The venom of the spider Macrothele raveni induces apoptosis in the myelogenous leukemia K562 cell line

Spider venoms are a rich source of bioactive compounds with therapeutic potential. In traditional Chinese medicine, spiders and spider venoms have been used in the treatment of various ailments. In the present study, the venom of the spider Macrothele raveni potently suppressed cell growth in the myelogenous leukemia K562 cell line in a dose and time-dependent manner with an IC(50) of 5.1 μg/mL. The venom also had a low inhibitory effect on human lymphocytes with an IC(50) of approximately 36.4 μg/mL, indicating that the venom is relatively selective for leukemic cells. Venom treated K562 cells showed typical morphological indicators of apoptosis including condensation of nuclei and fragmentation of DNA. Annexin V-FITC and propidium iodide dual staining further demonstrated that the venom had potent apoptogenic activity. Venom treatment induced caspase 3 and caspase 8 activation in K562 cells and promoted PARP cleavage. The present results indicate that the venom of the spider M. raveni potently and selectively suppresses the growth of K562 cells by inducing apoptosis via caspase 3 and caspase 8 mediated signaling pathways.     

Immunochemical analysis of the determinant recognized by a monoclonal antibody (MBr1) which specifically binds to human mammary epithelial cells

A monoclonal antibody (MBr1) raised against a membrane preparation (CM) of a human breast cancer line (MCF-7) and characterized as mammary gland epithelium associated (S. Mènard, E. Tagliabue, S. Canevari, G. Fossati, and M. I. Colnaghi. Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res., 43:1295-1300, 1983), was used to biochemically define and partially purify its target antigen. The antigenic activity recognized by MBr1 was unaffected by treatment of MCF-7 cells with trypsin, protease K, or Vibrio cholerae neuraminidase and by heating at 100 degrees but was abolished by treatment with methanol. Since this behavior suggested a glycolipid nature of the MBr1-defined antigen, total lipids were obtained by chloroform:methanol or tetrahydrofuran:phosphate buffer extractions from crude membrane preparations of MCF-7 cells and of breast cancer surgical specimens. Total absorption of MBr1 activity was found by breast cancer lipid extracts, whereas no absorbing capability was detected with a series of highly purified acid and neutral glycolipids or with normal and neuraminidase-treated red blood cells of human, ox, and sheep species. The same pattern of inhibition of MBr1-binding activity was obtained with total lipid extract and both phases after diethyl ether partition. However, when the three extracts were chromatographed on diethylaminoethyl-Sepharose, the antigenic activity was recovered only in the neutral glycolipid fractions. Periodate oxidation of MCF-7 crude membrane preparation abolished MBr1-binding activity, suggesting that the carbohydrate portion of the molecule may constitute the antigenic determinant.     

Characterization of platelet aggregation induced by human breast carcinoma and its inhibition by snake venom peptides,trigramin and rhodostomin

Summary MCF-7 cells, a metastatic human breast carcinoma line, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma (PRP). MCF-7 tumor cell-induced platelet aggregation (TCIPA) was almost blocked by apyrase (0.5 U/ml) and completely inhibited by hirudin (5 U/ml). This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 µM), but was limited by cell pretreatment with phospholipase A₂. MCF-7 cell suspension caused marked, dose-dependent decrease in plasma recalcification times using normal, Factor VIII-deficient, and Factor IX-deficient human plasma. This effect was potentiated in cell lysates but was inhibited in intact cells preincubated with sphingosine. MCF-7 cell suspension did not affect the recalcification time of Factor VII-deficient plasma. Taken together, these data suggest that MCF-7 TCIPA arises from MCF-7 tisssue factor activity expression. Trigramim and rhodostomin, RGD-containing snake venom peptides which antagonized the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented MCF-7 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against human tissue factor, platelet membrane glycoprotein IIb/IIIa and Ib prevented MCF-7 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC₅₀, 0.1 µM) and rhodostomin (IC₅₀, 0.03 µM), were about 5,000 and 18,000 times, respectively, more potent than GRGDS (IC₅₀, 0.54 mM).     

