Prostaglandin F_(2α)synthase promotes oxaliplatin resistance in colorectal cancer through prostaglandin F_(2α)-dependent and F_(2α)-independent mechanism

BACKGROUND Oxaliplatin(Oxa)is the first-line chemotherapy drug for colorectal cancer(CRC),and Oxa resistance is crucial for treatment failure.Prostaglandin F_(2α)synthase(PGF 2α)(PGFS),an enzyme that catalyzes the production of PGF_(2α),is involved in the proliferation and growth of a variety of tumors.However,the role of PGFS in Oxa resistance in CRC remains unclear.AIM To explore the role and related mechanisms of PGFS in mediating Oxa resistance in CRC.METHODS The PGFS expression level was examined in 37 pairs of CRC tissues and paracancerous tissues at both the mRNA and protein levels.Overexpression or knockdown of PGFS was performed in CRC cell lines with acquired Oxa resistance(HCT116-OxR and HCT8-OxR)and their parental cell lines(HCT116 and HCT8)to assess its influence on cell proliferation,chemoresistance,apoptosis,and DNA damage.For determination of the underlying mechanisms,CRC cells were examined for platinum-DNA adducts and reactive oxygen species(ROS)levels in the presence of a PGFS inhibitor or its products.RESULTS Both the protein and mRNA levels of PGFS were increased in the 37 examined CRC tissues compared to the adjacent normal tissues.Oxa induced PGFS expression in the parental HCT116 and HCT8 cells in a dosedependent manner.Furthermore,overexpression of PGFS in parental CRC cells significantly attenuated Oxainduced proliferative suppression,apoptosis,and DNA damage.In contrast,knockdown of PGFS in Oxa-resistant HCT116 and HCT8 cells(HCT116-OxR and HCT8-OxR)accentuated the effect of Oxa treatment in vitro and in vivo.The addition of the PGFS inhibitor indomethacin enhanced the cytotoxicity caused by Oxa.Treatment with the PGFS-catalyzed product PGF_(2α)reversed the effect of PGFS knockdown on Oxa sensitivity.Interestingly,PGFS inhibited the formation of platinum-DNA adducts in a PGF_(2α)-independent manner.PGF_(2α)exerts its protective effect against DNA damage by reducing ROS levels.CONCLUSION PGFS promotes resistance to Oxa in CRC via both PGF_(2α)-dependent and PGF_(2α)-independent mechanisms.

Keap1/Nrf2信号通路在非小细胞肺癌氧化应激机制中的作用

目的检测非小细胞肺癌(NSCLC)组织中Kelch样环氧氯丙烷相关蛋白-1(Keap1)、核因子E2相关因子2(Nrf2)蛋白表达水平,分析其与临床病理参数、氧化应激指标的相关性,为临床治疗提供潜在靶点。方法选取2017年4月至2020年4月郑州市第三人民医院收治的100例NSCLC患者为研究对象,免疫组化法检测并比较癌组织、癌旁组织中Keap1、Nrf2蛋白表达水平;比较不同临床病理参数患者Keap1、Nrf2蛋白表达水平;比较不同Keap1、Nrf2蛋白表达患者血清超氧化物歧化酶(SOD)、诱导型一氧化氮合酶(iNOS)、丙二醛(MDA)水平,并采用Spearman法分析SOD、i NOS、MDA与临床病理参数的相关性,采用Pearson法分析SOD、iNOS、MDA与Keap1、Nrf2蛋白水平的的相关性;比较不同Keap1、Nrf2蛋白表达患者的生存率。结果癌组织、癌旁组织Keap1蛋白阳性率分别为77.00%、53.00%,Nrf2蛋白阳性率分别为74.00%、45.00%,Keap1蛋白OD值分别为0.41±0.07、0.33±0.05,Nrf2蛋白OD值分别为0.39±0.06、0.31±0.06,癌组织Keap1、Nrf2蛋白阳性率及OD值明显高于癌旁组织,差异均有统计学意义(P<0.05);Keap1蛋白阳性表达与病理分级、T分期呈正相关(r=0.569、0.574,P<0.01),Nrf2蛋白阳性表达与病理分级、T分期呈正相关(r=0.527、0.539,P<0.01);Keap1蛋白阳性者、阴性者的血清SOD水平分别为(86.78±9.14)U/m L、(115.07±12.13)U/m L,MDA水平分别为(4.42±0.82)mmol/L、(3.24±0.56)mmol/L,i NOS水平分别为(22.74±4.31)U/m L、(15.59±3.02)U/mL,Nrf2蛋白阳性者、阴性者血清SOD水平分别为(84.94±9.12)U/mL、(117.06±12.37)U/mL,MDA水平分别为(4.48±0.85)mmol/L、(3.21±0.52)mmol/L,iNOS水平分别为(23.02±4.28)U/mL、(15.64±3.10)U/mL,Keap1、Nrf2蛋白阳性者血清SOD水平明显低于阴性者,MDA、iNOS水平明显高于阴性者,差异均有统计学意义(P<0.05);Keap1、Nrf2蛋白表达与SOD呈负相关(r=-0.612、-0.614,P<0.01),与MDA、iNOS呈正相关(r_(Keap1)=0.609、0.614,P<0.01;r_(Nrf2)=0.610、0.608,P<0.01);Keap1、Nrf2蛋白阳性表达者3年生存率为85.71%、83.78%,明显低于阴性表达者的95.65%、100.00%,差异均有统计学意义(P<0.05)。结论NSCLC组织中Keap1、Nrf2蛋白表达水平升高,且与病理分级、T分期密切相关,该信号通路活化可参与氧化应激反应过程,且对预判患者预后具有一定临床意义。

