Aldehyde dehydrogenase 1, a potential marker for cancer stem cells in human sarcoma

Tumors contain a small population of cancer stem cells (CSC) proposed to be responsible for tumor maintenance and relapse. Aldehyde dehydrogenase 1 (ALDH1) activity has been used as a functional stem cell marker to isolate CSCs in different cancer types. This study used the Aldefluor® assay and fluorescence-activated cell sorting (FACS) analysis to isolate ALDH1^high cells from five human sarcoma cell lines and one primary chordoma cell line. ALDH1^high cells range from 0.3% (MUG-Chor1) to 4.1% (SW-1353) of gated cells. Immunohistochemical staining, analysis of the clone formation efficiency, and xCELLigence microelectronic sensor technology revealed that ALDH1^high cells from all sarcoma cell lines have an increased proliferation rate compared to ALDH1^low cells. By investigating of important regulators of stem cell biology, real-time RT-PCR data showed an increased expression of c-Myc, β-catenin, and SOX-2 in the ALDH1^high population and a significant higher level of ABCG2. Statistical analysis of data demonstrated that ALDH1^high cells of SW-982 and SW-1353 showed higher resistance to commonly used chemotherapeutic agents like doxorubicin, epirubicin, and cisplatin than ALDH1^low cells. This study demonstrates that in different sarcoma cell lines, high ALDH1 activity can be used to identify a subpopulation of cells characterized by a significantly higher proliferation rate, increased colony forming, increased expression of ABC transporter genes and stemness markers compared to control cells. In addition, enhanced drug resistance was demonstrated.     

Aldehyde dehydrogenase activity helps identify a subpopulation of murine adipose-derived stem cells with enhanced adipogenic and osteogenic differentiation potential

AIM To identify and characterize functionally distinct subpopulation of adipose-derived stem cells(ADSCs).METHODS ADSCs cultured from mouse subcutaneous adipose tissue were sorted fluorescence-activated cell sorter based on aldehyde dehydrogenase(ALDH) activity, a widely used stem cell marker. Differentiation potentials were analyzed by utilizing immunocytofluorescece and its quantitative analysis.RESULTS Approximately 15% of bulk ADSCs showed high ALDH activity in flow cytometric analysis. Although significant difference was not seen in proliferation capacity, the adipogenic and osteogenic differentiation capacity was higher in ALDHHi subpopulations than in ALDHLo. Gene set enrichment analysis revealed that ribosome-related gene sets were enriched in the ALDHHi subpopulation. CONCLUSION High ALDH activity is a useful marker for identifying functionally different subpopulations in murine ADSCs. Additionally, we suggested the importance of ribosome for differentiation of ADSCs by gene set enrichment analysis.     

Lgr5 expression as stem cell marker in human gastric gland and its relatedness with other putative cancer stem cell markers

To explore Lgr5 as the possible stem cell marker in human gastric tissue, 259 normal gastric tissues and dissected gastric adenocarcinoma were analyzed by immunohistochemistry, immunofluorescence double staining and qRT-PCR. The results demonstrated that Lgr5 was expressed in the bottom of the normal gastric gland units, and showed a differential expression in gastric adenocarcinoma with varying differentiation. Lgr5 and Bmi1 were co-expressed within the same cells of gastric glands. CD26 +, CD44 +, ALDH1 + and CD133 + cells co-existed with Lgr5 + cells in the stem cell zone of adjacent normal gastric mucosa, and they were detectable in gastric adenocarcinoma but behaved differently. We concluded that Lgr5 may be the adult stem cell marker in human gastric epithelium; Lgr5 and Bmi1 may belong to the same stem cell population; Lgr5, CD26, CD44, ALDH1, and CD133 may be functionally-associated.     

CD44~+CD24~(-/low)与ALDH1~+作为乳腺癌干细胞标志物之异同

越来越多的研究表明,CD44+CD24-/low与ALDH1+都是乳腺癌干细胞标志物。CD44+CD24-/low细胞与ALDH1+细胞具有很多相同的性质,但又有不同的性质和特点。该文主要就CD44+CD24-/low与ALDH1+之间的相同点、两者在乳腺癌干细胞特性方面的差异及其与乳腺癌基因亚型、预后、转移和耐药之间的不同关系等方面作一综述。     

ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome

Application of stem cell biology to breast cancer research has been limited by the lack of simple methods for identification and isolation of normal and malignant stem cells. Utilizing in vitro and in vivo experimental systems, we show that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase activity (ALDH) have stem/progenitor properties. These cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential and greatest growth capacity in a xenotransplant model. In breast carcinomas, high ALDH activity identifies the tumorigenic cell fraction, capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. In a series of 577 breast carcinomas, expression of ALDH1 detected by immunostaining correlated with poor prognosis. These findings offer an important new tool for the study of normal and malignant breast stem cells and facilitate the clinical application of stem cell concepts.     

Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer

Aldehyde dehydrogenase 1 (ALDH1) has been considered to be a marker for cancer stem cells. However, the role of ALDH1 in head and neck squamous cell carcinoma (HNSCC) has yet to be determined. In this study, we isolated ALDH1-positive cells from HNSCC patients and showed that these HNSCC-ALDH1⁺ cells displayed radioresistance and represented a reservoir for generating tumors. Based on microarray findings, the results of Western blotting and immunofluorescent assays further confirmed that ALDH1⁺-lineage cells showed evidence of having epithelial-mesenchymal transition (EMT) shifting and endogenously co-expressed Snail. Furthermore, the knockdown of Snail expression significantly decreased the expression of ALDH1, inhibited cancer stem-like properties, and blocked the tumorigenic abilities of CD44⁺CD24⁻ALDH1⁺ cells. Finally, in a xenotransplanted tumorigenicity study, we confirmed that the treatment effect of chemoradiotherapy for ALDH1⁺ could be improved by Snail siRNA. In summary, it is likely that ALDH1 is a specific marker for the cancer stem-like cells of HNSCC.     

Surface antigenic profiling of stem cells from human omentum fat in comparison with subcutaneous fat and bone marrow

Omentum fat derived stem cells have emerged as an alternative and accessible therapeutic tool in recent years in contrast to the existing persuasive sources of stem cells, bone marrow and subcutaneous adipose tissue. However, there has been a scanty citation on human omentum fat derived stem cells. Furthermore, identification of specific cell surface markers among aforesaid sources is still controversial. In lieu of this existing perplexity, the current research work aims at signifying omentum fat as a ground-breaking source of stem cells by surface antigenic profiling of stem cell population. In this study, we examined and compared the profiling of cell surface antigenic expressions of hematopoietic stem cells, mesenchymal stem cells, cell adhesion molecules and other unique markers such as ABCG2, ALDH and CD 117 in whole cell population of human omentum fat, subcutaneous fat and bone marrow. The phenotypic characterization through flowcytometry revealed the positive expressions of CD 34, CD 45, CD 133, HLADR, CD 90, CD 105, CD 73, CD 29, CD 13, CD 44, CD 54, CD 31, ALDH and CD 117 in all sources. The similarities between the phenotypic expressions of omentum fat derived stem cells to that of subcutaneous fat and bone marrow substantiates that identification of ultimate source for curative therapeutics is arduous to assess. Nevertheless, these results support the potential therapeutic application of omentum fat derived stem cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-012-9427-4) contains supplementary material, which is available to authorized users.     

Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90α was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.     

Aldehyde Dehydrogenase 1 (ALDH1) Is a Potential Marker for Cancer Stem Cells in Embryonal Rhabdomyosarcoma

Cancer stem cells (CSCs) are defined as a small population of cancer cells with the properties of high self-renewal, differentiation, and tumor-initiating functions. Recent studies have demonstrated that aldehyde dehydrogenase 1 (ALDH1) is a marker for CSCs in adult cancers. Although CSCs have been identified in some different types of pediatric solid tumors, there have been no studies regarding the efficacy of ALDH1 as a marker for CSCs. Therefore, in order to elucidate whether ALDH1 can be used as a marker for CSCs of pediatric sarcoma, we examined the characteristics of a population of cells with a high ALDH1 activity (ALDH1^high cells) in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children. We used the human embryonal RMS (eRMS) cell lines RD and KYM-1, and sorted the cells into two subpopulations of ALDH1^high cells and cells with a low ALDH1 activity (ALDH1^low cells). Consequently, we found that the ALDH1^high cells comprised 3.9% and 8.2% of the total cell population, respectively, and showed a higher capacity for self-renewal and tumor formation than the ALDH1^low cells. With regard to chemoresistance, the survival rate of the ALDH1^high cells was found to be higher than that of the ALDH1^low cells following treatment with chemotherapeutic agents for RMS. Furthermore, the ALDH1^high cells exhibited a higher degree of pluripotency and gene expression of Sox2, which is one of the stem cell markers. Taken together, the ALDH1^high cells possessed characteristics of CSCs, including colony formation, chemoresistance, differentiation and tumor initiation abilities. These results suggest that ALDH1 is a potentially useful marker of CSCs in eRMS.     

