Polo-like kinase 1 as a biomarker predicts the prognosis and immunotherapy of breast invasive carcinoma patients

Invasive breast carcinoma(BRCA)is associated with poor prognosis and high risk of mortality.Therefore,it is critical to identify novel biomarkers for the prognostic assessment of BRCA.Methods:The expression data of polo-like kinase 1(PLK1)in BRCA and the corresponding clinical information were extracted from TCGA and GEO databases.PLK1 expression was validated in diverse breast cancer cell lines by quantitative real-time polymerase chain reaction(qRT-PCR)and western blotting.Single sample gene set enrichment analysis(ssGSEA)was performed to evaluate immune infiltration in the BRCA microenvironment,and the random forest(RF)and support vector machine(SVM)algorithms were used to screen for the hub infiltrating cells and calculate the immunophenoscore(IPS).The RF algorithm and COX regression model were applied to calculate survival risk scores based on the PLK1 expression and immune cell infiltration.Finally,a prognostic nomogram was constructed with the risk score and pathological stage,and its clinical potential was evaluated by plotting calibration charts and DCA curves.The application of the nomogram was further validated in an immunotherapy cohort.Results:PLK1 expression was significantly higher in the tumor samples in TCGA-BRCA cohort.Furthermore,PLK1 expression level,age and stage were identified as independent prognostic factors of BRCA.While the IPS was unaffected by PLK1 expression,the TMB and MATH scores were higher in the PLK1-high group,and the TIDE scores were higher for the PLK1-low patients.We also identified 6 immune cell types with high infiltration,along with 11 immune cell types with low infiltration in the PLK1-high tumors.A risk score was devised using PLK1 expression and hub immune cells,which predicted the prognosis of BRCA patients.In addition,a nomogram was constructed based on the risk score and pathological staging,and showed good predictive performance.Conclusions:PLK1 expression and immune cell infiltration can predict post-immunotherapy prognosis of BRCA patients.

Mouse KL2 is a unique MTSE involved in chromosome-based spindle organization and regulated by multiple kinases during female meiosis

Microtubule-severing enzymes(MTSEs)play important roles in mitosis and meiosis of the primitive organisms.However,their roles in mammalian female meiosis,which accounts for over 80%of gamete-originated human reproductive diseases,remain unexplored.In the current study,we reported that katanin-like 2(KL2)was the only MTSE concentrating at chromosomes.Furthermore,the knockdown of KL2 significantly reduced the chromosome-based increase in the microtubule(MT)polymer,increased aberrant kinetochore-MT(K-MT)attachment,delayed meiosis,and severely affected normal fertility.We demonstrated that the inhibition of aurora B,a key kinase for correcting aberrant K-MT attachment,significantly eliminated KL2 expression from chromosomes.Additionally,KL2 interacted with phosphorylated eukaryotic elongation factor-2 kinase,and they competed for chromosome binding.Phosphorylated KL2 was also localized at spindle poles,with its phosphorylation regulated by extracellular signal-regulated kinase 1/2.In summary,the current study reveals a novel function of MTSEs in mammalian female meiosis and demonstrates that multiple kinases coordinate to regulate the levels of KL2 at chromosomes.

Vaccinia-related kinase 2 variants differentially affect breast cancer growth by regulating kinase activity

Genetic information is transcribed from genomic DNA to mRNA,which is then translated into threedimensional proteins.mRNAs can undergo various post-transcriptional modifications,including RNA editing that alters mRNA sequences,ultimately affecting protein function.In this study,RNA editing was identified at the 499th base(c.499)of human vaccinia-related kinase 2(VRK2).This RNA editing changes the amino acid in the catalytic domain of VRK2 from isoleucine(with adenine base)to valine(with guanine base).Isoleucine-containing VRK2 has higher kinase activity than the valine-containing VRK2,which leads to an increase in tumor cell proliferation.Earlier we reported that VRK2 directly interacts with dystrobrevin-binding protein(dysbindin)and results in reducing its stability.Herein,we demonstrate that isoleucine-containing VRK2 decreases the level of dysbindin than valinecontaining VRK2.Dysbindin interacts with cyclin D and thereby regulates its expression and function.The reduction in the level of dysbindin by isoleucine-containing VRK2 further enhances the cyclin D expression,resulting in increased tumor growth and reduction in survival rates.It has also been observed that in patient samples,VRK2 level was elevated in breast cancer tissue compared to normal breast tissue.Additionally,the isoleucine form of VRK2 exhibited a greater increase in breast cancer tissue.Therefore,it is concluded that VRK2,especially dependent on the 167th variant amino acid,can be one of the indexes of tumor progression and proliferation.

