Stimulated Cell Proliferation Research Articles

Evaluation of Growth Hormone’s Effects on the Activation of Monocyte Cell Line THP-1 and U937

Growth hormone (GH) is a peptide hormone secreted by the pituitary gland and stimulates cell proliferation and division to promote tissue growth. In addition, the development of the immune system and the regulation of the immune response have been reported. Along with the research results related to the inflammatory control function, a correlation between the severe symptoms of COVID-19 in the elderly and a decrease in the secretion of growth hormone has recently been suggested. Therefore, this research team studied the effect of growth hormone on the proliferation and inflammatory cytokine secretion of monocyte cell lines THP-1 and U937. As a result, it was confirmed that the proliferation of THP-1 and the secretion of TNF-α and IL-1β induced by LPS were decreased by growth hormone. On the other hand, the proliferation of U937 cell line was increased by growth hormone treatment, whereas the secretion of TNF-α and IL-1β was hardly affected. In the future, the analysis of the GH receptors expressed by the two cell lines and the study of the molecular mechanism of GH action are required. In addition, it is considered that the cell stimulation technique using Poly(I:C) needs to be established more accurately.

In Vitro Cytotoxicity and Genotoxicity Screening of Cuscuta Arvensis Beyr. and Achillea Wilhelmsii C. Koch

Plant-based compounds have been used for medicinal purposes since ancient times, as easily accessible and low-cost treatment options. Despite the widespread belief that plants are quite safe and devoid of side effects, scientific studies have revealed the toxicity potential of active components of plants on healthy cells. The present study was designed to investigate in vitro cytotoxicity and genotoxicity potential of Achillea wilhelmsii C. Koch and Cuscuta arvensis Beyr., which are frequently used in traditional medicine. In this context, cytotoxicity evaluation of the extracts was performed by MTT (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Our cytotoxicity results indicated that the extract from A. wilhelmsii did not affect the viability of fibroblasts at any of the concentrations, but rather significantly stimulated cell proliferation from a concentration of 25 µg/mL. On the other hand, the extract from C. arvensis significantly reduced the viability of fibroblasts at all concentrations tested. In the second part of this research, the DNA damaging potential of the extracts was investigated by in vitro comet assay at non-cytotoxic concentrations. A. wilhelmsii extract caused a significant increase in the percentage of DNA in the tail (%TDNA), which is considered an indicator of DNA damage, only at the highest concentration, while C. arvensis extract did not significantly affect %TDNA at concentrations tested. The results of the present study indicated that the methanolic extract from A. wilhelmsii may be considered safe up to a concentration of 100 μg/mL, however, the cytotoxicity potential of C. arvensis may be a factor limiting its safe use.

Discovery of New Triterpenoids Extracted from Camellia oleifera Seed Cake and the Molecular Mechanism Underlying Their Antitumor Activity.

Theasaponin derivatives, which are reported to exert antitumor activity, have been widely reported to exist in edible plants, including in the seed cake of Camellia oleifera (C.), which is extensively grown in south of China. The purpose of this study was to isolate new theasaponin derivatives from C. seed cake and explore their potential antitumor activity and their underlying molecular mechanism. In the present study, we first isolated and identified four theasaponin derivatives (compounds 1, 2, 3, and 4) from the total aglycone extract of the seed cake of Camellia oleifera by utilizing a combination of pre-acid-hydrolysis treatment and activity-guided isolation. Among them, compound 1 (C1) and compound 4 (C4) are newly discovered theasaponins that have not been reported before. The structures of these two new compounds were characterized based on comprehensive 1D and 2D NMR spectroscopy and high-resolution mass spectrometry, as well as data reported in the literature. Secondly, the cytotoxicity and antitumor property of the above four purified compounds were evaluated in selected typical tumor cell lines, Huh-7, HepG2, Hela, A549, and SGC7901, and the results showed that the ED50 value of C4 ranges from 1.5 to 11.3 µM, which is comparable to that of cisplatinum (CDDP) in these five cell lines, indicating that C4 has the most powerful antitumor activity among them. Finally, a preliminary mechanistic investigation was performed to uncover the molecular mechanism underlying the antitumor property of C4, and the results suggested that C4 may trigger apoptosis through the Bcl-2/Caspase-3 and JAK2/STAT3 pathways, and stimulate cell proliferation via the NF-κB/iNOS/COX-2 pathway. Moreover, it was surprising to find that C4 can inhibit the Nrf2/HO-1 pathway, which indicates that C4 has the potency to overcome the resistance to cancer drugs. Therefore, C1 and C4 are two newly identified theasaponin derivatives with antitumor activity from the seed cake of Camellia oleifera, and C4 is a promising antitumor candidate not only for its powerful antitumor activity, but also for its ability to function as an Nrf2 inhibitor to enhance the anticancer drug sensitivity.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV))/Bacterial Cellulose (BC) Biocomposites for Potential Use in Biomedical Applications.