Reversing the Intractable Nature of Pancreatic Cancer by Selectively Targeting ALDH-High,Therapy-Resistant Cancer Cells

Human pancreatic ductal adenocarcinoma (PDAC) is a cancer with a dismal prognosis. The efficacy of PDAC anticancer therapies is often short-lived; however, there is little information on how this disease entity so frequently gains resistance to treatment. We adopted the concept of cancer stem cells (CSCs) to explain the mechanism of resistance and evaluated the efficacy of a candidate anticancer drug to target these therapy-resistant CSCs. We identified a subpopulation of cells in PDAC with CSC features that were enriched for aldehyde dehydrogenase (ALDH), a marker expressed in certain stem/progenitor cells. These cells were also highly resistant to, and were further enriched by, treatment with gemcitabine. Similarly, surgical specimens from PDAC patients showed that those who had undergone preoperative chemo-radiation therapy more frequently displayed cancers with ALDH strongly positive subpopulations compared with untreated patients. Importantly, these ALDH-high cancer cells were sensitive to disulfiram, an ALDH inhibitor, when tested in vitro. Furthermore, in vivo xenograft studies showed that the effect of disulfiram was additive to that of low-dose gemcitabine when applied in combination. In conclusion, human PDAC-derived cells that express high levels of ALDH show CSC features and have a key role in the development of resistance to anticancer therapies. Disulfiram can be used to suppress this therapy-resistant subpopulation.     

Celastrol targets proteostasis and acts synergistically with a heat-shock protein 90 inhibitor to kill human glioblastoma cells

Glioblastoma multiforme is a devastating disease of the central nervous system and, at present, no effective therapeutic interventions have been identified. Celastrol, a natural occurring triterpene, exhibits potent anti-tumor activity against gliomas in xenograft mouse models. In this study, we describe the cell death mechanism employed by celastrol and identify secondary targets for effective combination therapy against glioblastoma cell survival. In contrast to the previously proposed reactive oxygen species (ROS)-dependent mechanism, cell death in human glioblastoma cells is shown here to be mediated by alternate signal transduction pathways involving, but not fully dependent on, poly(ADP-ribose) polymerase-1 and caspase-3. Our studies indicate that celastrol promotes proteotoxic stress, supported by two feedback mechanisms: (i) impairment of protein quality control as revealed by accumulation of polyubiquitinated aggregates and the canonical autophagy substrate, p62, and (ii) the induction of heat-shock proteins, HSP72 and HSP90. The Michael adduct of celastrol and N-acetylcysteine, 6-N-acetylcysteinyldihydrocelastrol, had no effect on p62, nor on HSP72 expression, confirming a thiol-dependent mechanism. Restriction of protein folding stress with cycloheximide was protective, while combination with autophagy inhibitors did not sensitize cells to celastrol-mediated cytotoxicity. Collectively, these findings imply that celastrol targets proteostasis by disrupting sulfyhydryl homeostasis, independently of ROS, in human glioblastoma cells. This study further emphasizes that targeting proteotoxic stress responses by inhibiting HSP90 with 17-N-Allylamino-17-demethoxygeldanamycin sensitizes human glioblastoma to celastrol treatment, thereby serving as a novel synergism to overcome drug resistance.     

ALDH1蛋白表达与胶质瘤干细胞体外分化相关性的研究

目的探讨乙醛脱氢酶1（ALDH1）蛋白表达与胶质瘤干细胞体外分化相关性。 方法将原代培养的胶质瘤干细胞分为分化组与对照组,分化组细胞使用10%的胎牛血清诱导,对照组细胞继续在无血清环境中培养,通过免疫荧光细胞化学染色和Western blot观察两组细胞ALDH1蛋白的表达情况,分别使用Wilcoxon符号秩检验和配对样本t检验分析两组ALDH1阳性细胞率和相对蛋白含量的差异。 结果分化组细胞呈完全贴壁生长,异型性明显,对照组细胞呈团状聚集,形态较为均一。两组的ALDH1阳性细胞率分别为：分化组18.78%?±?6.03%,对照组81.23%±?3.19%;ALDH1相对蛋白含量分别为：分化组0.035±0.009,对照组0.390±0.108。两组的阳性细胞率和相对蛋白含量比较差异具有统计学意义（Z = -2.666,P = 0.008;t = -10.637,P = 0.000）。 结论本实验通过半定量研究进一步证实了在体外培养状态下,ALDH1主要存在于较为原始的肿瘤细胞中,分化后几乎不表达,提示ALDH1作为可能的胶质瘤干细胞标志物仍有进一步研究的价值。     

CD90 is identified as a candidate marker for cancer stem cells in primary high-grade gliomas using tissue microarrays

