bluuebunny/crossref_metadata_2025 huggingface dataset:
Slow Release of Iron into Blood from Aspergillus Oryzae Compared to Ferrous Sulfate in Humans
Authors: Reddy Manju B. , Armah Seth M. , Bian Yilin
Published: November 2023

Iron fortification and supplementation are the usual strategies employed to prevent or treat anemia, the most common world‐wide nutritional problem. Although used extensively and effectively, the ferrous sulfate supplement is known to cause adverse side effects and have presented problems with compliance. Ferrous sulfate is rapidly absorbed and the bolus of iron enters blood rather quickly possibly leading to higher concentrations of non‐transferrin bound iron which induces oxidative stress. The objective of this study was to determine how quickly iron enters into blood stream from the iron‐enriched Asperigillus oryzae (AspironTM, ASP) in contrast to ferrous sulfate. Iron‐enriched ASP was produced by using a proprietary technology developed by Cura Global Health, Inc. by growing the fungi with ferrous sulfate, dried, and ground to a fine powder. Eighteen healthy, female subjects (18–35 y), non‐pregnant, non‐anemic, and with low iron status (ferritin ≤30 μg/L), completed the study. A randomized, double blind, cross‐over experimental design was used with three treatments. Subjects were randomized to consume a test meal with three iron supplements (10 mg iron as FeSO4 and ASP as well as 20 mg iron as ASP) in the form of capsules during three separate visits: Blood samples were collected after consuming iron supplement capsules with a meal (rice and vegetables with orange juice) at 0, 30, 60, 90, 120, 150, 180, 210 and 240 min. The area under the curve (AUC) was calculated for FeSO4 and the two ASP doses based on changes in serum iron concentrations from baseline. Ferritin and hemoglobin concentrations were measured at the beginging of study but C‐reactive protein was measured at the beginning of each treatment. The average ferritin and hemoglobin concentrations were 18.3 μg/L and 131 g/L, respectively. The mean AUC of FeSO4 (1674.5 ± 1548.4) was significantly higher (p<0.05) than the AUCs of 10 mg Aspiron (868.7 ± 480.7; p<0.05) and 20 mg Aspiron (899.9 ± 771.4). After reaching their maximum iron levels (~120 min), serum iron levels with FeSO4 declined while both Aspiron doses seemed to remained constant. CRP concentrations were low (1.6 mg/L) didn't vary among the treatments. Based on the slow iron release results in our study, we can speculate that the ASP iron might be absorbed by a different mechanisms similar to ferritin iron or it might be digested slowly requiring longer time for iron to be released in to blood. Our results suggest strong potential for ASP's use as a supplement in polulations where ferrous sulfate supplementaion is of concern.

Support or Funding Information

Cura Global Health, Inc.
Selenoprotein T Positively Regulates α‐synuclein Expression and Cell Proliferation in SK‐N‐SH Cells
Authors: Zhu Jian‐Hong , Zhu Hui , Shao Zi‐Qiang , Sun Sheng‐Nan , Zhang Xiong
Published: November 2023

The exclusive retention of selenium (Se) in brain during Se deficiency implicates an important role of this element in brain function and relevant disorders such as Parkinson's disease (PD). While the biological function of Se is mostly through selenoproteins, we have previously screened selenotranscriptome in five brain regions in a chronic mouse model of PD and identified some unique changes of selenoproteins. Amongst includes the expression of selenoprotein T (SelenoT) reduced in the substantia nigra, but increased in the cortex and pons of PD mice. In this study, we aimed to further understand the role of SelenoT in PD and the underlying mechanisms. Analysis in peripheral blood mononuclear cells showed an elevated level of SelenoT mRNA in PD patients compared with the controls. By using MPP+‐treated SK‐N‐SH neuronal cells as a PD cellular model, we found that MPP+ treatment increased SelenoT mRNA and protein expression. Results from siRNA knockdown and overexpression showed that SelenoT positively regulated cell proliferation and the mRNA and protein expression of α‐synuclein, a key molecule involved in PD pathogenesis. Knockdown of SelenoT led to elevated cellular Ca2+ level, but reduced levels of p53 and reactive oxygen species, which may involve in the regulation of SelenoT on α‐synuclein expression. Results from siRNA knockdown also showed a reduction in cyclin‐dependent kinase 4 (CDK4), but an increased activity in CDK2, p‐38 and p‐JNK1/2, which is implicated the regulation of cell proliferation. Taken together, our data suggest SelenoT is responsive to PD inducer and may play a role in the pathogenesis of PD. Further work are needed to clarify the specific targets of SelenoT, as well as its role in PD by using conditional knockout animal models.
Se and Zn nutritional status of collegiate female athletes by exercise type
Authors: Okhee Lee
Published: November 2023

Most collegiate athletes usually carry out hard training in order to promote exercise performance, hence the production of free radicals and ROS will increase through the higher energy metabolism and oxygen ingestion. Therefore, the requirements of antioxidant nutrients might be increased compared to sedentary people and be different by exercise type. However, the nutritional status of Se and Zn, antioxidant micro‐minerals, have been rarely reported among Korean collegiate players, possibly because of their analysis difficulties. In this study, we evaluated the nutritional status of Se and Zn level, and their relationship between their dietary intake and serum levels by exercise type. The collegiate athletic females of judo, aerobics, and basketball and sedentary females were recruited. And Se and Zn levels in serum were measured using NAA method and their dietary intake were calculated by using 24‐hour recall method and food mineral database of Korean Nutrition Society, respectively. Average serum level of Se was in range of 9.7~12.2ug/dL and lowest in judo players and tends to be higher in basketball player compared to sedentary. Average Zn level in serum was in range of 81.8~105.0 ug/dL and lower in judo, aerobic players compared to sedentary and basketball players. The dietary intake of Se was higher than the recommend amount of KDRI Se in all groups, and the highest among basketball players who ingested double amount of RNI Se of KDRI. The dietary Zn intake was higher in three exercise group compared to sedentary collegiate female. The rank of dietary Se and Zn levels by exercise type didn't correspond to those of serum levels, respectively. Despite of higher dietary intake of Se, it's nutritional status was lower in female judo player than sedentary female. It is suggested that higher Se intake may be necessary in judo female players, which should be confirmed with a wide and deep evaluation studies.

