Equol: a comparison of the effects of the racemic compound with that of the purified S-enantiomer on the growth, invasion, and DNA integrity of breast and prostate cells in vitro

It has been postulated that the R- and S-equol enantiomers have different biological properties given their different binding affinities for the estrogen receptor. S-(-)equol is produced via the bacterial conversion of the soy isoflavone daidzein in the gut. We have compared the biological effects of purified S-equol to that of racemic (R and S) equol on breast and prostate cancer cells of varying receptor status in vitro. Both racemic and S-equol inhibited the growth of the breast cancer cell line MDA-MB-231 (> or = 10 microM) and the prostate cancer cell lines LNCaP (> or = 5 microM) and LAPC-4 (> or = 2.5 microM). The compounds also showed equipotent effects in inhibiting the invasion of MDA-MB-231 and PC-3 cancer cells through matrigel. S-equol (1, 10, 30 microM) was unable to prevent DNA damage in MCF-7 or MCF-10A breast cells following exposure to 2-hydroxy-4-nonenal, menadione, or benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. In contrast, racemic equol (10, 30 microM) prevented DNA damage in MCF-10A cells following exposure to 2-hydroxy-4-nonenal or menadione. These findings suggest that racemic equol has strong antigenotoxic activity in contrast to the purified S-equol enantiomer implicating the R-, rather than the S-enantiomer as being responsible for the antioxidant effects of equol, a finding that may have implications for the in vivo chemoprotective properties of equol.     

Daidzein,R-(+)equol and S-(−)equol inhibit the invasion of MDA-MB-231 breast cancer cells potentially via the down-regulation of matrix metalloproteinase-2

Purpose

Soy isoflavones may inhibit tumor cell invasion and metastasis via their effects on matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The current study investigates the effects of daidzein, R- and S-equol on the invasion of MDA-MB-231 human breast cancer cells and the effects of these compounds on MMP/TIMP expression at the mRNA level.

Methods

The anti-invasive effects of daidzein, R- and S-equol (0, 2.5, 10, 50 μM) on MDA-MB-231 cells were determined using the Matrigel invasion assay following 48-h exposure. Effects on MMP-2, MMP-9, TIMP-1 and TIMP-2 expression were assessed using real-time PCR. Chiral HPLC analysis was used to determine intracellular concentrations of R- and S-equol.

Results

The invasive capacity of MDA-MB-231 cells was significantly reduced (by approximately 50–60 %) following treatment with 50 μM daidzein, R- or S-equol. Anti-invasive effects were also observed with R-equol at 2.5 and 10 μM though overall equipotent effects were induced by all compounds. Inhibition of invasion induced by all three compounds at 50 μM was associated with the down-regulation of MMP-2, while none of the compounds tested significantly affected the expression levels of MMP-9, TIMP-1 or TIMP-2 at this concentration. Following exposure to media containing 50 μM R- or S-equol for 48-h intracellular concentrations of R- and S-equol were 4.38 ± 1.17 and 3.22 ± 0.47 nM, respectively.

Conclusion

Daidzein, R- and S-equol inhibit the invasion of MDA-MB-231 human breast cancer cells in part via the down-regulation of MMP-2 expression, with equipotent effects observed for the parent isoflavone daidzein and the equol enantiomers.     

Equol Enhances Apoptosis-inducing Activity of Genistein by Increasing Bax/Bcl-xL Expression Ratio in MCF-7 Human Breast Cancer Cells