Post-translational modifications of prostaglandin-endoperoxide synthase 2 in colorectal cancer:An update

The biosynthesis of prostanoids is involved in both physiological and pathological processes. The expression of prostaglandin-endoperoxide synthase 2(PTGS2; also known as COX-2) has been traditionally associated to the onset of several pathologies, from inflammation to cardiovascular, gastrointestinal and oncologic events. For this reason, the search of selective PTGS2 inhibitors has been a focus for therapeutic interventions. In addition to the classic non-steroidal anti-inflammatory drugs, selective and specific PTGS2 inhibitors, termed coxibs, have been generated and widely used. PTGS2 activity is less restrictive in terms of substrate specificity than the homeostatic counterpart PTGS1, and it accounts for the elevated prostanoid synthesis that accompanies several pathologies. The main regulation of PTGS2 occurs at the transcription level. In addition to this, the stability of the mRNA is finely regulated through the interaction with several cytoplasmic elements, ranging from specificmicroR NAs to proteins that control mR NA degradation. Moreover, the protein has been recognized to be the substrate for several post-translational modifications that affect both the enzyme activity and the targeting for degradation via proteasomal and non-proteasomal mechanisms. Among these modifications, phosphorylation, glycosylation and covalent modifications by reactive lipidic intermediates and by free radicals associated to the proinflammatory condition appear to be the main changes. Identification of these post-translational modifications is relevant to better understand the role of PTGS2 in several pathologies and to establish a correct analysis of the potential function of this protein in diseases progress. Finally, these modifications can be used as biomarkers to establish correlations with other parameters, including the immunomodulation dependent on molecular pathological epidemiology determinants, which may provide a better frame for potential therapeutic interventions.

Decaprenyl diphosphate synthase subunit 2 as a prognosis factor in hepatocellular carcinoma

AIM:To investigate the involvement of decaprenyl diphosphate synthase subunit 2(PDSS2) in development and progression of human hepatocellular carcinoma(HCC).METHODS:PDSS2 protein expression was examined in well-and poorly differentiated HCC tumor samples.The levels of PDSS2 expression were compared with clinical features and prognosis of HCC patients.The effects of PDSS2 on cell proliferation,cell cycle,apoptosis,cell migration,and invasion in HCC Hep G2 cells were also investigated.RESULTS:PDSS2 was downregulated in poorly differentiated cancer samples compared with welldifferentiated tumor samples,and the expression level was markedly lower in HCC tissues than in histologically normal tissue adjacent to the cancer.Reduced protein expression was negatively associated with the status of HCC progression.In addition,overexpression of PDSS2dramatically suppressed cell proliferation and colony formation,and induced apoptosis in Hep G2 cells by inducing G1-phase cell-cycle arrest.The migration and invasion capabilities of Hep G2 cells were significantly decreased following PDSS2 overexpression.CONCLUSION:Decreased PDSS2 expression is an unfavorable prognostic factor for HCC,and PDSS2 has potent anticancer activity in HCC tissues and Hep G2cells.

