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Inflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan.

Abstract Background Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with both anti-tumorigenic and pro-tumorigenic activity, affecting tumor cell biology, the balance between cell survival and death. The final effect of TNFα is dependent on the type of malignant cells, with the potential to arrest cancer progression. Methods In order to explain the diverse cellular response to TNFα, we engineered melanoma and colorectal carcinoma cell lines stably overexpressing this cytokine. Results Under the TNFα overexpression, significant upregulation of two genes was observed: proinflammatory cytokine IL6 gene in melanoma cells A375 and gene for pro-apoptotic ligand TRAIL in colorectal carcinoma cells HT29, both mediated by TNFα/TNFR1 signaling. Malignant melanoma line A375 displayed also increased autophagy on day 3, followed by premature senescence on day 6. Both processes seem to be interconnected, following earlier apoptosis induction and deregulation of mitochondrial functions. We documented altered mitochondrial status, lowered ATP production, lowered mitochondrial mass, and changes in mitochondrial morphology (shortened and condensed mitochondria) both in melanoma and colorectal carcinoma cells. Overexpression of TNFα was not linked with significant affection of the subpopulation of cancer stem-like cells in vitro. However, we could demonstrate a decrease in aldehyde dehydrogenase (ALDH) activity up to 50%, which is associated with to the stemness phenotype. Conclusions Our in vitro study of direct TNFα influence demonstrates two distinct outcomes in tumor cells of different origin, in non-epithelial malignant melanoma cells of neural crest origin, and in colorectal carcinoma cells derived from the epithelium.

Background: Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with both anti-tumorigenic and pro-tumorigenic activity, affecting tumor cell biology, the balance between cell survival and death. The final effect of TNFα is dependent on the type of malignant cells, with the potential to arrest cancer progression.Methods: In order to explain the diverse cellular response to TNFα, we engineered melanoma and colorectal carcinoma cell lines stably overexpressing this cytokine.Results: Under the TNFα overexpression, significant upregulation of two genes was observed: proinflammatory cytokine IL6 gene in melanoma cells A375 and gene for pro-apoptotic ligand TRAIL in colorectal carcinoma cells HT29, both mediated by TNFα/TNFR1 signaling. Malignant melanoma line A375 displayed also increased autophagy on day 3, followed by premature senescence on day 6. Both processes seem to be interconnected, following earlier apoptosis induction and deregulation of mitochondrial functions. We documented altered mitochondrial status, lowered ATP production, lowered mitochondrial mass, and changes in mitochondrial morphology (shortened and condensed mitochondria) both in melanoma and colorectal carcinoma cells. Overexpression of TNFα was not linked with significant affection of the subpopulation of cancer stem-like cells in vitro. However, we could demonstrate a decrease in aldehyde dehydrogenase (ALDH) activity up to 50%, which is associated with to the stemness phenotype.Conclusions: Our in vitro study of direct TNFα influence demonstrates two distinct outcomes in tumor cells of different origin, in non-epithelial malignant melanoma cells of neural crest origin, and in colorectal carcinoma cells derived from the epithelium. [ABSTRACT FROM AUTHOR]

Abstract Background Mesenchymal stromal cells (MSCs) become an attractive research topic because of their crucial roles in tissue repair and regenerative medicine. Foreskin is considered as a valuable tissue source containing immunotherapeutic MSCs (FSK-MSCs). Results In this work, we used aldehyde dehydrogenase activity (ALDH) assay (ALDEFLUOR™) to isolate and therefore characterize subsets of FSK-MSCs. According to their ALDH activity, we were able to distinguish and sort by fluorescence activated cell sorting (FACS) two subsets of FSK-MSCs (referred as ALDH+ and ALDH−). Consequently, these subsets were characterized by profiling the gene expression related to the main properties of MSCs (proliferation, response to hypoxia, angiogenesis, phenotype, stemness, multilineage, hematopoiesis and immunomodulation). We thus demonstrated by Real Time PCR several relevant differences in gene expression based on their ALDH activity. Conclusion Taken together, this preliminary study suggests that distinct subsets of FSK-MSCs with differential gene expression profiles depending of ALDH activity could be identified. These populations could differ in terms of biological functionalities involving the selection by ALDH activity as useful tool for potent therapeutic applications. However, functional studies should be conducted to confirm their therapeutic relevance.

