P21 Cip1 Expression Research Articles

Obovatol from Magnolia obovata inhibits vascular smooth muscle cell proliferation and intimal hyperplasia by inducing p21Cip1

AimsObovatol is isolated from Magnolia obovata leaves and this active component has various pharmacological properties such as anti-oxidant, anti-platelet, anti-fungal and anti-inflammatory activities. In the present study, we investigated the inhibitory effects of obovatol on in vitro vascular smooth muscle cell (VSMC) proliferation and in vivo neointimal formation in a rat carotid artery injury model. Methods and resultsObovatol (1–5μM) exerted concentration-dependent inhibition on platelet-derived growth factor (PDGF)-BB-induced rat VSMC proliferation, without exhibiting any cellular toxicity or apoptosis, as determined by cell count, [3H]thymidine incorporation and Annexin-V-binding analyses. Treatment with obovatol blocked the cell cycle in G1 phase by down-regulating the expression of cyclins and CDKs, and selectively up-regulating the expression of p21Cip1, a well-known CDK inhibitor. Effects of perivascular delivery of obovatol were assessed 14 days after injury. The angiographic mean luminal diameters of the obovatol-treated groups (100μg and 1mg: 0.78±0.06 and 0.77±0.07AU, respectively) were significantly larger than that of the control group (0.58±0.07AU). The obovatol-treated groups (100μg and 1mg: 0.14±0.04 and 0.09±0.03mm2, respectively) showed significant reduction in neointimal formation versus the control group (0.17±0.02mm2). Immunohistochemical staining demonstrated strong expression of p21Cip1 in the neointima of the obovatol-treated groups. ConclusionsThese data suggest that obovatol inhibits VSMC proliferation by perturbing cell cycle progression, possibly due to activation of p21Cip1 pathway. These results also show that obovatol may have potential as an anti-proliferative agent for the treatment of restenosis and atherosclerosis.

Connexin-43 Regulates the Cell Cycle Entry of Hematopoietic Stem Cells within the Stem Cell Niche.

Abstract 1500Poster Board I-523Bone marrow (BM) osteoblasts and stromal (O/S) cells are crucial in the establishment of the hematopoietic niches in the BM. Connexin 43 (Cx43) is expressed by BM stromal cells and by hematopoietic stem cells and progenitors (HSC/P) and is overexpressed in the BM endosteal space upon administration of chemotherapy or radiotherapy. We have previously reported that Cx43 is critical in fetal liver and in BM hematopoiesis. Since Cx43 is expressed by both HSC and the hematopoietic microenvironment, we dissected out the cellular mechanisms responsible for Cx43 function in the BM. We analyzed the hematopoiesis of mice deficient in Cx43 in the O/S cells (Collagen 1α-Creflox/flox; O/S-Cx43-deficient) or in the hematopoietic cells (Vav1-Creflox/flox; H-Cx43-deficient). Upon basal conditions, analysis of the HSC compartment of H-Cx43-deficient mice showed a ∼30% decreased content of immunophenotypically defined long-term HSC (LT-HSC) in BM of H-Cx43KO mice compared with their WT littermates, whereas there was not significant variation in the ST-HSC population content. The reduced LT-HSC population in H-Cx43KO mice was associated with a modest increased quiescence (∼12% increase of LT-HSC in G0). Interestingly, the expression of cyclin D1 and p21cip1 in the H-Cx43KO LT-HSC were 50% reduced and 4-fold increased, respectively, suggesting a decreased ability to enter cell cycle. While we found no significant engraftment difference in primary recipients of competitive repopulation assays, we found a marked reduction (>50%) in the engraftment ability of LT-HSC Cx43-deficient cells when transplanted into secondary recipients. When submitted to stress by 5-fluorouracil (5-FU) administration, H-Cx43KO mice showed a severely decreased hematopoietic recovery of peripheral blood (PB) counts for neutrophils and platelets accompanied with a marked reduction in the BM cellularity and hematopoietic progenitor content on day +14 after treatment. This defect was associated with a dramatic decreased (∼75 %) in the proliferation of the Cx43-deficient, LT-HSC population by 48 hours post-5-FU administration and a relative decrease of the expansion of the ST-HSC/MPP pool as early as 6 days post-5-FU administration. Interestingly, O/S-Cx43-deficient mice also showed severely delayed hematological recovery after 5-FU administration, with reduction in cellularity and hematopoietic progenitor content, suggesting that the increased hematopoietic toxicity induced by 5-FU in the context of Cx43 deficiency may depend on HSC-to-O/S Cx43 homotypic communication. This communication would be responsible of control of the G1 restriction checkpoint in LT-HSC. In summary, our findings suggest that Cx43 expression plays a crucial role controlling the LT-HSC pool size and fitness in response to stress. Disclosures:Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

