Evaluation of Antiproliferative Activities and Action Mechanisms of Extracts from Two Species of Ganoderma on Tumor Cell Lines

Background. N-Isopentenyladenosine (iPA) is a member of the cytokinins, a family of plant hormones that regulate plant cell growth and differentiation. iPA is present in mammalian cells in a free form, as a mononucleotide in the cytoplasm, or in a tRNA-bound form. Kinetin Riboside (KR) is devoid of cyclin-dependent kinase inhibitory activity and displayed potent antiproliferative activity against various human cancer cell lines and induced apoptosis in human myeloid leukemia cells. Earlier research has demonstrated that KR antiproliferative and apoptogenic activities are antagonized by pharmacological inhibitors of adenosine kinase (ADK), suggesting that KR bioactivation through metabolic conversion into the nucleotide form is essential for KR cytotoxicity. Aims. Apoptotic mechanism of iPA and KR led us to study in details the dosedependent cell cycle arrest and apoptogenic effect of iPA and KR on MCF-7 breast and HCT-15 colon cell lines. On the other hand, cell cycle progression or apoptosis can be affected by activation of cell cycle checkpoints in response to DNA damage. We have also studied the interaction of iPA with DNA& BSA as a complementary information related to iPA antiproliferative activity. Methods. Human breast cancer MCF-7 and HCT-15 colon cencer cells were supplied from ATCC. Growth activity of iPA and KR in vitro was evaluated by the Sulforhodamine B (SRB) assay. Apoptosis and cell cycle profile were assessed by flow cytometry. To explore the structural changes of macromoleules on addition of ligands, UV–vis absorbance spectra of macromoleules were measured at different concentrations of ligands. Results. We have calculated an IC50 of 12.2 μmol/L for iPA and IC50 of 15 μmol/L for KR against MCF-7 and same IC50 of 2.5 μmol/L for both iPA and KR against HCT-15. The cell morphology of treated cells was affected by iPA and KR treatment and loss of adhesion, rounding, cell shrinkage and detachment from the substratum. The MCF-7 cell cycle analysis by flow cytometry showed that there was a prominent increase in the amount of sub-G1/G0 phase by iPA treatment. At the concentration of 5 μM, KR led to decrease in the percentage of cells in G0/ G1 phase, which and increase percentage of cells in S and G2/M phase. Our result from structural analysis showed interaction of iPA with DNA and the binding constant value KiPA–DNA=4.4 x 10 M together with the shift of UV-vis absorbance suggest that iPA interacts at DNA surface. The BSA binding studies showed that iPA is located along the polypeptide chains with overall affinity constant of KiPABSA=4.9 x 10 M and these spectral changes were caused by compound–BSA complex formation, in which compound iPA was strongly bound at the hydrophobic positions of the BSA supported by the hydrophobic interaction. Conclusion. We concluded that the iPA and KR have cytotoxic effect on human MCF-7 and HCT-15 cells with loss of adhesion, rounding, cell shrinkage and detachment from the substratum in treated cells. Cell cycle analysis showed an indication of the inhibition of cell growth through a mechanism of apoptosis. Our result from structural analysis showed interaction of iPA with DNA suggest that iPA interacts at DNA surface and research is required in order to demonstrate that this surface-binding effect may be related to DNA damage in MCF-7 cells.

Purpose : This study aimed to evaluate the possible cytotoxic activity of the essential oil of the Eugenia uniflora leaves on normal and tumoral human cells by colorimetric cell viability assay, based on the use of tetrazolium salt (XTT). Methods : To achieve the cytotoxicity it was used normal human lung fibroblast cell line GMO7492A and the evaluation of antiproliferative activity was performed on three tumor cell lines, as follows: human glioblastoma (MO59J), human cervical adenocarcinoma (HeLa) and human breast adenocarcinoma (MCF-7). For the determination of cytotoxic concentration to the normal line, 12 concentrations were evaluated being from 2.44 to 5000 µg/mL. In the evaluation of antiproliferative activity were tested 8 different concentrations of the extract (3.91 to 500 µg/mL). Results : The results in the normal line GM07492A showed that concentrations higher or equal to 39.1 µg/mL are significantly different of the negative control. In the evaluation of antiproliferative activity on tumor cell lines MO59J, HeLa and MCF-7 was observed that the concentration of 125 µg/mL showed a cytotoxic effect on these lines being significantly different from the negative control. The results from the evaluation of the antiproliferative activity in different tumor cell lines of E. uniflora oil was not selective for the tested cell types. Conclusion : E. uniflora oil did not show cytotoxic activity in concentrations lower than 39.1 µg/mL, however, the values found to IC 50 for tumor cells were superior, concluding that the oil has no selectivity for tumor cells tested.

Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stress-activated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.

13-cis Retinoic Acid Induces Apoptosis and Cell Cycle Arrest in Human SEB-1 Sebocytes

Antiproliferative activity of goniothalamin enantiomers involves DNA damage, cell cycle arrest and apoptosis induction in MCF-7 and HB4a cells

To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.

Aims. Study the effect of Abnormal Savda Munziq (ASMq) ethanol extract on the proliferation, apoptosis, and correlative gene, expression in colon cancer cells (Caco-2) to elucidate the molecular mechanisms responsible for the anticancer property of Abnormal Savda Munziq. Materials and Methods. ASMq ethanol extract was prepared by a professional pharmacist. Caco-2 cells were treated with different concentration of ASMq ethanol extract (0.5–7.5 mg/mL) for different time intervals (48 and 72 h). Antiproliferative effect of ASMq ethanol extract was determined by MTT assay; DNA fragmentation was determined by gel electrophoresis assay; cell cycle analysis was detected by flow cytometer; apoptosis-related gene expression was detected by RT-PCR assay. Results. ASMq ethanol extract possesses an inhibition effect on Caco-2 cells proliferation, induction of cell apoptosis, cell cycle arrest in sub-G1 phase, and downregulation of bcl-2 and upregulation of Bax gene expression. Conclusion. The anticancer mechanism of ASMq ethanol extract may be involved in antiproliferation, induction of apoptosis, cell cycle arrest, and regulation of apoptosis-related gene expression such as bcl-2 and Bax activity pathway.

A number of proteins involved in cell growth control, including members of the Ras family of GTPases, are modified at their C terminus by a three-step posttranslational process termed prenylation. The enzyme isoprenylcysteine carboxylmethyl-transferase (Icmt) catalyzes the last step in this process, and genetic and pharmacological suppression of Icmt activity significantly impacts on cell growth and oncogenesis. Screening of a diverse chemical library led to the identification of a specific small molecule inhibitor of Icmt, cysmethynil, that inhibited growth factor signaling and tumorigenesis in an in vitro cancer cell model (Winter-Vann, A. M., Baron, R. A., Wong, W., dela Cruz, J., York, J. D., Gooden, D. M., Bergo, M. O., Young, S. G., Toone, E. J., and Casey, P. J. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 4336-4341). To further evaluate the mechanisms through which this Icmt inhibitor impacts on cancer cells, we developed both in vitro and in vivo models utilizing PC3 prostate cancer cells. Treatment of these cells with cysmethynil resulted in both an accumulation of cells in the G(1) phase and cell death. Treatment of mice harboring PC3 cell-derived xenograft tumors with cysmethynil resulted in markedly reduced tumor size. Analysis of cell death pathways unexpectedly showed minimal impact of cysmethynil treatment on apoptosis; rather, drug treatment significantly enhanced autophagy and autophagic cell death. Cysmethynil-treated cells displayed reduced mammalian target of rapamycin (mTOR) signaling, providing a potential mechanism for the excessive autophagy as well as G(1) cell cycle arrest observed. These results identify a novel mechanism for the antitumor activity of Icmt inhibition. Further, the dual effects of cell death and cell cycle arrest by cysmethynil treatment strengthen the rationale for targeting Icmt in cancer chemotherapy.

