Effective chemopreventive activity of genistein against human breast cancer cells

Chemopreventive and cytotoxic effect of genistein against human breast cancer cell lines was investigated. Genistein inhibited cell proliferation in estrogen receptor-positive (MCF-7) and estrogen receptor-negative (MDA-MB-231) human breast carcinoma cell lines. Cytochrome P450 (CYP) 1A1-mediated ethoxyresorufin O-deethylase (EROD)activity was inhibited by genistein in a concentration-dependent manner. Genistein significantly inhibited 12-Otetradecanoylphorbol-13-acetate (TPA)-induced cyclooxygenase-2 activity and protein expression at the concentrations of 10 (p < 0.05), 25 (p < 0.05) and 50 mM (p < 0.01). In addition, ornithine decarboxylase (ODC) activity was reduced to 53.8 % of the control after 6 h treatment with 50 mM genistein in MCF-7 breast cancer cells. These results suggest that genistein could be of therapeutic value in preventing human breast cancer.     

Selective fraction of Scutellaria baicalensis and its chemopreventive effects on MCF-7 human breast cancer cells

Based on our previous observation, the whole Scutellaria baicalensis extract (SbE) did not show significant breast cancer cell inhibitory effect. In this study, we isolated a baicalin-deprived-fraction (SbF1) of Scutellaria baicalensis, and baicalin-fraction (SbF3), and evaluated their anti-breast cancer properties using MCF-7 cells. The content of four flavonoids in extract/fractions were determined using high performance liquid chromatography. Analytical data showed that in SbF1, the major constituents are baicalein and wogonin, while SbF3 only contains baicalin. The antiproliferative effects of fractions and SbE were assayed using modified trichrome stain method. SbF1 showed significant antiproliferative effect. Treated with 100 μg/ml of SbF1 for 72 h inhibited MCF-7 cell growth by 81.6%, while in the same treatment concentration, SbF3 increased cell growth by 22.6%. SbF1 was recognized as an active fraction of SbE. The effects of four flavonoids in SbE, scutellarin, baicalin, baicalein and wogonin, were determined, and data showed that baicalein and wogonin significantly inhibited MCF-7 cell growth. In contrast, in certain concentrations, scutellarin and baicalin increased cancer cell growth. The effects of SbF1 on cell cycle and apoptosis were assayed using flow cytometry. SbF1 arrested MCF-7 cells in S- and G2/M-phases, and significantly increased induction of cell apoptosis. These combined phytochemical and biological data provide evidence for further chemopreventive studies of the baicalin-deprived SbE on breast cancer.     

Cancer chemopreventive effects of lactic acid bacteria

Lactic acid bacteria (LAB) provide several potential health and nutritional benefits, including improving the nutritional value of food, controlling serum cholesterol levels, and controlling some types of cancer. Numerous in vitro, in vivo, human, and epidemiological studies have provided evidence of the chemopreventive effects of LAB on colon, bladder, liver, breast, and gastric cancers. These effects act via diverse mechanisms, including alteration of the gastrointestinal microflora, enhancement of the host's immune response, and antioxidative and antiproliferative activities. This review discusses the recent progresses on the chemopreventive effects of LAB on specific cancer types and the underlying molecular mechanisms.     

Evaluation of sphinganine and sphingosine as human breast cancer chemotherapeutic and chemopreventive agents

No comparative study of the effects of sphingolipid metabolites on proliferation and differentiation in normal human breast epithelial cells versus stem cells and tumorigenic cells has been reported. The purpose of this study was to evaluate the chemotherapeutic and chemopreventive potential of sphingoid bases (sphingosine and sphinganine) using a novel cell culture system of normal human breast epithelial cells (HBEC) developed from breast tissues of healthy women obtained during reduction mammoplasty (Type I HBEC with stem cell characteristics and Type II HBEC with basal epithelial cell phenotypes) and transformed tumorigenic Type I HBEC. The results show that sphinganine inhibited the growth and induced apoptosis of transformed tumorigenic Type I HBEC more potently than sphingosine (IC(50) for sphinganine 4 microM; sphingosine 6.4 microM). Both sphinganine and sphingosine at high concentrations (8-10 lM) arrested the cell cycle at G(2)/M. Sphinganine inhibited the growth and caused death of Type I HBEC more strongly than sphingosine. In comparison, Type II HBEC (normal differentiated cells) were less sensitive to the growth-inhibitory effects of sphingoid bases than Type I HBEC (stem cells) or transformed tumorigenic Type I HBEC, suggesting that sphingoid bases may serve as chemotherapeutic agents. At concentrations (0.05, 0.1, and 0.5 microM) that are below the growth-inhibitory range, sphingoid bases induced differentiation of Type I HBEC to Type II HBEC, as detected morphologically and via expression of a tumor suppressor protein, maspin, which is a marker of Type II HBEC. Thus, sphingoid bases may function as chemotherapeutic as well as chemopreventive agents by preferentially inhibiting cancer cells and eliminating stem cells from which most breast cancer cells arise.     

