Aldehyde Dehydrogenase Assay Research Articles

Novel therapeutic approaches targeting cancer stem cells: Unveiling new frontiers in breast cancer treatment.

Breast cancer remains the leading cause of mortality among women with cancer. This article delves into the intricate relationship between breast cancer and cancer stem cells (CSCs), emphasizing advanced methods for their identification and isolation. The key isolation techniques, such as the mammosphere formation assay, surface marker identification, Side Population assay, and Aldehyde Dehydrogenase assay, are critically examined. Furthermore, the review analyzes CSC-specific molecular signaling pathways, focusing on actionable targets like CD44/CD24, Nanog, and Oct4. The potential of targeted therapies and small molecules that disrupt these pathways is explored. Additionally, the review highlights immunotherapy strategies against CSCs, focusing on resistance mechanisms and the critical role of precision medicine. The study investigates how precision medicine enhances therapeutic outcomes by targeting specific CSC biomarkers. This comprehensive analysis offers insights into recent advancements and emerging strategies in breast cancer treatment, pointing toward future therapeutic innovations.

Aldehyde Dehydrogenase Enzyme in Relation to Cord Blood stem cells Potency

Abstract Introduction/Objective In umbilical cord blood transplantation (UCBT), the number of cluster of differentiation (CD)34+ cells and colony-forming units (CFUs) in the cord blood (CB) graft positively correlate with patient survival. Since aldehyde dehydrogenase (ALDH) activity is high in hematopoietic stem cells, the number of ALDH-bright (ALDHbr) cells was examined in comparison with the number of CD34+ cells and CFUs for the quality assessment of CB units, Aim and objectives; to correlate the aldehyde dehydrogenase (ALDH) enzyme assay with colony-forming unit–granulocyte/macrophage (CFU-GM) assay before cryopreservation of cord blood buffy coats (CBBC), in order to investigate the hypothesis that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. Methods/Case Report This study was carried out at Benha University Hospital on Fifty (50) samples of Human Umbilical Cord Blood which were processed for mononuclear cells (MNC) separation by density gradient centrifugation method using Ficoll-Paque media that layered on the diluted blood. In parallel, 1 million nucleated cells were sampled and prepared for flow cytometry for analysis of ALDH activity according to the manufacturer’s instructions. Results (if a Case Study enter NA) CD34+ ALDH+ (%) (Of MNC) showed significant positive correlation with ALDH++ (%) (Of CD34+) and numbers of total CFU/40000 cells. Also ALDH++ (%) (Of CD34+) showed significant positive correlation with numbers of total CFU/40000 cells. Conclusion The ALDH assay excludes nonviable and apoptotic cells, and therefore correlates well with CFU enumeration compared to the number of viable CD34+ cells. We propose that the ALDH assay might replace the CFU-GM method in CBU potency measurements.

Mammosphere formation assay optimization in the characterization of cancer stem cells of the primary breast tumor

Background/Purpose: Breast cancer is the most frequently diagnosed cancer and the leading cause of death from cancer among females worldwide. Breast cancer tumors that feature breast cancer stem cells (BCSCs) are known to cause drug resistance and metastasis. Culturing BCSCs from primary tumors as mammospheres is both difficult and costly. Therefore, the ability to form BCSC mammospheres in-vitro has become essential in assessing their characterization. Flow cytometric analysis of surface markers and measurement of aldehyde dehydrogenase (ALDH) activity are among other methods used to evaluate cancer cells' stem cell activity. 
 Methods:The research material consisted of BCSCs isolated from the tumor tissues collected from two patients with invasive ductal carcinoma breast tumors. Subsequently, several stem cell surface markers, i.e., cluster of differentiation 44 (CD44), CD24, and CD133, were analyzed using flow cytometry during the third passaging of the cells. ALDH assay is performed with negative control verapamil incubated cells. Two mammosphere forming methods, i.e., low attachment and agar-coated wells together with medium seeded in three different cell concentrations, were compared. 
 Results: CD44+, CD24- and CD133+antibody expressions showed that these cells could be tumor-initiating CSCs. ALDH assay results also indicated that these cells possessed stem cell features. In addition, the results of the mammosphere assay revealed that agar-coated wells at a concentration of 7000 cells/cm2 had more prominent floating features and viable spheres.
 Conclusion: The findings of this study supported the hypothesis that agar-coated culture dishes in mammosphere culture would increase the mammosphere formation efficiency (MFE) value and revealed the importance of the number of cells in elucidating the nature of BCSCs.

