Expression Levels Of CDK4 Research Articles

Effect of PKA Gene on Acute Lymphoblastic Leukemia in Children and Its Mechanism

To explore the effect of PKA gene on acute T lymphocyte leukemia cells in children and its mechanism. Jurkat and Sup-T1 cells were divided into 2 group: control group (Jurkat and Sup-T1 cells treated with non-specific siRNA) and transfected group (Jurkat and Sup-T1 cells transfected with PKA siRNA). The effects of down-regulating the expression of PKA gene on the viability, proliferotion, migration and cell cycle distribution of Jurkat and Sup-T1 cells in 2 groups were analyzed by CCK-8 assay, transwell experiment, cell colony-formation test and flow cytometry; the cyclin-related protein levels after transfection with PKA siRNA were detected by Western blot. It was revealed that the expression of PKA in Jurkat and Sup-T1 cells decreased to different degree after siRNA transfection(P<0.05). CCK-8 assay showed that the proliferation of Jurkat and Sup-T1 cells in the transfected group was slower than that in the control group(P<0.05). Transwell experiment showed that the migration and invasion ability of Jurkat cells in the transfection group was weaker than that in the control group (P<0.05). The cell colong formation number of Jurkat and Sup-T1 cells in the transfection group decreased significantly (P<0.05). The cell level of Jurkat and Sup-T1 cells in G0/G1 phase increased after tansfection (P<0.05). Western blot assay revealed that the expression levels of CDK2, CyclinD1 and p-Rb in the Jurkat and Sup-T1 cells of the transfection group were suppressed (P<0.05). The down-regulating PKA gene expression can decrease the proliferation and migration of tumor cells, and also can restrict the cell proliferation through related cell cycle proteins.

Sperm-borne miR-449b influences cleavage, epigenetic reprogramming and apoptosis of SCNT embryos in bovine

Accumulating evidence indicates the absence of paternally derived miRNAs, piwiRNAs, and proteins may be one important factor contributing to developmental failure in somatic cell cloned embryos. In the present study, we found microRNA-449b (miR-449b) was highly expressed in sperm. Target gene predictions and experimental verification indicate that several embryonic development-related genes, including CDK6, c-MYC, HDAC1 and BCL-2, are targets of miR-449b. We therefore investigated the role of miR-449b using somatic cell nuclear transfer (SCNT) embryo model. Bovine fetal fibroblasts, expressing miR-449b through a doxycycline (dox) induced expression system were used as nuclear donor cells for SCNT. The results showed that miR-449b expression in SCNT embryos significantly enhanced the cleavage rate at 48 h after activation and the levels of H3K9 acetylation at the 2-cell to 8-cell stages, meanwhile, significantly decreased the apoptosis index of blastocysts. In addition, we verified miR-449b could regulate the expression levels of CDK6, c-MYC, HDAC1 and BCL-2. In conclusion, the present study shows that miR-449b expression improves the first cleavage division, epigenetic reprogramming and apoptotic status of bovine preimplantation cloned embryos.

Cell cycle regulation and apoptosis mediated by p53 in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei

Although hypoxic aquatic environments cause negative effects on shrimp, these animals can withstand somewhat hypoxia, but the cellular mechanisms underlying this capacity are still poorly understood. In humans, mild hypoxia causes the induction of many proteins to allow cell survival. In contrast, apoptosis is induced during severe hypoxia leading to cell death. p53 is a key transcription factor that determines cells fate towards cell cycle arrest or induction of apoptosis in humans. The aim of this work was to study the role of p53 in cell cycle regulation and apoptosis in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei. p53 was silenced by RNAi and afterwards the shrimp were exposed to hypoxia. Cdk-2 was used as indicator of cell cycle progression while caspase-3 expression and caspase activity were analyzed as indicators of apoptosis. p53 levels in hepatopancreas were significantly higher at 48 h after hypoxic treatment. Increased expression levels of Cdk-2 were found in p53-silenced shrimp after 24 and 48 h in the normoxic treatments as well as 48 h after hypoxia, indicating a possible role of p53 in cell cycle regulation. In response to hypoxia, unsilenced shrimp showed an increase in caspase-3 expression levels, however an increase was also observed in caspase activity at 24 h of normoxic conditions in p53-silenced shrimps. Taken together these results indicate the involvement of p53 in regulation of cell cycle and apoptosis in the white shrimp in response to hypoxia.

