Inhibition of cyclin D1 expression by cyclin D1 shRNAs in human oral squamous cell carcinoma cells is associated with increased cisplatin chemosensitivity

Cyclin D1 is a well-known cell cycle regulator. Recently, its pro-survival function has been revealed in several tumors. Because increasing expression of cyclin D1 is a common event in oral squamous cell carcinoma (OSCC) and has been correlated with cisplatin resistance, we investigated if cyclin D1 inhibition could increase cisplatin chemosensitivity of OSCC. Five cyclin D1 shRNAs were prepared and 3 were selected for subsequent experiments. IC50 values for cisplatin were determined by an MTT assay. Cisplatin-induced apoptosis and cell cycle block were investigated. A tumor transplantation model was generated to examine the cisplatin sensitivity of Tca/cisplatin after in vivo cyclin D1 silencing. The role of nuclear factor-kappaB (NF-kappaB) and cyclin-dependent kinase 4 (CDK4) in cyclin D1-mediated cisplatin resistance was also examined. The most effective shRNA resulted in 84.51% knockdown of the cyclin D1 protein level. After the transfection with the 2 most effective shRNAs, the cisplatin IC50 decreased from 5.88 microg/ml to 1.36 microg/ml and 2.47 microg/ml, although overexpression of cyclin D1 rendered OSCC cells more resistant to cisplatin treatment (IC50 increased from 6.43 microg/ml to 12.24 microg/ml). This decreasing IC50 was correlated with the down-regulation of cisplatin-induced NF-kappaB activity in cyclin D1 knockdown cells, and was independent of CDK4 function. In vivo tumor transplantation models also confirmed a cisplatin-sensitizing effect of cyclin D1 shRNA in OSCC. A TUNEL assay validated the increase in apoptosis as induced by cisplatin in cyclin D1 knockdown cells. Cyclin D1 may be an important target for future therapy in patients with OSCC.     

A potential mechanism for fumonisin B(1)-mediated hepatocarcinogenesis: cyclin D1 stabilization associated with activation of Akt and inhibition of GSK-3beta activity

Fumonisin B(1) (FB(1)) is a worldwide corn contaminant and has been epidemiologically linked to the high incidence of human esophageal cancer in South Africa and China. FB(1) is hepatocarcinogenic in rats by an unknown mechanism. Inhibition of ceramide synthase and disruption of membrane phospholipids have been shown to be mechanisms of toxicity. Here we show overexpression of cyclin D1 protein in both preneoplastic and neoplastic liver specimens obtained from a long-term feeding study of FB(1) in rats. In rats fed FB(1) short-term, cyclin D1 protein levels in liver were increased up to five-fold in a dose-responsive manner. Northern blot analysis demonstrated no increase in mRNA levels of cyclin D1. 2D electrophoresis of cyclin D1 protein in FB(1)-treated samples showed a distinct pattern of migration (presence of less negatively charged form of the protein) that differed from controls. Recently, it has been shown that phosphorylation of cyclin D1 by glycogen synthase kinase 3beta (GSK-3beta) on a single threonine residue (Thr-286) positively regulates proteosomal degradation of cyclin D1. In FB(1)-treated samples we detected GSK-3beta phosphorylated on serine 9; activated protein kinase B (Akt) appears to be responsible for this activity-inhibiting phosphorylation. These findings suggest that overexpression of cyclin D1 results from stabilization due to a lack of phosphorylation mediated by GSK-3beta. We also observed an increase in cyclin dependent kinase 4 (Cdk4) complexes with cyclin D1 in FB(1)-treated samples; additionally, elevated Cdk4 activity was shown by increased phosphorylation of the retinoblastoma protein. In summary, the activation of Akt leads to increased survival, inhibition of GSK-3beta activity and post-translational stabilization of cyclin D1, all events responsible for disruption of the cell cycle G(1)/S restriction point in hepatocytes. This is the first report suggesting the mechanism by which FB(1) acts as a carcinogen.     

