Inhibition of Tumor-associated Fatty Acid Synthase Hyperactivity Induces Synergistic Chemosensitization of HER-2/neu-Overexpressing Human Breast Cancer Cells to Docetaxel (taxotere)

The lipogenic enzyme fatty acid synthase (FAS) is differentially overexpressed and hyperactivated in a biologically aggressive subset of breast carcinomas and minimally in most normal adult tissues, rendering it an interesting target for antineoplastic therapy development. Recently, a molecular connection between the HER -2/ neu (c- erb B-2) oncogene and FAS has been described in human breast cancer cells. Here, we examined the relationship between breast cancer-associated FAS hyperactivity and HER -2/ neu -induced breast cancer chemoresistance to taxanes. Co-administration of docetaxel (Taxotere) and the mycotoxin cerulenin, a potent and non-competitive inhibitor of FAS activity, demonstrated strong synergism in HER -2/ neu -overexpressing and docetaxel-resistant SK-Br3 cells, modest synergism in moderately HER -2/ neu -expressing MCF-7 cells, and it showed additive effects in low HER -2/ neu -expressing and docetaxel-sensitive MDA-MB-231 cells. Sequential exposure to cerulenin followed by docetaxel again yielded strong synergism in SK-Br3 cells, whereas antagonistic and moderate synergistic interactions were observed in MCF-7 and MDA-MB-231 cells, respectively. Importantly, inhibition of FAS activity dramatically decreased the expression of HER -2/ neu oncogene in SK-Br3 breast cancer cells. To the best of our knowledge this is the first study demonstrating that FAS is playing an active role in HER -2/ neu -induced breast cancer chemotherapy resistance.     

Inhibition of Fatty Acid Synthase (FASN) synergistically enhances the efficacy of 5-fluorouracil in breast carcinoma cells

The lipogenic enzyme fatty acid synthase (FASN) is differentially overexpressed and hyperactivated in a biologically aggressive subset of breast carcinomas and minimally in most normal adult tissues, rendering it an interesting target for anti-neoplastic therapy development. We previously reported that the FASN blockade can induce a synergistic chemosensitization of breast cancer cells to microtubule interfering agents (MIAs) such as docetaxel, paclitaxel and vinorelbine. Upon pharmacological inhibition of FASN activity using the natural antibiotic cerulenin [(2S,3R)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide], we evaluated the role of FASN-catalyzed endogenous fatty acid biogenesis on the sensitivity of SK-Br3, MCF-7 and MDA-MB-231 breast cancer cell lines to the anti-metabolite 5-fluorouracil (5-FU). Cells were exposed simultaneously to cerulenin and 5-FU, sequentially to 5-FU followed by cerulenin or cerulenin followed by 5-FU. Cell viability was determined by MTT assays and the increase in 5-FU-induced cell growth inhibition was measured by dividing 5-FU IC30 and IC50 values (i.e., 30% and 50% inhibitory concentrations, respectively) that were obtained in the absence of cerulenin by those in its presence. Co-exposure to cerulenin enhanced 5-FU efficacy up to 20-, 81-, and 58-times in SK-Br3, MCF-7 and MDA-MB-231 cells, respectively. Pre-treatment with cerulenin followed by the addition of 5-FU increased 5-FU efficacy up to 31-, 87-, and 126-times in SK-Br3, MCF-7 and MDA-MB-231 cells, respectively. Pre-treatment with 5-FU followed by the addition of cerulenin augmented 5-FU efficacy up to 107-, 20-, and 18-times in SK-Br3, MCF-7 and MDA-MB-231 cells, respectively. When isobologram transformations of multiple dose-response analyses were performed to detect in vitro synergy, we concluded that the nature of the interaction between cerulenin and 5-FU in individual breast cancer cells lines generally exhibited sequence-dependency. Thus, while synergism was mainly observed when breast cancer cells were exposed to 5-FU prior to cerulenin, moderate synergism or additive interactions was obtained either when the chemical FASN blocker preceded 5-FU or when both drugs were concurrently administered. Of note, no antagonist interactions occurred upon any schedule of combined treatment with cerulenin and 5-FU. Our current findings revealing a schedule-dependent synergistic interaction between 5-FU and cerulenin represents, to the best of our knowledge, the first evidence that FASN-catalyzed de novo FA biogenesis plays a key role in regulating breast cancer cell response to antimetabolite-based therapies.     

