Expression Of Cyclin Research Articles

Laminaria japonica Peptides Suppress Liver Cancer by Inducing Apoptosis: Possible Signaling Pathways and Mechanism.

The anticancer properties of Laminaria japonica peptides (LJPs) have never been studied. Here, we extracted LJPs from fresh seaweed and explored their anti-liver cancer activity (in vivo and in vitro). LJPs were isolated/purified by HPLC-ESI-MS. HepG2 cell apoptosis and cell cycle were evaluated. MTT assays were used to examine the cytotoxicity of LJPs. Caspase activation of caspases 3 and 9, cleaved caspases 3 and 9, and cleaved PARP was examined by Western blotting. The PI3K/AKT pathway and the phosphorylation states of MAPKs (p38 and JNK) were examined. We found that the LJP-1 peptide had the most antiproliferative activity in H22 cells in vitro. LJP-1 blocked H22 cells in the G0/G1 phase, accompanied by inhibition of cyclin expression. LJP-1 induced apoptosis through caspase activation and regulation of the ASK1/MAPK pathway. Concurrent in vivo studies demonstrated that LJP-1 significantly inhibited tumor growth and induced tumor cell apoptosis/necrosis. In conclusion, LJPs, particularly LJP-1, exert strong inhibitory effects on liver cancer growth in vivo and in vitro. LJP-1 induces HCC cell apoptosis through the caspase-dependent pathway and G0/G1 arrest. LJP-1 induces caspase-dependent apoptosis, in part by inhibiting PI3K, MAPK signaling pathways, and cell cycle proteins. LJP-1 has the potential to be a novel candidate for human liver cancer therapeutics.

Pygo1 Regulates the Behavior of Human Non-Small-Cell Lung Cancer via the Wnt/β-Catenin Pathway.

Abnormal activation of the classical Wnt pathway has been reported in non-small-cell lung cancer (NSCLC) previously. Pygo family genes, the core regulators of Wnt/β-catenin signaling, were also reported to be involved in tumorigenesis. However, the role of the homolog Pygo1 in human lung cancer remains unclear. In the current study, we demonstrated an association of increased Pygo1 expression with consistent high nuclear β-catenin signals across pathological tissue samples of early-stage human NSCLC. Overexpression of Pygo1 in lung cancer cells resulted in enhanced G1/S cell phase transformation, reduced apoptosis, and increased cell proliferation. These changes were accompanied by the downregulation of cell cycle-related proteins, such as RB, p16, p53, and p27Kip1, and increased expression of CyclinE1. Migration, wound healing, and colony formation assays revealed that Pygo1 overexpression enhanced the invasion and migration of lung cancer cells, increased the formation of clones, and suppressed E-cadherin expression. In addition, overexpression of Pygo1 in lung cancer cells led to an increase of β-catenin/TCF4 complex, as well as upregulated expression of target genes of β-catenin. In vivo experiments also revealed that Pygo1 overexpression promoted the tumorigenicity of a xenograft tumor model, while Wnt inhibition partially blocked the effect of Pygo1 overexpression. In conclusion, Pygo1 affects human NSCLC via the canonical Wnt/β-catenin pathway, which provides new clues for lung cancer pathology.

Expression of Cyclin D1 in Cutaneous Xanthogranuloma and Solitary Reticulohistiocytoma.

Expression of Cyclin D1 in Cutaneous Xanthogranuloma and Solitary Reticulohistiocytoma.

ODP338 Neuromedin B Receptor Antagonist Suppresses ACTH Secretion and Cell Proliferation in Human and Mouse Corticotroph Adenoma

