Inhibition Of Cell Cycle Progression Research Articles

Hydroxy Piperlongumines: Synthesis, Antioxidant, Cytotoxic Effect on Human Cancer Cell Lines, Inhibitory Action and ADMET Studies

AbstractPiperlongumine, an anticancer natural product, was sequentially demethylated to obtain mono, di and trihydroxy piperlongumines. The hydroxy piperlongumines were evaluated for cytotoxic effect on five human cancer cell lines (kidney, skin, cervical, breast, and lung) in vitro. Results from cytotoxic and cell growth inhibition studies showed monohydroxy piperlongumine (MHPL) was the most active of the series, exhibited potent cytotoxic effect on all the tested cell lines. Flow cytometry analysis shows MHPL inhibited cell cycle progression of HeLa cells in the S phase, indicated that it inhibits the DNA replication process. Fluorescence staining indicated MHPL treated cells were apoptotic. MHPL upregulated the apoptotic genes (caspase‐3, −8, Bax, and p53) and downregulated anti‐apoptotic gene (Bcl‐2). The antioxidant study revealed that hydroxy piperlongumines are effective antioxidants. For MHPL, all the molecular descriptors were within the range of 95 % of approved drugs suggesting high druglikeliness. Our results suggest that MHPL could be developed as a potential lead molecule for cancer therapy.

PI3K p110α Blockade Enhances Anti-Tumor Efficacy of Abemaciclib in Human Colorectal Cancer Cells.

Simple SummaryColorectal cancer (CRC) is the third most common cancer and the second highest cause of cancer related mortality worldwide. Especially, the survival of advanced CRC patients who were failed to achieve durable remission after the anti-angiogenic and anti-epithelial growth factor receptor agents are still poor. The aim of our study was to investigate the anti-tumor activity of the CDK4/6 inhibitor, abemaciclib, as a single agent and to identify an optimal combination agent with abemaciclib in CRC cell lines. We confirmed that abemaciclib monotherapy showed anti-tumor activity and combination therapy with abemaciclib and BYL719 demonstrated synergistic effects in CRC cell lines. Moreover, our study suggested that PIK3CA mutation could be a predictive marker for efficacy of abemaciclib and BYL719 combination therapy. These findings provide novel insight into a possible therapeutic strategy for patients with relapsed and refractory CRC.Targeting cell cycle regulation in colorectal cancer has not been fully evaluated. We investigated the efficacy of the CDK4/6 inhibitor, abemaciclib, and confirmed a synergistic interaction for PI3K p110α and CDK dual inhibition in colorectal cancer cell lines. Caco-2 and SNU-C4 cell lines were selected to explore the mechanism of action for and resistance to abemaciclib. In vitro and in vivo models were used to validate the anti-tumor activity of abemaciclib monotherapy and BYL719 combination therapy. Abemaciclib monotherapy inhibited cell cycle progression and proliferation in Caco-2 and SNU-C4 cells. CDK2-mediated Rb phosphorylation and AKT phosphorylation appeared to be potential resistance mechanisms to abemaciclib monotherapy. Abemaciclib/BYL719 combination therapy demonstrated synergistic effects regardless of PIK3CA mutation status but showed greater efficacy in the PIK3CA mutated SNU-C4 cell line. Growth inhibition, cell cycle arrest, and migration inhibition were confirmed as mechanisms of action for this combination. In an SNU-C4 mouse xenograft model, abemaciclib/BYL719 combination resulted in tumor growth inhibition and apoptosis with tolerable toxicity. Dual blockade of PI3K p110α and CDK4/6 showed synergistic anti-tumor effects in vivo and in vitro in human colorectal cancer cell lines. This combination could be a promising candidate for the treatment of patients with advanced colorectal cancer.

Crosstalk Between AR and Wnt Signaling Promotes Castration-Resistant Prostate Cancer Growth.