Identification of organoselenium compounds that possess chemopreventive properties in human prostate cancer LNCaP cells

The process of cancer development consists of three sequential stages termed initiation, promotion, and progression. Oxidative stress damages DNA and introduces mutations into oncogenes or tumor suppressor genes, thus contributing to cancer development. Cancer chemoprevention is defined to prevent or delay the development of cancer by the use of natural or synthetic substances. In the present study, we synthesized a series of organoselenium compounds and evaluated their possible chemopreventive properties in human prostate cancer LNCaP cells. Among 42 organoselenium compounds tested, two compounds, 3-selena-1-dethiacephem 13 and 3-selena-1-dethiacephem 14 strongly activated the Nrf2/ARE (antioxidant response element) signaling and thus markedly increased expression of heme oxygenase-1 (HO-1), a phase II antioxidant enzyme. Translocation of Nrf2 to the nucleus preceded HO-1 protein induction by two compounds. The intracellular ROS level was strongly reduced immediately after treatment with these compounds, showing that they are potent antioxidants. Finally, both compounds inhibited cell growth via cell cycle arrest. Our findings suggest that compounds 13 and 14 could not only attenuate oxidative stress through Nrf2/ARE activation and direct ROS scavenging but also inhibit cell growth. Thus, these compounds possess the potential as pharmacological agents for chemoprevention of human prostate cancer.     

Prolactin-inducible proteins in human breast cancer cells

The mechanism of action of prolactin in target cells and the role of prolactin in human breast cancer are poorly understood phenomena. The present study examines the effect of human prolactin (hPRL) on the synthesis of unique proteins by a human breast cancer cell line, T-47D, in serum-free medium containing bovine serum albumin. [35S]Methionine-labeled proteins were analysed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Treatment of cells with hPRL (1-1000 ng/ml) and hydrocortisone (1 microgram/ml) for 36 h or longer resulted in the synthesis and secretion of three proteins having molecular weights of 11,000, 14,000, and 16,000. Neither hPRL nor hydrocortisone alone induced these proteins. Of several other peptide hormones tested, only human growth hormone, a hormone structurally and functionally similar to hPRL, could replace hPRL in causing protein induction. These three proteins were, therefore, referred to as prolactin-inducible proteins (PIP). Each of the three PIPs was purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and specific antibodies were generated to them in rabbits. By immunoprecipitation and immunoblotting (Western blot) of proteins secreted by T-47D cells, it was demonstrated that the three PIPs were immunologically identical to one another. In addition, the 16-kDa and 14-kDa proteins (PIP-16 and PIP-14), and not the 11-kDa protein (PIP-11), incorporated [3H]glycosamine. Furthermore, 2-deoxyglucose (2 mM) and tunicamycin (0.5 micrograms/ml), two compounds known to inhibit glycosylation, blocked the production of PIP-16 and PIP-14, with a concomitant increase in the accumulation of PIP-11. These results indicate PIP-16 and PIP-14 are glycosylated variants of PIP-11. Finally, in vitro translation of poly(A)+ messenger RNA followed by immunoprecipitation revealed a 12.5-kDa protein, possibly the precursor form of PIPs. In addition, T-47D cells treated with hPRL plus hydrocortisone contained 10-fold more mRNA for PIPs than control cells, suggesting that the hormones' action is at the level of gene expression. Our finding represents a first demonstration of prolactin regulation of gene expression in human target cells. The human breast cancer cells, T-47D, appear to be an excellent model to afford future studies on the molecular action of prolactin and on the possible role of prolactin in human breast cancer.     

