Salicylic acid directly binds to ribosomal protein S3 and suppresses CDK4 expression in colorectal cancer cells.

Colorectal cancer is a significant cause of morbidity and represents a serious public health issue in many countries. The development of a breakthrough preventive method for colorectal cancer is urgently needed. Aspirin has recently been attracting attention as a cancer preventive drug, and its inhibitory effects on the development of various cancers have been reported in several large prospective studies. However, the underlying molecular mechanisms have not yet been elucidated in detail. In the present study, we attempted to identify the target proteins of aspirin using a chemical biology technique with salicylic acid, the main metabolite of aspirin. We generated salicylic acid-presenting FG beads and purified salicylic acid-binding proteins from human colorectal cancer HT-29 cells. The results obtained showed the potential of ribosomal protein S3 (RPS3) as one of the target proteins of salicylic acid. The depletion of RPS3 by siRNA reduced CDK4 expression and induced G1 phase arrest in human colorectal cancer cells. These results were consistent with the effects induced by the treatment with sodium salicylate, suggesting that salicylic acid negatively regulates the function of RPS3. Collectively, the present results show the potential of RPS3 as a novel target for salicylic acid in the protective effects of aspirin against colorectal cancer, thereby supporting RPS3 as a target molecule for cancer prevention.

Oncogenic PAX6 elicits CDK4/6 inhibitor resistance by epigenetically inactivating the LATS2-Hippo signaling pathway.

Intrinsic resistance to CDK4/6 inhibitors hinders their clinical utility in cancer treatment. Furthermore, the predictive markers of CDK4/6 inhibitors in gastric cancer (GC) remain incompletely described. Here, we found that PAX6 expression was negatively correlated with the response to palbociclib in vitro and in vivo in GC. We observed that the PAX6 expression level was negatively correlated with the overall survival of GC patients and further showed that PAX6 can promote GC cell proliferation and the cell cycle. The cell cycle is regulated by the interaction of cyclins with their partner serine/threonine cyclin-dependent kinases (CDKs), and the G1/S-phase transition is the main target of CDK4/6 inhibitors. Therefore, we tested whether PAX6 expression was correlated with the GC response to palbociclib. We found that PAX6 hypermethylates the promoter of LATS2 and inactivates the Hippo pathway, which upregulates cyclin D1 (CCND1) expression. This results in a suppressed response to palbociclib in GC. Furthermore, we found that the induction of the Hippo signaling pathway or treatment with a DNA methylation inhibitor could overcome PAX6-induced palbociclib resistance in GC. These findings uncover a tumor promoter function of PAX6 in GC and establish overexpressed PAX6 as a mechanism of resistance to palbociclib.

Longitudinal tumor fraction trajectories predict risk of progression in metastatic HR+ breast cancer patients undergoing CDK4/6 treatment.

Despite improved clinical outcomes, intrinsic or acquired resistance to CDK4/6 inhibitor treatment has limited the success of this treatment in HR+ HER2- metastatic breast cancer patients. Biomarkers are urgently needed, and longitudinal biomarker measurements may harbor more dynamic predictive and prognostic information compared to single time point measurements. The aim of this study was to explore the longitudinal evolution of circulating tumor fractions within cell-free DNA assessed by an untargeted sequencing approach during CDK4/6 therapy and to quantify the potential association between longitudinal z-score measurements and clinical outcome by using joint models. Forty-nine HR+ HER2- metastatic breast cancer patients were enrolled, and z-score levels were measured at baseline and during 132 follow-up visits (median number of measurements per patient = 3, 25th -75th percentile: 3-5, range: 1-8). We observed higher baseline z-score levels (estimated difference 0.57, 95% CI: 0.147-0.983, P-value = 0.008) and a constant increase of z-score levels over follow-up time (overall P-value for difference in log z-score over time = 0.024) in patients who developed progressive disease. Importantly, the joint model revealed that elevated z-score trajectories were significantly associated with higher progression risk (HR of log z-score at any time of follow-up = 3.3, 95% CI, 1.44-7.55, P = 0.005). In contrast, single z-score measurement at CDK4/6 inhibitor treatment start did not predict risk of progression. In this prospective study, we demonstrate proof-of-concept that longitudinal z-score trajectories rather than single time point measurements may harbor important dynamic information on the development of disease progression in HR+ HER2- breast cancer patients undergoing CDK4/6 inhibitor treatment.

