Hexosaminidase Assay Research Articles

Histodenz Separation of Lysosomal Subpopulations for Analysis of Chaperone-mediated Autophagy.

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, in which proteins are individually selected for lysosomal degradation. CMA degradation targets bear a pentapeptide consensus motif that is recognized by the cytosolic chaperone HSPA8 (Hsc70), which participates in the trafficking of the target to the lysosomal surface. From there, it is translocated into the lysosomal lumen, independent of vesicle fusion, in a process dependent upon the lysosomal transmembrane protein LAMP2A. There are limited tools for studying CMA in whole cells and tissues, and many of the best techniques for studying CMA rely on the preparation of lysosome enriched fractions. Such experiments include (1) the in vitro evaluation of CMA substrate uptake activity, (2) the characterization of changes to lysosomal resident and CMA regulatory proteins, and (3) lysosomal targetomics, i.e., the use of quantitative proteomics to characterize lysosomal degradation targets. Previous studies using discontinuous metrizamide gradients have shown that a subpopulation of liver lysosomes is responsible for the majority of CMA activity ("CMA+ "). These CMA+ lysosomes are low density and have higher levels of MTORC2 relative to the "CMA- " lysosomes, which are high density and have higher levels of MTORC1. Because of safety concerns surrounding metrizamide, however, this compound is difficult to obtain, and it is impractically expensive. Here, we have provided protocols for isolation of lysosomal subpopulations for CMA-related analyses from mouse liver using Histodenz, a safe and affordable alternative to metrizamide. Supplementary protocols show how to perform CMA activity assays with appropriate statistical analysis, and how to analyze for lysosomal breakage/membrane integrity. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation of lysosomal subpopulations from mouse liver using discontinuous Histodenz gradients Alternate Protocol: Isolation of lysosomes from cultured cells using discontinuous Histodenz gradients Support Protocol 1: Verifying enrichment of lysosomal markers in lysosome-enriched fractions Support Protocol 2: Measuring in vitro uptake of CMA substrates Support Protocol 3: Measuring lysosomal membrane integrity by hexosaminidase assay.

MP14-18 MODELING BLADDER CANCER IN DOWN SYNDROME ANIMALS FOR DISCOVERY OF NOVEL THERAPEUTICS

You have accessJournal of UrologyCME1 Apr 2023MP14-18 MODELING BLADDER CANCER IN DOWN SYNDROME ANIMALS FOR DISCOVERY OF NOVEL THERAPEUTICS Binh Vo, Erika Abbott, Carolyn Vivian, Xena Moore, Ganeshkumar Rajendran, Scott Weir, Katie Dennis, Benjamin Woolbright, and John Taylor Binh VoBinh Vo More articles by this author , Erika AbbottErika Abbott More articles by this author , Carolyn VivianCarolyn Vivian More articles by this author , Xena MooreXena Moore More articles by this author , Ganeshkumar RajendranGaneshkumar Rajendran More articles by this author , Scott WeirScott Weir More articles by this author , Katie DennisKatie Dennis More articles by this author , Benjamin WoolbrightBenjamin Woolbright More articles by this author , and John TaylorJohn Taylor More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Down syndrome (DS) is the most common chromosomal abnormality in humans and results in an increased risk for hematologic cancers, despite a paradoxically lower rate of adult solid tumors. Epidemiological evidence supports the idea that DS patients have a lower risk for bladder cancer (BCa) in spite of reduced environmental exposures. We sought to evaluate potential protective mechanisms against cancer formation in the Ts65Dn mouse model of DS, and hypothesized Ts65Dn mice would be protected against (BCa) formation and progression. METHODS: Tumors were initiated in Ts65Dn and WT mice using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Ts65Dn or WT animals were given 0.05% BBN for 16 weeks before sacrifice. Bladders were weighed at time of death. For Reactome pathways, Gene Set Enrichment Analysis (GSEA) was performed after RNA-seq. Proliferation was assessed using the hexosaminidase assay on HTB-9, HTB-5 and T24 BCa cells. HTB-9 cells (5×105) xenografts were implanted in the flank of nude mice in 50% Matrigel/saline and allowed to develop over two weeks. When mean tumor sizes per cage reached 50-100 mm3 determined using calipers, treatment with 25 mg/kg barasertib, i.p., Q1D for 5 days per week was started. Tumors were excised and weighed on the final day. RESULTS: After 16 weeks of 0.05% BBN exposure, Ts65Dn mice had smaller bladder tumors which trended towards lower stage tumors compared to WT mice. Although Ds mice and patients are known to have higher expression of anti-angiogenic proteins as such DSCR1, we did not see a decrease in angiogenesis in the Ds tumors, indicating potentially novel mechanisms of protection. RNA-sequencing indicated pathways associated with the cell cycle were most affected in the Ts65Dn mice, including downregulation of the cell cycle regulator Aurora Kinase B (AURKB). We evaluated drug sensitivity in a set of BCa cell lines using the AURKB specific inhibitor barasertib-HQPA. HTB-9 cells were highly susceptible to barasertib (IC50 ∼30-50 nM) whereas T24/HTB-5 cells had dramatically higher IC50 values (∼50 µM). Drug efficacy was assessed by evaluating phosphorylation of the AURKB substrate histone H3. Phospho-histone H3 levels were reduced in all cell lines and inhibition of a known transporter of barasertib using the compound valspodar minimally enhanced barasertib toxicity in T24 cells, indicating the potential for a novel mechanism of resistance. CONCLUSIONS: Our results indicate Ts65Dn mice have reduced tumor formation when exposed to BBN. Using Ts65Dn mice as a model may be a means for finding new drug targets as demonstrated by our results targeting AURKB. Source of Funding: Leo and Anne Albert Institute for Bladder Cancer and Institute for Advancing Medical Innovation at KUMC © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e189 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Binh Vo More articles by this author Erika Abbott More articles by this author Carolyn Vivian More articles by this author Xena Moore More articles by this author Ganeshkumar Rajendran More articles by this author Scott Weir More articles by this author Katie Dennis More articles by this author Benjamin Woolbright More articles by this author John Taylor More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract 5150: MIF-2 in bladder cancer: potential therapeutic target

