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Aim: A series of pyridopyrimidine derivatives 5-20 was designed, synthesized and examined for antitumor activity using four types of malignant cells. Materials & methods: Cervical cancer (HeLa), hepatic cancer (HepG-2), breast cancer (MCF-7) and colon cancer (HCT-166) cells, as well as normal human lung fibroblast cells (WI-38) were used to determine the cytotoxicity. Results: Pyrazol-1- yl pyridopyrimidine derivative 5 was found to be the most active compound against three malignant cells Hela, MCF-7 and HepG-2 with IC 50 values of 9.27, 7.69 and 5.91 μM, respectively, related to standard Doxorubicin. Moreover, compounds 5 and 10 showed good inhibition against cyclin dependent kinase (CDK4/cyclin D1) and epidermal growth factor (EGFR) enzymes.

Background: Hydroxytyrosol is one of the phenolic compounds of olive oil and can induce anticancer effects on colorectal cancer cells., Objective: The aim of the present study was to evaluate the free hydroxytyrosol and nano-capsulated hydroxytyrosol effects on the cell cycle arrest in HT-29 colorectal cancer cell line., Methods: The nano-capsulated hydroxytyrosol was synthesized in poly lactide-co-glycolide-co-polyacrylic acid (PLGA-PAA) copolymer. MTT assay was performed to evaluate the anti-proliferative and anti-tumor effects of the free hydroxytyrosol and nano-capsulated hydroxytyrosol. Finally, the relative expression of CDKN1A, CDKN1B, and CCND1 genes was evaluated in control and treated colorectal cancer cells by using Real-Time PCR., Results: The obtained results from the MTT assay showed that the cytotoxic effects of the nano-capsulated hydroxytyrosol on the colorectal cancer cell line (IC50= 6PPM) were significantly more than free hydroxytyrosol (IC50= 12PPM) after 72h. Also, nano-capsulated hydroxytyrosol showed more significant effects on the upregulation of CDKN1A and CDKN1B genes and down-regulation of the CCND1 gene in colorectal cancer cells., Conclusion: In conclusion, the present study showed that hydroxytyrosol led to the death of colorectal cancer cells through cell cycle arrest. Also, the PLGA-PAA copolymer dramatically caused to increase the cytotoxic effects of the hydroxytyrosol on the colorectal cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The expanding clinical application of CDK4- and CDK6-inhibiting drugs in the managements of breast cancer has raised a great interest in testing these drugs in other neoplasms. The potential of combining these drugs with other therapeutic approaches seems to be an interesting work-ground to explore. Even though a potential integration of CDK4 and CDK6 inhibitors with radiotherapy (RT) has been hypothesized, this kind of approach has not been sufficiently pursued, neither in preclinical nor in clinical studies. Similarly, the most recent discoveries focusing on autophagy, as a possible target pathway able to enhance the antitumor efficacy of CDK4 and CDK6 inhibitors is promising but needs more investigations. The aim of this review is to discuss the recent literature on the field in order to infer a rational combination strategy including cyclin-D1/CDK4-CDK6 inhibitors, RT, and/or other anticancer agents targeting G1-S phase cell cycle transition.

Objective: Kaempferol has been reported to play an anti-tumor role in various human cancers, while its role in gallbladder cancer (GBC) is unclear., Materials and Methods: We found that kaempferol significantly inhibited the growth, invasion and migration, meanwhile induced apoptosis through cells arrested at G0/G1 phase of GBC cell lines, including GBC-SD and SGC996 cells in vitro., Results: Kaempferol promoted the release of cytochrome C from the mitochondria to cytoplasm, the activation of c-caspase-3 and c-caspase-9 and increased the expression levels of pro-apoptotic factor Bax, meanwhile decreased the expression levels of anti-apoptotic factor Bcl-2. In addition, the expression levels of CDK4, CDK6 and cyclin D1, which are members of the CDK4/CDK6/cyclin D1 signaling pathway, were also decreased by kaempferol. Moreover, kaempferol could efficiently prevent tumor progression of GBC in the xenograft in vivo., Conclusions: Our results demonstrated that kaempferol suppressed GBC progression through activation of the CDK4/CDK6/cyclin D1 signaling pathway, suggesting that it might be a potential anti-tumor agent for clinical treatment of GBC.

Background: Paeoniflorin (Pae) possesses anti-tumor activity in various malignancies. However, it is unclear whether Pae plays a sensitizer role in breast cancer (BC) and the molecular mechanisms involved in this process. Our oligonucleotide microarray revealed that microRNA (miR)-15b is the most significantly downregulated miRNA in MCF-7/4-hydroxytamoxifen (4-OHT) cells treated with Pae. This paper summarized the relevance of Pae in BC cell endocrine resistance to tamoxifen (Tam) and the molecular mechanisms involved miR-15b expression., Materials and Methods: 4-OHT-resistant BC cell lines were developed and treated with different concentrations of Pae. Flow cytometry, lactose dehydrogenase activity, caspase-3 activity, colony formation, and EdU assays were carried out to assess the impact of Pae on BC cells. Differentially expressed miRNAs in BC cells treated with Pae were analyzed by microarray. Targeting mRNAs of screened miR-15b as well as the binding of forkhead box O1 (FOXO1) to the cyclin D1 (CCND1) promoter sequence were predicted through bioinformatics analysis. Finally, the expression of β-catenin signaling-related genes in cells was detected by Western blotting., Results: Pae (100 μg/mL) inhibited the clonality and viability of BC cells, while enhancing apoptosis in vitro. Pae also repressed miR-15b expression. Overexpression of miR-15b restored the growth and resistance of BC cells to 4-OHT. Moreover, Pae promoted FOXO1 expression by downregulating miR-15b, thereby transcriptionally inhibiting CCND1 and subsequently blocking β-catenin signaling., Conclusion: Pae inhibits 4-OHT resistance in BC cells by regulating the miR-15b/FOXO1/CCND1/β-catenin pathway., Competing Interests: All authors declare that they have no conflicts of interest in this work., (© 2021 Wang et al.)

Background: The quest for strong, safe and cost-effective natural antiproliferative agents that could reduce cancer has been the focus now a days. In this regard, the organosulfur compounds from garlic (Allium sativum L.), like Diallyl Sulfide (DAS) and Diallyl Disulfide (DADS), have been shown to exhibit potent antiproliferative and anticancer properties in many studies. However, the potential of these compounds against viral oncoproteins in cervical cancer has not been fully elucidated yet., Objective: The objective of this study was to analyze the antiproliferative and apoptotic properties of DADS and DAS in HPV16+ human cervical cancer Caski cell line., Methods: Caski (cervical cancer cells) were cultured and followed by the treatment of various concentrations of organosulphur compounds (DADS and DAS), cell viability was measured by MTT assay. The apoptotic assay was performed by DAPI and Hoechst3342 staining. Reactive Oxygen Species (ROS) was estimated by DCFDA staining protocol. The distributions of cell cycle and apoptosis (FITC-Annexin V assay) were analyzed by flow cytometry. Finally, gene expression analysis was performed via quantitative real time PCR., Results: Our results showed that DAS and DADS exerted a significant antiproliferative effect on Caski cells by reducing the cell viability and inducing a dose-related increment in intracellular ROS production along with apoptosis in Caski cells. DAS and DADS also induced cell cycle arrest in G0/G1 phase, which was supported by the downregulation of cyclin D1 and CDK4 and upregulation of CDK inhibitors p21WAF1/CIP1 and p27KIP1 in Caski cells. Additionally, DAS and DADS lead to the downregulation of viral oncogene E6 and E7 and restoration of p53 function., Conclusion: Thus, this study confirms the efficacy of both the organosulfur compounds DADS and DAS against cervical cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Purpose: Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC., Experimental Design: Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients., Results: We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro . Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant., Conclusions: Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations., (©2019 American Association for Cancer Research.)