Detection of LAMA2 c.715C>G:p.R239G mutation in a newborn with raised creatine kinase: A case report

BACKGROUND We report a rare case of primary clinical presentation featuring elevated creatine kinase(CK)levels in a neonate,which is associated with the LAMA2 gene.In this case,a heterozygous mutation in exon5 of the LAMA2 gene,c.715C>G(resulting in a change of nucleotide number 715 in the coding region from cytosine to gua-nine),induced an amino acid alteration p.R239G(No.239)in the patient,repre-senting a missense mutation.This observation may be elucidated by the neonatal creatine monitoring mechanism,a phenomenon not previously reported.CASE SUMMARY We analysed the case of a neonate presenting solely with elevated CK levels who was eventually discharged after supportive treatment.The chief complaint was identification of increased CK levels for 15 d and higher CK values for 1 d.Ad-mission occurred at 18 d of age,and despite prolonged treatment with creatine and vitamin C,the elevated CK levels showed limited improvement.Whole exo-me sequencing revealed the presence of a c.715C>G mutation in LAMA2 in the newborn,correlating with a clinical phenotype.However,the available informa-tion offers insufficient evidence for clinical pathogenicity.CONCLUSION Mutations in LAMA2 are associated with the clinical phenotype of increased neonatal CK levels,for which no specific treatment exists.Whole genome sequen-cing facilitates early diagnosis.

Interleukin-1 receptor associated kinase 2 is a functional downstream regulator of complement factor D that controls mitochondrial fitness in diabetic cardiomyopathy

Diabetic cardiomyopathy is a disorder of the cardiac muscle that affects patients with diabetes.The exact mechanisms underlying diabetic cardiomyopathy are mostly unknown,but several factors have been implicated in the pathogenesis of the disease and its progression towards heart failure,including endothelial dysfunction,autonomic neuropathy,metabolic alterations,oxidative stress,and alterations in ion homeostasis,especially calcium transients[1].In Military Medical Research,Jiang et al.[2]sought to determine the functional role of complement factor D(Adipsin)in the pathophysiology of diabetic cardiomyopathy.

Crosslink among cyclin-dependent kinase 9,ATP binding cassette transporter G2 and Beclin 1 in colorectal cancer

Colorectal cancer(CRC)ranks third in the number of cancers mainly because of the inability to diagnose it at an early stage.The pathogenesis of CRC is complicated,which is the result of the complex interaction of multiple genetic and environmental factors.Currently,one of the main treatments for CRC is chemotherapy.But the primary cause of CRC treatment failure is drug resistance.The expression of cyclin-dependent kinase 9(CDK9)was correlated with elevated autophagy levels in colon cancer,and high expression of CDK9 indicates a poor prognosis in CRC.The incidence of autophagy and the expressions of Beclin 1 and ATP binding cassette transporter G2 are different in left and right colon cancer,and autophagy may be involved in the occurrence of chemotherapy resistance.In this article,the roles of CDK9,ATP binding cassette transporter G2 and Beclin 1 in CRC were elucidated,emphasizing the linkages among them and providing potential therapeutic targets of CRC.

Polo-like kinase 1 expression is a prognostic factor in human colon cancer

AIM：To clarify the expression patterns and prognostic implications of the mitotic regulator Polo-like kinase 1 （PLK1） in colon cancer. METHODS： Expression of PLK1 was investigated by immunohistochemistry （158 cases） and immunoblotting in tissue of colon adenomas and adenocarcinomas. PLK1 expression patterns were correlated with clinicopathological parameters and patient prognosis. In addition, expression of PLK1 was evaluated by immunoblot and PCR in colon carcinoma cell lines, and coexpression of PLK1 with the proliferation marker Ki-67 was investigated. RESULTS： Weak PLK1 expression was observed in normal colon mucosa and adenomas. In contrast, 66.7% of carcinomas showed strong expression of PLK1. Overexpression of PLK1 correlated positively with Dukes stage （P〈0.001）, tumor stage （P = 0.001） and nodal status （P〈0.05）. Additionally, PLK1 expression was a prognostic marker in univariate survival analysis （P〈0.01） and had independent prognostic significance （RR = 3.3, P = 0.02） in patients with Iocoregional disease. Expression of PLK1 mRNA and protein was detected in all cell lines investigated. Coexpression of PLK1 and Ki-67 was observed in the majority of colon cancer cells, but a considerable proportion of cells showed PLK1 positivity without Ki-67 expression. CONCLUSION： PLK1 is a new prognostic marker for colon carcinoma patients and may be involved in tumorigenesis and progression of colon cancer. Strategies focusing on PLK1 inhibitionin vivo might therefore represent a promising new therapeutic approach for this tumor entity. 2005 The WIG Press and Elsevier Inc. All rights reserved

Overexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patients

AIM： To elucidate the role of overexpressed polo-like kinasel （PLK1）in hepatocellular carcinoma （HCC）. METHODS： We prospectively collected clinicopathological, immunohistochemical and semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） data from 135 HCC patients undergoing successful hepatectomy. The correlations between PLK1 mRNA expression and clinicopathologic variables were analyzed by Mann-Whitney U test. Prognostic factors were identified by univariate and multivariate analyses. RESULTS： Immunohistochemical results showed overexpression of PLK1 was mainly found in tumor tissues compared with tumor-free tissue. A similar mRNA result was obtained by semi-quantitative RT-PCR. A total of 111 samples were positive for PLK1 mRNA expression. The positive expression was correlated with venous invasion, tumor nodules and Edmondson grade. Furthermore, 1, 3, 5-year survival rates in the positive expression group were significantly lower than the negative control group. Multivariate analysis showed that positive PLK1 expression was an independent risk factor for HCC. CONCLUSION： PLK1 could be a potential biomarker for diagnosis and therapy for HCC.

Polo-like kinase 1,a new therapeutic target in hepatocellular carcinoma

AIM： To investigate the role of polo-like kinase 1 （PLK1） as a therapeutic target for hepatocellular carcinoma （HCC）. METHODS： PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA （siRNA） was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, and cell proliferation using 3-（4,5-dim ethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2（4-sulf ophenyl）-2H-tetrazolium （MTS） and bromodeoxyuridine （BrdU） assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end label- ing （TUNEL） assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progres- sion was compared with controls. RESULTS： RT-PCR showed that PLK1 was overexpre- ssed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells, siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLKl-treated mice, but not in controls. CONCLUSION： Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G path- way.

Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC),often diagnosed at advanced stages without curative therapies,is the fifth most common malignant cancer and the second leading cause of cancer-related mortality.Polo-like kinase 1(PLK1)is activated in the late G2 phase of the cell cycle and is required for entry to mitosis.Interestingly,PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome.Baculoviral inhibitor of apoptosis repeatcontaining 5(BIRC5)is also highly overexpressed in HCC and plays key roles in this malignancy.AIM To determine the expression patterns of PLK1 and BIRC5,as well as their correlation with p53 mutation status and patient clinical outcome.METHODS The expression patterns of PLK1 and BIRC5,and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset.Cell viability,cell apoptosis,and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155,alone or in combination.The in vivo efficacy of volasertib and YM155,alone or in combination,was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice.RESULTS Our bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated.The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations.High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome.PLK1 inhibitors(volasertib and GSK461364)or a BIRC5 inhibitor(YM155)selectively targeted Huh7 cells with mutated p53,but not HepG2 cells with wild-type p53.The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway.The efficacy of volasertib and YM155,alone or in combination,was validated in vivo in a Huh7-derived xenograft model.CONCLUSION PLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

Thymoquinone and Poloxin are slow-irreversible inhibitors to human Polo-like kinase 1 Polo-box domain

Objective： To provide a kinetic model（s） and reveal the mechanism of thymoquinone and Poloxin blocking an emerging anti-cancer target, human Polo-like kinase 1 （hPlkl） Polo-box domain （PBD）. Methods： The binding kinetics was determined by using a fluorescence polarization based assay. The putative mechanism was examined with a competition test. Results： Thymoquinone follows a one-step binding with an association rate constant （k1） of 6.635× 10^3 L.mol^-1 min^-1.Poloxin fit a two-step binding with a dissociation constant （Ki） of 118 μmol/L for the intermediate complex and its isomerization rate （k4） of 0.131 5 minJ to form an irreversible adduct. No significant dissociation was observed for either ligand up to 13 h. The inhibitors responded insignificantly to the presence of Michael donors as hPIkl-PBD competitors. Conclusion： Thymoquinone and Poloxin are slow-tight ligands to the hPlkl-PBD with kinetic models distinct from each other. Michael addition as the mechanism is excluded.