Autophagy Inhibition Sensitizes Renal Tubular Epithelial Cell to G1 Arrest Induced by Transforming Growth Factor beta (TGF-ß).

BACKGROUND Cell cycle arrest and autophagy have been demonstrated to be involved in various transforming growth factor (TGF)-ß-mediated phenotype alterations of tubular epithelial cells (TECs) and tubulointerstitial fibrosis. But the relationship between cell cycle arrest and the autophagy induced by TGF-ß has not been explored well. MATERIAL AND METHODS The effects of autophagy inhibition on TGF-ß-induced cell cycle arrest in TECs were explored in vitro. Human kidney-2 (HK-2) cells were stimulated by TGF-ß with or without a combined treatment of autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (Baf). RESULTS Autophagy inhibition by CQ or Baf promotes the suppression of growth in TGF-ß-treated HK-2 cells, as detected by the Cell Counting Kit-8 (CCK-8) method. In addition, CQ or Baf stimulation enhances G1 arrest in TGF-ß treated HK-2 cells, as investigated using propidium iodide (PI) staining and flow cytometry, which was further confirmed by a decrease in the expression of phosphorylated retinoblastoma protein (p-RB) and cyclin-dependent kinase 4 (CDK4). The upregulation of p21 induced by CQ or Baf may mediate an enhanced G1 arrest in TGF-ß treated HK-2 cells. Western blot analysis showed that TGF-ß-induced expression of extracellular matrix fibronectin was notably upregulated in the presence of autophagy inhibitors. CONCLUSIONS Inhibition of autophagy sensitizes the TECs to G1 arrest and proliferation suppression induced by TGF-ß that contributes to the induction of tubulointerstitial fibrosis.

Apigenin, a flavonoid constituent derived from P. villosa, inhibits hepatocellular carcinoma cell growth by CyclinD1/CDK4 regulation via p38 MAPK-p21 signaling.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide. Apigenin was widely used in HCC treatment; however, the detailed mechanisms have not been clarified. We isolated, characterized, and identified Apigenin from the P. villosa plant using ethanol-extracted, semi-preparative HPLC and NMR. MTT was used to detect the cytotoxicity of Apigenin in HepG2, SMMC-7721 and Huh-7 cell lines. The cell cycle changes of Apigenin on HepG2 using flow cytometry and the key molecules of cell cycle regulation by RT-qPCR and Western blot. Apigenin was ethanol-extracted and semi-preparative HPLC was used for isolation and purification. The compounds were identified and the results showed Apigenin was one of the bioactive compounds. Apigenin exhibited relatively high cytotoxicity in HepG2, SMMC-7721, and Huh-7. Cell cycle analysis showed that Apigenin could induce G1 arrest in HepG2 in a dose-dependent manner. CyclinD1 was up-regulated and CDK4 was down-regulated upon Apigenin treatment, which indicated that Apigenin could block cell cycle progression at the G1 phase though the regulation of CDK4 and CyclinD1 expression. In conclusion, the present findings might provide new insights about the implication of Apigenin and P. villosa in cancer therapy.

Revealing the action mechanisms of dexamethasone on the birth weight of infant using RNA-sequencing data of trophoblast cells.