Glioma is the most common highly malignant primary brain tumor. MicroRNA-519d-3p exerts important effects in several tumors, but its functional role in glioma remained poorly understood. In this study, we found miR-519d-3p expression was significantly decreased in glioma tissues and cell lines. Moreover, the in vitro experiments showed that overexpression of miR-519d-3p suppressed cell proliferation and induced cell cycle G0/G1 phase arrest using MTT and flow cytometry assays in glioma cell lines, U87 and U251. Mechanistically, Cyclin D1 (CCND1) was predicted and confirmed as the direct target genes of miR-519d-3p using luciferase report assay. In addition, knockdown of CCND1 imitated the suppressive effects of miR-519d-3p on cell proliferation and cell cycle progression. Furthermore, restoration of CCND1 reversed the effects of miR-519d-3p overexpression in glioma cells. Taken together, these data demonstrate that suppression of CCND1 by miR-519d-3p might be a therapeutic target for glioma. Abbreviations miR-519d-3p: microRNA-519d-3p; CCND1: Cyclin D1; ATCC: American Type Culture Collection; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PI: propidium iodide; WT: wild type; MUT: mutant type; SD: standard deviation.

RNA interference offers therapeutic opportunities for the clinical targeting of otherwise undruggable oncogenes. However RNAi can have off-target effects that considerably increase treatment risks. To manage these side effects and allow an easy subtraction of their activity in healthy tissues, we present here the TAG-RNAi approach where cells that are not designated targets do not have the mRNA tag. Using TAG-RNAi we first established the off-target signatures of three different siRNAs specific to the Cyclin D1 oncogene by RNA-sequencing of cultured cancer cells expressing a FLAG-HA-tagged-Cyclin D1. Then, by symmetrical allografts of tagged-cancer cells and untagged controls on the left and right flanks of model mice, we demonstrate that TAG-RNAi is a reliable approach to study the functional impact of any oncogene without off-target bias. Finally we show, as examples, that mutation-specific TAG-RNAi can be applied to downregulate two oncogenic mutants, KRAS-G12V or BRAF-V600E, while sparing the expression of the wild-type proteins. TAG-RNAi will thus avoid the traditional off-target limitations of RNAi in future experimental approaches.

Purpose: The aim of this study was evaluating the cytotoxic and radiosensitizing effects of Ursolic Acid (UA) and Kamolonol Acetate (KA) on HCT116 cell line and finally investigating the functional role of NF-κB and CCND1 genes in the radiosensitizing activity of UA and KA., Materials and Method: The cytotoxic effects of UA and KA by MTT assay was evaluated on HCT-116. Clonogenic assay was performed to investigate of radiosensitizing effects of UA and KA on HCT116. To assessment the expression levels of NF-κB and CCND1 genes, real-time PCR method was used., Results: The results of MTT assay revealed that UA and KA have cytotoxic effects on HCT116 cell line. According to clonogenic assay, survival fraction of treated cells with UA and KA has been decreased compared to the survival fraction of untreated cells. UA and KA lead to the decrease in the expression level of NF-κB. Synergistic effect of radiosensitizing agents with radiation was only approved for UA and 2 Gy of radiation., Conclusion: Based on our study, UA and KA have cytotoxic effects on HCT116 cell line. Furthermore, UA may lead to radiosensitization of human colorectal tumor cells by NF-κB1 and CCND1signaling pathways., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Differentiation-inducing factor-1 (DIF-1) has been reported to inhibit the proliferation of various mammalian cells by unknown means, although some possible mechanisms of its action have been proposed, including the activation of glycogen synthase kinase-3 (GSK-3). Here, we report an alternative mechanism underlying the action of DIF-1 in human breast cancer cell line MCF-7, on which the effects of DIF-1 have not been examined previously. Intragastric administration of DIF-1 reduced the tumor growth from MCF-7 cells injected into a mammary fat pad of nude mice, without causing adverse effects. In cultured MCF-7, DIF-1 arrested the cell cycle in G 0 /G 1 phase and suppressed cyclin D1 expression, consistent with our previous results obtained in other cell species. However, DIF-1 did not inhibit the phosphorylation of GSK-3. Investigating an alternative mechanism for the reduction of cyclin D1, we found that DIF-1 reduced the protein levels of signal transducer and activator of transcription 3 (STAT3). The STAT3 inhibitor S3I-201 suppressed cyclin D1 expression and cell proliferation and the overexpression of STAT3 enhanced cyclin D1 expression and accelerated proliferation. Differentiation-inducing factor-1 did not reduce STAT3 mRNA or reduce STAT3 protein in the presence of cycloheximide, suggesting that DIF-1 inhibited STAT3 protein synthesis. Seeking its mechanism, we revealed that DIF-1 inhibited the activation of 70 kDa and/or 85 kDa ribosomal protein S6 kinase (p70 S6K /p85 S6K ). Inhibition of p70 S6K /p85 S6K by rapamycin also reduced the expressions of STAT3 and cyclin D1. Therefore, DIF-1 suppresses MCF-7 proliferation by inhibiting p70 S6K /p85 S6K activity and STAT3 protein synthesis followed by reduction of cyclin D1 expression., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Emergence of drug-resistant mutations in the course of myeloma cell evolution and subsequent relapse of myeloma appears to be currently inevitable in most patients. To remedy this situation, we are trying to develop therapeutic small guide RNAs (sgRNAs) based on tRNase Z L -utilizing efficacious gene silencing (TRUE gene silencing), an RNA-mediated gene expression control technology. We designed two sets of double heptamer-type sgRNA, which target the human BCL2 mRNA. Both sets of double heptamer-type sgRNA reduced viability of human myeloma cell lines, RPMI-8226 and KMM-1. We also performed a mouse xenograft experiment to examine how the double heptamer-type sgRNA DHa1(BCL2)/DHa2(BCL2) can reduce the growth of KMM-1 cells in vivo. Median survival periods of the sgRNA cohorts were greater than that of the control cohort by 11-43 days. Furthermore, we designed two sets of double heptamer-type sgRNA, which target the human CCND1 mRNA, and both sets synergistically reduced RPMI-8226 cell viability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Osteosarcoma is a highly invasive primary malignancy of bone. Magnolol is biologically active, which shows antitumor effects in a variety of cancer cell lines. However, it has not been elucidated magnolol's effects on human osteosarcoma cells (HOC). This study aimed to determine antitumor activity of magnolol and illustrate the molecular mechanism in HOC. Magnolol showed significant inhibition effect of growth on MG-63 and 143B cells and induced apoptosis and cell cycle arrest at G0/G1. In osteosarcoma cells, magnolol upregulated expressions of proapoptosis proteins and suppressed expressions of antiapoptosis proteins. Additionally, under the pretreatment of pifithrin-a (PFT-a, a p53 inhibitor), the magnolol-induced apoptosis was significantly reversed. The results above indicated that magnolol induces apoptosis in osteosarcoma cells may via G0/G1 phase arrest and p53-mediated mitochondrial pathway., (© 2019 Wiley Periodicals, Inc.)