Regulation of NADPH oxidase (Nox2) by lipid rafts in breast carcinoma cells

Oxidative stress has emerged as an important pathogenic factor in the development of breast cancer. Cholesterol-rich membrane rafts or lipid rafts (LRs) are reported to play an important role in oxidative stress-induced signal transduction. NADPH oxidase-dependent reactive oxygen species (ROS) production is implicated in oxidative stress in human mammary epithelial cells. In the present study, we determined the expression and regulation of membrane-bound subunits by LRs in human breast cancer cells. We report that basal levels of gp91phox and p22phox are expressed in breast cancer cells. We demonstrate for the first time that disruption of LRs resulted in the downregulation of NADPH oxidase subunits in breast cancer cells. Cholesterol depletion by 10 mM methyl-β-cyclodextrin (MβCD) translocated both gp91phox and p22phox out of LRs. Moreover, lipid raft disruption decreased NADPH oxidase activity (21.1 ± 0.5% in MCF-7 and 28.9 ± 1.0 in BT-549 cells), which was reversed by cholesterol repletion (95%). Therefore, the results suggest that the integrity of LRs plays an important role in the regulation of NADPH oxidase activity in breast cancer cells.     

An N-myristoylated protein kinase C-α pseudosubstrate peptide that functions as a multidrug resistance reversal agent in human breast cancer cells is not a P-glycoprotein substrate

 Protein kinase C-α (PKC-α) activation is an important contributing factor in human breast cancer MCF-7 MDR cell drug resistance. We recently reported the use of N-myristoylated PKC-α pseudosubstrate peptides with potent PKC-α inhibitory activity as reversal agents of drug resistance in MCF-7 MDR cells. The peptides potently inhibit phosphorylation of the PKC-α substrates P-glycoprotein (P-gp), raf kinase and PKC-α itself in MCF-7 MDR cells in association with a severalfold induction of intracellular uptake of P-gp substrate chemotherapeutics and a statistically significant twofold increase in cellular chemosensitivity. We now report that the N-myristoylated PKC-α pseudosubstrate peptide N-myristoyl-RFARKGALRQKNV (P3) is not a P-gp substrate in MCF-7 MDR cells based on a comparison of the cellular uptake of [¹²⁵I]-radiolabeled P3 in MCF-7 MDR vs MCF-7 WT cells. The extent of cellular uptake of the radiolabeled peptide in the drug-resistant cell line MCF-7 MDR was either greater than or equivalent to the uptake in the parental drug-sensitive MCF-7 WT cell line over a time course of 30 min to 6 h, and across a peptide concentration range of 25 – 100 μM. Additionally, treatment of the MCF-7 MDR cells with verapamil (VPL), a known P-gp efflux inhibitor, had no effect on the cellular accumulation of radiolabeled P3. Our results provide direct evidence that the N-myristoylated pseudosubstrate peptide is taken up equivalently by drug-sensitive and MDR cancer cells and therefore has potential value as an MDR reversal agent that operates by a novel mechanism. Received: 27 August 1996 / Accepted: 14 January 1997     

姜黄素抑制乳腺癌MCF-7细胞增殖及其相关的氧化应激机制

目的：探讨姜黄素抑制乳腺癌MCF7细胞增殖及其相关的氧化应激机制。方法：用不同浓度(5～40 μmol/L) 的姜黄素作用于乳腺癌细胞株MCF7细胞,以MTT法检测在6、12、24、48 h的细胞增殖率,流式细胞术检测细胞凋亡率,DCFHDA染色流式检测细胞内活性氧(reactive oxygen species ROS)生成变化,黄嘌呤氧化酶法检测氧化物歧化酶（superoxide dismutase,SOD）,可见光分光光度计法检测过氧化氢酶（catalase, CAT）活性。结果：低浓度(5、10 μmol/L) 姜黄素作用后MCF7细胞增殖受抑制（P<0.01）,但无凋亡发生;伴随细胞内ROS短暂升高后随时间逐步下降,SOD、CAT先下降后逐步提高。高浓度（20、40 μmol/L）姜黄素作用后细胞增殖抑制率明显增加（P<0.01）,细胞出现大量凋亡;伴随细胞内ROS即刻明显升高,随时间延长无明显下降;SOD、CAT明显下降（P<0.01）。结论：姜黄素具有剂量依赖的抗氧化与促氧化双重作用,不同浓度的姜黄素通过对乳腺癌细胞内氧化还原状态的调节参与了其抗肿瘤细胞增殖作用的机制。     

