Mechanism Of Cell Growth Inhibition Research Articles

Genistein induces cell growth inhibition in prostate cancer through the suppression of telomerase activity

To clarify the mechanism of the anticancer effect of genistein, we examined the effect of genistein on telomerase activity in prostate cancer cells. We hypothesized that genistein may exert its anticancer effect by modifying telomerase activity in prostate cancer cells. Prostate cancer (LNCaP) cells were cultured with genistein and the number of viable cells was counted. Growth medium was also collected to measure prostate-specific antigen (PSA) concentration. Polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay and reverse transcriptase (RT)-PCR analysis were performed to investigate telomerase activity and the expression of human telomerase reverse transcriptase (hTERT), c-myc and p21 mRNA. To examine the possibility that hTERT transcriptional activity is modulated by genistein, transient cell transfection studies were performed by using luciferase reporter assay. Telomere repeat amplification protocol (TRAP) assay and PCR analysis of hTERT were performed in androgen independent cells, DU-145. Cell growth of LNCaP was inhibited by genistein and PSA secretion was similarly reduced. In TRAP assay, the telomerase activity of LNCaP cells was reduced by genistein. Reverse transcriptase-PCR analysis revealed that the expression of hTERT and c-myc mRNA was down-regulated by genistein, whereas p21 mRNA increased in response to genistein. Luciferase reporter assay revealed that genistein reduced the transcriptional activity of hTERT. In DU-145 cells, telomerase activity and the expression of hTERT mRNA were also reduced by genistein. The current study elucidated the molecular mechanism of cell growth inhibition by genistein. The antiproliferative effects of genistein seem to be exerted on the hTERT transcriptional activity via different molecular pathways.

Adenoviral p16/CDKN2 gene transfer to malignant glioma: Role of p16 in growth, invasion, and senescence

In gliomas, a high frequency of homozygous p16 gene deletions have been demonstrated, which are believed to be linked with malignant progression. The aim of this study was to assess the role of p16 in growth, invasion, and senescence. The human glioma cell lines U87 MG and U373 MG were transduced with Ad-p16, and cell viability was assessed by trypan blue staining. To examine the mechanism of cell growth inhibition, cell cycle analyses and annexin assays were performed. The invasive potential of Ad-p16 transduced cells was evaluated using a Matrigel invasion assay, and trimolecular complex (MMP-2/MT1-MMP/TIMP-2) synthesis was proven by zymography and Western blotting. To establish the link between p16 and cell senescence, we stained for Senescence-Associated beta-galactosidase activity. A cell proliferation assay demonstrated that Ad-p16 treatment significantly inhibits cell growth. Moreover, this cell growth inhibition was induced by cell cycle arrest, not by apoptosis. In vitro treatment of malignant glioma cells with Ad-p16 significantly decreased their invasive potential by Matrigel invasion assay. However, we were unable to demonstrate any differences in the constitutive productions and secretions of MMP-2, MT1-MMP, and TIMP-2, among the mock-treated, Ad-lacZ-transduced, and Ad-p16-transduced cells. p16 expression caused an enlargement of all cells, and these were morphologically similar to senescent cells. Staining for Senescence-Associated beta-galactosidase activity showed that the enlarged cells stained positively. Taken together these data strongly suggest that the anti-cancer effect of p16 is modulated by p16-mediated cell cycle arrest and by the induction of senescence.

Peroxisome proliferator-activated receptor gamma ligand troglitazone induces cell cycle arrest and apoptosis of hepatocellular carcinoma cell lines.