Increased Ratios of Internal Organ Volume to Body Mass in Chuvash Polycythemia and HIF-2α Repression of p21Cip Expression through C-MYC.

Abstract 5039Chuvash polycythemia, the first hereditary disease associated with dysregulated oxygen sensing, is characterized by homozygous germ-line loss-of-function mutation of VHL (598C>T) resulting in elevated hypoxia inducible factor (HIF)-1 and HIF-2 levels, increased red cell mass and propensity to thrombosis. Organ size is determined by the size and number of cells, and its molecular mechanisms are not fully elucidated. Previous work from several groups demonstrated that cell proliferation is regulated in opposite directions by HIF-1α and HIF-2α. HIF-1α inhibits cell proliferation by displacing C-MYC from the promoter of the cyclin-dependent kinase inhibitor, p21Cip, and inducing its expression, while HIF-2α promotes MYC activity and cell proliferation. We determined the volumes of the liver, spleen, and kidneys and the diameter of the head of the pancreas by computerized tomography in 30 individuals with Chuvash polycythemia and 30 age- and sex-matched Chuvash controls and calculated the ratio of organ volume (or diameter) to body mass. The subjects with Chuvash polycythemia had larger ratios of the liver (P<0.01), spleen (P<0.01), kidneys (P<0.05) and head of the pancreas (P=0.05) than Chuvash controls. To test if HIF contributes to this phenotype, we examined Hif1a+/− mice and wild type littermates. We found that Hif1a+/− mice had larger median ratios of liver to body mass (factor of 1.5, P<0.005) and pancreas to body mass (factor of 1.3, P<0.05) than wild type mice. A hepatocyte size analysis revealed that there was no significant difference between Hif1a+/− and wild type mice. To identify how HIF contributes this phenotype, we performed gene expression profiles in the livers of Hif1a+/− and wild type littermates. The profiling results and subsequent individual confirmation showed that compared to wild type mice, Hif1a+/− mice had 2-fold higher HIF-2α mRNA levels (P<0.05) and 9.5-fold lower hepatic p21Cip mRNA levels (P<0.05). Our results showed that relative to body mass, liver, spleen, kidney and pancreas volumes are increased in Chuvash polycythemia. At least in the liver, this phenotype may result from increased HIF-2α and decreased p21Cip1 levels, leading to increased hepatocyte proliferation. DisclosuresNo relevant conflicts of interest to declare.

BMP-4 Induction of Arrest and Differentiation of Osteoblast-Like Cells via p21CIP1and p27KIP1Regulation

Cell cycle regulation by differentiation signals is critical for eukaryote development. We investigated the roles of bone morphogenetic protein (BMP)-4, an important stimulator of osteoblast differentiation and bone formation, in regulating cell cycle distribution in four osteoblast-like cell lines and mouse primary osteoblasts, and the underlying mechanisms. In all cells used, BMP-4 induced G(0)/G(1) arrest. The molecular basis of the BMP-4 effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 cells. BMP-4 induced p21(CIP1) and p27(KIP1) expressions and hence cell differentiation but had no effects on the expressions of cyclins A, B1, D1, and E, cyclin-dependent protein kinase-2, -4, and -6. Using specific small interfering RNA (siRNA), we found that BMP-4-induced G(0)/G(1) arrest, and p21(CIP1) and p27(KIP1) expressions were mediated by BMP receptor type IA (BMPRIA)-specific Sma- and Mad-related protein (Smad)1/5. BMP-4 induced transient phosphorylations of ERK; transfection of MG63 cells with ERK2, but not ERK1, -specific siRNA inhibited the BMP-4-induced responses in MG63 cells. Pretreatment of MG63 cells with Arg-Gly-Asp-Ser, which blocks the cell-extracellular matrix interaction, or transfection with beta(3) integrin-specific siRNA inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. BMP-4 induced transient increases in associations of beta(3)-integrin with focal adhesion kinase and Shc, the dominant-negative mutants of which inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. Our results indicate that BMP-4 induces G(0)/G(1) arrest and hence differentiation in osteoblast-like cells through increased expressions of p21(CIP1) and p27(KIP1), which are mediated by BMPRIA-specific Smad1/5. The extracellular matrix/beta(3) integrin/ focal adhesion kinase/Shc/ERK2 signaling pathway is involved in these BMP-4-induced responses in osteoblast-like cells.

