Assessment of biological activity and UPLC–MS based chromatographic profiling of ethanolic extract of Ochradenus arabicus

Natural products from wild and medicinal plants, either in the form of crude extracts or pure compounds provide unlimited opportunities for new drug leads owing to the unmatched availability of chemical diversity. In the present study, the cytotoxic potential of crude ethanolic extract of Ochradenus arabicus was analyzed by MTT cell viability assay in MCF-7 adenocarcinoma breast cancer cells. We further investigated its effect against oxidative stress induced by anticancer drug doxorubicin. In addition, Ultra Performance Liquid Chromatography–Mass Spectrometry (UPLC–MS) based chromatographic profiling of crude extract of O. arabicus was performed. The MTT assay data showed that the extract is moderately toxic to the MCF-7 cells. However, its treatment alone does not induce oxidative stress while doxorubicin increases the level of oxidative stress in MCF-7 cells. Whereas, simultaneous treatment of plant extract and doxorubicin significantly (p < 0.05) decreased the level of intracellular reactive oxygen species (ROS) and lipid peroxidation while an increase in the reduced glutathione and superoxide dismutase activity was observed in time and dose dependent manner. Hence, our finding confirmed cytotoxic and antioxidant potential of crude extract of O. arabicus in MCF-7 cells. However, further investigations on O. arabicus as a potential chemotherapeutic agent are needed. The analysis of bioactive compounds present in the plant extracts involving the applications of common phytochemical screening assays such as chromatographic techniques is discussed.     

Characterization and biological activity of the main flavonoids from Swiss Chard (Beta vulgaris subspecies cycla)

The molecular components of a phenolic fraction (P2), obtained from liquid chromatography of a Swiss Chard (Beta vulgaris subsp. cycla) extract, were identified using HPLC-ESI-MS/MS. The primary P2 components were: vitexin-2'O-rhamnoside, its demethylated form 2'-xylosylvitexin, isorhamnetin 3-gentiobioside, and rutin. P2 "in toto" and the single components were characterized for antioxidant capacity, antimitotic activity on MCF-7 human breast cancer cells and for toxicity to human lymphocytes and macrophages. P2 inhibited MCF-7 cell proliferation (IC(50) value = 9 microg/ml) without inducing apoptosis, showed no toxicity to human lymphocytes and slight toxicity to macrophages. Vitexin-2'O-rhamnoside strongly inhibited DNA synthesis in MCF-7 cells, whereas 2'-xylosylvitexin and isorhamnetin 3-gentiobioside were activators; combinations of activators and inhibitors maintained the over-all inhibitory effect.     

Antiproliferative activity of chloroformic extract of Persian Shallot, Allium hirtifolium, on tumor cell lines

Allium hirtifolim (Persian Shallot) belongs to Allium genus (Alliaceae family). We investigated the in vitro effects of chloroformic extract of A. hirtifolium and its Allicin on the proliferation of HeLa (cervical cancer), MCF7 (human, caucasion, breast, adenocarcinoma) and L929 (mouse, C3H/An, connective) cell lines. Our results showed that components of A. hirtifolium might inhibit proliferation of tumor cell lines. This inhibition in HeLa and MCF-7 cells was dose-dependent. The presence of Allicin was evaluated by TLC method in bulbs and the extract of A. hirtifolium was analyzed by HPLC. MTT test was performed 24, 48 and 72 h after cell culture. A significant decrease in cell lines was observed in HeLa and MCF-7 as compared to L929 cell lines. DNA fragmentation analysis revealed a large number of apoptotic cells in treated HeLa and MCF-7 cell groups, but no effects in L929 cells. Therefore A. hirtifolium might be a candidate for tumor suppression.     

Metabolic Profiling Reveals Sphingosine-1-Phosphate Kinase 2 and Lyase as Key Targets of (Phyto-) Estrogen Action in the Breast Cancer Cell Line MCF-7 and Not in MCF-12A

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.     

