Cell Mitogenesis Research Articles

Prognostic significance of hepatocyte growth factor in non-metastatic colorectal cancer

Hepatocyte growth factor (HGF), produced by mesenchymal cells, stimulates mitogenesis and angiogenesis in tumor cells. Tumor cells of some solid tumors do not secrete HGF. The aim of the study was to evaluate the prognostic significance of HGF expression in tumor tissue in colorectal cancer (CRC). The study included 50 patients with stage II-III colorectal cancer; they underwent radical surgical treatment, followed by adjuvant chemotherapy according to the FOLFOX/XELOX regimen. In primary tumor samples, quantitative PCR was used to assess the level of HGF expression. Statistical processing of the obtained data was carried out using STATISTICA 13.0, BioStat v.7.1., and Jamovi 1.6.8 software. The study aims to study a new marker. Comparison of characteristics in the case of non-normal distribution was carried out using the nonparametric Mann–Whitney U test. Cox and Kaplan–Meier linear regression tests were used to analyze progression-free survival. When discussing the results, we used our previously obtained data on the level of expression of TGF-β and CXCL8 in the tumor tissue. As a result of the studies, it was found that in 60% of tumor samples HGF was not expressed, but it was significantly higher than in the resection line samples. Analysis of relapse-free survival in patients with CRC according to the level of HGF expression (predicted level by proportional hazards assessment – 0.7) showed that the median survival in groups 1 (HGF expression more than 0.7) and 2 (HGF expression less than 0.7) was 23.3 and 62.9 months, respectively (long rank test p = 0.215). It was shown that the level of HGF mRNA in CRC tumors does not depend on age, stage of the disease, and sensitivity to FOLFOX/XELOX chemotherapy. The expression level is significantly reduced in tumors with a KRAS mutation and increased in those with a BRAF mutation, in poorly differentiated tumors. Using the level of HGF expression in the tumor tissue of patients with non-metastatic CRC before the start of chemotherapy to assess the prognosis of the relapse-free period is only possible in conjunction with the expression of TGF-β, CXCL8 in the tissue and the level of CEA in the blood of these patients.

Lysophosphatidic Acid Stimulates Mitogenic Activity and Signaling in Human Neuroblastoma Cells through a Crosstalk with Anaplastic Lymphoma Kinase.

Lysophosphatidic acid (LPA) is a well-documented pro-oncogenic factor in different cancers, but relatively little is known on its biological activity in neuroblastoma. The LPA effects and the participation of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) in LPA mitogenic signaling were studied in human neuroblastoma cell lines. We used light microscopy and [3H]-thymidine incorporation to determine cell proliferation, Western blot to study intracellular signaling, and pharmacological and molecular tools to examine the role of ALK. We found that LPA stimulated the growth of human neuroblastoma cells, as indicated by the enhanced cell number, clonogenic activity, and DNA synthesis. These effects were curtailed by the selective ALK inhibitors NPV-TAE684 and alectinib. In a panel of human neuroblastoma cell lines harboring different ALK genomic status, the ALK inhibitors suppressed LPA-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), which are major regulators of cell proliferation. ALK depletion by siRNA treatment attenuated LPA-induced ERK1/2 activation. LPA enhanced ALK phosphorylation and potentiated ALK activation by the ALK ligand FAM150B. LPA enhanced the inhibitory phosphorylation of the tumor suppressor FoxO3a, and this response was impaired by the ALK inhibitors. These results indicate that LPA stimulates mitogenesis of human neuroblastoma cells through a crosstalk with ALK.

Rapidly derived equimolar Ca: P phasic bioactive glass infused flexible gelatin multi-functional scaffolds – A promising tissue engineering

The study aims to design and fabricate an ultra-easier multi-functional biomedical polymeric scaffold loaded with unique equimolar Ca:P phasic bioactive glass material (BG). Gelatin (G) – 45S5 bioactive glass (BG) scaffolds were synthesized via a simple laboratory refrigerator with higher biocompatibility and cytocompatibility. The results proved that BG has enhanced bio-mineralization of the scaffolds and results support that the G: BG (1:2) ratio is the more appropriate composition. Brunauer-Emmett-Teller (BET) study confirms the higher surface area for pure Gelatin and G: BG (1:2). Scanning Electron Microscopic images display the precipitation of hydroxycarbonate apatite layer over the scaffolds on immersing it in simulated body fluid. Alkaline phosphate activity proved that G: BG (1:2) scaffold could induce mitogenesis in MG-63 osteoblast cells, thus helping in hard tissue regeneration. Sirius red collagen deposition showed that higher content bioactive glass incorporated Gelatin polymeric scaffold G: BG (1:2) could induce rapid collagen secretion of NIH 3T3 fibroblast cell line that could help in soft tissue regeneration and earlier wound healing. The scaffolds were also tested for cell viability using NIH 3T3 fibroblast cell lines and MG 63 osteoblastic cell lines through methyl thiazolyl tetrazolium (MTT) assay. Thus, the study shows a scaffold of appropriate composition G: BG (1:2) can be a multifunctional material to regenerate hard and soft tissues.