Pharmacological and small interference RNA-mediated inhibition of breast cancer-associated fatty acid synthase (oncogenic antigen-519) synergistically enhances Taxol (paclitaxel)-induced cytotoxicity

The relationship between breast cancer-associated fatty acid synthase (FAS; oncogenic antigen-519) and chemotherapy-induced cell damage has not been studied. We examined the ability of C75, a synthetic slow-binding inhibitor of FAS activity, to modulate the cytotoxic activity of the microtubule-interfering agent Taxol (paclitaxel) in SK-Br3, MDA-MB-231, MCF-7 and multidrug-resistant MDR-1 (P-Glycoprotein)-overexpressing MCF-7/AdrR breast cancer cells. When the combination of C75 with Taxol in either concurrent (C75 + Taxol 24 hr) or sequential (C75 24 hr --> Taxol 24 hr) schedules were tested for synergism, addition or antagonism using the isobologram and the median-effect plot analyses, co-exposure of C75 and Taxol mostly demonstrated synergistic effects, whereas sequential exposure to C75 followed by Taxol mainly showed additive or antagonistic interactions. Because the nature of the cytotoxic interactions was definitely schedule-dependent in MCF-7 cells, we next evaluated the effects of C75 on Taxol-induced apoptosis as well as Taxol-activated cell death and cell survival-signaling pathways in this breast cancer cell model. An ELISA for histone-associated DNA fragments demonstrated that C75 and Taxol co-exposure caused a synergistic enhancement of apoptotic cell death, whereas C75 pre-treatment did not enhance the apoptosis-inducing activity of Taxol. Co-exposure to C75 and Taxol induced a remarkable nuclear accumulation of activated p38 mitogen-activated protein kinase (p38 MAPK), which was accompanied by a synergistic nuclear accumulation of the p53 tumor-suppressor protein that was phosphorylated at Ser46, a p38 MAPK-regulated pro-apoptotic modification of p53. As single agents, FAS blocker C75 and Taxol induced a significant stimulation of the proliferation and cell survival mitogen-activated protein kinase extracellular signal-regulated kinase (ERK1/ERK2 MAPK) activity, whereas, in combination, they interfered with ERK1/ERK2 activation. Moreover, the combined treatment of C75 and Taxol inactivated the anti-apoptotic AKT (protein kinase B) kinase more than either agent alone, as evidenced by a synergistic down-regulation of AKT phosphorylation at its activating site Ser(473) without affecting AKT protein levels. To rule out a role for non-FAS C75-mediated effects, we finally used the potent and highly sequence-specific mechanism of RNA interference (RNAi) to block FAS-dependent signaling. Importantly, SK-Br3 and multi-drug resistant MCF-7/AdrR cells transiently transfected with sequence-specific double-stranded RNA oligonucleotides targeting FAS gene demonstrated hypersensitivity to Taxol-induced apoptotic cell death. Our findings establish for the first time that FAS blockade augments the cytotoxicity of anti-mitotic drug Taxol against breast cancer cells and that this chemosensitizing effect is schedule-dependent. We suggest that the alternate activation of both the pro-apoptotic p38 MAPK-p53 signaling and the cytoprotective MEK1/2 --> ERK1/2 cascade, as well as the inactivation of the anti-apoptotic AKT activity may explain, at least in part, the sequence-dependent enhancement of Taxol-induced cytotoxicity and apoptosis that follows inhibition of FAS activity in breast cancer cells. If chemically stable FAS inhibitors demonstrate systemic anticancer effects of FAS inhibition in vivo, these findings may render FAS as a valuable molecular target to enhance the efficacy of taxanes-based chemotherapy in human breast cancer.     

The role of the polyamine catabolic enzymes SSAT and SMO in the synergistic effects of standard chemotherapeutic agents with a polyamine analogue in human breast cancer cell lines

Introduction

Polyamine analogues have demonstrated significant activity against human breast cancer cell lines as single agents as well as in combination with other cytotoxic drugs. This study evaluates the ability of a polyamine analogue N ¹,N ¹¹-bis(ethyl)norspermine (BENSpm) to synergize with six standard chemotherapeutic agents, 5-fluorouracil (FU), fluorodeoxyuridine, cis-diaminechloroplatinum(II) (C-DDP), paclitaxel, docetaxel, and vinorelbine.