Non-genomic action of resveratrol on androgen and oestrogen receptors in prostate cancer: modulation of the phosphoinositide 3-kinase pathway

Prostate cancer represents a major concern in human oncology and the phytoalexin resveratrol (RES) inhibits growth and proliferation of prostate cancer cells through the induction of apoptosis. In addition, previous data indicate that in oestrogen-responsive human breast cancer cells, RES induces apoptosis by inhibition of the phosphoinositide-3-kinase (PI3K) pathway. Here, using androgen receptor (AR)-positive LNCaP and oestrogen receptor alpha (ERalpha)-expressing PC-3 prostate tumour cells, we have analysed whether the antiproliferative activity of RES takes place by inhibition of the AR- or ERalpha-dependent PI3K pathway. Although RES treatment (up to 150 microM) decreased AR and ERalpha protein levels, it did not affect AR and ERalpha interaction with p85-PI3K. Immunoprecipitation and kinase assays showed that RES inhibited AR- and ERalpha-dependent PI3K activities in LNCaP and PC-3, respectively. Consistently, lower PI3K activities correlated with decreased phosphorylation of downstream targets protein kinase B/AKT (PKB/AKT) and glycogen synthase kinase-3 (GSK-3). GSK-3 dephosphorylation could be responsible for the decreased cyclin D1 levels observed in both cell lines. Importantly, RES markedly decreased PKB/AKT phosphorylation in primary cultures from human prostate tumours, suggesting that the mechanism proposed here could take place in vivo. Thus, RES could have antitumoral activity in androgen-sensitive and androgen-non-sensitive human prostate tumours by inhibiting survival pathways such as that mediated by PI3K.     

Fangchinoline Inhibits Cell Proliferation Via Akt/GSK-3beta/cyclin D1 Signaling and Induces Apoptosis in MDA-MB-231 Breast Cancer Cells

Fangchinoline (Fan) inhibits cell proliferation and induces apoptosis in several cancer cell lines. The effectsof Fan on cell growth and proliferation in breast cancer cells remain to be elucidated. Here, we show that Faninhibited cell proliferation in the MDA-MB-231 breast cancer cell line through suppression of the AKT/Gsk-3beta/cyclin D1 signaling pathway. Furthermore, Fan induced apoptosis by increasing the expression of Bax(relative to Bcl-2), active caspase 3 and cytochrome-c. Fan significantly inhibited cell proliferation of MDAMB-231 cells in a concentration and time dependent manner as determined by MTT assay. Flow cytometryanalysis demonstrated that Fan treatment of MDA-MB-231 cells resulted in cell cycle arrest at the G1 phase,which correlated with apparent downregulation of both mRNA and protein levels of both PCNA and cyclin D1.Further analysis demonstrated that Fan decreased the phosphorylation of AKT and GSK-3beta. In addition, Fanup-regulated active caspase3, cytochrome-c protein levels and the ratio of Bax/Bcl-2, accompanied by apoptosis.Taken together, these results suggest that Fan is a potential natural product for the treatment of breast cancer.     

Delayed mammary gland involution in MMTV-AKT1 transgenic mice.

AKT1/protein kinase Balpha is a protein-serine/threonine kinase that regulates multiple targets involved in cell survival and cell cycle progression in a variety of cell types including breast cancer cells. To explore the role of Akt1 in mammary gland function and tumorigenesis, transgenic mice were generated that express human AKT1 under the control of the MMTV promoter. Virgin transgenic mice did not exhibit a dominant phenotype, but upon cessation of lactation, a notable delay in involution occurred compared to age-matched non-transgenic mice. The delay in involution coincided with increased hyperplasia as evidenced by an increased number of binucleated epithelial cells and a marked elevation in cyclin D1 expression in mammary epithelium. The delayed involution phenotype corresponded to increased phosphorylation of Thr308 in AKT1 and Ser136 in BAD, but not phosphorylation of Ser21 in GSK-3alpha. There was no evidence of mammary dysplasia or neoplasia during the lifespan of multiparous transgenic mice. These data suggest that AKT1 is involved in cell survival in the lactating and involuting mammary gland, but that overexpression of AKT1 alone is insufficient to induce transformation.     