Macrophage migration inhibitory factor mediates viability and apoptosis of PVM/Ms through PI3K/Akt pathway

Macrophage migration inhibitory factor (MIF) plays an important role in hearing function; however, the underlying mechanism remains indistinct. PVM/Ms from the stria vascularis of lateral wall of cochlea in young and aged mice were isolated, and the mRNA and protein expression levels were detected. MIF was knocked down or overexpresssed in vitro, and transfection was performed in vivo. Cell viability and apoptosis were determined by MTT assay and flow cytometry analysis, respectively. The hearing ability was tested by the auditory brain stem response. The results showed that MIF expression was significantly downregulated in aged mice. In aged mice, the viability of PVM/Ms significantly decreased, but the apoptotic number markedly increased. MIF knockdown in PVM/Ms in vitro significantly inhibited cell viability and induced cell apoptosis, but MIF overexpression showed contrasting results. Further studies showed that MIF knockdown in young mice resulted in serious hearing loss, but MIF overexpression in aged mice restored the hearing. Si-MIF inhibited the viability and induced apoptosis of PVM/Ms from young mice, whereas Ad-MIF induced the viability and inhibited apoptosis of PVM/Ms from aged mice. Moreover, MIF effectively altered the expression levels of CDK1, BRAF, p-ERK1/2, p-PI3K, and p-Akt. Furthermore, ERK inhibitor PD98059 or PI3K inhibitor LY294002 significantly reversed the effects of Si-MIF on PVM/Ms from young mice, whereas ERK activator EGF or PI3K activator IGF significantly reversed the effects of Ad-MIF on PVM/Ms from aged mice. Taken together, MIF mediates the viability and apoptosis of PVM/Ms, at least partially, through MAPK and/or PI3K/Akt pathway.

Abstract 2339: Downregulation of cell cycle genes in HNSCC by erufosine

Abstract Head and neck cancer ranks amongst the sixth most prevalent cancers worldwide and oral squamous cell carcinomas (OSCC) constitute 90% thereof. Erufosine is an ether-lipid-derived synthetic compound belonging to alkylphosphocholines (APCs), which has been shown to inhibit the proliferation of OSCC cells. It simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway, however, its exact mechanism of action is not fully understood. Here, we describe the activity of erufosine on the expression of cell cycle related genes in OSCC cells. The anti-proliferative effect of erufosine in cells of two human OSCC cell lines, HN-5 and SCC-61, was determined by MTT assay after 24h, 48h and 72h exposure. Based on these results, HN-5 cells were exposed to erufosine at concentrations corresponding to IC25, IC50 and IC75 concentrations, and then their mRNA was isolated and analyzed by Illumina Chip array. Gene expression modulation was confirmed qRT-PCR and Western blot. Gene set enrichment analysis was performed to identify core KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and GO (Gene Ontology) terms. In addition, erufosine’s effect on cell cycle distribution and colony formation was determined as well as its antineoplastic effect on respective xenografts in nude mice. As shown from TCGA data, cell cycle deregulation in HNSCC ranks amongst the top three out of 24 different types of cancers. In line with this, the median expression of cell cycle genes was higher than that of other genes within HNSCC. We also looked at the median gene expression of all CDKs and cyclins in 519 HNSCC patient samples from TCGA and found that the expression levels were higher than the global gene expression. This was in agreement with IHC staining of CCND1 in tumors from 30 South East Asian HNSCC patients. Furthermore, increased CCND1 and CDK6 expression levels from TCGA data had negative implication on patient survival. In our experimental results erufosine caused a dose dependent growth inhibition of OSCC cell lines and negative enrichment of genes related to the cell cycle process. Our microarray findings revealed that cyclins and CDKs were downregulated in a dose dependent manner in response to erufosine exposure. These findings were verified at both, mRNA and protein levels. Erufosine not only caused a massive G2M block but also inhibition of colony formation thus preparing the OSCC cells to undergo apoptosis. We are the first to show that erufosine inhibited tumor growth in vivo in a HNSCC xenograft model and caused downregulation of cyclinD1, CDK4 and CDK6 in lesions of the animals. These findings collectively show the potential of erufosine to be used as a cell cycle inhibitor in HNSCC progression and support the future evaluation of erufosine as a therapeutic approach in cancer treatment alone, or in combination. Citation Format: Shariq S. Ansari, Ashwini K. Sharma, Michael Zepp, Frank Bergmann, Rainer König, Martin R. Berger. Downregulation of cell cycle genes in HNSCC by erufosine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2339. doi:10.1158/1538-7445.AM2017-2339

Butein inhibits NF-κB, AP-1 and Akt activation in adult T-cell leukemia/lymphoma.