20(S)-Protopanaxadiol Induces Human Breast Cancer MCF-7 Apoptosis through a Caspase-Mediated Pathway

20(S)-Protopanaxadiol (PPD), a ginsenoside isolated from Pananx quinquefolium L., has been shown toinhibit growth and proliferation in several cancer cell lines. The aim of this study was to evaluate its anticanceractivity in human breast cancer cells. MCF-7 cells were incubated with different concentrations of 20(S)-PPDand cytotoxicity was evaluated by MTT assay. Occurrence of apoptosis was detected by DAPI and AnnexinV-FITC/PI double staining. Mitochondrial membrane potential was measured with Rhodamine 123. The Bcl-2and Bax expression were determined by Western blot analysis. Caspase activity was measured by colorimetricassay. 20(S)-PPD dose-dependently inhibited cell proliferation in MCF-7 cells, with an IC50 value of 33.3 μM at24h. MCF-7 cells treated with 20(S)-PPD presented typical apoptosis, as observed by morphological analysisin cell stained with DAPI. The percentages of annexin V-FITC positive cells were 8.92%, 17.8%, 24.5% and30.5% in MCF-7 cells treated with 0, 15, 30 and 60μM of 20(S)-PPD, respectively. Moreover, 20(S)-PPD couldinduce mitochondrial membrane potential loss, up-regulate Bax expression and down-regulate Bcl-2 expression.These events paralleled activation of caspase-9, -3 and PARP cleavage. Apoptosis induced by 20(S)-PPD wasblocked by z-VAD-fmk, a pan-caspase inhibitor, suggesting induction of caspase-mediated apoptotic cell death.In conclusion, the 20(S)-PPD investigated is able to inhibit cell proliferation and to induce cancer cell death bya caspase-mediated apoptosis pathway.     

Antitumor mechanism of action of a cyclopropyl antiestrogen (compound 7b) on human breast cancer cells in culture

 Cyclopropyl compound 7b [(Z)-1,1-dichloro2-[4-[2-(dimethylamino)ethoxy] phenyl]-2-(4-methoxy-phenyl)-3-cyclopropane] has been shown to be a pure antiestrogen in mouse uterine tissue. Antitumor activity was examined by evaluating the influence of 7b on the proliferation, estrogen receptor (ER) affinity and cell-surface morphology of ER-positive and ER-negative human breast cancer cells in culture. The antiproliferative potency of 7b was found to be equal to tamoxifen in ER-positive MCF-7 human breast cancer cells. Further, the antiproliferative activities of 7b and tamoxifen were reversed by coadministration of estradiol. Accordingly, the antiproliferative activity of compound 7b appears to be estrogen-mediated since it did not influence the growth of either ER-negative MDA-MB-231 human breast cells or A-549 human lung cancer cells in culture. An ER-dependent mechanism of action is also supported by the specific binding affinity of 7b for ER in MCF-7 cells. Further, a study of cell surface morphology using scanning electron microscopy (SEM) revealed that 7b reduced the density and distribution of microvilli (MV) on MCF-7 cells, which was reversed by coadministration of estradiol. Compound 7b did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7b inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on either ER-negative MDA-MB-231 cells or A-549 lung cancer cells. The results of this study confirm an antiestrogenic mechanism of action for 7b as previously observed in vivo and suggest that 7b would be effective in the treatment of estrogen-dependent breast cancer or as a prophylactic treatment for women with a high risk of breast cancer development. Received: 6 January 1995/Accepted: 9 October 1995     

Anti-Breast Cancer Activity on MCF-7 Cells of Melittin from Indonesia’s Apis cerana: An In Vitro Study

