Synergistic interaction of growth factors and albumin in regulating estradiol synthesis in breast cancer cells.

Estradiol 17 beta-hydroxysteroid dehydrogenase acts to convert estrone to the biologically active estrogen, estradiol, in breast tumors and MCF-7 breast cancer cells in vitro. In this study we have examined the ability of albumin to influence the effect of growth factors (insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha)) and cytokines (interleukin (IL)-1, IL-6) on estradiol 17 beta-hydroxysteroid dehydrogenase activity in MCF-7 cells. IGF-I (80 ng/ml) or albumin (30 micrograms/ml) stimulated estradiol 17 beta-hydroxysteroid dehydrogenase activity by 144% and 102% (p less than 0.01). The combination of IGF-I and albumin, however, produced a marked (704%) synergistic stimulation of estradiol 17 beta-hydroxysteroid dehydrogenase activity. EGF or TGF alpha failed to stimulate estradiol 17 beta-hydroxysteroid dehydrogenase activity and no synergism with albumin was detected. IL-1 (10 ng/ml), but not IL-6, also stimulated estradiol 17 beta-hydroxysteroid dehydrogenase activity and acted synergistically with albumin to stimulate enzyme activity. MCF-7 cells were shown to specifically bind 125I-albumin and binding is increased by pretreatment of cells with IGF-I (80 ng/ml) for 48 h. It is concluded that the synergism that results from treating MCF-7 cells with albumin and IGF-I may result from increased albumin uptake and subsequent biological effect.     

Effects of genistein and synergistic action in combination with eicosapentaenoic acid on the growth of breast cancer cell lines

Genistein, a prominent isoflavone in soy products, produced dose- and time-dependent in vitro growth inhibition at high concentrations (at least 185 microM) with an IC50 of 7.0-274.2 microM after 72 h incubation in four breast cancer cell lines (DD-762, Sm-MT, MCF-7 and MDA-MB-231) and one breast epithelial cell line (HBL- 100) of human and animal origin; it stimulated estrogen-receptor-positive MCF-7 cells at low concentrations (3.7 nM-37 microM). Genistein-exposed cells underwent apoptosis, confirmed by G2/M arrest followed by the appearance of a sub-G1 fraction in cell-cycle progression, and by a characteristic cell ultrastructure. The apoptosis cascade was due to up-regulation of Bax protein, down-regulation of Bcl-XL protein, and activation of caspase-3. Genistein acted in synergism with eicosapentaenoic acid (EPA), a fish oil component, on human breast cancer MCF-7 cells (genistein > 93.2 microM and EPA > 210.9 microM) and on MDA-MB-231 cells (genistein > 176.1 microM and EPA > 609.3 microM). Dietary intake of genistein in combination with EPA may be beneficial for breast cancer control.     

The regulation of apoptosis by activin and transforming growth factor-beta in early neoplastic and tumorigenic ovarian surface epithelium

Most ovarian neoplasms arise from the ovarian surface epithelium (OSE), and multiple growth factors have been implicated to influence the transformation from OSE. The present study was performed to investigate the role of activin and transforming growth factor-beta (TGFbeta) in normal and neoplastic OSE cells. An immortalized OSE cell line (IOSE-29) was generated from normal OSE by transfecting simian virus 40 large T antigen and was rendered tumorigenic after subsequent transfection with the E-cadherin gene (IOSE-29EC). The activin/inhibin subunits and activin receptors were expressed at both messenger ribonucleic acids and protein levels in these cells, suggesting that activin may have an autocrine role in neoplastic OSE cells. Treatments with activin (1-100 ng/mL) resulted in a significant decrease in cell proliferation in both IOSE-29 and IOSE-29EC cells, although we have shown that it stimulated the growth of ovarian cancer cells and had no effect on normal OSE. This inhibitory effect was attenuated with cotreatment with follistatin. Treatment with TGFbeta (0.1-10 ng/mL) also significantly decreased the proliferation of normal, IOSE-29, and IOSE-29EC cells in a dose-dependent manner. In addition, treatments with both activin and TGFbeta resulted in an increase in DNA fragmentation in IOSE-29EC cells in a dose-dependent manner. This apoptotic effect of activin was attenuated by cotreatment with follistatin. Treatment with TGFbeta (1 and 10 ng/mL) resulted in a significant decrease in Bcl-2 protein (up to 50%) in IOSE-29EC, whereas no difference was observed in Bax protein levels. Therefore, down-regulated Bcl-2 by TGFbeta may eventually induce apoptosis in IOSE-29EC cells. In contrast, no difference was observed in Bax and Bcl-2 protein expression after treatment with activin. In conclusion, the present study indicates that activin and TGFbeta inhibited growth and induced apoptosis in early neoplastic (IOSE-29) and tumorigenic OSE (IOSE-29EC) cells. Furthermore, antiapoptotic Bcl-2 protein was down-regulated by TGFbeta, whereas no difference was produced in Bax protein by activin or TGFbeta treatment or in Bcl-2 protein by activin. These results suggest that activin and TGFbeta may play a role in growth inhibition and induction of apoptosis in early neoplastic and tumorigenic stage of ovarian cancer.     

