p53 inhibits adriamycin-induced down-regulation of cyclin D1 expression in human cancer cells.

The tumor suppressor p53 gene product is an essential component of the cytotoxic pathway triggered by DNA-damaging stimuli such as chemotherapeutic agents and ionizing radiation. We previously demonstrated that adenovirus-mediated wild-type p53 gene transfer could enhance the cytotoxic actions of chemotherapeutic drugs both in vitro and in vivo; however, the molecular mechanism of this chemosensitization is still unclear. Cyclin D1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle. Here we show that infection with an adenovirus vector expressing the wild-type p53 gene (Ad-p53) caused an increase in cyclin D1 protein levels in human colorectal cancer cell lines DLD-1 and SW620; treatment with the anti-cancer drug adriamycin, however, down-regulated their cyclin D1 protein expression in a dose-dependent manner. The suppression of cyclin D1 expression following adriamycin treatment could be blocked by simultaneous Ad-p53 infection. Furthermore, DLD-1 and SW620 cells transfected with the cyclin D1 expression construct displayed increased sensitivity to adriamycin compared to that of the vector-transfected control. Our results suggest that ectopic wild-type p53 gene transfer results in increased cyclin D1 expression and, consequently, sensitizes human colorectal cancer cells to chemotherapeutic agents.     

Diltiazem inhibits transferrin receptor expression and causes G1 arrest in normal and neoplastic T cells.

Transferrin receptor expression is essential for the proliferation of both normal and malignant T cells. While transferrin receptor expression in normal T cells is tightly coupled to interleukin-2 receptor expression, transferrin receptor expression in malignant cells is usually constitutive and is released from this constraint. Temporally, the appearance of these membrane receptors is preceded by changes in the expression of the proto-oncogenes c-myc and c-myb. In addition, although an increase in the level of intracellular free calcium occurs early in the sequence of T-cell activation, the activation events dependent on this calcium flux have not been resolved. In the present study we report that diltiazem, an ion channel-blocking agent that inhibits calcium influx, arrested the growth in vitro of both normal and malignant human T cells in the G1 phase of the cell cycle. However, diltiazem did not inhibit the expression of c-myc or interleukin-2 receptor mRNA and protein in normal mitogen-activated T cells or the constitutive expression of c-myc and c-myb mRNA in malignant T cells (T acute lymphoblastic leukemia cells). In contrast, diltiazem prevented the induction of transferrin receptor (mRNA and protein) in normal T cells and caused a progressive loss of transferrin receptor (mRNA and protein) in malignant T cells. These data demonstrate that diltiazem can dissociate several growth-related processes normally occurring in G1 and thereby disrupt the biochemical cascade leading to cell proliferation.     

Tehranolide inhibits proliferation of MCF-7 human breast cancer cells by inducing G0/G1 arrest and apoptosis

Tehranolide, a novel natural sesquiterpene lactone with an endoperoxide group, bears a structural similarity to artemisinin and has been shown to inhibit cell growth. However, the underlying mechanisms of these activities remain obscure. The purpose of this study was to investigate the fundamental mechanisms by which tehranolide inhibits growth in MCF-7 cells. Cell growth was determined by using the MTT viability assay and counting cells. Apoptosis and cell-cycle progression were evaluated by means of Hoechst 33258 staining, flow cytometry with annexin-V/propidium iodide double staining, and ROS formation. The protein expression of Bax and Bcl-2 was demonstrated by Western blotting. Moreover, to determine the molecular mechanism whereby tehranolide mediates G0/G1 arrest, the expression of PI3K, p-PI3K, Akt, p-Akt, p27kip1, cyclin D1, and CDK4 was monitored. Cell proliferation was significantly inhibited by tehranolide in a dose- and time-dependent manner. This compound inhibited cell proliferation and induced G0/G1 arrest through the PI3K/Akt/cyclin D1 pathway. It also induced apoptosis and an increase in ROS. In addition, an increase in cytochrome c and Bax, as well as a decrease in Bcl-2, was observed. Moreover, blocking the CD95 receptor with an anti-CD95 antibody (ZB4) had no effect on tehranolide-mediated apoptosis. This study has yielded promising results, which show for the first time that tehranolide does inhibit the growth of cancer cells. The selective inhibition of cancer cell growth, the apoptosis induction via the mitochondrial pathway, and the G0/G1 arrest by modulating the PI3K/AKT signaling pathway and downregulating cyclin D1, which leads to the release of p27kip1 and the association of this inhibitor with the cyclin E/CDK2 complex, ultimately preventing cell-cycle progression from G1 to S phase, all serve to provide support for further studies of tehranolide as a possible anticancer drug in the clinical treatment of cancer.     