Ligand activation of peroxisome proliferator-activated receptor gamma (PPARgamma) results in the inhibition of proliferation of various cancer cells. The aim of this study is to investigate the mechanisms of cell growth inhibition of hepatocellular carcinoma (HCC) cell lines by the PPARgamma ligand, troglitazone. Six HCC cell lines were used to study the effects of troglitazone on cell growth by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, on cell cycle by flow cytometry, and on the cell cycle-regulating factors of late G1 phase by Western blotting. Apoptosis assays were performed by flow cytometry using membrane, nuclear, cytoplasmic, and mitochondrial markers. Caspase inhibitors were used to analyze the mechanisms of apoptosis induced by troglitazone. Troglitazone showed a potent dose-dependent effect on the growth inhibition of all six HCC cell lines, which were suppressed to under 50% of control at the concentration of 10 micromol/L. The growth inhibition was linked to the G1 phase cell cycle arrest through the up-expression of the cyclin-dependent kinase inhibitors, p21 and p27 proteins, and the hypophosphorylation of retinoblastoma protein. Troglitazone induced apoptosis by caspase-dependent (mitchondrial transmembrane potential decrease, cleavage of poly [adenosine diphosphate ribose] polymerase, 7A6 antigen exposure, Bcl-2 decrease, and activation of caspase 3) and caspase-independent (phosphatidylserine externalization) mechanisms. Our data suggest that ligand activation of PPARgamma by troglitazone or modified analogs of the thiazolidinedione class of drugs is a novel target for effective therapy against HCC, because of the significant antiproliferative and programmed cell death induction capabilities demonstrated by troglitazone.

Mechanism of Cell Growth Inhibition by Menadione

To demonstrate this hypothesis, logarithmically growinghuman hepatocarcinoma SK-Hep-1 cells were first incubat-ed with 100 µM of menadione for a period of 6, 12, or 24 h.Cells were then harvested and soluble extracts were assayedfor the expression levels of p53 using p53 monoclonalantibody. Interestingly, as shown in Figure 1 (lane 1),Western blotting showed that similar amounts of p53 wererecovered in immunoprecipitates from menadione-treatedand non-treated cells. In conjunction with the earlier obser-vation that the amount of p53 protein increases in responseto a variety of signals including DNA-damaging agentsthrough the production of reactive oxygen species (ROS),

Regulation of MCF-7 breast cancer cell growth by beta-estradiol sulfation.

Estrogen stimulation is an important factor in human breast cancer cell growth and development. Metabolism of beta-estradiol (E2), the major endogenous human estrogen, is important in regulating both the level and activity of the hormone in breast tissues. Conjugation of E2 with a sulfonate moiety is an inactivation process since the sulfate ester formed by this reaction can not bind and activate the estrogen receptor. In human tissues including the breast, estrogen sulfotransferase (EST, SULT1E1) is responsible for high affinity E2 sulfation activity. EST is expressed in human mammary epithelial (HME) cells but not in most cultured breast cancer cell lines, including estrogen responsive MCF-7 cells. Stable expression of EST in MCF-7 cells at levels similar to those detected in HME cells significantly inhibits cell growth at physiologically relevant E2 concentrations. The mechanism of cell growth inhibition involves the abrogation of responses observed in growth factor expression in MCF-7 cells following E2 stimulation. MCF-7 cells expressing EST activity did not show a decrease in estrogen receptor-alpha levels, nor a characteristic increase in progesterone receptor or decrease in transforming growth factor-beta expression upon exposure to 100 pM or 1 nM E2. The lack of response in these MCF-7 cells is apparently due to the rapid sulfation and inactivation of free E2 by EST. These results suggest that loss of EST expression in the transformation of normal breast tissues to breast cancer may be an important factor in increasing the growth responsiveness of preneoplastic or tumor cells to estrogen stimulation.

COMPARE: a web accessible tool for investigating mechanisms of cell growth inhibition

For more than 10 years the National Cancer Institute (NCI) has tested compounds for their ability to inhibit the growth of human tumor cell lines in culture (NCI screen). Work of Ken Paull [J. Natl. Cancer Inst. 81 (1989) 1088] demonstrated that compounds with similar mechanism of cell growth inhibition show similar patterns of activity in the NCI screen. This observation was developed into an algorithm called COMPARE and has been successfully used to predict mechanisms for a wide variety of compounds. More recently, this method has been extended to associate patterns of cell growth inhibition by compounds with measurements of molecular entities (such as gene expression) in the cell lines in the NCI screen. The COMPARE method and associated data are freely available on the Developmental Therapeutics Program (DTP) web site ( http://dtp.nci.nih.gov/). Examples of the use of COMPARE on these web pages will be explained and demonstrated.