Materials and methods

Four human breast cancer cell lines (MDA-MB-231, MCF-7, Hs578t, and T47D) and one immortalized, non-tumorigenic mammary epithelial cell line (MCF-10A) were used for in vitro combination studies with BENSpm and cytotoxic drugs. Xenograft mice models generated with MDA-MB-231 cells were used for in vivo studies with BENSpm and paclitaxel.

Results and conclusion

BENSpm exhibited synergistic inhibitory effect on cell proliferation in combination with 5-FU or paclitaxel in human breast cancer cell lines (MDA-MB-231 and MCF-7) and was either antagonistic or less effective in the non-tumorigenic MCF-10A cell line. Synergism was highest with 120 h concomitant treatment or pre-treatment with BENSpm for 24 h followed by concomitant treatment for 96 additional hours. Since the cytotoxic effects of many polyamine analogues and cytotoxic agents are believed to act, in part, through induction of the polyamine catabolic enzymes SSAT and SMO, the role of these enzymes on synergistic response was evaluated in MDA-MB-231 and MCF-7 treated with BENSpm and 5-FU or paclitaxel. Combination treatments of BENSpm with 5-FU or paclitaxel resulted in induction of SSAT mRNA and activity in both cell lines compared to either drug alone, while SMO mRNA and activity were increased only in MDA-MB-231 cells. Induction was greater with BENSpm/paclitaxel combination than BENSpm/5-FU. Further, RNAi studies demonstrated that both SSAT and SMO play a significant role in the response of MDA-MB-231 cells to treatment with BENSpm and 5-FU or paclitaxel. In MCF-7 cells, only SSAT appears to be involved in the response to these treatments. In an effort to translate combination studies from in vitro to in vivo, and to form a basis for clinical setting, the in vivo therapeutic efficacy of BENSpm alone and in combination with paclitaxel on tumor regression was evaluated in xenograft mice models generated with MDA-MB-231 cells. Intraperitoneal exposure to BENSpm or taxol singly and in combination for 4 weeks resulted in significant inhibition in tumor growth. These findings help elucidate the mechanisms involved in synergistic drug response and support combinations of polyamine analogues with chemotherapeutic agents which could potentially be used in the treatment of breast cancer.     

Combination of standard cytotoxic agents with polyamine analogues in the treatment of breast cancer cell lines.

Polyamines are essential for cell growth and differentiation. Structural polyamine analogues have been shown to have antitumor activity in experimental models including breast cancer. The ability of polyamine analogues to alter activity of cytotoxic chemotherapeutic agents in breast cancer models has not been evaluated. This study evaluates the ability of two polyamine analogues, N1-ethyl-N11-[(cyclopropyl)methyl]-4,8-diazaundecane (CPENSpm) and N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm) to synergize with cytotoxics in five human breast cancer cell lines. Antagonism, additivity, or synergy of the combinations was determined using the median effect/combination index model. The chemotherapeutic agents chosen, cis-diaminechloroplatinum(II), doxorubicin, 5-fluorouracil, fluorodeoxyuridine, 4-hydroperoxycyclophosphamide, paclitaxel, docetaxel, and vinorelbine, all have antitumor activity in breast cancer and represent a spectrum of mechanisms. Three treatment schedules of polyamine analogue and cytotoxic were tested in MCF-7 and MDA-MB-468 lines, demonstrating a schedule-dependence of synergistic growth inhibition. Cytotoxic agent alone for 24 h followed by polyamine analogue alone for 96 h resulted in the most synergistic combinations and the greatest synergy. This schedule was then tested in three additional breast cancer lines, and several synergistic combinations were again identified. Two cytotoxics, vinorelbine and the fluoropyrimidines, showed the most promise in combination with the polyamine analogues. They were able to synergize with one or both polyamine analogues in most of the breast cancer cell lines. CPENSpm was also able to synergize with virtually all of the cytotoxics in the estrogen receptor alpha-positive MCF-7 and T-47D lines. These preclinical data demonstrate a treatment schedule and combinations of polyamine analogues and cytotoxics that will be important to study mechanistically and clinically for breast cancer.     