Human T-cell leukemia virus type 1(HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma(ATLL) but there is no effective treatment for HTLV-1-associated diseases. Herein, we determined the effect of butein, a bioactive plant polyphenol, on cell growth, apoptosis and signaling pathways in HTLV-1-infected T-cell lines and on tumor growth in SCID mice. Treatment with butein caused a decrease in viability of HTLV-1-infected T-cell lines. Tcells cultured with butein showed obvious apoptosis morphology, and cleavage of poly(ADP-ribose) polymerase with activation of caspase-3, -8 and -9. Pretreatment of cells with caspase inhibitor partially blocked butein-induced inhibition of cell viability. Butein also resulted in cell cycle arrest at G1phase. Butein markedly downregulated the protein expression levels of CDK4, CDK6, cyclin D1, cyclin D2, cyclin E, survivin, XIAP, c-IAP2 and phospho-pRb. Butein also inhibited i) total and phospho-protein levels of IκB kinase (IKK)α and IKKβ, ii) degradation and phosphorylation of IκBα, iii) JunB and JunD, iv) total and phospho-protein levels of Akt, v) phosphorylation of RelA, vi)heat shock protein90, and vii) DNA-binding activity of NF-κB and AP-1. In mice harboring ATLL xenograft tumors, butein caused a significant inhibition of tumor growth and reduced serum levels of soluble interleukin-2 receptorα chain and soluble cluster of differentiation30. Considered together, the results indicated that butein has antiproliferative and proapoptotic properties through the suppression of NF-κB, AP-1 and Akt signaling in HTLV-1-infected Tcells, both invitro and invivo, suggesting its therapeutic potential against HTLV-1-associated diseases including ATLL.

PPeOp from Omphalia lapidescens Schroet induces cell cycle arrest and inhibits the migration of MC-4 gastric tumor cells.

The aim of the present study was to investigate the effect of purified Omphalia lapidescens protein (pPeOp) extracted by polyvinylpyrrolidone from the fungus Omphalia lapidescens Schroet on the proliferation and cell cycle progression of MC-4 human gastric tumor cells. Using polyvinylpyrrolidone, pPeOp was extracted from O. lapidescens Schroet. MC-4 cells were cultured with 30, 60 or 90 µg/ml pPeOp, with 5-fluorouracil used as a positive control. Survival rates of treated cells were significantly decreased compared with those of the untreated control group in a dose-dependent manner. Using flow cytometric analysis, cells treated with pPeOp were demonstrated to arrest in S phase and exhibit abnormal G0/G1 and G2/M phase cell cycle distribution. In addition, a wound healing assay demonstrated that pPeOp significantly inhibited the migration of MC-4 cells. The mRNA and protein expression levels of cyclin D1/cyclin-dependent kinase (CDK) 4, cyclin B/CDK1, cyclin A/CDK2, matrix metalloproteinase (MMP)-2 and MMP-9 were determined using reverse transcription-quantitative polymerase chain reaction analysis and western blotting. The mRNA expression level of CDK4 and cyclin A was significantly increased compared with the untreated control; however, cyclin D1, CDK1, CDK2, cyclin B, MMP-2, and MMP-9 exhibited a significantly decreased mRNA expression level, indicating that there is a negative association between concentration and cyclin D1 expression levels. The expression of the cycle arrest-associated proteins and migration-associated proteins examined were similar to the observed mRNA expression levels. In conclusion, pPeOp was identified to inhibit migration of and cause S phase cell cycle arrest in MC-4 cells.