Apoptosis and necrosis of human breast cancer cells by an aqueous extract of garden cress (Lepidium sativum) seeds

Conventional treatments for breast cancer are costly and have serious side effects. Non-conventional natural treatments have gained wide acceptance due to their promise of a cure with minimal or no side effects, but little scientific evidence exists. One such common remedy is the seed of the Lepidium sativum plant. Presented here is the first reported use of the aqueous extract of Lepidium sativum seeds on breast cancer cells. The ability of the extract to induce apoptosis and necrosis in the human breast cancer cell line MCF-7, compared to normal human skin fibroblasts (HFS), was determined by morphological changes in the cells using light microscopy, DNA fragmentation assay, and florescent stains (Annexin V and propidium iodide) using flow cytometry and fluorescent microscopy. Apoptosis was induced in both cells, and more in MCF-7, when they were treated with 25% and 50% extract, while necrosis was observed mainly after exposure to elevated extract concentrations (75%). DNA fragmentation resulted for both cells, in a time and dose-dependent manner. Both cells, at all extract concentrations, showed no significant differences in the number of living, dead, apoptotic, and necrotic cells. Finally, the results may indicate that apoptotic changes in MCF-7 may be independent of caspase-3, which is involved in apoptosis and is lacking in MCF-7 cells.     

Knocking down Wnt9a mRNA levels increases cellular proliferation

Wnts are secreted lipid-modified signaling proteins. Activation of Wnt signalling in many tissues has also been associated with cancer. In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a small double-stranded RNA corresponding to exon sequences in the mRNA. In this study we used pAVU6+27 vectors, which have SalI and XbaI clone sites, to construct the siRNA expression vectors for human Wnt9a. Two kinds of small interfering RNA inserts were designed, synthesized and visually tested for efficacy by in situ hybridization, the results demonstrated that in the cells, transfected with U6+27 cassettes with anti-Wnt9a hairpin siRNA inserts, dramatically reduced Wnt9a signals were observed as compared to the untransfected cells. The results of flow cytometry analysis showed that the cell proliferation was promoted after lowering expression of the human Wnt9a in MCF-7 cells by RNAi, but was inhibited after over-expression of human Wnt9a. These results suggests the expression level of human Wnt9a in MCF-7 that breast cancer may play a role in adjusting the rate of cellular proliferation.     

Differential effects of soybean and fenugreek extracts on the growth of MCF-7 cells

The effect of aqueous and ethanol extracts of soybean and fenugreek on the growth of MCF-7 cells, an estrogen receptor positive breast cancer cell line, has been examined in this study. Soybean is well known for the presence of phytoestrogens and fenugreek is reported to have medicinal use including anticancer properties. In a dose dependent manner soybean aqueous and ethanol extract promoted the growth and DNA synthesis in MCF-7 cells. On the contrary ethanol extract of fenugreek decreased the cell viability and induced early apoptotic changes such as flipping of phosphatidylserine and decrease of mitochondrial membrane potential. Degradation of cellular DNA into fragments comprising multiples of approximately 180-200 base pair was also observed. Cell cycle analysis by flow cytometry showed the presence of a subG1 apoptotic population which was more prominent at higher concentrations along with cell cycle arrest at G2/M phase. Our experiments show that while the soybean extract acts as a promoter of MCF-7 cell growth, the fenugreek extract induces apoptosis.     

Enhanced production of tissue-type plasminogen activator by estradiol in a novel type variant of human breast cancer MCF-7 cell line

ES-1 cells, which showed a higher sensitivity to the cytocidal action of estradiol were isolated from a human breast cancer MCF-7 cell line. Growth of ES-1 cells was inhibited by a dose of 17-beta estradiol that stimulated the growth of the parental MCF-7 cells. Proteins secreted from MCF-7 and ES-1 cells when cultured with 17-beta estradiol were compared by sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis (SDS-PAGE). Addition of estradiol to culture medium enhanced secretion of a protein of molecular mass of 52 kDa in media for both MCF-7 and ES-1 cell lines, but the secretion of a second 67 kDa protein was enhanced about 10-fold only in ES-1 cells. The analysis by SDS-PAGE of culture medium immunoprecipitated with anti-tissue-type plasminogen activator (t-PA) antibody demonstrated that the band of 67 kDa protein specifically secreted from estradiol-treated ES-1 cells contained t-PA. Zymography assays, quantitative immunoreactive assays, and Northern analysis showed about 5-fold specific increase by estradiol of t-PA with molecular mass of 65-70 kDa in ES-1 but not in its parental MCF-7 cells. Cellular level of the plasminogen activity was also specifically enhanced in ES-1 cells by estradiol, but only a slightly in MCF-7 cells. By contrast, another urokinase-type PA (u-PA) with molecular weight of 55 kDa showed very low level activity in both MCF-7 and ES-1 cell lines in the presence of estradiol. Formation of t-PA mRNA was specifically enhanced in ES-1 cells when ES-1 cells were treated for more than 12 h with 10(-8) M 17-beta estradiol. Estradiol did not elongate the lifetime of t-PA mRNA in ES-1 cells. A unique phenotype of ES-1 cells in response to estradiol is discussed in relation to activating expression of the t-PA gene.     