Synergism between the anticancer actions of 2-methoxyestradiol and microtubule-disrupting agents in human breast cancer

2-Methoxyestradiol (2-MeO-E(2)), a well-known nonpolar endogenous metabolite of 17beta-estradiol, has strong antiproliferative, apoptotic, and antiangiogenic actions in vitro and in vivo at pharmacologic concentrations. We determined in the present study whether 2-MeO-E(2) can enhance the anticancer actions of paclitaxel or vinorelbine (two commonly used microtubule-disrupting agents) in several human breast cancer cell lines, including the estrogen receptor-positive MCF-7 and T-47D cells and the receptor-negative MDA-MB-435s and MDA-MB-231 cells. 2-MeO-E(2) in combination with paclitaxel or vinorelbine exhibited a synergistic anticancer effect in these human breast cancer cells in vitro, and this synergistic effect was more pronounced when each of the drugs was used at relatively low concentrations. Additional experiments using female athymic BALB/c nu/nu mice showed that p.o. administration of 2-MeO-E(2) at 30 mg/kg body weight, once a week for 6 weeks, markedly enhanced the activity of paclitaxel or vinorelbine against the growth of the estrogen receptor-negative MDA-MB-231 human breast cancer xenografts in these animals. By contrast, combination of 2-MeO-E(2) with 5-fluorouracil only had a partial additive effect against the growth of these cell lines in culture, and no synergistic effect was observed. Interestingly, when doxorubicin was used in combination with 2-MeO-E(2), the antiproliferative effect of 2-MeO-E(2) was somewhat antagonized by doxorubicin when it was present at high concentrations. Our results showed that 2-MeO-E(2) at nontoxic or subtoxic doses selectively enhanced the effects of certain microtubule-disrupting agents (such as paclitaxel and vinorelbine) against the growth of the receptor-negative human breast cancer cells in culture and also in athymic nude mice.     

Pharmacological inhibition of fatty acid synthase (FAS): a novel therapeutic approach for breast cancer chemoprevention through its ability to suppress Her-2/neu (erbB-2) oncogene-induced malignant transformation

We designed our experiments to evaluate whether fatty acid synthase (FAS), a lipogenic enzyme linked to tumor virulence in population studies of human cancer, is necessary for the malignant transformation induced by Her-2/neu (erbB-2) oncogene, which is overexpressed not only in invasive breast cancer but also in premalignant atypical duct proliferations and in ductal carcinoma in situ of the breast. To avoid the genetic complexities associated with established breast cancer cell lines, we employed NIH-3T3 mouse fibroblasts engineered to overexpress human Her-2/neu coding sequence. NIH-3T3/Her-2 cells demonstrated a significant upregulation of FAS protein expression, which was dependent on the upstream activation of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/AKT pathways. Remarkably, pharmacological FAS blockade using the mycotoxin cerulenin or the novel small compound C75 completely suppressed the state of Her-2/neu-induced malignant transformation by inhibiting the ability of NIH-3T3/Her-2 cells to grow under either anchorage-independent (i.e., to form colonies in soft agar) or low-serum monolayer conditions. Moreover, NIH-3T3/Her-2 fibroblasts were up to three times more sensitive to chemical FAS inhibitors relative to untransformed controls as determined by MTT-based cell viability assays. In addition, pharmacological FAS blockade preferentially induced apoptotic cell death of NIH-3T3/Her-2 fibroblasts, as determined by an ELISA for histone-associated DNA fragments and by the terminal deoxynucleotidyltransferase (TdT)-mediated nick end labeling assay (TUNEL). Interestingly, the degree of Her-2/neu oncogene expression in a panel of breast cancer cell lines was predictive of sensitivity to chemical FAS inhibitors-induced cytotoxicity, while low-FAS expressing and chemical FAS inhibitors-resistant MDA-MB-231 breast cancer cells became hypersensitive to FAS blockade when they were engineered to overexpress Her-2/neu. Our observations strongly suggest that inhibition of FAS activity may provide a new molecular avenue for chemotherapeutic prevention and/or treatment of Her-2/neu-related breast carcinomas.     