Cytosine arabinoside (Ara-c) is a pyrimidine anti-metabolite that is capable of interfering with cellular proliferation by inhibiting DNA synthesis. Each inhibitor of cyclin-dependent kinase 4 (INK4) family member has the ability to bind to cyclin-dependent kinase 4 (CDK4) and inhibit the formation of the cell cycle-dependent CDK4/cyclin D1 complex, subsequently leading to cell cycle arrest in the G1/S phase. In this study, the expression of INK4 family genes in kidney cancer and the impact of these genes on patient prognosis were examined. Additionally, the effects of INK4 family genes and Ara-c on cell proliferation and tumor formation and development were examined. Finally, a potential association between Ara-c-induced cell cycle arrest and INK4-associated gene expression was evaluated. An upregulation of INK4 family genes was found to be positively correlated with the prognosis of patients with kidney cancer. Both the INK4 family genes and Ara-c were shown to induce cell cycle arrest and inhibit tumor formation and development. Moreover, Ara-c-induced cell cycle arrest was found to be associated with an Ara-c-induced upregulation of INK4 family gene expression, which ultimately inhibited the formation of the CDK4/cyclin D1 complex. These findings suggested that an upregulation of INK4 family genes has a positive effect on kidney cancer prognosis and can inhibit the formation and development of tumors. Moreover, Ara-c was shown to promote the upregulation of INK4 family genes, at the same time, Ara-c could directly regulate the cell cycle-dependent genes CDK4 and cyclin D1 ( CCND1 ), independent of the INK4 family genes.

The cyclinD1 is an emerging potent therapeutic drug target in the treatment of ovarian cancer. CyclinD1, Cdks phosphorylation regulates cell cycle and controls transcription. For this reason, CyclinD1 have been subject to extensive cell cycle- related research, and consequently various therapeutic inhibitor drugs have been developed to these protein targets. In the present study we identified that the expression levels of Bcl-2, Bax, caspase 8, 9 and cyclinD1 using Q-PCR method in SKOV3 cell lines treated with Isochamanetin. The viability and migratory inhibition ability also studied to know the mode of cell death. Further the expression levels of Bcl-2, caspase8,9, Cytochrome C and CyclinD1 were significantly down regulated in SKOV3 cancer cells treated with isochamanetin, a specific binding molecule to CyclinD1. The therapeutic molecules found by a high through put insilicoscreen of this pocket exhibit cytostatic nature and reduce protein levels of cell cycle. The novel structural site on CyclinD1, which is well conserved and inhibits the SKOV3 cells from G0-G1 to S phase cell cycle progression. The current result suggests that isochamanetin serves as potent binding agent to the cyclinD1.

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), as the novel iron chelator, has been reported to inhibit the tumorigenesis and progression of various cancer cells. However, whether Dp44mT has anticancer effects in colon cancer cells is still unknown. Here, we investigated the antitumor action of Dp44mT in colon cancer and its underlying mechanisms, and the connections between Dp44mT and N-myc downstream-regulated genes 1(Ndrg1). We used cell viability, migration and invasion assay, flow cytometry, western blot and qRT-PCR to examine the anticancer effects of Dp44mT and Ndrg1. We found that Dp44mT suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells and over-expression of Ndrg1 also suppressed cell viability, migration, invasion and induced apoptosis of colon cancer cells. Dp44mT attenuated the TGF-β1-induced EMT in colon cancer cells, and Dp44mT could up-regulate Ndrg1 expression level. Overexpression of Ndrg1 attenuates the TGF-β1-induced EMT, Dp44mT and Ndrg1 suppressed EMT through activation of Wnt/β catenin signaling pathway. In conclusion, our data demonstrated that Dp44mT/Ndrg1 have effective anticancer capability in colon cancer cells and that may represent a promising treatment strategy for human colon cancer., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Caroli disease represents a hepatic manifestation of autosomal recessive polycystic kidney disease, and belongs to a class of cholangiociliopathies. The role of Hedgehog signaling, a major pathway regulated by primary cilia, in biliary cystogenesis in Caroli disease remains unknown. Using the polycystic kidney (PCK) rat as an animal model of Caroli disease, this study investigated the involvement of Hedgehog signaling in its pathogenesis. In vitro experiments revealed that PCK cholangiocytes overexpressed Smoothened, Gli1, and Gli1's target molecule cyclin D1. The nuclear expression of Gli1, Gli2, and Gli3 was observed in PCK cholangiocytes by immunocytochemistry. An immunohistochemical analysis using liver sections confirmed the overexpression of Smoothened and cyclin D1, and the nuclear expression of the Gli proteins in the biliary epithelium of PCK rats as well as human Caroli disease. The treatment of PCK cholangiocytes with cyclopamine inhibited cell proliferative activity that was associated with the inhibition of nuclear translocation of Gli1 and Gli2, and reduced cyclin D1 expression. The in vivo administration of cyclopamine to PCK rats decreased abnormally elevated serum liver enzymes, and significantly attenuated bile duct dilation as well as kidney cyst formation. These results suggest that cholangiocyte hyperproliferation is causally associated with the aberrant activation of Hedgehog signaling, and the inhibition of the signaling has potential as a therapeutic strategy for biliary cystogenesis in Caroli disease., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Steroid sulfatase is detectable in most hormone-dependent breast cancers. STX64, an STS inhibitor, induced tumor reduction in animal assay. Despite success in phase І clinical trial, the results of phase II trial were not that significant. Breast Cancer epithelial cells (MCF-7 and T47D) were treated with two STS inhibitors (STX64 and EM1913). Cell proliferation, cell cycle, and the concentrations of estradiol and 5α-dihydrotestosterone were measured to determine the endocrinological mechanism of sulfatase inhibition. Comparisons were made with inhibitions of reductive 17β-hydroxysteroid dehydrogenases (17β-HSDs). Proliferation studies showed that DNA synthesis in cancer cells was modestly decreased (approximately 20%), accompanied by an up to 6.5% in cells in the G0/G1 phase and cyclin D1 expression reduction. The concentrations of estradiol and 5α-dihydrotestosterone were decreased by 26% and 3% respectively. However, supplementation of 5α-dihydrotestosterone produced a significant increase (approximately 35.6%) in the anti-proliferative effect of sulfatase inhibition. This study has clarified sex-hormone control by sulfatase in BC, suggesting that the different roles of estradiol and 5α-dihydrotestosterone can lead to a reduction in the effect of sulfatase inhibition when compared with 17β-HSD7 inhibition. This suggests that combined treatment of sulfatase inhibitors with 17β-HSD inhibitors such as the type7 inhibitor could hold promise for hormone-dependent breast cancer., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cell cycle activation has been associated with varying types of neurological disorders including brain injury. Cyclin D1 is a critical modulator of cell cycle activation and upregulation of Cyclin D1 in neurons contributes to the pathology associated with traumatic brain injury (TBI). Mitochondrial mass is a critical factor to maintain the mitochondrial function, and it can be regulated by different signaling cascades and transcription factors including NRF1. However, the underlying mechanism of how TBI leads to impairment of mitochondrial mass following TBI remains obscure. Our results indicate that augmentation of CyclinD1 attenuates mitochondrial mass formation following TBI. To elucidate the molecular mechanism, we found that Cyclin D1 interacts with a transcription factor NRF1 in the nucleus and prevents NRF1's interaction with p300 in the pericontusional cortex following TBI. As a result, the acetylation level of NRF1 was decreased, and its transcriptional activity was attenuated. This event leads to a loss of mitochondrial mass in the pericontusional cortex following TBI. Intranasal delivery of Cyclin D1 RNAi immediately after TBI rescues transcriptional activation of NRF1 and recovers mitochondrial mass after TBI., (Published by Elsevier Inc.)