Abstract Objective We previously reported that Neuromedin B (NMB) is expressed in murine pituitary corticotrophs under adrenal insufficiency (1). Because NMB is also expressed in several cancer cells and stimulates ACTH secretion, we hypothesized that NMB is related to corticotroph adenoma cell proliferation and hormone secretion. To examine this hypothesis, we investigated the effects of a NMB receptor (NMBR) antagonist on AtT-20 cells, a tumor xenograft model and patient-derived corticotroph adenoma cells. Methods 1. NMB and NMBR expression in human pituitary adenoma: We performed real-time qPCR and immunostaining on human pathological specimens of corticotroph and non-functioning pituitary adenomas to investigate NMB and NMBR expression. 2. Experiments in AtT-20 cells: We extracted RNAs, proteins and mediums from AtT-20 cells after incubation with NMBR antagonist PD168368, and performed real-time qPCR, western blotting and ELISA analyses. We also performed WST-1 assay to investigate cell proliferation. 3. Experiments in a tumor xenograft model: AtT-20 cells in Matrigel were injected subcutaneously into BALB/c-nu mice. A week after inoculation, we administered 1.2 mg/kg PD168368 intraperitoneally once daily for 14 days. Upon completion of treatment, tumors were measured and cardiac blood was collected. 4. Experiments in patient-derived corticotroph adenoma cells: We isolated surgically resected human corticotroph adenoma cells from patients who underwent trans-sphenoidal surgery and investigated mRNA expression and medium ACTH secretion after incubation with PD168368. Statistical analysis: Comparisons between two groups were made by unpaired Student t test. Multiple groups were compared using one-way ANOVA followed by Dunnett's test for comparison with control group. Statistical significance was defined as p < 0. 05. Results 1. NMB and NMBR expression levels were significantly higher in human corticotroph adenomas (13 and 33 times higher, respectively) than in non-functioning adenomas in the qPCR analyses. Immunostaining confirmed higher expression of NMB and NMBR in corticotroph adenoma. 2. Treatment with 100 nM PD168368 significantly suppressed Pomc mRNA and protein expression in AtT-20 cells by 22% and 25% respectively compared control group. Medium ACTH secretion, mRNA and protein expression of cyclin E1 and cell proliferation were also suppressed by PD168368. 3. Mice treated with PD168368 had significantly lower tumor growth rate, plasma ACTH and corticosterone than control group (70 vs 161%, 97 vs 180 pg/ml, 813 vs 1045 ng/ml, respectively). 4. Treatment with PD168368 significantly suppressed POMC mRNA expression (12-31%) in 4 out of 6 patient-derived corticotroph adenoma cells. Medium ACTH secretion was also suppressed (14-53%) in 3 out of 4 cases which could be evaluated. Cyclin E mRNA expression were also suppressed in 3 out of 4 cases in which POMC mRNA expression were suppressed. Conclusions NMBR antagonist may represent a potential treatment for Cushing disease, which effect may be mediated by decreased cyclin E expression. Reference: (1) Kameda H et al., Endocrinology 2014;155(7): 2492–9. Presentation: No date and time listed

Geniposide inhibits cell proliferation and migration in human oral squamous carcinoma cells via AMPK and JNK signaling pathways

Iridoids are a special class of cyclopentanoid monoterpenes, which exhibit a wide range of biological effects. The present study aimed to investigate the potential effects of three iridoids genipin, geniposide and geniposidic acid on three human oral squamous cell carcinoma (OSCC) cell lines HSC-2, SCC-9 and A253 in addition to studying the possible underlying mechanisms. Cell viability assay revealed that geniposide treatment significantly suppressed the proliferation of all three cancer cell lines. In addition, geniposide induced SCC-9 cell cycle arrest at the G2/M phase (flow cytometry) through downregulation of cyclin-dependent kinase 2 and Cyclin A2 expression (western blot analysis), whilst also inducing cell apoptosis (flow cytometry and acridine orange/ethidium bromide staining) by dissipating the mitochondrial membrane potential (flow cytometry), and upregulating the expression of cleaved caspase-3 and cleaved poly-ADP ribose polymerase (western blot analysis). A wound-healing assay indicated that geniposide impaired SCC-9 cell migration by increasing the expression of E-cadherin (western blot analysis), whilst suppressing the expression of MMP-2 (western blot analysis). Western blot analysis also demonstrated that geniposide induced autophagy in SCC-9 cells by upregulating the expression of Beclin-1 and light chain 3-II. Mechanistically, geniposide activated the 5'-AMP-activated protein kinase signaling pathway and inhibited the JNK signaling pathway in SCC-9 cells (western blot analysis). The present results indicated that geniposide is able to inhibit the proliferation and migration of the tongue squamous carcinoma cell line SCC-9, suggesting a potential strategy for OSCC treatment.

