M2型丙酮酸激酶及其在肿瘤生长中的作用

肿瘤细胞的快速增殖导致它和正常细胞比较需要更多的代谢原料。为维持其自身的高度增殖,肿瘤细胞往往过表达M2型丙酮酸激酶（pyruvate kinase subtypeM2,PKM2）。在肿瘤的糖代谢通路中,二聚体形式的PKM2（抑制型）促进葡萄糖中间产物进行生物大分子合成代谢,而四聚体结构的PKM2（激活型）则促进葡萄糖的氧化磷酸化为细胞提供能量,二聚体PKM2在肿瘤细胞中占主要地位。PKM2的过表达以及PKM2二聚体、四聚体之间的转化使肿瘤细胞能在各种氧含量和营养环境下生存并增殖,与肿瘤的发生和扩散有密切的关系。     

M2型丙酮酸激酶与Warburg效应在肺癌中的研究进展

靶向肿瘤代谢途径是肺癌治疗的新兴策略之一。Warburg效应是肿瘤代谢的主要特征,表现为肿瘤细胞在有氧条件下通过糖酵解获得能量。最近研究表明,M2型丙酮酸激酶作为糖酵解途径关键酶的异常表达与肺癌的增殖、转移、诊断、治疗及预后密切相关,是肺癌治疗的潜在靶点。     

M2型丙酮酸激酶在肿瘤代谢中的作用

M2型丙酮酸激酶（PKM2）是糖酵解途径的一个关键限速酶,在多种恶性肿瘤细胞中高表达.在肿瘤糖代谢通路中,PKM2可以通过在高活性的四聚体和低活性的二聚体之间相互转化,促进肿瘤的糖酵解和细胞增殖.肿瘤细胞通过多种方式调节PKM2的表达和酶活性如转录调节、变构调节和翻译后修饰调节.PKM2在肿瘤发生和肿瘤代谢中的重要作用使它有望成为肿瘤治疗的新靶点.     

MicroRNAs介导丙酮酸激酶影响肿瘤细胞的糖代谢

肿瘤细胞在氧含量充足的情况下仍倾向于糖酵解,被称之为Warburg效应,这是肿瘤细胞的重要特征之一,现在认为它是癌基因和抑癌基因共同作用的结果.目前研究表明microRNAs能够在转录后水平参与相关基因的表达,通过多种途径调节肿瘤细胞的糖代谢方式.其中丙酮酸激酶参与糖酵解的最后一个阶段,不同亚型的表达水平差异能调控糖代谢方式.本文着重探讨microRNAs调控丙酮酸激酶的作用机制,及其对肿瘤细胞产生的影响,为临床治疗提供理论基础.     

M2型丙酮酸激酶的抗肿瘤作用研究进展

摘 要：糖酵解（Warburg效应）是肿瘤细胞的重要特征，其相比于线粒体氧化磷酸化来说更高效地产生能量及大量的中间产物用于生物合成，还可抑制活性氧的生成。M2型丙酮酸激酶（pyruvate kinase M2，PKM2）多以低活性的二聚体形式存在于肿瘤细胞等快速增殖细胞中，作为催化糖酵解最后一个步骤的限速酶，是肿瘤细胞Warburg效应的一个关键调节因子。PKM2在肿瘤细胞中也发挥着重要的非代谢作用。PKM2被诱导转移入细胞核后，通过磷酸化特定的核蛋白后激活诸多基因的转录，促进肿瘤的生长。基于这些研究成果，该文综述了PKM2在肿瘤生长中的作用，并对PKM2的抑制剂和激活剂的抗肿瘤疗效分别进行讨论，并预测PKM2可能成为癌症治疗的潜在靶点。     

肿瘤M2型丙酮酸激酶在恶性胸腔积液中的诊断价值

目的 探讨肿瘤M2型丙酮酸激酶(Tu M2-PK)对鉴别结核性与肺癌性胸腔积液的临床价值.方法 50例恶性、48例结核性胸腔积液患者,采用酶联免疫吸附法(ELISA)测定血清和胸腔积液中的Tu M2-PK和CEA的水平.结果 肺癌性胸腔积液患者血清和胸腔积液中Tu M2-PK,CEA的表达水平与结核性胸腔积液患者比较有...     