Dexamethasone (DEX) could induce low birth weight of infant, and low birth weight has close associations with glucocorticoid levels, insulin resistance, hypertension, and metabolic syndrome in adulthood. This study was designed to reveal the action mechanisms of DEX on the birth weight of infant.Using quantitative real-time polymerase chain reaction (qRT-PCR), trophoblast cells of human placenta were identified and the optimum treatment time of DEX were determined. Trophoblast cells were treated by DEX (DEX group) or ethanol (control group) (each group had 3 samples), and then were performed with RNA-sequencing. Afterward, the differentially expressed genes (DEGs) were identified by R package, and their potential functions were successively enriched using DAVID database and Enrichr method. Followed by protein-protein interaction (PPI) network was constructed using Cytoscape software. Using Enrichr method and TargetScan software, the transcription factors (TFs) and micorRNAs (miRNAs) targeted the DEGs separately were predicted. Based on MsigDB database, gene set enrichment analysis (GSEA) was performed.There were 391 DEGs screened from the DEX group. Upregulated SRR and potassium voltage-gated channel subfamily J member 4 (KCNJ4) and downregulated GALNT1 separately were enriched in PDZ (an acronym of PSD-95, Dlg, and ZO-1) domain binding and Mucin type O-glycan biosynthesis. In the PPI network, CDK2 and CDK4 had higher degrees. TFs ATF2 and E2F4 and miRNA miR-16 were predicted for the DEGs. Moreover, qRT-PCR analysis confirmed that SRR and KCNJ4 were significantly upregulated.These genes might affect the roles of DEX in the birth weight of infant, and might be promising therapeutic targets for reducing the side effects of DEX.

SKLB188 inhibits the growth of head and neck squamous cell carcinoma by suppressing EGFR signalling.

BACKGROUND: Overexpression of epidermal growth factor receptor (EGFR) occurs in approximately 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. Thus, targeting EGFR is a promising strategy for treatment of HNSCC. Several small molecule EGFR inhibitors have been tested in clinical trials for treatment of HNSCC, but none of them are more effective than the current chemotherapeutic drugs. Thus, it is urgently needed to develop novel EGFR inhibitors for HNSCC treatment. METHODS: By screening an in-house focused library containing approximately 650 000 known kinase inhibitors and kinase inhibitor-like compounds containing common kinase inhibitor core scaffolds, we identified SKLB188 as a lead compound for inhibition of EGFR. The anticancer effects of SKLB188 on HNSCC cells were investigated by in vitro cell growth, cell cycle and apoptosis assays, as well as in vivo FaDu xenograft mouse model. Molecular docking, in vitro kinase profiling and western blotting were performed to characterise EGFR as the molecular target. RESULTS: SKLB188 inhibited HNSCC cell proliferation by inducing G1 cell cycle arrest, which was associated with downregulating the expression of Cdc25A, cyclins D1/A and cyclin-dependent kinases (CDK2/4), and upregulating the expression of cyclin-dependent kinase (CDK) inhibitors (p21Cip1 and p27Kip1), leading to decreased phosphorylation of Rb. SKLB188 also induced caspase-dependent apoptosis of HNSCC cells by downregulating the expression of Mcl-1 and survivin. Molecular docking revealed that SKLB188 could bind to the kinase domain of EGFR through hydrogen bonds and hydrophobic interactions. In vitro kinase assay showed that SKLB188 inhibited the activity of a recombinant human EGFR very potently (IC50=5 nM). Western blot analysis demonstrated that SKLB188 inhibited the phosphorylation of EGFR and its downstream targets, extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) and Akt in the cells. In addition, SKLB188 dose-dependently inhibited FaDu xenograft growth in nude mice, and concurrently inhibited the phosphorylation of Erk1/2 and Akt in the tumours. CONCLUSIONS: SKLB188 potently inhibits the growth of HNSCC cells in vitro and in vivo by targeting EGFR signalling. The results provide a basis for further clinical investigation of SKLB188 as a targeted therapy for HNSCC. Our findings may open a new avenue for development of novel EGFR inhibitors for treatment of HNSCC and other cancers.

Hydrogen sulfide accelerates cell cycle progression in oral squamous cell carcinoma cell lines.