Nitric oxide synthase 2 gene polymorphisms are associated with prostatic volume in Korean men with benign prostatic hyperplasia

The precise aetiology of benign prostatic hyperplasia （BPH） remains unclear; however, it is known that immunological inflammatory processes have a role in the pathogenesis of BPH initiation and progression. Nitric oxide synthase 2 （NOS2） inducible expression is closely correlated with prostatic disease, including prostate cancer and BPH. The aim of this study was to investigate the relationship between NOS2 polymorphisms and BPH. With a cohort of 205 controls and 229 BPH subjects, we genotyped three single nucleotide polymorphisms （SNPs） in the NOS2 gene, including rs2779248 （promoter, -278 T/C）, rs 10459953 （5＇-untranslated region） and rs2297518 （exon 16, missense, Ser608Leu）, using direct sequencing and restriction fragment length polymorphism. The genotypic and allelic frequencies between control and BPH subjects were compared, and the associations among the BPH subjects were analyzed. SNPStats, SNPAnalyzer and HelixTree programmes were used to analyze SNPs. There was no association on SNPs between control and BPH subjects. When BPH subjects were analyzed, there was no association on SNPs between the low and high prostate-specific antigen groups. However, one SNP （rs 10459953, odds ratio [OR] = 0.44, 95% confidence interval [CI] = 0.29-0.65, P 〈 0.0001, in codominant model; OR = 0.23, 95% CI = 0.12-0.46, P 〈 0.0001, in dominant model; and OR = 0.46, 95% CI = 0.24-0.86, P = 0.015, in recessive model） was associated with prostatic volume in BPH. We detected a strong association in genotype frequencies of NOS2 SNP （rs10459953） between subjects with small and large prostatic volume in BPH. The result suggests that NOS2 may be associated with prostatic volume in BPH.

Nerve growth factor and inducible nitric oxide synthase expression in the mesencephalon and diencephalon, as well as visual-and auditory-related nervous tissues, in a macaque model of type 2 diabetes

The present study detected distribution and expression of nerve growth factor and inducible nitric oxide synthase in the mesencephalon and diencephalon, as well as visual- and auditory-related nervous tissues, in a macaque model of type 2 diabetes using immunohistochemistry. Results showed that nerve growth factor expression decreased, but inducible nitric oxide synthase expression increased, in the mesencephalon and diencephalon, as well as visual- and auditory- related nervous tissues. These results suggested that nerve growth factor and inducible nitric oxide synthase play an important role in regulating the development of diabetic visual- and auditory-related diseases.

Cloning,expression,purification and bioinformatic analysis of 2-methylcitrate synthase from Mycobacterium tuberculosis

Objective:To clone,express and purify 2—methylcitrale synthase(Rv1131) gene of Mycobacterium tuberculosis(M.tuberculosis) and to study its structural characteristics using various bioinformatics tools.Methods:Rv1131 gene was amplified In polymerase chain reaction using M.tuberculosis H37 Rv genomic DNA and cloned into pGEM-T easy vector and sequenced.The gene was sub-cloned in pET28 c vector,expressed in Escherichia coli RL22(E.coli BL21)(DE3) cells and the recombinant protein was identified by Western blotting.The protein was purified using Nickel affinity chromatography and the structural characteristics like sub-cellular localization.presence of transmembrane helices and secondary structure of the protein were predicted by bioinformaties tools.Tertiary structure of the protein and phylogenetic analysis was aiso established by in silico analysis.Results:The expression of the recombinant protein(Rv1131) was confirmed by western blotting using anti-HIS antibodies and the protein was purified from the soluble fraction.In silico analysis showed that the protein contains no signal peptide and transmembrane helices,Active site prediction showed that the protein has histidine and aspartie acid residues at 242.281 & 332 positions respectively.Phylogenetic analysis showed100%homology with major mycobacterial species.Secondary structure predicts 2-methyleitrate synthase contain 51.9%;alpha-helk.8.7%extended strand and 39.4%random coils.Tertiary structure of the protein was also established.Conclusions:The enzyme 2-methylcitrate synthase from M.tuberculosis H37 Rv has been successfully expressed and purified.The purified protein will further be utilized to develop assay methods lor screening new inhibitors.

Lrrk2 R1628P转基因小鼠尿液外泌体诱导帕金森病样病理变化

目的:探讨Lrrk2 R1628P转基因小鼠尿液外泌体能否诱导帕金森病(PD)样病理改变。方法:提取野生型(WT)和Lrrk2 R1628P转基因小鼠尿液外泌体,使用免疫印迹法检测尿液外泌体标记物TSG101和CD81,Y216位点磷酸化糖原合成酶激酶-3β(pGSK-3β-216)表达水平。在C57小鼠纹状体中分别注射WT小鼠(C57-WT组,n=8)和Lrrk2 R1628P小鼠尿液外泌体(C57-Lrrk2组,n=8)。注射前3天,注射后1、3、6个月,利用平衡木、挂线、转棒和旷场实验对小鼠进行行为学分析,免疫组化法分析脑组织中磷酸化α-突触核蛋白(pα-syn)的沉积情况,免疫印迹法检测脑组织中pα-syn的表达。结果:Lrrk2 R1628P小鼠尿液外泌体TSG101和CD81表达水平与WT小鼠比较,差异无统计学意义(P>0.05),pGSK-3β-216表达水平高于WT小鼠(P<0.001)。C57-Lrrk2组小鼠出现PD样行为学表型(P<0.001),皮层、纹状体和黑质中有pα-syn沉积,脑组织中pα-syn表达水平为(0.44±0.06),C57-WT组则无表达(P<0.001)。结论:小鼠纹状体中注射Lrrk2 R1628P转基因小鼠的尿液外泌体能够诱导PD样病理改变。