Abstract Macrophage migration inhibitory factor (MIF1) is a pro-tumorigenic cytokine overexpressed in multiple cancers. Our previous data in a carcinogen (BBN) induced murine bladder cancer (BCa) model indicates genetic depletion of MIF1 induces stage arrest. Pharmacological inhibition of MIF1 with the inhibitor CPSI-1306 reduces tumor stage and bladder weights. These data indicate MIF1 is a therapeutic target in BCa. However, a second protein with significant homology to MIF1 was recently identified and termed MIF2. Both MIF1 and MIF2 bind and activate their receptor CD74 yielding significant functional homology and thus, targeting both proteins may be more efficacious. We hypothesize MIF2 recapitulates the pro-tumorigenic effects of MIF1 in BCa in a CD74 dependent manner. We assessed MIF1 and MIF2 by ELISA in serum from human patients with BCa and MIF1/2 expression were evaluated in TCGA. WT mice (C57Bl/6 background), MIF-/-, and CD74-/- mice were treated with 0.05% BBN for 16 weeks to induce BCa, and then switched to water. Tumors were allowed to develop until 20 weeks post initiation of BBN, a time frame consistent with muscle invasive tumor development. Some animals were given either vehicle, or a dual MIF1/MIF2 inhibitor 4-IPP (80mg/kg i.p. daily) for 4 weeks after cessation of BBN. We assessed bladder weights as a surrogate for tumor weight, tumor stage and immunohistochemistry for proliferation, angiogenesis and inflammation. In vitro cell proliferation was measured using the hexosaminidase assay and tautomerase activity was measured using two separate enzymatic assays for MIF tautomerase activity. MIF1 and MIF2 mRNA levels were elevated in TCGA. Analysis of serum from BCa patients indicated increased MIF1 and MIF2 in BCa patients, although this didn't reach statistical significance. 4-IPP treated C57Bl/6 animals had reductions in bladder weight, although tumor staging was similar between groups. 4-IPP treatment had no effect on bladder weights or tumor stage in CD74-/- mice supporting a role for CD74 mediated MIF signaling. To more fully assess the role of MIF2 directly, MIF1-/- mice were treated with 4-IPP to solely block MIF2 4-IPP treatment lead to major reductions in tumor stage, tumor incidence and bladder weight indicating inhibition of MIF2 may be more effective when MIF1 is chronically absent. Inflammation and proliferation trended lower, but did not reach statistical significance. MIF1 depletion did not affect MIF2 mRNA levels in bladder, heart or liver. In vitro treatment with 4-IPP substantially reduced proliferation of two high grade BCa cell lines (HTB-5, HTB-9) at concentrations consistent with doses found to inhibit recombinant MIF activity. These data indicate MIF1/2 enhance BCa tumorigenesis through activation of CD74. Combined MIF1/MIF2 may improve therapeutic targeting of MIF, especially during chronic MIF1 inhibition. Citation Format: Ganeshkumar Rajendran, Benjamin L. Woolbright, Erika Abbott, Austin Martin, Katie Dennis, John Arthur Taylor. MIF-2 in bladder cancer: potential therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5150.

Abstract 3867: Microsomal prostaglandin E synthase-1: A therapeutic target in bladder cancer