Support or Funding Information

2015 grant of Yongin university
Zinc Up‐regulates Nox1 Function by Increasing Mitochondrial ROS to Induce Senescence of Vascular Smooth Muscle Cells
Authors: Huang Jingwen , Feresin Rafaela G. , Salazar Gloria
Published: November 2023

Introduction

Zinc, an essential trace element, plays a significant role in the regulation of multiple cellular processes. We previously demonstrated that zinc overload up‐regulated reactive oxygen species (ROS) levels causing senescence. However, the source of ROS that mediates zinc effects was not determined.

Objectives

Thus, the purpose of this study was to investigate whether increased ROS levels and senescence induced by zinc is attributed to increased mitochondrial ROS (mtROS), ROS produced by NADPH oxidases (Nox) or both.

Methods

Vascular smooth muscle cells (VSMCs) treated with 50 μM zinc were incubated with 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCFDA) and MitoSox to measure hydrogen peroxide and mtROS, respectively. Western blots were used to determine the expression of Nox1, Nox4, antioxidant enzymes and the senescence markers, p21 and p53. After three days of the treatment, senescence was determined by measuring senescence associated β‐galactosidase (SA‐β‐gal) activity.

Results

We found that zinc overload increased the expression of Nox1, but not Nox4, and promoted the accumulation of zinc in the mitochondria causing increased mtROS generation. Blocking mtROS with MitoTEMPO reduced Nox1 expression and senescence induced by zinc.

Conclusions

Increased zinc levels promote mtROS generation, which up‐regulates Nox1 expression increasing cytosolic ROS levels and inducing senescence of VSMCs.

Support or Funding Information

This study was supported in part by the American Heart Association (14GRNT20180028), the American Federation for Aging Research (AFAR) and the Florida State University Council on Research and Creativity (COFRS).
The MTHFR 677TT Variant is Associated with Impaired Acute Blood Pressure Response to Sodium Ingestion
Authors: Murphy Melissa A , Breslin Paul A S , Miller Joshua W
Published: November 2023

Observational and genome‐wide association studies indicate that a common polymorphism (677C‐‐>T) in the FAD‐dependent enzyme, methylenetetrahydrofolate reductase (MTHFR), is associated with elevated blood pressure (BP) and increased risk of hypertension. This association is modified by riboflavin status and riboflavin supplements reduce BP in individuals homozygous for the variant form of the enzyme (677TT). However, little is known about the effect of the 677TT variant on dynamic responses of BP to vaso reactive stimuli. Previously, we demonstrated that systolic BP (SBP) drops in healthy adults within one hour after ingestion of a sodium chloride (NaCl) solution. To determine if MTHFR genotype impacts this acute BP response to NaCl ingestion, we tested 20 normotensive individuals (age 18–35y), 9 with the 677TT variant and 11 wild‐type controls (677CC) in a repeated measures design. On 6 different days, BP was measured in the subjects in the sitting position with a mercury brachial sphygmomanometer every 10 minutes for 60 minutes post ingestion of 475 mL of either a 157 mM NaCl solution or water with no NaCl. Plasma riboflavin was determined by HPLC. In all subjects there was an average of 3.1 ± 2.7 mmHg greater drop in SBP after ingesting the NaCl solution compared with water alone (p = 0.018). Additionally, the decrease in SBP was greater for 677CC individuals compared with 677TT individuals in both conditions: 677CC‐NaCl = −8.18 ± 3.62 mmHg vs. 677TT‐NaCl = −6.59 ± 2.15 mmHg and 677CC‐Water = −6.94 ± 2.01 mmHg vs. 677TT‐Water = −5.37 ± 2.78 mmHg (p = 0.013). Plasma riboflavin was not different between the 677CC and 677TT groups (14.4 ± 8.8 vs. 17.2 ± 11.9 nmol/L, respectively). These data indicate a lesser SBP response to NaCl ingestion, independent of riboflavin status, in the 677TT group, suggesting that acute regulation of BP in response to a vasoreactive stimulus may be impaired as a result of this gene variant.

Support or Funding Information

NIH DC02995
ZIP10 mediates renal MT2 production and attenuates oxidative stress
Authors: Hao Shoujin , Ding Gangqiang , Hao Hongbo , Tian Peiyao , Li Shixue , Zhao Hong , Darzynkiewicz Zbigniew , Ferreri Nicholas R.
Published: November 2023