丙酮酸脱氢酶与肿瘤

丙酮酸脱氢酶是丙酮酸生成乙酰辅酶A的关键酶,而乙酰辅酶A是葡萄糖进入三羧酸循环有氧氧化的首要原料。丙酮酸脱氢酶激酶可以抑制丙酮酸脱氢酶的活性。肿瘤细胞的总体丙酮酸脱氢酶激酶表达增加,造成丙酮酸脱氢酶活性降低,导致肿瘤细胞主要通过糖酵解的方式获取能量。恶性肿瘤糖酵解活跃能促进细胞增殖和抑制细胞凋亡,而形成肿瘤细胞糖酵解的微环境可保护肿瘤细胞逃避宿主免疫杀伤并减轻化疗药物损伤,还有利于肿瘤细胞的侵袭和转移。因此,丙酮酸脱氢酶在肿瘤的发生发展过程中起作用。事实上胚胎干细胞以及成体干细胞也主要是以糖酵解的方式获取能量。由此推断,丙酮酸脱氢酶的活性可能与肿瘤细胞的干性相关。     

腺病毒介导PKM2 RNAi对MCF7细胞影响的研究

目的 丙酮酸激酶(pyruvate kinase,PK)是糖酵解的关键酶之一,其M2型同工酶(PKM2)高表达于乳腺癌等肿瘤中.本研究探讨PKM2 RNAi对乳腺癌细胞MCF7系增殖、ATP产生、侵袭和细胞粘附能力的影响及其可能机制.方法 通过介导PKM2 shRNA构建重组腺病毒Ad-PKM2,用以感染人乳腺癌细胞系MCF7.病毒感染细胞24和48h后,收集细胞总蛋白行免疫印迹实验检测基因沉默的效果,CCK-8实验检测细胞增殖和粘附能力,Transwell实验检测细胞穿透人工基底膜能力,采用JC-1探针观察细胞线粒体活性并用试剂盒检测细胞ATP含量.结果 免疫印迹结果表明,腺病毒成功介导MCF7细胞发生PKM2 RNAi.病毒感染后细胞的增殖能力被抑制41.0％,P＜0.05.穿过人工基底膜的细胞减少50.2％;细胞的粘附能力下降34.7％,P＜0.05;细胞内的ATP含量从正常的90 μmol/L下降至68.3μmol/L(P＜0.05),而细胞线粒体活性并未受明显影响.结论 在乳腺癌MCF7细胞中,抑制PKM2蛋白的表达可有效抑制细胞的增殖、侵袭和粘附能力,其机制可能是减少了细胞的ATP能量供应.     

肿瘤型M2 丙酮酸激酶的生物学特性及临床应用

 肿瘤型M2丙酮酸激酶（TUM2-PK）是近年来研究较多的一种新型肿瘤标志物，在恶性肿瘤的早期诊断中显示较好的应用前景。本文主要就肿瘤型M2-PK的生物学特性及临床应用作一综述。     

M2型丙酮酸激酶非代谢功能在肿瘤治疗中的研究进展

丙酮酸激酶(pyruvate kinase,PK),作为糖酵解的关键酶之一,可以编码四个不同亚型的基因,其中M2型丙酮酸激酶(PKM2)主要表达在正常人类胚胎发育中,和组织修复、再生密切相关,随着研究的深入,PKM2在肿瘤组织中的作用受到越来越多的关注。PKM2除了代谢作用外,还可以通过PKM2抑制剂和激活剂变构调节四聚体和二聚体,二聚体状态的PKM2可以调节细胞核中的基因表达及细胞增殖。本文综述了PKM2表达调控,重点介绍了PKM2非代谢功能及在抗肿瘤治疗中的临床应用。     