Abstract Introduction and Objective: Prostaglandin E2 (PGE2) is a lipid-derived signaling agent that promotes tumorigenesis via increasing stemness, proliferation, and angiogenesis. PGE2 levels are elevated in multiple cancers, including bladder (BCa) where they may play a pathological role. PGE2 is generated in part by cyclooxygenase enzymes (COX-1/COX-2). Clinical trials using the COX-2 specific inhibitor celecoxib to prevent progression and recurrence of non-muscle invasive BCa report hazard ratio reductions; however, celecoxib fails to significantly outperform standard of care. These studies are dose-limited by cardiotoxicity present with COX inhibition, but have suggested higher doses of celecoxib may yield significant results. Microsomal PGE2 synthase-1 (mPGES-1) is one of two terminal enzymes (downstream of COX-1/2) associated with PGE2 production. Inhibition of mPGES-1 does not affect synthesis of the prostaglandins responsible for cardiotoxicity but still reduces PGE2 levels. Using a model of carcinogen induced BCa, we have previously established mPGES1-/- have significantly smaller bladder tumors by weight, reduced pathological stage, reduced PGE-2 metabolites, and reductions in percent tumor involvement in the bladder compared to WT mice. The purpose of this study was to further understand mPGES mediated tumorigenesis using in vitro cellular models. We hypothesized pharmacological inhibition with the mPGES-1 specific inhibitor MF63 would reduce PGE2 levels leading to reduced cellular proliferation and reduced PGE2 release in vitro. Methods: mPGES1 protein and RNA levels were measured in benign (UROtsa) or malignant BCa cell lines using western blotting and qPCR respectively. PGE2 levels were assessed by ELISA. Cell proliferation was assessed using the hexosaminidase assay and colony formation assays. Cell death was assessed using lactate dehydrogenase (LDH) release. Results: In vitro, BCa cell lines HT-1376, RT-4 and HTB-9 significantly upregulate mPGES1 as compared to a benign urothelial cell line (UROtsa). To assess the capacity of MF63 to effect PGE2 production, we treated HT-1376 and HTB-9 cells with MF63 at increasing concentrations and assessed PGE2 levels. MF63 significantly reduced PGE2 production after 24h in both cell lines. Furthermore, pharmacological inhibition of mPGES1 with MF63 at concentrations of 10µM and above significantly reduced cellular proliferation after 48 or 72 hours using both colony formation and the hexosaminidase assay to assess proliferation. Finally, concentrations of 50µM MF63 robustly induced cell death as measured by LDH release. Conclusions: These data show that inhibition of mPGES1 in vitro with the inhibitor MF63 results in decreases in PGE2 levels and reduced cellular proliferation, likely due in part to cell death induction. mPGES1 inhibitors are expected to have improved safety profiles and given our results, may have therapeutic potential in BCa. Citation Format: Benjamin Leeland Woolbright, Jordan Barney, Van Schloegel Schloegel, Erika Abbott, John Arthur Taylor. Microsomal prostaglandin E synthase-1: A therapeutic target in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3867.

Tumor M2-PK: A novel urine marker of bladder cancer.

PurposeBladder cancer is a “Warburg-like” tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment.MethodsHTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hexosaminidase assay. Western blot and Blue-native analysis was performed for protein expression of PKM2. Shikonin, an herb that is known to bind and inhibit PKM2, was utilized to determine if PKM2 has a role in glucose usage and cellular proliferation in bladder cancer cells by caspase activity assay. Institutional review board approval was obtained to collect healthy control and bladder cancer patient urine samples. The ScheBo M2-PK EDTA Plasma Test was performed on urine samples to assess urine Tumor M2-PK values.ResultsThe three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL). Similarly, low doses of glucose (25mg/dL) result in reduced proliferation. Increased cell growth in higher glucose concentration correlated with up-regulation of PKM2 protein expression. Shikonin, a PKM2 inhibitor, reduced cell proliferation and switched PKM2 isoforms from the dimer to tetramer. Lastly, dimer PKM2 (Tumor-M2PK) levels were assessed in the urine samples from bladder cancer (Bca) patients and healthy controls. Tumor M2-PK significantly correlated with the presence of BCa in our subjects.ConclusionsOur studies demonstrate the potential of PKM2, specifically the dimer (Tumor-M2PK) as a target of drug therapy and as a urinary marker for bladder cancer.

Abstract 2865: Ciclopirox olamine: A common antifungal agent that inhibits growth of esophageal tumor cells in vitro and in vivo

Abstract Esophageal carcinoma continues to carry a poor prognosis due presentation at advanced stages. Current chemotherapeutics often only prolong survival for months to a few years. Drug repositioning is an important pharmacologic strategy that involves investigation and implementation of known drugs for treatment of new diseases. This method significantly reduces the costs required for establishing the safety of a drug and is associated with higher rates of drug approval. Ciclopirox olamine (CPX) is an antifungal agent that has been on the market since the 1970s. The mechanism of action is believed to include disruptions of DNA repair and cell division signals. Recently, however, this drug has been studied for its antitumor properties demonstrated in human rhabdomyosarcoma, breast carcinoma, colon adenocarcinoma, bladder carcinoma, and hematologic malignancies. To date, there are no studies related to the effect of CPX on esophageal cancer. Here, we show that ciclopirox olamine causes growth inhibition of four esophageal cell lines: TE-10, SKGT4, FLO1 and ESO1. This growth inhibition is demonstrated by hexosaminidase assay performed after incubation with the drug for 24-72 h. The IC50 values range between 5-20µM depending on the cell line used. Cell death was also demonstrated by immunofluorescent staining with Hoschst and propidium iodide, which shows a decrease in the number of viable cells with increasing concentrations of CPX. Clonogenicity assay was also performed with each cell line and demonstrates a decrease in the ability of cells to form colonies after 24 and 48 hour treatment with CPX and subsequent incubation in standard media for 5-7 days. Cell cycle analysis demonstrates G0/G1 arrest in cells treated with CPX. The effects of CPX on the cell cycle are further supported by Western blot analysis showing a decrease in in CDK4 and CDK6, which are necessary for cell cycle progression from the G0/G1 phase. We have also found that treatment with CPX results in a decrease in β-catenin in TE10 cells, suggesting that the drug is affecting this pathway to cause growth inhibition of tumor cells. Finally, we used our ESO1 mouse esophageal squamous cell carcinoma cell line for a xenograft study in which tumor cells were injected into the flanks of mice. Mice treated with intraperitoneal injections of 300 mg/m2 of CPX had smaller tumor volumes compared to untreated controls. In summary, we have shown that CPX inhibits growth of esophageal tumor cells both in vitro and in vivo. Our data suggest that CPX induces cell cycle arrest of tumor cells. The mechanism of tumor cell inhibition may be related to downregulation of the WNT/β-catenin signaling pathway. In future studies, we plan to analyze the effects of CPX on other components in the WNT family from in vitro cells as well as tumor xenografts. Citation Format: Randi J. Ryan, Prabhu Ramamoorthy, Dharmalingam Subramaniam, Shrikant Anant, Scott Weir. Ciclopirox olamine: A common antifungal agent that inhibits growth of esophageal tumor cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2865.