Objective: Breast cancer is the most common case of cancers. Apitheraphy has been traditionally used for abundance diseases. This study aims to evaluate and compare the anti-breast cancer activity of melittin from Indonesia’s Apic cerana as a potential drug for treating breast cancer. Methods: Apis cerana bee venom (BV) was collected from a bee farm in Cikurutung, Bandung using an electrical venom device. The BV was then purified using the ÄKTA Start system and HiTrap™ SP HP cation exchange chromatography column. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify melittin based on its molecular mass and lowry’s protein assay to measure melittin concentration. Melittin cytotoxicity was measured with brine shrimp lethality test (BSLT), while MCF-7 breast cancer cells MTT assay was used to measure its anti-breast cancer activity, based on inhinition rate. Results: 95.432 μg/mL melittin is purified from 62.8 mg/L BV, using  cation exchange chromatography. Melittin in vitro analysis with MCF-7 MTT assay is used to determine anti-breast cancer activity in dose dependent manner. Furthermore, melttin BSLT result showed a LC50 16.67675 μg/mL. Therefore, the MTT assay  was conducted in 5, 10 and 15 μg/mL with MCF-7 inhibition values of 0.768 ± 0.014, 3.303 ± 0.011, and 35.714 ± 0.009 %, respectively. Conclusion: Indonesia’s Apis cerana has the potential to be used as a therapeutic peptide for breast cancer treatment.     

Antiproliferative Activity of Marrubium persicum Extract in the MCF-7 Human Breast Cancer Cell Line

Aim: Developing antitumor drugs from natural products is receiving increasing interest worldwide dueto limitations and side effects of therapy strategies for the second leading cause of disease related mortality,cancer. Methods: The antiproliferative activity of a methanolic extract from the aerial parts of Marrubiumpersicum extract was assessed with the MCF-7 breast cancer cell line using the MTT test for cell viability andcytotoxicity indices. In addition, antioxidant properties of the extract were evaluated by measuring its ability toscavenge free DPPH radicals. Moreover, the total phenolic and flavonoid content of the extract was determinedbased on Folin-Ciocalteu and colorimetric aluminum chloride methods. Results: The findings of the study forthe antiproliferative activity of the methanolic extract of M. persicum showed that growth of MCF-7 cells wasinhibited by the extract in a dose and time dependent manner, where a gradual increase of cytotoxicity effect hasbeen achieved setting out on 200 μg/mL concentration of the plant extract. The antioxidant assay revealed thatthe extract was a strong scavenger of DPPH radicals with an RC50 value of 52 μg/mL. The total phenolic andflavonoids content of the plant extract was 409.3 mg gallic acid equivalent and 168.9 mg quercetin equivalentper 100g of dry plant material. Conclusion: Overall, M. persicum possesses potential antiproliferative andantioxidant activities on the malignant MCF-7 cell line that could be attributed to the high content of phenolicsand flavonoids, and therefore warrants further exploration.     

BmKn-2 Scorpion Venom Peptide for Killing Oral Cancer Cells by Apoptosis

Scorpion venom peptides recently have attracted attention as alternative chemotherapeutic agents thatmay overcome the limitations of current drugs, providing specific cytotoxicity for cancer cells with an abilityto bypass multidrug-resistance mechanisms, additive effects in combination therapy and safety. In the presentstudy, BmKn-2 scorpion venom peptide and its derivatives were chosen for assessment of anticancer activities.BmKn-2 was identified as the most effective against human oral squamous cells carcinoma cell line (HSC-4) byscreening assays with an IC50 value of 29 μg/ml. The BmKn-2 peptide killed HSC-4 cells through induction ofapoptosis, as confirmed by phase contrast microscopy and RT-PCR techniques. Typical morphological featuresof apoptosis including cell shrinkage and rounding characteristics were observed in treated HSC-4 cells. Theresults were further confirmed by increased expression of pro-apoptotic genes such as caspase-3, -7, and -9but decrease mRNA level of anti-apoptotic BCL-2 in BmKn-2 treated cells, as determined by RT-PCR assay.In summary, the BmKn-2 scorpion venom peptide demonstrates specific membrane binding, growth inhibitionand apoptogenic activity against human oral cancer cells.     

Function analysis of estrogenically regulated protein tyrosine phosphatase gamma (PTPgamma) in human breast cancer cell line MCF-7

Protein tyrosine phosphatase gamma (PTPgamma) is a member of the receptor-like family of tyrosine phosphatases and has been implicated as a tumor suppressor gene in kidney and lung cancers. Based on our previous findings, we hypothesize that PTPgamma is a potential estrogen-regulated tumor suppressor gene in human breast cancer. To examine the effects of PTPgamma on growth of MCF-7 human breast cancer cells and compare the estrogenic responses of human breast cells with different PTPgamma expression levels, we established several stably transfected MCF-7 cell lines expressing different levels of PTPgamma, which were confirmed by RT-PCR and immunostaining. In our work, we used the antisense construct to breakdown endogenous PTPgamma level in MCF-7 cells. The results from doubling time assay suggested that PTPgamma is capable of inhibiting MCF-7 breast cancer cell growth. We further demonstrated that PTPgamma is able to inhibit anchorage-independent growth of breast cancer cells in soft agar and reduce the estrogenic responses of MCF-7 cell proliferation to estradiol-17beta (E(2)) and zeranol (Z, a nonsteroidal growth promoter with estrogenic activity). Our data suggest that PTPgamma may function as an important modulator in regulating the process of tumorigenesis in human breast.     