Degradation of cyclin D3 independent of Thr-283 phosphorylation

Cyclin D3 has been shown to play a major role in the regulation of cell cycle progression in lymphocytes. It is therefore important to understand the mechanisms involved in the regulation of this protein. We have previously shown that both basal and cAMP-induced degradation of cyclin D3 in Reh cells is dependent on Thr-283 phosphorylation by glycogen synthase kinase-3beta (GSK-3beta). We now provide evidence of an alternative mechanism being involved in the regulation of cyclin D3 degradation. Treatment of lymphoid cells with okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A), induces rapid phosphorylation and proteasomal degradation of cyclin D3. This degradation is not inhibited by the GSK-3beta inhibitors lithium or Kenpaullone, or by substitution of Thr-283 with Ala on cyclin D3, indicating that cyclin D3 can be degraded independently of Thr-283 phosphorylation and GSK-3beta activity. Interestingly, in vitro experiments revealed that PP1, but not PP2A, was able to dephosphorylate cyclin D3 efficiently, and PP1 was found to associate with His-tagged cyclin D3. These results support the hypothesis that PP1 constitutively keeps cyclin D3 in a stable, dephosphorylated state, and that treatment of cells with OA leads to phosphorylation and degradation of cyclin D3 through inhibition of PP1.     

Resveratrol causes WAF-1/p21-mediated G(1)-phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinoma A431 cells.

Resveratrol (trans-3,4',5,-trihydroxystilbene), a phytoalexin found in grapes, nuts, fruits, and red wine, is a potent antioxidant with cancer-preventive properties. The mechanism by which resveratrol imparts cancer chemopreventive effects is not clearly defined. Here, we demonstrate that resveratrol, via modulations in cyclin-dependent kinase (cdk) inhibitor-cyclin-cdk machinery, results in a G(1)-phase arrest of the cell cycle followed by apoptosis of human epidermoid carcinoma (A431) cells. Resveratrol treatment (1-50 microM for 24 h) of A431 cells resulted in a dose-dependent (a) inhibition of cell growth as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, (b) G(1)-phase arrest of the cell cycle as shown by DNA cell cycle analysis, and (c) induction of apoptosis as assessed by ELISA. The immunoblot analysis revealed that resveratrol treatment causes a dose- and time-dependent (a) induction of WAF1/p21; (b) decrease in the protein expressions of cyclin D1, cyclin D2, and cyclin E; and (c) decrease in the protein expressions of cdk2, cdk4, and cdk6. Resveratrol treatment was also found to result in a dose- and time-dependent decrease in kinase activities associated with all of the cdks examined. Taken together, our study suggests that resveratrol treatment of the cells causes an induction of WAF1/p21 that inhibits cyclin D1/D2-cdk6, cyclin D1/D2-cdk4, and cyclin E-cdk2 complexes, thereby imposing an artificial checkpoint at the G(1)-->S transition of the cell cycle. This series of events results in a G(1)-phase arrest of the cell cycle, which is an irreversible process that ultimately results in the apoptotic death of cancer cells. To our knowledge, this is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of cancer cells by resveratrol.     

UVA-induced cell cycle progression is mediated by a disintegrin and metalloprotease/epidermal growth factor receptor/AKT/Cyclin D1 pathways in keratinocytes

UVA (315-400 nm), which constitutes approximately 95% of the UV irradiation in natural sunlight, represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here, we show that a low, nonlethal dose of UVA induces dose-dependent cell cycle progression in human HaCaT keratinocytes. We found that UVA induced cyclin D1 accumulation, whereas siRNA knockdown of cyclin D1 blocked the UVA-induced cell cycle progression, indicating that this process is mediated by cyclin D1. UVA irradiation also induced AKT activation; when cells were incubated with phosphatidylinositol-3-OH kinase/AKT inhibitor or infected with dominant-negative AKT, cyclin D1 up-regulation, cell cycle progression, and proliferation were inhibited, suggesting that AKT activation is required for UVA-induced cell cycle progression. In contrast, extracellular signal-regulated kinase (ERK) was not activated by UVA exposure; incubation with ERK/mitogen-activated protein kinase inhibitor had no effect on UVA-induced cyclin D1 up-regulation and cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVA exposure. EGFR kinase inhibitor AG attenuated the UVA-induced AKT/cyclin D1 pathway and cell cycle progression, indicating that EGFR is upstream of AKT/cyclin D1 pathway activation. Furthermore, metalloprotease inhibitor GM6001 blocked UVA-induced cell cycle progression, and siRNA knockdown of a disintegrin and metalloprotease (ADAM)17 had a similar inhibitory effect, demonstrating that ADAM17 mediates the EGFR/AKT/cyclin D1 pathway and cell cycle progression to the S phase induced by UVA radiation. Identification of these signaling pathways in UVA-induced cell proliferation will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer.     

