ER阴性乳腺癌高表达乳腺癌干细胞标志物乙醛脱氢酶1

目的探讨85例乳腺浸润性导管癌中肿瘤干细胞标志物乙醛脱氢酶1(ALDH1)的表达情况及与临床病理特征的关系。方法采用免疫组化法检测乳腺组织芯片85例乳腺浸润性导管癌组织中ALDH1的表达并分析其临床意义。结果 ALDH1在雌激素受体(ER)阴性的乳腺癌中表达率(34.6%)显著高于ER阳性者(12.1%;P=0.024)。不同亚型乳腺癌中存在ALDH1表达的显著差异,其中三阴性乳腺癌中ALDH1的表达较高(50.0%,P=0.034)。ALDH1的表达在不同大小肿块的肿瘤中也存在差异,其中肿块小于2 cm的乳腺癌中ALDH1的表达率最高(50.0%;P=0.040)。而ALDH1的表达与患者年龄、肿瘤分级、淋巴结转移、疾病分期及PR、HER-2状态无关(P>0.05)。结论 ER阴性乳腺癌尤其是三阴性乳腺癌中ALDH1的表达率较高,提示ER阴性乳腺癌中存在较高比例的干细胞,进而有助于更好地理解其预后差的特点。     

乙醛脱氢酶1A1在胃癌组织中的表达及其临床意义

目的 探讨乙醛脱氢酶1A1（ALDH1A1）在胃癌组织中的表达及临床意义。方法 采用免疫组化法检测1072例胃癌组织及配对癌旁组织中ALDH1A1的表达,并分析其与临床病理特征及总生存时间（OS）的关系。结果 ALDH1A1在胃癌组织中的阳性表达率为49.9％（535/1072）,在癌旁组织中为43.3％（465/1072）,差异有统计学意义（P＜0.05）。 ALDH1A1在胃癌组织中的表达与年龄、浸润深度、分化程度、淋巴结转移及TNM分期有关（P＜0.05）。全组患者的中位OS为32.2个月。ALDH1A1阳性表达者为23.3个月,低于阴性表达者的60.4个月（P＜0.001）。Cox多因素分析显示,ALDH1A1表达、分化程度、TNM分期、年龄、淋巴结转移、癌栓、浸润深度和神经侵犯是影响OS的独立因素。结论ALDH1A1在胃癌组织中表达上调,且与胃癌患者预后相关,有可能成为反映胃癌预后新的肿瘤标志物。     

Identification of brain tumour initiating cells using the stem cell marker aldehyde dehydrogenase

Aldehyde dehydrogenase (ALDH) has been identified in stem cells from both normal and cancerous tissues. This study aimed to evaluate the potential of ALDH as a universal brain tumour initiating cell (BTIC) marker applicable to primary brain tumours and their biological role in maintaining stem cell status.Cells from various primary brain tumours (24 paediatric and 6 adult brain tumours) were stained with Aldefluor and sorted by flow cytometry. We investigated the impact of ALDH expression on BTIC characteristics in vitro and on tumourigenic potential in vivo.Primary brain tumours showed universal expression of ALDH, with 0.3–28.9% of the cells in various tumours identified as ALDH⁺. The proportion of CD133⁺ cells within ALDH⁺ is higher than ALDH cells. ALDH⁺ cells generate neurospheres with high proliferative potential, express neural stem cell markers and differentiate into multiple nervous system lineages. ALDH⁺ cells tend to show high expression of induced pluripotent stem cell-related genes. Notably, targeted knockdown of ALDH1 by shRNA interference in BTICs potently disturbed their self-renewing ability. After 3 months, ALDH⁺ cells gave rise to tumours in 93% of mice whereas ALDH cells did not. The characteristic pathology of mice brain tumours from ALDH⁺ cells was similar to that of human brain tumours, and these cells are highly proliferative in vivo.Our data suggest that primary brain tumours contain distinct subpopulations of cells that have high expression levels of ALDH and BTIC characteristics. ALDH might be a potential therapeutic target applicable to primary brain tumours.     