Anticancer activities of soy isoflavones, such as genistein and equol, a bioactive metabolite of daidzein, have been extensively studied because of possible involvement in the prevention of breast cancer. However, their interactions still remain unclear. We investigated here whether cytotoxic activity of genistein was enhanced by equol, using estrogen receptor positive MCF-7, HER2-positive SK-BR-3, and triple-negative MDA-MB-468 cell lines. Although cytotoxicity of genistein did not significantly differ between three subtypes of breast cancer cells, cytotoxic activities of genistein were significantly enhanced in combination with 50 μM equol in MCF-7 cells, but not in SK-BR-3 and MDA-MB-468 cells. In fluorescence activated cell sorting (FACS) analyses, MCF-7 cells were arrested at the G2/M by genistein but at G1/S by equol. Combination treatment arrested cells at G2/M but abolished equol-induced G1 block, indicating an antagonistic activity of genistein against equol in cell-cycle progression. Although apoptosis was not so evident with genistein alone, the combination made a drastic induction of apoptosis, accompanied by the increase of Bax/Bcl-xL expression ratio, without affecting the activities of Akt and mTOR. Taken together, these data suggest that enhancement of genistein activity by equol would be mainly mediated by augmented induction of apoptosis rather than arrest or delay of the cell cycle.     

Involvement of PPARalpha in the growth inhibitory effect of arachidonic acid on breast cancer cells

Epidemiological studies suggest that dietary PUFA may influence breast cancer progression. n-3 PUFA are generally known to exert antitumour effects, whereas reports relative to n-6 PUFA anti-carcinogen effects are controversial. Arachidonic acid (AA; 20:4n-6) and its metabolites have been shown to inhibit the growth of human breast cancer cell lines, even if the downstream mechanisms by which AA may influence carcinogenesis remain unresolved. We explored the molecular basis for AA influence on proliferation, signal transduction and apoptosis in two human breast cancer cell lines, MCF-7 and MDA-MB-231. In both cell lines AA inhibited cell growth in a dose-dependent manner, even if MDA-MB-231 was somewhat more growth-inhibited than MCF-7. AA decreased extracellular signal-regulated protein kinase 1/2 phosphorylation level, and positively modulated PPARgamma and PPARalpha expression, with only a slight effect against PPARbeta/delta. In addition, AA increased Bak (an apoptosis-regulating protein) expression and reduced procaspase-3 and -9 levels only in MDA-MB-231 cells, thus indicating that the growth inhibitory effect can be correlated with apoptosis induction. In both cell lines the use of a specific antagonist made it possible to establish a relationship between AA growth inhibitory effect and PPARalpha involvement. AA decreases cell proliferation most likely by inducing apoptosis in MDA-MB-231 cells, while in the MCF-7 cell line the growth inhibitory activity can be attributed to the inhibition of the signal transduction pathway involved in cell proliferation. In both cases, the results here presented suggest PPARalpha as a possible contributor to the growth inhibitory effect of AA.     

雌马酚通过抑制核转录因子-κB表达水平诱导人乳腺癌MDA-MB-231细胞凋亡

目的研究雌马酚对雌激素受体表达阴性的人乳腺癌细胞株MDA-MB-231的作用。方法用MTT法测定雌马酚干预对MDA-MB-231细胞活力的影响;光学显微镜观察雌马酚对细胞形态的影响;流式细胞术及免疫细胞化学法检测雌马酚对细胞凋亡的作用。结果雌马酚可以抑制MDA-MB-231细胞的活力(P<0.05)。雌马酚可抑制NF-κB的表达而诱导细胞凋亡。结论雌马酚可通过降低NF-κB的表达诱导MDA-MB-231凋亡。     

Cytotoxic Activity of Piper cubeba Extract in Breast Cancer Cell Lines

This study aimed to evaluate the cytotoxicity of a crude extract of Piper cubeba against normal and breast cancer cell lines. To prepare the extract, P. cubeba seeds were ground, soaked in methanol and dichloromethane and isolated by column chromatography. Fractions were tested for cytotoxicity effects on normal fibroblast (L929), normal breast (MCF-12A) and breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231). The most effective fraction was selected for DNA fragmentation assay to detect apoptotic activity. The results showed that the methanolic crude extract had a higher cytotoxic activity against MDA-MB-468 and MCF-7 than a dichloromethane crude extract. Then, the methanolic crude extract was separated into six fractions, designated A to F. Fraction C was highly active against breast cancer cell lines with an IC₅₀ value less than 4 μg/mL. Therefore, Fraction C was further separated into seven fractions, CA to CG. The ¹H-NMR profile showed that Fraction CE was long chain hydrocarbons. Moreover, Fraction CE demonstrated the highest activity against MCF-7 cells with an IC₅₀ value of 2.69 ± 0.09 μg/mL and lower cytotoxicity against normal fibroblast L929 cells with an IC₅₀ value of 4.17 ± 0.77 μg/mL. Finally, DNA fragmentation with a ladder pattern characteristic of apoptosis was observed in MCF-7, MDA-MB-468, MDA-MB-231 and L929 cells, but not in MCF-12A cells.     