The Novel Long Noncoding RNA TUSC7 Inhibits Proliferation by Sponging MiR-211 in Colorectal Cancer

Background/Aims: The novel long noncoding RNA (lncRNA) tumor suppressor candidate 7 (TUSC7) has been reported as a potential tumor suppressor, while the functional role of TUSC7 is still unknown in colorectal cancer (CRC). Here, we characterized TUSC7 expression profile in CRC patients and investigated its biological function and potential molecular mechanism. Methods: RNA isolation, qRT-PCR, cell counter kit-8 assay, cell cycle assay, EdU assay, and western blot were performed. Statistical analyses were performed using SPSS 18.0 software and p value < 0.05 was considered as statistically significant. Results: In a cohort of CRC patients, we found TUSC7 was significantly downregulated in CRC tissues compared with adjacent non-tumor tissues (P < 0.01). Patients with high expression of TUSC7 had better survival than those with low expression of TUSC7 (HR = 0.342, 95% CI: 0.120-0.972, P = 0.044). Cell count kit 8 and EdU assays showed that ectopic expression of TUSC7 in HCT116 and SW480 cells significantly inhibited cell proliferation rate. After silence of TUSC7 with small interfering RNA, cell proliferation rate increased. Flow cytometry analyses revealed cycles were arrested at G1 phase after TUSC7 overexpression. We found there were 2 binding sites of miR-211-3p within the sequence of TUSC7 and TUSC7 expression level was negatively correlated with miR-211-3p. TUSC7 overexpression increased the expression level of CDK6, which is a downstream target of miR-211-3p, in both RNA and protein level. Furthermore, luciferase reporter assay indicated that TUSC7 could sponge miR-211-3p. Conclusion: To summary, we demonstrated that TUSC7 is a potential tumor suppressor in CRC, and TUSC7 could inhibit CRC cell proliferation by completely sponging miR-211-3p.

Design, synthesis and biological evaluation of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamides as CDK1/Cdc2 inhibitors

A series of new (N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,3-diphenyl-1H-pyrazole-4-carboxamide derivatives (8–35) were designed, synthesized and evaluated as CDK1/Cdc2 inhibitors. Biological evaluation assays indicated that compounds 16 and 27 showed the most potent growth inhibitory activity against human cancer cell lines (MIAPaCa-2, MCF-7 and HeLa) with GI50 values ranging from 0.13 to 0.7 μM, compared with the positive control nocodazole (0.81–0.95 μM). Flow cytometric analysis revealed that these compounds induce cell cycle arrest in the G2/M phase and Western blot analysis suggested that compound treatment resulted in reduction of CDK1 expression levels in MCF-7 cell line. Moreover, the apoptosis inducing effect of the compounds was studied using Hoechst staining, Rhodamine 123 staining (MMP), carboxy-DCFDA staining (ROS), Annexin V-FITC assay. Based on these studies, two compounds 16 and 27 have been identified as promising new molecules that have the potential to be developed as leads.

The cell cycle-related genes as biomarkers for schizophrenia

BackgroundRecent studies suggest that genomic abnormalities such as single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) may elevate the risk of schizophrenia. Such genomic abnormalities often occur during chromosomal DNA replication in the S phase of cell cycle. In addition, several studies showed that abnormal expressions of several cell cycle-related genes are associated with schizophrenia. Therefore, here we compared mRNA expression levels of cell cycle-related genes in peripheral blood cells between patients with schizophrenia and healthy controls. MethodmRNA expression levels of cell cycle-related genes in peripheral blood cells from patients with schizophrenia and healthy controls were measured with quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). The discovery, replication and intervention studies with Q-RT-PCR were performed as follows: discovery (40 cases and 20 controls), replication (82 cases and 74 controls) and intervention (22 cases and 18 controls). ResultNine genes were identified in the discovery and replication stages as schizophrenia-associated genes. Moreover, the combination of mRNA expression levels of CDK4, MCM7 and POLD4 was identified as a potential biomarker for schizophrenia with multivariate logistic regression analysis. The intervention stage revealed that the mRNA expression levels of these three genes were significantly decreased in the acute state of schizophrenia, and CDK4 was significantly recovered in the remission state of schizophrenia. ConclusionThe combination of mRNA expression levels of three cell cycle-related genes such as CDK4, MCM7 and POLD4 is expected to be a candidate for useful biomarkers for schizophrenia. Especially, the mRNA expression changes of CDK4 may be potential as both trait and state markers for schizophrenia.

