Synergistic Effect of Squalene and Hydroxytyrosol on Highly Invasive MDA-MB-231 Breast Cancer Cells

Several studies relate Mediterranean diet and virgin olive oil (VOO) intake with lower risk of several chronic diseases, including breast cancer. Many of them described antitumor properties of isolated minor compounds present in VOO, but beneficial properties of VOO arise from the effects of all its compounds acting together. The aim of the present study was to test the antitumor effects of two minor compounds from VOO (hydroxytyrosol (HT) and squalene (SQ)) on highly metastatic human breast tumor cells (MDA-MB-231) when acting in combination. Both isolated compounds were previously analyzed without showing any antitumoral effect on highly invasive MDA-MB-231 breast cancer cells, but the present results show that HT at 100 µM, combined with different concentrations of SQ, could exert antitumor effects. When they are combined, HT and SQ are able to inhibit cell proliferation, promoting apoptosis and DNA damage in metastatic breast cancer cells. Therefore, our results suggest that the health-promoting properties of VOO may be due, at least in part, to the combined action of these two minor compounds.     

Tyrosol and hydroxytyrosol are absorbed from moderate and sustained doses of virgin olive oil in humans

OBJECTIVE: To investigate the absorption of tyrosol and hydroxytyrosol from moderate and sustained doses of virgin olive oil consumption. The study also aimed to investigate whether these phenolic compounds could be used as biomarkers of virgin olive oil intake. DESIGN AND INTERVENTIONS: Ingestion of a single dose of virgin olive oil (50 ml). Thereafter, for a week, participants followed their usual diet which included 25 ml/day of the same virgin olive oil as the source of raw fat. SETTING: Unitat de Recerca en Farmacologia. Institut Municipal d'Investigació Mèdica (IMIM). SUBJECTS: Seven healthy volunteers. RESULTS: An increase in 24 h urine of tyrosol and hydroxytyrosol, after both a single-dose ingestion (50 ml) and short-term consumption (one week, 25 ml/day) of virgin olive oil (P<0.05) was observed. Urinary recoveries for tyrosol were similar after a single dose and after sustained doses of virgin olive oil. Mean recovery values for hydroxytyrosol after sustained doses were 1.5-fold those obtained after a single 50 ml dose. CONCLUSIONS: Tyrosol and hydroxytyrosol are absorbed from realistic doses of virgin olive oil. With regard to the dose-effect relationship, 24 h urinary tyrosol seems to be a better biomarker of sustained and moderate doses of virgin olive oil consumption than hydroxytyrosol.     

Tocotrienols inhibit growth of ZR-75–1 breast cancer cells

The vitamin E component of palm oil provides a rich source of tocotrienols which have been shown previously to be growth inhibitory to two human breast cancer cell lines: responsive MCF7 cells and unresponsive MDA-MB-231 cells. Data presented here shows that the tocotrienol-rich fraction (TRF) of palm oil and individual fractions (alpha, gamma and delta) can also inhibit the growth of another responsive human breast cancer cell line, ZR-75-1. At low concentrations in the absence of oestrogen tocotrienols stimulated growth of the ZR-75-1 cells, but at higher concentrations in the presence as well as in the absence of oestradiol, tocotrienols inhibited cell growth strongly. As for MCF7 cells, α -tocopherol had no effect on growth of the ZR-75-1 cells in either the absence or presence of oestradiol. In studying the effects of tocotrienols in combination with antioestrogens, it was found that TRF could further inhibit growth of ZR-75-1 cells in the presence of tamoxifen (10 -7 M and 10 -8 M). Individual tocotrienol fractions (alpha, gamma, delta) could inhibit growth of ZR-75-1 cells in the presence of 10 -8 M oestradiol and 10 -8 M pure antioestrogen ICI 164 384. The immature mouse uterine weight bioassay confirmed that TRF could not exert oestrogen antagonist action in vivo. These results provide evidence of wider growth-inhibitory effects of tocotrienols beyond MCF7 and MDA-MB-231 cells, and with an oestrogen-independent mechanism of action, suggest a possible clinical advantage in combining administration of tocotrienols with antioestrogen therapy.     