AP-2γ promotes proliferation in breast tumour cells by direct repression of the CDKN1A gene

Overexpression of the activator protein (AP)-2gamma transcription factor in breast tumours has been identified as an independent predictor of poor outcome and failure of hormone therapy. To understand further the function of AP-2gamma in breast carcinoma, we have used an RNA interference and gene expression profiling strategy with the MCF-7 cell line as a model. Gene expression changes between control and silenced cells implicate AP-2gamma in the control of cell cycle progression and developmental signalling. A function for AP-2gamma in cell cycle control was verified using flow cytometry: AP-2gamma silencing led to a partial G1/S arrest and induction of the cyclin-dependent kinase inhibitor, p21cip/CDKN1A. Reporter and chromatin immunoprecipitation assays demonstrated a direct, functional interaction by AP-2gamma at the CDKN1A proximal promoter. AP-2gamma silencing coincided with acquisition of an active chromatin conformation at the CDKN1A locus and increased gene expression. These data provide a mechanism whereby AP-2gamma overexpression can promote breast epithelial proliferation and, coupled with previously published data, suggest how loss of oestrogen regulation of AP-2gamma may contribute to the failure of hormone therapy in patients.

Blockade of MGMT Expression by O6 Benzyl Guanine Leads to Inhibition of Pancreatic Cancer Growth and Induction of Apoptosis

We sought to determine whether administration of a MGMT blocker, O(6)-benzyl guanine (O(6)BG), at an optimal biological dose alone or in combination with gemcitabine inhibits human pancreatic cancer cell growth. Human pancreatic cancer L3.6pl and PANC1 cells were treated with O(6)BG, either alone or in combination with gemcitabine, and the therapeutic efficacy and biological activity of these drug combinations were investigated. O(6)BG sensitized pancreatic cancer cells to gemcitabine. Protein and mRNA expression of MGMT, cyclin B1, cyclin B2, cyclin A, and ki-67 were significantly decreased in the presence of O(6)BG. In sharp contrast, protein expression and mRNA message of p21(cip1) were significantly increased. Interestingly, O(6)BG increases p53-mediated p21(cip1) transcriptional activity and suppresses cyclin B1. In addition, our results indicate that p53 is recruited to p21 promoter. Furthermore, an increase in p21(cip1) and a decrease in cyclin transcription are p53 dependent. The volume of pancreatic tumors was reduced by 27% in mice treated with gemcitabine alone, by 47% in those treated with O(6)BG alone, and by 65% in those mice given combination. Immunohistochemical analysis showed that O(6)BG inhibited expression of MGMT and cyclins, and increased expression of p21(cip1). Furthermore, there was a significant decrease in tumor cell proliferation and an increase in tumor cell apoptosis. Collectively, our results show that decreased MGMT expression is correlated with p53 activation, and significantly reduced primary pancreatic tumor growth. These findings suggest that O(6)BG either alone or in combination with gemcitabine may provide a novel and effective approach for the treatment of human pancreatic cancer.