Correlation between cyclin-dependent kinase inhibitor p27kip1 and trastuzumab-resistance in gastric cancer

To investigate the correlation between cyclin-dependent kinase inhibitor p27kip1 and trastuzumab-resistance in gastric cancer. We selected HER2-overexpressed human gastric cancer cell line NCI-N87 to establish trastuzumab-resistant NCI-N87/TR cell line by stepwise exposure to different doses of trastuzumab. The 50% inhibitory concentration (IC(50)) of trastuzumab and resistance index (RI) were calculated or analyzed by MTT assay. The expression levels of cdk2 and p27kip1 were detected by Western blot. After the treatment with cdk2 inhibitor (Purvalanol A), the expression levels of relevant proteins in NCI-N87/TR cells were detected by Western blot, and the sensitivity to trastuzumab was analyzed by MTT assay. Compared with NCI-N87 cells, the expression of cdk2 was significantly increased in NCI-N87/TR cells (P<0.001), while the expression of p27kip1 showed a significant decrease (P<0.001). Restoration of the p27kip1 protein expression by cdk2 inhibitor (Purvalanol A) increased the sensitivity of NCI-N87/TR to trastuzumab. Down-regulation of p27kip1 might be a mechanism for triggering trastuzumab resistance to gastric cancer cell line NCI-N87.

Protein inhibitor of activated STAT xα depresses cyclin D and cyclin D kinase, and contributes to the inhibition of osteosarcoma cell progression.

Previous studies have shown that protein inhibitor of activated STAT (PIAs)xα is crucial in protein sumoylation and is associated with cancer cell progression. However, the mechanism underlying the inhibitory effect on cancer cells, which may assist in developing novel treatment strategies in cancer remains to be elucidated. In present study, the expression levels of PIAsxα from tissue samples of osteosarcoma and adjacent tissues from 25 patients were analyzed using reverse transcription-quantitative polymerase chain reaction, western blot and immunohistochemical analyses. In addition, techniques using an overexpression vector and small interfering (si)RNAs were used to examine the effect of PIAsxα on osteosarcoma cells. Finally, using xenograft U2-OS osteosarcoma cells overexpressing PIAsxα, the effect of PIAsxα on osteosarcoma formation was determined. The results revealed low expression of PIAsxα in osteosarcoma tissues. In addition, following overexpression of PIAsxα, the apoptotic rates were significantly increased. The rate of G2/M arrest was at the highest level in the overexpression group, compared with other groups assessed. Furthermore, the expression levels of cyclin D1 and cyclin D3 were inhibited following PIAsxα increase, indicating the repressive effects of PIAsxα on cell cycle. Accordingly, cyclin D kinase (CDK) genes, including CDK4, CDK6 and CDK8, increased markedly following treatment with PIAsxα siRNAs. The expression levels of CDK4, CDK6 and CDK8 decreased significantly in the overexpression group, compared to the other groups. Furthermore, high expression levels of PIAsxα inhibited tumor formation in a nude mouse model. Taken together, these findings provide evidence for the effects of PIAsxα and its mechanism on osteosarcoma progression, which offers novel insight into sumoylation and the cell cycle in osteosarcoma.

Loop Regulation Between microRNAs and Epigenetics Underlie microRNA Dysregulation in Multiple Myeloma and Is Associated with the Disease Progression