Involvement of the Opioid Peptide Family in Cancer Progression.

Peptides mediate cancer progression favoring the mitogenesis, migration, and invasion of tumor cells, promoting metastasis and anti-apoptotic mechanisms, and facilitating angiogenesis/lymphangiogenesis. Tumor cells overexpress peptide receptors, crucial targets for developing specific treatments against cancer cells using peptide receptor antagonists and promoting apoptosis in tumor cells. Opioids exert an antitumoral effect, whereas others promote tumor growth and metastasis. This review updates the findings regarding the involvement of opioid peptides (enkephalins, endorphins, and dynorphins) in cancer development. Anticancer therapeutic strategies targeting the opioid peptidergic system and the main research lines to be developed regarding the topic reviewed are suggested. There is much to investigate about opioid peptides and cancer: basic information is scarce, incomplete, or absent in many tumors. This knowledge is crucial since promising anticancer strategies could be developed alone or in combination therapies with chemotherapy/radiotherapy.

Involvement of the Orexinergic System in Cancer: Antitumor Strategies and Future Perspectives

Peptides promote the mitogenesis and migration of tumor cells, and cancer cells overexpress peptide receptors. The involvement of the orexinergic system in cancer is reviewed here, including thirteen cancer types (e.g., adrenocortical adenoma, breast, colon, gastric, liver, neuroblastoma, pancreas, prostate). An upregulation of the orexinergic system has been reported in many tumors, and orexin receptors (OXRs) mediate a dual effect: apoptosis in some tumors and a proliferative action in others. OXR antagonists or agonists are potential antitumor agents against tumors expressing OXRs. The complexities of the biological processes associated with the orexigenic system are also described in the review, as they may provide the basis for the development of new therapies: OXR dimerization/oligomerization, epigenetic mechanisms controlling the orexinergic system, possible biomarkers of this system for tumor risk/prognosis, protective effects mediated by orexins against chemotherapeutic drugs, the combination therapy of OXR antagonists/agonists with radiotherapy or chemotherapy, and the anti-inflammatory effects mediated by orexins. Taking these data into account, future therapeutic applications as well as research lines to be developed are also mentioned and discussed. This knowledge will allow for the development of antitumor strategies in the future.

Dietary Polyphenols and Obesity: A Review of Polyphenol Effects on Lipid and Glucose Metabolism, Mitochondrial Homeostasis, and Starch Digestibility and Absorption.

Obesity is a major global public health concern, limiting socio-economic development and human productivity. As studies focus on finding sustainable solutions to this challenge, polyphenols have shown promising results and have become a research focus. This is mainly because of associated lower risks of side effects with their use, compared to synthetic pharmaceuticals. In this study, the anti-obesity potentials of dietary polyphenols have been reviewed. Using a narrative approach, the biological activities of polyphenols and their influence on energy metabolism and mechanisms are discussed. Specifically, their roles in insulin-dependent glucose uptake, insulin sensitivity, lipid metabolism and storage in adipocytes, starch digestibility, and regulation of mitophagy and mitogenesis in muscle cells and adipocytes, were considered. After considering the major findings of many related studies, it was confirmed that polyphenols can prevent and ameliorate obesity by fighting insulin resistance (IR) induced by pro-inflammatory cytokines, scavenging reactive oxygen species (ROS) and limiting their effects, and by regulating the expression and/or activity of key enzymes along relevant pathways. More human studies are needed to reveal more about the anti-obesity effects of dietary polyphenols and their effective doses in humans.