TGF-β3 is expressed in taste buds and inhibits proliferation of primary cultured taste epithelial cells

Transforming growth factor-betas (TGF-betas), expressed in various tissues, play important roles in embryonic development and adult tissue homeostasis through their effects on cell proliferation, cell differentiation, cell death, and cell motility. However, expression of TGF-beta signaling components and their biological effect on taste epithelia has not been elucidated. We performed expression analysis of TGF-beta signaling components in taste epithelia and found that the TGF-beta3 mRNA was specifically expressed in taste buds. Type II TGF-betas receptor (TbetaR-II) mRNA was specifically expressed in the tongue epithelia including the taste epithelia. To elucidate the biological function of TGF-beta3 in taste epithelia, we performed proliferation assay with primary cultured taste epithelial cells. In the presence of TGF-beta3, percentage of BrdU-labeled cells decreased significantly, suggesting that the TGF-beta3 inhibited the proliferation of cultured taste epithelial cells through inhibiting cell-cycle entry into S phase. By quantitative reverse transcription-polymerase chain reaction assay, we found that the TGF-beta3 resulted in an increased level of expression of p15Ink4b and p21Cip1, suggesting that the TGF-beta3 inhibited the taste epithelial cell proliferation through inhibiting G1cyclin-Cdk complexes. Taken together, these results suggested that the TGF-beta3 may regulate taste epithelial cell homeostasis through controlling cell proliferation.

Fish Oil Supplementation Inhibits NNK-Induced Lung Carcinogenesis in the A/J Mouse

High intake of fish oil with a low omega-6 (n-6)/omega-3 (n-3) polyunsaturated fatty acid (PUFA) ratio has been suggested to protect against many chronic diseases. However, the effect of different ratios of dietary n-6 and n-3 PUFA on lung tumorigenesis has not been investigated. In this study, we examined the effect of a 4 mo dietary supplementation with corn oil (with a high n-6/n-3 ratio) and fish oil (with a low n-6/n-3 ratio) as compared with soybean oil (isocaloric control with the same n-6/n-3 ratio as the base diet) on tumor incidence and tumor prevalence in the A/J mouse model of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis. We found that dietary supplementation had no effect on overall lung tumor incidence, but fish oil supplementation was able to decrease lung tumor prevalence by 78% and 80% compared to groups receiving soybean oil and corn oil supplementation, respectively. The inhibitory effect of fish oil on lung tumor prevalence was associated with increased expressions of cell cycle inhibitor p21Cip1 and lipoxygenase isoform 15-LOX in the lungs. These data suggest that fish oil with a low ratio of n-6/n-3 PUFA could be beneficial in the prevention of lung carcinogenesis.

Activation of Phosphatidylinositol-3′-kinase/AKT Signaling Is Essential in Hepatoblastoma Survival

Hepatoblastoma represents the most frequent malignant liver tumor in childhood. The phosphatidylinositol-3'-kinase (PI3K)/AKT pathway is crucial in downstream signaling of multiple receptor tyrosine kinases of pathogenic importance in hepatoblastoma. Increased PI3K/AKT signaling pathway activity and activating mutations of PIK3CA, encoding a PI3K catalytic subunit, have been reported in different childhood tumors. The current study was done to analyze the role of PI3K/AKT signaling in hepatoblastoma. Immunohistochemical stainings of (Ser473)-phosphorylated (p)-AKT protein, its targets p-(Ser9)-GSK-3beta and p-(Ser2448)-mTOR, as well as the cell cycle regulators Cyclin D1, p27(KIP1), and p21(CIP1) were done and the PIK3CA gene was screened for mutations. In vitro, two hepatoblastoma cell lines treated with the PI3K inhibitor LY294002 were analyzed for AKT and GSK-3beta phosphorylation, cell proliferation, and apoptosis. Additionally, simultaneous treatments of hepatoblastoma with LY294002 and cytotoxic drugs were carried out. Most tumors strongly expressed p-AKT, p-GSK-3beta, and p-mTOR; subgroups showed significant Cyclin D1, p27(KIP1), and p21(CIP1) expression. One hepatoblastoma carried an E545A mutation in the PIK3CA gene. In vitro, PI3K inhibition diminished hepatoblastoma cell growth being accompanied by reduced AKT and GSK-3beta phosphorylation. Flow cytometry and 4', 6-diamidino-2-phenylindole stainings showed that PI3K pathway inhibition leads to a substantial increase in apoptosis and a decrease in cellular proliferation linked to reduced Cyclin D1 and increased p27(KIP1) levels. Simultaneous treatment of hepatoblastoma cell lines with LY294002 and cytotoxic drugs resulted in positive interactions. Our findings imply that PI3K signaling plays an essential role in growth control of hepatoblastoma and might be successfully targeted in multimodal therapeutic strategies.