In vitro inhibition of proliferation of estrogen-dependent and estrogen-independent human breast cancer cells treated with carotenoids or retinoids

Both estrogen-receptor (ER) positive MCF-7 and ER-negative Hs578T and MDA-MB-231 human breast cancer cells were treated with carotenoids (beta-carotene, canthaxanthin and lycopene) and retinoids (all-trans-, 9-cis- and 13-cis-retinoic acid and all-trans-retinol). Among carotenoids, beta-carotene significantly reduced the growth of MCF-7 and Hs578T cells, and lycopene inhibited the growth of MCF-7 and MDA-MB-231 cells. Canthaxanthin did not affect the proliferation of any of the three cell lines. All-trans- and 9-cis-retinoic acid significantly reduced the growth of both MCF-7 and Hs578T cells, whereas 13-cis-retinoic acid and all-trans-retinol had a significant effect only on MCF-7 cells. MCF-7 and Hs578T cells treated with all-trans-retinoic acid (all-t-RA) were further studied for the mechanism behind growth inhibition. Retinoic acid receptors alpha and gamma (RARalpha, gamma) in MCF-7 cells and RARalpha, beta and gamma in Hs578T cells were not induced by all-t-RA treatment at either the protein or mRNA level. Hs578T cells treated with all-t-RA had significantly more cells in the G0/G1 stage of the cell cycle, but the same was not observed for MCF-7 cells. All-t-RA induced a dose-dependent cell death in MCF-7 cells, which may be a necrotic phenomenon. These results demonstrate that ER status is an important, although not essential factor for breast cancer cell response to carotenoid and retinoid treatments, and the mode of action of all-t-RA in MCF-7 and Hs578T cells is not through the induction of RAR. Other mechanistic pathways that are either followed by or concomitant with growth inhibition are possible.     

Dietary Fatty Acids from Leaves of Clerodendrum Volubile Induce Cell Cycle Arrest,Downregulate Matrix Metalloproteinase-9 Expression,and Modulate Redox Status in Human Breast Cancer

The antiproliferative effect of the fatty acid components of Clerodendrum volubile leaves as well as its antioxidant effect on MCF-7 and MDA-MB-231 human breast cancer cell lines were investigated. Fatty acids extracted from C. volubile leaf oil were subjected to gas chromatography mass spectrometry (GCMS) analysis. The cells were cultured and treated with the fatty acids for 48 h, after which the antiproliferation effect was ascertained via MTT assay and cell viability analysis using BD fluorescence activated cells sorting (FACS) Calibur. Cell cycle was analyzed by flow cytometry on FACS Calibur. Western blotting was used in determining expression of proteins in the cell lines. The treated cell lines were assessed for reduced glutathione level, catalase, superoxide dismutase, and lipid peroxidation. The fatty acids significantly inhibited cell proliferation, arrested G0/G1 phase, downregulated the expression of MMP-9, and attenuated oxidative stress in of MCF-7 cell lines but had little or no effect on MDA-MB-231 cell lines. These results indicate the therapeutic potential of the fatty acids components of the leaves of C. volubile on human breast cancer, which may be explored further in drug development.     