Transcriptional regulation of human polo-like kinases and early mitotic inhibitors

Human polo-like kinases （PLK1-PLK4） have been implicated in mitotic regulation and carcinogenesis. PLK1 phosphorylates early mitotic inhibitor 1 （Emil） to ensure mitosis entry, whereas Emi2 plays a key role during the meiotic cell cycle. Transcription factor E2F is primarily considered to regulate the G1/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested. A gap still exists between the molecular basis of E2F and mitotic regulation. The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes. Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells. A reporter gene assay revealed that the putative promoter regions of PLK1, PLK3, and PLK4 genes were responsive to activators E2F, E2F1-E2F3. We further characterized the putative promoter regions of Emil and Emi2 genes, and these could be regulated by activators E2F and E2F1-E2F4, respectively. Finally, PLK1-PLK4, Emil, and Emi2 mRNA expression levels in human adult, fetal tissues, and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus. Collectively, these results indicate that E2F can integrate G1/S and G2/M to oscillate the cell cycle by regulating mitotic genes PLK and Emi, leading to determination of the cell fate.

Casein kinase 2 interacts with and phosphorylates ataxin-3

Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 （CK2）. Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase （GST） pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results （1） Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro. （2） In 293 cells, both wild type and expanded ataxin-3 interacted with CK2β, but not CK2α. （3） CK2 phosphorylated wild type and expanded ataxin-3. Conclusion Ataxin-3 is a substrate of protein kinase CK2.

Cloning of the Full-length Gene for Tobacco Ethylene Receptor NTHK2 and Characterization of Its Kinase Domain

Previously the partial sequence of an ethylene receptor gene NTHK2 was isolated from tobacco (Nicotiana tabacum L. var. Xanthi) plants and it was wound and drought inducible. In the present study full-length cDNA of NTHK2 was cloned by 5'-RACE method. NTHK2 gene has 3 216 bp, with 509 bp of 5'-non-coding region and 427 lip of 3'-non-coding region, and encodes an ethylene-receptor homolog of 760, amino acids. NTHK2 protein has a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. In the putative histidine kinase domain, the histidine at the phosphorylation site was replaced by an asparagine. To study the biochemical property of NTHK2, its kinase domain was expressed as a fusion protein with glutathione S-transferase (GST) using yeast Schizzosaccharomyces pombe as an expression system. In vitro kinase assay showed that NTHK2 kinase domain can autophosphorylate in the presence of Mg2+, indicating that NTHK2 may function as a kinase. Further studies will elucidate the function of NTHK2 in plant.

bFGF激活tyrosine kinase／PI3K／PLCγ增加血管内皮细胞[Mg^2＋]i的研究

目的探讨碱性成纤维细胞生长因子(basic fibroblastgrowth factor,bFGF)对人脐带静脉内皮细胞(human umbilicalvein endothelial cells,HUVECs)内游离镁离子浓度([Mg2+]i)的调节机制研究。方法我们采用荧光指示剂mag-fura-2,运用PTi阳离子测定系统动态测HUVECs的[Mg2+]i。结果经酪氨酸激酶阻断剂(tyrphostin A23和genistein)、3-磷脂酰肌醇激酶阻断剂(wortmannin和LY294002)、磷脂酶Cγ阻断剂(U73122)预处理,能阻断bF-GF诱导的[Mg2+]i增加。但经磷脂酶Cγ阻断剂无活性的类似物(U73343)和丝裂原活化蛋白激酶阻断剂(SB202190和PD98059)预处理,不能阻断bFGF诱导的[Mg2+]i增加。结论bFGF通过酪氨酸激酶/3-磷脂酰肌醇激酶/磷脂酶Cγ信号传递途径使细胞内的Mg2+库释放Mg2+,从而增加HUVECs的[Mg2+]i。

NIMA related kinase 2 promotes gastric cancer cell proliferation via ERK/MAPK signaling