The aim of this study was to obtain biocomposites consisting of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), bacterial cellulose (BC) and α-tocopherol by a melt processing technique for potential use in biomedical applications. The melt processing and roughness of biocomposites were evaluated and compared to sample without BC. The degradation rate of PHBV/BC biocomposites was measured in phosphate buffer saline (PBS) by determining the mass variation and evidencing of thermal and structural changes by differential scanning calorimetry (DSC) and attenuated total reflectance-Fourier transformed infrared spectrometry (ATR-FTIR). The cell viability, cell morphology, cell cycle distribution and total collagen content were investigated on murine NCTC fibroblasts. Overall, the adding of BC to polyester matrix led to an adequate melt processing of biocomposites and increased surface roughness and cytocompatibility, allowing the cells to secrete the extracellular matrix (collagen) and stimulate cell proliferation. Results showed that the PHBV/BC biocomposites were favorable for long-term degradation and could be used for the design of medical devices with controlled degradability.

A tale of two peripheral blood CD34+ cell counts: Evaluation of unexpectedly high values prior to peripheral stem cell collection

Hematopoietic stem cells (HSCs) form the foundation of treatment for many hematologic and neoplastic diseases. The ability to collect HSCs that have been released into peripheral blood (PB) rather than harvesting from bone marrow (BM) directly has dramatically improved the likelihood of successful collection while minimizing donor discomfort [1]. To facilitate the release of HSCs into PB, cytokines that stimulate cell proliferation and agents that directly antagonize stromal adherence factors are administered for several days pre-collection.

Transcription Factors STAT3 and MYC Are Key Players of Human Platelet Lysate-Induced Cell Proliferation.

Human platelet lysate (HPL) is an efficient alternative for animal serum supplements, significantly enhancing stromal cell proliferation. However, the molecular mechanism behind this growth-promoting effect remains elusive. The aim of this study was to investigate the effect of HPL on cell cycle gene expression in different human stromal cells and to identify the main key players that mediate HPL's growth-enhancing effect. RT-qPCR and an antibody array revealed significant upregulation of cell cycle genes in stromal cells cultured in HPL. As HPL is rich in growth factors that are ligands of tyrosine kinase receptor (TKR) pathways, we used TKR inhibitors and could significantly reduce cell proliferation. Genome profiling, RT-qPCR and Western blotting revealed an enhanced expression of the transcription factors signal transducer and activator of transcription 3 (STAT3) and MYC, both known TKR downstream effectors and stimulators of cell proliferation, in response to HPL. In addition, specifically blocking STAT3 resulted in reduced cell proliferation and expression of cell cycle genes. Our data indicate that HPL-enhanced cell proliferation can, at least in part, be explained by the TKR-enhanced expression of STAT3 and MYC, which in turn induce the expression of genes being involved in the promotion and control of the cell cycle.