The antigene strategy based on site-specific recognition of duplex DNA by triplex DNA formation has been exploited in a wide range of biological activities. However, specific triplex formation is mostly restricted to homo-purine strands within the target duplex DNA, due to the destabilizing effect of CG and TA inversion sites where there is an absence of natural nucleotides that can recognize the CG and TA base pairs. Hence, the design of artificial nucleosides, which can selectively recognize these inversion sites with high affinity, should be of great significance. Recently, we determined that 2-amino-3-methylpyridinyl pseudo-dC (3MeAP-ΨdC) possessed significant affinity and selectivity toward a CG inversion site and showed effective inhibition of gene expression. We now describe the design and synthesis of new modified aminopyridine derivatives by focusing on small chemical modification of the aminopyridine unit to tune and enhance the selectivity and affinity toward CG inversion sites. Remarkably, we have newly found that 2-amino-4-methoxypyridinyl pseudo-dC (4OMeAP-ΨdC) could selectively recognize the CG base pair in all four adjacent base pairs and form a stable triplex structure against the promoter sequence of the human gene including multiple CG inversion sites.

This study focuses on the protective effect of epigenetic silencing of CyclinD1 against spinal cord injury (SCI) using bone marrow-derived mesenchymal stem cells (BMSCs) in rats. Eighty-eight adult female Wistar rats were randomly assigned into the sham group, the control group, the si-CyclinD1 + BMSCs group and the BMSCs group. CyclinD1 protein and mRNA expressions after siRNA transfection were detected by Western blotting and qRT-PCR. The siRNA-CyclinD1 BMSCs were transplanted into rats in the si-CyclinD1 + BMSCs group using stereotaxic method 6 hr after SCI. Hindlimb locomotor performance was determined using inclined plane test and Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Expressions of glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) were detected by immunohistochemistry. Inclined plane and BBB scores in the control, si-CyclinD1 + BMSCs, and BMSCs groups were significantly lower than the sham group, but these scores were evidently decreased in the control group and increased in the si-CyclinD1 + BMSCs group compared with the BMSCs group. The repair degree of spinal cord tissues of rats in the si-CyclinD1 + BMSCs group was obvious than the BMSCs group. GFAP and NGF protein expressions were markedly decreased in the control, si-CyclinD1 + BMSCs and BMSCs groups when compared with the sham group. GFAP- and NGF-positive cells were significantly increased in the si-CyclinD1 + BMSCs group while decreased in the control group. Our study provides evidence that epigenetic silencing of CyclinD1 using BMSCs might accelerate the repair of SCI in rats., (© 2017 Wiley Periodicals, Inc.)

Immunoediting caused by antitumor immunity drives tumor cells to acquire refractory phenotypes. We demonstrated previously that tumor antigen-specific T cells edit these cells such that they become resistant to CTL killing and enrich NANOG high cancer stem cell-like cells. In this study, we show that synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, is overexpressed in immunoedited cells and upregulates NANOG by hyperactivating the cyclin D1-CDK4/6 axis. The SCP3-cyclin D1-CDK4/6 axis was preserved across various types of human cancer and correlated negatively with progression-free survival of cervical cancer patients. Targeting CDK4/6 with the inhibitor palbociclib reversed multiaggressive phenotypes of SCP3 high immunoedited tumor cells and led to long-term control of the disease. Collectively, our findings establish a firm molecular link of multiaggressiveness among SCP3, NANOG, cyclin D1, and CDK4/6 and identify CDK4/6 inhibitors as actionable drugs for controlling SCP3 high immune-refractory cancer. Significance: These findings reveal cyclin D1-CDK4/6 inhibition as an effective strategy for controlling SCP3 high immune-refractroy cancer. Cancer Res; 78(10); 2638-53. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Objective: To investigate the effects of miR-519d on the 5-fluorouracil resistance in colorectal cancer cells and to explore the mechanism., Materials and Methods: Colorectal cancer cells HCT116 and SW480 were transfected with miR519d-mimic or siCCND1 by transient transfection. The sensitivity of cells to 5-Fu was assayed by MTT assay. Dual luciferase assay was used to examine the effect of CCND1 on the sensitivity of cells to 5-Fu mediated by miR-519d., Results: MiR-519d was overexpressed in colorectal cancer cells after transient transfection with miR-519d mimics. Overexpression of miR-519d increased the sensitivity of colorectal cancer cells to 5-Fu. MiR-519d negatively regulated the expression of CCND1 via directly bound to CCND1 3`UTR. si-CCND1 could downregulate the CCND1 expression in colorectal cancer HCT116 and SW480 cells. si-CCND1 increased the sensitivity of colorectal cancer cells to 5-Fu., Conclusions: miR-519d inhibits the expression of CCND1 and then plays a role in alleviating 5-Fu resistance in colorectal cancer cells.

The ubiquitin-proteasome system (UPS) participates in both physiological and pathological processes through the posttranslational regulation of intracellular signal transduction pathways. F-box and WD-40 domain protein 11 (Fbxw11) is a component of the SCF (Skp1-Cul1-F-box) E3 ubiquitin ligase complex. Fbxw11 regulates various signal transduction pathways, and it may have pathological roles in tumorigenesis. However, the role of Fbxw11 in the development of leukemia and the underlying mechanisms remain largely unknown. In this study, Fbxw11 expression was aberrantly upregulated in patients with lymphocytic leukemia. Its expression was dramatically decreased in patients who achieved complete remission (CR) after chemotherapy. The high level of Fbxw11 expression in L1210 lymphocytic leukemia cells stimulated cell proliferation in vitro and tumor formation in vivo. The effects were mediated by the stimulation of cell cycle progression rather than the induction of apoptosis. Furthermore, a bioinformatics analysis suggested concomitant activation of the NF-κB and β-catenin/TCF signaling pathways, which were confirmed by reporter gene assays. Moreover, blocking experiments suggested the involvement of both pathways in the growth-promoting effects of Fbxw11. Our results reveal the role of Fbxw11 in lymphocytic leukemia cells and imply that Fbxw11 may serve as a potential molecular target for the treatment of lymphocytic leukemia.