Selected Articles from This Issue

Highlights| October 04 2022 Selected Articles from This Issue Author & Article Information Online ISSN: 1557-3125 Print ISSN: 1541-7786 ©2022 American Association for Cancer Research2022American Association for Cancer Research Mol Cancer Res (2022) 20 (10): 1469. https://doi.org/10.1158/1541-7786.MCR-20-10-HI Related Content A commentary has been published: Intronic Cis-Element DR8 in hTERT Is Bound by Splicing Factor SF3B4 and Regulates hTERT Splicing in Non–Small Cell Lung Cancer A commentary has been published: Distinct Transcriptional Programs in Ascitic and Solid Cancer Cells Induce Different Responses to Chemotherapy in High-Grade Serous Ovarian Cancer A commentary has been published: TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling View more A commentary has been published: EPAC Regulates Melanoma Growth by Stimulating mTORC1 Signaling and Loss of EPAC Signaling Dependence Correlates with Melanoma Progression View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record October 4 2022 Citation Selected Articles from This Issue. Mol Cancer Res 1 October 2022; 20 (10): 1469. https://doi.org/10.1158/1541-7786.MCR-20-10-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Cutaneous melanoma metastasis begins with lymphatic invasion, followed by hematogenous dissemination and distant organ metastasis. While Rodríguez, Castro Pérez, Setaluri, and co-authors have shown that exchange protein directly activated by cAMP (EPAC) drives growth and survival in primary melanoma tumors but not metastatic lesions, underlying signaling dynamics remain unclear. In this study, the investigators used genetically matched primary and metastatic melanoma cells and pharmacological and genetic EPAC modulation to query EPAC-regulated signaling in each case. The authors found that a pharmacological EPAC inhibitor, ESI-09, abrogates expression of cyclins, cyclin-dependent kinases (CDK), and CDK inhibitors uniquely in primary melanoma cells, and correspondingly disrupts cell cycle progression only in primary melanoma cells. Mechanistically, the authors demonstrated that while EPAC-mediated RAP1 activation and EPAC subcellular localization are similar in primary and metastatic melanoma cells, EPAC inhibition affects AKT and mTORC1 signaling and downstream metabolic features in primary but not metastatic cells. All together,... You do not currently have access to this content.

Tenglong Buzhong granules inhibits the growth of SW620 human colon cancer.

To observe the anticancer effects of the granular preparation of Tenglong Buzhong decoction (,TBD), i.e Tenglong Buzhong granules (, TBG), in human SW620 colon cancer. BALB/c nude mice were subcutaneously transplanted with SW620 cells, and treated with TBG (2.56 g/kg, once per day) and/or 5-Fu (104 mg/kg, once per week) for 21 d. Apoptosis, Caspase activities and cellular senescence were measured by commercial kits. The protein expression and phosphorylation were detected by Western blot or immunohistochemistry. TBG and 5-Fu inhibited tumor growth. The tumor inhibition rate of the TBG, 5-Fu, and TBG+5-Fu groups was 42.25%, 51.58%, and 76.08%, respectively. Combination of TBG and 5-Fu showed synergetic anti-cancer effects. TBG and 5-Fu induced apoptosis, activated caspase-3, -8, and -9, increased SMAC expression, inhibited XIAP expression. TBG induced cellular senescence, upregulated cyclin-dependent kinase inhibitor 1a (CDKN1a) and cyclin-dependent kinase inhibitor 2a (CDKN2a) expression, and inhibited phosphorylation of retinoblastoma-associated protein (RB) and expression of cyclin E1 (CCNE1) and cyclin-dependent kinases (CDK) 2. TBG also inhibited angiogenesis accompanied by downregulation of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). TBG inhibits SW620 colon cancer growth, induces apoptosis SMAC-XIAP-Caspases signaling, induces cellular senescence through CDKN1a/CDKN2a-RB-E2F signaling, inhibits angiogenesis by down-regulation of HIF-1α and VEGF, and enhances the effects of 5-Fu.

Pro-Apoptotic and Anti-Invasive Properties Underscore the Tumor-Suppressing Impact of Myoglobin on a Subset of Human Breast Cancer Cells.

