Cell Cycle Inhibitory Protein Research Articles

Baicalein suppresses the proliferation and invasiveness of colorectal cancer cells by inhibiting Snail‑induced epithelial‑mesenchymal transition.

Scutellaria baicalensis (S. baicalensis) is a plant that is widely used for medicinal purposes. Baicalein, one of the primary bioactive compounds found in S. baicalensis, is thought to possess antitumor activity, although the specific mechanisms remain unclear. Therefore, the present study aimed to evaluate the ability of baicalein to disrupt the proliferation and metastatic potential of colorectal cancer (CRC) cells; a rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometric method was employed for the identification of baicalein in an S. baicalensis aqueous extract and in rat plasma. To investigate the effects of baicalein, Cell Counting Kit-8 (CCK-8), western blotting, wound-healing and Transwell assays were performed. The data indicated that baicalein was absorbed into the blood and was able to effectively disrupt the proliferation, migration and invasion abilities of CRC cells in a dose- and time-dependent manner. Baicalein treatment was also revealed to decrease the expression of epithelial-mesenchymal transition (EMT)-promoting factors including vimentin, Twist1, and Snail, but to upregulate the expression of E-cadherin in CRC cells. The expression levels of cell cycle inhibitory proteins p53 and p21 also increased following baicalein treatment. In addition, Snail-induced vimentin and Twist1 upregulation, as well as E-cadherin downregulation, were reversed following treatment with baicalein. In conclusion, the results of the present study indicate that baicalein may suppress EMT, at least in part, by decreasing Snail activity.

Albumin promotes proliferation of G1 arrested serum starved hepatocellular carcinoma cells

Albumin is the most abundant plasma protein and functions as a transport molecule that continuously interacts with various cell types. Because of these properties, albumin has been exploited by the pharmaceutical industry to improve drug delivery into target cells. The immediate effects of albumin on cells, however, require further understanding. The cell interacting properties and pharmaceutical applications of albumin incentivises continual research into the immediate effects of albumin on cells. The HepG2/C3A hepatocellular carcinoma cell line is used as a model for studying cancer pathology as well as liver biosynthesis and cellular responses to drugs. Here we investigated the direct effect of purified albumin on HepG2/C3A cell proliferation in the absence of serum, growth factors and other serum originating albumin bound molecules. We observed that the reduced cell counts in serum starved HepG2/C3A cultures were increased by the inclusion of albumin. Cell cycle analysis demonstrated that the percentage of cells in G1 phase during serum starvation was reduced from 86.4 ± 2.3% to 78.3 ± 3.2% by the inclusion of albumin whereas the percentage of cells in S phase was increased from 6.5 ± 1.5% to 14.3 ± 3.6%. A significant reduction in the cell cycle inhibitor protein, P21, accompanied the changes in the proportions of cell cycle phases upon treatment with albumin. We have also observed that the levels of dead cells determined by DNA fragmentation and membrane permeabilization caused by serum starvation (TUNEL: 16.6 ± 7.2%, ethidium bromide: 13.8 ± 4.8%) were not significantly altered by the inclusion of albumin (11.6 ± 10.2%, ethidium bromide: 16.9 ± 8.9%). Therefore, the increase in cell number was mainly caused by albumin promoting proliferation rather than protection against cell death. These primary findings demonstrate that albumin has immediate effects on HepG2/C3A hepatocellular carcinoma cells. These effects should be taken into consideration when studying the effects of albumin bound drugs or pathological ligands bound to albumin on HepG2/C3A cells.

Yes-associated protein regulates podocyte cell cycle re-entry and dedifferentiation in adriamycin-induced nephropathy