Purpose: The present study aimed to investigate the molecular mechanisms underlying the anticancer properties of ginger extract (GE) in mice bearing solid Ehrlich carcinoma (SEC) and to evaluate the use of GE in combination with doxorubicin (DOX) as a complementary therapy against SEC., Methods: SEC was induced in 60 female mice. Mice were divided into four equal groups: SEC, GE, DOX and GE + DOX. GE (100 mg/kg orally day after day) and DOX (4 mg/kg i.p. for 4 cycles every 5 days) were given to mice starting on day 12 of inoculation. On the 28th day, blood samples were collected, mice were scarified, tumor volume was measured, and tumor tissues were excised., Results: The anti-cancer effect of GE was mediated by activation of adenosine monophosphate protein kinase (AMPK) and down-regulation of cyclin D1 gene expression. GE also showed pro-apoptotic properties as evidenced by elevation of the P53 and suppression of nuclear factor-kappa B (NF-κB) content in tumor tissue. Co-administration of GE alongside DOX markedly increased survival rate, decreased tumor volume, and increased the level of phosphorylated AMPK (PAMPK) and improved related pathways compared to DOX group. In addition, the histopathological results demonstrated enhanced apoptosis and absence of multinucleated cells in tumor tissue of GE + DOX group., Conclusion: AMPK pathway and cyclin D1 gene expression could be a molecular therapeutic target for the anticancer effect of GE in mice bearing SEC. Combining GE and DOX revealed a greater efficacy as anticancer therapeutic regimen.

Autophagy is a catabolic process for degrading dysfunctional proteins and organelles, and closely associated with cancer cell survival under therapeutic, metabolic stress, hypoxia, starvation and lack of growth factors, contributing to resistance to therapies. However, the role of autophagy in breast cancer cells is not well understood. In the present study, we investigated the role of autophagy in highly aggressive and metastatic triple negative breast cancer (TNBC) and non-metastatic breast cancer cells and demonstrated that the knockdown of autophagy-related genes (LC3 and Beclin-1) inhibited autophagy and significantly suppressed cell proliferation, colony formation, migration/invasion and induced apoptosis in MDA-MB-231 and BT-549 TNBC cells. Knockdown of LC3 and Beclin-1 led to inhibition of multiple proto-oncogenic signaling pathways, including cyclin D1, uPAR/integrin-β1/Src, and PARP1. In conclusion, our study suggests that LC3 and Beclin-1 are required for cell proliferation, survival, migration and invasion, and may contribute to tumor growth and progression of highly aggressive and metastatic TNBC cells and therapeutic targeting of autophagy genes may be a potential therapeutic strategy for TNBC in breast cancer.

Concurrent radio chemotherapy treatment prolongs the survival rate of patients with advanced cervical cancer; however, it has adverse side‑effects. β‑elemene, an active component of the traditional Chinese medicinal herb Curcuma zedoaria, is a promising alternative therapeutic drug for the treatment of advanced cervical cancer. The aim of the present study was to investigate the antitumor effects of β‑elemene in human cervical cancer SiHa cells and to determine its underlying therapeutic molecular mechanisms. Cell viability, cell cycle progression and apoptosis were detected using an MTT assay and flow cytometry analysis. Furthermore, the levels of cell migration and cell invasion were investigated using Transwell and wound healing assays. The expression levels of Cyclin‑dependent kinase inhibitor 2B (P15), Cyclin D1, cellular tumor antigen p53, apoptosis regulator Bcl‑2 (Bcl‑2), apoptosis regulator BAX (Bax), 72 kDa type IV collagenase (MMP‑2), matrix metalloproteinase‑9 (MMP‑9), β‑catenin, transcription factor 7 (TCF7), and Myc proto‑oncogene protein (c‑Myc) were analyzed via western blotting. The results revealed that β‑elemene inhibited the proliferation of SiHa cells in a dose and time‑dependent manner. Administration of β‑elemene induced G1 phase cell‑cycle arrest, as demonstrated by the upregulation of P15 expression and the downregulation of Cyclin D1 expression. Furthermore, the present study revealed that β‑elemene induced apoptosis in SiHa cells by enhancing the expression of p53 and Bax, and suppressing the expression of Bcl‑2. In addition, treatment with β‑elemene inhibited cell migration and invasion via downregulation of MMP‑2 and MMP‑9 expression levels. Western blotting demonstrated that β‑elemene reduced the expression levels of β‑catenin and its downstream target molecule TCF7, thus resulting in reduced levels of their target proteins, including c‑Myc, Cyclin D1, Bax and MMP‑2 in cervical cancer cells. The results of the present study suggested that β‑elemene may inhibit cell proliferation and invasion, in addition to inducing apoptosis, via attenuation of the Wnt/β‑catenin signaling pathway in cervical cancer cells.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G 0 /G 1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.

The compound 29-(4-methylpiperazine)-luepol (M22), a novel derivative of lupeol has shown anti-proliferative effects against the human non-small cell lung cancer A549 cell line. M22 showed significant anti-proliferative activity at 6.80 μM and increased accumulation of G1 cells and effectively suppressed expression of the G1 arrest-related genes cyclins D1 and E1, CDK2 and CDC25A. This was further confirmed by Western blotting demonstrating decreased cyclin D1 and CDC25A protein levels. Furthermore, M22 caused induction of apoptosis that downregulated the anti-apoptotic BCL-2 gene and increased expression of BAX, CASP3 and CASP9 as well as the APAF1 gene. The effect of caspase-induced apoptosis was confirmed by an increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP). Taken together, our findings indicated that M22 possessed potent anti-proliferative and apoptotic activities.

Background/aims: The long noncoding RNA homeobox (HOX) transcript antisense intergenic RNA (HOTAIR) has been demonstrated to be a vital modulator in the proliferation and metastasis of ovarian cancer cells, but its potential molecular mechanism remains to be elucidated. In the current study, we aimed to uncover the biological role of lncRNA HOTAIR and its underlying regulatory mechanism in the progression and metastasis of ovarian cancer., Methods: HOTAIR expression was detected by quantitative RT-PCR (qRT-PCR) and northern blotting. The SKOV3 ovarian cancer cell line was chosen for the subsequent assays. In addition, the molecular mRNA and protein expression levels were examined by qRT-PCR and western blotting. The competitive endogenous RNA (ceRNA) mechanism was validated by bioinformatics analysis and a dual luciferase reporter gene assay., Results: HOTAIR expression was significantly higher in ovarian carcinoma tissues and cell lines than in the control counterparts. Both CCND1 and CCND2 were downstream targets of miR-206. The inhibition of HOTAIR elevated the expression of miR-206 and inhibited the expression of CCND1 and CCND2. Moreover, CCND1 and CCND2 were highly expressed in ovarian cancer tissues, and their expression was positively correlated with HOTAIR expression. Finally, the functional assays indicated that the anticancer effects of miR-206 could be rescued by the simultaneous overexpression of either CCND1 or CCND2 in ovarian cancer., Conclusion: HOTAIR enhanced CCND1 and CCND2 expression by negatively modulating miR-206 expression and stimulating the proliferation, cell cycle progression, migration and invasion of ovarian cancer cells., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Antimicrobial textile products are developing rapidly as an important part of functional textiles. Silver nanoparticles (AgNPs) are nanotechnology products with antimicrobial properties. However, exposure to nanoparticles in daily life is an important issue for public health, still being updated. Aim was to evaluate the effects of AgNPs on the development of zebrafish embryos focusing on Wnt pathway, proliferation, oxidant-antioxidant status, and apoptosis. The expressions of ccnd1 and gsk3β were determined by RT-PCR, whereas β-catenin and proliferative cell antigen (PCNA) expressions were determined immunohistochemically. Lipid peroxidation, superoxide dismutase, and glutathione-S-transferase activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Oxidant status, apoptosis, immunohistochemical PCNA, and β catenin staining increased, whereas ccnd1 and antioxidant enzyme activities decreased in AgNPs-exposed embryos in a dose-dependent manner. Our results indicate the interaction of possible mechanisms that may be responsible for the toxic effects of AgNPs in zebrafish embryos., (© 2017 Wiley Periodicals, Inc.)