Inhibition of the CaM-kinases augments cell death in response to oxygen radicals and oxygen radical inducing cancer therapies in MCF-7 human breast cancer cells

Many cancer treatments induce cell death through lethal oxidative stress. Oxidative stress also induces the activation of the calcium/calmodulin-dependent kinases (CaM-Ks), CaM-KII and CaM-KIV. In turn, the CaM-Ks are known to induce the activation of antiapoptotic signaling pathways, such as Akt, ERK, and NF-kappaB in many different cell types. The aim of this study was to determine the role of CaM-Kinases in resistance to hydrogen peroxide and three oxidative stress-inducing cancer therapies in MCF-7 breast cancer cells. We found that oxidative stress induced CaM-Kinase activity in MCF-7 breast cancer cells and that CaM-K inhibition increased hydrogen peroxide-induced cell death in MCF-7 human breast cancer cells. When MCF-7 cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy in the presence of a CaM-K inhibitor a greater level of cell killing was observed than when cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy alone. In support of this finding, CaM-K inhibition increased hydrogen peroxide-induced apoptosis in MCF-7 cells, as determined by increased number of apoptotic cells, DNA fragmentation, and PARP cleavage. Pharmacological and molecular inhibition indicated that CaM-KII was participating in hydrogen peroxide-induced ERK phosphorylation in breast cancer cells indicating a potential mechanism by which this sensitization occurs. This is the first time that CaM-K inhibition is reported to sensitize cancer cells to reactive oxygen intermediate inducing cancer treatments.     

Phycosynthesis of Silver Nanoparticles Using Cladophora Glomerata and Evaluation of Their Ability to Inhibit the Proliferation of MCF-7 and L20B Cell Lines

Background: Nanotechnology is receiving greater attention these days as a result of its applications in numerous industrial, medical, and environmental fields. Objective: To synthesize silver nanoparticles with a green alga, Cladophora glomerata, and determine their inhibitory activity against tumor cell (MCF-7) and transgenic mouse cell (L20B) lines. Materials and Methods: Methanol extract was prepared from Cladophora glomerata and used as a safe factory for the synthesis of silver nanoparticles (AgNPs). UV-visible spectrophotometer, X-ray diffraction, scanning electron microscopy, and EDX analyses were used to characterize the biosynthesized AgNPs. The anti-tumor activity of the phycosynthesized AgNPs was tested against the MCF-7 and L20B cell lines. Furthermore, the bioactive compounds in the algal extract were determined by gas chromatography-mass spectroscopy (GC-MS). Results: The phycosynthesis produced clusters of spherical and polydispersed cuboidal pure AgNPs with an average size of 32 nm. The phycosynthesized AgNPs possess anti-cancer and anti-tumor activities on the MCF-7 and L20B cell lines, with significant anti-proliferation percentages of 52.8 and 65.8%, respectively, after 48 hours of treatment with 100 μg/ml AgNPs. Both treated cell lines showed a significant change in cellular shape and tissue detachment. The GC-MS analysis revealed the presence of a high proportion of octadecanoic acid (47.59%) and hexadecanoic acid (14.97%). Conclusion: Cladophora glomerata contains chemicals that improve the stabilization and reduction properties of the nanoparticles. It can be used as a safe, local, and natural source for the synthesis of AgNPs and can also be used as a benign factory for many other metal nanoparticles. The phycosynthesized AgNPs have anti-cancer and anti-tumor activities on the test cell lines and provide an insight into the potential for using them as a trend in cancer nanotherapy.     