Stability Program for Hematopoietic Progenitor Products from Apheresis (HPC-A) at an Academic Institution

We perform stability testing on HPC-A products stored in vapor phase nitrogen from 1 week to 5 years. Stability is defined as CD34 viability of 70% or higher (±40% from baseline), and negative sterility results at 14 days. Potency is defined as 21 days or less to ANC engraftment if infused. We performed a comparison on frozen products from deceased patients or soon to be transplanted products stored for more than 10 years (group 3, n=7) or less than 10 years (group 2, n=10). The objective of our analysis was to establish a method to predict stability and potency, with clinical and economic implications. Thirteen patients were consented on an IRB approved protocol, and request for exemption was presented to the IRB for the remaining. Freshly isolated samples (group 1, n=3) provided control data. The demographic characteristics of the cellular products were matched between groups, except that older samples were volume reduced using the COBE-2991 device, (p=0.007). Samples in group 2 were stored for a median time of 3.6 years (range 0.42, 7.1), and samples in group 3 for 12.31 years (range10.12-14.34), respectively. We observed a post-thaw TNC decrease of 30% in group 2 and 36% in group 3 (p=0.9). CD34 counts decreased by 6.5% in group 2, and 11.37% in group 3 (p=0.7). We observed a 26% reduction of viable CD34 cells in group 2 vs. 34% in group 3 (p=0.1). All but two patients in group 3 received the first transplant, with products stored for a median of 2 days in group 1, 9 days in group two, and 137 days in group 3 (p=0.03). Cellular composition was consistent among products. All products engrafted ANCs within 12 days (p=0.5) and PLT within 17 days (p=0.4). Four patients in group 2 and 1 patient in group 3 received a second transplant. Median storage for group 2 was 3.6 years (0.42-7.09), compared with 12.3 years (10.12-14.34) in group 3 (p=1). All second transplants achieved prompt engraftment of both ANCs and PLT (≤12 d, p=0.2; 0.046, resp.). Regarding the ability of lineage depleted CD34 stem cells to form colonies in vitro, we collected data from 13 independent hematopoietic colony-forming unit assays. A statistically significant reduction in colony formation for both groups 2 and 3 was observed when compared with group 1. Other authors have reported successful engraftment in the absence of colony formation in vitro. Therefore, we decided to perform a functional evaluation of the aldehyde dehydrogenase (ALDH) enzyme activity using a commercially available flow cytometry kit in alternative. As expected, ALDH functionality correlated with CD34 viability (Pearson's coefficient: 0.4). (Figure 1). HPC-A products stored longer than 10 years resulted in efficient engraftment after the infusion of standard doses of viable CD34 cells. Viability assessed with 7-AAD is a simple and essential measure to assess the stability of frozen products, and the ALDH assay is an efficient adjunct to assess the functionality of CD34 stem cells.

ATP-binding Cassette Transporters Substantially Reduce Estimates of ALDH-positive Cancer Cells based on Aldefluor and AldeRed588 Assays

Aldehyde dehydrogenase (ALDH) assays measure the accumulated fluorescence of enzyme products. However, cancer cells frequently co-express ALDH and ATP-binding cassette (ABC) transporters, which might mediate efflux of ALDH assay reagents. We demonstrate expression of active multidrug resistance protein1 (MDR1), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP) in CT26 cancer cells as well as expression of MRP and BCRP in HT29 cancer cells. Without transporter inhibition, only small portions of both cell types were estimated to be ALDH-positive based on Aldefluor and AldeRed588 assays. However, MK-571 (MRP inhibitor) and novobiocin (BCRP inhibitor) substantially increased the rate of ALDH-positive CT26 cells based on either Aldefluor or AldeRed588 assays. Verapamil (MDR inhibitor) did not influence assay results. MK-571 also substantially increased the rate of ALDH-positive HT29 cells. Limiting dilution assays demonstrated greater numbers of tumor-spheres formed by Aldefluor-positive compared to -negative CT26 cells selected in the presence of MK-571 or novobiocin but not in their absence. These results reveal that Aldefluor and AldeRed588 products are efficient substrates for MRP- and BCRP-mediated efflux and substantially reduce estimated ALDH positivity rates in cancer cells. These findings demonstrate that complete blockade of these transporters is important to ensure accurate ALDH assay results and to develop newer assay techniques.

