Potent genistein derivatives as inhibitors of estrogen receptor alpha-positive breast cancer

The estrogen receptor (ER) is a major target for the treatment of breast cancer cells. Genistein, a soy isoflavone, possesses a structure similar to estrogen and can both mimic and antagonize estrogen effects although at high concentrations it inhibits breast cancer cell proliferation. Hence, to enhance the anti-cancer activity of Genistein at lower concentrations, we have synthesized seven structurally modified derivatives of Genistein (MA-6, MA-8, MA-11, MA-19, MA-20, MA-21 and MA-22) based on the structural requirements for an optimal anti-cancer effect. Among those seven, three derivatives (MA-6, MA-8 and MA-19) showed high antiproliferative activity with IC(50) levels in the range of 1-2.5 μM, i.e., at much lower concentrations range than Genistein itself, in three ER-positive breast cancer cell lines (MCF-7, 21PT and T47D) studied. In our analysis, we noticed that at IC(50) concentrations, the MA-6, MA-8 and MA-19 Genistein derivatives induced apoptosis, inhibited ER-α messenger RNA expression and increased the ratio of ER-β to ER-α levels in a manner comparable to the parent compound Genistein. Of note, these three modified Genistein derivatives exerted their effects at concentrations 10-15 times lower than the parent compound, decreasing the likelihood of significant ER- α pathway activation, which has been a concern for Genistein. Hence these compounds might play a useful role in breast cancer chemoprevention.     

Molecular effects of the isoflavonoid genistein in prostate cancer

Differences in diet have been proposed to be at least partially responsible for the low rate of prostate cancer in Asian populations compared with men in Western countries. One of the compounds that occurs in a greater quantity in the Eastern diet is genistein, an isoflavonoid found in high concentrations in serum after ingestion of soy-rich foods. Extensive molecular studies have been performed to determine its potential health benefits. The mechanism of action of genistein is complex and includes several cellular pathways. In addition to its estrogenic and/or antiestrogenic activities, genistein has been reported to inhibit steroidogenesis and block several protein tyrosine kinases, including epidermal growth factor receptor and src tyrosine kinases. Moreover, it arrests the cell cycle, induces apoptosis, and has antiangiogenic and antimetastatic properties and antioxidant activity. Herein, we review the current literature on the molecular mechanisms of genistein in relation to its effects on prostate cancer cells.     

Carrier mediated action of platinum complexes on estrogen receptor positive tumors

Three 1,2-diaminoethane-dichloro-platinum(II) complexes linked to 5-hydroxy-2-(4-hydroxyphenyl)-3-methylindole by spacer groups of varying length were evaluated for cytostatic activity in estrogen receptor (ER) positive and negative tumor cells. In vitro, only the growth of ER positive MCF-7 mammary tumor cells was inhibited whereas hormone independent MDA-MB 231 cells did not respond. In vivo, a strong inhibitory effect was only observed in ER positive MXT mammary tumors of the mouse. The complex with a hexyl group as spacer reduced the tumor weight by 89% after 6 weeks of treatment. The R 3327 Dunning prostatic tumor of the rat, which also contains ER was inhibited, too. Generally, the effect in ER negative tumors was weak. These findings can be rationalized by the high binding affinities of the complexes for ER. By the mouse uterine weight test it was shown that the endocrine activity of the complexes is very low. Therefore, a mode of action different from that exerted by estrogens or antiestrogens has to be assumed.     