We previously showed that metallothioneins (MTs) induced by zinc supplementation contribute to beneficial effects in the context of diabetic nephropathy (DN). The contribution of Zip10 to the regulation of MTs in the kidney was determined in this study. Male db/db and non‐diabetic db/m mice (age: 10–12 wk) were randomized into different groups and ZIP10 mRNA abundance in freshly isolated renal tubules was determined by qRT‐PCR using isoform‐specific primers. ZIP10 mRNA abundance was approximately 50% less (p<0.05) in renal tubules from db/db mice compared to non‐diabetic db/m mice. MT2 mRNA abundance also was less by about 40% (p<0.05) in renal tubules from db/db mice compared to non‐diabetic db/m mice. Systemic and intrarenal oxidative stress was evaluated by examination of 8‐hydroxyde‐oxyguanosine (8‐OHdG). The results showed that the 8‐OHdG content in the kidney (6.3±1.3ng/mgDNA) and urine 8‐OHdG excretion (912.8 ± 33.4 ng/24h) from db/db mice was significantly higher than the 8‐OHdG content in kidney tissues (2.5±0.6ng/mgDNA) and urine 8‐OHdG excretion (382.2 ± 25.7 ng/24h) from the non‐diabetic db/m mice. To further investigate the damage of the renal tubules, we determined the levels of Kidney ischemia molecule (Kim)‐1 (db/db: 223.8±22.6ng/ml vs db/m: 112.7±11.6ng/ml) and neutrophil gelatinase‐associated lipocalin (NGAL) (db/db: 173.3±10.8ng/ml vs db/m: 82.5±4.4ng/ml) in the urine, which originate from damaged renal tubular epithelial cells. Collectively, these results show that the increased levels of Kim‐1 and NGAL are associated with reduced ZIP10 expression and MT2 levels in db/db mice. We then determined the contribution of ZIP10 to MT2 production in primary renal tubular epithelial cells isolated from wild type mice that were transfected with a ZIP10‐specific shRNA silencing lentivirus construct, psiLV‐U6‐Z10‐ex3. Specific knockdown of ZIP10 mRNA with psiLV‐U6‐Z10‐ex3 decreased MT2 expression by approximately 70% while the control lentivirus construct was without effect. The anti‐genotoxic effect of MTs was evident in prevention of DNA damage and micronucleus formation in renal tubular epithelial cells treated with MTs (mixture of MT1 and MT2). The MT2 gene silencing in renal tubular epithelial cells significantly increased reactive oxygen species generation and H2O2‐induced cytotoxicity. Taken together, these findings suggest that ZIP10 may be important for renal MT2 production and attenuation of oxidative stress in DN. Understanding the specific mechanisms by which ZIP10 contributes to renal MT2 production and diminution of kidney damage may provide novel insights for the treatment of DN.
Up regulation of Spleen Tyrosine Kinase in ballooned hepatocytes with Mallory‐Denk Bodies in Alcoholic Liver Disease
Authors: Afifiyan Nikoo , Samadzadeh Sara , Masouminia Maryam , Tillman Brittany , French Barbara A. , French Samuel W.
Published: November 2023

Liver injury from alcohol abuse causes balloon hepatocytes and MDB formation. Mallory‐Denk bodies (MDBs) are found in 70% to 75% of patients with alcoholic hepatitis (AH). MDBs are composed of intracellular aggregations of misfolded proteins in ballooned hepatocytes. To determine the mechanism of balloon degeneration and MDB formation in hepatocytes, we evaluated the role of spleen tyrosine kinase (Syk) which is associated with transmembrane receptors to mediate numerous signal transductions downstream of these receptors. Spleen tyrosine kinase is widely expressed in hematopoietic cells and modulates several signaling pathways including PIK3/AKT and p7056 pathways. These pathways are important in regulating the cell cycle and are actively involved in mediating cell adhesion. Dysregulation of the PI3K/Akt pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. In this study, liver biopsy sections fixed in formalin and embedded in paraffin (FFPE) were used from patients with alcoholic hepatitis (AH). The results show that the expression of Syk protein is up regulated by RNAseq and real time PCR analyses in the alcoholic hepatitis patients compared to controls. The results were also supported by using IHC fluorescent intensity staining morphometric system. Morphometric quantification of fluorescent intensity showed a two fold increase in Syk in the cells forming MDBs compared to surrounding normal hepatocytes. Interestingly, the upregulation of Syk protein is co‐localized with ubiquitinated proteins in the cytoplasm of balloon cell hepatocytes which have formed MDBs. This observation demonstrates the role of Syk as a potential multifunctional target in protein quality control mechanism of hepatocytes when ER stress is activated.
The Inhibitory Effects of HJC0416 on Liver Fibrogenesis in Activated Hepatic Stellate Cells
Authors: Nunez‐Lopez Omar , Wang Xiaofu , Graham Gabriel , Kandathiparampil Arline , Ye Na , Chen Haiying , Chen Haijun , Zhou Jia , Radhakrishnan Ravi S
Published: November 2023

Introduction

The STAT3 signaling pathway regulates activation of hepatic stellate cells (HSCs) and promotes its fibrogenic behavior. Using a fragment‐based drug design approach, our group has developed HJC0416, a novel orally bioavailable small‐molecule inhibitor of STAT3. HJC0416 was found to induce apoptosis and decreased tumor growth in breast cancer models. In the present study, we studied the antifibrogenic properties of HJC0416 in activated hepatic stellate cells (HSC), which are the major effector cells of liver fibrosis.

Methods

Human (LX‐2) and rat (HSC‐T6) hepatic stellate cell lines were used. Cell proliferation was measured by Alamar Blue assay. Cellular protein expression levels were determined by Western blotting and immunofluorescence. Cell cycle was assessed by flow cytometry. Yo‐pro‐1 staining was used to measure apoptosis.

Results

HJC0416 treatment significantly inhibited LX‐2 and HSC‐T6 cell proliferation in a dose‐dependent fashion, and induced apoptosis and S‐phase cell cycle arrest. HSC activation marker α‐smooth muscle actin was also attenuated. HJC0416 suppressed Tyr705‐phosphorylated STAT3 (pSTAT3) and impaired pSTAT3 nuclear translocation and transcriptional activity. STAT3 target genes cyclin D1 and c‐myc were down‐regulated by HJC0416. Static, a well‐known STAT3 inhibitor, confirmed that HSC proliferation was suppressed by STAT3 inhibition. Extracellular matrix (ECM) proteins collagen type I (Coll.I) and fibronectin (FN) are major components of hepatic scar, with TGFβ being the most potent stimulator for ECM production. Our data revealed that endogenous Coll.I and FN expression were suppressed by HJC0416 in a dose‐dependent manner; TGFβ‐stimulated Coll.I and FN production were prevented by pretreatment with HJC0416.