SiRNA特异性抑制cyclin D1的表达及其shRNA表达质粒的构建

目的 研究siRNA （small interfering RNA）对cyclin D1基因表达的抑制作用及shRNA表达质粒的构建.方法 化学合成针对cyclin D1基因的siRNA,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹检测cyclin D1 mRNA和蛋白的表达;合成特异性cyclin D1的RNA干扰寡核苷酸序列,采用克隆技术,将其插人Psilencer2.1-U6质粒表达载体,构建cyclin D1 shRNA（small hairpin RNA,shRNA）表达载体.结果 化学合成的siRNA转染MCF-7细胞,48 h 后cyclin D1基因和蛋白表达都明显降低;构建特异性针对cyclin D1的shRNA 表达质粒,酶切和测序结果显示,siRNA片段已成功插入Psilencer2.1-U6载体,载体构建成功.结论 siRNA可以有效抑制MCF-7细胞株中cyclin D1的表达,并成功构建其shRNA表达质粒.     

An alternatively spliced cyclin D1 isoform, cyclin D1b, is a nuclear oncogene

Glycogen synthase kinase-3beta-dependent phosphorylation of cyclin D1 at a conserved COOH-terminal residue, Thr-286, promotes CRM1-dependent cyclin D1 nuclear export at the G(1)-S boundary. Mutations that perturb the phosphorylation of cyclin D1 at Thr-286 contribute to cell transformation, although to date, no such mutations have been found in human cancers. Cyclin D1 (CCND1) undergoes alternative splicing leading to the production of an mRNA predicted to encode a unique cyclin D1 isoform, cyclin D1b, which lacks Thr-286. We have cloned and expressed cyclin D1b, and find that it retains the ability to bind to and activate CDK4. Unlike canonical cyclin D1a, cyclin D1b remains nuclear through the cell cycle where its constitutive expression facilitates cellular transformation. Using antisera specific for cyclin D1b, the protein was detected in a high percentage of esophageal cancer-derived cell lines and in primary esophageal carcinomas. Therefore, alternative splicing leads to expression of a nuclear, oncogenic cyclin D1 isoform that is expressed in human cancer.     

Identification of mutations that disrupt phosphorylation-dependent nuclear export of cyclin D1

Although cyclin D1 is overexpressed in a significant number of human cancers, overexpression alone is insufficient to promote tumorigenesis. In vitro studies have revealed that inhibition of cyclin D1 nuclear export unmasks its neoplastic potential. Cyclin D1 nuclear export depends upon phosphorylation of a C-terminal residue, threonine 286, (Thr-286) which in turn promotes association with the nuclear exportin, CRM1. Mutation of Thr-286 to a non-phosphorylatable residue results in a constitutively nuclear cyclin D1 protein with significantly increased oncogenic potential. To determine whether cyclin D1 is subject to mutations that inhibit its nuclear export in human cancer, we have sequenced exon 5 of cyclin D1 in primary esophageal carcinoma samples and in cell lines derived from esophageal cancer. Our work reveals that cyclin D1 is subject to mutations in primary human cancer. The mutations identified specifically disrupt phosphorylation of cyclin D1 at Thr-286, thereby enforcing nuclear accumulation of cyclin D1. Through characterization of these mutants, we also define an acidic residue within the C-terminus of cyclin D1 that is necessary for recognition and phosphorylation of cyclin D1 by glycogen synthase kinase-3 beta. Finally, through construction of compound mutants, we demonstrate that cell transformation by the cancer-derived cyclin D1 alleles correlates with their ability to associate with and activate CDK4. Our data reveal that cyclin D1 is subject to mutations in primary human cancer that specifically disrupt phosphorylation-dependent nuclear export of cyclin D1 and suggest that such mutations contribute to the genesis and progression of neoplastic growth.     