Background: MicroRNAs (miRs) are small non-coding RNAs of 19-25 bases in length that have the ability to modulate gene expression. Some miRs are involved in carcinogenesis and act as tumor suppressor genes (TSG). It has been shown that epigenetics and miRs play an important role in multiple myeloma (MM) progression; however, precise mechanism underlying miR dysregulation has not yet been fully elucidated. Transcriptional silencing of TSG in cancer cells is often associated with DNA methylation and carried out by DNMTs. miR-29 family directly targets DNMTs and promoters of miR-34 family are also methylated in cancers. In this study, we attempted to clarify the interaction between miR and epigenetics focusing on the miR-29 and miR-34 families and their associated genes to understand mechanism of miR dysregulation in MM.Methods: Bone marrow plasma cells from 123 MM patients, 57 MGUS patients, 20 control subjects and 9 MM cell lines were analyzed. This study was approved by the IRB of Gunma University and all patients provided their informed consent prior to enrollment. MiRs and their target gene mRNA values were determined by RQ-PCR. DNA methylation status was determined by methylation-specific PCR. Decitabine, nutlin-3, c-myc inhibitor 10058-F4, and miRNA-mimicTM were used.Result: We found a significantly reduced expression of miR-29a, -29b, -29c, -34a, -34b, and -34c in MM patients compared with MGUS patients and the control subjects (all: p<0.001). DNMT1, -3A and -3B were elevated in MM patients compared with MGUS patients and the control subjects (p<0.001, p<0.001, p=0.01, respectively). DNMT1 was inversely correlated with miR-29a and miR-29b (r=-0.419, p=0.005, r=-0.407, p=0.006, respectively). DNMT3A was inversely correlated with miR-29a, miR-29b and miR-29c (r=-0.315, p=0.04; r=-0.371, p=0.01; r=-0.315, p=0.04, respectively). DNMT3B was inversely correlated with miR-29a and miR-29b (r=-0.353, p=0.02; r=-0.358, p=0.02, respectively). The promoter regions of miR-34a and miR-34b/c were methylated in the MM cell lines, and the rate of methylation of these miRs were higher in MM patients (45.4%, 70.2%, respectively) compared with MGUS patients (15.8%, 26.3%, respectively) (p<0.001). There were significant positive correlations among the miRs expression levels: 29a-34a r=0.448, p<0.001; 29a-34b r=0.309, p=0.001; 29b-34a r=0.500, p<0.001; 29b-34b r=0.297, p=0.002). The expression level of TP53 and its downstream target p21 was higher in MM patients (p=0.004, p<0.001) compared with MGUS patients and the control subjects. The expression level of TP53 was positively correlated with p21 (r=0.33, p<0.001), but not with miR-34a, -b or -c, which were presumed to be upregulated by TP53. In the MM cell lines, nutlin-3, which accumulates TP53 protein, did not increase the expression of miR-34a, -b or -c; however, decitabine increased the expression of pri-miR-34a by 1.2-7.3 fold, pri-miR-34b by 3.3-7.1 fold and increased miR-34a and miR-34b/c by 1.3-2.3 fold and 1.2-5.4 fold, respectively, suggesting that miR-34 family transcription was suppressed by methylation. The transfection of miR-29a or -29b reduced DNMT3A and 3B expression by 0.5 fold and increased pri-miR-34 expression by 2-8 fold. Treatment with a c-myc inhibitor increased the expression of pri-miR-29a/b-1 by 6-8 fold, and miR-29b by 4-7 fold in the MM cell lines. Moreover, the c-myc inhibitor increased the expression of pri-miR-34a by 20-192 fold and miR-34a by 3-6 fold. The transfection of miR-34 reduced the expression of target genes CDK6, SIRT1 and c-myc by 0.4 to 0.6 fold. The expression levels of CDK6, SIRT1 and c-myc were significantly higher in MM patients compared with MGUS patients and the control subjects (p=0.04, p<0.0001, p<0.0001, respectively).Conclusion: We found a significant reduction in the miRs expression levels in MM patients and cell lines, which was partly associated with methylation. Correlations between miRs and related transcripts in patients and an in vitro study demonstrate a negative regulation loop where c-myc suppresses the expression of the miR-29 family, the miR-29 family suppresses DNMT, DNMT suppresses the miR-34 family through promoter methylation, and the miR-34 family suppresses c-myc. This mechanism might underlie the dysregulation of miRs in MM and the disruption of this loop mechanism may be a novel target for MM treatment. DisclosuresNo relevant conflicts of interest to declare.

Discovery of pyrrolospirooxindole derivatives as novel cyclin dependent kinase 4 (CDK4) inhibitors by catalyst-free, green approach

Aiming to develop a new target for the anticancer treatment, a series of 5′H-spiro[indoline-3,4′-pyrrolo [1,2-a]quinoxalin]-2-ones has been synthesized by simple, highly efficient and environmentally friendly method in excellent yields under catalyst-free conditions using ethanol as a green solvent. A simple filtration of the reaction mixture and subsequent drying affords analytically pure products. The synthesized derivatives were evaluated for their antiproliferative activity against five different human cancer cell lines, among the congeners compound 3n showed significant cytotoxicity against the human prostate cancer (DU-145). Flow cytometric analysis revealed that this compound induces cell cycle arrest in the G0/G1 phase and Western blot analysis suggested that reduction in Cdk4 expression level leads to apoptotic cell death. This was further confirmed by mitochondrial membrane potential ((ΔΨm), Annexin V-FITC assay and docking experiments. Furthermore, it was observed that there is an increase in expression levels of cyclin dependent kinase inhibitors like Cip1/p21 and Kip1/p27.