桑葚花色苷提取物对乳腺癌细胞凋亡及线粒体膜电位的影响

目的:观察桑葚花色苷提取物对人乳腺癌细胞株MDA-MB-453、MDA-MB-231和MCF-7细胞凋亡及线粒体膜电位的影响.方法:利用超声辅助乙醇萃取法提取桑葚花色苷,pH示差法测定提取物花色苷总含量,以50、100和150 mg/mL桑葚花色苷提取物作用三种乳腺癌细胞MDA-MB-231、MDA-MB-453和MCF-7 24h,采用Annexin V/PI双染流式细胞分析法检测细胞凋亡水平变化,JC-1探针染色激光共聚焦扫描显微镜观察MDA-MB-453细胞线粒体膜电位水平变化.结果:凋亡分析结果表明,桑葚花色苷提取物作用后三种乳腺癌细胞凋亡率均升高,显示出促凋亡效应,且具有剂量-效应关系,100和150 mg/mL组凋亡率显著升高(P＜0.05).激光共聚焦扫描显微镜检测结果显示,桑葚花色苷提取物作用24h,可使MDA-MB-453细胞线粒体膜电位显著下降,表现为红色/绿色荧光的比值显著降低(P＜0.05).结论:桑葚花色苷提取物可显著降低乳腺癌细胞线粒体膜电位,并促发细胞凋亡.     

The in vitro biological activity of Lepidium meyenii extracts

The biological activity of methanolic and aqueous extracts from dehydrated hypocotyls of Lepidium meyenii (Brassicaceae, vernacular name “maca”), was studied on rat hepatocytes and human breast cancer MCF-7 cells. The extracts did not exhibit cytotoxicity in hepatocyte primary cultures up to 10 mg/ml as measured by the MTT viability test, and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) leakage. Moreover, after 72 h, extracts inhibited LDH and AST leakage from the hepatocytes. When hepatocytes were intoxicated by t-butyl hydroperoxide, neither extract prevented oxidative damage. Both extracts showed weak antioxidant activity in the DPPH radical scavenging test with IC₅₀ values of 3.46 ± 0.16 and 0.71 ± 0.10 mg/ml, for aqueous and methanolic extracts, respectively. Thus, the observed effect on spontaneous enzyme leakage is probably mediated through mechanisms other than antioxidant activity. Both methanolic and aqueous extracts have shown estrogenic activity comparable with that of silymarin in MCF-7 cell line. Maca estrogenicity was exhibited in the range from 100 to 200 μg of extract per ml. The findings in the present study show that maca does not display in vitro hepatotoxicity. In contrast, a slight cytoprotective effect, probably not mediated by antioxidant capacity, was noted. Maca extracts exhibited estrogenic activity comparably to the effect of silymarin in MCF-7 cells.     

Effects of triptolide from Tripterygium wilfordii on ERα and p53 expression in two human breast cancer cell lines

The aim of the study was to discover possible differential cytotoxicity of triptolide towards estrogen-sensitive MCF-7 versus estrogen-insensitive MDA-MB-231 human breast cancer cells. Considering that MCF-7 cells express functional Estrogen receptor α (ERα) and wild-type p53, whereas MDA-MB-231 cells which are ERα-negative express mutant p53, the anti-proliferation effect of triptolide on MCF-7 and MDA-MB-231 cells were examined, the apoptotic effect and cell cycle arrest caused by triptolide were investigated, ERα and p53 expression were also observed in this paper. The results showed that the anti-proliferation effects were induced by triptolide in both cell lines. But the value of IC₅₀ in MCF-7 cells for its anti-proliferation effect was about one tenth of that in MDA-MB-231 cells, which indicated that the effect is more potent in MCF-7 cells. Condensed chromatin or fragmented nuclei could be found in MCF-7 cells treated with only 40 nM triptolide but in MDA-MB-231 cells they couldn’t be observed until the concentration reached to 400 nM. Triptolide induced significant S cell cycle arrest along with the presence of sub-G0/G1 peak in MDA-MB-231 cells, whereas there was only slightly S cell cycle arrest on cell cycle distribution in MCF-7 cells. The role of p53 in two breast cancer cells was examined, the results showed that the mutant p53 in MDA-MB-231 cells was suppressed and the wild-type p53 in MCF-7 was increased. Moreover, triptolide could down regulate the expression of ERα in MCF-7 cells. The results showed that triptolide is much more sensitive to ERα-positive MCF-7 cells than to ERα-negative MDA-MB-231 cells, and the sensitivity is significantly associated with the ERα and p53 status.     