CCAAT/enhancer-binding protein delta regulates mammary epithelial cell G0 growth arrest and apoptosis.

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPdelta gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPdelta is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPdelta induction is G0-specific in mouse mammary epithelial cells; C/EBPdelta gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPdelta antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPdelta-overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPdelta levels were rescued in AS1 cells by transfection with a C/EBPdelta "sense" construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPdelta plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.     

Trichostatin A inhibits beta-casein expression in mammary epithelial cells.

Many aspects of cellular behavior are defined by the content of information provided by association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. We have previously found that the minimal ECM- and Prl-responsive enhancer element BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous beta-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of beta-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM mediated rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types.     

Differential expression of cholinephosphotransferase in normal and cancerous human mammary epithelial cells

Membrane phospholipids as well as fatty acid profile of cell membrane phospholipids are altered in tumorigenicity and malignancy. Synthesis of total cellular phosphatidylcholine (PC) can be used as a marker for membrane proliferation in neoplastic mammary gland tissues. Cholinephosphotransferase (CPT), the terminal enzyme in the de novo synthesis of PC, has an important role in regulating the acyl group of PC in mammalian cells. In this study, the effect of neoplasia on CPT was examined. The gene shows an elevated expression in cancerous (11-9-14) breast epithelial cell line when compared to that of normal non-tumorigenic (MCF-12A) breast epithelial cell line. Four nucleotide substitutions are observed in the cancer cell line. Of these, three are null mutations, but the third one shows an interesting serine to tyrosine substitution (at amino acid position 89 of our partial sequence which corresponds to position 323 of the CPT sequence reported as NM_020244 in GenBank) in 11-9-14 cells. The tyrosine is present in the right context of KSELYQDT, which directs tyrosine phosphorylation at the tyrosine site. Biochemical approach also reveals a 1.5-fold stimulation in CPT activity in 11-9-14 cells compared to that of the MCF-12A cells.     

Differential expression of human tissue factor in normal mammary epithelial cells and in carcinomas.

BACKGROUND: Tissue factor (TF) is a glycoprotein which binds factor VIIa. The TF-VIIa complex serves as a potent initiator of the coagulation pathways. TF, an immediate early gene, may also play a role in cell growth. Expression of TF was correlated with some types of cancers. MATERIALS AND METHODS: Normal, immortalized, and tumor human mammary epithelial cells were used in the experiments. The differential display (DD) technique was used to identify genes differentially expressed in the cells. TF expression patterns were examined by Northern blot analysis, immunofluorescence staining of cultured cells, and immunohistochemical staining in human cryostat sections. RESULTS: In a 5-way display, an amplified polymerase chain reaction (PCR) product was found in normal and immortalized human mammary epithelial cells but not in the breast cancer cells. The PCR fragment was cloned and sequenced. The result showed that the fragment was identical to human tissue factor. Northern blot analysis showed that expression level of tissue factor mRNA remained high in growing, quiescent, and senescent normal mammary epithelial cells. Immunofluorescence staining also confirmed tissue factor expression pattern in the cell lines tested. Immunohistochemical staining showed that tissue factor was expressed in the normal luminal and myoepithelial cells of some ducts but not others. No staining was observed in invasive carcinoma cells. However, myoepithelial cell staining was seen in some residual ductal structures in invasive tumors. CONCLUSIONS: This study shows the use of DD to reveal the loss of TF expression pattern in human breast cancer cell lines. Immunohistochemical staining results showed breast carcinoma cells expressed little TF, if any, suggesting that TF is not required for breast tumor cell invasion. The results also indicated that TF expression was independent of the proliferation status of the expressing cells. The expression pattern of TF may be a meaningful marker in the development of breast cancer.     