miR-21在辐射敏感性不同的3种乳腺癌细胞株中的表达差异及意义

目的:研究miR-21在MDA-MB-435S、MCF-7、MDA-MB-231这3种辐射敏感性不同的乳腺癌细胞株中表达量的差异及其意义。方法:①采用实时定量PCR法分别检测3种乳腺癌细胞株中miR-21的表达量的差异。②时程关系:在3Gy的X射线分别照射3种乳腺癌细胞株后,分别于0、4、8、12、24 h,进行实时荧光定量PCR检测其miR-21的表达量。结果:①miR-21在MDA-MB-435S细胞株的表达量最低,在MCF-7型细胞株中的表达量处于中间水平,在MDA-MB-231型细胞株中的表达量最高。②时程关系:miR-21在MDA-MB-231型细胞株受照后表达量逐渐升高,在12 h达到峰值,24 h后基本恢复至照前水平;miR-21在MCF-7型细胞株受照后,表达量也逐步升高,8 h达到峰值,24 h后基本恢复至照前水平;mir-21在MDA-MB-435S型细胞株受照后,表达量一路下降,8 h达到最低值,24 h后,其表达量仍明显低于照前水平。结论:miR-21的表达量与乳腺癌细胞的辐射敏感性成反比。     

Anti-cancer activity of a novel palladium(II) complex on human breast cancer cells in vitro and in vivo

Anti-cancer effects of a newly-synthesized palladium(II) complex, [Pd(sac)(terpy)](sac)·4H₂O (sac = saccharinate, and terpy = 2,2′:6′,2″-terpyridine), were tested against human breast cancer cell lines, MCF-7 and MDA-MB-231. The Pd complex had a strong anti-growth effect in a dose- and time-dependent manner in vitro. This effect was also confirmed by the experiment performed on Balb/c mice in vivo. The IC₅₀ values were 0.09 μM for MDA-MB-231 and 3.05 μM for MCF-7. It was also very effective in disrupting the formation of MDA-MB-231 tubules on matrigel, indicative of a putative anti-invasive activity. It induced apoptosis via the cell death genes of DR4 and DR5. In conclusion, this newly-synthesized Pd (II) complex represents a potentially active novel drug for the breast cancer treatment.     

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.     

Wasabi-Derived 6-(Methylsulfinyl)Hexyl Isothiocyanate Induces Apoptosis in Human Breast Cancer by Possible Involvement of the NF-κB Pathways

6-(methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi (Wasabia japonica), which is a popular spice in Japan. 6-MSITC has been reported to inhibit the proliferation of breast cancer and melanoma cell lines. We inoculated 30 female Balb-nu/nu mice with MDA-MB-231 or -453 cells, and orally administered varying concentrations of 6-MSITC for 12 days following tumor growth. The tumor volumes and tumor weights from mice inoculated with MDA-MB-231 cells, and the tumor volumes of MDA-MB-453 cells were significantly inhibited by 6-MSITC on Days 9 and 11 after drug administration. DNA fragmentation, DNA ladder, and caspase 3/7 activity performed in vitro revealed that 6-MSITC induced apoptosis of MDA-MB-231, MDA-MB-453, and MCF-7 cells. Furthermore, nuclear factor-κB (NF-κB) expression in the nuclei and phosphorylation of inhibitor κBα (IκBα) was downregulated by 6-MSITC in a concentration-dependent manner; however, this activity was not observed in MCF-7 cells. Moreover, this downregulation of phosphorylated IκBα by 6-MSITC in MDA-MB-231 and -453 cells supports its inhibitory effects on NF-κB activity. The expression of phosphorylated AKT (pAKT) reduced by 6-MSITC was confirmed in MDA-MB-231 cells. Thus, we conclude that 6-MITC promotes apoptosis of breast cancer cells by inhibiting NF-kB and therefore releasing its control of the PI3K/AKT pathway.     