A soluble form of the murine common γ chain is present at high concentrations in vivo and suppresses cytokine signaling

AbstractThe common gamma-chain (γc) is a component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 and is essential for their signal transduction. Western blotting and a newly established enzyme-linked immunosorbent assay detected substantial constitutive levels (50-250 ng/mL) of soluble γc (sγc) in sera of murine inbred strains. It was demonstrated that purified immune cells, such as T, B, and natural killer cells, and macrophages released this protein after activation. Transfection experiments with cDNA encoding the full-length γc showed that shedding of the transmembrane receptor led to the release of sγc. The shedding enzymes, however, appeared to be distinct from those cleaving other cytokine receptors because inhibitors of metalloproteases (eg, TAPI) did not influence sγc release. In vivo, superantigen-induced stimulation of T cells enhanced sγc serum concentrations up to 10-fold within 6 hours. Because these findings demonstrated regulated expression of a yet unknown molecule in the immune response, further experiments were performed to assess the possible function(s) of sγc. A physiological role of sγc was indicated by its capacity to specifically inhibit cell growth induced by γc-dependent cytokines. Mutational analysis revealed that the C-terminus and the WSKWS motif are essential for the cytokine inhibitory effect of the sγc and for binding of the molecule to cytokine receptor-expressing cells. Thus, competitive displacement of the transmembrane γc by excess sγc is the most likely mechanism of cell growth inhibition. It was implied that naturally produced sγc is a negative modulator of γc-dependent cytokines.

Effect of black and green tea polyphenols on c-jun phosphorylation and H(2)O(2) production in transformed and non-transformed human bronchial cell lines: possible mechanisms of cell growth inhibition and apoptosis induction.

The biological activities of theaflavin (TF), theaflavin gallate (TFG) and theaflavin digallate (TFdiG) from black tea and (-)-epigallocatechin 3-gallate (EGCG) and (-)-epigallocatechin (EGC) from green tea were investigated using SV40-immortalized (33BES) and Ha-ras gene transformed (21BES) human bronchial epithelial cell lines. Growth inhibition and cell viability were measured by trypan blue dye exclusion assay following 24 h treatment with the tea polyphenols. TFdiG, EGC and EGCG displayed comparable inhibitory effects on the growth of 21BES cells, with estimated IC(50) values of 22-24 microM. TFG exhibited a lower inhibitory activity (IC(50) 37 microM) and TF was even less effective (IC(50) 47 microM) in this cell line. A similar effect was also observed in 33BES cells. These results suggest that the gallate structure of theaflavins is important for growth inhibition. Exposure of 21BES cells to 25 microM TFdiG, EGC and EGCG for 24 h led to induction of cell apoptosis/death as determined by the Annexin V apoptosis assay. With TFdiG treatment cell death occurred early, and quickly peaked at 8-12 h. Morphological observations showed that TFdiG-treated cells appeared irregular in shape, with cytoplasmic granules, suggesting a cytotoxic effect. On the other hand, EGC and EGCG showed a lag phase before a rapid increase in apoptosis between 16 and 24 h, without any marked morphological changes, which was similar to that induced by H(2)O(2). TFdiG, EGC and EGCG induced similar amounts of H(2)O(2) formation in 21BES cells. Exogenously added catalase significantly prevented EGC- and EGCG-induced cell apoptosis, but did not prevent TFdiG-induced cell death, suggesting that H(2)O(2) is involved in the apoptosis induced by EGCG and EGC, but not in TFdiG-induced cell death. EGCG and TFdiG were shown to decrease c-jun protein phosphorylation in 21BES cells. Such inhibition is expected to result in lowered AP-1 activity, which may contribute to the growth inhibitory activity of tea polyphenols.