Ginkgo biloba leaf extract effects on inducible nitric oxide synthase, Bcl-2, and Bax expression in rat models of spinal cord injury

BACKGROUND：Ginkgo biloba leaf extract exhibits neuroprotective effects in spinal cord injury. However, the mechanisms of action remain unclear. OBJECTIVE： To investigate inducible nitric oxide synthase （iNOS） and Bcl-2/Bax expression in the injured spinal cord, and to explore the neuroprotective mechanisms of ginkgo biloba leaf extract in rats with spinal cord injury. DESIGN, TIME AND SETTING： The randomized, controlled, cell molecular biology experiment was performed at Soochow University, China from March 2007 to March 2008. MATERIALS： A total of 120 healthy, adult Sprague Dawley rats were selected for this study. Rat models of moderate acute thoracic （T9） spinal cord injury were established using the modified Allen method. Shuxuening injection was obtained from Zhenbaodao Pharmaceutical Co., Ltd., China. Methylprednisolone was purchased from North China Pharmaceutical Co., Ltd. METHODS： All rats were equally and randomly divided into four groups. Only the spinal cord was exposed in the sham operation group rats. In the trauma group, rats were not treated with drugs following spinal cord injury. Rats in the hormone group were intraperitoneally injected with 30 mg/kg methylprednisolone following spinal cord injury. Rats in the ginkgo biloba leaf extract group were intraperitoneally infused with a 1.0 mL/kg Shuxuening injection per day. MAIN OUTCOME MEASURES： At 1 hour, as well as 1, 3, 5, 7, and 14 days after spinal cord injury, iNOS- and Bcl-2/Bax-positive cells were quantified with immunohistochemistry. Pathological changes were detected using hematoxylineosin staining under an optical microscope. RESULTS： Spinal cord injury in the ginkgo biloba leaf extract and hormone groups was milder compared with the trauma group. Demyelination was significantly ameliorated and the necrotic cavity was obviously reduced in the injured spinal cord of rats in the ginkgo biloba leaf extract and hormone groups at each time point. iNOS expression was increased in the injured spinal cord, and reached a peak at 5 days. The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract and hormone groups compared with the trauma group （P 〈 0.05-0.01）. The number of iNOS-positive cells was lower in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury （P 〈 0.05）. Bcl-2 expression reached a peak at 3 days, and Bax expression reached a peak at 5 days following rat spinal cord injury. Bcl-2 expression was increased, but Bax expression was decreased in the ginkgo biloba leaf extract and hormone groups compared with the trauma group （P 〈 0.05-0.01）. Bcl-2 expression was greater, but Bax expression was reduced in the ginkgo biloba leaf extract group compared with the hormone group at 7 and 14 days after spinal cord injury （P 〈 0.05）. CONCLUSION： Ginkgo biloba leaf extract exhibits neuroprotective effects by upregulating Bcl-2 expression, downregulating Bax expression, and significantly inhibiting high expressions of iNOS in the injured spinal cord. The neuroprotective effects of ginkgo biloba leaf extract are greater compared with methylprednisolone at 1 week after spinal cord injury.