Vitamin E analog alpha-TEA, methylseleninic acid, and trans-resveratrol in combination synergistically inhibit human breast cancer cell growth

Alpha-tocopherol ether-linked acetic acid analog [2,5,7,8-tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid (alpha-TEA)] is a novel form of vitamin E effective at killing cancer cells but not normal cells. alpha -TEA alone and together with methylseleninic acid (MSA) and trans-resveratrol (t-RES) were investigated for ability to induce apoptosis, DNA synthesis arrest, and cellular differentiation and inhibit colony formation in human MDA-MB-435-F-L breast cancer cells in culture. The 3 agents alone were effective in inhibiting cell growth by each of the 4 different assays, and 3-way combination treatments synergistically inhibited cell proliferation in each assay in comparison to individual treatments. Furthermore, combinations of alpha -TEA, t-RES, and MSA significantly enhanced levels of apoptosis in human breast (MDA-MB-231, MCF7, and T47D) and prostate (LnCaP, PC-3, and DU-145) cancer cell lines as well as in immortalized but nontumorigenic MCF10A cells but not primary cultures of human mammary epithelial cells. Western immunoblotting confirmed the induction of apoptosis in that the 3 agents induced poly(adenosine diphosphate-ribose) polymerase cleavage, with earlier detection and more complete cleavage seen in the combination treatment. Mechanistic studies showed combination treatments to inhibit cell proliferation via downregulation of cyclin D1 and induce apoptosis via activation of caspases 8 and 9 and downregulation of prosurvival proteins FLIP and survivin. In summary, the combination of alpha-TEA, MSA, and t-RES is more effective than single treatments for inhibiting cell proliferation, inducing cellular differentiation, and inducing cell death by apoptosis in human cancer cells in culture.     

Unsymmetrical methylene derivatives of indoles as antiproliferative agents

Indole-3-carbinol is a natural product which has been shown to reduce the incidence of spontaneous and carcinogen-induced mammary tumours in animals. Eighteen unsymmetrical methylene derivatives of indoles were prepared by reaction of Mannich bases of 7-hydroxycoumarins with substituted indoles in acetic or propionic anhydride. The synthesised molecules were tested in vitro against the MCF7 and MDA-MB-231 breast cancer cell lines by MTT and cell count assays. Results from 16 tested compounds showed that 60% of them exerted some effects against the MDA-MB-231 compared to about 30% towards the MCF7. Among all, the 3-(7'-acetoxy-4-methylcoumarin-8'-yl)methyl-2-methylindole resulted the most effective in both cell lines, compared to indole-3-carbinol. In conclusion, these preliminary results report that some of these compounds might be promising potential antiproliferative agents.     

Modulation of CYP1A1, CYP1A2 and CYP1B1 Expression by Cabbage Juices and Indoles in Human Breast Cell Lines

Epidemiological studies have shown that consumption of cabbage and sauerkraut is connected with significant reduction of breast cancer incidences. Estrogens are considered a major breast cancer risk factor and their metabolism by P450 enzymes substantially contributes to carcinogenic activity. The aim of this study was to investigate the effect of cabbage and sauerkraut juices of different origin on the expression profile of the estrogen metabolism key enzymes (CYP1A1, CYP1A2, CYP1B1) in breast cell lines MCF7, MDA-MB-231, and MCF10A. The effects of cabbage juices were compared with that exerted by indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM). The treatment with cabbage juices or indoles for 72?h affected the expression of CYP1 family genes in cell-type dependent manner. Their induction was found in all cell lines, but the ratio of CYP1A1 to CYP1B1 was 1.22- to 10.6-fold in favor to CYP1A1 in MCF7 and MCF10A cells. Increased levels of CYP1A2 in comparison with CYP1B1 were also observed in MCF7 cells. In contrast, in MDA-MB-231 cells CYP1B1 was preferentially induced. Since the cell lines investigated differ in invasion capacity, these results support epidemiological observations and partly explain the mechanism of the chemopreventive activity of white cabbage products.     