Conclusion

HJC0416 inhibits HSC activation via the STAT3 signaling pathway. HJC0416 represents a promising anti‐hepatic fibrogenic agent.

Support or Funding Information

This work was supported by grants P50 CA097007, P30 DA028821, R21 MH098344 (JZ) from the National Institutes of Health, Cancer Prevention Research Institute of Texas award, R. A. Welch Foundation Chemistry and Biology Collaborative Grant (JZ) from the Gulf Coast Consortia, and John Sealy Memorial Endowment Fund and the Center for Addiction Research (JZ) from the University of Texas Medical Branch.
Characterization of cellular senescence mechanisms in alcoholic liver injury
Authors: Meng Fanyin , Lorenzo Sugeily Ramos , Francis Heather , Glaser Shannon , Alpini Gianfranco
Published: November 2023

Background

Cellular senescence is a stress‐responsive program limiting the proliferation of damaged cells and leading to stable cell‐cycle arrest. Accumulation of senescent hepatocytes may contribute to loss of functional hepatic mass and lead to liver decompensation and fibrosis. The current study aimed to characterize the functional role of cellular senescence during alcoholic‐induced hepatitis.

Methods

Senescence related gene expression was assessed using a Cellular Senescence PCR Array and/or real‐time PCR analysis in ethanol‐ and LPS‐treated normal human hepatocytes (N‐Heps) and cholangiocytes (HiBEC), as well as in liver specimen from mice fed alcohol for 5 weeks relative to control liver tissue. Cellular senescence and viability were measured by SA‐β‐gal Activity and MTS assay. The upstream modulators of senescence were defined in ethanol/LPS treated hepatocytes and cholangiocytes in vitro, and in TLR‐4 knockout mice in vivo.

Results

We identified that 5 week of ethanol feeding significantly increased total liver histopathology score and hepatocellular senescence by PCR array and SA‐β‐gal assay. The up‐regulation of hepatic senescence initiators, PAI‐1 and EGR1, were further verified by real‐time PCR assay. Treatment of N‐Heps and HiBECs with ethanol (86 mM) and LPS (20 ng/ml) for 72 hr significantly increased PAI‐1 and EGR1 expression, along with the enhanced SA‐β‐gal activity and reduced cellular viability detected by MTS assay. Silencing of PAI‐1 decreased ethanol and LPS‐induced senescence in both N‐Heps and HiBECs, whereas inhibition of EGR1 only reduced the senescence and increased viability in N‐Hep cells. Interestingly, silencing of PAI‐1 and EGR1 also reduced the pro‐fibrotic markers, α‐SMA and MMP‐9 in N‐Hep cells, whereas silencing of LPS receptor TLR4 decreased these markers in the same group of N‐Hep cells, suggesting LPS‐mediated cellular senescence during alcohol‐induced liver fibrosis. Furthermore, the expression of TLR4 and the verified LPS related senescence markers, including E2F1, ID1 and IGFBP3 were significantly altered in ethanol‐fed mouse liver specimens compared to controls. TLR4 knockout mice displayed less sensitivity to alcoholic injury, along with reduced PAI‐1 and EGR1 levels and recovered expressions of α‐SMA and MMP‐9.

Summary and Conclusion

Our results show that PAI‐1 and EGR1 are the critical regulator of LPS induced cellular senescence and alcoholic hepatitis. The findings provide new insight into the function of LPS regulated cellular senescence and increase opportunities for the development of novel treatment paradigms for the management of alcoholic liver diseases.

Support or Funding Information

VA Merit Review Awards and NIH/NIDDK R01 Grants
Lipocalin 2 is a Yap‐beta‐catenin target and a biomarker of disease burden in a murine model of hepatoblastoma
Authors: Bell Danielle Elizabeth , Tao Junyan , Preziosi Morgan , Pradhan Tirthadipa , Monga Satdarshan P
Published: November 2023

Hepatoblastoma is the most common type of pediatric liver malignancy. Despite advances in therapy, metastatic disease is still associated with a poor prognosis. Aberrations in the Wnt/b‐catenin signaling pathway due to mutations in CTNNB1 are the most prevalent mutations findings in these tumors. Recently mutations in the nuclear translocation of the Yes‐Associated‐Protein Pathway (YAP) has also been reported in hepatoblastomas. In fact b‐catenin‐Yap co‐activation was observed in 80% of hepatoblastoma patients. Hydrodynamic tail vein injection of active upregulated‐beta‐catenin and active‐YAP plasmids in mice also led to hepatoblastoma tumors. Gene array analysis using mRNA isolated from these mice tumors from tumor‐bearing livers had identified lipocalin 2 as one of the upregulated genes, with increased binding sites to both TCF4 and TEAD, which are transcription factors for beta‐catenin and YAP respectively. Lipocalin 2 is a small molecule protein being increasingly studied in the field of cancer. Previously we had shown that indeed human hepatoblastoma tumors showed notable lipocalin‐2 (LCN2) expression by immunohistochemistry. To address if the increased LCN2 expression observed in humans correlated with the mouse model of hepatoblastoma, liver lysates from wild type livers and mouse hepatoblastoma tumors were analyzed. The tumor‐bearing livers were found to have a 40‐fold increased lipocalin 2 expression by real time PCR. We next used ELISA to quantify the amount of serum lipocalin 2 in these animals. We saw increased values of Lipocalin 2 serum levels in mice with tumors compared to the wild type. In fact, in fact we found that serum lipocalin 2 levels was an indicator of the tumor burden in these mice. The liver weight to body weight ratio of the mice had a direct positive correlation with serum lipocalin 2. To address that lipocalin 2 was regulated by Wnt and Yap signaling, we preformed western blot analysis on human hepatoblastoma HepG2 cells, that had been transfected with beta‐catenin or Yap silencing RNA. Protein levels of lipocalin 2 were decreased in following beta‐catenin knockdown of both, although it was more pronounced upon beta‐catenin suppression of cells. Additionally, western blot preformed on liver lysates from hepatocyte specific beta‐catenin knock out mice, also showed decreased levels of lipocalin 2. In conclusion, we have shown lipocalin 2 to be regulated by beta‐catenin and Yap signaling in hepatoblastoma and may be useful as a biomarker of the disease burden in a hepatoblastoma mouse model. There is an implication that this may be a potential serum marker in human patients as well. The fluctuations of lipocalin 2 levels in the serum may be a direct result of alterations in beta‐catenin signaling and could be participating to oncogenesis.
Assessing the role of hepatocyte‐specific Wnts in hepatocellular carcinoma
Authors: Preziosi Morgan , Monga Satdarshan
Published: November 2023