The expression of myoglobin (MB), well known as the oxygen storage and transport protein of myocytes, is a novel hallmark of the luminal subtype in breast cancer patients and correlates with better prognosis. The mechanisms by which MB impacts mammary tumorigenesis are hitherto unclear. We aimed to unravel this role by using CRISPR/Cas9 technology to generate MB-deficient clones of MCF7 and SKBR3 breast cancer cell lines and subsequently characterize them by transcriptomics plus molecular and functional analyses. As main findings, loss of MB at normoxia upregulated the expression of cell cyclins and increased cell survival, while it prevented apoptosis in MCF7 cells. Additionally, MB-deficient cells were less sensitive to doxorubicin but not ionizing radiation. Under hypoxia, the loss of MB enhanced the partial epithelial to mesenchymal transition, thus, augmenting the migratory and invasive behavior of cells. Notably, in human invasive mammary ductal carcinoma tissues, MB and apoptotic marker levels were positively correlated. In addition, MB protein expression in invasive ductal carcinomas was associated with a positive prognostic value, independent of the known tumor suppressor p53. In conclusion, we provide multiple lines of evidence that endogenous MB in cancer cells by itself exerts novel tumor-suppressive roles through which it can reduce cancer malignancy.

Co-targeting CDK2 and CDK4/6 overcomes resistance to aromatase and CDK4/6 inhibitors in ER+ breast cancer

Resistance to aromatase inhibitor (AI) treatment and combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy (ET) are crucial clinical challenges in treating estrogen receptor-positive (ER+) breast cancer. Understanding the resistance mechanisms and identifying reliable predictive biomarkers and novel treatment combinations to overcome resistance are urgently needed. Herein, we show that upregulation of CDK6, p-CDK2, and/or cyclin E1 is associated with adaptation and resistance to AI-monotherapy and combined CDK4/6i and ET in ER+ advanced breast cancer. Importantly, co-targeting CDK2 and CDK4/6 with ET synergistically impairs cellular growth, induces cell cycle arrest and apoptosis, and delays progression in AI-resistant and combined CDK4/6i and fulvestrant-resistant cell models and in an AI-resistant autocrine breast tumor in a postmenopausal xenograft model. Analysis of CDK6, p-CDK2, and/or cyclin E1 expression as a combined biomarker in metastatic lesions of ER+ advanced breast cancer patients treated with AI-monotherapy or combined CDK4/6i and ET revealed a correlation between high biomarker expression and shorter progression-free survival (PFS), and the biomarker combination was an independent prognostic factor in both patients cohorts. Our study supports the clinical development of therapeutic strategies co-targeting ER, CDK4/6 and CDK2 following progression on AI-monotherapy or combined CDK4/6i and ET to improve survival of patients exhibiting high tumor levels of CDK6, p-CDK2, and/or cyclin E1.

Effect of acetone fraction of Ottelia alismoides on the G2/M cell cycle arrest and apoptosis in the human carcinoma cell lines

Ethnopharmacological relevanceThe North-eastern parts of India have immense therapeutic floras, Ottelia alismoides is an aquatic plant that has been in use for a long time in traditional medicine for treating diseases like cancer, tuberculosis, diabetes, febrifuge, hemorrhoids, and rubefacient. In lung and skin carcinoma cells with a high rate of proliferation and metastasis including drug resistance and non-specific target activity, generates important challenges towards their treatment strategy. Thus, finding novel therapeutic targets to treat lung and skin cancer progression is essential to enhance the patients' survival with treatment. Aim of the studyThe purpose of this study was to evaluate the apoptotic potential of acetone extract of O. alismoides (L.) Pers. (OA-AC) and to identify the compounds responsible for this effect, HRLC-MS-QTOF analysis of the extract has been undertaken along with in-silico molecular docking analysis of the identified compounds. Materials and methodsA549 and A431 cells were treated with acetone extract of O. alismoides (OA-AC) at 24 h and 48 h exposure and cell cycle phase distribution was evaluated and also apoptosis induction activity was evaluated by OA-EtBr staining and Mitochondrial outer membrane potential assay. Western blotting was performed for the evaluation of apoptotic protein expression. At last, the HR-LCMS of OA-AC was analyzed to identify the compounds responsible for the apoptotic activity of the extract. ResultsThe cell cycle phase distribution analysis in A549 and A431 cells at 24hrs exposure with 10 μg/mL and 25 μg/mL of OA-AC showed a potent arrest or blockage at the G2/M phase of the cell cycle with reduced expression of cyclin B and p-Cdc2. At 48 h exposure, apoptosis was observed in these cancer cells with elevated expression of Bax, p21 and cleaved caspase 3 and reduced expression of the Bcl2. ConclusionAO-EtBr staining of these cancer cells reveals that the death induced by OA-AC was apoptotic in nature with depolarization of mitochondrial membrane due to loss or damage of the mitochondrial membrane. The HRLC-MS-QTOF analysis of OA-AC depicted 14 major isolable compounds and molecular docking analysis displayed 4 compounds that might act as an inhibitor of cyclin B for G2/M phase arrest that leads to apoptotic induction in the cells.