Vitamin E Analog α -TEA,Methylseleninic Acid,and Trans-Resveratrol in Combination Synergistically Inhibit Human Breast Cancer Cell Growth

Alpha-tocopherol ether-linked acetic acid analog [2,5,7,8-tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid (α -TEA)] is a novel form of vitamin E effective at killing cancer cells but not normal cells. α -TEA alone and together with methylseleninic acid (MSA) and trans-resveratrol (t-RES) were investigated for ability to induce apoptosis, DNA synthesis arrest, and cellular differentiation and inhibit colony formation in human MDA-MB-435-F-L breast cancer cells in culture. The 3 agents alone were effective in inhibiting cell growth by each of the 4 different assays, and 3-way combination treatments synergistically inhibited cell proliferation in each assay in comparison to individual treatments. Furthermore, combinations of α -TEA, t-RES, and MSA significantly enhanced levels of apoptosis in human breast (MDA-MB-231, MCF7, and T47D) and prostate (LnCaP, PC-3, and DU-145) cancer cell lines as well as in immortalized but nontumorigenic MCF10A cells but not primary cultures of human mammary epithelial cells. Western immunoblotting confirmed the induction of apoptosis in that the 3 agents induced poly(adenosine diphosphate-ribose) polymerase cleavage, with earlier detection and more complete cleavage seen in the combination treatment. Mechanistic studies showed combination treatments to inhibit cell proliferation via downregulation of cyclin D1 and induce apoptosis via activation of caspases 8 and 9 and downregulation of prosurvival proteins FLIP and survivin. In summary, the combination of α -TEA, MSA, and t-RES is more effective than single treatments for inhibiting cell proliferation, inducing cellular differentiation, and inducing cell death by apoptosis in human cancer cells in culture.     

L-Carvone Induces p53, Caspase 3 Mediated Apoptosis and Inhibits the Migration of Breast Cancer Cell Lines

A wide variety of natural compounds exists that possesses significant cytotoxic as well as chemopreventive activity through induction of apoptosis in cancer cells. The antiproliferative and apoptotic effect of L-carvone, an active component of spearmint (Mentha spicata) was studied on breast cancer (MCF 7 and MDA MB 231) and normal (MCF 10A) cell lines, and insight into its mechanism of action was attained. L-carvone inhibited proliferation of MCF 7 (IC₅₀ 1.2 mM) and MDA MB 231 cells (IC₅₀ 1.0 mM) and inhibited the migration of breast cancer cell lines. L-carvone induced apoptosis as observed by nuclei fragmentation and the presence of apoptotic bodies in DAPI, AnnexinV/propidium iodide, and TUNEL assays. L-carvone exposure arrested MCF 7 cells in S phase of the cell cycle. DNA damage caused by L-carvone was apparent from the increased tail moment in COMET assay, which could be induced by an increase in ROS that was measured using a fluorescence probe. Glutathione levels were also increased. The increased level of p53, Bad, cleaved caspase 3, and cleaved PARP explained p53 and caspase-mediated apoptosis.     

Virgin olive oil phenols inhibit proliferation of human promyelocytic leukemia cells (HL60) by inducing apoptosis and differentiation

Although epidemiologic evidence and animal studies suggest that olive oil may prevent the onset of cancer, the components responsible for such an effect and their mechanisms of action remain largely unknown. In the present study, we investigated the effect of a virgin olive oil phenol extract (PE) on proliferation, the cell cycle distribution profile, apoptosis, and differentiation of the human promyelocytic cell line HL60. PE inhibited HL60 cell proliferation in a time- and concentration-dependent manner, as demonstrated by the viable cell count and 3-[4,5-dimethyl(thiazol-2-yl)]-3,5-diphenyltetrazolium bromide (MTT) metabolism. Cell growth was completely blocked at a PE concentration of 13.5 mg/L; apoptosis was also induced as detected by fluorescence microscopy and flow cytometry. Determination of the cell cycle distribution by flow cytometry revealed an accumulation of cells in the G(0)/G(1) phase. Two compounds isolated from PE, the dialdehydic forms of elenoic acid linked to hydroxytyrosol (3,4-DHPEA-EDA) and to tyrosol (pHPEA-EDA), were shown to possess properties similar to those of PE; they account for a part of the powerful effects exerted by the complex mixture of compounds present in PE. The concentrations of the different compounds in PE were determined by HPLC, and the purity of 3,4-DHPEA-EDA and pHPEA-EDA was ascertained by NMR. Treatment with PE induced a differentiation in HL60 cells, which subsequently acquired the ability to produce superoxide ions and reduce nitroblue tetrazolium to formazan. These results support the hypothesis that polyphenols play a critical role in the anticancer activity of olive oil.     