BACKGROUND NIMA related kinase 2(NEK2) is closely related to mitosis, and it is currently considered to be over-expressed frequently in many poorly prognostic cancers.However, the effect of the up-regulated NEK2 on cellular signaling in tumors,such as gastric cancer(GC), is con-fusing.AIM To determine the role of the up-regulation of NEK2 in GC.METHODS To investigate the pathological significance of NEK2 in GC, the expression pattern of NEK2 in GC was investigated based on the 'Oncomain' database and compared between 30 pairs of cancer samples and adjacent tissues. The coexpression of NEK2 and ERK in GC was analyzed using The Cancer Genome Atlas(TCGA) database and confirmed in clinical samples by quantitative realtime PCR(qRT-PCR), and the survival curve was also plotted. Western blot or qRT-PCR was used to analyze the effect of NEK2 on the phosphorylation levels of ERK and c-JUN in two GC cell lines(BGC823 and SGC7901) with NEK2 overexpression, and the expression of the downstream effector cyclin D1.Furthermore, CCK8, EdU incorporation assay, and flow cytometry were used to detect the proliferative ability of BGC823 and SGC7901 cells with stably silenced ERK.RESULTS NEK2 was significantly up-regulated in human GC tissues. ERK was significantly associated with NEK2 expression in human clinical specimens, and combined overexpression of NEK2 and ERK potentially forecasted a poor prognosis andsurvival in GC patients. NEK2 knockdown in GC cells inhibited ERK and c-JUN phosphory-lation and reduced the transcription of cyclin D1. More interestingly,NEK2 can rescue the inhibition of cellular viability, proliferation, and cell cycle progression due to ERK knockdown.CONCLUSION Our results indicate that NEK2 plays a carcinogenic role in the malignant proliferation of GC cells via the ERK/MAPK signaling, which may be important for treatment and improving patient survival.

Ca2+/calmodulin-dependent protein kinase II regulates colon cancer proliferation and migration via ERK1/2 and p38 pathways

AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys

Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Sphingosine kinase 1 is upregulated with lysophosphatidic acid receptor 2 in human colorectal cancer

AIM: To examine the expression of SphK1, an oncogenic kinase that produces sphingosine 1-phosphate (S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid (LPA) receptor overexpressed in various cancers, in human colorectal cancer.METHODS: Real-time reverse-transcription polymerase chain reaction was used to measure the mRNA expression of SphK1, LPAR2, and the three major S1P receptors in 27 colorectal cancer samples and corresponding normal tissue samples. We also examined the correlation between the expression of SphK1 and LPAR2.RESULTS: Colorectal cancer tissue in 22 of 27 patients had higher levels of SphK1 mRNA than in normal tissue. In two-thirds of the samples, SphK1 mRNA expression was more than two-fold higher than in normal tissue. Consistent with previous reports, LPAR2 mRNA expression in 20 of 27 colorectal cancer tissue samples was higher compared to normal tissue samples. Expression profiles of all three major S1P receptors, S1PR1, S1PR2, and S1PR3, varied without any trend, with no significant difference in expression between cancer and normal tissues. A highly significant positive correlation was found between SphK1 and LPAR2 expression [Pearson’s correlation coefficient (r) = 0.784 and P < 0.01]. The mRNA levels of SphK1 and LPAR2 did not correlate with TNM stage.CONCLUSION: Our findings suggest that S1P and LPA may play important roles in the development of colorectal cancer via the upregulation of SphK1 and LPAR2, both of which could serve as new therapeutic targets in the treatment of colorectal cancer.

Sodium Butyrate Induces Apoptosis of Human Colon Cancer Cells by Modulating ERK and Sphingosine Kinase 2

Objective To investigate the role of extracellular signal-regulated kinase （ERK） in apoptosis of human colon cancer （HCT116） cells. Methods After the HCT116 cells were pretreated with specific ERK inhibitor （U0126） or specific siRNA and exposed to 10 mmol/L sodium butyrate （NaBT） for 24 h, their apoptosis was detected by flow cytometry, levels of SphK2 and ERK protein were measured by Western blot, and translocation of SphK2 was assayed by immunofluorescence microscopy. Results The U0126 and siRNAs specific for SphK2 blocked the export of SphK2 from nuclei to cytoplasm and increased the apoptosis of HCT116 cells following NaBT exposure. Over-expression of PKD decreased NaBT-induced apoptosis of HCT116 cells, which was reversed by U0126. Furthermore, transfection of HCT116 cells with constitutively activated PKD plasmids recovered the UO126-blocked export of SphK2. Conclusion ERK regulates the export of SphK2 and apoptosis of HCT116 cells by modulating PKD. Modulation of these molecules may help increase the sensitivity of colon cancer cells to the physiologic anti-colon cancer agent, NaBT.