Experience in a Public Cord Blood Bank Using a Segment-Based Aldehyde Dehydrogenase Assay as a Biomarker for Umbilical Cord Blood Potency

Background: AMR-001, an autologous CD34+ cell product derived from mini-marrow harvest, is currently undergoing Phase II trials to treat acute myocardial infarction (AMI). At the time of AMR-001 infusion, it is believed that the infarct-region stromal derived factor-1 (SDF-1) levels are peaked. It was found that improvement in cardiac perfusion and infarct size correlated with the mobility potential of CD34+ cells, as mediated by a SDF-1 gradient. We have initiated a study to identify potential microRNAs (miRNAs) and proteins that may be used as biomarkers that correlate to CD34+ cell migratory potential. Methods: In vitro transwell migratory assays were performed on purified CD34+ cells derived from bone marrow of healthy donors. After 4 hours at 37 C, CD34+ cells that migrated into the lower chamber in the presence of SDF-1, the non-mobilized cells in the upper chamber, and untreated cells were harvested. The miRNA expression profile was analyzed (Sistemic, Ltd) using microarray slides (Agilent). A biotin label-based antibody array (RayBiotech) was used for the detection of 1000 proteins. Results: Hierarchical clustering analysis of the miRNA data showed that mobilized cells grouped separately from the non-mobilized/untreated cells. Sixty-three miRNAs were upregulated in the mobilized samples compared to non-mobilized/untreated samples, including two miRNAs which have a reported pro-angiogenic role in migratory cells. Twenty-six proteins had higher expression in mobilized cells compared to non-mobilized cells. Several of the identified proteins have a role in migration or angiogenesis. Conclusion: Analysis of the miRNA and protein profiles of the CD34+ cells identified a number of miRNAs/proteins that represent possible markers for a migratory phenotype. qPCR and ELISA assays will be performed to verify the specific miRNAs and proteins identified. This approach will enable the development of a biomarker assay for migratory potential of AMR-001.

The aldehyde dehydrogenase cord blood potency assay excludes early apoptotic cells.

Cord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic stem cell (HSC) transplantation. The manipulations affect HSC functionality, that is, induce apoptosis and reduce viability. HSC content, commonly expressed as CBU potency, that is, the expected ability of a CBU to restore hematopoiesis, is traditionally approximated through viable CD34+ cells and the colony-forming unit (CFU) cell cultivation assay. Alternative approaches, for example, the aldehyde dehydrogenase (ALDH) enzyme-based assay, are also forthcoming. We hypothesized that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. To investigate this, we designed a protocol for simultaneous staining of viable and apoptotic CD34+ and ALDH+ cells using 7-aminoactinomycin (7-AAD) and annexin V, in frozen-thawed CBUs. Results were correlated with results from the colony-forming unit-granulocyte/macrophage (CFU-GM) assay. Samples from 57 CBUs were thawed and simultaneously analyzed for CD34+ cells, ALDH+ cells, viability (7-AAD), and apoptosis (annexin V) using flow cytometry. Enumeration of CFUs was also performed. No nonviable and few apoptotic cells (mean 0.7%) were identified in the ALDH+ population compared to the viable CD34+ population (mean 3.6%). The total number of ALDH+ cells correlated better than viable CD34+ cells (r = 0. 72 vs. r = 0.66; p < 0.0001) with the results of the CFU assay. The ALDH assay excludes nonviable and apoptotic cells, and therefore correlates better with CFU enumeration compared to the number of viable CD34+ cells. We propose that the ALDH assay might replace the CFU-GM method in CBU potency measurements.

Macrophage conditioned medium promotes colorectal cancer stem cell phenotype via the hedgehog signaling pathway.

