bluuebunny/crossref_metadata_2025 huggingface dataset:
Xylobiose ameliorates hyperglycemia and dyslipidemia via regulating hepatic lipogenic genes in db/db mice
Authors: Kim Eunju , Lim Eunjin , Jung Sangwon , Yoo Sang‐Ho , Kim Yuri
Published: November 2023

Diabetes mellitus (DM) is a metabolic disorder in glucose and fat metabolism and has become a major public concern in worldwide. Xylobiose consists of two molecules of xylose and is a major disaccharide in a well‐known sweetener, xylo‐oligosaccharide. Although it has been reported the hypoglycemic effect of xylo‐oligosaccharide, the physiological effect of xylobiose on blood glucose and lipogenesis has not been evaluated. The objective of study was to evaluate the hypoglycemic and hypolipogenic effect of xylobiose in db/db mice. Five‐ week old male C57BL/KsJ‐db/db mice were randomized into 3 groups as follows: (1) a control group of C57BL/6 mice that received AIN93G (Ctrl), (2) a diabetic control group of C57BL/KsJ‐db/db mice that received AIN93G (DB), and (3) a group that supplemented with 5% xylobiose as part of the total sucrose content of the diet (XB5). Mice were fed the experimental diets for 6 weeks. Supplementation of xylobiose reduced food and water intake and improved glucose tolerance by reducing the levels of oral glucose tolerance test area under the curve, fasting blood glucose, HbA1c, insulin, and HOMA‐IR. These results indicate xylobiose attenuates hyperglycemia by regulating insulin and blood glucose metabolism. In addition, xylobiose supplementation significantly decreased the levels of TG, total cholesterol, and LDL‐C. Histopathologically, fat accumulation in liver tissues was reduced in XB 5 group. Expressions of lipogenic genes, including, FAS, PPARγ, SREBP‐1C, SREBP‐2, ACC, and HMG‐CoA reductase in liver tissues were significantly down‐regulated by xylobiose supplementation. Furthermore, xylobiose supplementation suppressed the mRNA expressions of pro‐inflammatory cytokines, TNF‐α, IL‐1β, IL‐6, and MCP‐1 and phosphorylation of three MAPK, such as JNK/SAPK, p38, and ERK (1/2). Taken together, xylobiose exhibits considerable anti‐diabetic, hypolipogenic, and anti‐inflammatory alternation in db/db mice.

Support or Funding Information

This research was supported by High Value‐added Food Technology Development Program (Project number: 313024‐03‐2‐HD040), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea, and Brain Korea 21 Plus (Project No. 22A20130012143)
Effects of Stevioside on oxidative DNA damage in liver and kidney of High Fat Diet Induced type 2 diabetes in Rats
Authors: Rotimi Solomon Oladapo , Rotimi Oluwakemi A , Obi Patience O. , Onuzulu Chinonye D , Ogunlana Olubanke O. , Dokunmu Titilope M. , Iyanda‐Joel Wisdom O
Published: November 2023

Type 2 diabetes mellitus (T2DM) is the most prevalent form of diabetes and it has been reported to be associated with oxidative stress‐induced cellular dysfunction including diabetic nephropathy. Stevioside (STV), a natural non‐caloric sweetener refined from the leaves of Stevia rebaudiana Bertoni, has been reported for its insulinotropic and antihyperlipemic effects. In order to investigate the influence of STV on oxidative stress and oxidative DNA damage, high fat‐low streptozocin rat model of T2DM were treated orally with 0.125mg/Kg, 0.25mg/Kg and 0.50mg/Kg body weight of STV for 21days. The levels of plasma insulin and dipeptidyl peptidase‐4 (DPP IV) were determined using enzyme‐linked immunosorbent assay while other biomarkers of T2DM, organ function, oxidative stress and lipid profile were assayed spectrophotometrically. DNA damage in the liver and kidney was determined by assessing the internucleosomal DNA fragmentation pattern on agarose gel electrophoresis. STV treatment resulted in decrease in the levels of fasting plasma glucose, insulin and DPP IV as well as in the activities of plasma amylase and kidney angiotensin‐converting enzyme. STV also significantly (p<0.05) improved plasma lipid profile and oxidative stress in the liver and kidney of the diabetic rats, with rats treated with 0.50mg/kg STV having the lowest levels of malondialdehyde and nitric oxide in liver and kidney. There was also a concomitant decrease in the fragmentation of genomic DNA in the liver and kidney of the diabetic rats. This ability of STV, administered orally, to prevent oxidative DNA damage in the liver and kidney of type 2 diabetic rats should contribute to its use in the management of T2DM.
Novel Nutritional Formula Protects Against Cellular Damage
Authors: Wood Steve M , Gray Russell , Hester Shelly N , Mastaloudis Angela F , Kern Dale , Namkoong Jin , Draelos Zoe D
Published: November 2023