A COMPARITIVE AND PROGNOSTIC STUDY OF CIRCULATING TUMOR CELLS AND VEGFR-2 IN DIFFERENT HISTOLOGICAL GRADES OF ORAL SQUAMOUS CELL CARCINOMA

Background And Objectives: Oral squamous cell carcinoma (OSCC) is the fourth leading cancer and the eighth leading cause of cancer - related death worldwide. Despite improvements in diagnosis and therapeutic concepts, the 5 year overall survival rate has not improved significantly over the last 25 years and remains around 56%. Circulating tumor cells are cancer cells detached from a primary or secondary tumor and entered the circulation, these serve as a clinical biomarker for diagnostic, prognostic and pharmacological purposes. Neoangiogenesis is essential for the growth and metastasis of solid tumors.VEGFR-2 is one of the major mediator of endothelial cell mitogenesis, proliferation and survival. Inhibition of angiogenesis by disruption of VEGFR-2 pathways, this could suppress tumors` growth by limiting their blood supply. Hence, the present study was carried out to isolate and study circulating tumor cells in different grades of oral squamous cell carcinoma from the peripheral venous blood of the patient. To assess the histological grading of oral squamous cell carcinoma by using H and E sections of the biopsy sample and to assess the immunohistochemical expression of VEGFR-2 / FLK1 in circulating tumor cells and in biopsy samples. Material and Methods: The present study consisted of 60 paraffin embedded blocks of histologically diagnosed cases of 15 well differentiated OSCC, 15 moderately differentiated OSCC, 15 poorly differentiated OSCC and 15 cases of normal epithelium. Out of 45 OSCC cases, 20 OSCC patients’ blood samples were collected and subjected to CTCs isolation using Pluriselect beads. Tissue sections of 4µm thickness of paraffin embedded tissue blocks were subjected to VEGFR-2 staining. Expression of VEGFR-2, were counted randomly in 5 high power fields(40X).Quantitative analysis of VEGFR-2 was done using image analysis (image progress software) and data obtained was subjected to statistical analysis using ANOVA and Unpaired t test. Results: Higher mean expression for VEGFR-2 was seen in PDSCC followed by MDSCC, WDSCC and the control group. The difference in mean VEGFR-2 expression was found to be statistically significant between WDSCC & MDSCC (P< 0.049) as well as between WDSCC &PDSCC (P<0.000) and between MDSCC& PDSCC (P<0.001).The circulating tumor cells (CTCs) were found in 3/20(15%) patients of OSCC. Conclusion: The expression of the VEGFR-2 was higher in PDSCC as compared to MDSCC, WDSCC and normal epithelium respectively.Circulating tumor cells were found in 3 patients of OSCC.

Chronic SSRI Treatment, but Not Norepinephrine Reuptake Inhibitor Treatment, Increases Neurogenesis in Juvenile Rats

There has been growing recognition that major depressive disorder is a serious medical disorder that also affects children. This has been accompanied by an increased use of antidepressant drugs in adolescents; however, not all classes of antidepressants are effective in children and adolescents. There is an increasing need to understand the differences in antidepressant action in different developmental stages. There are some data indicating that the behavioral effect of chronic antidepressant treatment in adult rodents is dependent on hippocampal neurogenesis; however, it is not known which classes of antidepressant drugs induce hippocampal neurogenesis in adolescent rodents. Three classes of antidepressant drugs were tested in two age groups of Sprague Dawley rats, pre-adolescent (postnatal days 11–24) and adolescent (postnatal days 21–34): monoamine oxidase inhibitors (MAOIs); selective serotonin reuptake inhibitors (SSRIs); serotonin norepinephrine reuptake inhibitors (SNRIs); and tricyclic antidepressants (TCAs). To address which classes of antidepressant drugs might alter the rate of mitogenesis in neural progenitor cells in an adolescent rodent model, adolescent Sprague Dawley rats were treated with the thymidine analog 5-bromo-deoxy-2′-uridine (BrdU) on postnatal days 21 and 22 and antidepressant drugs or vehicle for 14 days (postnatal days 21–34). To address which classes of antidepressant drugs might alter the rate of neurogenesis, postnatal day-21 Sprague Dawley rats were treated with antidepressant drugs or vehicle for 14 days (postnatal days 21–34) and BrdU on postnatal days 33 and 34. In both experimental paradigms, BrdU-positive cells in the subgranular zone and the granule cell layer were counted. Newborn neurons were identified in the neurogenic paradigm by identifying cells expressing both the neuronal specific marker NeuN and BrdU using confocal microscopy. Only the SSRI fluoxetine significantly altered the basal mitogenic and neurogenic rates in adolescent rats. Treatment with the monoamine oxidase inhibitor (MAOI) tranylcypromine (TCP) and the TCA desipramine did not alter the rate of hippocampal neurogenesis in the adolescent rats. This is consistent with human clinical observations, where only SSRIs have efficacy for treatment of depression in patients under the age of 18. In pre-adolescent rats, postnatal days 11–24, none of the drugs tested significantly altered the basal mitogenic or neurogenic rates. All of the classes of antidepressant drugs are known to induce hippocampal neurogenesis in adult rats. The mechanisms of action underlying this developmental difference in antidepressant drug action between juveniles and adults are not known.