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, and the third most common cancer‐related mortality. Additionally, liver fibrosis or cirrhosis is a prerequisite to HCC in 70–90% of cases. Treatment options are limited, and HCC incidence continue to rise each year, it is of utmost importance to better understand mechanisms of liver injury and HCC to provide better options for patients. Overexpression of Wnts have been observed in different cancers including HCC, and we hypothesize hepatocyte‐specific Wnts affect HCC progression. We used hepatocyte‐specific Wntless knockout animals (KO) which are unable to secrete Wnts from hepatocytes, and subjected them to a strict regimen of diethylnitrosame and carbon tetrachloride (DEN/CCl4) to induce hepatic injury, fibrosis, and cancer. Importantly, all tumors will be β‐catenin wild‐type, representing a majority of patients and making our study of Wnts relevant. At 3 months we noted no differences in injury or tumor nodules between control (CON) and KO. At 5 months all animals had HCC and in KO we noted increased cytoplasmic β‐catenin, increased target gene expression, and frequent CK19+ foci. We predict hepatocytes secrete noncanonical Wnts to prevent β‐catenin activation and tumor dedifferentiation, and we hypothesize KO will have more metastatic, dedifferentiated, and advanced tumors at 7 months. By identifying the cell type responsible for Wnt secretion and their conclusive roles in HCC, we will be providing a direction to develop therapies that minimize toxicity through selective inhibition of tumor‐promoting Wnts while leaving other Wnt signaling intact.
Absence of Class A Scavenger Receptor Distinguishes a Phenotype of Tumor Associated Macrophages in Classic Hodgkin's Lymphoma
Authors: Yuan Youzhong , Holthoff Emily , Post Jacqueline , James Jennifer , Post Ginell R. , Post Steven R.
Published: November 2023

Tumor‐associated macrophages (TAMs) modulate the development and progression of various cancers. The correlation between adverse clinical prognosis of classic Hodgkin's lymphoma (cHL) patients and the number of TAMs, as defined by CD68, CD163, or CSF1R immunoreactivity in diagnostic lymph node sections indicates that TAMs have an important role in cHL. Although these markers are widely used to identify TAMs, they are detected at all stages of monocyte/macrophage differentiation. In contrast, the Class A scavenger receptor [SR‐A, CD204] is a glycoprotein with an expression profile that is restricted to tissue macrophages. Although SR‐A expression in cHL has not been reported, SR‐A immunoreactivity is inversely correlated with patient prognosis in various carcinomas. To explore the possibility that SR‐A would better correlate with tumor macrophages and therefore be a specific prognostic marker in cHL, lymph node samples from patients with cHL were immunostained with antibodies to CD68 and SR‐A. As additional controls, a subset of cases were also stained with CD14 and CD163. Our results showed robust CD68, CD163, and CD14 stained TAMs in sections from cHL patients, regardless of morphologic subtype. In contrast, most of the TAMs identified with CD68 were negative for SR‐A, with SR‐A staining being limited to macrophages in the fibrous collagenous bands characteristic of nodular sclerosis cHL. Importantly, SR‐A and CD68 staining patterns were similar in recurrent cHL involving the liver, T‐cell‐histiocyte rich lymphoma, and in lymph node metastases of colon carcinoma. The novel finding that, in lymph nodes from cHL patients, SR‐A expression is absent in cells that stain with other monocyte/macrophage markers suggests that these cells may not be fully differentiated macrophages, or that SR‐A is specifically down‐regulated in cHL. In either case, the absence of SR‐A defines a unique phenotype of TAMs in cHL.
Inhibition of Aggressive Natural Killer Cell Leukemia Cell Functions by Inhibitors of the Mevalonate Pathway and Its Products
Authors: Carlson Whitney , Pradhan Prajakta , Steele Timothy
Published: November 2023

Aggressive natural killer cell leukemia is a rare form of leukemia characterized by an increase in proliferation of malignant natural killer cells. Aggressive natural killer cell leukemia is one of the deadliest cancers known. Survival time is normally weeks to a few months once diagnosed. Aggressive natural killer cell leukemia is highly resistant to treatment, making it important to find a treatment for natural killer cell leukemia. In this study, effects of a geranylgeranyl transferase inhibitor, a farnesyl transferase inhibitor, and Rho GTPase inhibitors were investigated on the aggressive natural killer cell line YT‐INDY. Cancer cells are capable of upregulating mevalonate metabolism for protein prenylation and cholesterol biosynthesis. The farnesyl transferase inhibitor Tipifarnib and geranylgeranyl transferase inhibitor GGTI‐286 inhibit mevalonate metabolism by blocking the farnesylation and geranylgeranylation of proteins, respectively. We investigated the effects of GGTI‐286 and Tipifarnib on cell proliferation, cell cycle progression, and activation of the ERK MAP kinase pathway. Rho GTPases are small molecules that, when active, regulate functions such as progression through the cell cycle, cell growth, and transcription. Upregulation of Rho GTPases have been found in certain cancers. We determined whether the Rac1 inhibitors NSC 23766 and EHT 1864, cdc42 inhibitors ZCL 278 and ML 141 and ROCK inhibitors GSK429286A and Y‐27632 could inhibit YT‐INDY cell proliferation and ERK MAP kinase activation. The results of our investigations showed that Tipifarnib and GGTI‐286 were potent inhibitors of YT‐INDY proliferation (Tipifarnib IC50 = 7.2 uM, and GGTI‐286 IC50 = 8.0 uM), cell cycle progression and ERK MAP kinase activation. Among the Rho GTPase inhibitors, we found that the ROCK inhibitors (GSK429286A and Y‐27632) and Rac1 inhibitors (NSC 23766 and EHT 1864) produced significant inhibition of YT‐INDY proliferation. We concluded from these investigations that inhibitors of the mevalonate pathway or products of the mevalonate pathway can interrupt YT‐INDY cell growth, suggesting that this approach could lead to more effective treatment of this devastating cancer.