Human cells possess protection and repair mechanisms that may be described as “aging defense mechanisms—ADMs.” Nutrients are important cofactors for cellular function and protection; similarly, natural ingredients, including phytonutrients, have been shown to support key ADMs. The purpose of this study was to determine the effects of a novel nutritional supplement—NNS (containing fish oil, vitamins K2 and D, alpha‐lipoic acid, co‐enzyme Q10, quercetin, citrus bioflavonoids—naringin and hesperidin, d‐limonene, resveratrol, carotenoids—lycopene, lutein, and astaxanthin, purple corn and rosemary extracts) on ADMs (i.e. cellular protection). Forty healthy, nonsmoking men and women between the ages of 40 and 75 years of age with Fitzpatrick skin types I and II were recruited to participate. Cellular injury was induced by 1 minimal erythema dose (MED), 2MED, and 3 MED solar simulated ultraviolet (UV) radiation exposure to non‐sun exposed skin. Assessments at baseline and 8 weeks post supplementation included: skin erythema at all three MED doses and biopsy of the 3MED site to determine # of apoptotic cells. In addition, total skin carotenoid levels were assessed non‐invasively using the BioPhotonic Scanner (Nu Skin Enterprises, Provo, Utah). There was a dramatic effect of the NNS on ADMs following 8 weeks supplementation. Decreases in skin erythema at all three MED doses: 1MED (p=0.019), 2MED (p<0.001), and 3MED (p=0.001) were observed. Supplementation with the NNS led to a significant reduction in the mean number of apoptotic cells, 11.6 at baseline vs. 5.7 cells/mm2 at the 3MED dose at week 8, suggesting that the NNDS protected against both uv‐induced DNA damage and apoptosis. Finally, skin carotenoid levels increased from 28,600 Raman Intensity Units (RIUs) to 38,775 RIUs (p<0.001) indicative of an increase in antioxidant protection in the skin tissue post‐supplementation. In conclusion, 8 weeks supplementation with the NNS supported key ADMs related to cellular health and function including protection against UV induced cellular damage, apopotosis, inflammation and erythema.

Support or Funding Information

The study was funded by Nu Skin Enterprises and registered on ClinicalTrials.gov (#NCT02525224)
Cyanidin, a Natural Anti‐Glycation Agent, Inhibits Methylglyoxal‐ and Glucose‐Mediated Protein Glycation in vitro
Authors: Suantawee Tanyawan , Cheng Henrique , Adisakwattana Sirichai
Published: November 2023

Introduction

Protein glycation and advanced glycation end products (AGEs) formation are associated with developing macrovascular and microvascular complications of diabetes mellitus. Cyanidin, a natural anthocyanidin abundant in various fruits and vegetables, has been reported a number of beneficial health effects attributed to its antioxidant properties. However, there are no studies related to the antiglycation activity of cyanidin.

Objective

The aim of the study was to investigate the inhibitory activity of cyanidin on in vitro protein glycation in bovine serum albumin (BSA) models; BSA/methylglyoxal (MG) and BSA/glucose systems.

Methods

The various concentrations of cyanidin were incubated with BSA and 1 mM methylglyoxal or 0.50 M glucose for 14 days at 37°C, pH 7.4. The fluorescent AGEs and non‐fluorescent Nɛ‐(carboxymethyl) lysine (CML) was assessed using a spectrofluorometer and an enzyme‐linked immunosorbent assay (ELISA). The protein oxidation was measured using the level of protein carbonyl content and protein thiol groups. The MG‐trapping capacity was performed by using high‐performance liquid chromatography (HPLC).

Results

The results demonstrated that cyanidin (0.125–1 mM) significantly inhibited the formation of fluorescent AGEs in BSA/MG (9.10–43.48%) and BSA/glucose (8.95–32.93%) systems in a concentration‐dependent manner. Correspondingly, cyanidin (1 mM) significantly decreased the level of CML in BSA/MG (55.77%) and BSA/glucose (49.42%) systems. Cyanidin prevented oxidative protein damage by protecting the depletion of protein thiol groups. Interestingly, cyanidin directly trapped MG and then significantly inhibited the formation of AGEs (5.92–36.95%).

Discussion

These results demonstrated that cyanidin was capable to inhibit MG‐and glucose‐mediated protein glycation and oxidative‐induced protein damage in BSA model. It suggested that cyanidin inhibited MG‐induced formation of AGEs related to its trapping ability to MG.

Conclusion

Cyanidin might be a new promising antiglycation agent for preventing or ameliorating AGEs‐mediated diabetic complications.
Specialist Program in Pathobiology: An undergraduate program in mechanisms of disease
Authors: Gotlieb Avrum , Templeton Douglas
Published: November 2023

A specialist undergraduate program was developed in Pathobiology at the University of Toronto in order to teach current concepts in mechanisms of disease to students selected from a large undergraduate life science student population in the Faculty of Arts and Science. The goal of the program was not to train laboratory professionals or to be a pre‐med course. New faculty appointments supported by the university provost, Faculty of Medicine and department were required. The faculty of the Department of Laboratory Medicine and Pathobiology was expanded to include a faculty of both basic and clinical scientists who were able to teach a unique curriculum consisting of one full and ten half courses on diverse topics in pathobiology, including cardiovascular pathobiology, neurodegenerative disease, cancer, inflammation, and immunopathology. Following foundation courses in the various life sciences in second year of the Arts and Science curriculum, the Pathobiology courses are introduced in third and fourth year. The courses are small with no more than 30 students in a class which permits more discussion and opportunities for mentoring of students. Students are given didactic lectures and prepare reports and grant applications focused on topics in pathobiology. Reading the current literature on pathogenesis of disease is required. There are hands‐on research opportunities through project course work and twelve‐week summer student placements in graduate faculty research laboratories where students are supervised by faculty, interact with graduate students and carry out a project which they report upon in poster format. Many students utilized these opportunities to select labs for graduate studies. Fourteen classes of Hons. B.Sc. students have graduated from the program. Outcomes were tracked for students graduating between 2007 and 2012 and outcomes for 99 students (70% of available graduates) were determined for the year following graduation. Of these, 52 went to medical school (including three into MD/PhD programs), 31 to graduate life sciences research, 10 to other health care‐related pursuits (e.g., pharmacy, public health), and six to other non‐medical careers. Two former students are currently residents in anatomic pathology. The undergraduate specialist (honours) program in Pathobiology requires a dedicated research faculty who are able to teach at the intersection of basic pathobiology and clinical disease and provides students with an excellent background and preparation for careers in basic discovery and clinical biomedical research.
“ATF6 Promotes Early Stem Cell Development”
Authors: Kroeger Heike , Chiang Wei‐Chieh , Wiseman R Luke , Lin Jonathan H.
Published: November 2023