化疗对三阴性乳腺癌CD44+CD24-/low细胞亚群的影响

目的:观察化疗对三阴性乳腺癌CD44+CD24-/low细胞亚群的影响。方法:用0.2、1、5倍药物血浆峰浓度(peak plasma concentration,PPC)的紫杉醇、多西紫杉醇、氟尿嘧啶、表柔比星、长春瑞滨和吉西他滨分别作用人三阴性乳腺癌MDA-MB-231细胞24、48和72h后,MTT法检测细胞生长情况;FCM法检测1倍PPC的各药物作用48h以及细胞复苏后CD44+CD24-/low细胞含量的变化;另外,FCM法检测10例转移性三阴性乳腺癌患者在化疗前后,外周血中CD44+CD24-/low细胞的含量变化。结果:6种药物均可抑制MDA-MB-231细胞增殖。1倍PPC的各药作用后,CD44+CD24-/low细胞含量未明显升高,以氟尿嘧啶组含量降低最为明显(P<0.05)。转移性三阴性乳腺癌患者在化疗后,外周血中CD44+CD24-/low细胞含量降低(P<0.05)。结论:化疗药物紫杉醇、多西紫杉醇、氟尿嘧啶和表柔比星可降低MDA-MB-231细胞株中CD44+CD24-/low细胞亚群的含量。化疗可使转移性三阴性乳腺癌患者外周血中CD44+CD24-/low细胞含量降低。     

Comparative analysis of xanafide cytotoxicity in breast cancer cell lines

Xanafide, a DNA-intercalating agent and topoisomerase II inhibitor, has previously demonstrated comparable cytotoxicity to the parent drug amonafide (NSC 308847). The current study was conducted to investigate further the anti-proliferative effects of xanafide in human breast cancer cell lines, in vitro and in vivo. The in vitro activity of xanafide against MCF-7, MDA-MB-231, SKBR-3 and T47D cell lines was compared to that of paclitaxel, docetaxel, gemcitabine, vinorelbine and doxorubicin. In MCF-7, xanafide demonstrated comparable total growth inhibition (TGI) concentrations to the taxanes and lower TGI values than gemcitabine, vinorelbine and doxorubicin. MCF-7 (oestrogen receptor (ER)+/p53 wild-type) was the most sensitive cell line to xanafide. MDA-MB-231 and SKBR-3 exhibited similar sensitivity to xanafide. T47 D (ER+/p53 mutated), showed no response to this agent. The in vivo activity of xanafide was further compared to that of docetaxel in MCF-7 and MDA-MB-231 cell lines using the hollow fibre assay. Xanafide was slightly more potent than docetaxel, at its highest dose in MCF-7 cell line, whereas docetaxel was more effective than xanafide in MDA-MB-231 cell line. Our results show that there is no relationship between sensitivity of these cell lines to xanafide and cellular levels of both isoforms of topoisomerase II and suggest that ER and p53 status and their crosstalk may predict the responsiveness or resistance of breast cancer patients to xanafide.     

Temporal and quantitative regulation of mitogen-activated protein kinase (MAPK) modulates cell motility and invasion.

We have shown that ER-negative and invasive human breast cancer cell lines MDA-MB-468 and MDA-MB-231 have constitutively higher mitogen activated protein kinase (ERK1&2/MAPK) when compared to the ER-positive and non-invasive MCF-7 human breast cancer cells. In MCF-7 cells, TGFalpha stimulation induced only transient MAPK activation, leading to a transient increase in cell migration. However, MDA 231 and MDA 468 cells, TGFalpha stimulation induced sustained MAPK activation, which correlated with enhanced cell motility and in vitro invasion. Serum stimulation activates ERK/MAPK activity persistently in both ER-positive and ER-negative breast cancer cells, leading to enhanced and sustained cell migration. Inhibition of MAPK activation by anti-sense MEK expression in MDA-MB-468 cells significantly inhibits cell migration and in vitro invasion. In contrast, MCF-7 cells expressing constitutively activated MEK show a significant increase in MAPK activity and cell migration, but this failed to enhance in vitro invasion. The kinetic profiles of MAPK activation and inhibition show a relationship between the duration and magnitude of MAPK activation and cell migration in both ER-positive and ER-negative human breast cancer cells. These studies show that cell motility is modulated by the magnitude and the duration of MAPK activation; but increased activation of MAPK may not be sufficient to allow in vitro invasion in non-invasive MCF-7 breast cancer cells.     