Abstract P5-14-06: Towards breast cancer prevention through reduced breast density: Suppressive effects of tamoxifen on normal breast epithelial cells

Abstract Background: Breast density is positively associated with risk of breast cancer and the local microenvironment in the normal breast is known to significantly impact breast cancer initiation and further progression. The selective estrogen receptor modulator tamoxifen, with beneficial clinical effects on breast cancer recurrence, has also been shown to reduce mammographic density potentially explaining the primary preventive effects of tamoxifen. Still, important gaps persist regarding the cellular and molecular basis of how tamoxifen modifies breast density and subsequently breast cancer risk. Purpose: To investigate the impact by tamoxifen on normal human breast epithelial cells (MCF-10A) with the additional ambition to mimic variations in breast density on the cellular level. Experimental design: Effects of tamoxifen on MCF-10A cells at different cellular densities were evaluated in vitro. Flow cytometry and the sulforhodamine-B assay were used to assess cell cycle distribution and proliferation. Cell adhesion to extracellular matrix proteins was analyzed using hexosaminidase assay. Modulation of integrin receptor levels was determined by Western immunoblotting. Results: Tamoxifen exposure resulted in impaired cell cycle progression of human breast epithelial cells with a dose-dependent G2/M-phase arrest and reduced cell proliferation. Irrespective of tamoxifen treatment, the percent cellular adhesion to fibronectin and collagen type I by MCF-10A cells was significantly greater in high-density models compared to low-density models. Following tamoxifen treatment the adhesion was reduced in all density models. Consistent with the tamoxifen-induced lower adhesion, the integrin α1 and β3 levels were reduced in a dose-dependent way. Conclusion: These data support the hypothesis that tamoxifen affects the normal breast epithelium and its adhesion capacity, which may contribute to the clinically observed decrease in breast density and a potential reduced risk of breast tumor establishment. Citation Format: Rosendahl AH, Puig Blasco L, Borgquist S. Towards breast cancer prevention through reduced breast density: Suppressive effects of tamoxifen on normal breast epithelial cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-14-06.

Abstract A26: Pimozide, an antipsychotic derivative, targets the STAT signaling pathway in osteosarcoma

Abstract Objective: Osteosarcoma (OS) is the most common pediatric bone cancer worldwide with an annual US incidence of 800. Chemotherapy and surgery have resulted in a marginal increase in survival of localized OS. However, that same treatment regimen has not been as successful for improving metastatic OS, as it yields an abysmal 5-year overall survival of less than 30% in patients afflicted with the disease. There have not been new successful standardized treatment agents in the last 2 decades for OS. Current standard practice still implements the same chemotherapeutic agents: cisplatin, doxorubicin, and methotrexate. These nonspecific agents have failed to yield optimal survival in metastatic OS patients. Hence, there is a need to utilize different agents that can specifically target signaling pathways involved in the pathogenesis of OS. One such pathway that has been found to be instrumental in the pathogenesis of OS is the JAK/STAT pathway, and the antipsychotic medication pimozide appears to be able to halt OS growth through inhibiting this pathway. Herein, we investigated the effects of pimozide on OS cancer growth and elucidated its mechanistic action through STAT signaling pathway. Methods: Human OS cell lines KHOS, MG63, and SJSA cell lines and normal osteoblast cells were used in the study. The effects of pimozide on cell proliferation were assessed by hexosaminidase and clonogenicity assays. Effects of pimozide on stem cells were evaluated by colonosphere formation. Furthermore, the effect of pimozide on OS cells was evaluated by live cell fluorescent staining. Western blot was utilized to evaluate the effect on STAT protein expression. Results: Pimozide treatment resulted in a dose- and time-dependent inhibition of proliferation and colony formation in all three osteosarcoma cell lines but not in osteoblast cells. It induces caspase 3 and 7 activity. Pimozide also showed reduced live cell fluorescent staining. Pimozide significantly reduced the number and size of spheroid formation. In addition, pimozide inhibits OS stem cell marker protein CD44 expression. Moreover, pimozide treatment decreased the expression of STAT5 signaling proteins on Western blot, which suggests that pimozide inhibits OS growth through STAT signaling pathway. Conclusion: Together, these data suggest that pimozide inhibits OS cancer growth by inhibiting the STAT signaling pathway. Pimozide could serve as a potential therapeutic agent for OS. Citation Format: Pablo Angulo, Katherine Chastain, Dharmalingam Subramaniam, Shrikant Anant. Pimozide, an antipsychotic derivative, targets the STAT signaling pathway in osteosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A26.