Stem cells have scant and simplified organelles. The endoplasmic reticulum (ER) is the organelle of protein assembly, protein folding and protein maturation. The Unfolded Protein Response (UPR) is the regulatory mechanism that supports ER function and homeostasis. We identified significant expansion of the ER and the UPR regulators, IRE1, PERK, and ATF6, during early human embryonic stem cell development. Interestingly, the ATF6 pathway, but not the IRE1 or PERK pathways, was found to be strongly activated during early differentiation. Over‐expression of the transcriptional activator bZIP domain of ATF6 led to a dramatic increase of ER in hESCs. By contrast, pharmacologic inhibition of ATF6 cleavage by S1P reduced ER growth. We obtained similar findings using induced pluripotent stem cells (iPSCs), generated from achromatopsia patients that carry the loss‐of‐function R342C mutation in ATF6. In mutant ATF6 iPS cells, growth of the ER was significantly impaired during early stem cell differentiation when compared to wild‐type ATF6 iPS cells. Last, we found that ATF6 expression enhanced the acquisition of germ layer (ectoderm, mesoderm, endoderm) markers while ATF6 inhibition impaired acquisition of differentiation markers. We further identified that ATF6 expression can be stimulated by the FGF2 signaling cascade during these early differentiation steps. In summary, we identify a novel function for ATF6 in stem cells to promote ER expansion coupled with differentiation. Our findings suggest defects in stem cell differentiation may contribute to the early pathologies seen in human patients bearing loss‐of‐function ATF6 mutations.
Modeling melanoma as in‐vitro artificial tumor tissues using natural 3D matrix materials
Authors: Lyden Timothy , Martin Mike
Published: November 2023

Since 2004, the University of Wisconsin‐River Falls Tissue and Cellular Innovation Center (TCIC) has been engaged in modeling a variety of artificial tissue constructs using several natural 3D matrix materials as well as hanging drop organoids. These constructs have included primary tissues of fetal origin and patient tumor samples as well as continuous cell lines of tumors and “normal” tissues from several species including human. Most recently, we have begun a series of studies to examine and model 3D tumor development of melanoma using the paired mouse cell lines B16F1 and B16F10. These lines represent low and high metastatic lineages respectively. In our preliminary studies with these cells we have employed the newly developed 3D Biomatrix hanging drop system to generate tumor organoid structures. Additionally, we have also employed a natural 3D decellularized extracellular matrix material to generate large‐scale, ~0.5 cm3, artificial tumor structures. These structures displayed 2 distinct morphological arrangements as the tissue underwent progressive developmental changes. These two arrangements include multiple branching projections or large rounded and smooth hemispherical projections. In some cases, the branching projections arise from similar rounded regions of the overall artificial tumors. Melanin containing cells are present within the branching structures and some, but not all of the rounded features. Individual highly pigmented and dendritic cells can be observed within the developing tumor structure in early stages, but become indistinguishable in later and more darkly pigmented structures. To date, viable artificial melanoma tumors have been maintained for more than 30 days. Throughout this time after an initial colonization and outgrowth period of approximately 1–1.5 weeks, the main tumor mass generated and released branching processes which then established new explanted monolayers. These structures also parallel organoids generated in hanging drop cultures. Taken as a whole, these studies demonstrate that 3D artificial tissues generated from B16F1 and B16F10 cell lines provide a viable and complex model of melanoma tumor tissues in vitro.
INDOLEAMINE2,3‐DIOXYGENASE INHIBITION DECREASES VEGF‐A EXPRESSION IN HUMAN BLADDER CANCER CELLS AND DIMINISHES ANGIOGENESIS IN COCULTURE WITH HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC)
Authors: Malta Camila Soares , de Oliveira Brito Rodrigo Barbosa , Matos Yves Teles , de Souza Diego Mota , Matheus Luiz Henrique Gomes , Dellê Humberto
Published: November 2023

Indoleamine 2,3‐dioxygenase (IDO) is an enzyme with immunomodulatory properties and it is expressed in the placental tissue, therefore, it has a role in protecting the embryo against the maternal immune system. The expression of IDO has been found in several types of cancer, including bladder cancer, being related to tumor development. In addition, IDO is expressed in tumor vessels, hypothesizing that IDO could be involved in the tumoral angiogenesis process.

The aim was to verify if IDO has an influence on the angiogenesis process induced by bladder cancer cells underwent to hypoxia, analyzing the effect of IDO inhibition on VEGF expression and in vitro angiogenesis.

In the first step, human bladder cancer cells (T24, ATCC) were cultured under normoxia or hypoxia for 1h, 8h, 24h or 48h, in order to identify the better period to induce high VEGF‐A expression (qPCR). The hypoxia was induced by AnaeroGen System (O2<1%, Oxoid). Then, the T24 cells were treated with the IDO inhibitor (MT, 1‐methyl‐D‐tryptophan) to analyzed the effect of MT on VEGF‐A expression in hypoxia condition. HIF‐1alpha was also analyzed by qPCR and Western Blot (WB).In the second step, T24 cells were cocultured with matrigel‐coated HUVEC cells (ATCC) under hypoxia for 48h, with or without MT, and the vascular tubes were counted. MTT method was used to evaluate the viability of the MT‐treated HUVEC cells.