Mesenchymal stromal cells (MSCs) are promising tools in regenerative medicine and targeted therapies. Although different origins have been described, there is still huge need to find a valuable source harboring specific subpopulations of MSCs with precise therapeutic functions. Here, we isolated by fluorescence activated cell sorting technique, two populations of Wharton's jelly (WJ)-MSCs based on their aldehyde dehydrogenase (ALDH) activity. Two different ALDH activities (low vs. high) were thus observed. We then analyzed their gene expression profile for stemness, phenotype, response to hypoxia, angiogenesis, hematopoietic support, immunomodulation and multilineage differentiation abilities (osteogenesis, adipogenesis, and chondrogenesis). According to ALDH activity, many differences in the mRNA expression of these populations were noticed. In conclusion, we provide evidences that WJ harbors two distinct populations of MSCs with different ALDH activity. These populations seem to display specific functional competences that may be interesting for concise therapeutic applications. [ABSTRACT FROM AUTHOR]

Adipose-derived stem cells (ADSCs) are abundant and readily obtained, and have been studied for their clinical applicability in regenerative medicine. Some surface antigens have been identified as markers of different ADSC subpopulations in mice and humans. However, it is unclear whether functionally distinct subpopulations exist in dogs. To address this issue, we evaluated aldehyde dehydrogenase (ALDH) activity--a widely used stem cell marker in mice and humans--by flow cytometry. Approximately 20% of bulk ADSCs showed high ALDH activity. Compared to cells with low activity (ALDHLo), the high-activity (ALDHHi) subpopulation exhibited a higher capacity for adipogenic and osteogenic differentiation. This is the first report of distinct ADSC subpopulations in dogs that differ in terms of adipogenic and osteogenic differentiation potential. [ABSTRACT FROM AUTHOR]

The leaves of Sasa quelpaertensis Nakai were extracted with 80% ethanol and further partitioned with n-hexane, chloroform, ethyl acetate, n-butanol, and aqueous fractions to evaluate the biological activity through assessment via various in vitro assays, including total phenol content; 1,1-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazothiazoline-6-sulfornic acid (ABTS) radical scavenging; reducing power; α-glucosidase and tyrosinase inhibitory; and alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity assays. The highest activity was found in the ethyl acetate fraction for all assays and showed stronger DPPH radical scavenging, reducing power, and tyrosinase inhibitory activity than the positive controls (butylated hydroxytoluene, α-tocopherol, and arbutin, respectively). When compared to the ethyl acetate fraction, the n-butanol fraction had lower rates, but it still demonstrated relatively high activity. The activity of the n-hexane fraction was high for DPPH and ABTS radical scavenging activity and contained significant amounts of phenol content, whereas the chloroform fraction possessed the highest reducing power, tyrosinase inhibitory, and ADH and ALDH activity, despite having the lowest phenol content when compared to the other fractions. These findings clearly indicate that S. quelpaertensis Nakai leaves can be a good natural source of antioxidants and tyrosinase inhibitors, as well as ADH and ALDH activity inducers, suggesting that may have potential for treating various diseases and improving human health. [ABSTRACT FROM AUTHOR]

The malignant cells of childhood acute lymphoblastic leukemia (ALL) do not form a homogenous entity but a collection of differently maturated blasts. The most immature leukemia cells may be more resistant to therapy than the bulk of more differentiated blasts. We studied 42 patients with childhood ALL treated according to the ALL-BFM 2000 protocol. At diagnosis, we determined the immunophenotype and the aldehyde dehydrogenase (ALDH) activity of the leukemic cells. Additionally, we investigated the expression of CD34, CD38 and CD45 to define a population of immunophenotypically immature cells (CD34+/CD38−/CD45−/low). We then studied levels of minimal residual disease (MRD) after induction therapy (day 33) to determine therapy response. Including all cases ( n = 42), there was no correlation between ALDH positive cells, CD34+/CD38−/CD45−/low cells and MRD levels. A subset of 18 ALLs displayed a more mature phenotype with low-ALDH positivity (< 1%). Analyzing this cohort, ALDH positive blasts overlapped with the CD34+/CD38−/CD45−/low population. The initial rate of ALDH positivity correlated with MRD levels at day 33 of therapy ( r = 0.61, P < .01). We conclude that in pediatric ALL, ALDH positivity as a marker of immaturity and stemness has prognostic significance only in phenotypically mature cases when the ALDH activity is not a property of the majority of the leukemic blasts. In case of an immature ALL phenotype, ALDH activity might be an inherent characteristic of the whole leukemia and is not limited to a more immature subpopulation that could confer to resistance and increased MRD-levels during therapy. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies have examined the therapeutic effect of endothelial progenitor cells (EPCs) during the chronic phase of cerebral infarction in rats; however, few studies have investigated the effects of EPCs during the acute phase of infarction. In this study, we evaluated the therapeutic effect of EPCs with low aldehyde dehydrogenase activity (Alde-Low EPCs) in rats with acute cerebral infarction, and our results provide insight that may help to identify a therapeutic mechanism of EPCs for acute cerebral infarction. The administration of Alde-Low EPCs into rats with acute cerebral infarction results in the accumulation and migration of the Alde-Low EPCs into the infarct area and the subsequent decrease of infarct volume. Moreover, we found that the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) signaling pathway may regulate the accumulation of Alde-Low EPCs. The transplantation of Alde-Low EPCs may represent a potential treatment strategy for acute cerebral infarction. [Copyright &y& Elsevier]