Differential anti-tumor activity of coriolus versicolor (Yunzhi) extract through p53- and/or Bcl-2-dependent apoptotic pathway in human breast cancer cells

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells, but the underlying mechanism has not been fully elucidated. The present study aimed to evaluate the in vitro anti-tumor activity of a standardized aqueous ethanol extract prepared from CV on four breast cancer cell lines using MTT assay, and test whether the mechanism involves apoptosis induction and modulation of p53 and Bcl-2 protein expressions using cell death detection ELISA, p53 and Bcl-2 ELISAs respectively. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of three breast tumor cell lines, with ascending order of IC50 values: T-47D, MCF-7, MDA-MB-231, while BT-20 cells were not significantly affected. Tumoricidal activity of the CV extract was found to be comparable to a chemotherapeutic anti-cancer drug, mitomycin C. Nucleosome productions in apoptotic MDA-MB-231, MCF-7 and T-47D cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of CV extract. Expression of p53 protein was significantly upregulated only in T-47D cells treated with the CV extract in a dose- and time-dependent fashion, but not in MCF-7 (except at 400 mug/ml after 16 h) and MDA-MB-231 cells. The CV extract significantly induced a dose-dependent downregulation of Bcl-2 protein expression in MCF-7 and T-47D cells, but not in MDA-MB-231 cells. These results suggested that apoptosis induction, differentially dependent of p53 and Bcl-2 expressions, might be the possible mechanism of CV extract-mediated cytotoxicity in human breast cancer cells in vitro.     

乳腺癌干细胞富集与鉴定的初步研究

目的:通过从MCF-7、ZR-75-1、MDA-MB-231乳腺癌细胞系中培养富集及鉴定乳腺癌干细胞(breast cancer stem cell,BCSC),寻找培养与富集乳腺癌干细胞的方法。方法:贴壁培养MCF-7、ZR-75-1、MDA-MB-231细胞系,倒置显微镜观察各细胞形态;流式细胞仪分别分选收集CD44-CD24-、CD44-CD24+、CD44+CD24-及 CD44+CD24+ 细胞,其中CD44+CD24-为乳腺癌干细胞,其余三类为对照组;MTT法计数细胞,绘制MCF-7、ZR-75-1、MDA-MB-231细胞系生长曲线;MCF-7细胞系进行无血清悬浮培养1个周期,流式细胞仪检测分子表面标记物CD44+CD24-含量,贴壁培养的CD44+CD24-乳腺癌干细胞为对照组;将分选的MCF-7（CD44+CD24-）和分选的其余MCF-7细胞（非CD44+CD24-）进行干性成球实验,鉴定CD44+CD24-干性表达。结果:MCF-7、MDA-MB-231细胞系富含表面标志物CD44-CD24-的乳腺癌细胞;ZR-75-1细胞系富含分子表面标志物CD44+CD24+的乳腺癌细胞;生长曲线显示MCF-7、ZR-75-1、MDA-MB-231均呈持续增长,MDA-MB-231细胞生长较MCF-7、ZR-75-1细胞快;通过无血清悬浮培养CD44+CD24-乳腺癌干细胞由19.4%富集到88.9%;成球实验中CD44+CD24-表型细胞成球数量较分选的其余MCF-7细胞（非CD44+CD24-表型）明显增多,成球率分别为（36.5±1.7）%,（1.1±0.5）%。结论:流式细胞仪可成功分选出分子表面标志物为CD44+CD24-的乳腺癌干细胞;CD44+CD24-可能不是乳腺癌干细胞唯一的表面标志物;MDA-MB-231细胞系较MCF-7、ZR-75-1细胞系生长快;无血清悬浮培养法可简便、高效地富集乳腺癌干细胞;CD44+CD24-乳腺癌干细胞干性表达较强。     

Exogenous supplementation with omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA; 22:6n-3) synergistically enhances taxane cytotoxicity and downregulates Her-2/neu (c-erbB-2) oncogene expression in human breast cancer cells.