Effects of perinatal exposure to bisphenol A on induction of prostate cancer in Sprague Dawley rats by MNU and testosterone

Perinatal and neonatal exposure to bisphenol A (BPA) has been linked to enhancement of prostate carcinogenesis in rats induced by combined treatment with estradiol and testosterone, but human data are lacking. This study aimed to determine the effects of perinatal BPA exposure on induction of prostate cancer in rats by sequential treatment with N-methyl-N-nitrosamine (MNU) and continuous low dose administration of testosterone. Pregnant Sprague Dawley rats were exposed to BPA administered by subcutaneous Alzet minipumps at doses of 2.5 or 25 µg/kg body weight/day from gestational day 9 until postnatal day 28 when pups were weaned providing exposure of offspring in utero and via the mother’s milk. At 10–12 weeks of age, one male offspring per litter was treated with an intraperitoneal injection of MNU after hormonal stimulation of prostatic cell proliferation followed two weeks later by subcutaneous insertion of Silastic implants containing testosterone until the termination of the study 57–58 weeks after MNU injection. The perinatal BPA exposure did not significantly affect the incidence of prostate carcinomas which was slightly lower in exposed rats (33–23 %) than in control animals (40 %). Carcinomas in all accessory sex glands combined were also insignificantly less frequent in exposed (46–48 %) than in control rats (60 %). The incidence of malignant tumors at any site in the body was significantly lower in exposed rats (81–65 %) than in controls (93 %). In conclusion, perinatal BPA exposure did not significantly modify prostate cancer induction by MNU plus testosterone in rats, unlike the enhancement of prostate carcinogenesis induced by treatments involving estradiol administration. Which of the two models of prostate carcinogenesis is more relevant for the human situation is unclear at present.

Quercetin can affect porcine ovarian cell functions and to mitigate some of the effects of the environmental contaminant toluene

The present in vitro experiments aimed to examine the effects of the plant polyphenol quercetin and the environmental contaminant toluene on basic ovarian cell functions, including the ability of quercetin to be a natural protector against the adverse effects of toluene. The influence of toluene, quercetin, and their combination on proliferation (accumulation of PCNA), apoptosis (accumulation of bax) and release of progesterone, testosterone and insulin-like growth factor I (IGFI) by cultured porcine ovarian granulosa cells was investigated. Toluene stimulated cell proliferation and inhibited progesterone, IGF-I and testosterone release but did not affect apoptosis. Quercetin, when administered alone, inhibited cell proliferation, apoptosis, IGF-I and testosterone release and stimulated progesterone output. When administered in combination with toluene, quercetin mitigated toluene's effects on proliferation and on progesterone release and induced toluene to exhibit a pro-apoptotic effect. These observations demonstrate the direct effects of both quercetin and toluene on basic ovarian functions and a protective effect of quercetin against the effects of toluene. Therefore, quercetin-containing plants could be regulators of porcine reproduction and natural protectors against the adverse effects of the environmental contaminant toluene.

Extraction and characterization of hyaluronic acid from the eyeball of Nile Tilapia (Oreochromis niloticus)

Hyaluronic acid (HA) is a biopolymer of enormous value aggregation for in general industry. The vitreous humor of the eyeball from Nile tilapia contains appreciable amounts of hyaluronic acid. In this sense, the aim of this work was to extract and characterize hyaluronic acid from the eyeball of the Nile tilapia for biomedical applications, adding value to fish industry residues. The characterization by infra-red (FTIR), 13C nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC) confirmed that hyaluronic acid was obtained. The gel permeation chromatography (GPC) showed that the obtained material presents a low molecular mass (37 KDa). Thermogravimetry (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD) analysis showed that the materials present a thermal stability superior to the commercial hyaluronic acid from Streptococcus equi, with a partially crystalline character. The cytotoxicity assay (MTT method) with fibroblast cells (L929) demonstrated that the extracted biopolymer besides not being cytotoxic, was able to stimulate cell proliferation. Therefore, the hyaluronic acid extracted from this source of residue constitutes a product with biotechnological potential, which has adequate quality for wide biomedical applications.

Downregulation of Sirt6 by CD38 promotes cell senescence and aging.