IntroductionProstate cancer (PCa) is the most commonly diagnosed cancer and the third leading cause of cancer-related death in males in the United States. Despite the initial efficacy of androgen deprivation therapy in prostate cancer (PCa) patients, most patients progress to castration-resistant prostate cancer. However, the mechanisms underlying the androgen-independent progression of PCa remain largely unknown.MethodsIn this study, we established a PCa cell line (LNCaP-AI) by maintaining LNCaP cells under androgen-depleted conditions. To explore the cellular and molecular mechanisms of androgen-independent growth of PCa, we analyzed the gene expression patterns in androgen-independent prostate cancer (AIPC) compared with that in androgen-dependent prostate cancer (ADPC). KEGG pathway analysis revealed that Wnt signaling pathways were activated after androgen deprivation therapy (ADT). In vitro experiments showed that the inhibition of Wnt pathway reduced AIPC cell growth by inhibiting cell cycle progression and promoting apoptosis. Furthermore, WNT5A, LEF1 were identified as direct targets of AR by chromatin immunoprecipitation (ChIP) assay and public ChIP-seq datasets analysis.ResultsIn the present study, we found a regulatory mechanism through which crosstalk between androgen receptor (AR) and Wnt signals promoted androgen-independent conversion of PCa. The Wnt pathway was inhibited by androgen in androgen-dependent prostate cancer cells, but this blocking effect was not elicited in androgen-independent prostate cancer (AIPC) cells. Moreover, Wnt pathway genes WNT5A and LEF1 were directly downregulated by AR. In vitro experiments showed that inhibition of the Wnt pathways repressed AIPC cell growth by inhibiting cell cycle progression and promoting apoptosis. We found that WNT5A and LEF1 were downregulated in low-grade PCa but upregulated in metastatic PCa.ConclusionIn summary, we revealed that crosstalk between AR and Wnt signaling pathways promotes androgen-independent growth of PCa, which may provide novel therapeutic opportunities for castration-resistant prostate cancer.

The opposing roles of the mTOR signaling pathway in different phases of human umbilical cord blood-derived CD34+ cell erythropoiesis

As an indispensable, even lifesaving practice, red blood cell (RBC) transfusion is challenging due to several issues, including supply shortage, immune incompatibility, and blood‐borne infections since donated blood is the only source of RBCs. Although large‐scale in vitro production of functional RBCs from human stem cells is a promising alternative, so far, no such system has been reported to produce clinically transfusable RBCs due to the poor understanding of mechanisms of human erythropoiesis, which is essential for the optimization of in vitro erythrocyte generation system. We previously reported that inhibition of mammalian target of rapamycin (mTOR) signaling significantly decreased the percentage of erythroid progenitor cells in the bone marrow of wild‐type mice. In contrast, rapamycin treatment remarkably improved terminal maturation of erythroblasts and anemia in a mouse model of β‐thalassemia. In the present study, we investigated the effect of mTOR inhibition with rapamycin from different time points on human umbilical cord blood‐derived CD34+ cell erythropoiesis in vitro and the underlying mechanisms. Our data showed that rapamycin treatment significantly suppressed erythroid colony formation in the commitment/proliferation phase of erythropoiesis through inhibition of cell‐cycle progression and proliferation. In contrast, during the maturation phase of erythropoiesis, mTOR inhibition dramatically promoted enucleation and mitochondrial clearance by enhancing autophagy. Collectively, our results suggest contrasting roles for mTOR in regulating different phases of human erythropoiesis.

PHTHALATES AND HYPEROSMOTIC STRESS FORCE LOSS OF GROWTH AND PROGRESSION PAST G1 PHASE IN FUCCI EMBRYONIC STEM CELLS

Determine whether various stresses including positive control hyperosmotic sorbitol, hormonal cortisol, PFOA (Perfluorooctanoic acid) and phthalates slow growth and suppress cell cycle progression measured by FUCCI (Fluorescence Ubiquitination Cell Cycle Indicator) embryonic stem cells (ESC) to test for effects on growth and stemness/differentiation balance.

Cadmium inhibits cell cycle progression and specifically accumulates in the maize leaf meristem.