Human mesenchymal stromal cell therapy for damaged cochlea repair in nod-scid mice deafened with kanamycin

BackgroundKanamycin, mainly used in the treatment of drug-resistant-tuberculosis, is known to cause irreversible hearing loss. Using the xeno-transplant model, we compared both in vitro and in vivo characteristics of human mesenchymal stromal cells (MSCs) derived from adult tissues, bone marrow (BM-MSCs) and adipose tissue (ADSCs). These tissues were selected for their availability, in vitro multipotency and regenerative potential in vivo in kanamycin-deafened nod-scid mice. MethodsMSCs were isolated from informed donors and expanded ex vivo. We evaluated their proliferation capacity in vitro using the hexosaminidase assay, the phenotypic profile using flow-cytometry of a panel of surface antigens, the osteogenic potential using alkaline phosphatase activity and the adipogenic potential using oil-red-O staining. MSCs were intravenously injected in deafened mice and cochleae, liver, spleen and kidney were sampled 7 and 30 days after transplantation. The dissected organs were analyzed using lectin histochemistry, immunohistochemistry, polymerase chain reaction (PCR) and dual color fluorescence in situ hybridization (DC-FISH). ResultsMSCs showed similar in vitro characteristics, but ADSCs appeared to be more efficient after prolonged expansion. Both cell types engrafted in the cochlea of damaged mice, inducing regeneration of the damaged sensory structures. Several hybrid cells were detected in engrafted tissues. DiscussionBM-MSCs and ADSCs showed in vitro characteristics suitable for tissue regeneration and fused with resident cells in engrafted tissues. The data suggest that paracrine effect is the prevalent mechanism inducing tissue recovery. Overall, BM-MSCs and ADSCs appear to be valuable tools in regenerative medicine for hearing loss recovery.

Honokiol affects melanoma cell growth by targeting the AMP-activated protein kinase signaling pathway

BackgroundMalignant melanoma is an aggressive form of skin cancer with limited effective therapeutic options. Melanoma research concentrates on maximizing the effect on cancer cells with minimal toxicity to normal cells. AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis and has been shown to control tumor progression regulating the cell cycle, protein synthesis, and cell growth and/or survival. Honokiol (HNK) is a biphenolic compound derived from Magnolia officinalis, a plant that has been used in traditional Chinese and Japanese medicine for the treatment of various pathological conditions. Recent studies have shown that HNK has antitumor activity with relatively low toxicity. In this study, we demonstrated that the growth inhibitory effects of HNK on melanoma and melanoma cancer stem cells were mediated through the activation of AMPK and hence AMPK signaling in melanoma cells. MethodsWe determined the effects of HNK treatment on various melanoma cell lines. HNK-induced cell growth inhibitory effects were determined using hexosaminidase assay. Protein expression studies were done by immunoblotting. Primary spheroid assay was used to assess stemness by growing single suspension cells in ultralow attachment plates. ResultsHNK is highly effective in inhibiting melanoma cells by attenuating protein kinase B/mammalian target of rapamycin and AMPK signaling. HNK showed significant inhibition of the spheroid-forming capacity of melanoma cells and, hence, stemness. HNK significantly decreased the number and size of melanospheres in a dose-dependent manner. Western blot analyses showed enhanced phosphorylation of AMPK in melanoma cells. Furthermore, HNK decreased the cellular adenosine triphosphate pool in a dose-dependent manner with maximum effects observed at 48 hours. ConclusionsThe results suggest that HNK can target melanoma cells and mark them for cell death through AMPK signaling. Further studies are warranted for developing HNK as an effective chemopreventive/therapeutic agent in melanoma.

Abstract 5274: Wnt inhibitory factor 1 reduces the growth and migration of human adenoid cystic carcinoma cells