BackgroundThere is conflicting data on the role of macrophages in colorectal cancer (CRC); some studies have shown that macrophages can exert an anti-tumor effect whereas others show that macrophages are tumor promoting. We sought to determine the role of conditioned medium (CM) from macrophages, in particular classically activated macrophages, on the development of the CSC phenotype in CRC cells, which is believed to mediate tumor growth and chemoresistance.MethodsMurine (CT26) and human (HCP-1) CRC cell lines were treated with CM from lipopolysaccharide (LPS)-activated murine macrophages. The CSC population was assessed using the sphere-forming assay and aldehyde dehydrogenase assay. Chemoresistance studies were performed using the MTT assay. CSC transcription factors and SHH protein were analyzed by Western blotting.ResultsThe results showed that LPS-activated macrophage CM induced the CSC phenotype in CRC cells. Further studies showed that the CSC phenotype was mediated by the sonic hedgehog (SHH)-Gli signaling pathway, which is known to drive self-renewal; these effects were blocked by depletion of SHH in macrophage CM. In addition, LPS-activated macrophage CM enhanced chemoresistance.ConclusionsLPS-activated macrophages play an active role in promoting the CSC phenotype through activation of the SHH-Gli signaling pathway in CRC cells.

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood Hematopoietic Stem Cell Assessment.

Objective:Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for prediction of engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method.Materials and Methods:In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units.Results:Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 post-thaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92)x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant.Conclusion:In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques.

정제봉독의 ADH와 ALDH 활성 효과

본 연구에서는 서양종꿀벌의 일벌에서 채집하여 정제한 정제봉독이 알코올 분해능에 관련이 있는 ADH와 ALDH 효소 활성에 미치는 영향을 알아보고자 하였다. ADH와 ALDH 활성은 in vitro kit를 사용하여 측정하였다. 그 결과 정제봉독 처리에 의해 ADH와 ALDH 효소 활성이 크게 증가하는 것으로 확인되었다. ADH 효소는 1mg/ml 이상의 정제봉독을 처리했을 경우 양성대조구(2mg/ml) 대비 88.6±7.34%의 활성도를 보였으며, ALDH 효소 역시 1mg/ml 이상의 정제봉독에서 양성대조구(2mg/ml) 대비 94.6±0.57%의 활성도를 나타내었다. 이상의 결과로 정제봉독은 숙취 해소능에 영향을 미치는 지표물질인 ADH와 ALDH 효소 활성을 크게 증가시키는 것으로 사료되었으며, 향후 추가시험을 통해 숙취해소 작용을 갖는 식의약품 소재로 사용 될 수 있을 것으로 생각된다.

Abstract 2887: Macrophage conditioned medium promotes colon cancer stem cell phenotype via the hedgehog signaling pathway

Abstract Background: Contradicting studies have shown that increased macrophages infiltration in colorectal cancer (CRC) could either result in anti-tumoral or in pro-tumoral effects. Because of these inconsistencies, we sought to determine the role of conditioned medium from macrophages, in particular classically activated macrophages, on the development of the CSC phenotype in CRC cells, which is believed to mediate tumor growth and chemoresistance. Methods: Murine (CT26) and human (HCP-1) CRC cell lines were treated with conditioned media (CM) from lipopolysaccharide (LPS)-activated murine macrophages. Viability studies were performed using the MTT assay, annexin V-FITC assay, and Western blot analysis. The CSC population was assessed using the sphere-forming assay and aldehyde dehydrogenase assay. Results: The results showed that 1) murine LPS-activated macrophage CM induced apoptosis in murine and human CRC cells, 2) murine and human LPS-activated macrophage CM secreted paracrine factors that promoted the CSC phenotype in murine and human CRC cells via the Sonic hedgehog (SHH)-Gli signaling pathway, which is known to drive self-renewal and 3) these effects were blocked by depletion of SHH in macrophage CM. Conclusions: Our data show that LPS-activated macrophages play an active role in promoting the CSC phenotype through activation of the SHH-Gli signaling pathway. Citation Format: Fan Fan, Rui Wang, Delphine R. Boulbes, Huiyuan Zhang, Stephanie S. Watowich, Rajat Bhattacharya, Ling Xia, Xiang-Cang Ye, Lee M. Ellis. Macrophage conditioned medium promotes colon cancer stem cell phenotype via the hedgehog signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2887. doi:10.1158/1538-7445.AM2017-2887