Podocytes are terminally differentiated cells with little proliferative capacity. The high expression levels of cell cycle inhibitory proteins, including p21, p27, and p57, play an important role in maintaining the low level of proliferation of mature podocytes. In the present study, we aimed to explore the role of yes-associated protein (YAP) signalling in adriamycin-induced podocyte re-entry into the cell cycle and dedifferentiation. Proliferating cell nuclear antigen (PCNA)-, cyclin-dependent kinase 4 (CDK4)-, and Cyclin D1-positive podocytes were found in mice with adriamycin-induced nephropathy. In vitro, adriamycin administration increased the percentage of cells in S phase and the upregulation of mesenchymal-related marker proteins. CDK4 and cyclin D1 were significantly up-regulated after incubation with adriamycin. Overexpression of YAP in podocytes promoted their entry into the cell cycle; up-regulated cyclin D1, desmin, and snail2 expression and down-regulated Wilms’ tumour 1 (WT1) and nephrin production. Recombinant murine FGF-basic induced podocytes to re-enter the cell cycle, inhibited WT1 and nephrin, and increased desmin and snail2 expression. Pretreating podocytes with verteporfin, an inhibitor of YAP/ TEA domain transcription factor (TEAD), decreased the adriamycin-induced overexpression of cyclin D1 and reduced the ratio of S-phase podocytes. This result was further verified by knocking down YAP expression using RNA interference. In conclusion, adriamycin induced podocytes to re-enter the cell cycle via upregulation of CDK4 and cyclin D1 expression, which was at least partly mediated by YAP signalling. Re-entry into the cell cycle induced the over-expression of mesenchymal markers in podocytes.

In Utero Exposure to Gestational Diabetes Alters DNA Methylation and Gene Expression of CDKN2A/B in Langerhans Islets of Rat Offspring.

Objective DNA methylation, a major epigenetic reprogramming mechanism, contributes to the increased prevalence of type 2 diabetes mellitus (T2DM). Based on genome-wide association studies, polymorphisms in CDKN2A/B are associated with T2DM. Our previous studies showed that gestational diabetes mellitus (GDM) causes apoptosis in β-cells, leading to a reduction in their number in pancreatic tissue of GDM-exposed adult rat offspring. The aim of this study was to examine the impact of intrauterine exposure to GDM on DNA methylation, mRNA transcription, as well as protein expression of these factors in the pancreatic islets of Wistar rat offspring. Our hypothesis was that the morphological changes seen in our previous study might have been caused by aberrant methylation and expression of CDKN2A/B. Materials and Methods In this experimental study, we delineated DNA methylation patterns, mRNA transcription and protein expression level of CDKN2A/B in the pancreatic islets of 15-week-old rat offspring of streptozotocin-induced GDM dams. We performed bisulfite sequencing to determine the DNA methylation patterns of CpGs in candidate promoter regions of CDKN2A/B. Furthermore, we compared the levels of mRNA transcripts as well as the cell cycle inhibitory proteins P15 and P16 in two groups by qPCR and western blotting, respectively. Results Our results demonstrated that hypomethylation of CpG sites in the vicinity of CDKN2A and CDKN2B genes is positively related to increased levels of CDKN2A/B mRNA and protein in islets of Langerhans in the GDM offspring. The average percentage of CDKN2A promoter methylation was significantly lower in GDM group compared to the controls (P<0.01). ConclusionWe postulate that GDM is likely to exert its adverse effects on pancreatic β-cells of offspring through hypomethylation of the CDKN2A/B promoter. Abnormal methylation of these genes may have a link with β-cell dysfunction and diabetes. These data potentially lead to a novel approach to the treatment of T2DM.

Abstract 894: Inhibition of Long Noncoding RNA lncExACT1 Induces Physiological Cardiac Hypertrophy and Protects Against Pathological Hypertrophy

Long noncoding-RNAs (lncRNAs) are critical regulators of cardiac development as well as pathological hypertrophy and heart failure (HF). However, their roles in exercise-induced physiological hypertrophy are unclear. Here, we used RNAseq to identify a novel class of cardiac lncRNAs that are dynamically regulated by exercise. We call these l ong n on c oding Ex ercise A ssociated C ardiac T ranscripts (lncExACTs). Among them, lncExACT1, a highly conserved lncRNA, is down-regulated in exercised hearts but upregulated in transverse aortic constriction (TAC)-induced pathological hypertrophy and HF. In primary neonatal cardiomyocytes (CMs), transfection of LNA antisense oligonucleotide complementary to lncExACT1 (GapmeR) was sufficient to inhibit lncExACT1 expression and increase CM size with a gene expression pattern consistent with physiological hypertrophy (increased PCG1α and α/βMHC ratio, all p &lt;0.05 vs . control). In contrast, lentiviral overexpression of lncExACT1 in primary CMs induced a pathological gene expression pattern (decreased PCG1α and β/αMHC ratio, with increased ANP and BNP, all p &lt;0.05 vs . control). In vivo, GapmeR treatment for two weeks reduced cardiac lncExACT1 expression (0.5-fold at 2 weeks), increased ventricular wall thickness and fractional shortening (FS, p =0.034 vs . control), increased heart weight to tibial length ratio (HW/TL) and α/βMHC. In contrast, injection of AAV9-lncExACT1 increased cardiac lncExACT1 (6-fold at 5 weeks) increased HW/TL as well as βMHC and ANP. Further, lncExACT1 acted as a sponge for microRNA-222 (a microRNA we previously reported is necessary for physiological cardiac growth) thereby increasing expression of microRNA-222 targets, including p27, a cell cycle inhibitor protein. Moreover, GapmeR injection reduced TAC-induced increase of interventricular septal end diastole (IVSd) and left Ventricular Posterior Wall Dimensions (LVPWd) without affecting FS. We conclude that inhibition of lncExACT1 is sufficient to induce physiological hypertrophy and protect against pathological hypertrophy, while induction of lncExACT1 promotes pathological hypertrophy. lncExACT1 appears to mediate these effects, at least in part, by acting as competing endogenous RNA for microRNA-222.