Autophagy promotes radiation-induced senescence but inhibits bystander effects in human breast cancer cells

Ionizing radiation induces cellular senescence to suppress cancer cell proliferation. However, it also induces deleterious bystander effects in the unirradiated neighboring cells through the release of senescence-associated secretory phenotypes (SASPs) that promote tumor progression. Although autophagy has been reported to promote senescence, its role is still unclear. We previously showed that radiation induces senescence in PTTG1-depleted cancer cells. In this study, we found that autophagy was required for the radiation-induced senescence in PTTG1-depleted breast cancer cells. Inhibition of autophagy caused the cells to switch from radiation-induced senescence to apoptosis. Senescent cancer cells exerted bystander effects by promoting the invasion and migration of unirradiated cells through the release of CSF2 and the subsequently activation of the JAK2-STAT3 and AKT pathways. However, the radiation-induced bystander effects were correlated with the inhibition of endogenous autophagy in bystander cells, which also resulted from the activation of the CSF2-JAK2 pathway. The induction of autophagy by rapamycin reduced the radiation-induced bystander effects. This study reveals, for the first time, the dual role of autophagy in radiation-induced senescence and bystander effects.     

Tranilast enhances the anti-tumor effects of tamoxifen on human breast cancer cells in vitro

Background

Tamoxifen is the most widely used anti-estrogen for the treatment of breast cancer. Studies show that the combination therapy with other substances that helps the activity of tamoxifen. The objective of this study was to evaluate the effect of tamoxifen when used in combination with tranilast on human breast cancer cells.

Results

Two MCF-7 and MDA-MB-231 human breast cancer cell lines were treated with tamoxifen and/or tranilast. The cell viability and cytotoxicity was assessed using MTT and LDH assays; the apoptotic effects were examined by TUNEL assay, acridine orange/ethidium bromide staining and DNA laddering, also the expression levels of bax and bcl-2 genes were detected by real-time RT-PCR. The mRNA expression of TGF-β ligands and receptors examined using real-time RT-PCR and TGF-β1 protein secretion levels were also evaluated by ELISA assay. Inhibitory effect of these drugs on invasion and metastasis were tested by wound healing and matrigel invasion assay.We found that combination of these drugs led to a marked increase in growth and proliferation inhibition compared to either agent alone. Furthermore, bax and bcl-2 affected by tamoxifen and/or tranilast and resulted in a significant increase in bax and decrease in bcl-2 mRNA expression. In addition, treatment with tamoxifen and/or tranilast resulted in significant decreased in TGF-β1, 2, 3, TGF-βRI and II mRNA and TGF-β1 protein levels while TGF-βRIII mRNA level was increased and invasion was also inhibited.

Conclusions

These findings indicate that tranilast, by synergistic effect, enhances the activity of tamoxifen and the TGF-β pathway is a target for this combination therapy, therefore; we propose that this combined treatment may be suitable selection in prevention of breast cancer.     

Estrogenic effects of two derivatives of icariin on human breast cancer MCF-7 cells

The aims of the present study were to determine the estrogenic activities of icariin (ICA) and its derivatives and their structure–estrogenic activity relationship. Therefore, icaritin (ICT) and desmethylicaritin (DICT) were derived from ICA. The estrogenic activities of ICA, ICT and DICT were examined by cell proliferation and progestogen receptor mRNA expression of estrogen-receptor-positive MCF-7 cells. Current studies exhibited that ICT and DICT both markedly enhanced the proliferation of MCF-7 cells; as compared to estradiol (100%), their relative proliferative effects (RPE) were 90% and 94%, respectively. Cell proliferation induced by ICT and DICT was completely antagonized by ICI182,780. ICT and DICT increased progestogen receptor (PR) at mRNA levels at 48 h after treatment, although the effects were not as prominent as 17β-estradiol (E₂). These phenomena were not observed with ICA. Results demonstrate that ICT and DICT (nonconjugated forms) possess estrogen-like activity; however, ICA appears to have no estrogenicity in the MCF-7 cell line model in vitro.     