Magnesium depletion causes growth inhibition,reduced expression of cyclin D1, and increased expression of P27KIP1 in normal but not in transformed mammary epithelial cells

In this study, we have evaluated the effects of extracellular magnesium restriction on the growth and cell cycle parameters of normal (HC11) and transformed (MCF-7) breast epithelial cell lines. Cells were incubated in medium with different concentrations of Mg²⁺ (from 0.5 to 0 mM) and the growth rates were determined by [³H]-thymidine incorporation and cell counting. The growth of the HC11 cells was drastically inhibited by Mg²⁺ depletion whereas the MCF-7 cells were only slightly inhibited (about 50% and 15%, respectively, after incubation in 0.05 mM Mg for 48 h). Cell cycle analyses showed a decrease in the percentage of cells in the S phase when both cell lines were incubated at low Mg²⁺ concentration. However, while the percentage of cells in both the G0/G1 and G2/M phases was increased in the HC11 cells, only the percentage of cells in the G2/M phase was increased in the MCF-7 cell line. Extracellular magnesium depletion was associated with increased expression of the cyclin-dependent kinase inhibitor p27^Kip1 and decreased expression of cyclin D1 in the HC11 but not in the MCF-7 cells. We also demonstrated that Mg²⁺ depletion does not inhibit kinase activities in the normal HC11 cells and that Mg²⁺-restricted HC11 cells are still responsive to the epidermal growth factor (EGF)- and insulin-mediated stimulation of cell growth. These data suggest that normal but not transformed mammary epithelial cells are inhibited by extracellular Mg²⁺ restriction and that this effect might be mediated by changes in the levels of expression of both cyclin D1 and p27^Kip1. J. Cell. Physiol. 180:245–254, 1999. © 1999 Wiley-Liss, Inc.     

Narirutin downregulates lipoxygenase-5 expression and induces G0/G1 arrest in triple-negative breast carcinoma cells

BackgroundTriple-negative breast cancer (TNBC) accounts for 20% of breast cancer that does not express HER2, progesterone and estrogen receptors. It is associated with a high mortality rate, morbidity, metastasis, recurrence, poor prognosis and resistance to chemotherapy. Lipoxygenase-5 (LOX-5), cyclooxygenase-2 (COX-2), cathepsin-D (CATD), ornithine decarboxylase (ODC) and dihydrofolate reductase (DHFR) are involved in breast cancer carcinogenesis; hence, there is a pressing need to identify novel chemicals that targets these enzymes. Narirutin, a flavanone glycoside abundantly present in citrus fruits, is reported to have immune-modulatory, anti-allergic and antioxidant potential. Still, the cancer chemopreventive mechanism against TNBC has not been explored.MethodsIn vitro experiments, enzyme activity, expression analysis, molecular docking and MD simulation were carried out.ResultsNarirutin suppressed the growth of MDA-MB-231 and MCF-7 in a dose-proportional manner. The pronounced effect with >50% inhibition was observed in SRB and MTT assays for MDAMB-231 cells. Unexpectedly, narirutin suppressed the proliferation of normal cells (24.51%) at 100 μM. Further, narirutin inhibits the activity of LOX-5 in cell-free (18.18 ± 3.93 μM) and cell-based (48.13 ± 7.04 μM) test systems while moderately affecting COX-2, CATD, ODC and DHFR activity. Moreover, narirutin revealed a down-regulation of LOX-5 expression with a fold change of 1.23. Besides, MD simulation experiments confirm that narirutin binding forms a stable complex with LOX-5 and improves the stability and compactness of LOX-5. In addition, the prediction analysis demonstrates that narirutin could not cross the blood-brain barrier and did not act as an inhibitor of different CYPs.Conclusions and significanceNarirutin could be a potent cancer chemopreventive lead for TNBC, further paving the way for synthesizing novel analogues.     