VEGF‐A expression increased in a time‐depending fashion under hypoxia, reaching the higher value at 48h (relative expression of 13.16±0.83 vs. 1.00±0.51 in normoxia; p<0.05). The transcriptional expression (mRNA) of HIF‐1alpha was not altered by hypoxia, however, an expressive increase was observed by WB (qualitative analysis). Treatment with MT significantly reduced the VEGF‐A expression on T24 cells and decreased HIF‐1alpha by WB. When T24 cells were cocultured with HUVEC, a significant reduction of vascular tube formation was observed under hypoxia (31.57±3.02 tubes/field in non‐treated cells vs. 18.08±2.06 tubes/field in MT‐treated cells; p<0.05). The MTT analysis revealed that MT treatment significantly reduced the HUVEC viability (reduction of 30%; p<0.05).

These preliminary results demonstrated that IDO modulates angiogenesis in bladder cancer, being a possible target to alternative therapeutic strategies.
Renal subcapsular space of Balb/c nude mice as a route to evaluate differentiation of human bladder carcinoma T24 cells subpopulations
Authors: Souza Diego Mota , Matheus Luiz Henrique Gomes , de Oliveira Brito Rodrigo Barbosa , Matos Yves Silva Teles , Malta Camila Soares , Ferreira Janaina Mendes , Silva Chrisna Souza , Dellê Humberto
Published: November 2023

Recurrence of bladder cancer after resections with progression to more aggressive forms are critical to promote cancer‐specific death and metastasis. Subpopulations of cancer cells, including cancer stem‐like cells, have been found inside the bladder tumors, having different abilities for malignancy and chemotherapy resistance. Subpopulations have been described and isolated from a culture of T24 cells, which represent major human epithelial bladder cancer cell with clinical relevance. However, in situ influences can not be assessed by another way except by animal model. Since T24 cells are not tumorigenic in nude mice when inoculated subcutaneously and technical limits exist in orthotopic procedures, we evaluated the renal subcapsular space as an alternative route to analyze these cells in vivo.

Balb/c nude mice underwent renal subcapsular inoculation of 1×106 T24 cells, suspended in two different volumes of PBS (50 μl and 10 μl). Four weeks post‐inoculation, grafted kidneys were harvested and fixed for histology and immunohistochemistry analysis. Other tissues were investigated for metastasis.

Inoculation of the cells in a volume of 50 μl was unsuccessful. However, a very high success rate was reached when 10μl volume was used (87.5%). It was possible to identify the formation of a pseudo‐bladder structure on renal subcapsular space, as well as a clear cellular differentiation, which was immunohistochemically characterized for epithelial to mesenchymal transition (cytokeratin for epithelial cells and vimentin for mesenchymal cells). Subpopulations of T24 cells were found infiltrating the renal parenchyma toward the medulla, besides surrounding renal vessels.

Renal subcapsular inoculation is an effective route to analyze subpopulations and differentiation of T24 cells in vivo.

Support or Funding Information

FAPESP (2012/04423‐0)
Cigarette Smoke‐Induced Bladder Cancer Initiation
Authors: McHowat Jane , Marentette John
Published: November 2023

Tobacco smoking is a major risk factor associated with the development and metastasis of bladder cancer. Muscle‐invasive bladder cancer with a high risk of metastasis is the most common cause of mortality amongst bladder cancer patients, with a 5‐year survival rate below 10%. Cigarette smoking and increased metastasis correlate, but the underlying mechanism is unknown. Platelet activating factor (PAF) is a potent inflammatory mediator that we have shown to be involved in attachment of tumor cells to the endothelium lining the vasculature following cigarette smoke extract exposure. Attachment to endothelial cells occurs through the interaction between the tumor cell PAF‐receptor (PAFR) and PAF on the endothelial cell surface. The PAF/PAFR interaction facilitates transendothelial cell migration of tumor cells into the circulation to establish secondary tumor sites. PAF has also been shown to promote growth of the primary tumor via angiogenesis, allowing for increased nutrient supply to the developing tumor. Pigment epithelium‐derived factor (PEDF) is an anti‐angiogenic protein that has been shown to be down regulated in higher grade tumors. Preliminary experiments in which we exposed mice to cigarette smoke for 4 weeks indicated signs of urothelial cell hyperplasia, increased basal PAF expression, reduced PEDF expression and nuclear localization of β‐catenin. These observed changes indicate that PAF accumulation and PEDF down regulation could increase growth of primary bladder tumors as well as increase the potential for metastasis. This work highlights two potentially exciting therapeutic targets for managing bladder cancer: targeting the PAF‐PAFR interaction or increasing expression of PEDF.
Implications For Platelet Activating Factor Receptor Antagonism For Managing Bladder Cancer Progression
Authors: McHowat Jane , Marentette John
Published: November 2023