2型糖尿病患者血清内皮型一氧化氮合酶、网膜素1水平预测糖尿病肾脏疾病发生的价值研究

目的 分析2型糖尿病患者血清内皮型一氧化氮合酶(endothelial nitric oxide synthase,e NOS)、网膜素1(Omentin-1)水平对糖尿病肾脏疾病(diabetic kidney disease,DKD)发生的预测价值。方法 选取2020年5月至2023年2月石家庄市第二医院收治的2型DKD患者124例作为DKD组、单纯2型糖尿病患者125例作为对照组。收集所有患者的临床指标;酶联免疫吸附法检测患者血清eNOS、Omentin-1水平;Pearson法分析DKD患者血清eNOS、Omentin-1和临床指标的相关性;多因素Logistic回归模型分析影响2型糖尿病患者发生DKD的危险因素;受试者工作特征曲线分析2型糖尿病患者血清eNOS、Omentin-1水平对DKD发生的预测价值。结果 与对照组比较,DKD组患者血清总胆固醇[(4.75±0.91)mmol/L比(4.48±0.96)mmol/L]、糖化血红蛋白[(9.32±1.25)%比(8.56±1.23)%]、24 h尿蛋白定量[(2.78±0.31)g比(0.15±0.02)g]、尿微量白蛋白(urinary microalbumin,mAlb)[(273.12±20.41)mg/L比(23.08±3.35)mg/L]、血肌酐(serum creatinine,Scr)[(209.53±22.59)μmol/L比(73.52±9.21)μmol/L]水平显著升高,估算肾小球滤过率(estimatedglomerularfiltrationrate, eGFR)[(72.52±13.51)mL·min^(-1)·(1.73m^(2))^(-1)比(107.34±10.27)m L·min^(-1)·(1.73 m^(2))^(-1)]、血清e NOS[(24.48±3.37)U/L比(33.82±4.52)U/L]、Omentin-1[(28.75±4.42)μg/L比(43.63±6.78)μg/L]水平显著降低(P<0.05);DKD患者血清eNOS与Omentin-1水平呈正相关(r=0.674,P<0.05);血清eNOS、Omentin-1与24 h尿蛋白定量、m Alb、Scr呈负相关(r=-0.456、-0.551、-0.503、-0.527、-0.497、-0.495, P<0.05),与eGFR呈正相关(r=0.523、 0.602,P<0.05);24 h尿蛋白定量、mAlb、Scr是影响2型糖尿病患者发生DKD的危险因素(P<0.05),eGFR、e NOS、Omentin-1是影响2型糖尿病患者发生DKD的保护因素(P<0.05);血清eNOS、Omentin-1两者联合预测2型糖尿病患者发生DKD的曲线下面积(area under curve,AUC)为0.942,分别高于血清e NOS(AUC=0.882)、Omentin-1(AUC=0.862)各自单独预测2型糖尿病患者发生DKD的AUC(P<0.05)。结论 DKD患者血清eNOS与Omentin-1水平均呈低表达,二者联合检测对2型糖尿病患者发生DKD有一定预测价值。

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma:Correlation with microvessel density

AIM: Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins. Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide.Both have constitutive and inducible isoforms. The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis, tumorigenesis and inflammatory processes. This study was to clarify their role in pancreatic adenocarcinomas.METHODS: We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocarcinomas of different grade and stage. The results were compared with microvessel density and clinicopathological data.RESULTS: Twenty-one (52.5%) of the cases showed iNOS expression, 15 (37.5%) of the cases were positive for COX-2.The immunoreaction was heterogeneously distributed within the tumors. Staining intensity was different between the tumors. No correlation between iNOS and COX-2 expression was seen. There was no relationship with microvessel density.However, iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression. There was no correlation with other clinicopathological data.CONCLUSION: Approximately half of the cases expressed iNOS and COX-2. These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas. Due to a low prevalence of COX-2 expression, chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.

GSK3/Nrf2调控的生物节律在机体衰老中的规律

背景:生物节律(昼夜节律)紊乱是一个典型的与衰老有关的问题,维持生物节律的正常运作可能是一种很有前景的抗衰老策略。核转录因子NF-E2相关因子2的表达具有生物节律;糖原合成酶激酶3系统代表了一个“调节阀”,它控制核转录因子NF-E2相关因子2水平的细微振荡。抗氧化基因转录水平的昼夜变化可以影响生物体对氧化应激的反应,但是糖原合成酶激酶3/NF-E2相关因子2在调节机体衰老中的具体分子机制仍令人困惑。目的:拟通过对该领域文献的回顾,寻找糖原合成酶激酶3/核转录因子NF-E2相关因子2调控的生物节律在机体衰老中的一般规律。方法:文献资料法通过对有关“糖原合成酶激酶3、核转录因子NF-E2相关因子2、生物节律以及衰老”等相关文献进行检索、查阅和筛选,为全文的分析奠定理论基础。对比分析法通过对所得到文献进行阅读分析,比较文献之间的异同点,为论点提供合理的理论支撑。通过对文献的进一步对比分析,理清相关指标间的关系,为全文的分析明确思路。结果与结论:①糖原合成酶激酶3可通过对节律基因的调节间接调控核转录因子NF-E2相关因子2的表达;②糖原合成酶激酶3和核转录因子NF-E2相关因子2是抗衰老程序的组成部分,且与生物节律相关;③并且糖原合成酶激酶3/核转录因子NF-E2相关因子2参与多种代谢途径,包括与衰老相关疾病(2型糖尿病和癌症)和神经退行性疾病相关的代谢途径。

ATP Synthase β-subunit Abnormality in Pancreas Islets of Rats with Polycystic Ovary Syndrome and Type 2 Diabetes Mellitus