OBJECTIVE: To investigate the cell cycle regulator role of the third gaseous transmitter hydrogen sulfide (H2 S) in three oral SCC cell lines by using NaHS, a donor of H2 S. METHODS: The synchronized oral squamous cell carcinoma cell lines (Cal27, GNM, and WSU-HN6) were treated with different concentrations of NaHS and then subjected to cell proliferation, cell cycle, and Western blot analyses. RESULTS: The CCK-8 assay results showed that the exogenously administered H2 S donor, NaHS, induced CAL-27, and GNM cell proliferation in a concentration-dependent manner, and the cell cycle analysis indicated that NaHS accelerated cell cycle progression of the synchronized CAL-27, GNM, and WSU-HN6 cells. Western blot analysis revealed that the cell cycle regulatory genes RPA70 and RB1 were significantly down-regulated and that proliferating cell nuclear antigen (PCNA) and CDK4 were markedly up-regulated by NaHS at specific time points in the cell cycle. In addition, our results indicated that the phosphorylation of Akt and Erk1/2 was involved in exogenous H2 S-induced oral SCC cell proliferation. CONCLUSIONS: H2 S is a potential pro-proliferative factor of human oral SCC cells that accelerates the progression of the SCC cell cycle; thus, H2 S plays a deleterious role in oral SCC cancer development.

A combination of STI571 and BCR-ABL1 siRNA with overexpressed p15INK4B induced enhanced proliferation inhibition and apoptosis in chronic myeloid leukemia.

p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.

Inhibition of proliferation and induction of G1-phase cell-cycle arrest by dFMGEN, a novel genistein derivative, in lung carcinoma A549 cells.

Genistein (GEN) is a molecule of great interest as a potent chemopreventive agent against atherosclerosis and cancer. However, the bioavailability of GEN is very low in vivo. Our previous study showed that a GEN derivative, 7-difluoromethyl-5,4'-dimethoxygenistein (dFMGEN) has a better bioavailability than GEN in vivo. In this study, we further evaluated the efficacy of dFMGEN as a candidate for cancer therapy. We demonstrated that dFMGEN treatment decreased the viability of A549 cells in a concentration- and time-dependent manner and induced cell-cycle arrest at the G(1) phase. G(1) phase arrest was correlated with a significant reduction of Cdk4 and cyclin D1 protein level. Further studies showed that cyclin-dependent kinase (Cdk)4 and cyclin D1 protein-level decrease was caused by Cdk inhibitors p15, p21, and p27 level increase, and decreased protein level directly suppressed Rb protein phosphorylation and E2F-1 expression, then cell-cycle progression was arrested. Finally, we also found that dFMGEN has a dosage effect in suppressing tumor growth in vivo, and that dFMGEN was well tolerated by animals. In summary, our results suggest that dFMGEN has therapeutic potential for the treatment of human lung cancer.

Capsaicin induces cycle arrest by inhibiting cyclin-dependent-kinase in bladder carcinoma cells.

OBJECTIVE: Capsaicin is a specialized agonist of transient receptor potential vanilloid type 1 Ca2(+) channel, a member of the vanilloid receptor family of cation channels. We aimed to investigate the effects of capsaicin on the proliferation and cell death of human bladder cancer cells. METHODS: Human bladder cancer cell line 5637 was cultured and the expression of transient receptor potential vanilloid type 1 verified by immunofluorescence and Western blot. Cells were given different disposals (different capsaicin concentration with/without pre-treating with capsazepine; capsazepine, acting as a competitive antagonist of capsaicin) to observe cell viability, cell cycle and cell death by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and flow cytometry. The apoptosis indexes, such as intracellular production of reactive oxygen species and mitochondrial membrane potential were assessed to elucidate the potential mechanism of capsaicin effects in the cells. RESULTS: Capsaicin decreased the viability of 5637 cells in a dose-dependent way. The flow cytometry outcome showed that capsaicin blocked the cell cycle in the G0/G1 period. The Western blot of cyclin-dependent-kinase involved in G1/S transfer verified this. Meanwhile, increased reactive oxygen species production and decreased mitochondrial membrane potential were detected in capsaicin-treated groups. CONCLUSIONS: Capsaicin induces cell death through increased reactive oxygen species and decreased mitochondrial membrane potential. Furthermore, capsaicin inhibits the proliferation of 5637 bladder carcinoma cells by cycle arrest with the inhibition of CDK2, CDK4 and CDK6.

