[Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic rats].

This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis revealed that there were significant differences in the percentage of S + G2M phase, a value of MTT and the expression rate of PCNA protein in three groups (P <0.01). Sense CyclinD1 eukaryotic expression vectors could have a positive effect on the proliferation of ASMC, however the antisence one have a negative effect, which implicated that CyclinD1 might contribute to the process of airway smooth muscle cell proliferation.

Estrogen receptor-α36 is involved in icaritin induced growth inhibition of triple-negative breast cancer cells.

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.

Glucagon phosphorylates serine 552 of ß-catenin leading to increased expression of cyclin D1 and c-Myc in the isolated rat liver.

In the last 20 years the prevalence of metabolic disorders, in particular type 2 diabetes (T2D), has more than doubled. Recently, a strong link between T2D and cancer, in particularly liver cancer has been reported. However, the mechanism connecting the development of type 2 diabetes and cancer remains unknown. One of the biggest drivers of liver cancer is alterations in the Wnt/ß-catenin pathway. In this study, we aimed to identify the effect of glucagon on ß-catenin in the isolated rat liver. We found glucagon, which is substantially raised in patients with T2D, rapidly phosphorylates ß-catenin on serine 552 that is associated with increased expression of genes cyclin D1 (CCND1) and c-Myc (MYC), which are known to be involved in liver cancer. This finding may explain the increased risk of liver cancer in people with T2D.

Lentivirus­delivered nemo­like kinase small interfering RNA inhibits laryngeal cancer cell proliferation in vitro.

Laryngeal squamous cell carcinoma is the most common form of head and neck squamous cell carcinoma. Multiple approaches have been applied to treat this type of cancer; however, no significant improvement in survival rate has been achieved. In the present study, the role of nemo­like kinase (NLK) in human laryngeal carcinoma Hep­2 cells was investigated. NLK has been identified as an important regulator of cell growth, patterning and cell death in a variety of organisms. Lentivirus­mediated­shRNA was employed to silence endogenous NLK expression. Downregulation of the expression of NLK following lentivirus infection was confirmed using reverse transcription quantitative polymerase chain reaction and western blot analysis. The effects of NLK downregulation on Hep­2 cell proliferation and cell cycle progression were analyzed using an MTT assay and flow cytometry, respectively. Downregulation of NLK also inhibited tumorigenesis and regulated the expression of cell cycle protein expression levels. Therefore, it was hypothesized that NLK is necessary for cell survival and tumorigenesis in laryngeal cancer cells. Furthermore, the absence of NLK may lead to cancer cell death. Collectively, the results of the present study demonstrated that the lentivirus­mediated targeted disruption of NLK may be a promising therapeutic method for the treatment of laryngeal cancer.

Resveratrol regulates the cell viability promoted by 17ß-estradiol or bisphenol A via down-regulation of the cross-talk between estrogen receptor α and insulin growth factor-1 receptor in BG-1 ovarian cancer cells.

Endocrine disrupting chemicals (EDCs) and estrogens appear to promote development of estrogen-dependent cancers, including breast and ovarian carcinomas. In this study, we evaluated the cell viability effect of BPA on BG-1 human ovarian cancer cells, along with the growth inhibitory effect of resveratrol (trans-3,4,5-trihydroxystilbene; RES), a naturally occurring phytoestrogen. In addition, we investigated the underlying mechanism(s) of BPA and RES in regulating the interaction between estrogen receptor alpha (ERα) and insulin-like growth factor-1 receptor (IGF-1R) signals, a non- genomic pathway induced by 17ß-estradiol (E2). BPA induced a significant increase in BG-1 cell growth and up-regulated mRNA levels of ERα and IGF-1R. In parallel with its mRNA level, the protein expression of ERα was induced, and phosphorylated insulin receptor substrate-1 (p-IRS-1), phosphorylated Akt1/2/3, and cyclin D1 were increased by BPA or E2. However, RES effectively reversed the BG-1 cell proliferation induced by E2 or BPA by inversely down-regulating the expressions of ERα, IGF-1R, p-IRS-1, and p-Akt1/2/3, and cyclin D1 at both transcriptional and translational levels. Taken together, these results suggest that RES is a novel candidate for prevention of tumor progression caused by EDCs, including BPA via effective inhibition of the cross-talk of ERα and IGF-1R signaling pathways.