Abstract 3356: Characterization and enrichment of head and neck cancer stem cells suggests differences between HPV+ and HPV- subtypes of HNSCC cells

Abstract Head and neck squamous cell carcinomas (HNSCC) are the sixth most prevalent type of malignancy worldwide. Despite recent advances in multidisciplinary treatments, the overall survival and quality of life of patients with advanced HNSCC have not improved considerably over the past decade. Recent reports have demonstrated that tumors contain a small subpopulation of cells called cancer stem-like cells (CSCs), which are responsible for tumor maintenance and metastasis. CSCs have the ability to self-renew and are responsible for tumorigenesis, progression, metastasis, and relapse after treatment. Recent studies demonstrate that even established HNSCC cell lines contain a definite sub-population of CSC providing us with the opportunity to discerning their roles in progression, treatment, and relapse of the disease. In vitro Sphere Forming Assays (using ultra-low attachment plate and serum free media) were employed as the key method of CSC enrichment. qPCR analyses, aldehyde dehydrogenase (ALDH) activity, Side Population Phenotype assay (SPP) were used to confirm the results of the primary analyses. To confirm an in vivo CSC phenotype, xenograft tumor formation, through subcutaneous injection in immunocompromised mice was done. In vitro sphere forming assay followed by subsequent passaging of the sphere enriched cells (SECs) have successfully demonstrated the existence and enrichment of a CSC sub-population in HNSCC cell lines. Findings suggest a differential existence of CSCs related to the HPV status of the cell. ALDH assay and SPP assay confirmed the results of sphere formation. qPCR analyses of the SECs have confirmed enrichment in several stemness marker genes (Oct-4, Sox2, Nanog, Bmi-1, cMyc, KLF4, Beta catenin etc.), also following the identical differential pattern. Various EMT markers genes (Vimentin, Fibronectin, E-cadherin, N-cadherin, Snail, Slug, Twist, ZO-1 etc.) were also assessed, portraying a significant level of expression in CSC populations. SECs subcutaneously injected in immunocompromised mice formed tumors with as few as 50 cells. We have demonstrated that a definite subpopulation of CSCs exists in HNSCC cell lines. Significant differences were seen between the HPV+ and HPV- subtypes. Analyses of EMT marker genes and other molecular pathways in CSC confirm the differences between HPV+ and HPV- cells too following the similar pattern. In vivo tumor xenograft formation establishes the heightened tumorigenicity of CSCs/SECs. Citation Format: Anirban Chatterjee, Kwangok Nickel, Randall J. Kimple. Characterization and enrichment of head and neck cancer stem cells suggests differences between HPV+ and HPV- subtypes of HNSCC cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3356.

The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is necessary for ethanol production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.

Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation of adhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In strains without adhE, we note changes in biochemical activity, product formation, and growth.

Abstract 2699: Identification and characterization of active components of antrodia cinnamomea mycelia on targeting cancer initiating cells

Abstract Background: Cancer initiating cells (CICs) are a subtype of cancer cells, which have the enhanced stemness properties and tumorigenic abilities. CICs are also considered to render resistance of chemotherapy, then, result in recurrence and metastasis. Recent researches and our discovery show that elevated activity of aldehyde dehydrogenase (ALDH) is closely related to CICs. Development of drugs that can specific target cancer initiating cells as a therapeutic method to against the disease would benefit for treatment of cancer patients. Methods: We first used the ALDH assay to screen for purified compounds of the myceluim from Antrodia Cinnamomea with inhibitory activity in cancer cells. Then, we find out 4-acetylantroquinonol B (4-AAQN B) significantly down regulated the ALDH activity after its administration. Further, the anticancer activity of 4-AAQN B by targeting CICs will be identified both in vitro and in vivo. Results: Our results showed that ALDH activity and stemness marker CD44, uPAR, CCN1 significantly decreased after 4-AAQN B administration. We also observed the decreased cell viability in cancer cells. On note, 4-AQQN B reduced ROS high cells, which are considered as activated CICs. Conclusion: The anticancer activity of has been reported before. However, we are the first group to confirm that 4-AAQN B can selectively target CICs. The abovementioned findings provide an alternative therapeutics for future cancer treatment in combination with conventional chemotherapy. Citation Format: Meng-Chia Wu, Jeng-Fan Lo. Identification and characterization of active components of antrodia cinnamomea mycelia on targeting cancer initiating cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2699. doi:10.1158/1538-7445.AM2014-2699