Intracrine PTHrP protects against serum starvation-induced apoptosis and regulates the cell cycle in MCF-7 breast cancer cells.

PTHrP is secreted by breast cancer cells in vivo and in vitro. In the breast cancer cell line MCF-7, PTHrP overexpression is associated with increased mitogenesis. We used this cell line to study the mechanism for the proliferative effects of PTHrP. Clonal MCF-7 lines were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization signal (NLS). Mutation of the NLS negated the proliferative effects and nuclear trafficking of PTHrP, indicating that increased mitogenesis is mediated via an intracrine pathway. Cells overexpressing wild-type PTHrP were enriched in G2 + M stage of the cell cycle compared with cells overexpressing NLS-mutated PTHrP, indicating an intracrine role for PTHrP in cell cycle regulation. Wild-type PTHrP also protected MCF-7 cells from serum starvation-induced apoptosis. Cells overexpressing wild-type PTHrP showed significantly greater cell survival than cells overexpressing NLS-mutated PTHrP. The ratios of the apoptosis-regulating proteins Bcl-2 to Bax and Bcl-x(L) to Bax were higher in cells overexpressing wild-type, but not NLS-mutated, PTHrP compared with control cells. These findings suggest that the proliferative effects of PTHrP in breast cancer cells are mediated through regulation of the cell cycle and apoptosis, and that controlling PTHrP production in breast cancer may be therapeutically useful.     

The antiepidermal growth factor receptor agent gefitinib (ZD1839/Iressa) improves antihormone response and prevents development of resistance in breast cancer in vitro

Although many estrogen receptor-positive breast cancers initially respond to antihormones, responses are commonly incomplete with resistance ultimately emerging. Delineation of signaling mechanisms underlying these phenomena would allow development of therapies to improve antihormone response and compromise resistance. This in vitro investigation in MCF-7 breast cancer cells examines whether epidermal growth factor receptor (EGFR) signaling limits antiproliferative and proapoptotic activity of antihormones and ultimately supports development of resistance. It addresses whether the anti-EGFR agent gefitinib (ZD1839/Iressa; TKI: 1 mum) combined with the antihormones 4-hydroxytamoxifen (TAM: 0.1 mum) or fulvestrant (Faslodex; 0.1 mum) enhances growth inhibition and prevents resistance. TAM significantly suppressed MCF-7 growth over wk 2-5, reducing proliferation detected by immunocytochemistry and fluorescence-activated cell sorter cell cycle analysis. A modest apoptotic increase was observed by fluorescence-activated cell sorter and fluorescence microscopy, with incomplete bcl-2 suppression. EGFR induction occurred during TAM response, as measured by immunocytochemistry and Western blotting, with EGFR-positive, highly proliferative resistant growth subsequently emerging. Although TKI alone was ineffective on growth, TAM plus TKI cotreatment exhibited superior antigrowth activity vs. TAM, with no viable cells by wk 12. Cotreatment was more effective in inhibiting proliferation, promoting apoptosis, and eliminating bcl-2. Cotreatment blocked EGFR induction, markedly depleted ERK1/2 MAPK and protein kinase B phosphorylation, and prevented emergence of EGFR-positive resistance. Faslodex plus TKI cotreatment was also a superior antitumor strategy. Thus, increased EGFR evolves during treatment with antihormones, limiting their efficacy and promoting resistance. Gefitinib addition to antihormonal therapy could prove more effective in treating estrogen receptor-positive breast cancer and may combat development of resistance.     