A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A growing body of studies has documented the pathological influence of impaired alternative splicing (AS) events on numerous diseases, including cancer. In addition, the generation of alternatively spliced isoforms is frequently noted to result in drug resistance in many cancer therapies. To gain comprehensive insights into the impacts of AS events on cancer biology and therapeutic developments, this paper highlights recent findings regarding the therapeutic routes of targeting alternative-spliced isoforms and splicing regulators to treatment strategies for distinct cancers., Competing Interests: The authors declare no conflict of interest.

Context: 4-Nerolidylcatechol (4-NRC) has showed antitumor potential through apoptosis. However, its apoptotic mechanisms are still unclear, especially in leukemic cells., Objectives: To evaluate the cytotoxic potential of 4-NRC and its cell death pathways in p53-null K562 leukemic cells., Materials and Methods: Cytotoxicity of 4-NRC (4.17-534.5 μM) over 24 h of exposure was evaluated by MTT assay. 4-NRC-induced apoptosis in K562 cells was investigated by phosphatidylserine (PS) externalization, cell cycle, sub-G1, mitochondrial evaluation, cytochrome c, cyclin D1 and intracellular reactive oxygen species (ROS) levels, and caspase activity analysis., Results: IC 50 values obtained were 11.40, 27.31, 15.93 and 15.70 μM for lymphocytes, K562, HL-60 and Jurkat cells, respectively. In K562 cells, 4-NRC (27 μM) promoted apoptosis as verified by cellular morphological changes, a significant increase in PS externalization and sub-G1 cells. Moreover, it significantly arrested the cells at the G0/G1 phase due to a reduction in cyclin D1 expression. These effects of 4-NRC also significantly promoted a reduction in mitochondrial activity and membrane depolarization, accumulation of cytosolic cytochrome c and ROS overproduction. Additionally, it triggered an increase in caspases -3/7, -8 and -9 activities. When the cells were pretreated with N-acetyl-l-cysteine ROS scavenger, 4-NRC-induced apoptosis was partially blocked, which suggests that it exerts cytotoxicity though not exclusively through ROS-mediated mechanisms., Discussion and Conclusion: 4-NRC has antileukemic properties, inducing apoptosis mediated by mitochondrial-dependent mechanisms with cyclin D1 inhibition. Given that emerging treatment concepts include novel combinations of well-known agents, 4-NRC could offer a promising alternative for chemotherapeutic combinations to maximize tumour suppression.

Myelodysplastic syndromes (MDS) are a group of heterogeneous diseases characterized by poorly formed blood cells. We wanted to elucidate the underlying molecular mechanism to better determine pathogenesis, prognosis, diagnosis, and treatment for patients with MDS. We compared gene expression levels between normal and MDS tissue samples by immunohistochemical analysis. We studied the proliferation, survival, and migration of MDS cells using the EDU assay, colony formation, and transwell assays. We assessed the apoptotic rate and cell cycle status using flow cytometry and Hoechst staining. Finally, we evaluated RNA and protein expressions using polymerase chain reaction and Western blots, respectively. We found that resveratrol suppressed SKM-1 (an advanced MDS cell line) proliferation in a dose-dependent manner. Consistent with this finding, the EDU and colony formation assays also showed that resveratrol inhibited SKM-1 growth. Moreover, flow cytometry and Hoechst 33258 staining demonstrated that resveratrol induced apoptosis and a change in cell cycle status in SKM-1 cells, while the transwell assay showed that resveratrol reduced the migratory ability of SKM-1 cells. Resveratrol also decreased the expression of CCND1 (a gene that encodes the cyclin D1 protein) and increased expressions of KMT2A [lysine (K)-specific methyltransferase 2A] and caspase-3, suggesting that resveratrol exerts its effect by regulating CCND1 in SKM-1 cells. In addition, a combination of resveratrol and the PI3K/AKT inhibitor LY294002 exhibited a stronger inhibitory effect on the SKM-1 cells, compared with resveratrol alone. Our study proved that resveratrol suppresses SKM-1 growth and migration by inhibiting CCND1 expression. This finding provides novel insights into the pathogenesis of MDS and might help develop new diagnosis and treatment for patients with MDS.

Regulation of cell proliferation is necessary for immune responses, tissue repair, and upkeep of organ function to maintain human health. When proliferating cells complete mitosis, a fraction of newly born daughter cells immediately enter the next cell cycle, while the remaining cells in the same population exit to a transient or persistent quiescent state. Whether this choice between two cell-cycle pathways is due to natural variability in mitogen signalling or other underlying causes is unknown. Here we show that human cells make this fundamental cell-cycle entry or exit decision based on competing memories of variable mitogen and stress signals. Rather than erasing their signalling history at cell-cycle checkpoints before mitosis, mother cells transmit DNA damage-induced p53 protein and mitogen-induced cyclin D1 (CCND1) mRNA to newly born daughter cells. After mitosis, the transferred CCND1 mRNA and p53 protein induce variable expression of cyclin D1 and the CDK inhibitor p21 that almost exclusively determines cell-cycle commitment in daughter cells. We find that stoichiometric inhibition of cyclin D1-CDK4 activity by p21 controls the retinoblastoma (Rb) and E2F transcription program in an ultrasensitive manner. Thus, daughter cells control the proliferation-quiescence decision by converting the memories of variable mitogen and stress signals into a competition between cyclin D1 and p21 expression. We propose a cell-cycle control principle based on natural variation, memory and competition that maximizes the health of growing cell populations.

Scope: Boswellia serrata (BS) resin is a popular dietary supplement for joint nourishment. In this study, we investigated the chemopreventive effects of dietary BS extract and its impact of gut microbiota on azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colitis-associated colon cancer in mice., Methods and Results: Male ICR mice were injected with AOM and 2% DSS via drinking water. The mice were fed with 0.25 or 0.5% BS extract, and colonic tissue were collected at 15 weeks. The main effective components of BS supercritical CO 2 extraction were analyzed by LC-MS/MS are boswellic acids. We found that treatment with BS extract significantly reduce the colonic tumor formation. Western blot and histological analysis revealed that dietary BS extract could markedly reduce the inflammation associated protein levels expression. Furthermore, BS extract reduced cell proliferation via inhibiting phosphorylation level of protein kinase B (Akt), glycogen synthase kinase 3β (GSK3β), and downregulation of cyclin D1. In addition, BS extract also altered the composition of gut microbiota by enhancing the proportion of Clostridiales and reducing the percentage of Bacteroidales., Conclusion: In summary, BS extract decreased the protein levels of inflammative enzymes such as inducible nitric oxide synthase and cyclooxygenase-2 in colonic mucosa. It also mediated Akt/GSK3β/cyclin D1 signaling pathway and altered the composition of gut microbiota to alleviate tumor growth. Taken together, this study suggests that BS extract has great potential to suppress colon tumorigenesis., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: Hydroxytyrosol (HT), a polyphenol from olives, is a potential anticancer agent. This study was designed to evaluate the anticancer activity of HT against prostate cancer cells, and the mechanism thereof., Methods and Results: Treatment of LNCaP and C4-2 prostate cancer cells with HT resulted in a dose-dependent inhibition of proliferation. This was in contrast to HT's ineffectiveness against normal prostate epithelial cells RWPE1 and PWLE2, suggesting cancer-cell-specific effect. HT induced G1/S cell cycle arrest, with inhibition of cyclins D1/E and cdk2/4 and induction of inhibitory p21/p27. HT also induced apoptosis, as confirmed by flow cytometry, caspase activation, PARP cleavage, and BAX/Bcl-2 ratio. It inhibited the phosphorylation of Akt/STAT3, and induced cytoplasmic retention of NF-κB, which may explain its observed effects. Finally, HT inhibited androgen receptor (AR) expression and the secretion of AR-responsive prostate-specific antigen., Conclusion: Castration-resistant prostate cancers retain AR signaling and are often marked by activated Akt, NF-κB, and STAT3 signaling. Our results establish a pleiotropic activity of HT against these oncogenic signaling pathways. Combined with its nontoxic effects against normal cells, our results support further testing of HT for prostate cancer therapy.