Decreased nicotinamide adenine dinucleotide (NAD+) levels accompany aging. CD38 is the main cellular NADase. Cyanidin-3-O-glucoside (C3G), a natural inhibitor of CD38, is a well-known drug that extends the human lifespan. We investigated mechanisms of CD38 in cell senescence and C3G in antiaging. Myocardial H9c2 cells were induced to senescence with D-gal. CD38 siRNA, C3G and UBCS039 (a chemical activator of Sirt6) inhibited D-gal-induced senescence by reducing reactive oxygen species, hexokinase 2 and SA-β-galactosidase levels. These activators also stimulated cell proliferation and telomerase reverse transcriptase levels, while OSS-128167 (a chemical inhibitor of Sirt6) and Sirt6 siRNA exacerbated the senescent process. H9c2 cells that underwent D-gal-induced cell senescence increased CD38 expression and decreased Sirt6 expression; CD38 siRNA and C3G decreased CD38 expression and increased Sirt6 expression, respectively; and Sirt6 siRNA stimulated cell senescence in the presence of C3G and CD38 siRNA. In D-gal-induced acute aging mice, CD38 and Sirt6 exhibited increased and decreased expression, respectively, in myocardial tissues, and C3G treatment decreased CD38 expression and increased Sirt6 expression in the tissues. C3G also reduced IL-1β, IL-6, IL-17A, TNF-α levels and restored NAD+ and NK cell levels in the animals. We suggest that CD38 downregulates Sirt6 expression to promote cell senescence and C3G exerts an antiaging effect through CD38-Sirt6 signaling.

Prognostic Outcome of Mesenchymal Transition Biomarkers in Correlation with EGFR Expression in Epithelial Ovarian Carcinoma Patients

CD44 is an epithelial-mesenchymal transition (EMT) surface receptor that regulates the interactivity between the cells and the extracellular matrix, thereby promoting cell migration. The epidermal growth factor receptor (EGFR) family is a trans-membrane kinase-related protein. It regulates cell adhesion proteins, which may promote cell proliferation and invasiveness. Mesenchymal epithelial transition (MET) is another EMT receptor that stimulates cell proliferation, invasion, survival, and angiogenesis. This study aimed to evaluate the prognostic impact of CD44, EGFR expressions, and MET gene amplification in epithelial ovarian cancer (EOC). This is a retrospective cohort study, including 85 cases of EOC. CD44 and EGFR expressions were evaluated in both epithelial and stromal cells by immunohistochemistry. Tumor cells also underwent a cytogenetic analysis using fluorescent in situ hybridization (FISH) to detect MET gene amplification. High CD44 expression in tumors was significantly associated with serous subtypes (P=0.001), peritoneal deposits (P=0.002), and advanced stage (P=0.002). EGFR high tumor expression demonstrated a significant association with lymph node metastasis (P=0.038) and the advanced stage of EOC (P=0.016). Increased copy number of the MET gene was significantly associated with partial therapy response (P=0.030). CD44 and EGFR tumor high expression was associated with poor overall survival (OS). In addition, MET gene gain in tumors was associated with a shorter OS (P=0.000). EMT biomarkers (CD44 and MET) and EGFR expression in EOC are independent prognostic factors for OS. MET gene increase copy number was detected in cases of serous neoplasm and associated with poor survival and minimal therapy response.