3-D nanocomposite scaffolds: Tissue engineering for bone reconstruction

A challenge to the orthopedic surgeons in veterinary and human fields is the treatment of delayed union, malunion and nonunion. Apart from renovation of arrangement and constant fixation, in many cases, adjunctive measures such as bone-grafting or the use of bone-graft substitutes, are of dominant significance. In general bone-graft materials comprise one or more components: an osteoconductive matrix, which acts as a scaffold to new bone growth; osteoinductive proteins, backing the mitogenesis of indistinguishable cells; and osteogenic cells, which can form bone in the proper environment. Our review focuses on the currently existing bone graft and graft substitutes for the novel therapeutic approaches in the clinical situation of orthopedic surgery. This review is based on an extensive literature search of different composite scaffolds developed as bone regenerative therapies. The settlement and drawbacks of different composite scaffold developed techniques, the properties of generally used ceramics and polymers, and the properties of presently investigated synthetic composite grafts. To follow, an exhaustive review of in vivo models is used to test composite scaffolds in segmental bone defects (SBDs) to serve as a guide to design suitable translational studies and to recognize the challenges that require to be overcome in scaffold design for successful transformation. This includes the formative of the anatomical position within the animals, selecting the accurate study period, and an overview of scaffold presentation evaluation.

Evaluation of Midkine Expression in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma: A Cross-Sectional Study

Abstract Introduction Oral cancer accounts for 3% of all malignancies diagnosed worldwide annually. Midkine (MK) plays a role in oncogenesis and tumor progression, including cell migration, mitogenesis, antiapoptosis, and angiogenesis. Aim and Objective To study and compare MK expression in various grades of oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Methodology This is a cross-sectional study where 60 archival specimens of OSCC, OED, and control were taken for immunohistochemical examination. Antibody MK (A-9) SC-46701 mouse monoclonal (200µg/mL, Santacruze Biotechnology) was used for immunohistochemical staining. The stained sections were observed under the objective (100 × ) of research microscope (DM 1000 LED) with computer-assisted image analyzer (Leica Application Suit V-4.1). Quantitative analysis was done to count MK reactive cells in all groups. Statistical analysis was performed using Kruskal–Wallis H test with posthoc Bonferroni's test for multiple group comparisons, and p-value < 0.05 was considered as statistically significant. Results MK expression was observed in OSCC and OED and was absent in control. The distribution of average percentage positivity was significantly higher in OED compared with OSCC and control. Statistically significant correlation was found between the size of the tumor and increased MK expression. Conclusion MK protein is overexpressed in OED and OSCC, and can be used as biological marker for early detection.

Lipid rafts integrity is essential for prolactin-induced mitogenesis in mouse embryonic stem cells.

Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constraints and regulations applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using two-dimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergent-free preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency, we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs act in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.

Aprepitant Promotes Caspase-Dependent Apoptotic Cell Death and G2/M Arrest through PI3K/Akt/NF-κB Axis in Cancer Stem-Like Esophageal Squamous Cell Carcinoma Spheres.

The antagonists of the neurokinin-1 receptor (NK1R) are known for their anti-inflammatory, anxiolytic, antiemetic, and anticancer activities. Aprepitant, a nonpeptide NK1R antagonist, is used in nausea and vomiting, the most common side effects of cancer chemotherapy in patients. It has been established that NK1R activation by substance P (SP), which links cancer promotion and progression to a neurokinin-mediated environment, became one mechanism that corresponds to the mitogenesis of tumor cells. Therefore, this study is aimed at explaining and evaluating the anticancer impacts of aprepitant on esophageal squamous cancer cell (ESCC) spheres by using in vitro experiments, such as resazurin, ROS, annexin-V binding, RT-PCR, and Western blot analysis. As a result, we showed that aprepitant had strong antiproliferative and cytotoxic effects on ESCC cell spheres. Also, aprepitant caused significant G2-M cell cycle arrest depending on concentration increase. Further, exposure of cells to this agent resulted in caspase -8/-9-dependent apoptotic pathway activation by modifying the expression of genes involved in apoptosis. Besides, treatment of the cells by aprepitant abrogates of the PI3K/Akt pathway, as shown by reducing the level of Akt, induces apoptotic cell death. In summary, pharmacological inhibition of NK1R with aprepitant seems to have a significant chance of treating ESCC as a single agent or in conjunction with other chemotherapeutic drugs.