Acyclic retinoid synergises with valproic acid to inhibit growth in human hepatocellular carcinoma cells

A malfunction of retinoid X receptor-α (RXRα) due to phosphorylation is associated with the development of hepatocellular carcinoma (HCC) and acyclic retinoid (ACR), which targets RXRα, can prevent the development of second primary HCC. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, induces apoptosis and cell cycle arrest in cancer cells. VPA can also enhance the sensitivity of cancer cells to retinoids. The present study examined the possible combined effects of ACR plus VPA in HepG2 human HCC cell line. The combination of 5 μM ACR and 1 mM VPA, about the IC 25 value for both compounds, synergistically inhibited the growth of HepG2 cells without affecting the growth of Hc normal human hepatocytes. The combined treatment with ACR plus VPA also acted synergistically to induce apoptosis and G 0–G 1 cell cycle arrest in HepG2 cells. This combination further exerted a synergistic inhibition of the phosphorylation of RXRα, ERK, Akt and GSK-3β proteins and caused an accumulation of acetylated histones H3 and H4 proteins. VPA enhanced the ability of ACR to raise the cellular levels of RARβ and p21 CIP1. The combination of these agents markedly increased both the RARE and RXRE promoter activities in HepG2 cells. These results suggest that ACR and VPA cooperatively increase the expression of RARβ and p21 CIP1, while inhibiting the phosphorylation of RXRα, and these effects were associated with induction of apoptosis and the inhibition of cell growth in HepG2 cells. This combination might therefore be an effective regimen for the chemoprevention and chemotherapy of HCC.

Cip/Kip cyclin-dependent protein kinase inhibitors and the road to polyploidy

Cyclin-dependent kinases (CDKs) play a central role in the orderly transition from one phase of the eukaryotic mitotic cell division cycle to the next. In this context, p27Kip1 (one of the CIP/KIP family of CDK specific inhibitors in mammals) or its functional analogue in other eukarya prevents a premature transition from G1 to S-phase. Recent studies have revealed that expression of a second member of this family, p57Kip2, is induced as trophoblast stem (TS) cells differentiate into trophoblast giant (TG) cells. p57 then inhibits CDK1 activity, an enzyme essential for initiating mitosis, thereby triggering genome endoreduplication (multiple S-phases without an intervening mitosis). Expression of p21Cip1, the third member of this family, is also induced in during differentiation of TS cells into TG cells where it appears to play a role in suppressing the DNA damage response pathway. Given the fact that p21 and p57 are unique to mammals, the question arises as to whether one or both of these proteins are responsible for the induction and maintenance of polyploidy during mammalian development.

NORE1A Tumor Suppressor Candidate Modulates p21CIP1 via p53

NORE1A (RASSF5) is a proapoptotic Ras effector that is frequently inactivated by promoter methylation in human tumors. It is structurally related to the RASSF1A tumor suppressor and is itself implicated as a tumor suppressor. In the presence of activated Ras, NORE1A is a potent inducer of apoptosis. However, when expressed at lower levels in the absence of activated Ras, NORE1A seems to promote cell cycle arrest rather than apoptosis. The mechanisms underlying NORE1A action are poorly understood. We have used microarray analysis of an inducible NORE1A system to screen for physiologic signaling targets of NORE1A action. Using this approach, we have identified several potential signaling pathways modulated by NORE1A. In particular, we identify the cyclin-dependent kinase inhibitor p21(CIP1) as a target for NORE1A activation and show that it is a vital component of NORE1A-mediated growth inhibition. In primary human hepatocellular carcinomas (HCC), loss of NORE1A expression is frequent and correlates tightly with loss of p21(CIP1) expression. NORE1A down-regulation in HCC also correlates with poor prognosis, enhanced proliferation, survival, and angiogenic tumor characteristics. Experimental inactivation of NORE1A results in the loss of p21(CIP1) expression and promotes proliferation. The best characterized activator of p21(CIP1) is the p53 master tumor suppressor. Further experiments showed that NORE1A activates p21(CIP1) via promoting p53 nuclear localization. Thus, we define the molecular basis of NORE1A-mediated growth inhibition and implicate NORE1A as a potential component of the ill-defined connection between Ras and p53.