The TALLYHO/JngJ (TH) mouse is a novel polygenic model of type 2 diabetes and exhibits obesity, hyperglycemia (males), hyperinsulinemia, hyperlipidemia, and enlarged pancreatic islets. Since the kidney is damaged by hyperglycemia in other animal models, the present study aimed to determine the kidney phenotype of TH mice using immunoblot and histological analyses of the kidneys of 6-week-old (prediabetic) and 16-week-old TH mice. Interestingly, even 6-week-old male TH mice showed significant increases in kidney weight, compared to C57BL/B6 (B6) mice. Cuboidal parietal epithelium was observed in the Bowman's capsule in male TH mice at the prediabetic age. Water accumulated inside the kidneys of male TH mice in an age-dependent manner, but not in B6 mice. Since Swr/J mice are reported to develop diabetes insipidus and share 86.8% genotype homology with TH mice, the expression level of arginine vasopressin receptor 2 (AVPR2), a candidate protein for diabetes insipidus, was examined and determined to be significantly reduced in the kidneys of prediabetic male TH mice, compared to B6 mice. Aldehyde dehydrogenase (ALDH) activity in the kidneys of prediabetic male TH mice was significantly lower than that in age-matched male B6 mice, while there were no differences between female TH and B6 mice. These results suggest that the kidney phenotype of prediabetic TH mice occurs only in males, accompanied by a reduction in ALDH activity and AVPR2 expression. The kidney phenotype of male TH mice at a prediabetic age becomes evident before the onset of diabetes. [ABSTRACT FROM AUTHOR]

We have demonstrated previously that cord blood CD133+ cells isolated in the G0 phase of the cell cycle are highly enriched for haematopoietic stem cell (HSC) activity, in contrast to CD133+G1 cells. Here, we have analysed the phenotype and functional properties of this population in more detail. Our data demonstrate that a large proportion of the CD133+G0 cells are CD38 negative (60.4%) and have high aldehyde dehydrogenase activity (75.1%) when compared with their CD133+G1 counterparts (13.5 and 4.1%, respectively). This suggests that stem cell activity resides in the CD133+G0 population. In long-term BM cultures, the CD133+G0 cells generate significantly more progenitors than the CD34+G0 population (P<0.001) throughout the culture period. Furthermore, a comparison of CD133+G0 versus CD133+G1 cells revealed that multilineage reconstitution was obtained only in non-obese diabetic/SCID animals receiving G0 cells. We conclude that CD133+ cells in the quiescent phase of the cell cycle have a phenotype consistent with HSCs and are highly enriched for repopulating activity when compared with their G1 counterparts. This cell population should prove useful for selection and manipulation in ex vivo expansion protocols.Bone Marrow Transplantation (2009) 43, 627–635; doi:10.1038/bmt.2008.368; published online 10 November 2008 [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) are a subpopulation of cells within a heterogeneous tumor that have enhanced biologic properties such as increased capacity for self-renewal, increased tumorigenicity, enhanced differentiation capacity, and resistance to chemo- and radiotherapies. This unit describes protocols to isolate and characterize potential cancer stem cells from a solid tumor (oral cancer). This involves creating a single-cell suspension from tumor tissue, tagging the cell subpopulation of interest, and sorting cells into different populations. Finally, the sorted subpopulations can be evaluated for their ability to meet the functional requirements of a CSC, which primarily include increased tumorigenicity in an in vivo xenograft assay. Mastering the protocols in this unit will allow the researcher to study populations of cells that may have properties of CSCs.