Abstract B55: High expression of CDK6 protects breast cancer cells from fulvestrant-mediated apoptosis and is a predictor of resistance to fulvestrant treatment in estrogen receptor-positive metastatic breast cancer

Abstract Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a major clinical problem. Recently, the CDK4/6 inhibitor palbociclib combined with letrozole was approved for treatment of ER+ advanced breast cancer, and other CDK4/6 inhibitors are also being investigated in clinical trials in combination with different endocrine treatments, including fulvestrant. However, the role of CDK4/6 in endocrine resistance and their potential as predictive biomarkers of endocrine treatment response remains to be defined. We evaluated the expression of genes of the cyclin D/CDK4-6/p16INK4A/Rb pathway in a panel of endocrine-resistant breast cancer cell lines by gene array and found increased expression of CDK6 in two fulvestrant-resistant cell models vs. sensitive cells. Reduction of CDK6 expression by specific gene knockdown impaired fulvestrant-resistant cells growth and induced apoptosis by reducing p70 ribosomal S6 kinase 2 activity. Treatment with palbociclib re-sensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma phosphorylation. Evaluation of CDK6 expression levels in metastatic samples from breast cancer patients treated with fulvestrant (N = 45) showed that high levels of CDK6 correlated significantly with shorter progression-free survival (PFS) (p = 0.0006), while no association was observed between CDK6 levels and PFS in patients receiving other endocrine treatments (N = 41, p = 0.874). Our results indicate that upregulation of CDK6 may be an important mechanism in overcoming fulvestrant-mediated growth inhibition in breast cancer cells. Patients with advanced ER+ breast cancer exhibiting high CDK6 expression in the metastatic lesions show shorter PFS upon fulvestrant treatment and thus may benefit from the addition of CDK4/6 inhibitors in their therapeutic regimens. Citation Format: Carla L. Alves, Daniel Elias, Maria Lyng, Martin Bak, Anne Lykkesfeldt, Henrik J. Ditzel. High expression of CDK6 protects breast cancer cells from fulvestrant-mediated apoptosis and is a predictor of resistance to fulvestrant treatment in estrogen receptor-positive metastatic breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B55.

Prevention of Simvastatin-Induced Inhibition of Tendon Cell Proliferation and Cell Cycle Progression by Geranylgeranyl Pyrophosphate.

Statins have been reported to induce tendinopathy and even tendon rupture. The present study was designed to investigate the potential molecular mechanism underlying the adverse effect of simvastatin on tendon cells. An invitro tendon healing model was performed using tendon cells isolated from rat Achilles tendons. The viability of tendon cells and cell cycle progression were examined by the MTT assay and flow cytometric analysis, respectively. Immunofluorescent staining for Ki-67 was used to assess the proliferation activity of tendon cells. Western blot analysis and coimmunoprecipitation was used to determine the protein expression of cell cycle-related proteins. To investigate the potential mechanism underlying the effect of statins on tendon cells, mevalonate, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP) was added to simvastatin-treated tendon cells. Simvastatin inhibited the invitro tendon healing model and tendon cell proliferation in a dose-dependent manner. Immunofluorescent staining demonstrated reduced ki-67 expression in simvastatin-treated tendon cells. Furthermore, simvastatin induced cell cycle arrest at the G1 phase. The expression levels of cdk1, cdk2, cyclin A, and cyclin E were downregulated by simvastatin in a dose-dependent manner. The inhibitory effect of simvastatin was proved to mediate the reduction of mevalonate, and the addition of exogenous GGPP completely prevented the inhibitory effect of simvastatin on tendon cells. The present study demonstrated, for the first time, the molecular mechanism underlying simvastatin-induced tendinopathy or tendon rupture. GGPP was shown to prevent the adverse effect of simvastatin in tendon cells without interfering with its cholesterol-reducing efficacy.

Syntenin controls migration, growth, proliferation, and cell cycle progression in cancer cells.