Cell cycle effects of iron depletion on T-47D human breast cancer cells

T-47D human breast cancer cells grown in culture medium containing low concentrations of fetal calf serum (FCS) proliferated very slowly, with an accumulation of cells in the G2 phase of the cell cycle, increased polyploid cells, and increased expression of transferrin receptors. Cell proliferation was stimulated by the addition of human transferrin or ammonium ferric citrate to the medium. Growth inhibition and accumulation of G2-phase cells could also be produced in T-47D cells grown in medium containing 10% FCS by the addition of the iron chelator, desferrioxamine. It is concluded that cellular deprivation of iron and/or transferrin is the major cause of reduced proliferation rates and G2-phase arrest which accompany the culture of these cells in medium supplemented with low concentrations of FCS.     

Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice

Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγ^null (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4⁺ T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy.     

Tamoxifen induced membrane alterations in human breast cancer cells

It was previously shown that proliferation of CG-5 cells, an estrogen supersensitive variant of the MCF-7 cell line, is inhibited by tamoxifen starting three days after addition to culture medium. In this paper we report here on the effect of tamoxifen on CG-5 cell membrane morphology using scanning electron microscope. The addition of the antiestrogen to CG-5 cells causes marked alterations of cell surface, consisting of reduction in the length and the number of microvilli. Moreover, cells appear flattened and of reduced thickness. These modifications are more pronounced in cells plated at low density, but they are observed also in nearly confluent monolayers. In this case alterations are more evident in the center of cell islands, while at the periphery there is a certain mobility as reflected by the persistence of few microvilli and philopodia. These early effects of tamoxifen on cell membrane raise the possibility that parallel modifications in membrane function may occur which are responsible for alterations impairing transport mechanisms.     

Mitoxantrone affects topoisomerase activities in human breast cancer cells

The effects of mitoxantrone, an antineoplastic DNA intercalator, on topoisomerase I and II were studied in two human breast cancer cell lines. A large increase of topoisomerase I activity was found when cells were exposed to various doses of mitoxantrone. Maximal effect was achieved with 20 and 40 ng/mL in T47D and MCF-7 cells respectively. The enhancement on topoisomerase I activity seemed to be reversible, to be dependent on time of exposure to the drug and to require cellular integrity. Type II topoisomerase was inhibited in T47D cells after treatment for one hour with 10 ng/mL of mitoxantrone and enzyme activity was undetectable at higher doses (40 ng/mL). This inhibitory effect did not take place in vitro unless the concentration of the intercalator was increased to 400-500 ng/mL.     

Lectin of Abelmoschus esculentus (okra) promotes selective antitumor effects in human breast cancer cells

The anti-tumor effects of a newly-discovered lectin, isolated from okra, Abelmoschus esculentus (AEL), were investigated in human breast cancer (MCF7) and skin fibroblast (CCD-1059 sk) cells. AEL induced significant cell growth inhibition (63 %) in MCF7 cells. The expression of pro-apoptotic caspase-3, caspase-9, and p21 genes was increased in MCF7 cells treated with AEL, compared to those treated with controls. In addition, AEL treatment increased the Bax/Bcl-2 ratio in MCF7 cells. Flow cytometry also indicated that cell death (72 %) predominantly occurred through apoptosis. Thus, AEL in its native form promotes selective antitumor effects in human breast cancer cells and may represent a potential therapeutic to combat human breast cancer.     

Apoptotic and antiproliferative effects of silk protein sericin conjugated-AgNO3 nanoparticles in human breast cancer cells