This study investigated the abnormal expression of ATP synthase β-subunit（ATPsyn-β） in pancreas islets of rat model of polycystic ovary syndrome（PCOS） with type 2 diabetes mellitus（T2DM）,and the secretion function changes after up-regulation of ATP5 b.Sixty female SD rats were divided into three groups randomly and equally.The rat model of PCOS with T2 DM was established by free access to the high-carbohydrate/high-fat diet,subcutaneous injections of DHEA,and a single injection of streptozotocin.The pancreas was removed for the detection of the ATPsyn-β expression by immunohistochemical staining,Western blotting and reverse transcription-PCR（RT-PCR）.The pancreas islets of the rats were cultured,isolated with collagenase Ⅴ and purified by gradient centrifugation,and the insulin secretion after treatment with different glucose concentrations was tested.Lentivirus ATP5 b was successfully constructed with the vector of GV208 and transfected into the pancreas islets for the over-expression of ATPsyn-β.The insulin secretion and intracellular ATP content were determined after transfection of the PCOS-T2 DM pancreas islets with Lenti-ATP5 b.The results showed that the expression of ATPsyn-β protein and m RNA was significantly decreased in the pancreas of PCOS-T2 DM rats.The ATP content in the pancreas islets was greatly increased and the insulin secretion was improved after the up-regulation of ATPsyn-β in the pancreas islets transfected with lenti-ATP5 b.These results indicated that for PCOS,the ATPsyn-β might be one of the key factors for the attack of T2 DM.

Inducible nitric oxide synthase expression is related to angiogenesis,bcl—2 and cell proliferation in hepatocellular carcinoma

In this study,we examined the expression of inducible nitric oxide synthase(iNOS) and vascular endothelial growth factor(VEGF) by immunohistochemical staining in 76 tissue sections collected from bepa-tocellular carcinoma(HCC) patients undergoing hepatectomy.Microvascular density (MVD) was determined by counting endothelial cells immunostained using anti-CD34 antibody.We performed DNA-flow cytometric analyses to elucidate the impact of iNOS and VEGF expression on the cell cycle of HCC.Most of the HCC cells that invaded stroma were markedly immunostained by iNOS antibody.The iNOS stain intensity of the liv-er tissue close to the tumor edge was stronger than that of HCC tissue,and the strongest was the hepatocytes colser to the tumor tissue.However,iNOS expression in 10 normal hepatic samples was undetectable.VEGF positive expression ratio was 84.8% in iNOS positive expression cases,and the ratio was 35.3% in negative cases.There was significant correlation(P=0.000) between iNOS and VEGF expression.Moreover,iNOS expression was significantly associated with bcl-2 and MVD,but without p53 expression.DNA-flow cytometric analyses showed that combined expression of iNOS and VEGF had significant impact on the cell cycle in HCC.PI(Proliferating Index) and SPF(S-phase fraction)in the combined positive expression of iNOS and VEGF group was significantly higher than that in the combined negative group.The present findings suggested that iNOS expression was significantly associated with angiogenesis,bcl-2 and cell proliferation of HCC.

Two farnesyl pyrophosphate synthases,GhFPS1–2,in Gossypium hirsutum are involved in the biosynthesis of farnesol to attract parasitoid wasps

Sesquiterpenoids play an import role in the direct or indirect defense of plants.Farnesyl pyrophosphate synthases(FPSs)catalyze the biosynthesis of farnesyl pyrophosphate,which is a key precursor of farnesol and(E)-β-farnesene.In the current study,two FPS genes in Gossypium hirsutum,GhFPS1 and GhFPS2,were heterologously cloned and functionally characterized in a greenhouse setting.The open reading frames for full-length GhFPS1 and GhFPS2 were each 1029 nucleotides,and encoded two proteins of 342 amino acids with molecular weights of 39.4 kDa.The deduced amino acid sequences of GhFPS1–2 showed high identity to FPSs of other plants.Quantitative real-time PCR analysis revealed that GhFPS1 and GhFPS2 were highly expressed in G.hirsutum leaves,and were upregulated in methyl jasmonate(MeJA)-,methyl salicylate(MeSA)-and aphid infestation-treated cotton plants.The recombinant proteins of either GhFPS1 or GhFPS2 plus calf intestinal alkaline phosphatase could convert geranyl diphosphate(GPP)or isopentenyl diphosphate(IPP)to one major product,farnesol.Moreover,in electrophysiological response and Y-tube olfactometer assays,farnesol showed obvious attractiveness to female Aphidius gifuensis,which is an important parasitic wasp of aphids.Our findings suggest that two GhFPSs are involved in farnesol biosynthesis and they play a crucial role in indirect defense of cotton against aphid infestation.