Cyclin D1 has become a potential target for anti-tumor therapy. Recently, a novel human anti‑cyclin D1 single‑chain variable fragment (AD5) was identified, which demonstrated specific binding activity to cyclin D1 and exhibited anti‑tumor effects. However, the detailed characteristics of AD5 remain unclear. In the present study, the structure and activity of AD5 in the presence of copper II (Cu2+) or iron III (Fe3+) metal ions was investigated by fluorescence spectroscopy, synchronous fluorescence and enzyme‑linked immunosorbent assay. Cu2+ and Fe3+ were able to bind to AD5 and quench the fluorescence intensity of AD5 primarily by static quenching, which slightly altered the conformation of AD5 at temperatures of 293, 298 and 303 K; however, these temperatures demonstrated different effects on the activity of AD5. These results may be of value for the clinical application of anti-cyclin D1 single chain antibodies in the future.

Objectives: Herbal drugs are popularly emerging as complementary and alternative medicines in cancer patients because of their cost effectiveness and minimal side-effects. The extract of Operculina turpethum (OT) is known to have antipyretic, anti-inflammatory and purgative properties. Since it is popularly known have antiinflammatory activity, we investigated its anti-tumor activity on four oral squamous cell carcinoma cell lines (OSCC) namely, (SCC-4, KB, SCC-9 and SCC-25)., Design: Antitumor activities of Operculina turpathum extract (OTE) was investigated by MTT and clonogenic assay, effect on cell cycle and apoptosis induction by Annexin-V/propidium iodide (PI) staining and flow cytometry and invasive potential of the tumor was determined by matrigel assay. The expression of various proteins involved in these mechanisms was analysed by western blotting., Results: OTE specifically inhibited the growth and colony formation of OSCC cells in a dose-dependent manner via inhibiting NF-κB and its downstream target COX-2. It further arrested cell cycle at G0/G1 phase by inhibiting cyclin-D1 and induced early apoptosis by up-regulating P53 in OSCC cells. It also limits the invasion capacity of OSCC cells by up to 55-60%., Conclusions: OTE shows antitumor activities in OSCC cells by inhibiting NF-κB, COX-2 and cyclin D1 and upregulation of p53 expression. It may be developed as a safe and promising alternative chemopreventive/chemotherapeutic agent for oral cancer., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A sub-class of ER-negative breast cancer that is negative for ER, PR and HER2 expression known as triple-negative breast cancer (TNBC) is highly malignant and lacks effective treatment. Recently, it has been reported that an isoform of estrogen receptor-alpha ER-α36 is expressed and plays a critical role in development of TNBC. ER-α36 forms a positive regulatory loop with epidermal growth factor receptor (EGFR), which promotes malignant growth of TNBC cells. Thus, ER-α36 has been proposed as an important target for development of novel drugs for TNBC. In this study, we evaluated the effects of icaritin, a prenylflavonoid derivant purified from Epimedium Genus, on growth of TNBC cells and examined the possible underlying mechanisms. Our study demonstrated that icartin decreased both ER-α36 and EGFR protein expression, and induced apoptosis in TNBC MDA-MB-231 and MDA-MB-453 cells. We also found that icaritin inhibited ER-α36-mediated MAPK/ERK pathway and cyclin D1 induction by estrogen. Our results thus indicated that icaritin has a potential to be developed into a novel therapeutic agent for human TNBC., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers in China, but the underlying molecular mechanism of ESCC is still unclear. Involvement of microRNAs has been demonstrated in cancer initiation and progression. Despite the reported function of miR-503 in several human cancers, its detailed anti-oncogenic role and clinical significance in ESCC remain undefined. In this study, we examined miR-503 expression by qPCR and found the downregulation of miR-503 expression in ESCC tissue relative to adjacent normal tissues. Further investigation in the effect of miR-503 on ESCC cell proliferation, migration, and invasion showed that enhanced expression of miR-503 inhibited ESCC aggressive phenotype and overexpression of CCND1 reversed the effect of miR-503-mediated ESCC cell aggressive phenotype. Our study further identified CCND1 as the target gene of miR-503. Thus, miR-503 functions as a tumor suppressor and has an important role in ESCC by targeting CCND1., (Copyright © 2017 Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China. Production and hosting by Elsevier B.V. All rights reserved.)

PTEN is one of the most frequently mutated genes in human cancers. The frequency of PTEN alterations is particularly high in endometrial carcinomas. Loss of PTEN leads to dysregulation of cell division, and promotes the accumulation of cell cycle complexes such as cyclin D1-CDK4/6, which is an important feature of the tumour phenotype. Cell cycle proteins have been presented as key targets in the treatment of the pathogenesis of cancer, and several CDK inhibitors have been developed as a strategy to generate new anticancer drugs. Palbociclib (PD-332991) specifically inhibits CDK4/6, and it has been approved for use in metastatic breast cancer in combination with letrazole. Here, we used a tamoxifen-inducible Pten knockout mouse model to assess the antitumour effects of cyclin D1 knockout and CDK4/6 inhibition by palbociclib on endometrial tumours. Interestingly, both cyclin D1 deficiency and palbociclib treatment triggered shrinkage of endometrial neoplasias. In addition, palbociclib treatment significantly increased the survival of Pten-deficient mice, and, as expected, had a general effect in reducing tumour cell proliferation. To further analyse the effects of palbociclib on endometrial carcinoma, we established subcutaneous tumours with human endometrial cancer cell lines and primary endometrial cancer xenografts, which allowed us to provide more translational and predictive data. To date, this is the first preclinical study evaluating the response to CDK4/6 inhibition in endometrial malignancies driven by PTEN deficiency, and it reveals an important role of cyclin D-CDK4/6 activity in their development. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Amifostine, 2-(3-aminopropyl) aminoethyl phosphorothioate, is a broad-spectrum cytoprotective agent used to treat nuclear radiation and chemical weapon injuries. Recently, amifostine has been shown to have a profound biological influence on tumor cells. To examine the effects and mechanisms underlying the effects of amifostine on human acute megakaryocytic leukemia, we evaluated the efficacy of amifostine against Dami cells and observed a cell cycle arrest in G 2 /M phase. Amifostine treatment also induced cell apoptosis of Dami cells which corresponds to formal studies. Through whole-genome microarray and bioinformatics analyses, we found that amifostine affected the gene expression of CCND1, BCL2, and CASP3 which revealed the mechanism amifostine acted on Dami cells. Thus, CCND1-BCL2 Gene Network is predicted to be a direct target of amifostine treating human acute megakaryocytic leukemia, which may provide a novel potential target for the therapy of several subtypes of human AML., (© 2016 John Wiley & Sons A/S.)