弓形虫ROP16蛋白对人乳腺癌MCF-7细胞增殖、周期和凋亡的影响

目的 探讨弓形虫Ⅰ型(RH)ROP16蛋白在人乳腺癌MCF-7细胞增殖、周期及凋亡方面的作用。方法 以空载体(MCF-7-HBLV)和过表达ROP16蛋白的慢病毒(HBLV-RH ROP16)分别感染MCF-7细胞,嘌呤霉素筛选出稳定表达ROP16的细胞株,Real time PCR、Western blot法检测MCF-7细胞中ROP16 mRNA和蛋白表达水平。CCK-8和流式细胞术检测细胞增殖、周期和凋亡。Western blot法检测细胞周期及凋亡相关蛋白表达。结果 相比MCF-7-HBLV(空载体组)和MCF-7细胞组(对照组),MCF-7-RH ROP16细胞组中ROP16 mRNA和蛋白表达升高;细胞增殖率降低(P<0.01);S期细胞比例、细胞凋亡率升高(P<0.01)。促凋亡因子Bax、P53、Caspase-9、Caspase-3及细胞周期蛋白依赖激酶抑制因子P21表达增高(P<0.01);抗凋亡蛋白Bcl-2及细胞周期蛋白A1(CyclinA1)、周期蛋白依赖性激酶2(CDK2)的表达均下降(P<0.01)。结论 弓形虫Ⅰ型(RH)ROP1...     

Angiotensin II stimulation of Na+/K+ATPase activity and cell growth by calcium-independent pathway in MCF-7 breast cancer cells

Here we demonstrated, by RT-PCR analysis, the expression of both angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in a breast cancer epithelial cell line, MCF-7. Ang II was not able to affect the intracellular Ca2+ concentration in Fura-2 loaded cells suggesting that AT1-mediated phospholipid hydrolysis is not involved in its intracellular transduction pathway. Ang II modulated the activity of the Na+/K+ATPase in a dose- and time-dependent manner and was mitogenic, with a dose-dependent (1-1000 nM) proliferative effect and a maximal response at 100 nM. Both Na+/K+ATPase activation and stimulation of proliferation were mediated by binding of Ang II to AT1, as the effects were completely blocked by DuP 753, a specific AT1 antagonist. CGP 42112, an AT2 antagonist, did not affect Ang II actions. The main conclusion of this study is that Ang II exerts its effects on cell proliferation and Na+/K+ATPase in breast cancer epithelial cells, MCF-7, via AT1 activation independently of the Ca(2+) signalling mechanism.     

PTEN对乳腺癌多药耐药MCF-7/ADR细胞凋亡的促进作用及机制

目的观察野生型PTEN基因促进乳腺癌多药耐药细胞MCF-7/ADR凋亡的作用并探讨其机制。方法采用脂质体介导法将真核表达载体pEGFP-C1-PTEN及突变载体pEGFP-C1-PTEN-C124S转染人乳腺癌多药耐药MCF-7/ADR细胞。MTT法观察细胞对阿霉素的敏感性和耐药倍数,流式细胞技术检测细胞凋亡率,Western blot检测Bcl-2和Caspase-3蛋白。结果转染组的凋亡率为36.86%,高于对照组（3.75%）和C124S组（4.00%）（P均〈0.05）。转染组对阿霉素的耐药倍数显著降低（t=4.77,P〈0.05）,其半数致死剂量IC50值为对照组的26.1%。转染后细胞内Bcl-2表达降低,且检测到Caspase-3活性裂解片段。结论PTEN基因可能通过下调Bcl-2表达及活化Caspase-3来促进乳腺癌多药耐药MCF-7/ADR细胞凋亡,增加其对阿霉素的药敏性。     