Functional domains of CCN1 (Cyr61) regulate breast cancer progression

CCN1 plays diverse roles in cellular proliferation, survival, migration and angiogenesis. We determined the relationship between CCN1 protein expression and clinical factors that are important for the classification of breast cancer. CCN1 contains four functional domains; the contribution of each of the structural domains to the biological properties of CCN1 in breast cancer was investigated. We performed immunohistochemistry for CCN1 on a breast cancer tissue array, and conducted a detailed statistical analysis on the relationship between CCN1 protein expression and clinical factors that are important for the classification of breast cancer. The structure-function relationship was examined using four mutant constructs in which one of the modules (DM1-DM4) had been deleted. MCF-7 breast cancer cells were stably transfected with these constructs and their biological activity was tested in comparison to full-length CCN1. Staining of CCN1 in tumors was positively correlated with AJCC disease stage. A strong association also was found between lymph node involvement and high CCN1 expression in patients with invasive breast cancer; there was a significant increase in the breast cancer expression of CCN1 in patients with positive lymph nodes (P=0.004), and the levels of CCN1 correlated with the number of positive lymph nodes (P=0.0006). Deletion of module 4 rendered CCN1 unable to either bind heparin or associate with the extracellular matrix. Furthermore, MCF-7/DM4 cells demonstrated reduced cell spreading, migration and proliferation, indicating that module 4 of the protein is important for its ability to promote these activities. These findings indicate that CCN1 is involved throughout the clinical progression of breast cancer to an invasive phenotype. The multimodular structure of CCN1 enables it to fulfill multiple functions that may contribute to the different stages of cancer development, raising the prospect that specific regions of CCN1 could be targeted for therapeutic benefit to inhibit particular aspects of malignancy in breast cancer.     

无血清悬浮法培养MCF-7细胞系并筛选该细胞系中肿瘤干细胞相关亚群的初步研究

目的：研究无血清培养基悬浮培养MCF-7乳腺癌细胞系,筛选并鉴定MCF-7乳腺癌细胞系中的肿瘤干细胞相关亚群。方法：应用乳腺癌培养基在无血清的条件下悬浮培养MCF-7乳腺癌细胞系。通过无血清培养筛选乳腺癌细胞系肿瘤干细胞相关亚群,将其接种于含血清培养基,观察分化。应用单克隆形成实验、表面标志检测、HOECHST33342染色检测来确定培养出的细胞中肿瘤干细胞的比例及其培养后肿瘤干细胞含量的变化。结果：乳腺癌MCF-7细胞系中有约2.12%的肿瘤细胞在无血清培养基中能够存活、增殖,形成自由漂浮的细胞球。细胞球可连续传代,若重新接种于含血清培养基中可重新贴壁分化,贴壁分化后细胞形态与直接在含血清培养基中培养的MCF-7无明显差别。流式细胞仪表面标志检测,细胞球中约含83.13%表达CD24^-CD44^＋的细胞,HOECHST33342染色提示细胞球中约含10.06%的侧亚群（sidepopulation）细胞。结论：MCF-7细胞系可在含生长因子的无血清培养基中悬浮生长,并维持细胞系。该细胞系中含有比例较低的具有增殖和分化能力的乳腺癌干细胞相关亚群。表面标志以及HOECHST33342检测差异提示细胞球中仅约1/8细胞具有干细胞的功能。     

Activation of inositol phospholipid signaling and Ca2+ efflux in human breast cancer cells by bombesin

Members of the bombesin-related family of peptides (BRPs) are mitogenic for a variety of cell types; however, a role for these peptides has not been previously described in human breast cancer. Early membrane receptor signal transduction mechanisms associated with bombesin action include phospholipase C-mediated inositol phospholipid hydrolysis and the elevation of cytosolic Ca2+ levels. We have investigated a potential role for BRPs in breast cancer by studying their effect on phospholipid hydrolysis, 45Ca2+ efflux, and cell growth in the human breast cancer cell line MCF-7. Bombesin stimulated a dose-dependent increase in the hydrolysis product inositol monophosphate during 1 h with a half-maximal effect around 1 nM. A transient increase in inositol trisphosphate in response to bombesin was also apparent at 2 min. Two distinct bombesin receptor antagonists inhibited this bombesin-induced phospholipid hydrolysis. Both bombesin- and gastrin-releasing peptide also stimulated a dose-related increase in inositol phosphate production in T47D cells, a different human breast cancer cell line. The efflux of 45Ca2+ from prelabeled MCF-7 cells was also stimulated by bombesin. This apparent cellular Ca2+ mobilization was partly dependent on extracellular Ca2+ and was inhibited by Ni2+. Despite this activation of putative mitogenic signaling pathways, bombesin had no effect on either proliferation or DNA synthesis in MCF-7 cells. These data implicate a functional role for BRPs in human breast cancer.     