电穿孔法介导醛脱氢酶基因与多药耐药基因的转移和表达

背景与目的：将不同类型的耐药基因同时导入造血细胞,以扩大耐药范围和进行体内选择是基因治疗的重要策略,这类载体基因片段较长,进行基因转移有一定的难度。本研究旨在寻找安全有效的长片段基因转移G1Na-AIM以NdeⅠ酶切线性化,电穿孔法转导PA317细胞,经长春新碱（VCR）和4－氢过氧化环磷酰胺（4－HC）选择后,应用Southern blot法确定原病毒的整合,逆转录聚合酶链反应（RT－PCR）和流式细胞术（FCM）分析转移基因的表达,筑巢式聚合酶链反应（nested PCR)检验转移系统的安全性。结果：电穿孔法有效介导了ALDH1与MDR1基因的同时转移,Southern blot法证实ALDH1与MDR1基因稳定整合至宿主细胞基因组,RT－PCR检测到转移基因的转录,FCM测定下游基因MDR1蛋白表达增高4倍,转导率达98％。nested PCR未检测到辅助病毒（env)。结论：电穿孔法安全有效地介导了ALDH1与MDR1基因的共移和高表达。     

乙醛脱氢酶1与乳腺癌耐药蛋白在乳腺癌中的表达及相关性研究

背景与目的：乳腺癌患者对化疗药物耐药是导致化疗失败的主要原因。有研究证实,肿瘤干细胞标志物乙醛脱氢酶1(aldehyde dehydrogenase 1,ALDH1)与某些抗癌药物(如环磷酰胺、顺铂等)的耐药有关,并发现经这些药物治疗后的患者癌灶细胞中ALDH1的含量较治疗前高。乳腺癌耐药蛋白(breast cancerresistance protein,BCRP)不仅在正常组织中表达,更高表达于治疗后的乳腺癌中,提示这可能与肿瘤耐药机制相关。关于在乳腺癌患者中两者是否存在共表达的研究很少,本研究主要探讨ALDH1、BCRP与临床病理特征的关系及两种蛋白表达之间的相关性。方法：采用免疫组化法检测乳腺浸润性导管癌组织石蜡切片中ALDH1与BCRP的表达,研究并探讨它们与临床病理特征的关系以及两者表达之间是否有相关性。结果：ALDH1、BCRP在乳腺癌及癌旁乳腺组织中表达差异有统计学意义(χ²=14.685,P=0.000;χ²=12.243,P=0.000)。ALDH1的表达与患者年龄、病理分期、腋窝淋巴结转移、组织学分级及雌激素受体(estrogen receptor,ER)、孕激素受体(progesterone receptor,PR)、人类表皮生长因子受体(human epidermal growth factor receptor,HER-2)状态均无关(P>0.05);BCRP的表达与HER-2高表达有关,HER-2高表达组的癌组织中BCRP阳性表达率较高,差异有统计学意义(χ²＝5.289,P＝0.021)。BCRP的表达与患者年龄、肿瘤分化程度、腋窝淋巴结转移、病理分期及ER、PR状态无关(P>0.05);乳腺癌肿瘤干细胞标志物ALDH1与BCRP无相关性(r=-0.039,P=0.786)。结论：ALDH1可能是区别于其他耐药蛋白的一种独立的生物学因子,参与乳腺癌的化疗耐药或肿瘤的侵袭、转移等恶性生物学行为。     

Vasodilator oxyfedrine inhibits aldehyde metabolism and thereby sensitizes cancer cells to xCT‐targeted therapy

The major cellular antioxidant glutathione (GSH) protects cancer cells from oxidative damage that can lead to the induction of ferroptosis, an iron‐dependent form of cell death triggered by the aberrant accumulation of lipid peroxides. Inhibitors of the cystine‐glutamate antiporter subunit xCT, which mediates the uptake of extracellular cystine and thereby promotes GSH synthesis, are thus potential anticancer agents. However, the efficacy of xCT‐targeted therapy has been found to be diminished by metabolic reprogramming that affects redox status in cancer cells. Identification of drugs for combination with xCT inhibitors that are able to overcome resistance to xCT‐targeted therapy might thus provide the basis for effective cancer treatment. We have now identified the vasodilator oxyfedrine (OXY) as a sensitizer of cancer cells to GSH‐depleting agents including the xCT inhibitor sulfasalazine (SSZ). Oxyfedrine contains a structural motif required for covalent inhibition of aldehyde dehydrogenase (ALDH) enzymes, and combined treatment with OXY and SSZ was found to induce accumulation of the cytotoxic aldehyde 4‐hydroxynonenal and cell death in SSZ‐resistant cancer cells both in vitro and in vivo. Microarray analysis of tumor xenograft tissue showed cyclooxygenase‐2 expression as a potential biomarker for the efficacy of such combination therapy. Furthermore, OXY‐mediated ALDH inhibition was found to sensitize cancer cells to GSH depletion induced by radiation therapy in vitro. Our findings thus establish a rationale for repurposing of OXY as a sensitizing drug for cancer treatment with agents that induce GSH depletion.     