Experience in a public cord blood bank using a segment-based aldehyde dehydrogenase assay as a biomarker for umbilical cord blood potency

Experience in a public cord blood bank using a segment-based aldehyde dehydrogenase assay as a biomarker for umbilical cord blood potency

Experience in a Public Cord Blood Bank Using a Segment-Based Aldehyde Dehydrogenase Assay As a Biomarker for Umbilical Cord Blood Potency

Experience in a Public Cord Blood Bank Using a Segment-Based Aldehyde Dehydrogenase Assay As a Biomarker for Umbilical Cord Blood Potency

Mir-199b, a Novel Tumor Suppressor miRNA In Acute Myeloid Leukemia With Prognostic Implications In FAB-M5 Subtype

Acute myeloid leukemia (AML) manifests a marked heterogeneity in both response to therapy and patient survival, observations that likely reflect its varied pathogenesis. Dysregulation of miRNAs that can act as tumor suppressors or oncogenes can result in tumorigenesis. One miRNA of interest is human miR-199b-5p, which is an intragenic miRNA encoded in the Dynamin 1 gene. Previously we demonstrated that miR-199b was significantly downregulated in AML and targets Podocalyxin and Discoidin Domain Receptor 1. Herein we investigated the functional role of miR-199b in AML and its prognostic implications. We first examined the relative miR-199b expression in steady state hematopoiesis and showed CD33+ myeloid progenitors had the highest miR-199b expression (p<0.03). In order to test whether suppression of miR-199b results in myeloproliferation, human bone marrow derived CD34+ cells were transduced with anti-miR-199b or control lentivirus particles. To examine the effect of miR-199b silencing on colony forming abilities, a CFU assay was performed. At day 16, silencing of miR-199b in CD34+ cells resulted in significant increases in CFU-GM colonies. We then investigated the effect of decreased miR-199b on hematopoietic stem cells (HSC) proliferation via aldehyde dehydrogenase assay. Anti-miR-199b CD34+ cells exhibited a significant increase in HSC numbers compared to control CD34+ cells demonstrating a proliferative advantage for HSC with decreased miR-199b. Via The Cancer Genome Atlas (TCGA), we analyzed the molecular and clinical characteristics of 166 AML cases to investigate a prognostic role for miR-199b. The Kaplan-Meier curves for high and low expression values of miR-199b and the observed distribution of miRNA expression revealed the highly expressed group had significantly better survival outcomes (p<0.016, log rank test). In addition, miR-199b expression varied by cytogenetic risk category in the least squared mean plot, with significantly higher miR-199b expression for the favorable category compared to the intermediate and poor categories combined (p<0.0001). Therefore, low expression of miR-199b predicts worse survival outcome and higher cytogenetic risk in AML. Additionally, there was significant difference between miR-199b expression across the AML subtypes with particularly low expression found in the M5 subtype. Furthermore, M5 subtype showed a poor prognosis with a one-year survival rate of only 25%, compared with 51% survival in the overall sample (p<0.024). With AML patients grouped into M5 versus other subtypes to look at the dichotomized miR-199b variable, we found that all of the M5 patients have low miR-199b expression (p<0.0001). Due to miR-199b significantly correlating with M5 subtype, the M5 cell line THP-1 was utilized to determine the epigenetic regulation of miR-199b in AML. Recently, epigenetic alterations such as histone modifications and DNA methylation have been shown to deregulate miRNA expression. Therefore, treatment of THP-1 cells for 24 hours with HDAC inhibitors AR-42 (2uM), Panobinostat (0.7uM), or Decitabine (demethylating agent, 5uM) showed miR-199b expression was significantly elevated upon AR-42 and Panobinostat treatment (p<0.0002 and p<0.0001 respectively). Furthermore, AR-42 and Panobinostat treated cells showed drastic apoptosis in both treatments. Thus, epigenetic regulation of miR-199b appears to be via histone modification. To further understand the hematopathological consequences of decreased miR-199b in the onset and development of myeloid leukemia, we employed a transduce / transplant mouse model. The Lin-Sca-1+Kit+ (LSK) population was isolated from Ly5.2 C57Bl6 mice, transduced with anti-miR-199b or control lentivirus particles, and transplanted into Ly5.1 C57Bl6 recipient mice. Short term (3.5 weeks) peripheral blood analysis revealed that loss of miR-199b results in significant increase of % neutrophils (p<0.001), % monocytes (p<0.04), and % eosinophils (p<0.02). Further analysis to determine the role of miR-199b in leukemogenesis is ongoing via this mouse model. Taken together, for the first time our results demonstrate that in vivo loss of miR-199b can lead to myeloproliferation and in vitro HDAC inhibitors can restore miR-199b-5p expression and promote apoptosis. Lastly, low miR-199b-5p in AML patients leads to worst overall survival and has prognostic significance for FAB-M5 subtype. Disclosures:No relevant conflicts of interest to declare.