A High Omega-3 Fatty Acid Diet Mitigates Murine Pancreatic Precancer Development

Objective To investigate the synergistic proliferation-inhibiting and apoptosis-inducing effects of recombinant human-derived interleukin-24 (rhIL-24) and recombinant human-derived decorin (rhDCN) on human hepatocellular carcinoma cells HepG2.Methods Cellular growth and morphological changes of HepG2 cells were observed under the inverted microscope at 48 h after being transiently transfected with pcDNA3.1 (+)-IL-24 and pcDNA3.1 (+)-DCN by Lipofectamine.The proliferation-inhibiting effects of IL-24 and DCN on HepG2 cells,respectively and jointly,were observed with MTT assay at 24 h,48 h and 72 h post-transfection.Apoptosis and cell-cycle of HepG2 cells were analyzed by flow cytometry at 48 h post-transfection.Results Compared to control groups,the cells of target gene groups presented typically changes of proliferation inhibition and apoptotic morphology,which occurred obviously in co-transfection group.The results of MTT assay showed that at 48 h and 72 h post-transfection,the profiferation-inhibiting rates in the group of cells co-transfected with IL-24 and DCN were (31.88±6.57) ％ and (36.83±3.76) ％,respectively,displaying significant difference with those of other groups (P ＜ 0.01).The results of flow cytometry showed that IL-24 and DCN can induce HepG2 cells apoptosis to some extent.Compared to the early apoptosis rate of cells of control groups,plasmid (2.98±0.72) ％,blank cell (3.50±0.92) ％,IL-24 (20.01±1.08) ％ and DCN (22.20±0.91) ％,a statistically remarkable apoptosis rate,(32.56±0.90) ％,can be seen in the group of cells treated with IL-24 and DCN jointly (P ＜ 0.01).The result of cell cycle analysis revealed that,compared to control groups,the proportion of cells was higher in the phase of G2/M in the IL-24 group (11.24±0.35) ％ and in the phase of G0/G1 in the DCN group (77.93±0.67) ％.The proportions of cells in the phases of G2/M increased to (71.36±0.60) ％ and that of G0/G1 statistically increased to (10.39±0.67) ％ in the group of cells co-transfected with IL-24 and DCN (P ＜ 0.01).Conclusions Combinatorial treatment of HepG2 cells with IL-24 and DCN can exert stronger synergistic proliferation-inhibiting effect and apoptosisinducing activity-in comparison to single therapies.IL-24 and DCN can induce cell cycle arrest on HepG2 cells,occurred in the phase of G2/M and G0/G1,respectively.Promoting effect of cell cycle arrest in the phase of both G2/M and G0/G1 can be seen on HepG2 cells co-transfected with IL-24 and DCN,which maybe the possible mechanism of the synergistic proliferation-inhibiting and apoptosis-inducing effect. Key words: Liver neoplasms; Interleukin-24; Decorin; Cell, HepG2; Gene therapy; Cell proliferation; Apoptosis; Cell cycle arrest

Adaphostin (NSC680410), a small molecule congener of tyrphostin AG957, has been demonstrated previously to have significant anti-proliferative effects in several leukemia models. However, this effect of adaphostin in adherent cells/solid tumor models has not been examined. In this study, we investigated the anti-proliferative effects of adaphostin in the human prostate cancer cell line PC-3. Specifically, we explored the potential molecular mechanism(s) by which adaphostin elicits its anti-proliferative effect(s). We demonstrate that adaphostin inhibits the proliferation of PC-3 cells by inducing a G(1) phase cell cycle arrest. This adaphostin-induced G(1) arrest was associated with an increase in the expression of p21 and p27 and a decrease in the expression of G(1)-specific cyclins (cyclin A, D1, and D3) and cyclin-dependent kinases 4 and 6. Consequently, a dramatic decrease in the phosphorylation of retinoblastoma protein was also observed. Additionally, we found that adaphostin treatment induced a decrease in the phosphorylation of nucleophosmin, a major nuclear phosphoprotein, and that this decreased phosphorylation was a result of the p21- and p27-mediated inactivation of cyclin E-cyclin-dependent kinase 2 complex kinase activity. Furthermore, we have determined that the adaphostin-mediated cell cycle arrest of PC-3 cells is dependent upon activation of the p38 MAPK. We also demonstrate that the hepatocyte growth factor receptor-c-Met is involved in the adaphostin-mediated signaling events that regulate p38 MAPK. Taken together, these results identify for the first time a signaling cascade of adaphostin-mediated G(1) phase-specific cell cycle arrest in PC-3 cells. These findings suggest that the tyrphostin member has a broader spectrum of activity than originally predicted.