A boomy century of periodontal regeneration with biological mediators

With the advanced and improved knowledge of bone regeneration on the molecular level, some of key molecules that alter the complicated physiological method were identified, and are already in scientific use or beneath research to enhance bone restore. Of those molecules, BMPs were the maximum considerably studied, as they are robust osteoinductive elements. They result in the mitogenesis of mesenchymal stem cells (MSCs) and different osteoprogenitors, and their differentiation in the direction of osteoblasts. Other growth factors except BMPs which have been implicated through out the bone regeneration, with one-of-a-kind features with respect of cell proliferation, chemotaxis and angiogenesis, are also being investigated or are presently getting used to reinforce bone restore, which include platelet-derived growth factor, transforming growth factor- β, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor, amongst others. One present day technique to enhance bone regeneration and soft-tissue recovery with the aid of using nearby application of growth factors is the use of platelet-rich plasma, an extent of the plasma fraction of autologous blood with platelet concentrations above baseline, that is wealthy in most of the aforementioned molecules. This overview focuses and target on the biological mediators that regulates key cellular events which have a capacity to induce the method of tissue repair and regeneration.

Alpelisib-Induced Hyperglycemia- A Small Case Series

The phosphoinositide-3-kinase (PI3K) family consists of highly conserved enzymes that are key intermediaries in signal transduction regulating cell survival and mitogenesis. This axis has been implicated in many types of cancers. In 2019 Alpelisib (Piqray), a reversible inhibitor specific to the PI3Kα subunit, in combination with fulvestrant, was approved to treat hormone receptor positive, human epidermal growth factor receptor 2 (Her2) negative, PI3K mutated breast cancer. This approval followed SOLAR-1 trial results showing a 35% risk reduction in cancer progression or death with alpelisib-fulvestrant compared to placebo in a cohort with PI3K mutated breast cancer. PI3K also plays a critical role in the insulin signaling pathway and alpelisib has been shown to cause a dose dependent rise in plasma glucose, insulin and c-peptide. Here we present a case series of severe hyperglycemia induced by alpelisib.A 44-year-old woman with ER+/Her2(-)/PI3K positive metastatic breast cancer was started on alpelisib. Previously, HbA1C was 5.4%. Hyperglycemia developed and HbA1c rose to 9.0% within 6 months of alpelisib 300mg daily. She started metformin and empagliflozin, which she was unable to tolerate due to nausea and vomiting. Her self-monitored blood glucoses were 300-400mg/dL within hours after her morning alpelisib dose. We discontinued empagliflozin when she developed metabolic acidosis with an increased anion gap. However, prior to any dose reduction, oncology discontinued alpelisib due to evidence of cancer progression. A week later, her glucoses normalized.Second case is of a 64-year-old woman with stage IV ER+/Her2(-)/PI3K mutated breast cancer with bony metastases, who was started on alpelisib 250mg. Her prior HbA1C was 5.5%. Ten days after initiation of alpelisib, she developed grade 3 hyperglycemia (blood glucoses 200-500mg/dL). She was started on metformin 2000mg with alpelisib dosed at noon. However, she noted a marked rise in blood glucose in the afternoon, few hours following alpelisib dose. Thus, moving the alpelisib to bedtime allowed better control of glycemia by using overnight basal insulin.Similarly, a 37-year-old woman with a history of ER+/HER2(-) stage IV metastatic breast cancer to the liver, with PI3K mutation was found to have acute, severe hyperglycemia with blood glucose of 300mg/dL, despite HbA1C being only 4.7%. This was attributed to initiation of alpelisib 2 days prior to admission. Given the severity of her insulin resistance (requiring > 100 units of insulin daily), alpelisib dose was reduced from 300mg to 150mg/day. On discharge, she was placed on metformin, dulaglutide, and basal and prandial insulins. Her HbA1C rose to 9.4% within 3 months of alpelisib initiation.This case series demonstrate the unique challenges in managing alpelisib induced reversible hyperglycemia.