Although CD90 has been identified as a marker for various kinds of stem cells including liver cancer stem cells (CSCs) that are responsible for tumorigenesis, the potential role of CD90 as a marker for CSCs in gliomas has not been characterized. To address the issue, we investigated the expression of CD90 in tissue microarrays containing 15 glioblastoma multiformes (GBMs), 19 WHO grade III astrocytomas, 13 WHO grade II astrocytomas, 3 WHO grade I astrocytomas and 8 normal brain tissues. Immunohistochemical analysis showed that CD90 was expressed at a medium to high level in all tested high-grade gliomas (grade III and GBM) whereas it was barely detectable in low-grade gliomas (grade I and grade II) and normal brains. Double immunofluorescence staining for CD90 and CD133 in GBM tissues revealed that CD133(+) CSCs are a subpopulation of CD90(+) cells in GBMs in vivo. Flow cytometry analysis of the expression of CD90 and CD133 in GBM-derived stem-like neurospheres further confirmed the conclusion in vitro. The expression levels of both CD90 and CD133 were reduced along with the loss of stem cells after differentiation. Furthermore, the limiting dilution assay demonstrated that the sphere formation ability was comparable between the CD90(+)/CD133(+) and the CD90(+)/CD133(-) populations of GBM neurospheres, which is much higher than that of the CD90(-)/CD133(-) population. We also performed double staining for CD90 and a vascular endothelial cell marker CD31 in tissue microarrays which revealed that the CD90(+) cells were clustered around the tumor vasculatures in high-grade glioma tissues. These findings suggest that CD90 is not only a potential prognostic marker for high-grade gliomas but also a marker for CSCs within gliomas, and it resides within endothelial niche and may also play a critical role in the generation of tumor vasculatures via differentiation into endothelial cells.     

High Expression of hTERT and Stemness Genes in BORIS/CTCFL Positive Cells Isolated from Embryonic Cancer Cells

BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3–5% of total). The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2) and cancer stem cell marker genes (CD44 and ALDH1) compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.     

The enzymatic activity of human aldehyde dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) is detected by Aldefluor, inhibited by diethylaminobenzaldehyde and has significant effects on cell proliferation and drug resistance

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.     

Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH^Lo cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH^Hi population, or whether all ALDH^Hi cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH^Hi cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH^Hi cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH^Lo population can develop ALDH^Hi populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH^Hi cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in defined medium favouring stem cell characteristics. Although ALDH^Hi status enriches for holoclone formation, this activity may be mediated by a minority of ALDH^Hi cells.     

Cancer Stem Cells Persist in Many Cancer Cell Lines

Both stem cells and cancer cells are thought to be capable of unlimited proliferation. Paradoxically, however, some cancers seem to contain stem-like cells (cancer stem cells). To help resolve this paradox, we investigated whether established malignant cell lines, which have been maintained over years in culture, contain a subpopulation of stem cells. We have shown that four cancer cell lines contain a small side population (SP), which, in many normal tissues, is enriched for stem cells of the tissue. We have also shown that SP cells in C6 glioma cell line, but not non-SP cells, can generate both SP and non-SP cells in culture and are largely responsible for the in vivo malignancy of this cell line. We propose that many cancer cell lines contain a minor subpopulation of stem cells that is enriched in a SP, can be maintained indefinitely in culture, and is crucial for their malignancy.     

Inhibition of breast cancer growth via miR-7 suppressing ALDH1A3 activity concomitant with decreasing breast cancer stem cell subpopulation

Breast cancer patients with high expression of aldehyde dehydrogenases (ALDHs) cell population have higher tolerability to chemotherapy since the cells posses a characteristic of breast cancer stem cells (BCSCs) that are resistant to conventional chemotherapy. In this study, we found that the ALDH-positive cells were higher in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in both BT549 and MDA-MB-231 cell lines but microRNA-7 (miR-7) level was lower in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in MDA-MB-231 cells. Moreover, miR-7 overexpression in MDA-MB-231 cells decreased ALDH1A3 activity by miR-7 directly binding to the 3′-untranslated region of ALDH1A3; while the ALDH1A3 expression was downregulated in MDA-MB-231 cells, the expressions of CD44 and Epithelium Specific Antigen (ESA) were reduced along with decreasing the BCSC subpopulation. Significantly, enforced expression of miR-7 in CD44⁺CD24⁻ESA⁺BCSC markedly inhibited the BCSC-driven xenograft growth in mice by decreasing an expression of ALDH1A3. Collectively, the findings demonstrate the miR-7 inhibits breast cancer growth via suppressing ALDH1A3 activity concomitant with decreasing BCSC subpopulation. This approach may be considered for an investigation on clinical treatment of breast cancers.