Esculetin induces cell cycle arrest and apoptosis in human colon cancer LoVo cells.

We investigated the anti-cancer effects of ESC in human colon cancer LoVo cells. Cell counting assay results showed that ESC inhibited the proliferation of LoVo cells. Cell cycle arrest results showed that cell cycle was arrested during the G0/G1 phase in the ESC-treated LoVo cells. Western blot results showed that the cell cycle inhibitory proteins p53, p27, and p21 were increased, and cyclin D1, the cell cycle progressive protein, was decreased. Sp1 is a transcription factor regulating cell proliferation, was decreased in the ESC-treated LoVo cells. Annexin V/propidium iodide staining results showed that ESC induces apoptosis in LoVo cells. Western blot results showed that Bax, cleaved caspase -3, -7, -9, and poly(ADP-ribose) polymerase, which are proapoptotic proteins, were increased and the antiapoptotic protein Bcl-2 was decreased. Taken together, ESC induced apoptosis and has an anti-cancer effect in LoVo cells.

Abstract 293: Inhibition of BET proteins augments nab-paclitaxel-gemcitabine-based chemotherapy response in preclinical models of pancreatic cancer

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) often presents with metastatic activity, leading to an extremely poor prognosis. Nanoparticle albumin-bound paclitaxel (NPT) in combination with gemcitabine (NPT+GEM) is a standard treatment for PDAC patients, resulting in a survival of ~8.5 months. Bromodomain and extraterminal domain (BET) proteins are epigenetic regulators of gene expression and are involved in cancer pathogenesis. Targeted inhibition of BET protein is currently under investigation for several cancers. We hypothesize that BET protein pathway inhibition by iBet-762 will enhance nab-paclitaxel-gemcitabine-based standard chemotherapy response in PDAC. METHODS: In vitro cell proliferation assays were performed using WST-1 reagent. Protein expressions were determined by Western Blot analysis. In vivo animal survival and tumor growth experiments were performed in NOD-SCID mice. RESULTS: BET inhibitor (iBET-762) and standard chemotherapy (NPT+Gem) had a dose-dependent in vitro growth inhibitory effect on several PDAC cell lines tested. Inhibition in cell proliferation at 1 μM drug concentration in NPT+Gem, iBET-762 and NPT+Gem+iBet-762 treatment groups was 64%, 27%, 76% in AsPC-1; 43%, 13%, 69% in Panc-1; and 42%, 51%, 75% in MIA PaCa-2 cells. Immunoblot analysis demonstrated that iBET-762 decreased the expression of oncogenic proteins c-Myc, β-catenin, vimentin and phospho-AKT, while increasing the expression of apoptosis-related proteins such as cleaved PARP-1 and cleaved caspase-3 and cell cycle inhibitor proteins P21 and P27. In subcutaneous xenografts, compared to controls, NPT+Gem and iBet-762 decreased tumor growth by 72% and 57%, respectively (p&amp;lt;0.02). Importantly, the combination therapy group (NPT+Gem+iBet-762) had an additive effect on tumor growth inhibition (98%, p&amp;lt;0.0001). In a peritoneal dissemination model, median animal survival compared to controls (21 days) was increased by NPT+Gem (33 days, a 57% increase) and iBet-762 (30 days, a 43% increase) therapy. This was further increased in the combination therapy group NPT+Gem+iBET-762 (44 days, a 110% increase). CONCLUSION: These findings demonstrate that nab-paclitaxel-gemcitabine-based standard chemotherapy response can be enhanced through specific inhibition of BET proteins by iBET-762 in preclinical models of PDAC. The data support the potential of this combinatorial therapeutic strategy for clinical PDAC therapy. Citation Format: Niranjan Awasthi, Sandeep Singh, Ross D. McCauley, Johann RE Schwarz, Margaret A. Schwarz, Roderich E. Schwarz. Inhibition of BET proteins augments nab-paclitaxel-gemcitabine-based chemotherapy response in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 293.