Lead identification of conformationally restricted β-lactam type combretastatin analogues: synthesis, antiproliferative activity and tubulin targeting effects

The synthesis and study of the structure-activity relationships of a series of rigid analogues of combretastatin A-4 are described which contain the 1,4-diaryl-2-azetidinone (β-lactam) ring system in place of the usual ethylene bridge present in the natural combretastatin stilbene products. The 1,4-diaryl-2-azetidinones are unsubstituted at C-3, or contain methyl substituent(s) at C-3. The most potent compounds 12d and 12e display antiproliferative activity at nanomolar concentrations when evaluated against the MCF-7 and MDA-MB-231 human breast carcinoma cell lines. 12d exerts antimitotic effects through an inhibition of tubulin polymerisation and subsequent G2/M arrest of the cell cycle in human MDA-MB-231 breast cancer cells, with similar activity to that of CA-4. These novel β-lactam compounds are identified as potentially useful scaffolds for the further development of antitumour agents which target tubulin.     

Pepper Seed Extract Suppresses Invasion and Migration of Human Breast Cancer Cells

This study was performed to determine the antimetastatic activities of chili pepper seed on human breast cancer cells. The water extract of chili pepper seeds was prepared and it contained a substantial amount of phenols (131.12 mg%) and no capsaicinoids. Pepper seed extract (PSE) suppressed the proliferation of MDA-MB-231 and MCF-7 cells at the concentration of 10, 25, and 50 μg/ml (MDA-MB-231: IC₅₀ = 20.1 μg/ml, MCF-7: IC₅₀ = 14.7 μg/ml). PSE increased the expression level of E-cadherin up to 1.2-fold of the control in MCF-7 cells. PSE also decreased the secretion of matrix metalloproteinase (MMP)-2 and MMP-9 in MDA-MB-231 and MCF-7 cells at the concentration of 25 and 50 μg/ml. PSE treatment significantly suppressed the invasion of MDA-MB-231 and MCF-7 cells in a dose-dependent manner. The motility of cancer cells was apparently retarded in the wound healing assay by the PSE treatment. Although our data collectively demonstrate that PSE inhibits invasion and migration of breast cancer cells, further study is needed to identify specific mechanisms and bioactive components contributing to antimetastatic effects of chili pepper seed.     

Anticancer activities of an anthocyanin-rich extract from black rice against breast cancer cells in vitro and in vivo

Anthocyanins widely present in human diet and have a variety of health effects. This study investigates the anticancer effects of an anthocyanin-rich extract from black rice (AEBR) on breast cancer cells in vitro and in vivo. AEBR reduced the viability of breast cancer cell lines MCF-7 (ER(+), HER2/neu(-)), MDA-MB-231 (ER(-), HER2/neu(-)), and MDA-MB-453 (ER(-), HER2/neu(+)) and induced apoptosis in MDA-MB-453 cells via the intrinsic pathway in vitro by activating caspase cascade, cleaving poly (ADP-ribose) polymerase (PARP), depolarizing mitochondrial membrane potential, and releasing cytochrome C. Oral administration of AEBR (100 mg/kg/day) to BALB/c nude mice bearing MDA-MB-453 cell xenografts significantly suppressed tumor growth and angiogenesis by suppressing the expression of angiogenesis factors MMP-9, MMP-2, and uPA in tumor tissue. Altogether, this study suggests the anticancer effects of AEBR against human breast cancer cells in vitro and in vivo by inducing apoptosis and suppressing angiogenesis.     

Twist及其相关调控基因在不同转移性人乳腺癌细胞中的差异表达

目的:观察不同转移潜能人乳腺癌细胞株中Twist及其相关调控基因mRNA表达的差异。方法:培养低侵袭性人乳腺癌细胞株MCF-7和高转移性人乳腺癌细胞株MDA-MB-435S,采用RT-PCR法检测两种细胞株中Twist、脆性组氨酸三联体(FHIT)、皮性钙黏附素(E-cadherin)、细胞凋亡相关基因Mcl-1、Bcl-2和Bax mRNA表达的差异。结果:MCF-7中FHIT、E-cadherin、Bax mRNA的表达显著高于MDA-MB-435S细胞,Twist mRNA在MDA-MB-435S细胞中的表达明显高于MCF-7,Mcl-1、Bcl-2 mRNA在两种细胞株中的表达无明显差异。结论:Twist、FHIT、E-cadherin、Bax mRNA表达差异可能与人乳腺癌细胞的不同转移潜能有关。