USP2a is a deubiquitinase responsible for stabilization of cyclin D1, a crucial regulator of cell-cycle progression and a proto-oncoprotein overexpressed in numerous cancer types. Here we report that lithocholic acid (LCA) derivatives are inhibitors of USP proteins, including USP2a. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3β-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

It has been shown that hypoxia stimulation promotes chondrocytes autophagy partly through HIF-1α, miR-146a and Bcl-2 progressively, and this mechanism represented the connection among hypoxia, miR-146a and autophagy, and provides a possible therapeutic strategy for osteoarthritis. However, the interaction between miR-146a and Bcl-2 is still unclear. Here in a hypoxic environment, we quantified the three reported miR-146a targets: two inflammation related targets Traf6, IRAK1; and the only reported target in chondrocytes Smad4. We confirmed the regulative function of miR-146a between hypoxia and these genes, and explored the Bcl-2 expression and autophagy level under extrinsic up-regulation of these three gene separately. All the three genes were down-regulated by hypoxia. Surprisingly, Traf6 and IRAK, but not the unique Smad4 in chondrocytes, were restored by antagomiR-146a. Both Ad-Traf6 and Ad-IRAK1 reinstated hypoxia or miR-146a repressed Bcl-2. However, Ad-Smad4 did not affect Bcl-2 in hypoxia or normoxia. The autophagy level showed a reverse variability compared to Bcl-2. Taken together, our results provided evidence that Smad4, the unique reported target for miR-146a in chondrocytes is unusually not involved in the chondrocytes autophagy, while the Traf6 and IRAK1 are the new targets for miR-146a in chondrocytes during autophagy.

Direct co-operation between sensitiser molecules BAD and NOXA in mediating apoptosis suggests that therapeutic agents which sensitise to BAD may complement agents which sensitise to NOXA. Dynamic BH3 profiling is a novel methodology that we have applied to the measurement of complementarity between sensitiser BH3 peptide mimetics and therapeutic agents. Using dynamic BH3 profiling, we show that the agent TG02, which downregulates MCL-1, sensitises to the BCL-2-inhibitory BAD-BH3 peptide, whereas the BCL-2 antagonist ABT-199 sensitises to MCL-1 inhibitory NOXA-BH3 peptide in acute myeloid leukaemia (AML) cells. At the concentrations used, the peptides did not trigger mitochondrial outer membrane permeabilisation in their own right, but primed cells to release Cytochrome C in the presence of an appropriate trigger of a complementary pathway. In KG-1a cells TG02 and ABT-199 synergised to induce apoptosis. In heterogeneous AML patient samples we noted a range of sensitivities to the two agents. Although some individual samples markedly favoured one agent or the other, in the group as a whole the combination of TG02 + ABT-199 was significantly more cytotoxic than either agent individually. We conclude that dynamic NOXA and BAD BH3 profiling is a sensitive methodology for investigating molecular pathways of drug action and complementary mechanisms of chemoresponsiveness.

Human diploid fibroblasts (HDFs) proliferation in culture has been used as a model of aging at the cellular level. Growth arrest is one of the most important mechanisms responsible for replicative senescence. Recent researches have been focusing on the function of vitamin E in modulating cellular signaling and gene expression. Therefore, the aim of this study was to elucidate the effect of palm γ-tocotrienol (vitamin E) in modulating cellular aging through p16 INK4a pathway in HDF cells. Primary culture of senescent HDFs was incubated with 70 μM of palm γ-tocotrienol for 24 hours. Silencing of p16 INK4a was carried out by siRNA transfection. RNA was extracted from the different treatment groups and gene expression analysis was carried out by real-time reverse transcription polymerase chain reaction. Proteins that were regulated by p16 INK4a were determined by western blot technique. The finding of this study showed that p16 INK4a mRNA was overexpressed in senescent HDFs, and hypophosphorylated-pRb and cyclin D1 protein expressions were increased (p < 0.05). However, downregulation of p16 INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions (p < 0.05) by γ-tocotrienol led to modulation of the cell cycle regulation during cellular aging. In conclusion, senescent HDFs showed change in biological process specifically in cell cycle regulation with elevated expression of genes and proteins which may contribute to cell cycle arrest. Palm γ-tocotrienol may delay cellular senescence of HDFs by regulating cell cycle through downregulation of p16 INK4a and hypophosphorylated-pRb and cyclin D1 protein expressions.

Western-style diet (WD) and dysbiosis are known to be associated with colonic inflammation, which contributes to carcinogenesis. Metformin (Met) exerts anti-inflammatory effects to induce AMP-activated protein kinase (AMPK), resulting in suppressed protein synthesis and reduced cell proliferation. Probiotic VSL#3 (V) modifies microbial composition. We investigated the chemopreventive mechanisms of Met and V in WD-induced colitis-associated colon carcinogenesis. Male BALB/c mice were randomly divided into five groups: a control diet (CD) group, WD group, WD+ Met (250mg/kg/day) group, WD+V (1.3 million bacteria/day) group, and WD+Met+V group. All mice were exposed to azoxymethane (10mg/kg) followed by 2% dextran sodium sulfate (DSS) for 7 days. Using HCT-116 human colon cancer cell line, expression of AMPK, extracellular signal-regulated kinase (ERK), cyclin D1, and Bcl-2 was investigated and cell cycle arrest was assessed. WD enhanced the severity of colitis and tumor growth compared with CD. The combination of Met and V significantly ameliorated colitis and tumor growth by inhibiting macrophage infiltration and maintaining epithelial integrity. In vitro assays showed that the combination therapy promoted late apoptosis by inhibiting cyclin D1 and Bcl-2 and activating pro-apoptotic ERK. A combination therapy with Met and V attenuates tumor growth in a mouse model of WD-induced colitic cancer, suggesting that this strategy could be useful for the chemoprevention of colon cancer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The Janus kinase-signal transducers and activators of transcription signaling pathway (JAK/STAT pathway) have displayed a critical role in tumor development and progression in multiple malignancies. Previous studies showed that inhibition of JAK/STAT signaling blocked cell growth and metastasis in cancer cells, however, the antitumor effects of JAK inhibitor AG490 on gallbladder cancer (GBC) have not been reported. Our present study aimed to investigate the effects and associated mechanisms of JAK inhibitor AG490 on cell growth, invasive potential and apoptosis in GBC cells (GBC-SD and SGC-996) indicated by MTT, cell colony formation, Transwell and flow cytometry. As a consequence, we found that JAK2 inhibitor AG490 inhibited cell growth and invasion, and induced cell apoptosis and cycle arrest in GBC-SD and SGC-996 cells. Furthermore, the expression levels of p-JAK2, p-STAT3, VEGFC-/-D and cyclinD1 were downregulated, while p53 expression was upregulated in AG490-treated GBC cells indicated by Western blot assay. Therefore, our findings demonstrate that JAK inhibitor AG490 inhibits growth and invasion of GBC cells via blockade of JAK2/STAT3 signaling and provides the potential therapeutic strategy for the treatment of GBC patients.