CD20 positive cells are undetectable in the majority of multiple myeloma cell lines and are not associated with a cancer stem cell phenotype

Although new therapies have doubled the survival of multiple myeloma patients, this remains an incurable disease. It has been postulated that the so-called myeloma cancer stem cells would be responsible for tumor initiation and relapse but their unequivocal identification remains unclear. Here, we investigated in a panel of myeloma cell lines the presence of CD20(+) cells harboring a stem-cell phenotype. Thus, only a small population of CD20(dim+) cells (0.3%) in the RPMI-8226 cell line was found. CD20(dim+) RPMI-8226 cells expressed the plasma cell markers CD38 and CD138 and were CD19(-)CD27(-). Additionally, CD20(dim+) RPMI-8226 cells did not exhibit stem-cell markers as shown by gene expression profiling and the aldehyde dehydrogenase assay. Furthermore, we demonstrated that CD20(dim+) RPMI-8226 cells are not essential for CB17-SCID mice engraftment and show lower self-renewal potential than the CD20(-) RPMI-8226 cells. These results do not support CD20 expression for the identification of myeloma cancer stem cells.

Development of a sensitive long-wavelength fluorogenic probe for nitroreductase: A new fluorimetric indictor for analyte determination by dehydrogenase-coupled biosensors

Nitroreductase (NTR) is a flavin-containing enzyme that uses NADH as the electron source to reduce nitroaromatic compounds to the corresponding amines. Previous studies have shown that nitroreductase-targeted latent fluorophores exhibit low solubility in the aqueous media and fluoresce at lower wavelengths upon uncloaking, thus limiting their effective applications. Here, we have prepared a new switch-on long-wavelength latent fluorogenic substrate, NTRLF ( 4), for NTR. In the presence of NADH, NTR catalyzes the reduction of the nitroaromatic moiety in NTRLF ( 4), followed by the cascade reaction, 1,6-rearrangement-elimination reaction, cyclic urea formation, and concomitant ejects a long-wavelength fluorescence coumarin ( 8). However, this reaction was inhibited in the presence of nitroaromatic analogues. The fluorescence signal generated by the cascade reaction was specific and insensitive to various reductants. Accordingly, we propose that NTRLF and NTR in the presences of NADH constitute a useful switch-off high-throughput fluorescence sensor for screening nitroaromatic compounds. Furthermore, NTRLF in the NTR-coupled 3-hydroxybutyrate dehydrogenase and aldehyde dehydrogenase assay reactions was a sensitive fluorimetric indicator for the quantitatively measurement of 3-hydroxybutyrate and propionaldehyde, respectively within micromolar range. Our novel NTRLF and NTR-coupled dehydrogenase assay platform may thus be effectively applied for the quantitative estimation of a broad range of analytes.

Creation of a Segment-Based Aldehyde Dehydrogenase Assay as a Biomarker for Umbilical Cord Blood Potency

Creation of a Segment-Based Aldehyde Dehydrogenase Assay as a Biomarker for Umbilical Cord Blood Potency