Loss of ATM Impairs Proliferation of Neural Stem Cells Through Oxidative Stress-Mediated p38 MAPK Signaling

Ataxia-telangiectasia (A-T) is a genetic disorder caused by a mutation of the Atm gene, which controls DNA repair, cell cycling, and redox homeostasis. Even though oxidative stress has been implicated in the neurological anomalies in A-T, the effects of ATM loss on neural stem cell (NSC) survival has remained elusive. In this study, we investigated the effects of oxidative stress on NSC proliferation in an animal model for A-T neurodegeneration. We found that cultured subventricular zone neurosphere cells from Atm(-/-) mice show impaired proliferation, as well as intrinsic elevation of reactive oxygen species (ROS) levels, compared with those from Atm(+/+) mice. We also show that increasing the levels of ROS by H(2)O(2) treatment significantly reduces Atm(+/+) neurosphere formation and proliferation. In Atm(-/-) neurosphere cells, the Akt and Erk1/2 pathways are disrupted, together with enhanced activity of the p38 mitogen-activated protein kinase (MAPK). Treatment of these cells with the antioxidant N-acetyl-L-cysteine (NAC) or with a p38 MAPK inhibitor restores normal proliferation and reduced expression of p21(cip1) and p27(kip1) in the Atm(-/-) NSCs. These observations indicate that ATM plays a crucial role in NSC proliferation, by activating Akt and Erk1/2 pathways and by suppressing ROS-p38 MAPK signaling. Together, our results suggest that p38 MAPK signaling acts as a negative regulator of NSC proliferation in response to oxidative stress. These findings suggest a potential mechanism for neuronal cell loss as a result of oxidative stress in NSCs in progressive neurodegenerative diseases such as A-T.

Ionizing Radiation Induces ATM-independent Degradation of p21Cip1 in Transformed Cells

The cyclin-dependent kinase inhibitor p21(Cip1) plays an important role in the cellular response to DNA damage. In normal cells, genotoxic stress activates the ATM-p53 pathway that up-regulates the expression of p21(Cip1) leading to cell cycle arrest. However, we have found that in several neoplastic cell lines, ionizing radiation (IR) induces ubiquitin-dependent degradation of p21(Cip1). This process is independent of the ATM pathway as it occurs in immortalized A-T fibroblasts. Knockdown of Skp2, an F-box protein capable of regulating the normal turnover of p21(Cip1), does not prevent the IR-induced degradation. Instead, this process requires the Cul4-DDB1(Cdt2) E3 ligase as knockdown of either DDB1 or Cdt2 rescues p21(Cip1) degradation after IR. Mutating the proliferating cell nuclear antigen-binding site of p21(Cip1) also prevents its IR-induced degradation suggesting that the p21(Cip1)-proliferating cell nuclear antigen interaction is critical for this event. Although ectopic expression of a nondegradable p21(Cip1) did not by itself affect the clonogenic survival of HEK293 cells after IR, the degradation of p21(Cip1) and other targets of the Cul4-DDB1(Cdt2) E3 ligase may collectively contribute to the survival of neoplastic cells after ionizing radiation.