EGCG stabilizes p27kip1 in E2-stimulated MCF-7 cells through down-regulation of the Skp2 protein

Loss of p27Kip1 is associated with a poor prognosis in breast cancer. According to previous findings, a decrease in p27Kip1 levels is mainly the result of enhanced proteasome-dependent degradation mediated by its specific ubiquitin ligase subunit S-phase kinase protein 2 (Skp2). Epigallocatechin-3-gallate (EGCG), the main constituent of green tea, was found to stabilize p27Kip1 levels in breast cancer, but whether this effect is mediated through changes in Skp2 expression remains unclear. Here we investigated the mechanisms involved in EGCG's growth inhibition of estrogen-responsive human breast cancer MCF-7 cells. In our results, EGCG increased p27Kip1 and decreased Skp2 in a time- and dose-dependent manner, suggesting that p27Kip1 and Skp2 may be involved in the growth inhibition by EGCG in estrogen-stimulated MCF-7 cells. Interestingly, mRNA levels of p27Kip1 and Skp2 did not significantly change in estrogen-stimulated MCF-7 cells after EGCG treatments. Moreover, overexpression of Skp2 in MCF-7 cells prevented accumulation of p27Kip1 and promoted resistance to the antiproliferative effects of EGCG. This suggests that the down-regulation of the F-box protein Skp2 is the mechanism underlying p27Kip1 accumulation. Furthermore, both tamoxifen and paclitaxel significantly and synergistically enhanced the growth inhibition of MCF-7 cells by EGCG through the down-regulation of Skp2 protein. However, the down-regulation of Skp2 was not always correlate with the up-regulation of p27, suggesting that EGCG-dependent Skp2 down-regulation can influence cell growth in several ways. The therapeutic strategies designed to reduce Skp2 may therefore play an important clinical role in treatment of breast cancer cells.     

Pretreatment with AT-101 enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of breast cancer cells by inducing death receptors 4 and 5 protein levels

Purpose

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has been shown to induce extrinsic pathway of apoptosis in many types of cancer cells. AT-101, an (?)-enantiomer of gossypol, is a potent anticancer agent that is shown to be an inhibitor of Bcl-2/Bcl-XL. In this study, we searched whether pretreatment with either of these drugs would result in the enhancement of apoptosis through induction of death receptors and activation of mitochondrial pathways within breast cancer cells.

Methods

Human breast cancer (MCF-7 and MDA-MB-231) and normal breast cells (MCF-10A) were treated with drugs alone/in combination/sequentially. XTT cell viability assay was used to evaluate cytotoxicity. For showing apoptosis, both DNA Fragmentation and caspase 3/7 activity measurements were done. ELISA and Western blot analysis were done to assess DR4 and DR5 protein levels. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array.

Results

The sequential treatment of AT-101 followed by TRAIL resulted in significant synergistic cytotoxicity and apoptosis. Moreover, pretreatment of breast cancer cells with AT-101 and then with TRAIL caused enhancement of the expression levels of DR4 and DR5 in both cancer cell lines, suggesting that these cells were under strong apoptotic stimuli.

Conclusions

These findings all together, strongly suggest that pretreatment with AT-101 enhances TRAIL-induced death-inducing signaling complex resulting in the engagement of the mitochondrial pathway to apoptosis in breast cancer cells. These promising, preliminary results make AT-101 and TRAIL a novel combination treatment candidate for breast cancer.     