Human mammary cancer cell mutants with altered hormone receptor activity

We have recently isolated retinoic acid-resistant clones U-2 and U-3 from human breast cancer cell line MCF-7 (Ueda et al. (1985) Cancer Res. 45, 3332-3338). Growth of MCF-7 cells was found to be stimulated by estradiol but that of U-2 or U-3 was not. Cytosol from U-2 or U-3 cells contained no detectable estradiol receptor activity, whereas that from the parental MCF-7 cells showed estradiol receptor activity of 32 fmol/mg cytosol protein with a Kd of 2.6 X 10(-10) M by Scatchard analysis. Sucrose gradient centrifugation analysis of the cytosol fraction confirmed the presence of estradiol receptor activity in MCF-7 but not in U-2. Cytosol from MCF-7 and U-2 cells showed progesterone receptor activities of 106 fmol/mg protein with a Kd of 7.4 X 10(-10) M and 13 fmol/mg protein with a Kd of 9.9 X 10(-10) M, respectively. Addition of estradiol to the culture medium of the cells increased the level of progesterone receptor about 2-fold in MCF-7, but not in U-2. U-2 or U-3 cells showed about 5-fold higher resistance to an antiestrogen, tamoxifen, than MCF-7, and they were also 300- to 1,000-fold more resistant to other antiestrogens, epitiostanol and medroxyprogesterone, than MCF-7. The altered cellular sensitivity of U-2 or U-3 to the hormone antagonists is discussed in relation to the absence or presence of hormone receptors.     

Identification of DEAD-box RNA Helicase 6 (DDX6) as a Cellular Modulator of Vascular Endothelial Growth Factor Expression under Hypoxia

Vascular endothelial growth factor A (VEGF) is a crucial proangiogenic factor, which regulates blood vessel supply under physiologic and pathologic conditions. The VEGF mRNA 5′-untranslated region (5′-UTR) bears internal ribosome entry sites (IRES), which confer sustained VEGF mRNA translation under hypoxia when 5′-cap-dependent mRNA translation is inhibited. VEGF IRES-mediated initiation of translation requires the modulated interaction of trans-acting factors. To identify trans-acting factors that control VEGF mRNA translation under hypoxic conditions we established an in vitro translation system based on human adenocarcinoma cells (MCF-7). Cytoplasmic extracts of MCF-7 cells grown under hypoxia (1% oxygen) recapitulate VEGF IRES-mediated reporter mRNA translation. Employing the VEGF mRNA 5′-UTR and 3′-UTR in an RNA affinity approach we isolated interacting proteins from translational active MCF-7 extract prepared from cells grown under normoxia or hypoxia. Interestingly, mass spectrometry analysis identified the DEAD-box RNA helicase 6 (DDX6) that interacts with the VEGF mRNA 5′-UTR. Recombinant DDX6 inhibits VEGF IRES-mediated translation in normoxic MCF-7 extract. Under hypoxia the level of DDX6 declines, and its interaction with VEGF mRNA is diminished in vivo. Depletion of DDX6 by RNAi further promotes VEGF expression in MCF-7 cells. Increased secretion of VEGF from DDX6 knockdown cells positively affects vascular tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Our results indicate that the decrease of DDX6 under hypoxia contributes to the activation of VEGF expression and promotes its proangiogenic function.     