Two Pathways of p27Kip1 Degradation Are Required for Murine Lymphoma Driven by Myc and EBV Latent Membrane Protein 2A.

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A), expressed in EBV latency, contributes to Burkitt lymphoma (BL) development in a murine model by acting as a constitutively active B cell receptor (BCR) mimic. Mice expressing both LMP2A and MYC transgenes (LMP2A/λ-MYC) develop tumors significantly faster than mice only expressing MYC (λ-MYC). Previously, we demonstrated the cell cycle inhibitor p27Kip1 is present at significantly lower levels in LMP2A/λ-MYC mice due to increased posttranslational degradation. P27Kip1 degradation can occur in the cytoplasm following phosphorylation on serine 10 (S10) or in the nucleus via the SCFSkp2 complex, which depends on Cks1. We previously demonstrated an S10A knock-in of p27Kip1 (p27S10A/S10A) that prevented S10 phosphorylation failed to significantly delay tumor onset in LMP2A/λ-MYC mice. We also previously demonstrated that a Cks1 knockout partially delayed tumor onset in LMP2A/λ-MYC mice, but onset was still significantly faster than that in λ-MYC mice. Here, we have combined both genetic manipulations in what we call p27Super mice. LMP2A/λ-MYC/p27Super mice and λ-MYC/p27Super mice both displayed dramatic delays in tumor onset. Strikingly, tumor development in LMP2A/λ-MYC/p27Super mice was later than that in λ-MYC mice and not significantly different from that in λ-MYC/p27Super mice. The p27Super genotype also normalized G1-S-phase cell cycle progression, spleen size, and splenic architecture in LMP2A/λ-MYC mice. Our results reveal both major pathways of p27Kip1 degradation are required for the accelerated BL development driven by LMP2A in our BL model and that blocking both degradation pathways is sufficient to delay Myc-driven tumor development with or without LMP2A.IMPORTANCE BL is a cancer that primarily affects children. The side effects of chemotherapy highlight the need for better BL treatments. Many BL tumors contain EBV, and our goal is to determine what makes EBV-positive BL different from EBV-negative BL. This may lead to more specific treatments for both types. All cases of BL require overexpression of MYC Mice engineered to express EBV LMP2A along with MYC (LMP2A/λ-MYC mice) develop tumors much more quickly than mice only expressing MYC (λ-MYC mice). Blocking degradation of the cell cycle inhibitor protein p27Kip1 in LMP2A/λ-MYC mice causes tumors to develop later than in λ-MYC mice, showing that p27Kip1 degradation may play a larger role in EBV-positive BL than EBV-negative BL. Furthermore, our studies suggest the cell cycle is an attractive target as a treatment option for LMP2A-positive cancers in humans.

Curcumin Induces Apoptosis by regulating the cell cycle proteins in MDA‐MB‐231 Breast CancerCells