It is well known that cadmium (Cd) pollution inhibits plant growth, but how this metal impacts leaf growth processes at the cellular and molecular level is still largely unknown. In the current study, we show that Cd specifically accumulates in the meristematic tissue of the growing maize leaf, while Cd concentration in the elongation zone rapidly declines as the deposition rates diminish and cell volumes increase due to cell expansion. A kinematic analysis shows that, at the cellular level, a lower number of meristematic cells together with a significantly longer cell cycle duration explain the inhibition of leaf growth by Cd. Flow cytometry analysis suggests an inhibition of the G1/S transition, resulting in a lower proportion of cells in the S phase and reduced endoreduplication in expanding cells under Cd stress. Lower cell cycle activity is also reflected by lower expression levels of key cell cycle genes (putative wee1, cyclin-B2-4, and minichromosome maintenance4). Cell elongation rates are also inhibited by Cd, which is possibly linked to the inhibited endoreduplication. Taken together, our results complement studies on Cd-induced growth inhibition in roots and link inhibited cell cycle progression to Cd deposition in the leaf meristem.

Abstract 2541: MicroRNA-497~195 cluster suppresses acute lymphoblastic leukemia growth by targeting CCND3/CDK4 and inhibiting cell cycle progression

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood. Relapse is associated with poor prognosis, requiring further investigation on high-risk leukemias. MicroRNAs (miRNAs) are involved in the regulation of physiological processes. Deregulated miRNAs have been described to play a role in the initiation and progression of leukemia. Here, we investigated miRNA expression profiles comparing leukemia samples of patients with good and poor outcome, also characterized by slow or rapid engraftment, respectively, upon transplantation into NOD/SCID mice. We found downregulation of the miR-497~195 cluster in B-cell precursor (BCP)-ALL patient-derived xenograft samples with rapid engraftment and early relapse. Accordingly, in an independent cohort, patients with a high expression of miR-497 or miR-195 showed higher event-free survival. We identified promoter methylation as the mechanism responsible for miR-497~195 downregulation, as it was associated with low miR-497~195 levels in xenografts and treatment with the demethylating agent Decitabine increased miR-497~195 expression in BCP-ALL cell lines. To study the role of miR-497~195 in BCP-ALL, we stably overexpressed the cluster in three xenograft samples. MiR-497~195 overexpression impaired engraftment of leukemic cells in NOD/SCID mice, as compared to control cells, prolonging recipient survival. Gene expression profiling and ex vivo analysis of miR-497~195 overexpressing xenograft cells showed that inhibition of cell proliferation is a mechanism responsible for the miRNA cluster tumor suppressive role. Mechanistically, CDK4 and CCND3 were downregulated at mRNA and protein levels upon miR-497~195 overexpression, leading to decreased RB1 phosphorylation, reduced transcription of E2F target genes, and inhibition of the entry in S phase. CDK4 mediated G1/S transition is also inhibited by CDKN2A and CDKN2B, which are deleted in 20% of BCP-ALL. Deletion of CDKN2A/B and concomitant low miR-497~195 expression were particularly associated with a high proportion of early relapse cases in our cohort, indicating that impaired regulation of G1/S transition is critical for ALL aggressiveness. Importantly, targeting this pathway with the CDK4/CDK6 inhibitor Palbociclib impaired ex vivo growth of BCP-ALL xenograft samples, indicating a possible therapeutic strategy. Altogether, we showed that the tumor-suppressive cluster miR-497~195 is downregulated by epigenetic repression in BCP-ALL and that low expression is associated with early relapse and rapid engraftment. Low miR-497~195 expression might cooperate with deletion of CDKN2A/B promoting tumor progression, through loss of CCND3/CDK4-dependent control of cell cycle regulation. Citation Format: Elena Boldrin, Enrico Gaffo, Judith Boer, Salih Demir, Julia Zinngrebe, Rainer Claus, Christoph Plass, Monique L. den Boer, Klaus-Michael Debatin, Geertruy te Kronnie, Stefania Bortoluzzi, Lüder Hinrich Meyer. MicroRNA-497~195 cluster suppresses acute lymphoblastic leukemia growth by targeting CCND3/CDK4 and inhibiting cell cycle progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2541.