The scaffold protein syntenin abounds during fetal life where it is important for developmental movements. In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis. Depending on the cancer model analyzed, syntenin affects various signaling pathways. We previously have shown that syntenin allows syndecan heparan sulfate proteoglycans to escape degradation. This indicates that syntenin has the potential to support sustained signaling of a plethora of growth factors and adhesion molecules. Here, we aim to clarify the impact of syntenin loss-of-function on cancer cell migration, growth, and proliferation, using cells from various cancer types and syntenin shRNA and siRNA silencing approaches. We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7. We further documented that syntenin controls the presence of active β1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins. These data confirm that syntenin supports the migration and growth of tumor cells, independently of their origin, and further highlight the attractiveness of syntenin as potential therapeutic target.

Expression of MicroRNA-26a and Its Target Gene CDK6 in Extra-nodal NK/T-Cell Lymphoma

To explore the expression level of microRNA-26a and its target gene CDK6 in extranodal NK/T-cell lymphoma (ENKTCL) and their possible role in genesis and development of ENKTCL. Real time fluorescent quantitative PCR was used to detect the expression level of miR-26a in tissue of 15 patients with ENKTCL and 10 samples of normal NK cells. Maxvision immunohistochemistry technique was used to detect the expression level of CDK6 and miR-26a in tissue of 20 ENKTCL cases, 10 cases of proliferative lymphadenitis and 10 samples of normal lymph node, respectively. The possible role of miR-26a and its target gene CDK6 in genesis and development of ENKTCL were analyzed according to the clinical features of ENKTCL patients. The expression of miR-26a was significantly lower in ENKTCL than that in normal NK cells. The expression of CDK6 was significantly higher in ENKTCL group than that in group proliferative lymphadenitis and normal lymph node. Correlation analysis showed that there was significant negative correlation between miR-26a expression and CDK6 expression (r = -0.54, P = 0.04). Meanwhile, there were no correlation of miR-26a expression with age, sex, Ann Arbor stage, LDH level, B symptoms and IPI. Although, there were no correlation of CDK6 expression with age, sex, LDH level and B symptoms, there were positive correlation of CDK6 expression with Ann Arbor stage and IPI. that abnormal expression of miR-26a may participate in genesis and development of ENKTCL through regulating the expression of its target gene CDK6.

Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma.

Antiproliferative activity of rosamultic acid is associated with induction of apoptosis, cell cycle arrest, inhibition of cell migration and caspase activation in human gastric cancer (SGC-7901) cells

BackgroundGastric cancer is the second leading cause of cancer related deaths after lung cancer globally. Among natural products, natural triterpenes represent a structurally diverse group of organic compounds with potent antitumor activity. PurposeThe objective of the present research work demonstrated the antiproliferative and apoptotic activity of rosamultic acid, a natural triterpenoid, in human gastric cancer (SGC-7901) cells. Its effect on cellular morphology, cell cycle arrest, DNA fragmentation and expression levels of caspase-3, caspase-8 and caspase-9 were also determined. MethodsAntiproliferative activity of rosamultic acid was evaluated by MTT assay. Phase contrast, fluorescence microscopy as well as flow cytometry using Hoechst 33342, acridine orange/ethidium bromide and Annexin V-FITC as cellular probes were used to evaluate the induction of apoptosis by rosamultic acid. Protein level expressions were analyzed by western blot analysis. ResultsThe results revealed that rosamultic acid induced dose-dependent as well as time dependent cytotoxic effects in SGC-7901 gastric cancer cells. It also led to a reduction in clonogenic activity along with inhibiting the cell migration. Characteristic features of apoptosis induced by rosamultic acid were observed and quantified. Cell cycle arrest at sub-G1 phase was induced by rosamultic acid along with downregulation of expression levels of CDK4, CDK6 and cyclin D1. Rosamultic acid also significantly led to the activation of caspase-3, -8 and -9 during the 48 h treatment along with cleaving PARP in a dose-dependent manner. DNA fragmentation following rosamultic acid treatment was also observed in these cells. ConclusionThe current study strongly reveals that rosamultic acid inhibits gastric cancer proliferation by inducing apoptosis mediated through cell cycle arrest, downregulation of cell cycle related protein expressions, inhibition of cell migration, DNA damage, and activation of caspases.