Glycogen Synthase Kinase 3β Inhibitor (2'Z,3'E)-6-Bromo-indirubin-3'-Oxime Enhances Drug Resistance to 5-Fluorouracil Chemotherapy in Colon Cancer Cells

Objective： To explore the effects and mechanism of glycogen synthase kinase 3β （GSK-313） inhibitor （2＇Z,3＇E）-6-bromo-indirubin-3＇-oxime （BIO） on drug resistance in colon cancer cells. Methods： The colon cancer SW480 and SW620 cells were treated with BIO, 5-fluorouracil （5-FU） and BIO/5-FU, separately. Cell cycle distribution, apoptosis level and efflux ability of rhodamine 123 （Rh123） were detected by flow cytometry. The protein expressions of P-glycoprotein （P-gp）, multidrug resistance protein 2 （MRP2）, thymidylate synthase （TS）, β-catenin, E2F-1 and βcl-2 were detected by Western blot. β-catenin and P-gp were stained with double immunofluorescence and observed under a confocal microscope. Results： BIO up-regulated β-catenin, P-gp, MRP2 and TS, enhanced the efflux ability of Rh123, decreased Bcl-2 protein and gave the opposite effect to E2F-1 protein in SW480 and SW620 ceils. Furthermore, BIO significantly inhibited cell apoptosis, increased S and G2/M phase cells, and reduced the cell apoptosis induced by 5-FU in SW480 cells, whereas the effects were slight or not obvious in SW620 cells. Conclusion： GSK-3β was involved in drug resistance regulation, and activation of β-catenin and inhibition of E2F-1 may be the most responsible for the enhancement of 5-FU chemotherapy resistance induced by GSK-β inhibitor β10 in colon cancer.

Bcl-2 expression significantly correlates with thymidylate synthase expression in colorectal cancer patients

AIM: To examine the expression of thymidylate synthase (TS) and oncoprotein Bcl-2 in advanced colorectal cancer (CRC) patients,and to determine their mutual relationship,association to therapeutic response and impact on disease outcome.METHODS: Tumor samples from 67 patients with CRC,who were treated at advanced stage with either irinotecan alone or in combination with 5-fluorouracil/ leucovorin,were analyzed for expression of TS and Bcl-2 using immunohistochemistry.RESULTS: A significant linear correlation between lower expression levels of Bcl-2 and lower levels of TS expression was found (P = 0.033).Patients with high levels of both TS and Bcl-2 expression had a significantly longer disease-free survival (DFS) (42.6 mo vs 5.4 mo,n = 25) than those with low TS/Bcl-2 index (P = 0.001).Tumors with low levels of both TS and Bcl-2 were associated with a longer survival with metastasis (WMS) interval in the whole patients group (n = 67,P = 0.035).TS/Bcl-2 index was not significantly related to disease-specific survival.CONCLUSION: The present data suggest that CRC patients with low TS/Bcl-2 demonstrate a significantly shorter DFS and longer WMS.

王浆酸对人结肠癌SW620细胞增殖的抑制作用及其网络药理学分析

目的:基于网络药理学探讨王浆酸(10-HDA)对人结肠癌SW620细胞增殖和迁移的影响,阐明其相关分子机制。方法:利用中药系统药理学(TMSCP)数据库和中医药综合数据库(TCMID)以关键词“蜂王浆”进行检索得到10-HDA等活性成分及对应靶点,采用Swiss Target Prediction数据库预测小分子靶点。采用Gene Cards数据库和在线人类孟德尔遗传(OMIM)数据库以关键词“Colon Cancer”获得靶点,利用String数据库和Cytoscape 3.8.0软件构建蛋白-蛋白互作(PPI)网络,筛选核心靶点;利用Metascape数据库对基因本体(GO)功能富集和京都基因与基因组百科全书(KEGG)信号通路进行富集分析,筛选特有成分10-HDA进行体外活性实验。将生长状态良好的人结肠癌SW620细胞分为对照组和不同剂量(1、5、10、15和20 mmol·L^(-1))10-HDA组,采用MTT法检测各组细胞活性并计算细胞存活率。SW620细胞分为对照组、低剂量(5 mmol·L^(-1))10-HDA组、中剂量(10mmol·L^(-1))10-HDA组和高剂量(15mmol·L^(-1))10-HDA组,Hoechst33342染色法观察各组细胞形态表现,细胞划痕实验检测各组细胞划痕愈合率,流式细胞术检测各组不同细胞周期细胞百分率,生化法检测各组细胞中总抗氧化能力(T-AOC)和超氧化物歧化酶(SOD)活性,Western blotting法检测各组细胞中B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)、含半胱氨酸的天冬氨酸蛋白水解酶9(Caspase-9)、糖原合成酶激酶3β(GSK3β)、β-连环蛋白(β-catenin)和细胞周期蛋白D1(Cyclin D1)蛋白表达水平。结果:TCMSP数据库筛选得到蜂王浆6种活性成分,10-HDA治疗结肠癌核心靶点28个。GO功能富集分析主要涉及细胞增殖和细胞凋亡等信号通路;KEGG信号通路富集分析涉及细胞周期、前列腺癌、细胞衰老和p53等信号通路,GSK3β/β-catenin信号通路与细胞周期有密切关联。与对照组比较,5、10、15和20 mmol·L^(-1)10-HDA组细胞存活率呈剂量依赖性降低(P<0.05或P<0.01)。与对照组比较,不同剂量10-HDA组细胞中凋亡细胞数明显增多,细胞划痕愈合率明显降低(P<0.05或P<0.01),中和高剂量10-HDA组细胞中S期细胞百分率明显升高(P<0.05或P<0.01),不同剂量10-HDA组细胞中T-AOC和SOD活性明显降低(P<0.05或P<0.01)。与对照组比较,低剂量10-HDA组细胞中Bcl-2蛋白表达水平明显降低(P<0.01),GSK3β蛋白表达水平明显升高(P<0.05);与对照组比较,中和高剂量10-HDA组细胞中Bax、Caspase-3、Caspase-9和GSK3β蛋白表达水平明显升高(P<0.05或P<0.01),Bcl-2、β-catenin和Cyclin D1蛋白表达水平明显降低(P<0.01)。结论:10-HDA可明显抑制结肠癌细胞增殖和迁移,并可促进结肠癌细胞的凋亡和氧化水平,其作用机制可能与激活GSK3β/β-catenin信号通路有关。