Heparanase modulates the prognosis and development of BRAF V600E-mutant colorectal cancer by regulating AKT/p27Kip1/Cyclin E2 pathway

BRAF V600E-mutant colorectal cancer (CRC) is a rare subtype of colorectal cancer with poor prognosis. Compelling evidence indicates that the heparanase (HPSE) gene has multiple functions in cancer, however, its role in BRAF V600E-mutant CRC remains elusive. Differentially expressed genes between BRAF V600E-mutant and wild-type patients were explored by analyzing public data from The Cancer Genome Atlas and the Gene Expression Omnibus. Clinical samples of 172 patients with BRAF V600E-mutant CRC diagnosed at Zhongshan Hospital Fudan University were collected. Overall survival was analyzed using Kaplan–Meier curves and Cox regression models. Cell models and xenografts were utilized to investigate the effect of HPSE on tumor proliferation. HPSE was significantly highly expressed in the BRAF V600E-mutant group. High HPSE expression level was independently associated with inferior survival in the BRAF V600E-mutant cohort. HPSE knockdown impeded tumor proliferation of BRAF V600E-mutant CRC cells in vitro and in vivo. Mechanistically, HPSE silencing arrested cell cycle in G0/G1 phase by downregulating Cyclin E2 expression via the AKT/p27Kip1 pathway. These findings support a role for HPSE in promoting BRAF V600E-mutant CRC progression, which suggests it holds great promise as a prognostic biomarker and a potential therapeutic target for the aggressive CRC subtype.

Seleno-vs. thioether triazine derivatives in search for new anticancer agents overcoming multidrug resistance in lymphoma

Lymphomas are still difficult to treat even with modern therapies as, among others, multidrug resistance (MDR) is often counteracting a successful cancer therapy. P-gp/ABC-transporters are well-known for their crucial role in the main tumour MDR mechanism, eliminating drugs and cytotoxic substances from the cancer cell by efflux, and their modulators are promising for innovative therapy, but none has been approved in the pharmaceutical market yet. Herein, we have designed, synthesised and analysed 30 novel seleno- and thioether 1,3,5-triazine derivatives conducting comprehensive studies to evaluate their potential application in human JURKAT lymphoma cells. Among the new compounds, four (11, 12, 13 and 23) were much more effective than the reference inhibitor verapamil, being potent ABCB1 inhibitors already at 2 μM, while 5 and 15 showed very potent ABCB1 inhibitory activity only at 20 μM. Results of P-gp ATPase assays, supported with docking studies, indicated the competitive substrate mode of modulating action for 15, while ABCB1, ABCC1 and ABCG2 genes expression induction by 15 with q-PCR was confirmed. All compounds were evaluated for their cytotoxic and antiproliferative properties in both sensitive (PAR) and resistant (MDR) mouse T-lymphoma cell lines, and compound 15, also considering its promising ABCB1 inhibition properties, was revealed to be the best compound in terms of its cytotoxic effect (IC50: 16.73 μM) as well as concerning the antiproliferative effect (IC50: 5.35 μM) in MDR cells.Regarding the mechanistic studies looking at the cell cycle, the thioether 15 and selenium derivatives 26 and 29 were significantly effective in the regulation of cell cycle-related genes alone or in co-treatment with doxorubicin counteracting Cyclin D1 and E1 expression and increasing p53 and p21 levels, shedding first light on their mechanism of action. In summary, we explored the chemical space of seleno- and thioether 1,3,5-triazine derivatives with interesting activity against lymphoma. Especially compound 15 is worthy of being studied deeper to evaluate its precise mode of action further as well it can be improved regarding its potency and drug-likeness.