Cigarette smoking is the number one risk factor for bladder cancer development and epidemiological data suggests that nearly half of all bladder cancer patients have a history of smoking. In addition to stimulating growth of a primary tumor, it has been shown that there is a correlation between smoking and tumor metastasis. Platelet activating factor (PAF) is an inflammatory cell recruiter that is expressed on the endothelium during times of inflammation and through binding with the PAF‐receptor (PAFR) facilitates transendothelial migration of circulating inflammatory cells. PAF has been implicated in primary tumor growth and interactions with the PAFR have been shown to stimulate cellular growth. PAF is generated through hydrolysis of membrane phospholipids by the activity of calcium‐independent phospholipase A2 (iPLA2) which removes the sn‐‐2 fatty acid yielding a free fatty acid, arachidonic acid, and a lysophospholipid. Acetylation of the newly formed lysophospholipid results in PAF production. Under normal circumstances, PAF is maintained at a low concentration by PAF‐acetylhydrolase (PAF‐AH) resulting in the inactive lyso‐PAF. In the present study, we show that cigarette smoke extract (CSE) exposure of bladder cancer cells inhibits PAF‐AH activity and increases iPLA‐2 activity resulting in increased PAF accumulation. In addition to PAF accumulation, CSE increases PAFR expression. Furthermore, by immunohistochemical analysis of tumor biopsy samples from bladder cancer patients we detected increased PAF, iPLA2 and the PAFR in tumor regions when compared to surrounding tissue. This data highlights a pathway that is upregulated in tumor areas of bladder cancer patients and that is influenced by CSE which could facilitate primary tumor growth and increase metastatic potential. Targeting of the PAF‐PAFR interaction could serve as a beneficial therapeutic target for managing further growth of a developing tumor.
Lactate Dehydrogenase A (LDH‐A) May Not Be Primarily Responsible for Maintaining High Tumor Lactic Acid Levels in Aggressive Breast Cancer
Authors: Mack Nzinga , Mazzio Elizabeth , Soliman Karam
Published: November 2023

A well‐known characteristic of tumor cells is the propensity to produce excessive amounts of lactic acid when deprived of normal levels of oxygen, and also when exposed to normal oxygen levels, also referred to as the Warburg effect. High levels of lactate are believed to be associated with elevated expression of lactate dehydrogenase A (LDHA), a protein up‐regulated in diverse aggressive malignancies. In this study, shRNAs were used to permanently knock down the expression of LDHA in human MDA‐MB 231 and MDA‐MB 468 cells. LDHA knockdown was confirmed by both Western Blot and qPCR. Furthermore, metabolic analysis included assessment of changes in cell viability, glucose utilization, lactate levels and cellular energy production by quantification of ATP. Silencing of the LDHA gene and protein had no effect on lactate production, glucose utilization, cellular metabolic activity and ATP in MDA‐MB 231 and MDA‐MB 468 tumor cells. These findings show no relationship between LDH‐A expression in both cells lines examined and production of lactate, nor is there any correlation to changes in the way glucose is utilized to produce ATP. The results obtained with the MDA‐MB 468 cell line confirm data previously obtained with the MDA‐MB 231 cell line. The data also suggest that cells readily adapt to knockdown conditions to continue their normal metabolic activities. Further analysis will require genomic profiling to discover shifts in gene expression that are responsible for this sustained production of lactate.

Support or Funding Information

Supported by grants from the National Institute on Minority Health and Health Disparities, NIMHD G12 MD007582 and P20 MD006738.
PEDF Reduction Induced by Cigarette Smoke: A Potential Role for Breast Tumor Development
Authors: Kispert Shannon E , McHowat Jane
Published: November 2023

There are many risk factors for cancer development and subsequent metastasis which include lifestyle choices such as diet, alcohol, and cigarette smoking. Smoking has been linked to several cancers including breast cancer. Many cohort studies have described the link between patients with breast cancer and those with long term smoking history. Despite the claim of correlation, the mechanism by which cigarette smoke alters breast epithelial cells and contributes to tumor cell growth and metastasis remains undefined. Pigment epithelium‐derived factor (PEDF) is a potent anti‐angiogenic factor and is shown to have inhibitory effects in numerous cancers. PEDF is often decreased or absent in tumor tissue when compared to surrounding stroma and its absence has resulted in cellular atypia. Female PEDF knockout mice show histologic changes in the breast at six months of age consistent with atypical ductal hyperplasia. We have shown that cigarette smoke exposure of breast tumor cells resulted in decreased PEDF expression. In order to assess the effects of cigarette smoke on the breast in‐vivo, we exposed six month old female mice to cigarette smoke using the SCIREQ inExpose system for up to six months and harvested mammary tissue for histological and biochemical analysis. Histological analysis revealed changes that include atypical epithelial growth in mammary tissue from mice exposed to six months of cigarette smoke when compared to those exposed to room air only. Immunohistochemistry showed increases in expression of proteins commonly upregulated in breast cancer including Ki‐67, VEGF, and COX‐2 in mammary tissue exposed to six months of smoking when compared to room air controls. Smoke exposure also caused a decrease in PEDF expression when compared to room air tissue. We have shown here for the first time that cigarette smoke alters the histology of the mammary tissue in mice and alters expression of important markers involved in human breast tumor development and progression.

Support or Funding Information

Presidents Research Fund Saint Louis University School of Medicine
Androgen Receptor Represses the Expression of Minichromosome Maintenance Proteins: a Mechanism for Androgen Therapy in Castration Resistant Prostate Cancer
Authors: Valencia Kevin , Han Dong , Gao Shuai , Han Wanting , Cai Changmeng
Published: November 2023