Silencing stathmin gene expression by survivin promoter-driven siRNA vector to reverse malignant phenotype of tumor cells

Stathmin gene overexpression has been shown to play an important role in maintenance of malignant phenotype in tumor cells, and the blocking efficacy and tumor specificity of this target has been concerned in clinical trails. In this report, we designed survivin promoter-driven siRNA eukaryotic expression vector that expressed the small interfering RNA targeting stathmin gene to selectively knock down the stathmin gene expression in two different kinds of tumor cell lines while sparing normal cell lines. The therapeutic potential of this recombinant vector was tested in human cervical cancer Hela cells and osteosarcoma SSOP-9607 cells, and in human umbilical vein endothelial cell line ECV304 cells as control. The siRNA vector- transfected Hela cells and SSOP-9607 cells revealed marked inhibition of stathmin expression and a dramatic growth inhibition comparing with ECV304 cells, parental-vector transfected cells and untransfected cells. Cell cycle analysis of siRNA vector transfected tumor cells by Flow Cytometry showed G(2)/M phase block, while morphologic analysis by TURNEL staining method showed marked increase of apoptosis. Our study indicates that survivin gene promoter-driven stathmin siRNA expression vector may have potential use in tumor gene therapy with targeted tumor gene silencing effect.     

Beta2-microglobulin signaling blockade inhibited androgen receptor axis and caused apoptosis in human prostate cancer cells

PURPOSE: beta2-Microglobulin (beta2M) has been shown to promote osteomimicry and the proliferation of human prostate cancer cells. The objective of this study is to determine the mechanism by which targeting beta2M using anti-beta2M antibody inhibited growth and induced apoptosis in prostate cancer cells. EXPERIMENTAL DESIGN: Polyclonal and monoclonal beta2M antibodies were used to interrupt beta2M signaling in human prostate cancer cell lines and the growth of prostate tumors in mice. The effects of the beta2M antibody on a survival factor, androgen receptor (AR), and its target gene, prostate-specific antigen (PSA) expression, were investigated in cultured cells and in tumor xenografts. RESULTS: The beta2M antibody inhibited growth and promoted apoptosis in both AR-positive and PSA-positive, and AR-negative and PSA-negative, prostate cancer cells via the down-regulation of the AR in AR-positive prostate cancer cells and directly caused apoptosis in AR-negative prostate cancer cells in vitro and in tumor xenografts. The beta2M antibody had no effect on AR expression or the growth of normal prostate cells. CONCLUSIONS: beta2M downstream signaling regulates AR and PSA expression directly in AR-positive prostate cancer cells. In both AR-positive and AR-negative prostate cancer cells, interrupting beta2M signaling with the beta2M antibody inhibited cancer cell growth and induced its apoptosis. The beta2M antibody is a novel and promising therapeutic agent for the treatment of human prostate cancers.     

Induction of apoptosis and G2/M cell cycle arrest by DCC.

The Deleted in Colorectal Cancer gene (DCC) encodes a cell surface receptor that belongs to the Ig superfamily. Inactivation of the DCC gene has been implicated in human tumor progression. However, little is known about the biological function of the DCC protein. In the present study, we demonstrated that expression of DCC activated caspase-3 and programmed cell death, or induced G2/M cell cycle arrest in tumor cells. In some cell lines, apoptosis was evident within 24 h of DCC expression. Timing of the appearance of apoptotic cells coincided with that of the cleavage of poly (ADP-ribose) polymerase, a substrate of caspase-3. Expression of the apoptosis inhibitory gene Bcl-2 was not able to abrogate the DCC-induced apoptosis. In the G2/M cycle arrest cells, cdk1 activity was inhibited. Our results suggest that the DCC protein may transduce signals resulting in activation of caspases or inhibition of Cdk1. These data provide a possible mechanism by which DCC suppresses tumorigenesis.     