Omega-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA; 22:6n-3) and other omega-3 and omega-6 PUFAs have raised interest as novel anticancer agents by exerting selective cytotoxic effects on human cancer cells without affecting normal tissues. Here, we examined the in vitro relationship between exogenous supplementation with DHA and breast cancer chemosensitivity to taxanes. We measured cell viability in the highly metastatic human breast cancer cell line MDA-MB-231 exposed sequentially to DHA followed by paclitaxel (Taxol) or docetaxel (Taxotere). As DHA by itself showed cytotoxic effects, possible synergistic interactions between DHA and taxanes were assessed, employing the combination index (CI) method and the isobologram analysis. Both methods showed a strong synergism (CI approximately 0.5; P<0.005) between DHA and taxanes in MDA-MB-231 cells. When the increase in taxanes efficacy was measured by dividing the IC50 values (50% inhibitory concentrations) obtained when the cells were exposed to taxanes alone by those after DHA pre-exposure, we found that DHA enhanced the cytotoxic activity of taxanes against MDA-MB-231 cells in a dose-dependent manner (up to 13- and 5-fold increase in Taxol and Taxotere efficacy, respectively). Importantly, sequential exposure to DHA followed by taxanes also yielded strong synergism in Her-2/neu (c-erbB-2)-overexpressing and taxanes-resistant SK-Br3 and BT-474 breast cancer cells. Moreover, exogenous supplementation with DHA significantly decreased the expression of Her-2/neu-codified p185(Her-2/neu) oncoprotein (up to 78% reduction in BT-474 cells). Our results provide experimental support to the hypothesis that omega-3 PUFAs can be used as modulators of tumor cell chemosensitivity and provide the rationale for in vivo preclinical investigation. In addition, this is the first study demonstrating that omega-3 PUFA DHA downregulates Her-2/neu oncogene expression in human breast cancer cells.     

Inhibitory effects of sea buckthorn procyanidins on fatty acid synthase and MDA-MB-231 cells

Fatty acid synthase (FAS) is overexpressed in many human cancers including breast cancer and is considered to be a promising target for therapy. Sea buckthorn has long been used to treat a variety of maladies. Here, we investigated the inhibitory effect of sea buckthorn procyanidins (SBPs) isolated from the seeds of sea buckthorn on FAS and FAS overexpressed human breast cancer MDA-MB-231 cells. The FAS activity and FAS inhibition were measured by a spectrophotometer at 340 nm of nicotinamide adenine dinucleotide phosphate (NADPH) absorption. We found that SBP potently inhibited the activity of FAS with a half-inhibitory concentration (IC₅₀) value of 0.087 μg/ml. 3-4,5-Dimethylthiazol-2-yl-2,3-diphenyl tetrazolium bromide (MTT) assay was used to test the cell viability. SBP reduced MDA-MB-231 cell viability with an IC₅₀ value of 37.5 μg/ml. Hoechst 33258/propidium iodide dual staining and flow cytometric analysis showed that SBP induced MDA-MB-231 cell apoptosis. SBP inhibited intracellular FAS activity with a dose-dependent manner. In addition, sodium palmitate could rescue the cell apoptosis induced by SBP. These results showed that SBP was a promising FAS inhibitor which could induce the apoptosis of MDA-MB-231 cells via inhibiting FAS. These findings suggested that SBP might be useful for preventing or treating breast cancer.     

Comparative studies on the polyamine metabolism and DFMO treatment of MCF-7 and MDA-MB-231 breast cancer cell lines and xenografts.

In order to characterize the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and xenografts, their growth kinetic parameters and some biochemical characteristics concerning the receptor status and polyamine metabolism were determined and compared. The doubling times calculated from the growth curves showed higher proliferation rate of MDA-MB-231 cells, both in culture (21 hours) and in xenograft (9.7 days), in comparison to the MCF-7 cells which had values of 32 hours and 11.6 days, respectively. Growth-dependent changes observed in the intracellular putrescine, spermidine and spermine concentrations indicated a higher activity of polyamine metabolism in the MDA-MB-231 cells and xenograft as well. However, biosynthetic key-enzyme ornithine decarboxylase activity (ODC, EC 4.1.1.17) showed neither characteristic differences between the two types of breast cancer, nor consistent relationship with their proliferation rate. Metabolic alterations of the MCF-7 and MDA-MB-231 cell lines grown in vitro were also reflected in the polyamine composition of their culture medium. Independently of their receptor status, both types of breast cancer were responsive to difluoromethylornithine (DFMO) treatment. DFMO inhibited the ODC activity totally and depleted the cellular polyamine levels. MCF-7 cells in culture were more sensitive to the antitumoral effect of DFMO than the MDA-MB-231 line, while the rate of growth inhibition did not differ significantly in the xenografts. The present results provided further evidence on the different polyamine metabolism of ER-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells in vitro and in vivo, suggesting a correlation of hormonal modulation with polyamines as a determinant group of biological response modifiers.     