Phosphorylation of cyclin D1 at Thr 286 during S phase leads to its proteasomal degradation and allows efficient DNA synthesis

Continuing proliferation requires regulation of cyclin D1 levels in each cell cycle phase. Growth factors stimulate high levels during G2 phase, which commits the cell to continue through G1 phase with sufficient cyclin D1 to initiate DNA synthesis. Upon entry into S phase, however, cyclin D1 levels rapidly decline. Our goal is to understand the mechanism and importance of this S-phase suppression. Here, we demonstrate that cyclin D1 levels decline during S phase due to reduced protein stability, without alterations in the rate of protein synthesis. This decline depends upon Thr 286, since mutation of this site eliminates the normal pattern of cyclin D1 suppression during S phase. As evidence that phosphorylation of Thr 286 is responsible for this decline, Thr 286 is shown to be more efficiently phosphorylated during S phase than in other cell cycle periods. Finally, high cyclin D1 levels during S phase are shown to inhibit DNA synthesis. This inhibitory activity presumably blocks the growth of cells with altered cyclin D1 expression characteristics. Abnormal stimulation of cyclin D1 might result in levels high enough to promote G1/S phase transition even in the absence of appropriate growth stimuli. In such cells, however, the levels of cyclin D1 would presumably be too high to be suppressed during S phase, resulting in the inhibition of DNA synthesis.     

miR-377-5p通过抑制AKT1/GSK-3β通路增强食管癌细胞TE-1的放射敏感性

目的 探究miR-377-5p对食管癌细胞TE-1放射敏感性的影响及机制。方法 TE-1细胞转染miR-377-5pmimic和miR-377-5pmimic NC构建过表达miR-377-5p细胞。对转染后的TE-1细胞照射后采用平板集落形成实验检测细胞放射生物学参数(D0、Dq、SF2),Transwell小室法检测细胞侵袭能力,细胞划痕实验检测细胞迁移能力,CCK8法检测细胞活力,流式细胞术检测细胞凋亡和细胞周期,免疫印迹检测AKT1和GSK-3β磷酸化水平。结果 2、4、6、8Gy照射的集落形成率均显著下降(P<0.05),且在同一剂量下miR-377-5pmimic组细胞集落形成率均显著低于miR-377-5pmimic NC组(P<0.05)。相比于miR-377-5pmimic NC组,miR-377-5pmimic组D0、Dq、SF2均显著下降(P<0.05),放射增敏比为1.34(D0值比);0 Gy照射后细胞侵袭、迁移、增殖能力显著下降,细胞凋亡水平显著上升,细胞周期被阻滞于G1期,AKT1和GSK-3β磷酸化水平显著下降(P<0.05);4 Gy照射后细胞侵袭、迁移、增殖能力下降,细胞凋亡水平显著上升,G1期显著延长,AKT1和GSK-3β磷酸化水平亦显著下降(均P<0.001)。结论 miR-377-5p能够增加食管癌细胞TE-1的放射敏感性,其机制可能是抑制AKT1/GSK-3β信号通路。     

PSAT1 regulates cyclin D1 degradation and sustains proliferation of non‐small cell lung cancer cells