Epigenetic silencing of the tumor suppressor cystatin M occurs during breast cancer progression

Cystatin M is a secreted inhibitor of lysosomal cysteine proteases. Several lines of evidence indicate that cystatin M is a tumor suppressor important in breast malignancy; however, the mechanism(s) that leads to inactivation of cystatin M during cancer progression is unknown. Inspection of the human cystatin M locus uncovered a large and dense CpG island within the 5' region of this gene (termed CST6). Analysis of cultured human breast tumor lines indicated that cystatin M expression is either undetectable or in low abundance in several lines; however, enhanced gene expression was measured in cells cultured on the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC). Increased cystatin M expression does not correlate with a cytotoxic response to 5-aza-dC; rather, various molecular approaches indicated that the CST6 gene was aberrantly methylated in these tumor lines as well as in primary breast tumors. Moreover, 60% (12 of 20) of primary tumors analyzed displayed CST6 hypermethylation, indicating that this aberrant characteristic is common in breast malignancies. Finally, preinvasive and invasive breast tumor cells were microdissected from nine archival breast cancer specimens. Of the five tumors displaying CST6 gene methylation, four tumors displayed methylation in both ductal carcinoma in situ and invasive breast carcinoma lesions and reduced expression of cystatin M in these tumors was confirmed by immunohistochemistry. In summary, this study establishes that the tumor suppressor cystatin M is a novel target for epigenetic silencing during mammary tumorigenesis and that this aberrant event can occur before development of invasive breast cancer.     

Syk对三氧化二砷诱导脑瘤细胞周期阻滞的影响

背景与目的：探讨Syk（Spleen tyrosine kiruase）的表达对As2O3在诱导脑瘤细胞周期阻滞过程中的作用。材料与方法：将syk基因整合至逆转录病毒载体pIND,与载体pVgRXR协同转染携带突变P53基因的脑瘤细胞U373,诱导Syk表达。用Western blot法分析Syk诱导细胞周期负性调控因子P21的表达;用流式细胞仪分析细胞DNA含量,用共聚焦显微镜观察Syk的表达对As2O3在诱导脑瘤细胞周期阻滞过程中的调节作用。结果：转染syk基因的U373细胞（U373 Syk-ind）在诱导剂PonA的作用下表达Syk蛋白。As2O3可使U373细胞周期停滞于G／M期,而表达Syk的U373细胞被As2O3阻滞于G／M期的细胞比例下降。Syk的表达可上调P21的表达。结论：As2O3可影响脑瘤细胞U373的细胞周期发展,将其阻滞于G／M期,但在诱导癌细胞凋亡的同时也产生了致瘤性,而Syk蛋白的表达可以使被阻滞的细胞比例下降,减少了As2O3治疗肿瘤中的副作用。     

DHCR24 gene expression is upregulated in melanoma metastases and associated to resistance to oxidative stress-induced apoptosis

The DHCR24 gene encoding for the 3beta-hydroxysterol delta24-reductase, an oxidoreductase involved in cholesterol biosynthesis, was isolated by subtractive hybridization as highly expressed in a short-term melanoma cell line derived from a cutaneous metastases (S/M2) compared to that obtained from the autologous primary tumor (S/P). DHCR24 (alias seladin-1, diminuto/dwarf1 homolog) has been reported to act as an antiapoptotic factor in neurons. Gene expression analysis by Northern blot confirmed that DHCR24 was 5-fold upregulated in S/M2 compared to S/P cells. High levels of DHCR24 gene expression were detected in 13/25 melanoma metastases and in 1/7 primary melanomas by real-time PCR, indicating that upregulation of this gene may occur in melanoma progression. In S/M2 cells, high DHCR24 gene expression associated with resistance to apoptosis triggered by oxidative stress induced by exposure to hydrogen peroxide. DHCR24 gene transfer was shown to protect melanoma cells from H2O2-induced cytotoxicity. Although higher cholesterol levels were shown in S/M2 cells compared to S/P cells, DHCR24 gene transfer did not increase cholesterol content. To evaluate whether DHCR24 acts as an antiapoptotic factor in melanoma metastases, the cytotoxic effect of chemotherapeutic agents was tested in DHCR24 transfectants and in the presence of a DHCR24 inhibitor, U18666A. High DHCR24 gene expression in transfectants did not result in a higher resistance to cytotoxic agents; treatment with U18666A was cytotoxic in S/P cells with a lower DHCR24 content and showed additive cytotoxic effect only when associated with H2O2 and not with cysplatin or etoposide, indicating that the DHCR24 protective effect is exerted through an oxidative stress-specific mechanism.     