Abstract 5030: Vitamin D exhibits therapeutic anti-tumor and anti-angiogenic potential by reducing VEGF levels and altering TIMP/MMP system in breast cancer

Abstract Background: Clinical and experimental observations suggest that Vitamin D deficiency associates with increased risk of breast cancer. Furthermore, studies concluded that Vitamin D precipitates in significant anti-neoplastic effects against breast, and inducing apoptosis and affecting angiogenesis in other types of cancer. However, the exact mechanism underlying this anti-angiogenic effect of Vitamin D is still elusive. This study aims to investigate the anti-cancer role of Vitamin D anti-cancer and its association with modulation of major factors involved in angiogenesis and metastasis in breast cancer cell lines. Experimental methods: The hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D3 (calcitriol), was used to evaluate anti-proliferative effect, inhibition of cell cycle progression and apoptosis induction in two breast cancer cell lines, MCF-7 and MDA-MB-231. Furthermore, impact of calcitriol on expression and activity of tissue inhibitors of metalloproteinases (TIMP), major groups of the matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), and tumor growth factor (TGF-β1) among others involved in tumor progression and angiogenesis were also examined. Results and Discussions: Calcitriol treatment (10 µM) significantly inhibited cell proliferation in a timely dependent manner. Both cell lines showed G1 cell cycle arrest, decreased levels of the cell death inhibitor, survivin, and apoptosis induction in response to calcitriol treatment. Proteome Profiler array analysis revealed that calcitriol resulted in reducing the levels of major pro-angiogenesis regulators, especially VEGF, TGF-β1, Urokinase-type plasminogen activator (uPA), and Amphiregulin. Analysis of mRNA and proteins expression upon calcitriol exposure showed an increase of TIMP1, TIMP2 and a decrease in MMP 2, 3 and 9 levels. Finally, quantitative analysis using ELISA revealed that calcitriol treatment reduced MMP2 and MMP9 activities. Conclusion: Our study demonstrates potential therapeutic property of calcitriol through inhibiting of pro-angiogenetic markers in breast cancer. Citation Format: Khuloud Bajbouj, Jasmin Shafarin, Ahmad Sawan, Ahmed Shehada, Walaa Eldin Elkhalifa. Vitamin D exhibits therapeutic anti-tumor and anti-angiogenic potential by reducing VEGF levels and altering TIMP/MMP system in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5030.

Abstract 5295: Impact of a multi-gene expression profile in predicting adjuvant trastuzumab outcome in HER2-positive breast cancer

Abstract Background Trastuzumab is the cornerstone of adjuvant therapy for HER2-positive breast cancer (BC), but up to 20% of patients relapse. Understanding resistance mechanisms could allow developing strategies to overcome this issue. We performed a case-control, retrospective study to compare the gene expression profiles (GEPs) of 27 patients who experienced disease relapse within 2 years from the start of adjuvant trastuzumab therapy, with those of 27 patients who were disease-free after at least 5 years. Here, we present the preliminary results on 12 cases and 12 controls, matched by age and hormone receptor status. Methods RNA was isolated from formalin-fixed paraffin-embedded primary tumors (FFPE) with AllPrep DNA/RNA FFPE Kit (Qiagen) and quantified by Nanodrop, before performing Nanostring gene expression profile. nCounter Breast Cancer 360 Panel (Nanostring Technologies) was used according to manufacturer's instructions. nSolver analysis software (Nanostring Technologies) was used to obtain normalized counts and to compare GEPs between cases and controls, considering as significant a Benjamini Yekutieli (BY) corrected p-value ≤0.05. Mann-Whitney median test was used to compare Ki67 score and Body Mass Index (BMI) between cases and controls. Results Out of 770 analyzed genes, none was significantly differentially expressed at BY p-value <0.05, probably due to the low number of cases. Five genes were differentially expressed with a BY p-value <0.1: CDKN1A (Log2 fold change -0.733, 95% CI: -1.06 − -0.409), GDF15 (Log2 fold change -1.92, 95% CI: -2.78 − -1.05), TFDP1 (Log2 fold change -0.567, 95% CI: -0.826 − -0.309) and ELF3 (Log2 fold change -1.52, 95%CI: -2.22 − -0.813) were downregulated in control patients, whereas TNFSF10 (Log2 fold change 1.27, 95% CI: 0.669 − 1.88) was upregulated. CDKN1A and TFDP1 are cell cycle regulators: the former encodes for p21, which inhibits cell cycle progression and is involved in DNA damage repair; the latter encodes a transcription factors that dimerizes with E2F, promoting cell cycle genes' transcription. GDF15 codes for a secreted ligand of the TGF-beta superfamily and acts as a pleiotropic cytokine involved in response to hypoxia, inflammation, acute injury and oxidative stress. ELF3 is a transcriptional activator involved in vascular inflammation and a critical downstream effector of HER2 pathway. TNFSF10 encodes a cytokine belonging to the TNF ligand family, inducing apoptosis in tumor cells. No differences were found between cases and controls considering Ki67 proliferation index and BMI. Conclusions Our preliminary results indicate that altered expression of genes involved in cell cycle, apoptosis, inflammation and stress response are involved in trastuzumab resistance. The validation of these results overall series is ongoing. The relations between GEPs and clinico-pathological features will be assessed. Citation Format: Sara Ravaioli, Roberta Maltoni, Francesca Pirini, Michela Palleschi, William Balzi, Elisabetta Petracci, Giovanni Martinelli, Andrea Rocca, Sara Bravaccini. Impact of a multi-gene expression profile in predicting adjuvant trastuzumab outcome in HER2-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5295.