Involvement of hepatocyte epidermal growth factor receptor mediated activation of mitogen-activated protein kinase signaling pathways in response to growth inhibition by a novel K vitamin

Compound 5 (Cpd 5), a synthetic K vitamin analogue, or 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, is a potent inhibitor of epidermal growth factor (EGF)-induced rat hepatocyte DNA synthesis and induces EGF receptor (EGFR) tyrosine phosphorylation. To understand the cellular responses to Cpd 5, its effects on the EGF signal transduction pathway were examined and compared to those of the stimulant, EGF. Cpd 5 induced a cellular response program that included the induction of EGFR tyrosine phosphorylation and the activation of the mitogen-activated protein kinase (MAPK) cascade. EGFR tyrosine phosphorylation was induced by Cpd 5 in a time- and dose-dependent manner. Coimmunoprecipitation studies demonstrated that both EGF and Cpd 5 induced tyrosine phosphorylation of EGFR was associated with increased amounts of adapter proteins Shc and Grb2, and the Ras GTP-GDP exchange protein Sos, indicating the formation of functional EGFR complexes. Although EGFR phosphorylation was induced both by the stimulant EGF and the inhibitor Cpd 5, the timing and intensity of activation by EGF and Cpd 5 were different. EGF activated EGFR transiently, whereas Cpd 5 induced an intense and sustained activation. Cpd 5-altered cells had a decreased ability to dephosphorylate tyrosine phosphorylated EGFR, providing evidence for an inhibition of tyrosine phosphatase activity. Both EGF and Cpd 5 caused an induction of phospho-extracellular response kinase (ERK), which was also more sustained with Cpd 5. Moreover, whereas Cpd 5 induced a striking translocation of phosphorylated ERK from cytosol to the nucleus, no significant nuclear translocation occurred after stimulation with EGF. The data suggest that this novel compound causes growth inhibition through antagonism of EGFR phosphatases and consequent induction of EGFR and ERK phosphorylation. This is supported by experiments with PD 153035 and PD 098059, antagonists of phosphorylation of EGFR and MAP kinase kinase (MEK), respectively, which both antagonized Cpd 5-induced phosphorylation and the inhibition of DNA synthesis. These results imply a mechanism of cell growth inhibition associated with intense and prolonged protein tyrosine phosphorylation. Protein tyrosine phosphatases may thus be a novel target for drugs designed to inhibit cell growth.

Selective inhibition of human papillomavirus-induced cell proliferation by (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine.

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) is a nucleoside phosphonate analog which in its active diphosphorylated form is known to inhibit herpesvirus DNA polymerase. In this study, we have demonstrated that, in a dose-dependent manner, this compound irreversibly suppressed proliferation of cells infected with human papillomavirus (HPV), which does not possess a viral DNA polymerase. To elucidate the mechanism of cell growth inhibition, cell cycle indicator-regulator expression, thymidine incorporation, transcript levels of apoptosis factors, and anabolic products of HPMPC following drug treatment were evaluated. HPMPC treatment reduced WAF1 (p21) levels independent of those of p53, while proliferating cell nuclear antigen increased. However, in comparison to controls, HPMPC-treated cells displayed a decrease in thymidine incorporation, indicating an inhibition of host DNA polymerase activity. In normal primary keratinocytes, HPMPC predominantly accumulated in the form of the choline adduct HPMPCp-choline. However, in HPV type 16-transformed keratinocytes, HPMPCpp was the most abundant anabolic product, with little HPMPCp-choline having formed. The data imply that an unrecognized viral factor is modulating the conversion of nucleotides, including HPMPC, to the triphosphorylated form.

P27(kip1) acts as a downstream effector of and is coexpressed with the beta1C integrin in prostatic adenocarcinoma.