Oxidative DNA damage is prevented by extracts of olive oil, hydroxytyrosol, and other olive phenolic compounds in human blood mononuclear cells and HL60 cells

Our aim in this study was to provide further support to the hypothesis that phenolic compounds may play an important role in the anticarcinogenic properties of olive oil. We measured the effect of olive oil phenols on hydrogen peroxide (H(2)O(2))-induced DNA damage in human peripheral blood mononuclear cells (PBMC) and promyelocytic leukemia cells (HL60) using single-cell gel electrophoresis (comet assay). Hydroxytyrosol [3,4-dyhydroxyphenyl-ethanol (3,4-DHPEA)] and a complex mixture of phenols extracted from both virgin olive oil (OO-PE) and olive mill wastewater (WW-PE) reduced the DNA damage at concentrations as low as 1 micromol/L when coincubated in the medium with H(2)O(2) (40 micromol/L). At 10 micromol/L 3,4-DHPEA, the protection was 93% in HL60 and 89% in PBMC. A similar protective activity was also shown by the dialdehydic form of elenoic acid linked to hydroxytyrosol (3,4-DHPEA-EDA) on both kinds of cells. Other purified compounds such as isomer of oleuropein aglycon (3,4-DHPEA-EA), oleuropein, tyrosol, [p-hydroxyphenyl-ethanol (p-HPEA)] the dialdehydic form of elenoic acid linked to tyrosol, caffeic acid, and verbascoside also protected the cells against H(2)O(2)-induced DNA damage although with a lower efficacy (range of protection, 25-75%). On the other hand, when tested in a model system in which the oxidative stress was induced by phorbole 12-myristate 13-acetate-activated monocytes, p-HPEA was more effective than 3,4-DHPEA in preventing the oxidative DNA damage. Overall, these results suggest that OO-PE and WW-PE may efficiently prevent the initiation step of carcinogenesis in vivo, because the concentrations effective against the oxidative DNA damage could be easily reached with normal intake of olive oil.     

The in vivo fate of hydroxytyrosol and tyrosol, antioxidant phenolic constituents of olive oil, after intravenous and oral dosing of labeled compounds to rats.

In vitro studies have shown phenolics in olive oil to be strong radical scavengers. The absorption and elimination of two radiolabeled phenolic constituents of olive oil, hydroxytyrosol and tyrosol were studied in vivo using rats. Compounds were administered intravenously (in saline) and orally (in oil- and water-based solutions). For both compounds, the intravenously and orally administered oil-based dosings resulted in significantly greater elimination of the phenolics in urine within 24 h than the oral, aqueous dosing method. There was no significant difference in the amount of phenolic compounds eliminated in urine between the intravenous dosing method and the oral oil-based dosing method for either tyrosol or hydroxytyrosol. Oral bioavailability estimates of hydroxytyrosol when administered in an olive oil solution and when dosed as an aqueous solution were 99% and 75%, respectively. Oral bioavailability estimates of tyrosol, when orally administered in an olive oil solution and when dosed as an aqueous solution were 98% and 71%, respectively. This is the first study that has used a radiolabeled compound to study the in vivo biological fates of hydroxytyrosol and tyrosol.     