Long Noncoding RNA Rps4l Mediates the Proliferation of Hypoxic Pulmonary Artery Smooth Muscle Cells

Pulmonary hypertension (PH) is a rare and fatal disorder involving the vascular remodeling of pulmonary arteries mediated by the enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs). Long noncoding RNAs are a subclass of regulatory molecules with diverse cellular functions, but their role in PH remains largely unexplored. We aimed to identify and determine the functions of long noncoding RNAs involved in hypoxia-induced PH and PASMC proliferation. RNA sequencing in a hypoxic mouse model identified hypoxia-regulated long noncoding RNAs, including Rps4l. Rps4l expression was significantly reduced in PH-model mice and hypoxic PASMCs. The subcellular localization of Rps4l was detected by RNA fluorescence in situ hybridization and quantification of nuclear/cytoplasmic RNA. Rps4l overexpression rescued pulmonary arterial hypertension features, as demonstrated by right ventricle hypertrophy, right ventricular systolic pressure, hemodynamics, cardiac function, and vascular remodeling. At the cellular level, Rps4l overexpression weakened cell viability and proliferation and suppressed cell cycle progression. Potential Rps4l-binding proteins were identified via RNA pull-down followed by mass spectrometry, RNA immunoprecipitation, and microscale thermophoresis. These results indicated that Rps4l is associated with and affects the stabilization of ILF3 (interleukin enhancer-binding factor 3). Rps41 further regulates the levels of HIF-1α and consequently leads to hypoxia-induced PASMC proliferation and migration. Our results showed that in hypoxic PASMCs, Rps4l expression decreases due to regulation by hypoxia. This decrease affects the proliferation, migration, and cell cycle progression of PASMCs through ILF3/HIF-1α. These results provide a theoretical basis for further investigations into the pathological mechanism of hypoxic PH and may provide insight for the development of novel treatments.

FBX8 promotes metastatic dormancy of colorectal cancer in liver

Patients with colorectal cancer (CRC) often develop malignant regrowth of metastatic dormant tumor cells in liver years after primary treatment. FBX8 is involved in suppressing tumor metastasis. Short-term chemotherapy experiments and liver metastasis mice model of orthotopic injection into the cecum were performed to construct the dormant models. GST-pull-down assay, Co-IP and immunofluorescence were used to confirm the bindings among FBX8 and its substrates. FBX8 upregulated the expression of epithelial and stemness markers, while downregulated the expression of mesenchymal and proliferative markers associated with tumor cell dormancy. FBX8 promoted the maintenance of metastatic dormancy of CRC cells. Mechanistically, FBX8 directly bound to HIF-1α, CDK4 and C-myc through its Sec7 domain and led to the ubiquitin degradation of these proteins, thereby inhibiting cell cycle progression, proliferation, angiogenesis, and metastasis. Clinically, FBX8 expression was negatively correlated with the HIF-1α, CDK4, and c-Myc in CRC tissues. Our study reveals a novel mechanism of FBX8 in regulating tumor metastatic dormancy in liver and provides new strategies for the treatment of CRC metastasis.

Macrophage NCOR1 Deficiency Ameliorates Myocardial Infarction and Neointimal Hyperplasia in Mice.