Andrographolide induces cell cycle arrest and apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes.

The pseudo-tumoral expansion of fibroblast-like synoviocytes is a hallmark of rheumatoid arthritis (RA), and targeting rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) may have therapeutic potentials in this disease. Andrographolide, a diterpenoid compound isolated from the herb Andrographis paniculata, has been reported to have potent anti-inflammatory activity. In the present study, we aimed to investigate the effects of andrographolide on human RAFLSs and the underlying molecular mechanism(s). RAFLSs were isolated from patients with RA and treated with or without various concentrations (i.e., 10, 20, and 30 µM) of andrographolide for 48 h. 3-[4,5-Dimethyl-2-yl]-2,5-diphenyl tetrazolium bromide assay revealed that andrographolide treatment decreased the proliferation of RAFLSs in a dose-dependent manner. Cell cycle analysis using propidium iodide (PI) staining showed a G0/G1 cell cycle arrest in andrographolide-treated RAFLSs. Immunoblotting analysis of key cell cycle regulators demonstrated that andrographolide treatment caused a dose-dependent increase in the expression of cell-cycle inhibitors p21 and p27 and a concomitant reduction of cyclin-dependent kinase 4. Exposure to andrographolide-induced apoptosis of RAFLSs measured by annexin V/PI double staining, which was coupled with promotion of cytochrome C release from mitochondria and activation of caspase-3. Moreover, andrographolide-treated RAFLSs displayed a significant decrease in the Bcl-2/Bax ratio compared to untreated cells. In conclusion, our data demonstrate that andrographolide exerts anti-growth and pro-apoptotic effects on RAFLSs, thus may have therapeutic potential for the treatment of RA.

Antiproliferative effects of artemisinin on human breast cancer cells requires the downregulated expression of the E2F1 transcription factor and loss of E2F1-target cell cycle genes.

Artemisinin, a sesquiterpene phytolactone derived from Artemisia annua, is a potent antimalarial compound with promising anticancer properties, although the mechanism of its anticancer signaling is not well understood. Artemisinin inhibited proliferation and induced a strong G1 cell cycle arrest of cultured MCF7 cells, an estrogen-responsive human breast cancer cell line that represents an early-stage cancer phenotype, and effectively inhibited the in-vivo growth of MCF7 cell-derived tumors from xenografts in athymic nude mice. Artemisinin also induced a growth arrest of tumorigenic human breast cancer cell lines with preneoplastic and late stage cancer phenotypes, but failed to arrest the growth of a nontumorigenic human mammary cell line. Concurrent with the cell cycle arrest of MCF7 cells, artemisinin selectively downregulated the transcript and protein levels of the CDK2 and CDK4 cyclin-dependent kinases, cyclin E, cyclin D1, and the E2F1 transcription factor. Analysis of CDK2 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK2 gene expression was accounted for by the loss of CDK2 promoter activity. Chromatin immunoprecipitation revealed that artemisinin inhibited E2F1 interactions with the endogenous MCF7 cell CDK2 and cyclin E promoters. Moreover, constitutive expression of exogenous E2F1 prevented the artemisinin-induced cell cycle arrest and downregulation of CDK2 and cyclin E gene expression. Taken together, our results demonstrate that the artemisinin disruption of E2F1 transcription factor expression mediates the cell cycle arrest of human breast cancer cells and represents a critical transcriptional pathway by which artemisinin controls human reproductive cancer cell growth.

Relationship between lunasin's sequence and its inhibitory activity of histones H3 and H4 acetylation.