BackgroundCurcumin, a natural polyphenolic compound found in turmeric, has potential cancertreatment efficacy, including apoptosis‐induction, in many cancer cell types. Cancer cells arecharacteristically anti‐apoptotic, highly proliferative andcontinue to live indefinitely. Controlof cell cycle progression and the initiation of apoptosis in breast cancercells is considered to be a potentially effective strategy for the prevention/treatment of the disease. The triple‐negative breast cancer cell line MDA‐MB‐231 does not express progesterone, estrogen, or human epidermal growth factor receptors, is highly aggressive and is difficult to treat. This study aims to elucidate the mechanism of curcumin‐induced apoptosis and its effects on cell cycle proteins in the MDA‐MB‐213 breast cancer cell line.MethodsApoptosis was measured by flow cytometric analysis of AnnexinV/Propidium Iodide expression. Cell cycle arrest was measured by Western Blot analyses of p21Waf‐1, p53 and retinoblastoma protein pRb, as well as the cyclin dependent kinases Cyclin B1, and Cyclin D1.ResultsFlow cytometry data suggest that curcumin induces both early and late stage apoptosis in MDA‐MB‐231 cells. Cell cycle inhibitory protein p21Waf‐1 was significantly increased in curcumin‐treated cells compared to untreated cells. Tumor suppressor protein p53 and Cyclin D1 were significantly down‐regulated (p=.0.0001 and p=0.014, respectively), pRb, another tumors uppressor protein, was unchanged, and Cyclin B1 was upregulated (p=0.0006).ConclusionGiven that upregulation of p21Waf‐1 can lead to apoptosis and can disrupt CDKs/cyclin complexes necessary for cell cycle progression, and that down‐regulation of Cyclin D1, which is required for progression through the G1/S phase and whose over expression is associated with most breast cancers, can lead to cell‐cycle arrest at G1, curcumin appears to be effective in both apoptosis induction, demonstrated also in the flow cytometric results, and in cell‐cycle arrest at G1. The upregulation of cyclinB1, which is involved in the G2/M phase transition, and the down‐regulation ofp53 by curcumin, indicate that additional mechanisms are involved with curcumin exposure. Our findings will aid in the development of future therapeutic strategies, including curcumin as a novel treatment supplement, to effectively induce both cell‐cycle arrest and apoptosis in breast tumors.Disclaimer: The views expressed in this manuscript are those of the author and do not reflect the official policy of the Department of Army, Department of Defense, or U.S. Government.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gypenoside L Inhibits Proliferation of Liver and Esophageal Cancer Cells by Inducing Senescence.

Senescence is an irreversible state of cell cycle arrest that can be triggered by multiple stimuli, such as oxygen reactive species and DNA damage. Growing evidence has proven that senescence is a tumor-suppressive approach in cancer treatment. Therefore, developing novel agents that modulate senescence may be an alternative strategy against cancer. In our study, we investigated the inhibitory effect of gypenoside L (Gyp-L), a saponin isolated from Gynostemma pentaphyllum, on cancer cell growth. We found that Gyp-L increased the SA-β-galactosidase activity, promoted the production of senescence-associated secretory cytokines, and inhibited cell proliferation of human liver and esophageal cancer cells. Moreover, Gyp-L caused cell cycle arrest at S phase, and activated senescence-related cell cycle inhibitor proteins (p21 and p27) and their upstream regulators. In addition, Gyp-L activated p38 and ERK MAPK pathways and NF-κB pathway to induce senescence. Consistently, adding chemical inhibitors efficiently counteracted the Gyp-L-mediated senescence, growth inhibition, and cell cycle arrest in cancer cells. Furthermore, treatment with Gyp-L, enhanced the cytotoxicity of clinic therapeutic drugs, including 5-fluorouracil and cisplatin, on cancer cells. Overall, these results indicate that Gyp-L inhibits proliferation of cancer cells by inducing senescence and renders cancer cells more sensitive to chemotherapy.

Ion Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered Proteins.

The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, κ14 and κ56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.

CCR2 Chemokine Receptors Enhance Growth and Cell-Cycle Progression of Breast Cancer Cells through SRC and PKC Activation.

Basal-like breast cancers are an aggressive breast cancer subtype, which often lack estrogen receptor, progesterone receptor, and Her2 expression, and are resistant to antihormonal and targeted therapy, resulting in few treatment options. Understanding the underlying mechanisms that regulate progression of basal-like breast cancers would lead to new therapeutic targets and improved treatment strategies. Breast cancer progression is characterized by inflammatory responses, regulated in part by chemokines. The CCL2/CCR2 chemokine pathway is best known for regulating breast cancer progression through macrophage-dependent mechanisms. Here, we demonstrated important biological roles for CCL2/CCR2 signaling in breast cancer cells. Using the MCF10CA1d xenograft model of basal-like breast cancer, primary tumor growth was significantly increased with cotransplantation of patient-derived fibroblasts expressing high levels of CCL2, and was inhibited with CRISP/R gene ablation of stromal CCL2. CRISP/R gene ablation of CCR2 in MCF10CA1d breast cancer cells inhibited breast tumor growth and M2 macrophage recruitment and validated through CCR2 shRNA knockdown in the 4T1 model. Reverse phase protein array analysis revealed that cell-cycle protein expression was associated with CCR2 expression in basal-like breast cancer cells. CCL2 treatment of basal-like breast cancer cell lines increased proliferation and cell-cycle progression associated with SRC and PKC activation. Through pharmacologic approaches, we demonstrated that SRC and PKC negatively regulated expression of the cell-cycle inhibitor protein p27KIP1, and are necessary for CCL2-induced breast cancer cell proliferation. IMPLICATIONS: This report sheds novel light on CCL2/CCR2 chemokine signaling as a mitogenic pathway and cell-cycle regulator in breast cancer cells.