BackgroundNCOR1 (nuclear receptor corepressor 1) is an essential coregulator of gene transcription. It has been shown that NCOR1 in macrophages plays important roles in metabolic regulation. However, the function of macrophage NCOR1 in response to myocardial infarction (MI) or vascular wire injury has not been elucidated.Methods and ResultsHere, using macrophage Ncor1 knockout mouse in combination with a mouse model of MI, we demonstrated that macrophage NCOR1 deficiency significantly reduced infarct size and improved cardiac function after MI. In addition, macrophage NCOR1 deficiency markedly inhibited neointimal hyperplasia and vascular remodeling in a mouse model of arterial wire injury. Inflammation and macrophage proliferation were substantially attenuated in hearts and arteries of macrophage Ncor1 knockout mice after MI and arterial wire injury, respectively. Cultured primary macrophages from macrophage Ncor1 knockout mice manifested lower expression of inflammatory genes upon stimulation by interleukin‐1β, interleukin‐6, or lipopolysaccharide, together with much less activation of inflammatory signaling cascades including signal transducer and activator of transcription 1 and nuclear factor‐κB. Furthermore, macrophage Ncor1 knockout macrophages were much less proliferative in culture, with inhibited cell cycle progression compared with control cells.ConclusionsCollectively, our data have demonstrated that NCOR1 is a critical regulator of macrophage inflammation and proliferation and that deficiency of NCOR1 in macrophages attenuates MI and neointimal hyperplasia. Therefore, macrophage NCOR1 may serve as a potential therapeutic target for MI and restenosis.

Novel scaffold hopping of potent benzothiazole and isatin analogues linked to 1,2,3-triazole fragment that mimic quinazoline epidermal growth factor receptor inhibitors: Synthesis, antitumor and mechanistic analyses

A series of benzothiazole/isatin linked to 1,2,3-triazole moiety and terminal sulpha drugs 5a-e and 6a-e were synthesized and evaluated for cytotoxic activity against a panel of cancer cell lines. The novel compounds showed variable IC50 range of activity and some of them were potent compared to reference drug. The promising compounds were subjected as postulated the mimicry proposal for quinazoline-based EGFR inhibitors for their inhibitory profile against EGFR TK enzyme. That data obtained revealed that most of these compounds were potent EGFR TK inhibitors at nanomolar concentrations. Among these, compounds 5a and 5b showed more potent activity on EGFR compared to erlotinib (IC50 103 and 104 versus 67.6 nM). Based upon the results, molecular docking analysis was performed on EGFR receptor and proved the strong contribution of fragments; benzothiazole, isatin, and triazole to the binding ATP pocket. When these selected compounds 5a and 5b were tested in an HepG2 model, they could effectively inhibited tumor growth, strongly induced cancer cell apoptosis, and suppressed cell cycle progression leading to DNA fragmentation. Well-DMET profile of the most active derivatives was presented and compared to the reference drugs. Taken together, we introduced novel triazole-sulpha drug hybrid for the first time as EGFR inhibitors and the results of our studies indicate that the newly discovered inhibitors have significant potential for anticancer treatment.

P14ARF: The Absence that Makes the Difference.

P14ARF is a tumor suppressor encoded by the CDKN2a locus that is frequently inactivated in human tumors. P14ARF protein quenches oncogene stimuli by inhibiting cell cycle progression and inducing apoptosis. P14ARF functions can be played through interactions with several proteins. However, the majority of its activities are notoriously mediated by the p53 protein. Interestingly, recent studies suggest a new role of p14ARF in the maintenance of chromosome stability. Here, we deepened this new facet of p14ARF which we believe is relevant to its tumor suppressive role in the cell. To this aim, we generated a monoclonal HCT116 cell line expressing the p14ARF cDNA cloned in the piggyback vector and then induced aneuploidy by treating HCT116 cells with the CENP-E inhibitor GSK923295. P14ARF ectopic re-expression restored the near-diploid phenotype of HCT116 cells, confirming that p14ARF counteracts aneuploid cell generation/proliferation.

Inhibition of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) stimulates osteoblastogenesis by potentiating bone morphogenetic protein 2 (BMP2) responses.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a pleiotropic enzyme involved in DNA repair, cell cycle control, and transcription regulation. A potential role for DNA-PKcs in the regulation of osteoblastogenesis remains to be established. We show that pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. Inhibition of DNA-PKcs inhibited cell cycle progression and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 response in osteoblasts and other mesenchymal cell types. Importantly, in vivo pharmacological inhibition of the kinase enhanced bone biomechanical properties. Bones from osteoblast-specific conditional Prkdc-knockout mice exhibited a similar phenotype of increased stiffness. In conclusion, DNA-PKcs negatively regulates osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.