Smad7 induces G0/G1 cell cycle arrest in mesenchymal cells by inhibiting the expression of G1 cyclins

The major Smad pathways serve in regulating the expression of genes downstream of TGFbeta signals. In this study, we examined the effects of sustained Smad7 expression in cultured cells. Interestingly, Smad7 caused various mesenchymal cells, including NIH3T3 fibroblast and ST2 bone-marrow stromal cells, to undergo a marked morphological alteration into a flattened cell shape, but kept them alive for as long as 60 days. Furthermore, Smad7 arrested the proliferation of the cells even before they reached confluence. These cells became quiescent in G0/G1 phase and accumulated a hypophosphorylated form of retinoblastoma. The cytostatic effect of Smad7 was closely associated with a preceding decrease in the levels of G1 cyclins, such as cyclin D1 and cyclin E. Accordingly, ectopic cyclin E was able to overcome the Smad7-induced arrest of proliferation. These results indicate that Smad7 functions upstream of G1 cyclins and suggest a novel role for Smad7 as an antiproliferative factor. In contrast to the growth of mesenchymal cells, that of epithelial cells was little susceptible to Smad7. The present findings raise the possibility that a link between Smad7 and the G1 to S phase transition may also contribute to the cell cycle control by certain Smad7-inducing stimuli in a cell-type-dependent fashion.     

Dysregulated expression of MIC-1/PDF in human prostate tumor cells

As a part of the study to identify genes associated with hormone-refractory stage of human prostate cancer, we have recently identified several genetic and epigenetic changes that seem to be associated with the progression of androgen-sensitive to androgen-independent prostate tumor cells. In the present study, we report a novel gene, macrophage inhibitory cytokine-1 (MIC-1) also known as prostate derived factor (PDF), that was highly expressed in androgen-independent LNCaP-C81 cells and its metastatic variant LNCaP-Ln3 compared to androgen-sensitive LNCaP-C33 cells. The MIC-1/PDF expression was dysregulated (very low to non-detectable) in the androgen-independent PC3 and DU145 cells. Interestingly, serum factors demonstrated a differential regulation of MIC-1/PDF in the androgen-sensitive and the androgen-independent cells of LNCaP cells. Immunohistochemical analysis on 15 prostatic adenocarcinomas showed a weak staining in the benign prostatic glandular area (intensity score 2.38+/-0.25; n=13), while the immunoreactivity was significantly stronger (p<0.05) in areas of adenocarcinoma (score 7.33+/-0.88; n=15). Altogether, these data suggest that the serum factors (including androgens and cytokines) might contribute to the regulation of the MIC-1/PDF gene that seems to be associated with the progression of prostate cancer.     

Differential expression of annexins I and II in normal and malignant human mammary epithelial cells

Abstract. Collagen-binding proteins ( CBPs ) of rat mammary tumors are identical to Ca²⁺-binding annexins [49]. We have now isolated a protein of 38 kDa from the human mammary tumor cell line ALAB by collagen type I affinity chromatography as well as by extraction of calcium-binding proteins. The 38-kDa band of both preparations was identified as annexin II (calpactin I) by its reaction with an annexin II-specific monoclonal antibody in Western blot analysis. Annexin I (lipocortin I) was not detectable in these cells. Two other human cell lines, the SV40-transformed cell line SV3 and cell line HBL-100, both established from normal mammary glands, were also positive for annexin II and negative for annexin I.
In vivo expression of annexins was investigated by immunohistological staining of normal and malignant human mammary tissue. The annexin II-specific mAb reacted with normal and tumor parenchyme whereas the annexin I-specific mAb reacted with acini and ductal myoepithelium of the normal mammary gland but showed no reaction with tumor tissue. Immunolocalization studies also showed annexin II expression in both normal and tumor stroma while only tumor stromal cells were found to be reactive with the antibody against annexin I. The differential expression of annexins in normal and malignant human mammary tissue suggests special functions of these proteins in the mammary gland.