Standard treatment for metastatic prostate cancer is androgen deprivation therapy (ADT). Though this is often efficacious, for many patients, this invariably leads to the development of a more aggressive form of prostate cancer that is Castration Resistant Prostate Cancer (CRPC). Previous studies have shown that restoration of Androgen Receptor (AR, a member of nuclear receptor transcription factor family) activity plays a critical role in prostate cancer development and progression to CRPC. We have reported that in prostate cancer AR can function as both transcription activator and repressor. The latter suppressive function of AR is mediated by the recruitment of hypophosphorylated Retinoblastoma Protein (Rb), which results in negative regulation of the expression of Minichromosome Maintenance Proteins (MCMs). This protein family is composed of six distinct subunits (MCM2‐7) that form pre‐replication complex and are essential for replication initiation. Preliminary data of AR ChIP‐seq and Rb ChIP‐seq in CRPC cells showed the co‐binding on the promoters of MCM genes. Therefore downregulation the expression and activity of MCMs may be a potential mechanism for Rb‐dependent AR growth suppressive function in CRPC. We hypothesized that in CRPC cells, the AR activity stimulated by androgen treatment can have a repressive effect on the function of MCMs through recruitment of Rb to promoters, can subsequently suppress DNA replication process, and can thereby repress cancer cell proliferation. We alter Rb expression and phosphorylation as well as the expression of MCMs in prostate cancer cell lines (VCaP, LNCaP, and C4‐2) and then measure DNA replication by labeling newly replicated DNA with halogenated nucleotide precursors (BrdU, CldU, IdU) in conjunction with the expression level of MCMs, to determine the role of MCMs in AR mediated growth repressive function in CRPC. This AR growth repressive function may be mediated by downregulation of the expression and activity of MCMs through recruitment of hypophosphorylated Rb.

Support or Funding Information

R00CA166507 (PI: Cai) 06/01/2015‐05/31/2017 (R00) NCI/NIH Molecular Basis of Androgen Receptor Mediated Gene Transcriptional Repression Objective: The aims of this project are to determine the molecular basis and functional importance for AR mediated repression of genes in prostate cancer cells. Aim 1. Determine the mechanisms of AR‐mediated repression on the subset of DNA synthesis and repair genes. Aim 2. Assess treatment with high dose androgen in combination with metabolic inhibitors on castration‐resistant PCa cell lines. Contact: Martinson O. Owusu 240‐276‐6297 martinson.owusu@mail.nih.gov
TIMP‐2: A Novel Biologic Therapy for Triple Negative Breast Cancer
Authors: Castro Nadia Pereira , Chowdhury Ananda , Jensen Sandra Mae , Cuttitta Frank , Stetler‐Stevenson William George , Solomon David
Published: November 2023

Triple‐negative breast cancer (TNBC) presents the poorest prognosis among the breast cancer subtypes, having no current standardized therapy. Here, we used an orthotopic mouse model that establishes spontaneous lung metastasis from JygMC(A) cells. These primary tumors resemble human TNBC both phenotypically and molecularly. Tissue inhibitor of metalloproteinase‐2 (TIMP‐2) belongs to a small family of low molecular weight proteins identified as endogenous metalloproteinase inhibitors. These proteins modulate growth, migration and invasion of tumor cells and inhibit tumor angiogenesis. The mechanisms involve both metalloproteinase‐dependent (inhibition of protease activity) and ‐independent activities. In our TNBC model, JygMC(A) cells were implanted into both sides of the 4th inguinal mammary fat pads of athymic nude mice (10 mice per experimental group). Ten days following tumor cell inoculation, a daily intraperitoneal (IP) injection of recombinant TIMP‐2 protein was given at doses ranging from 0.75/100ml to 6.0 mg/100ml. Tumor growth was measured biweekly by caliper measurement for a total of 4 weeks). In our TNBC model, preliminary results showed that recombinant TIMP‐2 protein inhibited tumor growth by 50% at day 34 post tumor cell injection at a dose concentration as low as 1.5 mg/100 ml. Taken together, our data show that recombinant TIMP‐2 contributes to reduction of mammary tumor size in this mouse model revealing a therapeutic potential of a biologic protein able to decrease mammary carcinoma progression. Further studies will examine the MMP‐dependent and MMP‐independent mechanisms responsible for reduced tumor growth in this model system. Moreover, these findings may lead to a more efficient clinical treatment strategy for TNBC.

Support or Funding Information

This work was funded by the NCI Center for Cancer Research Intramural Research Program Grant # ZIA SC009179‐24.
PAF/PAF‐R Expression and Effects of Cigarette Smoke Exposure in Human Breast Cancer
Authors: Kispert Shannon E , Schwartz Theresa , McHowat Jane
Published: November 2023

Platelet activating factor (PAF), a potent inflammatory lipid mediator, was first suggested to play a role in smoking‐induced injury when the plasma taken from tobacco smokers was determined to have higher concentrations of PAF than the plasma obtained from nonsmokers. Recent studies from our lab have demonstrated that cigarette smoke extract (CSE) inhibits PAF‐AH activity, the enzyme responsible for PAF catabolism and inactivation. PAF is not only an important mediator of inflammation but is also involved in many phases of cancer progression that include tumor growth, metastasis, and angiogenesis. PAF has been shown to trigger a variety of intracellular pathways including those involving tumor angiogenesis. Inhibition of PAF‐AH activity by cigarette smoke extract (CSE) enhanced endothelial cell surface expression of PAF and increased breast cancer cell adherence. When breast cancer cells were incubated with CSE, expression of the PAF receptor was increased and resulted in enhanced adherence to the endothelium. Conversely, when breast cancer cells were treated with the PAF receptor antagonist ginkgolide B, their adherence to lung endothelial cells was inhibited. Human breast cancer tissue showed higher PAF receptor expression via immunohistochemistry and immunofluorescence when compared with normal human breast tissue. PAF was also increased in human breast cancer tissues when compared with surrounding normal tissue. Thus, increased PAF receptor expression and PAF accumulation may contribute to breast tumor development and progression. Additionally, we propose that cigarette smoking may contribute to breast cancer via facilitation of the PAF‐PAF receptor interaction.