c-jun/AP-1 activation does not affect the antiproliferative activity of phenethyl isothiocyanate, a cruciferous vegetable-derived cancer chemopreventive agent.

Cruciferous vegetable-derived isothiocyanates (ITCs) display potent cancer chemopreventive activity, but also markedly stimulate oncogenic activator protein 1 (AP-1). AP-1 is well known to promote cell survival and proliferation. We examined the impact of AP-1 activation on antiproliferative activity of ITCs, using bladder cancer cells and phenethyl isothiocyanate (PEITC) as models. AP-1 transactivation induced by PEITC was almost completely suppressed by a dominant-negative c-jun (TAM67). However, suppression of AP-1 transactivation did not affect PEITC-induced apoptosis or cell-cycle arrest. Moreover, we previously showed that in response to ITC treatment c-jun was predominantly stimulated among AP-1 family members largely by c-jun N-terminal kinase (JNK) [Food Chem Toxicol 2005; 43: 1373-1380], but neither JNK inhibition nor forced expression of c-jun altered the antiproliferative activity of PEITC. In addition, cyclin D1, which is considered as an AP-1 target gene and promotes cell proliferation, was markedly elevated in PEITC-treated cells. Unexpectedly, neither TAM67 or JNK inhibition, nor forced c-jun expression had a significant impact on cyclin D1 induction by PEITC, indicating that c-jun/AP-1 does not play an important role in cyclin D1 induction by PEITC. In conclusion, despite the known role of c-jun/AP-1 as a stimulator of cell growth and proliferation, our data show that its activation does not diminish the antiproliferative activity of PEITC and is not responsible for cyclin D1 induction by PEITC.

Tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces cell proliferation in normal human bronchial epithelial cells through NFkappaB activation and cyclin D1 up-regulation.

Cigarette smoke contains several carcinogens known to initiate and promote tumorigenesis as well as metastasis. Nicotine is one of the major components of the cigarette smoke and the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific carcinogen. Here, we demonstrated that NNK stimulated cell proliferation in normal human bronchial epithelial cells (NHBE) and small airway epithelial cells (SAEC). Cells exposed to NNK resulted in an increase in the level of cyclin D1 protein (as early as 3-6 h). Increased phosphorylation of the Rb Ser(795) was detected at 6-15 h after NNK treatment and thereby promoted cells entering into the S phase (at 15-21 h). The increased cyclin D1 protein level was induced through activation of the transcription factor, nuclear factor kB (NFkappaB), in the NHBE cells. Treatment of the NHBE cells with PD98059, an ERK1/2 (extracellular signal-regulated protein kinase)-specific inhibitor, specifically suppressed the NNK-induced IkappaBalpha phosphorylation at position 32 of the serine residue, suggesting that the ERK1/2 kinase was involved in the IkappaBalpha phosphorylation induced by NFkappaB activation. To determine whether the NNK-induced NFkappaB activation and cyclin D1 induction were also observed in vivo, A/J mice were treated with NNK (9.1 mg) for 20 weeks and the results showed a significant induction of cyclin D1 and NFkappaB translocation determined by immunoblotting analyses. We further demonstrated that the nicotine acetylcholine receptor (nAchR), which contains the alpha3-subunit, was the major target mediating NNK-induced cyclin D1 expression in the NHBE cells. In summary, our findings demonstrate for the first time that NNK could stimulate normal human bronchial cell proliferation through activation of the NFkappaB, which in turn up-regulated the cyclin D1 expression.