Mechanisms of Androgen Receptor Agonist- and Antagonist-Mediated Cellular Senescence in Prostate Cancer.

The androgen receptor (AR) plays a leading role in the control of prostate cancer (PCa) growth. Interestingly, structurally different AR antagonists with distinct mechanisms of antagonism induce cell senescence, a mechanism that inhibits cell cycle progression, and thus seems to be a key cellular response for the treatment of PCa. Surprisingly, while physiological levels of androgens promote growth, supraphysiological androgen levels (SAL) inhibit PCa growth in an AR-dependent manner by inducing cell senescence in cancer cells. Thus, oppositional acting ligands, AR antagonists, and agonists are able to induce cellular senescence in PCa cells, as shown in cell culture model as well as ex vivo in patient tumor samples. This suggests a dual AR-signaling dependent on androgen levels that leads to the paradox of the rational to keep the AR constantly inactivated in order to treat PCa. These observations however opened the option to treat PCa patients with AR antagonists and/or with androgens at supraphysiological levels. The latter is currently used in clinical trials in so-called bipolar androgen therapy (BAT). Notably, cellular senescence is induced by AR antagonists or agonist in both androgen-dependent and castration-resistant PCa (CRPC). Pathway analysis suggests a crosstalk between AR and the non-receptor tyrosine kinase Src-Akt/PKB and the PI3K-mTOR-autophagy signaling in mediating AR-induced cellular senescence in PCa. In this review, we summarize the current knowledge of therapeutic induction and intracellular pathways of AR-mediated cellular senescence.

The anticancer activities of Vernonia amygdalina Delile. Leaves on 4T1 breast cancer cells through phosphoinositide 3-kinase (PI3K) pathway

Vernonia amygdalina Delile (Asteraceae) is used in traditional medicine to treat diabetes mellitus, and some research provides its activity to treat breast cancer. The aim of this study is to assess the anticancer activity of Vernonia amygdalina Delile leaves fractions on 4T1 breast cancer cells. Analysis of phytochemical compounds were carried out with LC-MS/MS. Cytotoxic activity was determined using the MTT method in the 4T1 cell line. Apoptosis, the cell cycle, and PI3K and mTOR profiles were analyzed with flow cytometry. The phytochemicals found were diterpene (ingenol-3-angelate) and some phenolics (chlorogenic acid and 4-methoxycinnamic acid), flavonoids (apigetrin, apigenin, luteolin, diosmetin, baicalin, rhoifolin, and scutellarin), and coumarines (7-hydroxycoumarine, 4-methylumbelliferone, and 4-methylumbelliferyl glucuronide). The results of the MTT assay showed that the IC50 values n-hexane fraction, ethylacetate fraction (EAF), and ethanol fractions were 1,860.54 ± 93.11, 25.04 ± 0.36, and 1,940.84 ± 96.37 μg/mL, respectively. EAF induced early and late apoptosis, inhibited cell cycle progression on the G2/M phase, and inhibited PI3K and mTOR expression. The EAF of Vernonia amygdalina Delile leaves showed anticancer activity on 4T1 breast cancer cells through induction of apoptosis, enhanced cell accumulation on G2/M phases in the cell cycle, and inhibited expression of PI3K and mTOR.

Antiproliferative and antiinflammatory coxib-combretastatin hybrids suppress cell cycle progression and induce apoptosis of MCF7 breast cancer cells.

In our study, some newly synthesized aryl-substituted pyrazole derivatives mimicking cis-diphenylethylene scaffold of two apoptotic inducing agents celecoxib and combretastatin A-4 were found to have strong antiproliferative as well as antiinflammatory activities. Among these coxib-combretastatin hybrids, two lead compounds 8 and 6c simultaneously inhibited prostaglandin E2 (PGE2) production in LPS-activated murine macrophage RAW 264.7 cells and suppressed cell cycle progression of MCF7 cells at G2/M or G0/G1 phases, but only compound 8 induced apoptosis via caspase-3 activation. Both the lead compounds showed good docking energies with both protein targets COX-2 and tubulin in the molecule interaction modeling. The cis-diphenylethylene scaffold of celecoxib or combretastatin A-4 as well as functional groups such as the ethyl ester group and the sulfonamide could be considered as potential key features for the dual activity of studied compounds meanwhile the trimethoxybenzene remained the crucial characterization of the newly derived compounds of combretastatins.