Impact of Freeze-Drying on Cord Blood (CB), Serum (S), and Platelet-Rich Plasma (CB-PRP) Preparations on Growth Factor Content and In Vitro Cell Wound Healing.

Blood-based preparations are used in clinical practice for the treatment of several eye disorders. The aim of this study is to analyze the effect of freeze-drying blood-based preparations on the levels of growth factors and wound healing behaviors in an in vitro model. Platelet-rich plasma (PRP) and serum (S) preparations from the same Cord Blood (CB) sample, prepared in both fresh frozen (FF) and freeze-dried (FD) forms (and then reconstituted), were analyzed for EGF and BDNF content (ELISA Quantikine kit). The human MIO-M1 glial cell line (Moorfield/Institute of Ophthalmology, London, UK) was incubated with FF and FD products and evaluated for cell migration with scratch-induced wounding (IncuCyte S3 Essen BioScience), proliferation with cyclin A2 and D1 gene expression, and activation with vimentin and GFAP gene expression. The FF and FD forms showed similar concentrations of EGF and BDNF in both the S and PRP preparations. The wound healing assay showed no significant difference between the FF and FD forms for both S and PRP. Additionally, cell migration, proliferation, and activation did not appear to change in the FD forms compared to the FF ones. Our study showed that reconstituted FD products maintained the growth factor concentrations and biological properties of FF products and could be used as a functional treatment option.

Long noncoding RNA-LINC00478 promotes the progression of clear cell renal cell carcinoma through PBX3.

Long noncoding RNAs play an important regulatory role in the development and progression of tumors. Our study found that LINC00478 was upregulated in clear cell renal cell carcinoma (ccRCC), so we made an in-depth exploration into its mechanism. In Caki-2 cells, we established the oe-LINC00478 cell line overexpressing LINC00478, and established underexpressing sh-LINC00478 cell line by short hairpinRNA silencing. The abilities of oe-LINC00478 cell invasion and metastasis were significantly enhanced, and the cell proliferative potential was also improved. The cellular expressions of PBX3, CDCA8, and CDK2 were upregulated, while in the sh-LINC00478 cells, the proliferative potential and metastatic and invasive abilities were weakened. Similarly, we established the PBX3-overexpressing oe-PBX3 cell line and the PBX3-underexpressing sh-PBX3 cell line, finding that the PBX3 overexpression enhanced the metastatic and invasive abilities of Caki-2 cells. When we overexpressed LINC00478 in PBX3-knockout Caki-2-PBX3- / - cells, no significant changes were noted in the metastatic or invasive ability. Through RNA pull-down and RNA-binding protein immunoprecipitation assays, we found that LINC00478 could facilitate the transcription-translation processes of PBX3 by binding to it, thus further promoting the expression of downstream cyclins to exert its action. In animal experimentation, the oe-LINC00478 and sh-LINC00478 Caki-2 cells were separately seeded, revealing that the tumor volume was significantly larger in the oe-LINC00478 group than in the sh-LINC00478 group. This study finds that by promoting the PBX3 transcription, LINC00478 can further regulate the expressions of downstream cyclins, thereby facilitating the metastasis and invasion of ccRCC.

Protective role of fermented whey and pumpkin extract against aflatoxin B1 and ochratoxin A toxicity in Jurkat T-cells

The beneficial effect of fermented whey and pumpkin extract rich in carotenoids was evaluated in Jurkat cells against aflatoxin B1 (AFB1) and ochratoxin A (OTA) cytotoxicity through a proteomic approach. The functional ingredients were added into mycotoxin contaminated bread formulation, which were digestedin vitro in order to simulate human intestinal absorption. Cell cultures were exposed during 7 days to these mycotoxins dissolved in: (a) 0.1% organic solvent (DMSO), (b) an intestinal digest of bread with pumpkin individually (PID) and (c) an intestinal digest of bread with pumpkin mixed with fermented whey (PID+WF). Extracted proteins were subjected to reduction and alkylation and subsequently a tryptic digestion in order to be analysed by liquid chromatography coupled with quadrupole time of flight (LC/MS-Q-TOF). Results obtained highlighted the beneficial role of functional ingredients employed through the identification of proteins involved in several biological processes and metabolic pathways, mainly antioxidant activity, nucleosome assembly and secretory senescence phenotype. Among proteins involved in antioxidant activity, peroxiredoxin 1 and 2 stand out. Comparing the different conditions investigated, a remarkable change was observed in their expression, ranging from a repression using the standard (DMSO 0.1%), to an overexpression when treated with the functional ingredients. Similarly, after PID and PID+WF treatment, histones’ expression implicated in the metabolic pathway of nucleosome assembly, such as H2A, H2B, H2C, H3 and H4, was increased. Furthermore, the expression of protein cyclin A2, which downregulation is involved in limiting carcinogenic cells growth, was lower in presence of both functional ingredients. Based on these findings, functional ingredients can act as protectors against genomic stress caused by mycotoxins, preventing the loss of vital cell functions and paralysing the growth of carcinogenic cells.

