) gene variants (including <I>RET</I> rearrangements and the Kinesin Family Member 5B <I>(KIF5B</I>)<I>-RET</I> fusion gene) using various methods including reverse transcription polymerase chain reaction (RT-PCR), transcript assays, fluorescence <I>in situ</I> hybridization (FISH), and immunohistochemistry (IHC). We also examined the protein expression of associated downstream signaling molecules to assess the effect of these variants on patient outcome.</P> <P><B>Method</B></P> <P>We constructed a tissue microarray (TMA) comprising 581 resected tumor tissues from patients with lung adenocarcinoma and analyzed the microarray by both FISH (using <I>RET</I> break-apart and <I>KIF5B-RET</I> SY translocation probes) and a commercial <I>RET</I> transcript assay. We evaluated the expression of RET and RET-related signaling molecules, including p-AKT and p-ERK, by TMA -based IHC staining.</P> <P><B>Results</B></P> <P>Among the 581 specimens, 51 (8.8%) specimens harbored <I>RET</I> rearrangements, including 12 cases (2.1%) carrying a <I>KIF5B-RET</I> fusion gene. Surprisingly, RET expression was lower in <I>KIF5B-RET</I> fusion gene-positive than in <I>RET</I> wild-type specimens. We detected activating <I>EGFR</I> mutations in 11 (21.6%) of the 51 <I>RET</I> variant-positive specimens. Among the <I>KIF5B-RET</I> fusion gene-positive specimens, p-ERK expression was significantly lower in the <I>EGFR</I> mutation subgroup showing RET expression than in the <I>EGFR</I> mutation subgroup that did not express RET. Similarly, the <I>RET</I> rearrangement group showed significant variation in the expression level of p-AKT (P = 0.028) and p-ERK, whose expression remarkably increased in specimens not expressing RET. The expression of p-ERK markedly increased in the <I>RET</I> rearrangement group regardless of RET expression.</P> <P><B>Conclusion</B></P> <P>This result suggests that a combination of RET and ERK inhibitors may be an effective treatment strategy for lung adenocarcinoma patients harboring <I>RET</I> variants.</P> <P><B>Highlights</B></P> <P> <UL> <LI> FISH is clinically applicable as well as RT-PCR for <I>RET</I> variants analysis. </LI> <LI> We detected the <I>EGFR</I> mutation and <I>KIF5B-RET</I> fusion gene in lung cancer. </LI> <LI> Expression of <I>RET-</I>associated signaling molecules varied in <I>RET</I> variant-positive groups. </LI> <LI> The <I>KIF5B-RET</I> fusion gene increased and inhibited p-ERK expression. </LI> <LI> RET and ERK inhibitors may be combined to treat lung adenocarcinoma with <I>RET</I> variants. </LI> </UL> </P>

Crystallization Processes and Magnetic Properties of Nanocomposite Nd_4.5Fe_77-xCrxB_18.5Amorphous Ribbons

Jung, Yun-Chul,Ohmori, Yasuya,Nakai, Kiyomichi,Hirosawa, Satoshi,Kanekiyo, Hirokazu 경상대학교 첨단소재연구소 2001 NANO 기술 심포지엄 Vol.2001 No.1

The effect of Cr addition on the crystallization processes and the magnetic properties of Nd4.5Fe77-xB18.5(x = 1 to 20at%) amorphous ribbons have been investigated by means of DTA, XRD and HRTEM. The crystallization sequences of the amorphous ribbons with 0 to 5% Cr can be expressed as: Am (amorphous)→Fe3B+[Am]→Nd2Fe23B3+[Am+Fe3B]→Nd2Fe14B+[Am+Fe3B+Nd2Fe23B3]→〈-Fe+[Fe3B+Nd2Fe23B3+Nd2Fe14B3+Nd2Fe14B]→[Fe3B+Nd2Fe14B+〈-Fe]→ NdFe4B4+[Fe3B+Nd2Fe23B+〈-Fe]. In the ribbons with Cr above 10%, the Cr1.65Fe0.35B0.96 and the (Cr, Fe)2B phases were formed instead of Nd2Fe23B3 and NdFe4B4, respectively. By the Addition of Cr, the crystallization temperature and the incubation time for the first crystallization increased and the formation of Nd2Fe23B3 was suppressed. With increasing the amounts of Cr content, the coercive force increased. However, the crystallization temperature and the incubation time for the first crystallization decreased in the 1% Cr ribbon.