Differential regulation of insulin-like growth factor-binding protein-3 protease activity in MCF-7 breast cancer cells by estrogen and transforming growth factor-beta1

We have examined the regulation of an insulin-like growth factor-binding protein-3 (IGFBP-3) protease secreted by MCF-7 human breast cancer cells using a ligand-binding assay that relies on the decrease in affinity for des(1-3)IGF-I that occurs when IGFBP-3 becomes proteolyzed. IGFBP-3 protease activity was not altered by treatment of MCF-7 cells with all-trans-retinoic acid, vitamin D, epidermal growth factor, platelet-derived growth factor, insulin, or forskolin. However, estradiol was a potent stimulator of IGFBP-3 protease activity, with a significant and maximal effect at 1 nM. This was prevented by cotreatment with tamoxifen, which had no significant effect in the absence of estradiol. By contrast, TGFbeta1 dose dependently inhibited the amount of protease activity secreted by MCF-7 cells, with complete reversal of IGFBP-3 degradation apparent in response to 10 ng/ml TGFbeta1. This study has demonstrated that estrogens and TGFbeta1, factors that are stimulatory and inhibitory, respectively, for MCF-7 cell growth, also stimulate and inhibit the production of an enzyme capable of proteolyzing the growth inhibitory protein IGFBP-3.     

乳腺癌细胞中HMGA2的表达及其反义寡核苷酸对乳腺癌细胞黏附侵袭的影响

目的观察HMGA2在乳腺癌细胞中的表达以及其反义寡核苷酸(ASODN)对乳腺癌细胞黏附和侵袭能力的影响。方法采用流式细胞术检测检测乳腺癌细胞株中HMGA2蛋白的表达;将HMGA2 ASODN及其对照序列(空白对照和错配序列)通过脂质体转染MCF-7细胞,参照V lodavsky实验方法和采用Boyden小室法检测细胞的黏附能力和侵袭能力。结果 HMGA2 ASODN能特异性下调MCF-7细胞HMGA2蛋白表达,而且能抑制其细胞黏附和侵袭能力。结论 HMGA2 ASODN抑制乳腺癌细胞黏附和侵袭能力具有可行性。     

Rationale for combining tamoxifen and interferon in the treatment of advanced breast cancer

Summary Several investigators have discussed the possible combination of tamoxifen and interferon (IFN) in the treatment of breast cancer patients. The rationale in combining these drugs is that IFN induces the expression of estrogen receptors and therefore increases the sensitivity of breast cancer cells toward the growth-inhibitory activity of tamoxifen. In this paper we review the literature on the IFN-mediated expression of estrogen receptors and the postulated synergism of tamoxifen and IFN in the growth inhibition of breast cancer cell lines. Our results indicate that neither type I nor type II IFN increases the expression of estrogen receptors in MCF-7 cells. Together with tamoxifen both type I and type II IFN mediate additive but not synergistic growth inhibition of MCF-7 cells. On the basis of these results it is feasible to test tamoxifen and IFN as combined therapy in breast cancer patients. Preliminary clinical data show that the combination of 30 mg tamoxifen and 2×10⁶ IU IFN- as daily doses may induce WHO grade 3 leukopenia and thrombopenia in patients who are pretreated with polychemotherapy.Abbreviations rIFN recombinant interferon - nIFN natural interferon     

Evidence for a biphasic apoptotic pathway induced by melatonin in MCF-7 breast cancer cells

Abstract:  Previous investigations demonstrated that melatonin exerts an oncostatic action on estrogen-responsive breast cancer, both in vitro and in vivo. Nevertheless, the pro-apoptotic effect of melatonin is still a matter of debate. An experimental study was undertaken to focus on melatonin-related apoptosis and to identify the apoptotic pathways involved. Whole cell-count, flow-cytometry analysis and proteins involved in apoptotic pathways [p53, p73, murine double minute 2 (MDM2), caspases-9,-7,-6, cleaved-poly ADP ribose polymerase (PARP), Bcl-2, Bax and apoptotic inducing factor (AIF)] were investigated in human MCF-7 breast cancer cells treated with physiological (1 nM) concentration of melatonin. Melatonin exerts a significant growth-inhibitory effect on MCF-7 cells, becoming evident after 72 hr and thereafter increasing linearly up to 144 hr. In this model, the growth-inhibition is transforming growth factor beta 1 (TGFβ1)-dependent and it might be reversed by adding an anti-TGFβ1 antibody. Melatonin induces a significant rise in apoptotic rate, at both 24 and 96 hr. The anti-TGFβ1 antibody almost completely suppresses melatonin-related late apoptosis; however, early apoptosis is unaffected. Early programmed cell death is associated with a significant increase in the p53/MDM2 ratio and in AIF release, without modifications in caspase activity or cleaved-PARP levels. Activated caspases-9 and -7 and cleaved-PARP increased significantly at 96 hr, concomitantly with a down-regulation of the Bcl-2/Bax ratio. These data suggest that two distinct apoptotic processes are triggered by melatonin in MCF-7 cells: an early, TGFβ1 and caspase-independent response, and a late apoptotic TGFβ1-dependent process in which activated-caspase-7 is likely to be the terminal effector.     