Abstract P054: Loss Of The Smooth-muscle-cell-angiotensin Ii-sensitive Lncrna Leads To Smc Hypertrophic And Hypertensive Remodeling Due To Cell Cycle Dysregulation.

Vascular smooth muscle cells (SMC) responses to increased levels of Ang II during hypertensive remodeling included enhanced vasoconstriction and hypertrophy due to the enlargement of the SMC on the vessel wall. Hypertrophic SMC usually present defects in cell division and cell polyploidy. However, the molecular mechanisms controlling SMC hypertrophy and polyploidy vs. hyperplasia are still not fully understood. Long-non-coding-RNAs (LncRNAs) are epigenetic regulators of gene expression influencing biological processes, including cell division fidelity. We discovered a novel lncRNA decreased in dedifferentiated SMC, the SMC-Ang II-Sensitive (SAS) lncRNA, which expression is reduced in response to Ang-II in cultured SMC and the aorta of hypertensive mice, suggesting a role in mediating hypertension-induced SMC hypertrophy. Publicly available transcriptional datasets revealed that SAS is preferentially expressed in SMC-rich tissues, including the aorta and renal artery, in humans and mice. Yet, the functional relevance of SAS in SMC has never been investigated. Knockdown of SAS reduces proliferation and migration in SMC treated with Platelet-Derived Growth Factor (PDGF-BB). SAS knockdown was also associated with distinct SMC hypertrophic morphological changes, including enlargement in cell size and polynucleation in vitro. We have generated a SAS KO mouse, and aortas from these mice present a higher number of polynucleated medial SMC than their WT littermates. These data correlate with a cell cycle arrest in G1 and senescence phenotype that SAS deficient SMC present due to dysregulation in cyclins expression. Furthermore, SAS defective cells show mitochondria hyperfusion and increased oxygen consumption that correlates with the observed senescence and arrest on the G1/S checkpoint, and it is exacerbated by treatment with Ang-II. Interestingly, treatment with Losartan, an Ang-II receptor inhibitor, rescues SAS expression on Ang-II treated SMC and diminishes SMC hypertrophy. Together, these observations suggest that a decrease in SAS causes SMC hypertrophy due to defects in cell cycle completion. SAS is a potent regulator of SMC morphology and is required for proper cell division and mitochondria organization.

491TiP A first-in-human phase I/II study of BLU-222, a potent, selective CDK2 inhibitor in patients with CCNE1-amplified or CDK4/6 inhibitor-resistant advanced solid tumors

Cyclins and cyclin-dependent kinases (CDK) regulate cell growth/proliferation. Several aggressive solid tumor types harbor cyclin E1 gene (CCNE1) amplification, while activated CDK4/6 complex increases expression of cyclin E1 and E2. CDK4/6 inhibitors (CDK4/6i) are effective in patients with estrogen receptor-positive, human epithelial growth factor receptor-2-negative breast cancer (ER+HER2- BC), but treatment resistance generally occurs. BLU-222 is an oral, investigational, potent, selective CDK2 inhibitor.

Antitumor Effect of Zinc Acetate in Hepatocellular Carcinoma Cell Lines via the Induction of Apoptosis.