The role of Algerian Ephedra alata ethanolic extract in inhibiting the growth of breast cancer cells by inducing apoptosis in a p53- dependent pathway

BackgroundEphedra alata, a member of the Ephedraceae family, was used to treat different diseases and it might be shown a strong efficacy to inhibit cancer cell lines.MethodsDue to the limited research available about this plant, the objective of this research was to evaluate the antioxidant, cytotoxic and apoptotic effects of Ephedra alata ethanolic extract (EAEE), against different human cancer cell lines.ResultsEAEE inhibited the growth of the liver (HepG2), breast (MCF-7), and colon cancer cells (Caco-2). MCF-7 cells with an IC₅₀ of 153 µg/ml, were the most sensitive to the extract. Furthermore, exploration using flow cytometry using Annexin V-FITC/PI assay demonstrated that EAEE caused death for all human cancer cells mainly through apoptosis. Very interestingly, qRT-PCR analysis using the ΔΔCt method revealed that four genes, Bax, p21, RB1, and TP53 were up-regulated in MCF-7 cells treated either with EAEE or S-FU drug. These findings let us believe that the mechanism by which EAEE kills breast cancer cells seems to be apoptosis via a P53-dependent manner, which involved intrinsic pathways through the induction of Bax, p21, and RB1.ConclusionsEAEE exhibits good biological properties in contradiction of HepG-2, MCF-7, and Caco-2 cell lines. This study appoints for the first time that EAEE increases the expression in MCF-7 cells of p53 and three more genetic traits that control cellular proliferation and apoptosis. Therefore, this plant could serve as a potential source to find new pro-apoptotic drugs for cancer treatment.     

Elevated levels of ERK2 in human breast carcinoma MCF-7 cells transfected with protein kinase Cα

Abstract. We investigated the effect of elevated levels of protein kinase Cα (PKCα) on cell proliferation in human breast carcinoma cells (MCF-7). MCF-7 cells transfected with either the pSV₂M(2)6 vector without the insert (MCF-7/Vector) or containing a full length cDNA encoding PKCα (MCF-7/PKCα) were compared. MCF-7/PKCα cells were found to have an increased proliferative rate with a doubling time of 15 h as compared to 42 h for MCF-7/Vector cells. Flow cytometry illustrated a greater percentage of MCF-7/PKCα cells in the S phase of the cell cycle. Western and Northern blot analyses demonstrated an increase in extracellular regulated protein kinase 2 (ERK2) gene expression in MCF-7/PKCα cells but no alteration of this gene expression in MCF-7/Vector cells. These results suggested that the elevated level of ERK2 which is also known as mitogen activated protein kinase is probably involved in the increase in MCF-7/PKCα cell proliferation.     

The influence of lead and arsenite on the inhibition of human breast cancer MCF-7 cell proliferation by American ginseng root (Panax quinquefolius L.)

American ginseng root (Panax quinquefolius) has a number of purported therapeutic effects, including inhibition of cancer cell proliferation. The ability of environmentally relevant heavy metals to alter ginseng effects on cancer cell growth was the subject of this study. A water extract of American ginseng root was applied alone or in combination with physiologically relevant doses of either lead (Pb) or arsenite to MCF-7 breast cancer cells in vitro and effects on cell proliferation were determined. Ginseng alone produced a significant dose-dependent inhibition of MCF-7 cell proliferation starting at 0.5 mg ml(-1). Treatment of MCF-7 cells with 2.5 microM arsenite significantly decreased MCF-7 cell proliferation (p < 0.01). When cells were treated with arsenite (1.25 or 2.5 microM) in combination with ginseng extract (0.5 mg ml(-1)), there was an apparent synergistic inhibition of cell proliferation. Treatment of MCF-7 breast cancer cells with 50 microM Pb significantly decreased cell proliferation relative to control (p < 0.01), and concomitant ginseng and Pb treatment did not lead to a further decrease. These results suggest that contaminant heavy metals, some of which have been detected in ginseng root extracts or commercial ginseng preparations, may alter the biological activity of ginseng.     

Cytotoxic Effects of the Benzophenanthridine Alkaloids Isolated from Eomecon chionantha on MCF-7 Cells and Its Potential Mechanism