Caveolin-1 Knockdown Decreases SMMC7721 Human Hepatocellular Carcinoma Cell Invasiveness by Inhibiting Vascular Endothelial Growth Factor-Induced Angiogenesis.

Background Recently, several studies have demonstrated that caveolin-1 overexpression is involved in apoptosis resistance, angiogenesis, and invasiveness in hepatocellular carcinoma (HCC). However, the mechanisms underlying caveolin-1-mediated tumor progression remain unclear. Methodogy. Lentiviral vectors were used to construct caveolin-1 small interfering RNA- (siRNA-) expressing cells. Secreted VEGF levels in SMMC7721 cells were evaluated by enzyme-linked immunosorbent assay (ELISA). SMMC7721 cell proliferation, cycle, apoptosis, and invasiveness were detected by MTT, flow cytometry, Annexin V-FITC/PI, and invasion assay, respectively. Phospho-eNOS levels in human umbilical vein endothelial cells (HUVECs) cocultured with SMMC7721 cell supernatants were analyzed by Western blot. Capillary-like tubule formation assay was performed to analyze endothelial tubular structure formation in HUVECs treated with supernatants from caveolin-1 siRNA-expressing SMMC7721 cells. SMMC7721 implantation and growth in nude mice were observed. Angiogenesis in vivo was analyzed by immunohistochemical angiogenesis assay. Results Caveolin-1 siRNA-expressing SMMC7721 cells secreted reduced levels of VEGF. Caveolin-1 RNAi also caused an inhibition of SMMC7721 cell proliferation and cell cycle progression that was accompanied by increased apoptosis. Supernatants from caveolin-1 siRNA-expressing SMMC7721 cells inhibited cell cycle progression and decreased phospho-eNOS levels in HUVECs. Endothelial tubular structure formation in HUVECs treated with supernatants from caveolin-1 siRNA-expressing SMMC7721 cells was considerably reduced. Caveolin-1 siRNA-expressing SMMC7721 cells also showed reduced tumorigenicity and angiogenesis induction in vivo. Conclusion Our results reveal a novel mechanism, whereby caveolin-1 positively regulates human HCC cell invasiveness by coordinating VEGF-induced angiogenesis.

Genotoxic effects and proteomic analysis on Allium cepa var. agrogarum L. root cells under Pb stress.

Ionic lead (Pb) in the environment has accumulated due to anthropogenic activities, causing a potential threat to plants and plant consumers. We conducted this study to reveal the molecular mechanism of Pb stress response in plants. The effects of Pb (5.0 and 15.0 μM) on mitosis, DNA replication, gene expression and proteins in root-tip cells of Allium cepa var. agrogarum L. were addressed. The results indicated that root growth was inhibited dramatically in Pb treatment groups. Chromosomal aberrations were observed and the mitotic index decreased during Pb treatments at different concentrations. The accumulation of reactive oxygen species (ROS) in onion roots was induced by Pb stress. Pb increased DNA damage and suppressed cell cycle progression. The above toxic effects got more serious with increasing Pb concentration and prolonging exposure time. A total of 17 proteins were expressed differentially between control and Pb exposure groups. Under Pb treatment, the decreased expression of Anx D1 indicated decreased defensive response; the decreased expression of SHMT1 indicated decreased respiration; the decreased expression of COMT2 indicated decreased response of other funtions; the increased expression of NDPK indicated increased transcription and protein synthesis; the increased expression of PR1 and CHI1 indicated increased pathogen invasion; the increased expression of ORC5 and MPK5 indicated the reduced DNA replicating activity; the decreased expression of POLD1 indicated the reduced DNA repair activity. Our results provide new insights at the proteomic level into the Pb-induced responses, defensive responses and toxic effects, and provide new molecular markers of the early events of plant responses to Pb toxicity.