Abstract Background: Adenoid cystic carcinoma (ACC) is a common malignancy that arises in major and minor salivary glands. It is well characterized by relatively high rate of local recurrence, and late onset of distant metastases, which cause high morbidity and mortality. Dysregulation in the Wingless-type (Wnt)/β-catenin pathway contributes to many human tumors. Here, we report that downregulation of an important Wnt antagonist, Wnt inhibitory factor 1 (WIF1), promotes ACC cell proliferation and migration. Aims: To determine the status of WIF1 and study the effects of WIF1 on human adenoid cystic carcinoma cell growth, migration, spheroid formation and senescence. Methods: Genomic DNA isolated from human adenoid cystic carcinoma cell lines (ACC52 and ACC112) was used for methylation-specific PCR after bisulfite modification. ACC52 and ACC112 cells were treated with a demethylating agent, 5-aza-2′-deoxycytidine (DAC) [50 µM for 4 days], and used for total RNA isolation and real-time RT-PCR to determine WIF1 mRNA expression. To determine the growth suppressive effects of WIF1, ACC52 and ACC112 cells were transiently transfected with either pCI blast (vector) or pCI blast-WIF1 and assessed for cell proliferation, migration, spheroid formation and senescence. Cell proliferation was determined at different time intervals (24, 48, 72 h) by hexosaminidase assay. Cell migration and spheroid formation were assessed by scratch assay and soft agar assay, respectively. Cellular senescence was determined using senescence β-galactosidase staining kit. Results: Methylation-specific PCR demonstrate that WIF1 is silenced by promoter methylation in ACC cells. DAC treatment increased the WIF1 mRNA expression by 2.3-fold and 3.8-fold in ACC52 and ACC112 cells, respectively compared with vehicle treatment. WIF1 significantly decreased the proliferation and migration of adenoid cystic carcinoma cells. Additionally, WIF1 significantly decreased the number of spheroids formed at 7 and 17 days. Furthermore, WIF1 induced a remarkable increase in cellular senescence in both the cell lines. Conclusions: We demonstrate that WIF1 is downregulated by promoter hypermethylation in human adenoid cystic carcinoma cells. Importantly, WIF1 inhibited the cell growth, migration and cancer stemness, while inducing cellular senescence. Our findings suggest that WIF1 is a potent growth inhibitor of adenoid cystic carcinoma cells and therefore could be a potential therapeutic agent for ACC. Grant support: This work was supported by the Adenoid Cystic Carcinoma Research Foundation (LQ). LQ holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Ilangovan Ramachandran, Vengatesh Ganapathy, Lurdes Queimado. Wnt inhibitory factor 1 reduces the growth and migration of human adenoid cystic carcinoma 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 5274. doi:10.1158/1538-7445.AM2014-5274

FRI0342 Degranulation of Mast Cells is Enhanced by Direct Contact with Rheumatoid Arthritis Synovial Fibroblasts but not Dermal Fibroblasts

BackgroundA number of studies have shown that synovial mast cells are activated, and elicit a pro-inflammatory role in rheumatoid arthritis (RA). Synovial mast cells are located around the microvasculature, but...

Adult Sandhoff Disease With 2 Mutations in the HEXB Gene Presenting as Brachial Amyotrophic Diplegia

Sandhoff disease is a rare autosomal recessive metabolic disorder of GM2 gangliosides. It is caused by a lack of functional N-acetyl-β-D-glucosaminidase A and B because of mutations in the HEXB gene. We describe a 55-year-old woman with adult Sandhoff disease presenting as brachial amyotrophic diplegia. The assay of total hexosaminidase involving A and B showed decreased level of these activities. Hex-A was 4.6 nmol·min·mL (normal: 7.0-20.0 nmol·min·mL) and Hex-B was 0.1 nmol·min·mL (normal: 1.0-10.0 nmol·min·mL), respectively. Analysis of HEXB gene demonstrated 2 point mutations that were located at the exon 5 (c.619A>G) and exon 11 (c.1250C>T). Compound heterozygosity of these 2 mutations may trigger the development of distinct adult Sandhoff disease phenotype. Sandhoff disease should be considered in the differential diagnosis of lower motor neuron disease, such as brachial amyotrophic diplegia, even if the age at onset is more than 50 years.

발효처리한 당귀의 항알레르기 효능에 대한 연구

Objectives : Allergy is an immune dysfunction caused by degranulation from mast cells in the early phase of allergic disease. The purpose of this study was to investigate the anti-allergic effect of fermented Angelicae gigantis Radix in human mast cell line, HMC-1. Method : The Angelicae gigantis Radix was fermented by Lactobacillus acidophilus. The cell toxicity of fermented Angelicae gigantis Radix(FAGR) was determined by MTT assay. The release of <TEX>${\beta}$</TEX>-hexosaminidase from HMC-1 stimulated by phorbol-12-myristate 13-acetate (PMA) plus A23187 was determined by <TEX>${\beta}$</TEX>-hexosaminidase assay. Also, the concentrations of cytokines (interleukin-<TEX>$1{\beta}$</TEX>, -6, -8 and tumor necrosis factor-alpha) were measured by enzyme-linked immunosorbent assay. The gene expression of COX-2 from HMC-1 stimulated by phorbol-12-myristate 13-acetate (PMA) plus A23187 was determined by reverse transcription polymerase chain reaction. The release of histamine on substance P-stimulated HMC-1 was measured by histamine assay. Result : The FAGR suppressed the release of <TEX>${\beta}$</TEX>-hexosaminidase, a marker of degranulation, from HMC-1 stimulated by PMA plus A23187. The FAGR inhibited the production of interleukin-<TEX>$1{\beta}$</TEX>, -6, -8 and tumor necrosis factor-alpha. The FAGR inhibited the expression of COX-2 mRNA. The FAGR suppressed the release of histamine on substance P-stimulated HMC-1. Conclusion : These results provide that FAGR may be beneficial in the treatment of allergic inflammatory disease.