Support or Funding Information

Our laboratory gratefully acknowledges the financial support of this research by the Iowa Osteopathic Education and Research Foundation.
Neuropilin Expression in Podocytes: The Implications for SEMA3 Anti‐Cancer Therapy
Authors: Nzikoba Ellah Nifashe , Zessler Astrid , Li Xiaoran , Briscoe David , Adam Rosalyn M. , Schumacher Valerie , Bielenberg Diane R.
Published: November 2023

Neuropilins (Nrp) are transmembrane receptors in endothelial and carcinoma cells that bind to angiogenic ligands including vascular endothelial growth factor (VEGF) to promote neovascularization, the sprouting of new blood capillaries providing oxygen and nutrients to the tumor. Additionally, Nrp binds to an alternate family of proteins, Class 3 Semaphorins (SEMA3), which compete with VEGF and inhibit angiogenesis. Targeting of either Nrp1 or Nrp2 with neutralizing antibodies or with exogenous SEMA3 proteins has successfully inhibited cancer progression in preclinical trials. Our goal was to investigate the expression, localization and function of Nrps within the kidney to examine potential therapeutic off‐target effects. We hypothesized that podocytes express Nrp1 but not Nrp2 and that the Nrp1 ligand, SEMA3A, would induce proteinuria while the Nrp2 ligand, SEMA3F, would not. Using immunoblotting, immunohistochemistry and X‐gal staining of kidneys from wildtype (Nrp2+/+) and transgenic mice (Nrp2+/lacz, Nrp2−/−), we determined that Nrp1 is highly expressed in the kidney and localized to tubular epithelial cells and glomerular podocytes. Nrp2, on the other hand, is weakly expressed in the kidney and localized to endothelial cells between tubules. Glomeruli are devoid of Nrp2 expression. In order to examine the effect of Nrp2 deletion on renal filtration function, we examined the protein content of urine from Nrp2+/+ and Nrp2−/− mice and found that both genotypes had similar protein filtration. Alternately, mice were treated intravenously with adenovirus encoding SEMA3 proteins. Livers from mice infected with these adenoviruses secreted SEMA3 proteins systemically. Mice exposed to SEMA3 proteins showed increased protein content in the urine. Taken together, these results may explain why NRP1 and VEGF combination antibody treatment caused severe proteinuria in cancer patients in a human Phase Ib clinical trial. Our data suggests that systemic therapies targeting NRP2 (anti‐NRP2 antibodies) may cause less toxicity than those targeting NRP1 yet still maintain anti‐tumor efficacy.

Support or Funding Information

Ellah Nzikoba was funded by the ASIP SROPP program and the study was funded by DK104641 (DRB, RMA).
HLA‐DR Positive Acute Promyelocytic Leukemia (APL)
Authors: Mendoza Alejandro S , Qing Xin , Dungo Maria , Lasky Joseph , Panosyan Eduard , Cai Junchao
Published: November 2023

BACKGROUND

Acute promyelocytic leukemia (APL) with t(15;17)(q22;q21)/PML‐RARα is a subtype of acute myeloid leukemia(AML) with distinct morphologic and immunophenotypic characteristics. It is a highly aggressive disease that requiresrapid diagnosis and early intervention. Compared with other types of AML, APL typically displaysa triad of absent or weak CD34, absent HLA‐DR, and positive CD117. HLA‐DR positive APL is extremely rareand its clinical and pathological features have not been reported. A total of 45 cases of APL with t(15,17)/PMLRARα were diagnosed at Harbor‐UCLA Medical Center from year 2006 to 2015. Among them, only two cases were positive for HLA‐DR by flow cytometry immunophenotyping. Here we describe the clinical, morphologic, immunophenotypic, and cytogenetic features of these two cases.

RESULTS AND DISCUSSION

The patients in both cases presented with anemia, thrombocytopenia, and decreased WBC count. In case A, the peripheralblood smear showed many myeloblasts and only occasional abnormalpromyelocytes. Although a thin Auer rod was found in rare blasts, neitherthe thick Auer rods that typically seen in APL nor faggot cells were identified. While case B showed characteristic morphologic features of hypergranular variant of APL in the bone marrow, no blasts orabnormal promyelocytes were identified in the peripheral bloodsmear. Therefore, APL should not be excluded even when abnormalpromyelocytes are not present in the peripheral blood smears. In addition, MPO cytochemical stain performed on the bone marrow aspiratesmear of case A did not show uniform strong staining pattern that is commonly observed in APL. Besides HLA‐DR, CD34 was also expressed on the blasts of case A. These atypical morphologic and immunophenotypic features may be attributed to the additional cytogenetic abnormalities found in case A.

CONCLUSION

HLA‐DR‐positive APL is very rare and represents about 4% of all APL cases in our study. Due to its rare occurrence, continuous reporting of HLA‐DR positive APL will bring to the awareness of the broad clinical and pathological spectrum of APL, provide better clinicopathologic correlation, and facilitate rapid diagnosis and thus promptinitiation of appropriate management of this aggressive malignant tumor.