Prostate-specific antigen modulates the expression of genes involved in prostate tumor growth

Prostate-specific antigen (PSA) is a serine protease that is widely used as a surrogate marker in the early diagnosis and management of prostate cancer. The physiological relevance of tissue PSA levels and their role in prostate tumor growth and metastasis are not known. Free-PSA (f-PSA) was purified to homogeneity from human seminal plasma by column chromatography, eliminating hk2 and all known PSA complexes and retaining its protease activity. Confluent monolayers of prostate cancer cell lines, PC-3M and LNCaP, were treated with f-PSA in a series of in vitro experiments to determine the changes in expression of various genes that are known to regulate tumor growth and metastasis. Gene array, quantitative polymerase chain reaction (QPCR), and enzyme-linked immunosorbent assay (ELISA) results show significant changes in the expression of various cancer-related genes in PC-3M and LNCaP cells treated with f-PSA. In a gene array analysis of PC-3M cells treated with 10 muM f-PSA, 136 genes were upregulated and 137 genes were downregulated. In LNCaP cells treated with an identical concentration of f-PSA, a total of 793 genes was regulated. QPCR analysis reveals that the genes for urokinase-type plasminogen activator (uPA), VEGF, and Pim-1 oncogene, known to promote tumor growth, were significantly downregulated, whereas IFN-gamma, known to be a tumor-suppressor gene, was significantly upregulated in f-PSA-treated PC-3M cells. The effect of f-PSA on VEGF and IFN-gamma gene expression and on protein release in PC-3M cells was distinctly dose-dependent. In vivo studies showed a significant reduction (P = .03) in tumor load when f-PSA was administered in the tumor vicinity of PC-3M tumor-bearing BALB/c nude mice. Our data support the hypothesis that f-PSA plays a significant role in prostate tumor growth by regulating various proangiogenic and antiangiogenic growth factors.     

Increased manganese superoxide dismutase (SOD-2) is part of the mechanism for prostate tumor suppression by Mac25/insulin-like growth factor binding-protein-related protein-1

Increased expression of mac25/insulin-like growth factor binding-protein related protein-1 (IGFBP-rP1) in human breast and prostate epithelial cell lines results in the suppression of tumor growth. CDNA expression array analysis revealed increased manganese superoxide dismutase (SOD-2) expression in the mac25/IGFBP-rP1-transfected M12 human prostate cancer cell line compared to M12 control cells. SOD-2 has been postulated to be a tumor suppressor. SOD-2 was also increased in LNCaP cells stably transfected with mac25/IGFBP-rP1, but not in mac25/IGFBP-rP1-transfected PC-3 cells. Mac25 LNCaP cells had a marked decrease in tumor growth in nude mice compared to controls, but there was no difference in tumor growth in mac25 PC-3 cells compared to control. Phosphorylated Erk and Akt were increased in the M12 and LNCaP transfected mac25/IGFBP-rP1 cells but not PC-3 mac25. Inhibition of PI-3 kinase results in a marked decrease in viability of the M12-mac25 cells compared to M12 controls. Cells treated with H(2)O(2) result in an increase in phospho-ERK. Transfection of SOD-2 in M12 cells markedly decreased tumor growth, apoptosis, G1 delay in the cell cycle, and expression of senescence associated beta-galactosidase. These results suggest that one of the downstream mediators of the senescence-associated tumor suppression effect of mac25/IGFBP-rP1 is SOD-2.     

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.