Advanced glycation end-products reduce podocyte adhesion by activating the renin-angiotensin system and increasing integrin-linked kinase

The aim of this study was to investigate the effects of advanced glycation end-products (AGEs) on podocyte adhesion and the underlying mechanisms. Immortalized mouse podocytes were exposed to various conditions and podocyte adhesion was evaluated using a hexosaminidase assay. The expression levels of integrin-linked kinase (ILK) were measured by quantitative polymerase chain reaction (qPCR) and western blotting. Treatment with AGEs resulted in a significant, concentration-dependent reduction in podocyte adhesion (P<0.05) and an incremental rise in ILK expression up to a maximum of 100%. Pretreatment with losartan significantly prevented the upregulation of ILK and attenuated the loss of podocyte adhesion observed in podocytes exposed to AGEs (P<0.05). However, the adhesion of losartan-treated podocytes remained lower than that of the podocytes exposed to bovine serum albumin. The results indicate that AGEs reduce podocyte adhesion via the upregulation of ILK expression, which occurs partly through activation of the renin-angiotensin system in podocytes.

Honokiol induces cytotoxic and cytostatic effects in malignant melanoma cancer cells

BackgroundMelanomas are aggressive neoplasms with limited therapeutic options. Therefore, developing new therapies with low toxicity is of utmost importance. Honokiol is a natural compound that recently has shown promise as an effective anticancer agent. MethodsThe effect of honokiol on melanoma cancer cells was assessed in vitro. Proliferation and physiologic changes were determined using hexosaminidase assay and transmission electron microscopy. Protein expression was assessed by immunoblotting. ResultsHonokiol treatment inhibited cell proliferation and induced death. Electron microscopy showed autophagosome formation. Reduced levels of cyclin D1 accompanied cell-cycle arrest. Honokiol also decreased phosphorylation of AKT (known as protein kinase B) and mammalian target of rapamycin, and inhibited γ-secretase activity by down-regulating the expression of γ-secretase complex proteins, especially anterior pharynx-defective 1. ConclusionsHonokiol is highly effective in inhibiting melanoma cancer cells by attenuating AKT/mammalian target of rapamycin and Notch signaling. These studies warrant further clinical evaluation for honokiol alone or with present chemotherapeutic regimens for the treatment of melanomas.

Titanium alloy surface oxide modulates the conformation of adsorbed fibronectin to enhance its binding to α5β1 integrins in osteoblasts

Our laboratory has previously demonstrated that heat (600°C) or radiofrequency plasma glow discharge (RFGD) pretreatment of a titanium alloy (Ti6Al4V) increased the net negative charge of the alloy's surface oxide and the attachment of osteoblastic cells to adsorbed fibronectin. The purpose of the current study was to investigate the biological mechanism by which these surface pretreatments enhance the capacity of fibronectin to stimulate osteoblastic cell attachment. Each pretreatment was found to increase the binding (measured by ELISA) of a monoclonal anti-fibronectin Ig to the central integrin-binding domain of adsorbed fibronectin, and to increase the antibody's inhibition of osteogenic cell attachment (measured by hexosaminidase assay). Pretreatments also increased the binding (measured by ELISA) of anti-integrin IgG's to the α(5) and β(1) integrin subunits that became attached to fibronectin during cell incubation. These findings suggest that negatively charged surface oxides of Ti6Al4V cause conformational changes in fibronectin that increase the availability of its integrin-binding domain to α(5) β(1) integrins.

Abstract 256: Involvement of ERK and ROS in CLEFMA-induced apoptosis in lung cancer cells

Abstract CLEFMA (4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid) is novel synthetic molecule with potent growth inhibitory activity in human cancer cells. Our microarray gene expression analysis suggested that it induces oxidative stress in lung adenocarcinoma H441 cells but not in normal lung fibroblasts. That oxidative stress is a triggering mechanism for both apoptotic and autophagic cell death has been supported by several recent reports. Compared to normal cells, the cancer cells are more vulnerable to oxidative stress-induced cell death because of their high metabolic activity, increased likelihood of electron escape during oxidative phosphorylation and overburdened antioxidant system. In this study, we tested our hypothesis that CLEFMA-induced apoptotic cell death was dependent on the accompanying increase in oxidative stress in H441cells. Methods: Inhibition of cell growth by CLEFMA (10 µM) was estimated by MTT or hexosaminidase assay. ROS production, cell cycle and apoptotic cell population were assessed by flow cytometry. The phosphorylation levels of extracellular signal-regulated kinase (ERK1/2), p38 and JNK1/2 were examined by Western blotting. N-acetylcysteine (NAC, 5 mM) and PD98059 (5 mM) were used as ROS scavenger and ERK1/2 inhibitors, respectively. The molecular markers of apoptosis were analyzed by RT-PCR and immunobloting. Results: NCI-DTP program confirmed anticancer activity of CLEFMA in 60 human cancer cell lines. CLEFMA-treated H441 cells showed typical molecular signatures of apoptosis- upregulated pro-apoptotic and downregulated anti-apoptotic genes. There was time-dependent activation of caspases 3/7, PARP cleavage and BAX; the expression of anti-apoptotic XIAP1 and BCL-xL was reduced, whereas pro-apoptotic p53 was increased. Since BID was not activated, it appears that CLEFMA works through intrinsic apoptosis pathway. CLEFMA also induced sustained activation of ERK, p38 and JNK. The pharmacologic inhibition of ERK, but not that of p38 and JNK, resulted in decreased anti-proliferative activity of CLEFMA. ERK inhibition with PD98059 abrogated CLEFMA-induced apoptosis. The sustained ERK activation was found to be associated with the inhibition of dual specificity phosphatase, DUSP6, expression. Treatment with ROS scavenger NAC abrogated CLEFMA-induced cell death, ERK activation and pro-apoptotic biomarkers, and reinstated DUSP6 expression. Conclusion: From the results of this study, we conclude that CLEFMA-induces apoptosis by enhancing oxidative stress in the cancer cells, and that the accompanying prolonged ERK activation is required for CLEFMA-induced H441 cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 256. doi:1538-7445.AM2012-256