ITF-2 Is Disrupted via Allelic Loss of Chromosome 18q21, and ITF-2B Expression Is Lost at the Adenoma-Carcinoma Transition

The ubiquitously expressed basic helix-loop-helix transcription factor ITF-2B has an important role in differentiation processes, and its transcription is regulated by beta-catenin. The ITF-2 gene is located in the chromosomal region 18q21; allelic loss of this locus occurs in 70% of colorectal cancers. We analyzed the expression, regulation, and function of ITF-2B in colorectal carcinogenesis. The loss-of-heterozygosity (LOH) status of 18q21 and expression of ITF-2B were studied in colorectal carcinomas using polymerase chain reaction-based methods and immunohistochemistry. The biologic effects of ITF-2B were studied in colorectal cancer cells. Reporter gene assays and chromatin immunoprecipitation were utilized to analyze effects of ITF-2B on gene transcription. ITF-2B is strongly expressed in colon adenomas but frequently down-regulated in carcinomas because of LOH at 18q21. ITF-2B induces cell cycle arrest and regulates the expression of p21(Cip1) via newly identified E-boxes in the CDKN1A gene, independently of p53. Loss of ITF-2B expression correlates with loss of p21(Cip1) expression in primary colon carcinomas. Accumulation of mutations and allelic losses are driving forces of colorectal carcinogenesis. ITF-2B, which is up-regulated during early colorectal carcinogenesis because of loss of adenomatous polyposis coli, is a target for LOH on chromosome 18q, along with deleted in colorectal carcinoma and Smad4. This finding, along with the fact that ITF-2B is a regulator of the key cell cycle inhibitor p21(Cip1), indicates that ITF-2B is a tumor suppressor that has an important function at the adenoma to carcinoma transition.

Flavonoid-induced autophagy in hormone sensitive breast cancer cells

The activity of 8-prenylapigenin (8-PA) and its 3'-methoxylated analogue isocannflavin B (IsoB) was investigated in estrogen-dependent T47-D and estrogen-independent MDA-MB-231 human breast cancer cell lines. 8-PA showed a biphasic effect on T47-D cell proliferation, while no significant effect was observed on MDA-MB-231 cells. Conversely, IsoB exhibited only an inhibitory effect on T47-D cell proliferation, accompanied by the appearance of an intense intracytoplasmic vacuolization of autophagic origin. Moreover, biochemical analysis showed that IsoB reduced Akt phosphorylation and p21(Cip1) expression in T47-D cells. These data show that the prenylflavone moiety is a versatile platform for the induction and modulation of bioactivity.

Antiproliferative Action of Cudraflavone B, Isolated From Cudrania tricuspidata, Through the Downregulation of pRb Phosphorylation in Aortic Smooth Muscle Cell Proliferation Signaling

Cudrania tricuspidata has been proposed to possess anti-inflammatory, antioxidant, hepatoprotective, and antitumor activities. Although cudraflavone B, isolated from the root bark of C. tricuspidata, has a variety of pharmacological effects, its effects on rat aortic smooth muscle cells (RASMCs) are unclear. In the present study, cudraflavone B was found to inhibit cell proliferation and DNA synthesis in cultured RASMCs. Pretreatment with cudraflavone B (0.1-4 microM) suppressed platelet-derived growth factor-BB (PDGF-BB)-stimulated cell number in a concentration-dependent manner. The inhibition percentages were 19.7%, 36.4%, 52.3%, and 99.1% at concentrations of 0.1, 1, 2, and 4 microM, respectively. Moreover, cudraflavone B inhibited [H]-thymidine incorporation into DNA in RASMCs in response to 25 ng/mL PDGF-BB. PDGF-BB-stimulated DNA synthesis was significantly reduced by 15.9%, 31.7%, 43.1%, and 78.2% at concentrations of 0.1, 1, 2, and 4 muM, respectively. Thus, cudraflavone B blocked the PDGF-BB-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Furthermore, PDGF-BB-induced phosphorylation of retinoblastoma protein (pRb), the hyperphosphorylation of which is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by cudraflavone B. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDKs), we investigated the expression of CDK2, CDK4, cyclin E, and cyclin D1 and the CDK inhibitors p21 and p27. Treatment with cudraflavone B downregulated the cyclins and CDKs and upregulated the expression of p21 and p27, a CDK inhibitor. These findings suggest that cudraflavone B inhibits RASMC proliferation via the induction of p21 and p27 expression and subsequent cell cycle arrest with reduction of pRb phosphorylation at the G1-S phase.