Abstract 4915: Nerve growth factor (NGF) facilitates LOX expression and promotes tumor metastasis by inhibiting miR-149-5p in human chondrosarcoma cells

Abstract Chondrosarcomas are malignant bone tumors that commonly produce cartilaginous chondroid matrix and are associated with metastases in the lung, especially in patients with high-grade chondrosarcoma. Neurotrophins such as nerve growth factor (NGF) are recognized as being drivers of neurogenesis during development and regeneration, and their expression in human tumors can stimulate cancer cell growth. Importantly, the infiltration of nerves in the tumor microenvironment actively stimulates cancer cell mitogenesis, invasiveness and metastasis. Until now, it has been unclear as to the potential involvement of NGF in human chondrosarcoma metastasis. This study reports that NGF induces migration and invasion of chondrosarcoma cell lines JJ012 and SW1353 and significantly increasing LOX expression. Significant dose-dependent increases were observed in levels of LOX mRNA and protein expression in both cell lines. In immunohistochemistry analyses, levels of NGF and LOX expression in human chondrosarcoma cartilage correlated with tumor stage and were substantially higher than NGF and LOX expression in normal cartilage. NGF-induced stimulation of metastasis and increases in LOX expression were prevented by pretreatment of the cell lines with PI3K, Akt and mTOR inhibitors, or transfection with small interfering RNAs (siRNAs). Further analysis revealed that NGF induced PI3K/Akt/mTOR signaling and dose-dependently downregulated mature microRNA-149-5p (miR-149-5p), which targets the 3' untranslated region (3'-UTR) of LOX. Furthermore, JJ012 cells overexpressing NGF induced LOX expression and increased cell migration and invasion. In summary, NGF stimulates LOX expression and promotes chondrosarcoma cell migration and invasion by inhibiting miR-149-5p via the PI3K/Akt/mTOR signaling pathway. Citation Format: Anoop Thadevoos Louis, Chih-Yang Lin, Chih-Hsin Tang. Nerve growth factor (NGF) facilitates LOX expression and promotes tumor metastasis by inhibiting miR-149-5p in human chondrosarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4915.

The Effect of EGF on VEGF expression on Submandibular Salivary Gland of Albino Rats Receiving Doxorubicin

Background: Vascular endothelial growth factor (VEGF) has two prominent biological effects of inducing mitogenesis and migration of vascular endothelial cells and to produce permeability on the microvasculature. Epidermal growth factor (EGF), known by its mitogenic activity on different types of cells, could induce the proliferation and differentiation of the epithelial cells and stimulate its regeneration. Doxorubicin (DXR) is a widely used anticancer drug and is one of most commonly used chemotherapeutic drugs, however it may cause some serious complications such as toxicity in various tissues in the body.Aims: The present study aims to investigate the effect of EGF on the VEGF immunoexpression in submandibular salivary gland tissue of rats receiving Doxorubicin (DXR).Methods and Material: 30 male Albino rats, two months old and weighing 200-250 gm body weight, were divided equally into three groups as follows: Group I (control group). Group II: rats received 20 mg/kg body weight DXR as a single intra peritoneal injection. Whereas, group III: rats received the same dose of DXR and on the next day they were injected intraperitoneally with EGF in a daily dose of 10μg/kg body weight for 7 consecutive days.Submandibular salivary glands were dissected out and processed for histological investigation and immunohistochemical localization of VEGF in the glandular tissue.Statistical analysis used: ANOVA was used to compare the mean area percentage of VEGF immunostaining among the specimens of different groups followed by Tukey’s Multiple Comparison Test.Results: VEGF expression in submandibular salivary glands of rats significantly decreased after DXR injection. However, daily intraperitoneal injections of EGF restored normal levels of VEGF expression in the glandular tissue.Conclusions: EGF preserved glandular architecture after DXR injection and consequently the immunohistochemical expression of VEGF in the glands to approximately the normal level.

Cell proliferation analysis is a reliable predictor of lack of carcinogenicity: Case study using the pyrethroid imiprothrin on lung tumorigenesis in mice

In the mouse carcinogenicity study, an apparent increase in lung adenocarcinoma was observed in male mice at 7000 ppm. Based on the overall evaluation of toxicology, oncology, pathology and statistics, we concluded that the apparent increase in lung tumors is not relevant for evaluation of carcinogenicity of imiprothrin (Regul Toxicol Pharmacol, 105, 1–14, 2019). To investigate whether imiprothrin has any mitogenic effect on mouse Club cells, the present study examined its effects on replicative DNA synthesis of Club cells and lung histopathology in male mice treated with imiprothrin for 7 days at 3500 and 7000 ppm in the diet. Isoniazid, a known mouse lung mitogen and tumor inducer, was also examined at 1000 ppm in the diet as a positive control of Club cell mitogenesis and morphological changes. Neither imiprothrin nor isoniazid caused any necrotic changes in lung by light or electron microscopy. There were no increases observed in the bromodeoxyuridine (BrdU) labeling index in the imiprothrin groups, while there was a statistically significant increase in the BrdU labeling index in the isoniazid group. These findings demonstrate that imiprothrin does not induce mouse Club cell proliferation or morphologic changes, supporting our previous conclusion described above. Thus, imiprothrin should not be classified as a carcinogen. Furthermore, this study indicates that short-term studies focusing on cell proliferation can be reliable for predicting a lack of carcinogenic potential of test chemicals.