We aimed to verify antitumor effects of zinc acetate on hepatocellular carcinoma (HCC) in vitro. Five HCC cell lines (HepG2, Hep3B, Huh7, HLE and Alex) were used to evaluate the antitumor effects of zinc acetate. Cell viability was determined by the Cell Counting Kit-8 assay. The cell-cycle alteration was evaluated by a flow cytometric analysis and the detection of cell cycle-related proteins. Apoptosis was determined based on the caspase-cleaved cytokeratin 18 (cCK18) levels. The microRNAs (miRNAs) related to an antitumor effect of zinc acetate were identified using microarrays. Zinc acetate significantly inhibited the proliferation of HCC cells in a dose-dependent manner. The treatment with zinc acetate resulted in significantly increased cCK18 levels in the supernatant and enhanced the expression of heme oxygenase-1 (HO-1) in HCC cells. The flow cytometric analysis revealed an increase of HCC cells in the S and G2/M phases by the administration of zinc acetate, and the expressions of Cdk2 and cyclin E were increased. The miRNA expression profile of the HCC cells treated with zinc acetate was extremely different from that of the untreated HCC cells. These results suggest that the zinc acetate supplementation induces the apoptosis of HCC cells, but does not affect the cell cycle progression. Upregulation of HO-1 and the alteration of miRNAs' profile may be involved in antitumor effects of zinc acetate in HCC cells.

PH-Responsive PEGylated Niosomal Nanoparticles as an Active-Targeting Cyclophosphamide Delivery System for Gastric Cancer Therapy.

A PEGylated niosomal formulation of cyclophosphamide (Nio-Cyclo-PEG) was prepared using a central composite design and characterized in terms of drug loading, size distribution, and average size. The stability of formulations was also studied at different conditions. In vitro cytotoxicity of drug delivery formulations was assessed on gastric cancer cells using MTT assay. The mechanism of cytotoxicity was studied at the transcriptional level by real-time PCR on Caspase3, Caspase9, CyclinD, CyclinE, MMP-2, and MMP-9 genes, while apoptosis was investigated with flow cytometry. The anti-metastatic property was evaluated using the scratch method. Propidium iodide staining was used to study the cell cycle. The results indicated that the as-designed nanocarrier exhibited a controlled drug release pattern with improved nanoparticle stability. It was found that the living cancer cells treated with Nio-Cyclo-PEG showed a significant decrease in number when compared with the niosomal carrier without PEG (Nio-Cyclo) and free drug (Cyclo). Moreover, the drug-loaded nanocarrier induced planned death (apoptosis) in the cancer cells through the regulation of Caspase3, Caspase9, CyclinD, CyclinE, MMP-9, and MMP-2 gene expression, indicating that the Nio-Cyclo-PEG formulation could significantly inhibit the cell cycle at the sub G1 phase as well as prevent the migration of cancer cells. In conclusion, Nio-Cyclo-PEG as developed in this study could serve as an active-targeting drug delivery nanocarriers for gastric cancer therapy with high efficacy and minimal side effects on healthy tissues/cells.

In vivo assessment of simultaneous G1 cyclins silencing by a tumor-specific bidirectional promoter on the mammary tumor in nude mice.

Dysregulation of G1 cyclins (cyclins D1 A and E) expression contributes to the loss of standard cell cycle control during tumorigenesis. This study aims to evaluate the inhibitory effect of G1 cyclins in nude mice. The human breast cancer MDA-MB-231 cells were subcutaneously transplanted into the supra-femoral right side of female Balb/c-nude mice. The dual shRNA vector harboring G1 cyclins shRNAs (bipSUR) was intratumorally injected by the in vivo jetPEI transfection reagent for 2 weeks. We have evaluated tumor growth and tumor weight as parameters of tumor progression. Finally, necropsy, histopathological analysis, and immunodetection of G1 cyclins were assessed. Also, apoptosis induction in tumor tissues was evaluated by TUNEL assay. No toxicity and metastasis was observed in the tumor-bearing mice treated by the bipSUR. Tumor weight and volume were significantly lower in the bipSUR treated mice than untreated tumor-bearing mice and control. Histopathological observations revealed more apoptotic foci and lower mitotic cells in tumor sections in the treated mice than in control groups. A significant reduction of G1 cyclins at the protein level was indicated in the bipSUR treated mice than in other groups. Apoptosis in tumor tissues was remarkably induced in response to the bipSUR (42.53%). The bipSUR reduced the protein expression of G1 cyclins and exhibited an inhibitory effect on MDA-MB-231 xenograft mice through apoptosis induction. Further research is demanded to identify the protein partners of G1 cyclins involved in the cancer pathways. These may offer new insight into the biomedical function of G1 cyclins in breast cancer progression.