Neuroprotection and neuroregeneration of cornea and ocular surface

AbstractNowadays, it is important to select the most appropriate treatment regimen for individual patients with ocular surface pathology namely dry eye disease (DED) or Meibomian Gland Dysfunction (MGD), applying to evidence‐based medicine, in order to reduce or avoid visual impairment and to improve the quality‐of‐life related to these pathologies.We shall provide an etiological therapeutic strategy to DED with special approach to the type of dry eye and to improve that approach according to inflammation and neuroprotective factors. Also, patients with DED have high levels of inflammatory molecules in the tears and ocular surface.Eye drops are the most frequent treatment for most common early cases of DED. Coenzyme Q10, present in some eye drops, can be considered a candidate for promoting the healing of the cornea—not only due to its bioenergetic effect, but also has antiapoptotic, anti‐inflammatory and antioxidant effect, which can be directly related to its neuroprotective role.For moderate cases or those not satisfactory treated with eye drops, the IPL (Intense Pulsed Light) is one of the most recent and promising option. IPL has the potential to interfere with the inflammatory cycle in DED, by upregulating anti‐inflammatory cytokines, or downregulating proinflammatory cytokines. One way to better understand the effect of IPL in DED patients would be to study the signs and clinical symptoms of these patients relating them to the changes in the genes found in their conjunctival cells, evaluating changes in gene expression related to inflammation and neuroprotection response, after treatment with IPL.Finally, an option for severe cases of DED namely neuropathic corneal lesions and/or present in immunological affected patients is ENDORET® that promotes for example refractive corneal ulcers healing. ENDORET® is an eye drop developed in the field of regenerative medicine. Basically, it is a plasma enriched in growth factors, made from the patient's own blood to promote the repair of the tissues on the surface of the eye, which stimulates cell proliferation and optimizes the processes of healing and tissue regeneration.

CC chemokine receptor 2 (CCR2) expression promotes diffuse large B-Cell lymphoma survival and invasion

In recent years, CC chemokine receptor 2 (CCR2) has been found to be involved in tumor growth, angiogenesis, epithelial mesenchymal transition, metastasis, and immune escape. CCR2 overexpression was first identified as a poor prognostic predictor in diffuse large B-cell lymphoma (DLBCL) in our published article, but the mechanisms involved remain unknown. In this work, we collected data from another 138 patients with DLBCL data and verified the CCR2 expression level and its relationship to clinicopathological characteristics. Furthermore, we explored the possible mechanisms via in vitro and in vivo experiments. We showed that CCR2 overexpression was an independent prognostic marker and predicted shorter overall survival (OS) and progression-free survival (PFS) in patients with DLBCL. Blockade of CCR2 expression with a CCR2 antagonist inhibited tumor cell proliferation, migration, and anti-apoptosis ability in vitro by affecting the PI3K/Akt signaling pathway and the p38 MAPK signaling pathway. Furthermore, administration of a CCR2 antagonist decreased tumor growth and dissemination of DLBCL cells and increased survival time in the xenograft model. Our study demonstrates that CCR2 expression plays an important role in the development of DLBCL by stimulating cell proliferation, migration, and anti-apoptosis. Therefore, the inhibition of CCR2 may be a potential target for anticancer therapy in DLBCL.The authors show that CC chemokine receptor 2 (CCR2) overexpression is an independent prognostic marker for diffuse large B-cell lymphoma (DLBCL) and predicts overall survival and progression-free survival in these patients. Blockade of CCR2 signaling with a CCR2 antagonist inhibits tumor cell proliferation, migration, and apoptosis inhibition by activating the PI3K/Akt signaling pathway and inhibiting the p38 MAPK signaling pathway. Furthermore, administration of a CCR2 antagonist decreases tumor growth and dissemination of DLBCL cells and increases survival time in a xenograft model.

Baghdadite: A Novel and Promising Calcium Silicate in Regenerative Dentistry and Medicine.

For several years, ceramic biomaterials have been extensively utilized to rebuild and substitute for body tissues. Calcium silicates have been proven to exhibit excellent bioactivity due to apatite formation and cell proliferation stimulation, in addition to degradability at levels adequate for hard tissue formation. These ceramics' excellent biological characteristics have attracted researchers. Baghdadite is a calcium silicate incorporating zirconium ions that enhances human osteoblast multiplication and development, increasing mineralization, and ossification. It has currently received much interest in academic institutions and has been extensively explored in the form of permeable frameworks, varnishes, bone adhesive and gap fillings, microparticles, and nanospheres, particularly in a wide range of biomedical applications. This review article aims to summarize and analyze the most recent research on baghdadite's mechanical characteristics, apatite-forming capability, dissolution pattern, and physiochemical qualities as a scaffold for dentofacial tissuè regeneration purposes.

Nanofiber Fractionalization Stimulates Healing of Large Intestine Anastomoses in Rabbits.