Antitumor activity and drug interactions of proteasome inhibitor Bortezomib in human high-risk myelodysplastic syndrome cells

The purpose of this study was to investigate the antitumor effects and drug interactions of the proteasome inhibitor Bortezomib against high-risk myelodysplastic syndrome (MDS) cells in vitro and in vivo. The high-risk MDS-derived MUTZ-1 cell line and bone marrow mononuclear cells from primary high-risk MDS patients were used to examine antitumor activity and drug interactions for Bortezomib. Apoptotic proteins, including caspase and Bcl-2 family members, as well as the protein FLIP, were studied. Phosphoinositide 3-kinase (PI3K)/Akt and MAPK signaling pathways were also examined. The PI3K inhibitor LY294002 was used to examine the involvement of the PI3K/Akt signaling pathway in the induction of apoptosis. Cytarabine (AraC) and daunorubicin (DNR) were used to test for synergistic effects between Bortezomib and chemotherapeutic agents. SCID mice xenografted with MUTZ-1 cells were used for in vivo study. We found that Bortezomib could induce growth arrest and apoptosis in high-risk MDS cells in vitro and in vivo. The mechanisms were related to decreased activation of the PI3K/Akt survival signaling pathway, but not the MAPK pathway, and involved inhibition of the NF-κB activity and downregulation of the Bcl-2/Bax and FLIPL/FLIPS ratios, triggering the activation of caspase cascades. This phenomenon was inhibited by the PI3K inhibitor LY294002. Bortezomib also acted synergistically with the chemotherapeutic agents AraC and DNR, which are associated with the inhibition of NF-κB activity. Our results demonstrate that Bortezomib can induce growth arrest and apoptosis of high-risk MDS cells and had a synergistic effect with two chemotherapeutic agents. Our findings provide new insights for the treatment of high-risk MDS, using either Bortezomib alone, or in combination with conventional antineoplastic agents.     

Interleukin-4 stimulates papillary thyroid cancer cell survival: implications in patients with thyroid cancer and concomitant Graves' disease

IL-4, a pleiotropic cytokine mainly produced by activated helper T lymphocytes type 2 (Th2), is known to protect thyroid cells from autoimmune damage. Acting via its receptors (IL-4Ralpha), IL-4 has antiproliferative and apoptotic effects in many malignancies. Its effect in thyroid cancer is unknown. We found that surgical specimens of thyroid carcinomas express both IL-4Ralpha and IL-4 in the majority of cases. Thyroid glands affected by Graves' disease also express IL-4. We also studied a panel of eight thyroid cancer cell lines from different histotypes and found that thyroid cancer cells express high levels of IL-4Ralpha although they do not express IL-4. We then compared the biological effects of IL-4 in TPC-1, a thyroid cancer cell line, and in MCF-7 breast cancer cells. IL-4 very weakly stimulated thyroid cancer cell proliferation, but it was very effective in protecting thyroid cancer cells from apoptosis induced by staurosporin. The protective effect of IL-4 was similar in magnitude to that of IGF-I and was associated with up-regulation of the antiapoptotic molecule Bcl-2 and weak down-regulation of the proapoptotic molecule Bax. Moreover, IL-4 slightly potentiated the survival effect of IGF-I. In contrast, IL-4 reduced growth and induced apoptosis in MCF-7 cells. Taken together, these findings suggest that thyroid cancer cells receive significant protection from apoptosis by IL-4 produced in the thyroid gland by activated T lymphocytes when concomitant Graves' disease is present.