Hypoxia promotes the phenotypic change of aldehyde dehydrogenase activity of breast cancer stem cells

Stable breast cancer cell (BCC) lines are valuable tools for the identification of breast cancer stem cell (BCSC) phenotypes that develop in response to several stimuli as well as for studying the basic mechanisms associated with the initiation and maintenance of BCSCs. However, the characteristics of individual, BCC‐derived BCSCs varies and these cells show distinct phenotypes depending on the different BCSC markers used for their isolation. Aldehyde dehydrogenase (ALDH) activity is just such a recognized biomarker of BCSCs with a CD44⁺/CD24^? phenotype. We isolated BCSCs with high ALDH activity (CD44⁺/CD24^?/Aldefluor^pos) from a primary culture of human breast cancer tissue and observed that the cells had stem cell properties compared to BCSCs with no ALDH activity (CD44⁺/CD24^?/Aldefluor^neg). Moreover, we found Aldefluor^pos BCSCs had a greater hypoxic response and subsequent induction of HIF‐1α expression compared to the Aldefluor^neg BCSCs. We also found that knocking down HIF‐1α, but not HIF‐2α, in Aldefluor^pos BCSCs led to a significant reduction of the stem cell properties through a decrease in the mRNA levels of genes associated with the epithelial‐mesenchymal transition. Indeed, HIF‐1α overexpression in Aldefluor^neg BCSCs led to Slug and Snail mRNA increase and the associated repression of E‐cadherin and increase in Vimentin. Of note, prolonged hypoxic stimulation promoted the phenotypic changes of Aldefluor^neg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes.     

Tumor endothelial cells accelerate tumor metastasis

Tumor metastasis is the main cause of cancer‐related death. Understanding the molecular mechanisms underlying tumor metastasis is crucial to control this fatal disease. Several molecular pathways orchestrate the complex biological cell events during a metastatic cascade. It is now well known that bidirectional interaction between tumor cells and their microenvironment, including tumor stroma, is important for tumor progression and metastasis. Tumor stromal cells, which acquire their specific characteristics in the tumor microenvironment, accelerate tumor malignancy. The formation of new blood vessels, termed as tumor angiogenesis, is a requirement for tumor progression. Tumor blood vessels supply nutrients and oxygen and also provide the route for metastasis. Tumor endothelial cells, which line tumor blood vessels, also exhibit several altered phenotypes compared with those of their normal counterparts. Recent studies have emphasized “angiocrine factors” that are released from tumor endothelial cells and promote tumor progression. During intravasation, tumor cells physically contact tumor endothelial cells and interact with them by juxtacrine and paracrine signaling. Recently, we observed that in highly metastatic tumors, tumor endothelial cells interact with tumor cells by secretion of a small leucine‐rich repeat proteoglycan known as biglycan. Biglycan from tumor endothelial cells stimulates the tumor cells to metastasize. In the present review, we highlight the role of tumor stromal cells, particularly endothelial cells, in the initial steps of tumor metastasis.     

Aldehyde dehydrogenase activity in xenografted human brain tumor in nude mice. Preliminary results in human glioma biopsies

ALDH activity measured fluorimetrically using a high concentration of aliphatic aldehyde as substrate was studied in human glioblastomas grafted in nude mice. Compared with normal brain, ALDH activity is significantly increased in malignant glioma tissue, especially in the cytosolic subcellular fraction. Correlatively, in comparison with normal brain tissue, MDA levels were significantly reduced in whole homogenates and in cytosolic fractions of xenografted glioblastoma tissue. Preliminary results concerning human malignant glioma biopsies are in good agreement with our experimental data. In view of previous works, these results suggest a relationship between alterations in ALDH iso-enzymes activities and cytosolic aldehyde concentrations with respect to normal or tumoral cell growth.     