Daphnoretin, a novel protein kinase C activator, induces growth arrest irreversibly on Human Hepatoma HepG2 Cells

Daphnoretin, a dicoumarin type natural product originally isolated from plant Wikstroemia indica C. A. Mey has been shown as a protein kinase C (PKC) activator with novel structure. Previous studies demonstrated that daphnoretin treatment inhibited the expression of hepatitis B virus surface antigen (HBsAg) in human hepatoma Hep3B cells. In this study, we found that daphnoretin inhibited cell growth and induced a G1‐phase cell cycle arrest in human hepatoma HepG2 cells. Treatment of HepG2 cells with daphnoretin significantly up‐regulated the expression of p21cip1 and decreased the kinase activity but not the protein levels of cdk2/cyclin E complex in HepG2 cells. Unlike the reversible effect of phobol ester, a well known protein kinase C activator, 48 hour‐treatment of daphnoretin caused an irreversible growth arrest in HepG2 cells associated with persistent accumulation of p21 in HepG2 cells. The transcriptome and miRNA expression profile analysis indicates that daphnoretin may coordinately regulate a set of growth related genes at transcriptional and miRNA mediated translational level in cultured HepG2 cells.

Effects of spaceflight and modeled microgravity on the proliferation of vascular smooth muscle cells and β‐catenin signaling pathway

ObjectiveSpaceflight and simulated microgravity induced cardiovascular deconditioning. Vascular smooth muscle cell(VSMC) plays a critical role in the regulation of cardiovascular functions. To elucidate the effects of spaceflight and modeled microgravity on the proliferation of VSMC and its mechanisms, we examined protein expression of β‐catenin and its related signal molecular,MethodsVSMCs exposed to microgravity for 48h in Shenzhou 7 spacecraft were fixed in space and stained by immunofluorescence after reentry. For the VSMCs exposed to modeled microgravity, the proliferation and cell cycle were measured by AlarmarBlue and flow cytometry .The protein expression of signal molecular was detected by western blot and immunofluorescence staining.ResultsSpaceflight and modeled microgravity inhibited the proliferation of VSMCs. Modeled microgravity caused VSMCs to slightly accumulate in G0/1, decreased the expression of N‐cadherin, β‐catenin, cyclin D1 and phosphor‐Akt, but increased the expression of phosphor‐ GSK‐ 3β and p21Cip1. Spaceflight decreased the expression of N‐cadherin, β‐catenin, cyclin D1, but decreased the expression of p21Cip1.ConclusionSpaceflight and modeled microgravity decreased the proliferation of VSMCs, associated with the inhibition of β‐catenin signaling pathway. (Funded by China manned‐space project)

Down-regulation of the HGF/MET autocrine loop induced by celecoxib and mediated by P-gp in MDR-positive human hepatocellular carcinoma cell line

Many tumors are resistant to drug-induced cell-cycle arrest and apoptosis. We have reported that apoptosis can be restored in human multidrug-resistant (MDR) hepatocellular carcinoma cell lines by celecoxib. Here we show that P-glycoprotein (P-gp) mediates cell-cycle arrest and autophagy induced by celecoxib in human MDR overexpressing hepatocellular carcinoma cell line by down-regulation of the HGF/MET autocrine loop and Bcl-2 expression. Exposure of cells to a low concentration of celecoxib down-regulated the expression of mTOR and caused G1 arrest and autophagy, while higher concentration triggered apoptosis. Cell growth inhibition and autophagy were associated with up-regulation of the expression of TGFβ1, p16 INK4b, p21 Cip1 and p27 Kip1 and down-regulation of cyclin D1, cyclin E, pRb and E2F. The role of P-glycoprotein expression in resistance of MDR cell clone to cell-cycle arrest, autophagy and apoptosis was shown in cells transfected with MDR1 small interfering RNA. These findings demonstrate that the constitutive expression of P-gp is involved in the HGF/MET autocrine loop that leads to increased expression of Bcl-2 and mTor, inhibition of eIF2α expression, resistance to autophagy/apoptosis and progression in the cell-cycle. Since mTor inhibitors have been proposed in treatment of “drug resistant” cancer, these data may help explain the reversing effect of mTor inhibitors.