SCOPE: Dysfunction of histone acetyltransferases (HATs) or histone deacetylases (HDACs) involved in histones acetylation has been associated with cancer. Inhibitors of these enzymes are becoming potential targets for new therapies. METHODS AND RESULTS: This study reports by Western-Blot analysis, that peptide lunasin is mainly an in vitro inhibitor of histone H4 acetylation by P300/cAMP-response element-binding protein (CBP)-associated factor (PCAF), with IC50 values dependent on the lysine position sensitive to be acetylated (0.83 µM (H4-Lys 8), 1.27 µM (H4-Lys 12) and 0.40 µM (H4-Lys 5, 8, 12, 16)). Lunasin is also capable of inhibiting H3 acetylation (IC50 of 5.91 µM (H3-Lys 9) and 7.81 µM (H3-Lys 9, 14)). Studies on structure-activity relationship establish that lunasin's sequence are essential for inhibiting H4 acetylation whereas poly-D sequence is the main active sequence responsible for H3 acetylation inhibition. Lunasin also inhibits H3 and H4 acetylation and cell proliferation (IC50 of 181 µM) in breast cancer MDA-MB-231 cells. Moreover, this peptide decreases expression of cyclins and cyclin dependent kinases-4 and -6, implicated in cell cycle pathways. CONCLUSION: Results from this study demonstrates lunasin's role as modulator of histone acetylation and protein expression that might contribute on its chemopreventive properties against breast cancer.

Up-regulation of retinoblastoma protein phosphorylation in gingiva after cyclosporine A treatment: an in vivo and in vitro study.

BACKGROUND AND OBJECTIVE: Cyclosporine A can induce gingival cell proliferation; however, the precise molecular regulation of the proliferation is uncertain. Therefore, this study was carried out to examine, in vivo and in vitro, the expression of genes and proteins associated with gingival cell proliferation after treatment with cyclosporine A. MATERIAL AND METHODS: Forty Sprague Dawley rats with right maxillary posterior edentulous gingivae were assigned to a cyclosporine A group (30 mg/kg daily of cyclosporine A, administered orally) or a control group (administered mineral oil only). The animals were killed 4 wk after treatment. The edentulous gingivae were dissected out and analyzed for the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (Rb1) mRNA and/or protein, and phosphorylated Rb1 (pRb1), by real-time RT-PCR or immunohistochemistry. In human gingival fibroblast (HGF) cultures, the expression of PCNA, CDK4, cyclin D1 and Rb1 proteins and Rb1 phosphorylation were determined by western blotting after cyclosporine A treatment (0-10(4) ng/mL). RESULTS: Proliferating cell nuclear antigen and cyclin D1 mRNAs (Pcna and Ccnd1, respectively) were expressed more strongly in the gingivae of cyclosporine A-treated animals than in the gingivae of the controls. Immunohistochemical analyses showed that a greater number of gingival cells stained positive for cyclin D1, CDK4 and pRb1 in the cyclosporine A group than in the control group. Increased expression of cyclin D1, CDK4 and PCNA proteins was observed in HGFs after cyclosporine A treatment. The phosphorylation of Rb1 was enhanced in HGFs after treatment with cyclosporine A at concentrations of 10(2)-10(3) ng/mL. CONCLUSION: The increases in cyclin D1, PCNA and CDK4, together with the enhanced phosphorylation of Rb1, suggest that cyclosporine A promotes cell-cycle progression through the G(1)/S transition in the gingiva.

The activated aryl hydrocarbon receptor synergizes mitogen-induced murine liver hyperplasia.