Effects of Human Placental Amnion Derived Mesenchymal Stem Cells on Proliferation and Apoptosis Mechanisms in Chronic Kidney Disease in the Rat.

Background and ObjectivesThe feature of chronic kidney failure (CKF) is loss of kidney functions due to erosion of healthy tissue and fibrosis. Recent studies showed that Mesenchymal stem cells (MSCs) differentiated into tubular epithelial cells thus renal function and structures renewed. Furthermore, MSCs protect renal function in CKF. Therefore, we aimed to investigate whether human amnion-derived mesenchymal stem cells (hAMSCs) can repair fibrosis and determine the effects on proliferation and apoptosis mechanisms in chronic kidney failure.Methods and ResultsIn this study, rat model of CKF was constituted by applying Aristolochic acid (AA). hAMSCs were isolated from term placenta amnion membrane and transplanted into tail vein of rats. At the end of 30 days and 60 days of recovery period, we examined expressions of PCNA, p57 and Parp-1 by western blotting. Immunoreactivity of PCNA, Ki67, IL-6 and Collagen type I were detected by immunohistochemistry. Besides, apoptosis was detected by TUNEL. Serum creatinine and urea were measured. Expressions of PCNA and Ki67 increased in hAMSC groups compared with AA group. Furthermore, expressions of PARP-1 apoptosis marker and p57 cell cycle inhibitory protein increased in AA group significantly according to control, hAMSC groups and sham groups. IL-6 proinflammatory cytokine increased in AA group significantly according to control, hAMSCs groups and sham groups. Expressions of Collagen type I protein reduced in hAMSCs groups compared to AA group. After hAMSC treatment, serum creatinine and urea levels significantly decreased compared to AA group. After injection of hAMSC to rats, Masson’s Trichrome and Sirius Red staining showed fibrosis reduction in kidney.ConclusionsAccording to our results hAMSCs can be ameliorate renal failure.

Cadmium induces apoptotic program imbalance and cell cycle inhibitor expression in cultured human astrocytes

Cadmium is a highly neurotoxic heavy metal impairing neurogenesis and induces neurodegenerative disorders. Toxic concentrations of cadmium induce astrocytic apoptosis by depleting intracellular glutathione levels, elevating intracellular calcium levels, altering mitochondria membrane potentials, and activating JNK and PI3K/Akt signaling pathways. Cadmium suppresses cell proliferation in kidney epithelial cells, lung fibroblasts, and primary myelocytes; however, cadmium’s effects on proteins regulating oxidative stress, apoptosis, and cell proliferation in astrocytes are less known. The present study hypothesized that cadmium alters levels of antioxidant enzymes, apoptotic regulator proteins, and cell cycle inhibitor proteins, resulting in apoptosis and cell cycle arrest. Concentrations ≥20 μM cadmium induced apoptosis and led to intracellular changes including DNA fragmentation, reduced mRNA expression of antioxidant enzymes (i.e., catalase and glutathione S transferase-A4), downregulation of B-cell lymphoma 2 (Bcl-2), and upregulation of Bcl-2-associated X protein (Bax). Moreover, cadmium suppressed astrocytic proliferation by inducing S and G2/M phase cell cycle arrest and promoting p53, p21, and p27 expression. In conclusion, this study provides mechanistic insight into cadmium-induced cytotoxicity of astrocytes and highlights potential targets for prevention of cadmium-induced apoptosis and cell cycle arrest.

EGR1 is essential for deoxynivalenol-induced G2/M cell cycle arrest in HepG2 cells via the ATF3ΔZip2a/2b-EGR1-p21 pathway