The effect of seal oil on paclitaxel induced cytotoxicity and apoptosis in breast carcinoma MCF-7 and MDA-MB-231 cell lines

Some studies have suggested that omega-3 polyunsaturated fatty acids (PUFAs) have an inhibitory effect on the growth of cancer cells and therefore have the potential to increase the efficacy of cancer chemotherapeutic drugs. Considering that omega-3 PUFAs are present abundantly in harp seal oil, we investigated the effect of seal oil on the cytotoxicity and apoptosis induced by paclitaxel in 2 breast cancer cell lines, MCF-7 and MDA-MB-231, respectively. Cytotoxicity evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the concentration of paclitaxel that is required for 50% inhibition of cell growth in the presence of seal oil was significantly lower than that of paclitaxel alone. Apoptosis assessment based on morphological changes and DNA fragmentation results indicated that more cells treated with paclitaxel in combination with seal oil underwent apoptosis than with paclitaxel alone. Western blot analysis showed that the expression of B cell lymphoma-2 (Bcl-2) protein, an apoptosis inhibitory protein, in both cell lines was decreased more significant by paclitaxel in combination with seal oil than by paclitaxel alone. In addition, seal oil alone was found to induce apoptosis in both cell lines tested, which appeared to be due to the increased intracellular lipid peroxides produced. It is therefore concluded that paclitaxel in combination with seal oil demonstrated enhanced cytotoxicity and apoptosis in MCF-7 and MDA-MB-231 cells compared to paclitaxel alone, and the use of seal oil may be beneficial in the treatment of breast cancer.     

The Effect of Seal Oil on Paclitaxel Induced Cytotoxicity and Apoptosis in Breast Carcinoma MCF-7 and MDA-MB-231 Cell Lines

Some studies have suggested that ω-3 polyunsaturated fatty acids (PUFAs) have an inhibitory effect on the growth of cancer cells and therefore have the potential to increase the efficacy of cancer chemotherapeutic drugs. Considering that ω-3 PUFAs are present abundantly in harp seal oil, we investigated the effect of seal oil on the cytotoxicity and apoptosis induced by paclitaxel in 2 breast cancer cell lines, MCF-7 and MDA-MB-231, respectively. Cytotoxicity evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the concentration of paclitaxel that is required for 50% inhibition of cell growth in the presence of seal oil was significantly lower than that of paclitaxel alone. Apoptosis assessment based on morphological changes and DNA fragmentation results indicated that more cells treated with paclitaxel in combination with seal oil underwent apoptosis than with paclitaxel alone. Western blot analysis showed that the expression of B cell lymphoma-2 (Bcl-2) protein, an apoptosis inhibitory protein, in both cell lines was decreased more significant by paclitaxel in combination with seal oil than by paclitaxel alone. In addition, seal oil alone was found to induce apoptosis in both cell lines tested, which appeared to be due to the increased intracellular lipid peroxides produced. It is therefore concluded that paclitaxel in combination with seal oil demonstrated enhanced cytotoxicity and apoptosis in MCF-7 and MDA-MB-231 cells compared to paclitaxel alone, and the use of seal oil may be beneficial in the treatment of breast cancer.     

Olive oil phenolics: effects on DNA oxidation and redox enzyme mRNA in prostate cells

Hydroxytyrosol, tyrosol and caffeic acid effects on hydrogen peroxide-induced DNA damage, hydroperoxide generation and redox enzyme gene expression were studied in oxidative-stress-sensitive human prostate cells (PC3). Hydroxytyrosol led to lower levels of hydroperoxides, DNA damage, and mRNA levels of classic glutathione peroxidase (GPx) for all the studied concentrations. Only hydroxytyrosol was effective at low concentrations (10 microm). Tyrosol reduced DNA oxidation only at high (>50 microm) concentrations and increased hydroperoxides, GPx and phospholipid hydroperoxide GPx mRNA levels. Caffeic acid elicited effects between those of the other two phenolics. Results indicate that hydroxytyrosol is the only significant antioxidant phenolic in olive oil and may be the major component accounting for its beneficial properties. Tyrosol appeared to exhibit pro-oxidant effects (only at high concentrations) and caffeic acid was neutral. Both number and position of hydroxyl groups appear to play a role in the cellular effects of hydroxytyrosol.     