Implications of understanding cancer stem cell (CSC) biology in head and neck squamous cell cancer

Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer in the world. Effective therapeutic modalities such as surgery, radiation, chemotherapy and combinations of each are used in the management of this disease. Efforts are ongoing throughout the world to improve early detection and prevention of HNSCCs. Often, treatment fails to obtain total cancer cure and this is more likely with advanced stage disease. In recent years it appears that one of the key determinants of treatment failure may be the presence of cancer stem cells (CSC) that 'escape' currently available therapies. CSCs form a minute portion of the total tumour burden but may play a disproportionately important role in determining outcomes. Molecular mechanisms which underlie the genesis of CSCs are yet not fully understood and their detection within the total tumour bulk remains a challenge. Specific markers like Aldehyde dehydrogenase 1 (ALDH1), CD44 and Bmi-1 have shown early promising results both in CSC detection and in guiding treatment protocols. CSCs have been shown to be relatively resistant to standard treatment modalities. It is hoped that developing robust in vitro and in vivo experimental models of CSCs might provide a means of devising more effective therapeutic strategies.     

PTEN抑制人脐静脉内皮细胞ECV304的增殖和VEGF的表达

目的:探讨与张力蛋白同源的10号染色体缺失的磷酸酶基因(phosphatase and tensin hemology deleted on chromo-some ten gene,PTEN)对人脐静脉内皮细胞ECV304细胞增殖、凋亡,及血管内皮生长因子(vascular endothelial growth factor,VEGF)及其受体1(VEGF receptor 1,VEGFR1)的影响。方法:将携带有野生型PTEN及绿色荧光蛋白(green fluorescent protein,GFP)基因的腺病毒Ad-PTEN-GFP及空载体腺病毒Ad-GFP感染ECV304细胞,MTT实验、Hoechst3342染色法及流式细胞术检测ECV304细胞的增殖和凋亡。实时荧光定量PCR法检测Ad-PTEN-GFP感染后ECV304细胞中PTEN、VEGF和VEG-FR1 mRNA表达水平,ELISA检测ECV304细胞培养上清中VEGF的水平。鸡胚尿囊膜(chick chorioallantoic membrane,CAM)血管生长实验检测PTEN对鸡胚血管生长的影响。结果:与Ad-GFP相比,Ad-PTEN-GFP感染能明显抑制ECV304细胞的增殖,诱导细胞凋亡,5 d时增殖抑制率可达(50.38±5.42)%、细胞凋亡率达(73.3±5.3)%。当感染复数为100时,Ad-PTEN-GFP组ECV304细胞的VEGF及VEGFR1 mRNA表达水平分别为未感染组的(13.40±1.32)%及(46.12±5.20)%。同时,Ad-PTEN-GFP感染能够明显抑制CAM体内血管生长[血管指数(57.6±3.37)%vs(92.2±4.37)%,P<0.05]。结论:PTEN能显著抑制人脐静脉内皮细胞ECV304的增殖、促进其凋亡,其机制可能与抑制VEGF和VEGFR1表达,抑制血管新生有关。     

Circulating endothelial progenitor cells

Angiogenesis research investigates the formation of new blood vessels in wound healing, tumour growth and embryonic development. Circulating, bone marrow-derived endothelial progenitor cells (EPCs) were first described 8 years ago, yet the exact nature of these endothelial precursor cells remains unclear. The contributions of circulating EPCs to angiogenesis in tumours, ischaemic injury and other diseases as well as their usefulness in the repair of wounded hearts and limbs remain under intense investigation.     

Eliminating drug resistant breast cancer stem-like cells with combination of simvastatin and gamma-tocotrienol

Present study shows that drug resistant human breast cancer cells are enriched in cancer stem-like cells (CSCs) and express elevated levels of Stat-3 signaling mediators, which contribute to CSC enrichment. Simvastatin (SVA) and gamma-tocotrienol (γT3) eliminate enriched CSCs and suppress expression of Stat-3 signaling mediators via inhibition of the mevalonate pathway and activation of de novo ceramide synthesis pathway, respectively. Combination of SVA + γT3 at low doses enhanced these actions via inhibition of the mevalonate pathway. Data demonstrate that SVA and γT3 alone or in combination possess the ability to eliminate CSCs in drug resistant human breast cancer cells.     