Support or Funding Information

We declare no benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
Hemophagocytic lymphohistiocytosis in a patient with Legionella infection and bone marrow monotypic plasma cells in the setting of chronic lymphocytic leukemia
Authors: Nguyen Luan , Ebaee Anoosheh , Pham Long , Ghani Hassan , French Samuel W. , Qing Xin
Published: November 2023

Hemophagocytic lymphohistiocytosis (HLH) is a rare condition in which the body makes too many activated immune cells (macrophages and lymphocytes) that phagocytose hematologic cells. HLH can be inherited or acquired. Acquired HLH has been associated with: infection, immunosuppressive medications, autoimmune diseases, certain cancers, and metabolic diseases. Here, we report the case of a 50 year old male with a history of chronic lymphocytic leukemia Rai Stage IV status post chemotherapy and a recent admission for Legionella pneumonia who presented with worsening fever despite levofloxacin treatment and marked hypercalcemia. A bone marrow biopsy showed 20–30% lambda‐restricted plasma cells. Additionally, hemophagocytosis was found in the bone marrow, which prompted the clinician to consider HLH in the diagnosis. After further testing, the patient met the criteria for HLH with an H‐score of 234 (98.4% probability of patient having HLH). Despite steroid treatment, the patient's condition deteriorated and the patient died 6 days after bone marrow biopsy.
Use of Anchored Multiplex PCR Enrichment for Detection of Gene Fusions in Solid Tumors by Next Generation Sequencing
Authors: Green Donald C , Deharvengt Sophie J , de Abreu Francine B , Steinmetz Heather B , Peterson Jason D , Tsongalis Gregory J
Published: November 2023

Background

The known genomic complexities of human cancer are being used to combat the disease with novel targeted therapies. Next generation sequencing (NGS) technologies have been introduced to determine a human cancer profile that is then used to develop a therapeutic management strategy. To date most NGS assays assess SNVs, Indels and some CNVs in various cancer related genes. Gene fusions are another type of mutation that have recently been implicated in the diagnosis and treatment of human cancers. In this study we assessed the ability to detect gene fusions using an anchored multiplex PCR‐enriched NGS assay.

Methods

RNA was extracted from 15 formalin fixed, paraffin embedded tissue samples and 10 cell lines using the AllPrep DNA/RNA FFPE kit. cDNA synthesis and library preparation were performed using 200 ng of RNA. RNA quantity and quality were evaluated using the Qubit HS RNA Assay and the Archer PreSeq RNA QC Kit, respectively. Libraries were quantified using the KAPA Library Quantification Kit and sequenced on the Illumina MiSeq System using the Archer FusionPlexTM Solid Tumor panel which detects fusions in 53 genes. Data analysis was performed using the Archer Analysis platform (v4.0.11). We examined the limit of detection (LOD) by varying the amount of total RNA input (200‐2.5 ng), precision in two sequencing runs using two different operators, and accuracy by comparison of results to results from independent FISH and NGS assays.

Results

We established QC metrics that included: (1) tumor content, (2) RNA quantification, (3) RNA quality, (4) library quantification, and (5) post‐sequencing analysis. While NGS results could be obtained using 10 ng of input RNA, more robust results were obtained when we used higher RNA inputs. Accuracy was determined to be 100% with detection of for all known fusions in the samples used. The following fusions were present in these samples: EML4‐ALK, ETV6‐NTRK3, EWSR1‐FLI1, TPM3‐NTRK1, TNM4‐NRG1, FGFR2‐COL14A1, MBNL1‐RAF1, ETV6‐RUNX1, BRD4‐NUTM1, and TMPRSS2‐ERG. All positive (CCD6‐RET, EML4‐ALK, SLC34A2‐ROS1) and negative controls gave the expected results.

Conclusions

This study demonstrates the feasibility of using the Archer Solid Tumor FusionPlex assay for detection of gene fusions in human cancers. The assay detects fusions of any partner gene with the 53 genes in the panel and thus makes novel fusion discovery possible.
Anti‐proliferative Properties of Methanolic Extracts of Annona muricata in Colon, Lung and Skin Cancer Cell Lines
Authors: Robles Jose F , Muñoz Kevin , Rivera Nilka , Suarez Edu
Published: November 2023

Annona muricata, commonly known as Soursop, is a tree that grows in tropical climates. Traditionally, different parts of A. muricata are used to prepare teas and juices, which have been used as homemade remedies for diseases such as diabetes and cancer. Previous studies in our laboratory, showed that extracts obtained from A. muricata's leaves had anti‐proliferative activity towards HT‐29 colorectal adenocarcinoma cell. Based on these previous results, in this study we evaluate the effect of the Soursop's fruit peel extract on the proliferation of colorectal adenocarcinoma at 24‐, 48‐ and 72‐hours. In addition, we explored if this extract affects the proliferation of non‐malignant CCD‐1074sk (epithelial) and MRC‐5 (fibroblast) cell lines using the same experimental design. We hypothesized that Soursop's fruit peel extracts will inhibit proliferation of HT‐29 colorectal adenocarcinoma but will not show affect the studied non‐malignant cell lines. We prepared our samples by performing methanolic extractions of the fruit's peel. For the proliferation assays we seeded the HT‐29, CCD‐1074 and MCR‐5 cells in 96‐well plates and after an overnight recovery period, we applied five treatments consisting of concentrations of the extracts: 400ug/mL, 300ug/mL, 200ug/mL, 100ug/mL, and 50ug/mL and DMSO at 1,43% final concentration as our control vehicle. Our preliminary results on the HT‐29 cells showed that at 24‐hours post treatment, only the highest concentration (400 ug/mL) presented a significant inhibition (p<0.05) at 48‐hours we observed inhibition from 50ug/mL to 400ug/mL and at 72‐hours we observed inhibition at all experimental concentrations. For the non‐malignant cell lines CCD‐1097and MRC‐5 the extract did not affect the proliferation of these when compared to their respective vehicle controls

Our preliminary data supports our hypothesis, suggesting that A. muricata fruit's peel extracts has a selective anti‐proliferative effect towards malignant cells.