Epigenetic modulation of BRCA1 and BRCA2 gene expression by equol in breast cancer cell lines

S-Equol is a metabolite resulting from the conversion of daidzein, a soya phyto-oestrogen, by the gut microflora. The potential protective effects of equol in breast cancer are still under debate. Consequently, we investigated the effects of equol on DNA methylation of breast cancer susceptibility genes (BRCA1 and BRCA2) and oncosuppressors in breast cancer cell lines (MDA-MB-231 and MCF-7) and in a dystrophic breast cell line (MCF-10a) following exposure to S-equol (2?μm) for 3 weeks. We demonstrated by quantitative analysis of methylated alleles a significant decrease in the methylation of the cytosine phosphate guanine (CpG) islands in the promoters of BRCA1 and BRCA2 after the S-equol treatment in MCF-7 and MDA-MB-231 cells and a trend in MCF-10a cells. We also showed that S-equol increases BRCA1 and BRCA2 protein expression in the nuclei and the cytoplasm in MCF-7, MDA-MB-231 and MCF-10a cell lines by immunohistochemistry. The increase in BRCA1 and BRCA2 proteins was also found after Western blotting in the studied cell lines. In summary, we demonstrated the demethylating effect of S-equol on the CpG islands inside the promoters of BRCA1 and BRCA2 genes, resulting in an increase in the level of expressed oncosuppressors in breast cancer cell lines.     

Induction of apoptosis in human breast cancer cells by tocopherols and tocotrienols

The apoptosis-inducing properties of RRR-alpha-, beta-, gamma-, and delta-tocopherols, alpha-, gamma-, and delta-tocotrienols, RRR-alpha-tocopheryl acetate (vitamin E acetate), and RRR-alpha-tocopheryl succinate (vitamin E succinate) were investigated in estrogen-responsive MCF7 and estrogen-nonresponsive MDA-MB-435 human breast cancer cell lines in culture. Apoptosis was characterized by two criteria: 1) morphology of 4,6-diamidino-2-phenylindole-stained cells and oligonucleosomal DNA laddering. Vitamin E succinate, a known inducer of apoptosis in several cell lines, including human breast cancer cells, served as a positive control. The estrogen-responsive MCF7 cells were more susceptible than the estrogen-nonresponsive MDA-MB-435 cells, with concentrations for half-maximal response for tocotrienols (alpha, gamma, and delta) and RRR-delta-tocopherol of 14, 15, 7, and 97 micrograms/ml, respectively. The tocotrienols (alpha, gamma, and delta) and RRR-delta-tocopherol induced MDA-MB-435 cells to undergo apoptosis, with concentrations for half-maximal response of 176, 28, 13, and 145 micrograms/ml, respectively. With the exception of RRR-delta-tocopherol, the tocopherols (alpha, beta, and gamma) and the acetate derivative of RRR-alpha-tocopherol (RRR-alpha-tocopheryl acetate) were ineffective in induction of apoptosis in both cell lines when tested within the range of their solubility, i.e., 10-200 micrograms/ml. In summary, these studies demonstrate that naturally occurring tocotrienols and RRR-delta-tocopherol are effective apoptotic inducers for human breast cancer cells.     

UHRF1的高表达降低人乳癌细胞对X射线敏感性的研究

目的：了解基因UHRF1的高表达对乳癌细胞MDA-MB-231辐射敏感性的影响.方法：利用克隆形成实验观察细胞存活;流式细胞术测定细胞周期;利用DNA片段分析和Annexin V 试剂盒测定细胞凋亡;Western blot 测定蛋白表达变化;利用经典的染色体分析,观察染色体畸变（着丝粒环和双着丝粒）.结果：与对照相比, UHRF1转染可明显降低MDA-MB-231细胞对X射线的敏感性.利用UHRF1-siRNA抑制UHRF1的表达,可显著增强细胞的辐射敏感性.UHRF1的高表达,可诱导G2/M期阻滞,抑制细胞凋亡,下调促凋亡蛋白Bax,上调DNA损伤修复蛋白Ku70和 Ku80的表达水平,而且,能抑制X射线诱导的染色体畸变.结论：UHRF1可能通过影响凋亡和DNA损伤修复成为乳癌放疗的新靶标.