Integrins are a large family of transmembrane receptors that, in addition to mediating cell adhesion, modulate cell proliferation. The beta1C integrin is an alternatively spliced variant of the beta1 subfamily that contains a unique 48-amino acid sequence in its cytoplasmic domain. We have shown previously that in vitro beta1C inhibits cell proliferation and that in vivo beta1C is expressed in nonproliferative, differentiated epithelium and is selectively downregulated in prostatic adenocarcinoma. Here we show, by immunohistochemistry and immunoblotting analysis, that beta1C is coexpressed in human prostate epithelial cells with the cell-cycle inhibitor p27(kip1), the loss of which correlates with poor prognosis in prostate cancer. In the 37 specimens analyzed, beta1C and p27(kip1) are concurrently expressed in 93% of benign and 84%-91% of tumor prostate cells. Forced expression of beta1C in vitro is accompanied by an increase in p27(kip1) levels, by inhibition of cyclin A-dependent kinase activity, and by increased association of p27(kip1) with cyclin A. beta1C inhibitory effect on cell proliferation is completely prevented by p27(kip1) antisense, but not mismatch oligonucleotides. beta1C expression does not affect either cyclin A or E levels, or cyclin E-associated kinase activity, nor the mitogen-activated protein (MAP) kinase pathway. These findings show a unique mechanism of cell growth inhibition by integrins and point to beta1C as an upstream regulator of p27(kip1) expression and, therefore, a potential target for tumor suppression in prostate cancer.

Analogs of (A2'p)nA. Correlation of structure of analogs of ppp(A2'p)2A and (A2'p)2A with stability and biological activity.

Analogs of (A2'p)2A core and ppp(A2'p)2A were chemically synthesized and their susceptibility to phosphodiesterase degradation and ability to either activate an endonuclease or to inhibit cell growth were determined. The absence of the internal 3'-OH groups ((3'dA2'p)2A) resulted in a 5-fold increase in stability, but also in a 10-fold decrease in activity, as measured by (a) activation of an endonuclease in cell-free extracts and inhibition of protein synthesis in intact cells by the 5'-triphosphate species and (b) inhibition of DNA synthesis in synchronized cells by the core analogs. An uncharged derivative of this analog containing two methylphosphotriesters, although significantly more stable, was even less active. Additional deletion of the terminal 3'-OH ((3'd A2'p)23'dA) resulted in a further 6-fold increase in stability (30-fold overall increase in stability), as well as approximately a 2-fold increase in ability to inhibit cell growth, as compared to the natural 2'5' A core. The analog lacking a terminal 2'-OH as well as lacking the internal 3'-OH group ((3'dA2'p)22'dA) showed an overall 15-fold increased stability, yet showed very little activity in inhibiting cell growth. The most stable (120-fold increased overall stability) as well as most active analog was a xyloadenosine analog of 2'5' A core, (xyloA2'p)2xyloA. These results show that modification of the 3'-terminal OH appears to be most important in increasing 2'-5' A core stability as well as biological activity. However, the mechanism of cell growth inhibition by these 2'-5' A core analogs may involve pathways different from those utilized by the 2'-5' A-dependent endonuclease.

Inhibition of calcium and glucose uptake by murine leukemia L5178Y cells treated with antiserum.

Studies were performed to determine whether decreases in transport of calcium and glucose might be among the earliest changes triggered by the antigen-antibody reactions occurring on the cell surface of murine leukemia L5178Y cells after treatment with rabbit antisera. After treatment with antisera, in the absence of complement, these cells exhibited a decreased uptake of 45Ca, 2-deoxy[3H]glucose, and 3-0-methyl[3H]glucose. These changes occurred rapidly, within 2 minutes after the addition of antiserum, in contrast to the previously reported inhibitory effects of antiserum on DNA, RNA, and protein synthesis, which became demonstrable only after 4 to 8 hours. The kinetics of uptake of the radioactive substrates was biphasic, with a very rapid initial uptake followed by less rapid linear uptake. The precise mechanism of cell growth inhibition remains to be elucidated, but one of the initial effects of antiserum treatment may be a perturbation at the cell membrane such that transport of specific nutrients is decreased, resulting in the observed effects on macromolecular synthesis.