Deoxynivalenol (DON) is a type B trichothecene mycotoxin that exerts multiple toxic effects on plants, animals and humans. Several reports have shown that DON leads to G2/M cell cycle arrest. However, its molecular mechanism is still unclear. In this study, we showed that DON induced strong G2/M cell cycle arrest in HepG2 cells, and the cell cycle-inhibitory protein p21 was highly upregulated by DON. Further analysis showed that the cell cycle regulating gene EGR1 was highly induced by DON and that EGR1 knockdown abolished the upregulation of p21 and G2/M cell cycle arrest. Furthermore, we showed that the induction of EGR1 was regulated by the stress-responsive transcription factor ATF3. ATF3ΔZip2a/2b, which is a DNA binding domain truncated isoform of ATF3, was upregulated by DON. ATF3 knockdown weakened the expression induction of EGR1 and G2/M cell cycle arrest by DON. Moreover, the upregulation of ATF3ΔZip2a/2 highly depended on the enhanced presence of histones H3K9ac and H3K27ac. H3K9ac and H3K27ac were enriched at the promoter region of ATF3 following the DON treatment, and the knocking down of the genes responsible for H3K9ac and H3K27ac abolished the upregulation of ATF3 by DON. In summary, we found that DON induced G2/M cell cycle arrest by sequentially inducing the expression of ATF3ΔZip2a/2b, EGR1 and p21, and EGR1 played an essential role in this process, which is a novel molecular mechanism of cell cycle arrest by DON and is important for understanding its toxicology.

Multiple mechanisms explain how reduced KRP expression increases leaf size of Arabidopsis thaliana.

Although cell number generally correlates with organ size, the role of cell cycle control in growth regulation is still largely unsolved. We studied kip related protein (krp) 4, 6 and 7 single, double and triple mutants of Arabidopsis thaliana to understand the role of cell cycle inhibitory proteins in leaf development. We performed leaf growth and seed size analysis, kinematic analysis, flow cytometery, transcriptome analysis and mathematical modeling of G1/S and G2/M checkpoint progression of the mitotic and endoreplication cycle. Double and triple mutants progressively increased mature leaf size, because of elevated expression of cell cycle and DNA replication genes stimulating progression through the division and endoreplication cycle. However, cell number was also already increased before leaf emergence, as a result of an increased cell number in the embryo. We show that increased embryo and seed size in krp4/6/7 results from seed abortion, presumably reducing resource competition, and that seed size differences contribute to the phenotype of several large-leaf mutants. Our results provide a new mechanistic understanding of the role of cell cycle regulation in leaf development and highlight the contribution of the embryo to the development of leaves after germination in general.

Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline.

Cellular senescence, which is characterized by an irreversible cell-cycle arrest1 accompanied by a distinctive secretory phenotype2, can be induced through various intracellular and extracellular factors. Senescent cells that express the cell cycle inhibitory protein p16INK4A have been found to actively drive naturally occurring age-related tissue deterioration3,4 and contribute to several diseases associated with ageing, including atherosclerosis5 and osteoarthritis6. Various markers of senescence have been observed in patients with neurodegenerative diseases7-9; however, a role for senescent cells in the aetiology of these pathologies is unknown. Here we show a causal link between the accumulation of senescent cells and cognition-associated neuronal loss. We found that the MAPTP301SPS19 mouse model of tau-dependent neurodegenerative disease10 accumulates p16INK4A-positive senescent astrocytes and microglia. Clearance of these cells as they arise using INK-ATTAC transgenic mice prevents gliosis, hyperphosphorylation of both soluble and insoluble tau leading to neurofibrillary tangle deposition, and degeneration of cortical and hippocampal neurons, thus preserving cognitive function. Pharmacological intervention with a first-generation senolytic modulates tau aggregation. Collectively, these results show that senescent cells have a role in the initiation and progression of tau-mediated disease, and suggest that targeting senescent cells may provide a therapeutic avenue for the treatment of these pathologies.

Antifibrosis Effect of Novel Oridonin Analog CYD0618 Via Suppression of the NF-κB Pathway

BackgroundLiver fibrosis is characterized as excessive deposition of the extracellular matrix proteins, primarily by activated hepatic stellate cells (HSCs). NF-κB has been reported as one of the major mediators of HSC activation. Previously, our team reported that oridonin exhibited antihepatic fibrogenetic activity in vitro. In this study, we examined the effects of its novel derivative CYD0618 on HSC viability, apoptosis, and NF-κB signaling. MethodsCell proliferation of activated human and rat HSC lines LX-2 and HSC-T6 was measured using Alamar Blue Assay. Apoptosis was measured by a Cell Death Detection ELISA kit. Cellular proteins were determined by Western blots and immunofluorescence. ResultsCYD0618 significantly inhibited LX-2 and HSC-T6 cell proliferation in a dose-dependent manner. CYD0618 induced cell apoptosis in both cell lines. CYD0618 treatment increased cell cycle inhibitory protein p21, p27, and induced apoptosis marker cleaved poly (ADP-ribose) polymerase, while suppressing the expression of Collagen type 1. CYD0618 blocked lipopolysaccharide (LPS)-induced NF-κB p65 nuclear translocation and DNA binding activity and prevented LPS-induced NF-κB inhibitory protein IκBα phosphorylation and degradation. LPS-stimulated NF-κB downstream target cytokines IL-6 and MCP-1 were attenuated by CYD0618. Endogenous and LPS-stimulated NF-κB p65 S536 phosphorylation was inhibited by CYD0618 treatment. ConclusionsThe potent antihepatic fibrogenetic effect of CYD0618 may be mediated via suppression of the NF-κB pathway.