Modulation of EPS8 functions by phosphorylation

Objectives Epidermal growth factor receptor pathway substrate 8 (EPS8) is a scaffolding protein involved in regulating cell proliferation, actin dynamics, and receptor trafficking in human cells. EPS8 expression is increased in a range of human cancers, including head and neck squamous cell carcinoma (HNSCC). Previous studies have indicated that overexpression of EPS8 enhances mitogenesis and migration of tumor cells and is sufficient to convert nontumorigenic cells to a tumorigenic phenotype. The nonreceptor tyrosine kinase Src is reported to phosphorylate EPS8 at 4 tyrosine residues, although the impact of this on EPS8 function is unknown. The purpose of this study was to investigate the role of tyrosine phosphorylation of EPS8 at Src target sites in modulating biochemical functions, cell growth, and motility in HNSCC. Study Design Expression plasmids encoding EPS8 with amino acid substitutions to phenylalanine (F) at the 4 putative Src phosphorylation sites (Y485 F, Y525 F, Y602 F, Y774 F), and all 4 combined (4 F), were prepared by site-directed mutagenesis. To evaluate the effect of unphosphorylated EPS8 on downstream signals and biologic behavior, plasmids were transduced into a model cell line expressing a normal endogenous level of EPS8. Additionally, cells were treated with dasatinib, a Src inhibitor, to block phosphorylation of Src substrates. Expression of downstream targets was evaluated by Western blotting. Wound closure and proliferation assays were used to investigate the impact of these mutations on cell motility and growth. Results FOXM1, AURKA, and AURKB levels were decreased in cells expressing the nonphosphorylatable 4 F and Y602 F EPS8 mutants, whereas cells harboring the Y485 F, Y525 F, and Y774 F EPS8 mutants showed no differences in the expression of these proteins compared with controls. Moreover, dasatinib treatment resulted in a significant decrease in the expression of EPS8 downstream targets. In addition, both Y602 F and 4 F mutants elicited a significant reduction in tumor cell proliferation and migration. Conclusions The nonphosphorylatable Y602 F-EPS8 and 4 F-EPS8 mutants decreased the expression of EPS8 downstream targets, as well as reducing tumor cell growth and motility, implying a crucial role for phosphorylation of EPS8, principally at Y602, in mediating protumorigenic signal transduction.

Defibrotide Inhibits Endothelial Cell Injury Induced By Plasmas of Patients with Thrombotic Microangiopathies