Seven benzophenanthridine alkaloids ( 1–7 ) were obtained from the 75 % EtOH extract of Eomecon chionantha, and exhibited moderate biological activity against MCF-7 cells. 8,12-dimethoxysanguinarine ( 1 , DSG) strongly decreased the cell viability of MCF-7 cell lines with an IC₅₀ value of 7.12 μΜ. Based on RNA-sequencing measure and KEGG pathway enrichment analysis results, the significant differentially expressed genes (DEGs) were associated with Pathways in Cancer and PI3 K-AKT signaling pathways in DSG treated group. The potential molecular regulatory mechanisms underlying the effect of DSG induces necroptosis in MCF-7 cells via molecular docking, TEM analysis, and ROS measurement. Besides, DEGs of bone metastasis-related genes such as PI3 K, IGF1R, Notch, and Wnt mRNA were significantly downregulated in the DSG-treated group on MCF-7 cells. DSG might be selected as a bone metastasis proteins inhibitor of IL-1β, IL-6, IκBα, IGF1R, Notch, NF-κB, PTHrp, PI3 K, PKB/AKT, PTEN, TNF-α, and Wnt via molecular docking. DSG suppressed bone metastasis by regulating the expression levels of IL-1β, IL-6, PTH, CROSS, TP1NP, and OSTEOC on MCF-7 cells using ELISA measurement. Thus, our findings reveal that DSG could be a lead compound for suppressing tumor cells to bone metastasis in breast cancer cells.     

Endocrine characteristics of human breast epithelial cells, MCF-10F

MCF-10F is a spontaneously immortalized nontransformed human breast epithelial cell line which does not grow in soft agar or form tumors in nude mice. Though the presence of estrogen receptors has not been found in these cells, they can metabolize estradiol very efficiently. The present study describes the endocrine characteristics of this cell line with respect to growth response to estradiol and its metabolites, estradiol metabolism and aromatase activity. MCF-10F cells were growth stimulated by 16alpha-hydroxyestrone and estriol, whereas, estradiol and other estradiol metabolites did not affect cell proliferation. The constitutive level of 16alpha-hydroxyestrone, a metabolite of estradiol biotransformation that has been associated with enhanced carcinogenesis in several animal, cell and tissue culture models, was a hundredfold higher in the non-transformed MCF-10F cells than in the transformed MCF-7 cells. Treatment with the carcinogen, dimethylbenz(a)anthracene (DMBA), however, did not upregulate 16alpha-hydroxylation as was observed in transformed MCF-7 cells. MCF-10F cells also had no detectable aromatase activity though the level of 17-oxidation was unusually high as compared with MCF-7 cells. Our results using the non-transformed MCF-10F cells as a model system suggests that the presence of high level of 16alpha-hydroxyestrone, a metabolite previously shown to be associated with malignant phenotype, may not be sufficient for breast cancer transformation.     

巨噬细胞集落刺激因子经PI3K/AKT信号途径影响人乳腺癌MCF-7细胞糖代谢

本文探讨巨噬细胞集落刺激因子（M-CSF）对人乳腺癌MCF-7细胞糖代谢的影响及其机制. 构建胞质稳定转染 M-CSF的MCF-7细胞（MCF-7-M）;ATP检测试剂盒检测MCF-7和MCF-7-M细胞的ATP生成;葡萄糖测定试剂盒、乳酸测试盒检测MCF-7和MCF-7-M细胞的葡萄糖摄取和乳酸分泌情况;蛋白质印迹法检测在糖酵解抑制剂2-脱氧葡萄糖（2-DG）和氧化磷酸化抑制剂OLIG处理后,M-CSF对MCF-7细胞的糖酵解关键酶：己糖激酶2（HK2）、丙酮酸激酶M2（PKM2）及葡萄糖转运体1（GLUT-1）表达的影响;MTT法检测在ATP消耗剂3-溴丙酮酸（3-BrPA）处理后,MCF-7和MCF-7-M细胞对5-FU敏感性的变化. 结果发现：MCF-7-M细胞的ATP水平显著高于MCF-7细胞（P<0.05）;2-DG降低了MCF-7和MCF-7-M细胞的ATP水平,并且降低MCF-7-M细胞ATP的效果更明显（P<0.01）;MCF-7-M细胞的糖摄取能力和乳酸分泌量显著高于MCF-7细胞（P<0.01）,经API-2处理后,MCF-7和MCF-7-M细胞葡萄糖消耗和乳酸分泌量均显著减少（P<0.01）;MCF-7-M细胞GLUT-1、HK2和PKM2的表达显著高于MCF-7细胞（P<0.01）;LY294002和API-2均可抑制MCF-7-M细胞GLUT-1的表达（P<0.05）;用3-BrPA处理后,MCF-7-M和MCF-7细胞对5-FU的药物敏感性显著增强（P<0.01). 综上,得出结论： 胞质M-CSF通过诱导GLUT-1、HK2和PKM2的表达,活化MCF-7细胞糖酵解途径;PI3K/AKT信号通路参与胞质M-CSF活化MCF-7细胞的糖酵解途径.