Mechanisms of hepatocyte proliferation triggered by tissue loss are distinguishable from those that promote proliferation in the intact liver in response to mitogens. Previous studies demonstrate that exogenous activation of the aryl hydrocarbon receptor (AhR), a soluble ligand-activated transcription factor in the basic helix-loop-helix family of proteins, suppresses compensatory liver regeneration elicited by surgical partial hepatectomy. The goal of the present study was to determine how AhR activation modulates hepatocyte cell cycle progression in the intact liver following treatment with the hepatomitogen, 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP). Mice were pretreated with the exogenous AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 24h prior to treatment with TCPOBOP (3 mg/kg).). In contrast to the suppressive effects of AhR activation observed during compensatory regeneration, TCDD pretreatment resulted in a 30-50% increase in hepatocyte proliferation in the intact liver of TCPOBOP-treated mice. Although pretreatment with TCDD suppressed CDK2 kinase activity and increased the association of CDK2 with negative regulatory proteins p21Cip1 and p27Kip1, a corresponding increase in CDK4/cyclin D1 association and CDK4 activity which culminated in enhanced phosphorylation of retinoblastoma protein, consistent with the increased proliferative response. These findings are in stark contrast to previous observations that the activated AhR can suppress hepatocyte proliferation in vivo and reveal a new complexity to AhR-mediated cell cycle control.

Cell kinetics and gene expression changes in colorectal cancer patients given resistant starch: a randomised controlled trial.

OBJECTIVE: This study investigated the effects of oral supplementation of resistant starch (RS) on tumour cell and colonic mucosal cell kinetics and on gene expression in patients with colorectal cancer (CRC), and its potential role in colon cancer prevention. METHODS: 65 patients with CRC were randomised to treatment with RS or ordinary starch (OS) and were given starch treatment for up to 4 weeks. Pretreatment and post-treatment biopsies were obtained from the tumour and colonic mucosa, and the effects of the starch treatment on cell proliferation and expression of the cell cycle regulatory genes CDK4 (cyclin-dependent kinase 4) and GADD45A (growth arrest and DNA damage-inducible, alpha) were investigated. RESULTS: The proportion of mitotic cells in the top half of the colonic crypt was significantly lower following RS treatment (3.1 (1.5), mean (SEM)) as compared with OS treatment (13.7 (3.2)) (p = 0.028). However, there was no effect of RS treatment on crypt dimensions and tumour cell proliferation index. There was significant upregulation in expression of CDK4 (p<0.01) and downregulation in expression of GADD45A (p<0.001) in the tumour tissue when compared with macroscopically normal mucosa. Following RS treatment, CDK4 expression in tumours (0.88 (0.15)) was twofold higher than that in the OS group (0.37 (0.16)) (p = 0.02). The expression of GADD45A, which was downregulated in the presence of cancer, was significantly upregulated (p = 0.048) following RS treatment (1.41 (0.26)) as compared with OS treatment (0.56 (0.3)). However, there were no significant differences in the expression of these genes in the normal mucosa following starch treatment. CONCLUSIONS: Cell proliferation in the upper part of colonic crypts is a premalignant marker and its reduction by RS supplementation is consistent with an antineoplastic action of this food component. Differential expression of the key cell cycle regulatory genes may contribute to the molecular mechanisms underlying these antineoplastic effects of RS.

Humic acid induces G1 phase arrest and apoptosis in cultured vascular smooth muscle cells.

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for Blackfoot disease (BFD). In this study, the ability of HA to inhibit cell cycle progression and induce apoptosis in cultured smooth muscle cells (SMCs; A7r5) was investigated. Treatment of the SMCs at various HA concentrations (25-200 microg/mL) resulted in sequences of events marked by apoptosis, as shown by loss of cell viability, morphology change, and internucleosomal DNA fragmentation. HA-induced apoptotic cell death that is associated with loss of mitochondrial membrane potential (Delta Psi m), cytochrome c translocation, caspase-3, -8, and -9 activation, poly ADP-ribose polymerase (PARP) degradation, dysregulation of Bcl-2 and Bax, and upregulation of p53 and phospholyrated p53 (p-p53) in SMCs. Flow cytometry analysis demonstrated that HA blocked cell cycle progress in the G1 phase in SMCs. This blockade of cell cycle was associated with reduced amounts of cyclin D1, CDK4, cyclin E, CDK2, and hyperphosphorylated retinoblastoma protein (pRb) in a time-dependent manner. Apparent DNA strand breaks (DNA damage) were also detected in a dose-dependent manner using Single-cell gel electrophoresis assay (comet assay). Furthermore, HA induced dose-dependent elevation of reactive oxygen species (ROS) level in SMCs, and antioxidant vitamin C and Trolox effectively suppressed HA-induced DNA damage and dysregulation of Bcl-2/Bax. Our findings suggest that HA-induced DNA damage, cell cycle arrest, and apoptosis in SMCs may be an underlying mechanisms for the atherosclerosis and thrombosis observed in the BFD endemic region.