Support or Funding Information

Presidents Research Fund Saint Louis University
The Histone Deacetylase Sirt2 Regulates Slug in Basal‐like Breast Cancer
Authors: Zhou Wenhui , Kuperwasser Charlotte
Published: November 2023

Basal‐like breast cancer (BLBC) represents the most aggressive and deadliest breast cancer subtype, affecting 20% of breast cancer patients. Unlike other subtypes, few driver mutations have been identified and, thus, no targeted therapies have been established. We previously observed the transcriptional repressor Slug/SNAI2 is frequently overexpressed in diseased breast tissues of women with inherited mutations in BRCA1; this aberrant expression of Slug represents an important determinant in the pathogenesis of aggressive BLBC. The mechanism by which Slug becomes deregulated in cancer, however, remains unknown. Using a small molecular inhibitor screen, we identified Sirt2 as a master regulator of Slug. Sirt2 inhibition leads to rapid turnover and depletion of Slug protein, whereas Sirt2 overexpression stabilizes Slug and increases its level and activity. Mechanistically, Sirt2 binds to and deacetylates Slug protein at specific lysine residues to prevent its proteasomal degradation, thereby stabilizing Slug protein. Analysis from human breast cancer patients revealed that Sirt2 is frequently amplified, overexpressed and strongly correlated with Slug status in basal‐like tumors. Importantly, Sirt2 is induced following BRCA1 inhibition and inactivation of Sirt2 in BRCA1‐associated breast cancer leads to rapid Slug turnover and loss of pathological features that are clinically relevant to BLBC tumors. Collectively, these observations revealed a regulatory mechanism controlling Slug abundance, whereby dysregulated Sirt2 manifests into Slug overexpression in human cancer. As such, targeting Sirt2 may be a rational strategy to molecularly dampen the Slug hyperactivity and malignant behaviors characteristic of BLBC and those associated with BRCA1 mutation.

Support or Funding Information

This work was supported by grants from the Breast Cancer Research Foundation, the NIH/NICHD (R01HD073035), and the NIH/NCI (P01CA092644)
FSTL1 Inhibits Tumor Growth and Metastatic Progression via Blocking Proteolytic Activation of SPP1 in Lung Adenocarcinoma
Authors: Chiou Jean , Su Chia‐Yi , Lin Yuan‐Feng , Hsiao Michael
Published: November 2023

Follistatin‐related protein 1 (FSTL1) plays a critical role in lung development through the interaction with bone morphogenetic protein (BMP) 4 and transforming growth factor β1 (TGF‐β1) signaling pathways. Although the supplement of FSTL1 was appeared to inhibit the in vitro cell growth of lung cancer cells, the precise mechanism and its role in lung cancer aggressiveness remains unclear. Here we found that low‐level FSTL1 is extensively detected in tumor tissues and significantly correlates with poor prognosis in patients with non‐small cell lung cancer, particularly lung ademonocarcinoma. Moreover, the expression of FSTL1 was inversely associated with the in vitro migration ability of several lung adenocarcinoma cell lines. Robustly, the treatment with recombinant FSTL1 protein significantly decreased migration and invasion of highly metastatic CL1‐5 cells in a dose‐dependent manner. In contrast, the mobility of poorly metastatic CL1‐0 cells was dose‐dependently enhanced by neutralizing FSTL1 using its specific antibody. Furthermore, the enforced expression of exogenous FSTL1 gene suppressed but the knockdown of FSTL1 promoted the in vitro and in vivo metastatic potential of CL1‐5 and CL1‐0 cells, respectively. Importantly, the administration of FSTL1 effectively inhibited the lung metastasis of CL1‐5 cells in an orthotropic animal model. The mass spectrometric analysis of FSTL1‐interacting proteins revealed that FSTL1 is capable of binding to the secreted phosphoprotein 1 (SPP1), Furthermore our results showed that FSTL1 binds to the matrix metalloproteinase‐3/7 (MMP3/7)‐recognition site and thereby blocks the proteolytic activation of pro‐SPP1 and its downstream signaling to ultimately inhibit lung cancer cell invasion and metastasis.

These results suggest that the downregulation of predicts a poor prognosis in lung adenocarcinoma and the administration of FSTL1 could be a new therapeutic strategy to inhibit tumor growth and combat metastatic progression in lung adenocarcinoma via preventing the proteolytically processing of pro‐SPP1 into its active form.
Pharmacogenomic Analysis of Adenylate Kinase‐4 Gene Expression Signature Identifies Withaferin‐A as an Anti‐Metastatic Agent in Non‐Small Cell Lung Cancer
Authors: JAN YI‐HUA , HSIAO MICHAEL
Published: November 2023

Lung cancer remains the leading cause of cancer death in the world due to metastatic disseminations. So far, there are no effective therapies available to fight this fatal process. Previously, we identified adenylate kinase 4 (AK4) as a lung cancer progression marker that enhances the invasion and metastasis ability of lung cancer cells. Therefore, we queried connectivity map profiles of AK4‐associated gene expression signature and identified Withaferin‐A (WFA) as a potential agent to treat metastatic lung cancer. We found WFA potently inhibited invasion and migration of CL1‐5 and A549 cells up to 50 percent at low‐cytotoxic doses in vitro. WFA treatment also abolished the invasion activity induced by AK4 ectopic overexpression in CL1‐0 cells. Microarray analysis further revealed that differentially regulated genes upon WFA treatment were significantly enriched in gene sets annotated with cell movement and invasion of cancer cells. WFA treatment resulted in activation of stress response pathways as canonical pathway analysis indicated genes involved in ER and oxidative stress were also significantly enriched. In addition, upstream analysis showed AMPK was activated upon WFA treatment. We then validated AMPK activation was sufficient to suppress TGF‐beta‐mediated epithelial‐to‐mesenchymal transition through knockdown of AK4. Furthermore, in vivo therapeutic model showed that WFA administration significantly decreased lung orthotopic xenograft tumor growth and liver and kidney metastasis compared to the control group. These results identify WFA can be applied to treat metastatic lung cancer through repurposing approved drugs that reverse AK4‐associated gene expression signature.
Analysis of the acetylcholine‐signaling pathway reveals differences between normal and malignant lung tissue
Authors: Brown Kathleen C , Lau Jamie K , Thornhill Brent A , Perry Haley E , Jones Dennie V , Pulliam Joseph F , Martin Jeremiah , Dasgupta Piyali
Published: November 2023