Bombyx mori silk sericin is a globular-like protein that is used as an antioxidant, antibacterial, and antitumor agent. In this current research, we isolated sericin by degumming process and formation of sericin-AgNO₃ NPs confirmed by UV–vis spectra, SEM, EDX, FTIR, and XRD patterns. The sericin and sericin-AgNO₃ NPs mediated changes in human breast cancer cells were determined. The antiproliferative activity of sericin-AgNO₃ NPs was analyzed by MTT dye reduction assay. Alterations at molecular levels were investigated by qRT-PCR, while apoptotic effects were studied by nuclear DNA staining. After 72 h treatment, sericin and sericin-AgNO₃ NPs showed significant antiproliferative effects in MDA-MB-231 (26 %) and MCF-7 (41 %) cells. Expression modification showed prominent stimulation of cell cycle arrest and stress related genes such as cyclin-dependent kinase inhibitors (CDKN1A, CDKN1B), and GADD family genes. RT-PCR results of the GADD family include GADD45A, B, G, 34, 153 and cyclin-dependent kinase inhibitors (CDKN1A, 1B) showed pronounced induction of 3.1 to 19.8-folds in MCF-7 cell line while induction in MDA-MB-231 cell line was 2.5 to 34.3-folds. Nuclear DAPI staining showed significant induction of apoptosis and nuclear fragmentation in MDA-MB-231 cells at a concentration of 1 mg/mL for both sericin and sericin-AgNO₃ NPs. Meanwhile, in case of MCF-7 cells, after treatment with sericin and sericin-AgNO₃ NPs (1 mg/mL), the cells changed into a round shape and lost their original spindle outlook in dose-dependent manners. We concluded that sericin-AgNO₃ NPs have significant antiproliferative, apoptosis, and genetic profiling effects in both breast cancer cell lines at the highest concentration.     

The kinetics of methotrexate polyglutamation in human breast cancer cells

The polyglutamation kinetics of methotrexate (MTX) in MCF-7 human breast cancer cells have been formulated mathematically. The model takes account of glutamation and hydrolysis kinetics up through the pentaglutamate level, increased synthesis of dihydrofolate reductase following exposure to drug, reversible tight-binding to reductase, and membrane transport of polyglutamates. The glutamation, hydrolysis, and efflux parameters have been determined from fits to experimental MTX polyglutamate uptake and efflux data. The preferred substrate for folypolyglutamyl synthase in the intact cell appears to be MTX diglutamate, on average being two to three times as reactive as either the parent drug or the triglutamate. Hydrolysis rate constants range from 0.03 to 0.19 h-1, but no clear trend with chain length is observable given the large uncertainty of each parameter estimate. However, the efflux of MTX polyglutamates from MCF-7 cells does show a trend with chain length decreasing with increasing length as expected. The best characteristic time of MTX diglutamate efflux is 4.1 h, about one-third that of the higher polyglutamate species, in agreement with observations on the MDA.MB.436 breast cancer cell line. The model shows quantitative agreement with the fraction of MTX polyglutamates found still to be bound to reductase in MCF-7 cells following 24 h of efflux, and qualitative agreement with the time dependence of bound MTX-polyglutamate concentration profiles obtained on the ZR-75 breast cancer line.     

Anti-growth factor activities of benzothiophenes in human breast cancer cells

We have tested the effects of two Eli-Lilly compounds, LY 117, 018 and raloxifene, on E2-regulated and IGF-I-induced proliferation or AP-1 activity in human breast cancer cells. We now demonstrate that both molecules have strong antiestrogenic and anti-growth factor inhibitory effects in MCF7 cells. They were as potent as ICI 182, 780 and more efficient than OH-Tam to prevent estradiol action whereas their inhibition on IGF-I stimulation was less than with ICI 182, 780 and equivalent to that of OH-Tam. Moreover, raloxifene was the most efficient molecule to prevent IGF-I-induced AP-1 activity, with a significant effect observed with a concentration as low as 5 × 10⁻¹¹ M in the presence of IGF-I alone. Similar dose–response curves were obtained with a combined treatment of IGF-I and E2 with a 2 log shift. Their action on IGF-I-induced proliferation was completely abrogated in MCF7 transfectants in which the expression of an antiestrogen-regulated protein tyrosine phosphatase, PTPL1, was abolished by antisense RNA transfection. Accordingly, they were both able to dose-dependently regulate the expression of PTPL1 and to interfere with the PI3-K/Akt pathway by drastically decreasing Akt phosphorylation exclusively in wild-type PTPL1 expressing cells.

Our data altogether demonstrate that raloxifene has a potent inhibitory effect on IGF-I action, with a drastic effect on AP-1 triggered responses as well as on Akt phosphorylation, suggesting that it might be a useful therapeutic agent in tumors in which these signalling pathways become constitutively active.