Selection of radioresistant tumor cells and presence of ALDH1 activity in vitro

Background

Tumor resistance to radiotherapy has been hypothesized to be mediated by a tumor subpopulation, called cancer stem cells (CSCs). Based on the proposed function of CSCs in radioresistance, we explored the cancer stem cell properties of cells selected for radioresistance phenotype.

Materials and methods

A549 and SK-BR-3 cells were radioselected with four single doses of 4 or 3 Gy in intervals of 10-12 days and used for colony formation assay and γ-H2AX foci formation assay. Expression of putative stem cell markers, i.e. Sox2, Oct4, ALDH1, and CD133 were analyzed using Western blotting. A549 and SK-BR-3 cells sorted based on their ALDH1 activity were analyzed in clonogenic survival assays.

Results

Radioselected A549 and SK-BR-3 cells (A549-R, SK-BR-3-R) showed increased radioresistance and A549-R cells presented enhanced repair of DNA-double strand breaks. PI3K inhibition significantly reduced radioresistance of A549-R cells. Cell line specific differences in the expression of the putative CSC markers Sox2 and Oct4 were observed when parental and radioselected cells were compared but could not be directly correlated to the radioresistant phenotype. However, enzyme activity of the putative stem cell marker ALDH1 showed a correlation to radioresistance.

Conclusions

Subpopulations of pooled radioresistant colonies, selected by various radiation exposures were analyzed for the presence of putative stem cell markers. Although the pattern of Sox2, Oct4, and CD133 expression was not generally associated with radioresistance, presence of ALDH1 seems to be indicative for subpopulations with increased radioresistance.     

乙醛脱氢酶2基因多态性和饮酒习惯与肝癌的易感性

目的研究乙醛脱氢酶2(ALDH2)基因多态性和饮酒习惯与肝癌的易感性关系.方法在肝癌高发区江苏省泰兴市采用病例对照研究方法,病例为确诊的原发性肝癌(208例)新发病例,对照(208例)按1∶1配对,选取与病例同性别、年龄相差不超过2岁,与病例居住同村或同一街道的非肿瘤居民,调查了研究对象的饮酒习惯等因素,并采用聚合酶链反应-限制性片段长度多态性(PCR-PFLP)方法检测了他们的ALDH2基因型.结果 (1)肝癌组中的ALDH21*1、ALDH21*2、ALDH22*2基因型频率分别为 57.69%、30.77%、11.54% ,与对照组的64.25%、28.50%、7.25%相比,差异无显著性(χ2 =2.64, P=0.267).(2) 携带ALDH2变异基因型(ALDH21*2或ALDH22*2)的饮酒者与携带ALDH2野生(ALDH21*1)基因型的不饮者相比,若开始饮酒年龄＜25岁、每月饮酒量 >3000 g、饮酒年数 >23年和饮酒总量>3千克年时,前者患肝癌的危险性分别是后者的3.62 (95%CI=1.23～10.70)、3.51( 95%CI=1.38～8.95)、3.02 ( 95%CI=1.18～7.71)和3.30 (95%CI= 1.23～8.83)倍.(3)携带ALDH2变异基因型的饮酒者, 患肝癌的OR值与饮酒总量存在显著的剂量效应趋势(P值=0.044).(4)HBsAg阳性并携带ALDH2变异基因型且饮酒>3千克年者, 与HBsAg阴性并携带ALDH2(ALDH21*1)野生基因型且饮酒≤3千克年者相比,发生肝癌的危险性升高52.17倍(95%CI: 5.73～493.39).结论乙醛脱氢酶2基因多态性和大量饮酒与肝癌易感性间存在着明显的协同作用,提示携带ALDH2变异基因型的饮酒者减少酒精消耗量,将有助于降低其发生肝癌的危险性.     

Prospective identification of tumorigenic osteosarcoma cancer stem cells in OS99‐1 cells based on high aldehyde dehydrogenase activity

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines, of which a significantly higher ALDH activity was present in the OS99‐1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99‐1 cells were grown in NOD/SCID mice, we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDH^br cells) from the OS99‐1 xenografts were much less frequent, averaging 3% of the entire tumor population, compared to those isolated directly from the OS99‐1 cell line. ALDH^br cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDH^lo cells), generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDH^br cells illustrated the stem cell characteristics of self‐renewal, the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A, Nanog and Sox‐2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma‐initiating cells and may potentially have therapeutic implications for human osteosarcoma.