Effects of gamma-linolenic acid and oleic acid on paclitaxel cytotoxicity in human breast cancer cells

It has been suggested that dietary interventions may improve the effectiveness of cancer chemotherapy. We have examined the combined in vitro cytotoxicity of paclitaxel and the fatty acids gamma-linolenic acid (GLA, 18:3n-6) and oleic acid (OA, 18:1n-9) in human breast carcinoma MDA-MB-231 cells. The effect of fatty acids on paclitaxel chemosensitivity was determined by comparing IC(50) and IC(70) (50 and 70% inhibitory concentrations, respectively) obtained when the cells were exposed to IC(50) and IC(70) levels of paclitaxel alone and fatty acids were supplemented either before or during the exposure to paclitaxel. The 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine cell growth inhibition. GLA by itself showed antiproliferative effects, and a possible GLA-paclitaxel interaction at the cellular level was assessed by the isobologram and the combination-index (CI) methods. Isobole analysis at the isoeffect levels of 50 and 70% revealed that drug interaction was predominantly synergistic when GLA and paclitaxel were added concurrently for 24 h to the cell cultures. Interaction assessment using the median-effect principle and the combination-index (CI) method showed that exposure of MDA-MB-231 cells to an equimolar combination of concurrent GLA plus paclitaxel for 24 h resulted in a moderate synergism at all effect levels, consistent with the results of the isobologram analysis. When exposure to GLA (24 h) was followed sequentially by paclitaxel (24 h) only an additive effect was observed. The GLA-mediated increase in paclitaxel chemosensitivity was only partially abolished by Vitamin E, a lipid peroxidation inhibitor, suggesting a limited influence of the oxidative status of GLA in achieving potentiation of paclitaxel toxicity. When OA (a non-peroxidisable fatty acid) was combined with paclitaxel, an enhancement of chemosensitivity was found when OA was used concurrently with paclitaxel, although less markedly than with GLA. Pretreatment of MDA-MB-231 cells with OA for 24 h prior to a 24 h paclitaxel exposure produced greater enhancement of paclitaxel sensitivity at high OA concentrations than the concurrent exposure to OA and paclitaxel. The OA-induced sensitisation to paclitaxel was not due to the cytoxicity of the fatty acid itself. When these observations were extended to three additional breast carcinoma cell lines (SK-Br3, T47D and MCF-7), simultaneous exposure to GLA and paclitaxel also resulted in synergism. GLA preincubation followed by paclitaxel resulted in additivity for all cell lines. Simultaneous exposure to paclitaxel and OA enhanced paclitaxel cytotoxicity in T47D and MCF-7 cells, but not in SK-Br3 cells, whereas preincubation with OA failed to increase paclitaxel effectiveness in all three cell lines. For comparison, the effects of other fatty acids on paclitaxel chemosensitivity were examined: GLA was the most potent at enhancing paclitaxel cytotoxicity, followed by alpha-linolenic acid (ALA; 18:3n.3), eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), whereas linoleic acid (LA; 18:2n-6) did not increase paclitaxel toxicity. These findings provide experimental support for the use of fatty acids as modulators of tumour cell chemosensitivity in paclitaxel-based therapy.     

Antiestrogenic effects of Z-1, 1-dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane(5a) on human breast cancer cells in culture.

Compound 5a ([Z]-1, 1-Dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane) is a novel cyclopropyl compound which was shown to be a pure antiestrogen. In the present study, the antiproliferative activity of 5a was examined on estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells using the hemocytometric trypan blue exclusion method. Compound 5a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10(-9) to 10(-5) M, but did not alter the growth of MDA-MB-231 or A-549 cells. Co-administration of estradiol (10(-8) M) reversed the antiproliferative activity of 5a (10(-7) M) on MCF-7 cells. Further, an ER-dependent mechanism of action is supported by the specific ER binding of 5a in MCF-7 cells observed in this study. The influence of 5a on the cell surface morphology of MCF-7 and MDA-MB-231 cells was studied using scanning electron microscopy (SEM). Compound 5a at 10(-6) M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10(-8) M). This compound did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 5a and tamoxifen inhibited the growth of ER-prositive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells. The results of this study with human breast cancer cells suggest that 5a may be highly effective in the treatment of estrogen-dependent breast cancer and/or in the prophylactic treatment of women with a high risk of breast cancer development.