Future biological experiments include the confirmation of this preliminary data and to evaluate the effect of our extracts on cell migration and invasion. As well, we expect to perform separation and identification of the chemical components present in the extracts by using High Pressure Liquid Chromatography (HPLC‐MS) coupled with Mass Spectrometry to identify key components in the extracts observed effects.
Induction of Paclitaxel Resistance in Human Breast Cancer Cell by JNK/SAPK‐Inhibitory Kinase through MAPK14/RELA/PTGS2 Signaling
Authors: Lai Tsung‐Ching
Published: November 2023

Paclitaxel resistance is a difficult puzzle in metastatic cancer patients with chemotherapy. In order to find new therapeutic targets significantly associated with breast cancer paclitaxel‐resistance, we performed lentiviral kinome shRNA‐based high‐throughput screening in breast cancer cell line resistant to paclitaxel treatment and identified JNK/SAPK‐Inhibitory Kinase, (JIK) as a new target. Here we determined that cell with overexpressed and reduced the expression of JIK resulted the changes of taxol sensitivity of breast cancer cells in vitro and in vivo. JIK overexpression was also found to be significantly associated with breast cancer patient disease‐free and overall survivals. Expression microarray and phosphoprotein kinase array analysis were performed to determine JIK downstream pathways and associated genes that enhanced paclitaxel resistance. Our results showed JIK overexpression increased paclitaxel resistance through upregulation of the phosphorylation of p38 mitogen‐activated protein kinase (MAPK14), v‐rel avian reticuloendotheliosis viral oncogene homolog A (RELA) and prostaglandin‐endoperoxide synthase 2 (PTGS2). Treatment with the inhibitors of these proteins in JIK‐overexpressed cells induced chemosensitization effects of the paclitaxel‐resistant breast cancer cells. In summary, our results showed that JIK overexpression induced paclitaxel resistance through modulation MAPK14 and RELA phosphorylation in breast cancer cells. JIK might be a feasible therapeutic target to develop inhibitors to treat breast cancer patients resistant to paclitaxel treatment.
MED28 Regulates Proinflammatory Cytokine‐induced Epithelial–Mesenchymal Transition and Invasion in Human Breast Cancer Cells
Authors: Hsieh Nien‐Tsu , Huang Chun‐Yin , Li Chun‐I , Weng Yu‐Ting , Lee Ming‐Fen
Published: November 2023

MED28, one of the Mediator subunits, exhibits multiple cellular roles, including involved in transcriptional activation and as a merlin, Grb2, and cytoskeleton‐associated protein, which is highly expressed in several types of malignancy including breast and colorectal tumors. Chronic inflammation, as suggested as one of the hallmarks of cancer, may promote carcinogenesis. Inflammatory cytokines such as tumor necrosis factor‐alpha (TNFα) may mediate tumor initiation, promotion, and progression. Recently, we have identified that MED28 is involved in regulating cell growth, epithelial–mesenchymal transition (EMT), and migration in human breast cancer cells. The objective of this study is to investigate if MED28 is involved in the proinflammatory cytokine‐induced EMT in human breast cancer cells. In aggressive MDA‐MB‐231 cells, MED28 suppression attenuated the mesenchymal morphology and reduced the expression of several mesenchymal biomarkers; however, MED28 overexpression exhibited a reciprocal effect. In addition, TNFα upregulated the expression of the target genes of NFκB/p65 and enhanced an even more aggressive phenotype, whereas suppression of MED28 attenuated the effect. Moreover, pterostilbene, a resveratrol analogue, suppressed the expression of MED28, p‐p65, and p65 downstream target genes, which warrants further investigation for a putative translational application in breast cancer.

Support or Funding Information

This work was supported by the grants NSC102‐2320‐B‐309‐001‐MY3 and MOST105‐2320‐B‐309‐001 to M‐F Lee and MOST103‐2815‐C‐309‐012‐B to C‐I Li.
Internalization and trafficking of PD‐L1 in MDAMB231 breast cancer cells
Authors: Rudkouskaya Alena , Barroso Margarida
Published: November 2023

Immunotherapy has emerged as a very promising type of cancer treatment. However, despite successful clinical use of several monoclonal antibodies developed to target immune checkpoint inhibitors present in tumor cells or tumor‐infiltrating lymphocytes, there is very little known about their endocytic pathway in cancer cells. To elucidate this process we studied programmed cell death‐ligand 1 (PD‐L1) uptake in MDAMB231 breast cancer cells using both live imaging and immunofluorescent staining of fixed cells. We found that five minute pulse with 10 ug/mL PD‐L1 AF488 and transferrin‐AF555 (Tf‐AF555), followed by wash and live imaging, results in significant accumulation of PD‐L1 both at the plasma membrane and in the endosomes often also containing Tf‐transferrin receptor complexes (Tf‐TfR). To further investigate PD‐L1 trafficking, we performed one hour uptake of both PD‐L1AF555 and Tf‐AF647, fixed the cells and stained with Early Endosome Antigen1 (EEA1). Airyscan imaging of these cells showed that many large vesicles, characteristic to MDAMB231 cells, are positive for all three markers suggesting again that even at late stages PD‐L1 is trafficking along the endocytic pathway with Tf‐TfR complexes. These findings may help to develop non‐invasive quantitative imaging approach to monitor the spatial distribution and accumulation of PD‐L1 in the tumor xenografts in preclinical studies to advance cancer immunotherapy.

Support or Funding Information

This work was funded by NIH grant R01 EB019443.