Background: Inhibition of prostate cancer stem cells (PCSCs) is an efficient curative maintenance protocol for the prevention of prostate cancer. The objectives of this study were to assess the efficiency of koenimbin, a major biologically active component of Murraya koenigii (L) Spreng, in the suppression of PC-3 cells and to target PC-3-derived cancer stem cells (CSCs) through apoptotic and CSC signaling pathways in vitro., Materials and Methods: The antiproliferative activity of koenimbin was examined using MTT, and the apoptotic detection was carried out by acridine orange/propidium iodide (AO/PI) double-staining and multiparametric high-content screening (HCS) assays. Caspase bioluminescence assay, reverse transcription polymerase chain reaction (RT-PCR), and immunoblotting were conducted to confirm the expression of apoptotic-associated proteins. Cell cycle analysis was investigated using flow cytometry. Involvement of nuclear factor-kappa B (NF-κB) was analyzed using HCS assay. Aldefluor™ and prostasphere formation examinations were used to evaluate the impact of koenimbin on PC-3 CSCs in vitro., Results: Koenimbin remarkably inhibited cell proliferation in a dose-dependent manner. Koenimbin induced nuclear condensation, formation of apoptotic bodies, and G 0 /G 1 phase arrest of PC-3 cells. Koenimbin triggered the activation of caspase-3/7 and caspase-9 and the release of cytochrome c , decreased anti-apoptotic Bcl-2 and HSP70 proteins, increased pro-apoptotic Bax proteins, and inhibited NF-κB translocation from the cytoplasm to the nucleus, leading to the activation of the intrinsic apoptotic pathway. Koenimbin significantly ( P <0.05) reduced the aldehyde dehydrogenase-positive cell population of PC-3 CSCs and the size and number of PC-3 CSCs in primary, secondary, and tertiary prostaspheres in vitro., Conclusion: Koenimbin has chemotherapeutic potential that may be employed for future treatment through decreasing the recurrence of cancer, resulting in the improvement of cancer management strategies and patient survival., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Mesenchymal stromal cells (MSCs) have particular properties that allow their use as therapeutic strategies for several cell-based applications. Historically, bone marrow (BM)-MSCs are isolated by culture adherence since specific cell surface markers are yet to be developed. This original work aimed to identify and characterize isolating expanded BM-MSCs based on their aldehyde dehydrogenase (ALDH) activity known to be a hallmark of stem cells and relevant for their isolation. We thus isolated by fluorescence-activated cell sorting technology two functionally different populations of BM-MSCs depending on their ALDH activity (ALDH + and ALDH - ). Transcriptome analysis and profiling clearly demonstrated that both populations of BM-MSCs present distinct pattern of genes related to the main properties of MSCs (proliferation, response to hypoxia, angiogenesis, phenotype, stemness, multilineage, hematopoiesis, immunomodulation) in an ALDH activity dependent manner. Both BM-MSC populations look to significantly differ in terms of biological responses and functionalities. More functional analyses are needed to understand and characterize the properties of these ALDH populations. Collectively, our results highlight ALDH activity as a potential feature for isolating and segregating functional and/or competent subset of BM-MSC populations, which may account for better and more efficient therapeutic issue.

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 ALDH Hi subpopulations than in ALDH Lo . Gene set enrichment analysis revealed that ribosome-related gene sets were enriched in the ALDH Hi 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., Competing Interests: Conflict-of-interest statement: Authors have no conflicts of interest.

Adipose-derived stem cells (ADSCs) are abundant and readily obtained, and have been studied for their clinical applicability in regenerative medicine. Some surface antigens have been identified as markers of different ADSC subpopulations in mice and humans. However, it is unclear whether functionally distinct subpopulations exist in dogs. To address this issue, we evaluated aldehyde dehydrogenase (ALDH) activity-a widely used stem cell marker in mice and humans-by flow cytometry. Approximately 20% of bulk ADSCs showed high ALDH activity. Compared to cells with low activity (ALDH Lo ), the high-activity (ALDH Hi ) subpopulation exhibited a higher capacity for adipogenic and osteogenic differentiation. This is the first report of distinct ADSC subpopulations in dogs that differ in terms of adipogenic and osteogenic differentiation potential.