Differential suppression of proliferation in MCF-7 and MDA-MB-231 breast cancer cells exposed to alpha-, gamma- and delta-tocotrienols is accompanied by altered expression of oxidative stress modulatory enzymes

Tocotrienols belong to the vitamin E family of chemicals known to have potent anti-proliferative and apoptotic activities against a variety of cancer cells with little to no comparable influence on the normal cells. Whether tocotrienols control the expression of phase II antioxidant enzymes in the context of their anti-carcinogenic mechanisms has not been investigated. The present studies were performed to test whether the differential growth inhibition resulting from exposure to α-, γ- and δ-tocotrienols in estrogen receptor-positive human MCF-7 and estrogen receptor-negative MDA-MB-231 breast cancer cells might be accompanied by changes in phase II antioxidant enzymes. Cell proliferation and clonogenicity in both cell lines were significantly inhibited by γ- and δ-tocotrienols with little affect when cells were similarly exposed to α-tocotrienol, at doses up to 10 μM. The expression and activity of several antioxidant enzymes in 10 μM tocotrienol-treated cells were determined by Western blot and biochemical assays. In MDA-MB-231 cells, δ- was more active than α- or γ-tocotrienols in up-regulating glutathione peroxidase; however, the three tocotrienols had comparable activity in inducing thioredoxin. In MCF-7 cells, expression of quinone reductase 2 and thioredoxin was increased by γ- and δ-tocotrienols, whereas quinone reductase 1 was unaffected by exposure to the tocotrienols. The tocotrienols also did not affect the expression and activity of superoxide dismutase in both MCF-7 and MDA-MB-231 cells, but increased catalase activity concomitant with slight reduction in the catalase expression. In MDA-MB-231 cells, treatment by tocotrienols led to several fold increase of NRF2 expression marked by corresponding decrease in KEAP1 levels. By contrast, no significant change in NRF2 and KEAP1 levels was observed in MCF-7 cells. These studies demonstrate that different tocotrienols show distinct and selective activity in regulating the NRF2-KEAP1, in coordination with the induced expression of cytoprotective oxidative stress modulatory genes and regulation of proliferation in breast cancer cells.     

The histone deacetylase inhibitor sodium butyrate induces breast cancer cell apoptosis through diverse cytotoxic actions including glutathione depletion and oxidative stress

Sodium butyrate (NaBu), a potent histone deacetylase inhibitor, modulates the expression of a large number of genes. The purpose of this study was to determine whether this dietary agent could induce apoptosis in MCF-7 cells, a breast cancer cell line that lacks caspase-3 activity, and to identify the mechanisms that underlie NaBu toxicity in these cells. Cell viability assessed by the activity of mitochondrial succinate dehydrogenase (MTT assay) revealed a dose-dependent reduction of MCF-7 cellular growth in response to NaBu treatment. Restoring caspase-3 function by transfection did not modify NaBu toxicity in these cells. Following a 24-h exposure, NaBu-induced cell growth arrest in G2/M phase in a dose-dependent fashion in association with stable expression of CDC25A, a G1-specific regulator of the cell cycle. The anti-proliferative effects of NaBu were accompanied by diminished expression of p53. Similarly, mRNA encoding c-Myc, a well-known regulator of p53, was decreased in NaBu-treated cells, while p21(Waf1/Cip1) mRNA was increased. Furthermore, bax mRNA level was up-regulated whereas a decline in Bcl-2 both protein and mRNA levels were detected in NaBu-treated cells. Apoptosis was observed following a treatment with 2 mM NaBu, reflected by Annexin-V staining and by the cleavage of poly(ADP-ribose) polymerase, whereas DNA laddering was absent. Apoptosis was associated with a pronounced depletion of intracellular glutathione levels. Finally, NaBu treatment significantly increased the activities of several antioxidant enzymes, including glutathione reductase, glutathione peroxidase, and catalase. Together, these data suggest that the pro-apoptotic effects of NaBu observed in MCF-7 cells are associated with oxidative stress.