A current topic of ma jor interest in regenerative medicine is the development of novel materials for accelerated healing of sutures, and nanofibers seem to be suitable materials for this purpose. As various studies have shown, nanofibers are able to partially substitute missing extracellular matrix and to stimulate cell proliferation and differentiation in sutures. Therefore, we tested nanofibrous membranes and cryogenically fractionalized nanofibers as potential materials for support of the healing of intestinal anastomoses in a rabbit model. We compared cryogenically fractionalized chitosan and PVA nanofibers with chitosan and PVA nanofiber membranes designed for intestine anastomosis healing in a rabbit animal model. The anastomoses were biomechanically and histologically tested. In strong contrast to nanofibrous membranes, the fractionalized nanofibers did show positive effects on the healing of intestinal anastomoses in rabbits. The fractionalized nanofibers were able to reach deep layers that are key to increased mechanical strength of the intestine. Moreover, fractionalized nanofibers led to the formation of collagen-rich 3D tissue significantly exceeding the healing effects of the 2D flat nanofiber membranes. In addition, the fractionalized chitosan nanofibers eliminated peritonitis, significantly stimulated anastomosis healing and led to a higher density of microvessels, in addition to a larger fraction of myofibroblasts and collagen type I and III. Biomechanical tests supported these histological findings. We concluded that the fractionalized chitosan nanofibers led to accelerated healing for rabbit colorectal anastomoses by the targeted stimulation of collagen-producing cells in the intestine, the smooth muscle cells and the fibroblasts. We believe that the collagen-producing cells were stimulated both directly due to the presence of a biocompatible scaffold providing cell adhesion, and indirectly, by a proper stimulation of immunocytes in the suture.

Effects of Neutralization on the Physicochemical, Mechanical, and Biological Properties of Ammonium-Hydroxide-Crosslinked Chitosan Scaffolds

It has been reported that chitosan scaffolds, due to their physicochemical properties, stimulate cell proliferation in different tissues of the human body. This study aimed to determine the physicochemical, mechanical, and biological properties of chitosan scaffolds crosslinked with ammonium hydroxide, with different pH values, to better understand cell behavior depending on the pH of the biomaterial. Scaffolds were either neutralized with sodium hydroxide solution, washed with distilled water until reaching a neutral pH, or kept at alkaline pH. Physicochemical characterization included scanning electron microscopy (SEM), elemental composition (EDX), Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, thermogravimetric analysis (TGA), and mechanical testing. In vitro cytotoxicity was assessed via dental-pulp stem cells' (DPSCs') biocompatibility. The results revealed that the neutralized scaffolds exhibited better cell proliferation and morphology. It was concluded that the chitosan scaffolds' high pH (due to residual ammonium hydroxide) decreases DPSCs' cell viability.

Single Nucleotide Polymorphisms in Insulin-like Growth Factor 2 (IGF2) gene and their associations with body weight and growth rate traits in indigenous guinea fowls (Numida meleagris) of northern Ghana

Insulin-like Growth Factor 2 (IGF2) plays important roles in stimulating cell proliferation, differentiation and migration culminating its effects as a modulator of juvenile growth in animals. Although the gene that codes for IGF2 (IGF2) has been investigated as a candidate gene in several livestock species, there is no information on polymorphisms in IGF2 in guinea fowls (gIGF2), an important indigenous poultry species from West Africa. Therefore, this study sought to identify the Single Nucleotide Polymorphisms (SNPs) in three populations of indigenous guinea fowls in northern Ghana. Target genomic regions in gIGF2 were amplified and sequenced from 84 indigenous guinea fowls from Upper East Region (n = 17), former Northern Region (n = 22) and Upper West Region (n = 45) of Ghana together with domesticated French variety (n = 3). The sequences were aligned with the reference genomic sequence (domesticated French variety) of chromosome 6 (GenBank accession no., NC_034414.1) to identify SNPs. Statistical associations among the genotypes arising from the SNPs and juvenile growth traits were estimated using linear models. Two novel SNPs were identified in gIGF2 among the indigenous guinea fowls. An insertion of a Guanine (G) within a poly G motif of the intron following the third exon at the 13,955,730 bp location was identified in the majority (84.5%) of indigenous guinea fowls, while the wild type allele was observed in the minority of indigenous guinea fowls and in the domesticated French variety sampled. Also, a biallelic transition arising from the substitution of G by Adenine (A) at position 13,956,496 bp (13,956,496 G > A) located on the fourth exon, which codes for most of the extension peptide of prepro IGF2, was observed in the minority (11.9%) of indigenous guinea fowls. No significant associations among the genotypes arising from the two SNPs, with body weights and weekly growth rates, were identified.