NSCLC is characterized by aggressive clinical course, rapid doubling time and a dismal survival rate. The development of NSCLC is closely correlated with smoking habits. A small percentage of NSCLC cases also occur in non‐smokers. Targeted therapies like EGFR‐inhibitors, EML‐ALK4 inhibitors, and B‐Raf inhibitors show excellent therapeutic response in NSCLC patients who are non‐smokers. However, they do not work well in NSCLC patients who are smokers. Therefore, the identification of novel molecular targets in NSCLC patients who are exposed to cigarette smoke is urgently needed. Tobacco components like nicotine and NNK have been known to accelerate the progression of lung cancer. One of the major mechanisms underlying the proliferative actions of nicotine and NNK is that they upregulate the levels of the growth factor acetylcholine (ACh). This ACh binds back to its target receptors (on NSCLC cells) and stimulates the proliferation of NSCLC cells in an autocrine manner. The ACh signaling loop is amplified by exposure to tobacco components and cigarette smoke. With this background in mind, we aim to compare the ACh‐signaling machinery between normal lung cells and NSCLC cells. We find that both normal lung epithelial cells and NSCLC cells express all the components of the ACh‐signaling pathway, namely choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), acetylcholinesterase (AChE) and choline transporter (ChT). Some of the ACh‐signaling proteins like ChAT, VAChT and AChE show differences in expression and activity between normal lung tissue and NSCLC cells. We believe that such differences may form the basis of targeted therapies in NSCLC patients who are exposed to cigarette smoke.

Support or Funding Information

Funding for our study was supported by the an NIH R15‐AREA Grant (1R15HL113681‐01A1), ACTSI Grant from Marshall University, the Flight Attendant Medical Research Institute YCSA Grant (82115),and the West Virginia IDeA Network of Biomedical Research Excellence (P20RR016477 and P20GM103434).
Losartan Blocks the Recruitment of Mast Cells in the Lungs of Rats Subjected to Fat Embolism with or without a Second Hit with LPS
Authors: Poisner Alan , Hamidpour Sopheila , Ho Andrea , Skaria Priya , Fletcher Amanda , Simon Steve , Molteni Agostino
Published: November 2023

In a rat model of pulmonary fat embolism (FE) using triolein (T), we have shown that the long term histopathological effects of triolein‐induced FE extend to 10 weeks and are inhibited by losartan (LOS), an angiotensin II type 1 receptor blocker1 and augmented by a second hit with LPS2. These effects are associated with an increase in renin staining at 6 weeks3. Since other workers have implicated pulmonary mast cells (MC) in local renin‐angiotensin (RAS)‐mediated pulmonary pathology4, we examined whether FE in our model was associated with an increase in MC number and if their appearance would be blocked by LOS and modified by a second hit with LPS. 36 Sprague‐Dawley rats were treated with T (0.2 ml i.v.) or saline. After 6 weeks, half of the groups were given saline or LPS 0.1 ml (3 mg/kg) i.p., followed 1 hour later with saline or LOS (10 mg/kg i.p.), followed by LOS in the drinking water (50 mg/l). 4 weeks later (10 weeks after T), the animals were necropsied after isoflurane anesthesia. Lungs were fixed in 10% formalin and stained with H&E for morphometric scoring and for mast cells using CD11 (c‐kit). Portions of the lungs were frozen and stained for fat using Oil Red O. Two pathologists unaware of the slides’ identity took 10 photographs at random in each slide at 400× and then counted the mast cell number.

MC number increased in T‐treated rats compared to saline controls (57.2 +/− 2.5 vs 20.0 +/− 2.3) (mean+/− SEM) (p=0.005). The MC count when T was followed by LPS rose to 66.8 +/− 14.5 (p=0.001 compared to saline). LPS or LOS alone did not cause a significant increase in MC (35.0 +/− 8.5 and 28.2 +/− 2.6, respectively.) The increases in MC caused by T and T+LPS were both blocked by LOS: (T+LOS 14.9 +/− 2.9; T+LPS+LOS 32.5 +/− 4.6; p=0.75 and 0.99 compared to LOS alone.

The appearance of fat globules at 10 weeks was greatly reduced from what we reported for rats at 6 weeks3 but they were increased in T‐treated rats compared to saline controls.

The results support suggestions that MC‐associated renin through its eventual product angiotensin II plays an important role in the histopathological changes produced by FE and the second hit with LPS. In addition, the presence of fat globules in the lungs 10 weeks after the initial challenge with T suggests that their continued presence may be important for the enhanced presence of MC in response to low grade inflammation. A role for macrophage signaling in this sequence is also supported5.

Support or Funding Information

Mary Katherine Geldmacher Research Foundation, St. Louis, MO