Introduction: Defibrotide is a polydisperse mixture of predominantly single-stranded polydeoxyribonucleotide sodium salts derived from porcine intestinal mucosa. It is currently the only therapy FDA-approved to treat hematopoietic stem cell transplant (HSCT)-associated hepatic veno-occlusive disease (VOD) with multi-organ dysfunction. Its mechanisms of action are complex and incompletely understood. In vitro, defibrotide can: promote endothelial cell (EC) mitogenesis via interaction with basic fibroblast growth factor; inhibit platelet adhesion and aggregation; reduce calcineurin inhibitor-induced EC apoptosis; and block pro-inflammatory cytokine activity, in the absence of anticoagulant effects (Blood Adv 2018;2:1495-1509). Given the critical role of EC injury with impaired regulation of thrombo-fibrinolytic pathways in both transplant-associated VOD and thrombotic microangiopathies (TMA), defibrotide has been used off-label to treat TMAs post-allogeneic HSCT. Significant clinical responses occurred in the majority of cases not complicated by renal failure (Bone Marrow Transplant 2019;54:142-145). It is known that conditioning regimens for autologous HSCT involve activation of pro-inflammatory pathways in EC which can be blocked by defibrotide (Biol Blood Marrow Transplant 2011;17:497-506). We sought to determine whether clinically relevant concentrations of defibrotide could block activation of caspase 8 in EC, an early step in apoptotic injury, as induced by plasmas from patients with TMAs secondary to thrombotic thrombocytopenic purpura (TTP), cancer/chemotherapy, and allogeneic HSCT. Methods: We utilized plasmas from individuals with acute TMAs: 13 with TTP; 5 with an atypical hemolytic-uremic syndrome (HUS)-type of TMA in the setting of cancer chemotherapy, persisting after withdrawal of chemotherapy; and 10 with TMAs following alloHSCT, persisting after withdrawal of GvHD prophylaxis. The in vitro model system was previously published by our lab, documenting the ability of plasmas from acute TTP patients to activate caspase 8 in EC, followed by apoptotic EC death (Blood 2008;112:340-349). This effect was dependent on plasma-based proinflammatory cytokines capable of down-regulating EC cytoprotective factors. Briefly, 5 x 105 primary human neonatal microvascular endothelial cells of dermal origin were plated in 1ml culture medium/well of 12 well polystyrene plates (3.6cm2/well) pre-coated with 1% fibronectin. 2% (v/v) patient or control plasma was added, alone or with varying concentrations of defibrotide (0.5-10μg/ml), for 3hrs at 37ºC. Cells were then harvested and a commercial colorimetric kit (Millipore) was used to assess caspase 8 enzyme activity. Results: Initial dose-response curves showed a plateau effect for defibrotide-mediated inhibition of caspase 8 activation at 5μg/ml; subsequent experiments utilized that dose. To control for the possibility of direct inhibition of caspase 8 by defibrotide, we found that caspase 8 activity initiated by staurosporine (1μM) was unaffected by defibrotide (Fig.) >50% inhibition of caspase 8 was seen for 3/5 cancer/chemotherapy-linked TMA patient plasmas, 7/13 TTP plasmas, and 0/10 TA-TMA plasmas. >20% suppression of such activation was seen for 5/5 cancer/chemotherapy TMA plasmas, 12/13 TTP plasmas, and 4/10 TA-TMA plasmas. Representative examples are shown in the Fig., including defibrotide blocking caspase 8 activity induced by plasmas from a patient with cancer/chemotherapy linked TMA (patient 18) or TTP (patient 41), but not from another TTP patient (patient 17). Conclusions: Interventions with high degrees of therapeutic success are available for TMAs related to TTP and primary aHUS. However, TMAs complicate 20% of alloHSCT, and though half may resolve when GvHD immunoprophylaxis is discontinued, three year survival rates for those in whom the TMA persists are as low as 11%. Based on multiple case reports, defibrotide may represent an important resource in those cases, and in other TMAs refractory to conventional treatment. Further studies are required to see if defibrotide-mediated blockade of plasma-induced EC activation and injury which we demonstrate in vitro correlates with in vivo responses, and if the mechanism involves suppression of pro-inflammatory cytokine-mediated cell activation. Disclosures No relevant conflicts of interest to declare.

Massive Ovarian Growth in a Woman With Severe Insulin-Resistant Polycystic Ovary Syndrome Receiving GnRH Analogue

Ovarian hyperandrogenism from polycystic ovary syndrome (PCOS) and hyperinsulinemia from insulin resistance are modulators of ovarian follicle development. We report on a woman with PCOS and hyperandrogenism and severe insulin resistance from metabolic syndrome who received long-term GnRH analogue therapy preceding bilateral salpingo-oophorectomy for massive ovarian enlargement. Ovarian histological examination showed proliferating granulosa cells within antral follicles coexistent with serous cystadenofibromas, demonstrating a unique link between hyperinsulinemia and granulosa cell mitogenesis. A 30-year-old woman with PCOS with hyperandrogenism, severe insulin resistance from metabolic syndrome, and nonalcoholic steatohepatitis experienced abdominal pain from bilaterally enlarged ovaries. She had previously experienced a pulmonary embolism while taking oral contraceptives and hepatotoxicity from metformin and spironolactone therapies. Long-term GnRH analogue therapy to induce pituitary desensitization to GnRH successfully decreased gonadotropin-dependent steroidogenesis without improving insulin resistance. Despite GnRH analogue therapy, progressive ovarian enlargement in the presence of hyperinsulinemia from worsening metabolic function eventually required bilateral salpingo-oophorectomy for removal of massively enlarged ovaries. Histological examination showed both ovaries contained proliferating granulosa cells within antral follicles coexistent with serous cystadenofibromas. In women with PCOS and hyperinsulinemia from severe insulin resistance due to metabolic syndrome, granulosa cell proliferation within antral follicles can occur despite long-term GnRH analogue therapy, implicating hyperinsulinemia as a granulosa cell mitogen in the absence of gonadotropin-dependent ovarian function.