P62SQSTM1 Affects Interacting Substrates MDM2, MDMX and Casein Kinase 1alpha to Yield Protection of Beta-Catenin Along with p53 Silencing for Refractory AML

Interventions targeting individual driver mutations in AML have not overcome resistant disease. Missing has been definition of a signal hub that integrates multiple oncogenic drivers for a composite pathway to resistance, where hub abundance determines signal(s) strength. We hypothesized that AML signal hub is p62SQSTM1, a nonmutant-active signal scaffolding for multiple pathways, including NFkB and MAPK (Moscat, Cell. 2016). Genetic epistasis in AML cell lines validated origin from P62 of such signals. Oxidative stress resistance pathways (OXPHOS) driven by p62SQSTM1 may also join with pathway(s) for leukemic progenitor replication, involving HOXA and beta-catenin within resistant AML's, but this has not been established. We sought merging pathways to OXPHOS-mediated resistance also serving to guard replicative potential through HOXA and beta-catenin. AMLs from patients were tested prior to treatment, and later failure, of induction chemotherapy, when given along with targeted agent (for cases with Flt3 mutation). Resistant AML blasts were found to have tandem overexpression of HOXAs and OXPHOS effectors p62SQSTM1, NRF2, NQO1 (NADPH oxidoreductase), BCL-2 ranging from 2-5-fold log10 relative quantity (RQ-Taqman) above chemo-sensitive core-binding factor AML, which lacks HOXA expression. We hypothesized that disrupting the downstream pathway from p62SQSTM1 to OXPHOS ending on MDM2 and/or MDMX (Todoric, Cancer Cell, 2017) is an approach to affect broad cell extinction. Indeed, besides destabilization/destruction of wild-type p53 affected by MDM2, the paralogue MDMX exists in an induced tripartite complex with CK1alpha (CK1) and p53, whose activity for restraining p53 and protecting beta-catenin from phosphorylation/inactivation is disrupted upon stress-induced CK1 activation (Huang, EMBOJ, 2020; Ueda, Cancer Cell, 2021), which we affected by using a proteasome inhibitor (PI). We found by immunoblotting AML blast lysates following PI treatment in vitro with Ixazomib, CK1 translocates to the nucleus and phosphorylation (S33,S37,T41,S45)/inactivation and cytoplasmic export of beta-catenin ensued, accompanying cell apoptosis, thus implicating MDMX inactivation resulting from PI. Primary p53-wild-type AML blasts or isogenic cell lines were treated in vitro with graded doses of the MDM2 inhibitors Pevonedistat (PEVO) or Siremadlin, either added alone, or in combination with Ixazomib at pharmacokinetically-achievable dose ranges typical in patients. Among high-risk primary AML blasts with Flt3ITD alone, or ITD along with NUP98-NSD1 or mDNMT3A, or Flt3neg with mutant EP300, or with PRDM16-SKI fusion as drivers, synergy/cooperativity was observed between MDM2 inhibitor and PI Ixazomib for inhibition of 3H-thymidine incorporation and visible apoptosis in vitro. Full characterization within PRDM16-SKI blasts by immunoblotting demonstrated expected posttranslational modification (PTM) on beta-catenin by PI, whereas distinct PTM for beta-catenin followed PEVO application, and the combination demonstrated further heterogeneity. Treatment of these blasts with Ixazomib or the combination with PEVO also led to induction of p53 in tandem with the destruction of MDM2/MDMX PTM isoforms and nuclear translocation of CK1. Strong upregulation/nuclear translocation of NRF2 resulted as part of the apoptotic pathway. To further discriminate the requirement for, and the nature of PTMs in the functional outcomes, FLAG-tagged (S33-A, S37-A,T41-A) mutant beta-catenin isoform, also lacking lysine ubiquitin target residues, was transfected in a lentiviral vector into p53-mutant K562 cells as host, and cells were compared to the same cells transfected with non-mutant FLAG-tagged beta-catenin. PEVO/Ixazomib did not induce PTM in mutant FLAG-beta-catenin transfectants, but the expected PTMs occurred in wild-type FLAG-tagged beta-catenin transfectants. In this context, PEVO became a stimulator of cell proliferation of the cells bearing mutant PTM-resistant beta-catenin, thus implicating beta-catenin disruption involving these residues as a requisite for optimal AML cell inhibition with the combination. In addition, a combination of PEVO/Ixazomib was as effective for apoptosis as was Flt3 inhibitor among certain Flt3mutant primary AML blasts. A clinical test of this strategy is warranted in high-risk relapsed/refractory p53WT/p62SQSTM1-expressing AML.