Multiple nodes in the one‐carbon metabolism pathway play important regulatory roles in cancer cell growth and tumorigenesis. The specific biological functions of metabolic enzymes in regulating the signaling pathways that are associated with tumor cell growth and survival, however, remain unclear. Our current study found that phosphoserine aminotransferase 1 (PSAT1), an enzyme catalyzing serine biosynthesis, was significantly up‐regulated in non‐small cell lung cancer (NSCLC) and was involved in the regulation of E2F activity. Loss‐ and gain‐of‐function experiments demonstrated that PSAT1 promoted cell cycle progression, cell proliferation and tumorigenesis. Mechanistic study suggested that elevated PSAT1 led to inhibition of cyclin D1 degradation and subsequently an alteration in Rb‐E2F pathway activity, which in turn enhanced G1 progression and proliferation of NSCLC cells. Moreover, phosphorylation of cyclin D1 at threonine 286 by GSK‐3β was required for PSAT1‐induced blockage of cyclin D1 degradation. We also found that the activity of p70S6K mediated the effects of PSAT1 on GSK‐3β phosphorylation and cyclin D1 degradation. We further identified that PSAT1 was over‐expressed in NSCLC and predicted poor clinical outcome of patients with the disease. Correlation analysis showed that PSAT1 expression positively correlated with the levels of phosphorylated GSK‐3β, cyclin D1 and phosphorylated Rb in NSCLC primary tumors. These findings uncover a mechanism for constitutive activation of E2F via which unrestrained cell cycle progression occurs in NSCLC and may represent a prognostic biomarker and therapeutic target.     

Enhancing cisplatin sensitivity in MCF-7 human breast cancer cells by down-regulation of Bcl-2 and cyclin D1

MCF-7 human breast cancer cells are relatively resistant to cisplatin treatment compared to other breast cancer cell lines. In order to identify possible targets for sensitizing the breast cancer cells to cisplatin treatment protein expression levels and the phosphorylation status of 27 different signaling proteins were examined. MCF-7 cells expressed high levels of anti-apoptotic Bcl-2 protein relative to more cisplatin sensitive breast cancer cells. After cisplatin treatment a decrease in cyclin D1 was seen in all the breast cancer cells studied. Therefore, Bcl-2 and cyclin D1 were chosen as putative targets for increasing cell death and growth arrest induced by cisplatin, thereby enhancing the drug sensitivity in MCF-7. RNA interference, using Bcl-2- and cyclin D1- siRNAs sensitized MCF-7 cells to cisplatin treatment and by simultaneous knockdown of both Bcl-2 and cyclin D1 further sensitization was seen. This shows the potential of targeting both apoptotic- and cell cycle-regulating pathways to enhance the effect of chemotherapy.     

miR-377-5p enhances radiosensitivity of esophageal cancer cell line TE-1 by inhibiting AKT1/GSK-3β signaling pathway

Objective To investigate the effect and mechanism of miR-377-5p on the radiosensitivity of esophageal cancer cell line TE-1. Methods Esophageal cancer cell line TE-1 was transfected with miR-377-5p mimic and miR-377-5p mimic NC to construct the TE-1 cells overexpressing miR-377-5p. After the transfected TE-1 cells were exposed to ionizing radiation, radiobiological parameters(D0,Dq,SF2) were detected by plate colony formation assay. Cell invasion ability was assessed by Transwell chamber assay, Cell migration ability was evaluated by cell scratch assay, Cell viability was assessed by CCK8 assay. Cell apoptosis and cell cycle were detected by flow cytometry, Western blot. The phosphorylation levels of AKT1 and GSK-3βwere measured by Western blot. Results At the doses of 2, 4, 6, and 8 Gy, the colony formation rate in each group was significantly decreased (all P<0.05), and the colony formation rate at the same irradiation dose in the miR-377-5p mimic group was significantly lower than that in the miR-377-5p mimic NC group (P<0.05). Compared with the miR-377-5pmimic NC group, the D0, Dq and SF2 at 2Gy were decreased significantly in the miR-377-5pmimic group (all P<0.05), and the radiosensitization ratio(D0 ratio) was 1.34. After 0 Gy ionizing radiation, the invasion, migration and proliferation abilities of TE-1 cells were significantly decreased, the level of cell apoptosis was significantly increased, the cell cycle was arrested in G1 phase, and the phosphorylation levels of AKT1 and GSK-3β were significantly decreased in the miR-377-5pmimic group (all P<0.05). Following 4 Gy irradiation, cell invasion, migration, proliferation abilities were decreased, the level of cell apoptosis was increased significantly, the G1 phase was significantly extended and the phosphorylation levels of AKT1 and GSK-3β were also decreased significantly in the miR-377-5pmimic group (all P<0.001). Conclusion miR-377-5p can increase the radiosensitivity of esophageal cancer cell line TE-1, which may be due to the inhibition of the AKT1/GSK-3β signaling pathway.