Abstract 4887: Curcuminoid CLEFMA induces autophagy-associated apoptosis in lung cancer

Abstract CLEFMA (4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid) is a novel curcuminoid discovered in our laboratory. Earlier we showed that CLEFMA induces oxidative stress-dependent cell death in lung adenocarcinoma H441 cells. Although apoptosis is the desired mechanism for chemotherapy-induced cell death, the associated pathways are often defective in lung cancers resulting in high levels of chemoresistance. Therefore, drugs that induce alternate modes of cell death, such as autophagy, may be beneficial. Drug-induced hyperinduction of autophagy is classified as type 2 programmed cell death. In most circumstances, autophagy parallels apoptosis, and such composite cell death is described as “autophagy-associated apoptosis”. Whether autophagy plays a synergistic or antagonistic role in overall cell death response to anticancer drugs is an important focus in cancer research. In this study, we have investigated the interaction between autophagy and apoptosis in CLEFMA mediated cell death. Methods: The human non-small cell lung carcinoma (H441) cell line was treated with CLEFMA (10 µM) and the molecular mechanisms of cell death were investigated. Growth inhibition was assessed by MTT or hexosaminidase assay. Apoptosis was measured by flow cytometry of annexin V-stained cells, RT-PCR and immunoblotting for apoptosis-related biomarkers. Induction of autophagy was established by electron microscopy and immunobloting for LC3B, ATG, beclin1, AKT and mTOR. Autophagy was inhibited by 3-methyladenine (3-MA 1 mM) as well as chloroquine and E64D+pepstatin combination. The tumor suppressive efficacy of CLEFMA was demonstrated in H441 xenograft mice intraperitoneally receiving 0, 5 and 10 µg/day of CLEFMA for 32 days. The mice were imaged for F-18-fluorodeoxyglucose uptake in tumor tissue. Results: CLEFMA suppressed the viability of H441 cell line in a dose-dependent manner (IC50 = 6.25 µM). It induced cell cycle arrest at G2/M phase with accompanying accumulation of p21 protein. Caspase 3/7 immunoblotting and annexin V flow cytometry confirmed the induction of apoptosis. At the same time, we observed induction of LC3BII and suppression of AKT/mTOR phosphorylation. The suppression of autophagy significantly increased CLEFMA-induced cell death and increased caspase cleavage. In the xenograft model, 5 and 10 µg CLEFMA significantly reduced the tumor volume by 72% and 96.2%, respectively; the tumor uptake of F-18-fluorodeoxyglucose was also reduced. Conclusions: We demonstrate the efficacy of CLEFMA as a potent tumor suppressive agent. The mechanistic results suggest that autophagy is a survival mechanism in CLEFMA-treated lung cancer cells and it antagonizes apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4887. doi:1538-7445.AM2012-4887

Medium from mesangial cells incubated with aggregated IgA1 from IgA nephropathy patients reduces podocyte adhesion through activation of the renin angiotensin system

Podocyte injury plays an important role in glomerulosclerosis in IgA nephropathy (IgAN), and detachment from the glomerular basement membrane is the main cause of podocyte damage. In a previous study we found that medium from mesangial cells incubated with aggregated IgA1 (aIgA1) isolated from IgAN patients decreased podocyte adhesive capacity. However, the underlying mechanism remains unclear. Podocytes were incubated in medium from mesangial cells incubated with aIgA1 isolated from IgAN patients, enalaprilat (10-5 M) and chymostatin (20 μM), or with enalaprilat and chymostatin separately. Podocyte adhesive capacity was evaluated by cell counting and hexosaminidase assay. Expression of the renin angiotensin system was measured by real-time PCR, Western blot analysis and ELISA. Angiotensinogen, renin and angiotensin II type 1 and 2 receptors mRNA and protein expression, angiotensin-converting enzyme activity, and angiotensin II levels increased in podocyte lysates and conditioned culture media on exposure to mesangial medium containing aIgA1 from IgAN patients (P <0.05). Enalaprilat or chymostatin partly improved the reduced adhesive capacity of podocytes compared to cells exposed to mesangial medium (P <0.05), but it was still lower than for podocytes exposed to mesangial medium containing aIgA1 from healthy controls (P <0.05). Our findings indicate that activation of the renin angiotensin system in podocytes is partly involved in downregulation of adhesive capacity in podocytes by mesangial medium in IgA nephropathy.