Natural variation of GmFNSII-2 contributes to drought resistance by modulating enzyme activity in soybean

As an essential crop that provides vegetable oil and protein,soybean(Glycine max(L.)Merr.)is widely planted all over the world.However,the scarcity of water resources worldwide has seriously impacted on the quality and yield of soybean.To address this,exploring excellent genes for improving drought resistance in soybean is crucial.In this study,we identified natural variations of GmFNSII-2(flavone synthase II)significantly affect the drought resistance of soybeans.Through sequence analysis of GmFNSII-2 in 632 cultivated and 44 wild soybeans nine haplotypes were identified.The full-length allele GmFNSII-2^(C),but not the truncated allele GmFNSII-2^(A) possessing a nonsense nucleotide variation,increased enzyme activity.Further research found that GmDREB3,known to increase soybean drought resistance,bound to the promoter region of GmFNSII-2^(C).GmDREB3 positively regulated the expression of GmFNSII-2^(C),increased flavone synthase abundance and improved the drought resistance.Furthermore,a singlebase mutation in the GmFNSII-2^(C) promoter generated an additional drought response element(CCCCT),which had stronger interaction strength with GmDREB3 and increased its transcriptional activity under drought conditions.The frequency of drought-resistant soybean varieties with Hap 1(Pro:GmFNSII-2^(C))has increased,suggesting that this haplotype may be selected during soybean breeding.In summary,GmFNSII-2^(C) could be used for molecular breeding of drought-tolerant soybean.

Mitochondrial carrier homolog 2 increases malignant phenotype of human gastric epithelial cells and promotes proliferation,invasion,and migration of gastric cancer cells

BACKGROUND The precise role of mitochondrial carrier homolog 2(MTCH2)in promoting malignancy in gastric mucosal cells and its involvement in gastric cancer cell metastasis have not been fully elucidated.AIM To determine the role of MTCH2 in gastric cancer.METHODS We collected 65 samples of poorly differentiated gastric cancer tissue and adjacent tissues,constructed MTCH2-overexpressing and MTCH2-knockdown cell models,and evaluated the proliferation,migration,and invasion of human gastric epithelial cells(GES-1)and human gastric cancer cells(AGS)cells.The mito-chondrial membrane potential(MMP),mitochondrial permeability transformation pore(mPTP)and ATP fluorescence probe were used to detect mitochondrial function.Mitochondrial function and ATP synthase protein levels were detected via Western blotting.RESULTS The expression of MTCH2 and ATP2A2 in gastric cancer tissues was significantly greater than that in adjacent tissues.Overexpression of MTCH2 promoted colony formation,invasion,migration,MMP expression and ATP production in GES-1 and AGS cells while upregulating ATP2A2 expression and inhibiting cell apoptosis;knockdown of MTCH2 had the opposite effect,promoting overactivation of the mPTP and promoting apoptosis.CONCLUSION MTCH2 can increase the malignant phenotype of GES-1 cells and promote the proliferation,invasion,and migration of gastric cancer cells by regulating mitochondrial function,providing a basis for targeted therapy for gastric cancer cells.