Genes encoding growth-inhibitory proteins are postulated to be candidate tumor suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumors. Here we report the cloning and functional characterization of a novel human bone marrow stromal cell (BMSC)-derived growth inhibitor (BDGI) by large scale random sequencing of a human BMSC cDNA library. Human BDGI cDNA encodes a 477-amino acid residue protein that shares high homology with rat and mouse pregnancy-induced growth inhibitors. The C-terminal of BDGI is identical to a novel human pregnancy-induced growth inhibitor, OKL38. BDGI is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved family of BDGI-like proteins. BDGI overexpression inhibits the proliferation, decreases anchorage-dependent growth, and reduces migration of MCF-7 human breast cancer cells, whereas down-regulation of BDGI expression promotes the proliferation of MCF-7 and HeLa cervix epitheloid carcinoma cells. Interestingly, the inhibitory effect of BDGI on MCF-7 cells is more potent than that of OKL38. We demonstrate that BDGI induces cell cycle arrest in S phase and subsequent apoptosis of MCF-7 cells, which is likely to account for the antiproliferative effects of BDGI. This process may involve up-regulation of p27Kip1 and down-regulation of cyclin A, Bcl-2, and Bcl-xL. The inhibitory effect of BDGI on cell proliferation and the induction of apoptosis were also observed in A549 lung cancer cells but not HeLa cells. These results indicate that BDGI might be a growth inhibitor for human tumor cells, especially breast cancer cells, possibly contributing to the development of new therapeutic strategies for breast cancer.

A series of pyrazolone compounds with different substitution patterns have been synthesized using microwave-assisted methods and evaluated their in vitro antiproliferative activity against human lung adenocarcinoma cell lines (A549 and NCI-H522). Among the tested compounds, the pyrazolone P7 exhibited high antiproliferative activity against both A549 and NCIH522 cancer cell lines while being 10 times less cytotoxic to non-cancerous cells. Moreover, our compounds P7 and P11 exhibited higher antiproliferative activity and selectivity against A549 and NCIH522 cells compared with the clinically approved drugs Afatinib and Gefitinib. The cell cycle analysis showed that the compound P7 and P11 arrests the cell cycle at G0/G1 phase, whereas the compounds P13 and P14 involved in G2/M phase arrest. The results from antiproliferative activity screening, cell cycle analysis, and kinase profiling indicate that the suitably substituted 1,3-diarylpyrazolones exhibit high antiproliferative activity against non-small cell lung cancer cells.

Background:Physcion is an anthraquinone from rhubarb (rhizomes of Rheum tanguticum) and has been reported to have anti-inflammatory, hepatoprotective, antifungal, and anti-cancer activities. However, the growth inhibitory activity against human cancer cells and the underlying molecular mechanisms have been poorly determined. This study was designed to investigate the anti-proliferative activity of physcion by induction of cell cycle arrest and apoptosis in human MDA-MB-231 triple negative breast cancer cell line.Methods:MDA-MB-231 cells were treated with physcion, and the anti-proliferative activity was evaluated by the sulforhodamine B assay. The mechanisms of action for the growth inhibitory activity of physcion were evaluated by flow cytometry for cell cycle distribution, and by Western blot for the assessment of potential target proteins.Results:Physcion showed a significant anti-proliferative activity against MDA-MB-231 human breast cancer cells. Flow cytometric analysis indicated that physcion markedly induced the accumulation of cells in the G0/G1 phase and the increase of cell population in the sub-G1 phase. The G0/G1 cell cycle arrest by physcion was associated with the down-regulation of Cyclin D1, Cyclin A, CDK4, CDK2, c-Myc and phosphorylated Rb protein expressions. The increase of sub-G1 peak by physcion was closely correlated with the induction of apoptosis, which was confirmed by the induction of cleaved poly-(adenosine diphosphate ribose) polymerase, activation of Caspases, and suppression of Bid and Bcl-2 expression.Conclusions:The induction of G0/G1 cell cycle arrest and apoptosis might be one of the plausible mechanisms of actions for the anti-proliferative activity of physcion in human breast cancer cells.

Design, synthesis and evaluation of antiproliferative and antitubulin activities of 5-methyl-4-aryl-3-(4-arylpiperazine-1-carbonyl)-4H-1,2,4-triazoles