Fusobacterium nucleatum stimulates cell proliferation and promotes PD-L1 expression via IFIT1-related signal in colorectal cancer

Fusobacterium nucleatum (F. nucleatum) is enriched in colorectal cancer (CRC) tissues and a high amount of F. nucleatum was associated with an immunosuppressive tumor environment. PD-L1 is an important immune checkpoint expressed on tumor cells and promotes tumor immune escape. Whether PD-L1 is regulated by F. nucleatum is still unclear. We demonstrated that F. nucleatum promoted CRC progression and upregulated PD-L1 protein expression in CRC cell lines. Combined m6A-seq and RNA-seq identified m6A-modified IFIT1 mediating F. nucleatum induced PD-L1 upregulation. IFIT1 mRNA was modified with m6A modifications in 3’UTR and the m6A levels were altered by F. nucleatum treatment. Our results also indicated that IFIT1 served as a potential oncogene in CRC and regulated PD-L1 protein levels through altering PD-L1 ubiquitination. Clinical CRC data confirmed the correlation among F. nucleatum abundance, IFIT1 and PD-L1 expressions. Our work highlighted the function of F. nucleatum in stimulating PD-L1 expression through m6A-modified IFIT1 and provided new aspects for understanding F. nucleatum mediated immune escape.

Regulation of the Key Epithelial Cancer Suppressor miR-124 Function by Competing Endogenous RNAs.

A decrease in the miR-124 expression was observed in various epithelial cancers. Like a classical suppressor, miR-124 can inhibit the translation of multiple oncogenic proteins. Epigenetic mechanisms play a significant role in the regulation of miR-124 expression and involve hypermethylation of the MIR-124-1/-2/-3 genes and the effects of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) according to the model of competing endogenous RNAs (ceRNAs). More than 40 interactomes (lncRNA/miR-124/mRNA) based on competition between lncRNAs and mRNAs for miR-124 binding have been identified in various epithelial cancers. LncRNAs MALAT1, NEAT1, HOXA11-AS, and XIST are the most represented in these axes. Fourteen axes (e.g., SND1-IT1/miR-124/COL4A1) are involved in EMT and/or metastasis. Moreover, eight axes (e.g., OIP5-AS1/miR-124-5p/IDH2) are involved in key pathways, such as Wnt/b-catenin, E2F1, TGF-β, SMAD, ERK/MAPK, HIF-1α, Notch, PI3K/Akt signaling, and cancer cell stemness. Additionally, 15 axes impaired patient survival and three axes reduced chemo- or radiosensitivity. To date, 14 cases of miR-124 regulation by circRNAs have been identified. Half of them involve circHIPK3, which belongs to the exonic ecircRNAs and stimulates cell proliferation, EMT, autophagy, angiogenesis, and multidrug resistance. Thus, miR-124 and its interacting partners may be considered promising targets for cancer therapy.