Abstract 2794: A novel bis-aspirinyl selenazolidine compound AS-10 as potential colon cancer therapeutic

Abstract Despite rigorous screening and aggressive surgical and chemotherapeutic treatments, colorectal cancer (CRC) remains the third leading cause of cancer-related deaths in women and the second leading cause in men in the United States. It is expected to cause about 50,260 deaths during 2017 (American Cancer Society). The 5-year survival rate for patients with colorectal cancer (CRC) is 64%, although for patients who present with distant metastases at diagnosis the 5 year survival is only 12%. Therefore, there is an urgent need to develop effective therapeutic strategies that will enhance the survival and improve quality of life of CRC patients. Non-steroidal anti-inflammatory drugs (NSAIDs), particularly aspirin, have recently been shown to be effective in reducing colon cancer incidences and are proposed to be effective CRC chemopreventive agents. Through extensive structure activity relationship (SAR) studies based on designing selenium-NSAID hybrid small molecules, we have identified a small drug-like molecule, AS-10, which contains two aspirinyl groups flanked to a selenazolidine ring. AS-10 effectively inhibited the viability of CRC cells (HT29, RKO, and HCT116) with an EC50 in the range of 1 to 2.5 µM. Interestingly, AS-10 showed more selectivity towards killing cancer cells than normal mouse embryonic fibroblasts (MEFs) and was &amp;gt;50 times more potent than current standard of care, 5-FU, for in killing CRC cells. Cell cycle studies revealed AS-10 treated CRC cells to have G1 and G2/M phase cell cycle arrest. Further, increasing protein expression of cell cycle inhibitory protein p21 confirmed our cell cycle findings in AS-10 treated CRC cells. Since these CRC cell lines are p53 mutant cells, it was noteworthy to see p53 independent expression of p21. Moreover, we explored the possibility of apoptosis in these cell cycle arrested cells and found that AS-10 treatment increased casapse 3/7 activity, and cells positive for phosphatidylserine on the outer cell membrane surface indicative of apoptosis. In silico evaluation shows that AS-10 with cLogP 2.2 and MW 473 fits perfectly into the requirements of Lipinski's Rule-of-Five, formulated to determine the drug-likeness of a small molecule. In addition, in nude mice, AS-10 exhibited a high maximum tolerated dose (MTD) (~80 mg/kg, IP, three times per week). Furthermore, AS-10 (45 mg/kg, IP, three times per week) effectively inhibited tumor growth by ~70% in a nude mouse colon cancer xenograft model without any apparent systemic toxicity. Overall, AS-10 appears to be a promising small molecule worthy of further development as a therapeutic for CRC patients. Citation Format: Deepkamal N. Karelia, Manoj Pandey, Daniel Plano, Shantu Amin, Arun K. Sharma. A novel bis-aspirinyl selenazolidine compound AS-10 as potential colon cancer therapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2794.

Gomesin peptides prevent proliferation and lead to the cell death of devil facial tumour disease cells

The Tasmanian devil faces extinction due to devil facial tumour disease (DFTD), a highly transmittable clonal form of cancer without available treatment. In this study, we report the cell-autonomous antiproliferative and cytotoxic activities exhibited by the spider peptide gomesin (AgGom) and gomesin-like homologue (HiGom) in DFTD cells. Mechanistically, both peptides caused a significant reduction at G0/G1 phase, in correlation with an augmented expression of the cell cycle inhibitory proteins p53, p27, p21, necrosis, exacerbated generation of reactive oxygen species and diminished mitochondrial membrane potential, all hallmarks of cellular stress. The screening of a novel panel of AgGom-analogues revealed that, unlike changes in the hydrophobicity and electrostatic surface, the cytotoxic potential of the gomesin analogues in DFTD cells lies on specific arginine substitutions in the eight and nine positions and alanine replacement in three, five and 12 positions. In conclusion, the evidence supports gomesin as a potential antiproliferative compound against DFTD disease.