Enhancement of hypoxia-induced apoptosis of human breast cancer cells via STAT5b by momilactone B.

We have shown previously that hypoxia activates the cyclin D1 promoter via the Jak2/STAT5b pathway in breast cancer cells. Most solid tumors contain hypoxic components and overexpression of cyclin D1. The purpose of the present study was to investigate the molecular mechanism by which momilactone B exerts its inhibitory effects on breast cancer cells. Momilactone B, extracted from Korean rice hulls, suppressed hypoxia-induced increases in phospho-STAT5, STAT5b, cyclin D1, and cdk4 protein levels in human breast cancer cells. STAT5b expression was inhibited by siRNA experiments leading to decreased cyclin D1. The effects of momilactone B on cell growth and apoptosis-related gene expression were investigated in breast cancer cells under hypoxic conditions (2% O2). Bax and p21 expression was found to be up-regulated, whereas ppRb and bcl-2 were down-regulated in momilactone B-treated cells under hypoxic conditions. However, the p53 protein level did not change. Flow cytometry with Annexin-FITC staining showed that the number of apoptotic cells increased in hypoxic cells treated with momilactone B compared with untreated hypoxic cells. Furthermore, caspase activity increased upon treatment with momilactone B under hypoxic conditions. These results indicate that momilactone B inhibits the growth of breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through STAT5b and a caspase-3 dependent pathway. We suggest that momilactone B accelerates hypoxia-induced apoptosis of human breast cancer cells through STAT5b, and may represent an effective chemopreventive or therapeutic agent against breast cancer.

BRCA1 overexpression sensitizes cancer cells to lovastatin via regulation of cyclin D1-CDK4-p21WAF1/CIP1 pathway: analyses using a breast cancer cell line and tumoral xenograft model.

It is well established that statins display potent anticancer activity in several types of proliferating tumor cells. However, how to promote the sensitivity of statins to mammary cancer is yet to be completely deciphered. The purpose of this study was to investigate whether breast cancer susceptibility gene 1 (BRCA1) overexpression sensitizes mammary cancer cells to statins. MCF-7 cells, which have only one wild-type BRCA1 allele, were transfected with pcDNA3-beta-HA-hsBRCA1 plasmids via liposomes to reconstitute BRCA1 overexpression human breast cancer cell line, and tumoral xenografts with BRCA1 overexpression were subsequently established in BALB/c nude mice. Then, the inhibitory activity of lovastatin on cancer cells and tumoral xenografts, and the underlying mechanism involving in cell-cycle regulatory proteins were analyzed. The proliferative ability of MCF-7 cells treated with lovastatin was reduced compared to normal, and further decreased in the presence of excess BRCA1, detected by methyl thiazolyl tetrazolium and flow cytometry techniques in vitro or by 5-bromodeoxyuridine incorporation in vivo. Additionally, the mRNA and protein expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma protein (pRb), was further down-regulated under exposure to lovastatin in condition of BRCA1 overexpression, but the expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor (CDKI), was further up-regulated, both in vitro and in vivo detected with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Moreover, we found the further reduced volume of tumoral xenografts treated with lovastatin in the presence of BRCA1 overexpression. Our results suggest that BRCA1 overexpression sensitizes cancer cells to lovastatin via regulation of cyclin D1-CDK4-p21WAF1/CIP1 pathway, which will provide an innovative experimental framework to study control of breast cancer cell proliferation.