Molecular aspects of estrogen receptor variants in breast cancer

Summary Measurements of the estrogen receptor (ER) and the estrogen-induced progesterone receptor (PgR) are used by most clinicians as indicators of both overall prognosis and likelihood of response to endocrine therapy. Patients with ER+/PgR+ tumors have the highest likelihood of response; conversely, patients with ER-/PgR- tumors have the lowest likelihood of response. Unfortunately, most patients treated successfully with endocrine therapy eventually develop endocrine-resistant disease recurrence. In an effort to study potential mechanisms of endocrine resistance, we have studied discordant ER-/PgR+ tumors, in which the normally estrogen-regulated PgR gene is induced in the apparent absence of ER. Our laboratory has previously cloned, from ER-/PgR+ tumors, a variant ER mRNA precisely missing the sequence corresponding to ER exon 5, and has demonstrated that the truncated protein product translated from this variant RNA is capable of constitutively inducing the expression of an estrogen-responsive reporter gene in a yeast expression vector system (Fuqua et al, Cancer Res 51:105-109, 1991). In the present report we describe further experiments to characterize the activity and biological consequences of expression of this variant ER in human breast cancer cells. We have stably transfected MCF-7 human breast cancer cells with a mammalian expression vector for the exon 5 deletion variant ER. These transfected cells produce a truncated ER protein of the expected 40 kDa size. Cells expressing the exon 5 ER deletion variant constitutively express PgR, and manifest increased anchorage-independent colony formation in the absence of estrogen. Furthermore, the anchorage-dependent growth of these cells was not inhibited by the triphenylethylene antiestrogens tamoxifen or 4-hydroxytamoxifen, unlike MCF-7 cells transfected with a control plasmid, which were growth inhibited by both of these compounds. Interestingly, the pure antiestrogen ICI 164,384 did inhibit the growth of exon 5 ER deletion variant-expressing transfectants. The implications of these results with regard to the treatment of tamoxifen-resistant disease are discussed.     

Breast-related effects of selective estrogen receptor modulators and tissue-selective estrogen complexes

A number of available treatments provide relief of menopausal symptoms and prevention of postmenopausal osteoporosis. However, as breast safety is a major concern, new options are needed, particularly agents with an improved mammary safety profile. Results from several large randomized and observational studies have shown an association between hormone therapy, particularly combined estrogen-progestin therapy, and a small increased risk of breast cancer and breast pain or tenderness. In addition, progestin-containing hormone therapy has been shown to increase mammographic breast density, which is an important risk factor for breast cancer. Selective estrogen receptor modulators (SERMs) provide bone protection, are generally well tolerated, and have demonstrated reductions in breast cancer risk, but do not relieve menopausal symptoms (that is, vasomotor symptoms). Tissue-selective estrogen complexes (TSECs) pair a SERM with one or more estrogens and aim to blend the positive effects of the components to provide relief of menopausal symptoms and prevention of postmenopausal osteoporosis without stimulating the breast or endometrium. One TSEC combination pairing conjugated estrogens (CEs) with the SERM bazedoxifene (BZA) has completed clinical development and is now available as an alternative option for menopausal therapy. Preclinical evidence suggests that CE/BZA induces inhibitory effects on breast tissue, and phase 3 clinical studies suggest breast neutrality, with no increases seen in breast tenderness, breast density, or cancer. In non-hysterectomized postmenopausal women, CE/BZA was associated with increased bone mineral density and relief of menopausal symptoms, along with endometrial safety. Taken together, these results support the potential of CE/BZA for the relief of menopausal symptoms and prevention of postmenopausal osteoporosis combined with breast and endometrial safety.     

ER通路信号传导与选择性雌激素受体调节剂耐药机制研究

内分泌治疗是乳腺癌综合治疗中的重要措施之一,此疗法面临的最大难题是原发性、继发性耐药,其确切机制尚不清楚.雌激素受体(ER)信号传导途径及各辅助因子的分子调控是近年来研究的重点,现综述有关研究进展,以期为临床选择性ER调节剂(SERM)耐药病例的治疗及耐药逆转提供帮助.     

FuGENE6介导雌激素受体质粒HEGO5转染不同乳腺癌细胞株后受体的表达

背景及目的：基因转染技术是研究乳腺癌细胞雌激素受体(estrogen receptor,ER)的表达及其作用机制的重要手段。本研究检测不同人类乳腺癌细胞株以FuGENE6试剂为载体进行转染时的转染效率,以及ER质粒HEG05转染后ER的表达情况。方法：不同乳腺癌细胞株先用pEGFP—N1质粒转染,流式细胞术(flow cytometry,FCM)对转染效率进行测定。将转染效率高的细胞株分别再用HEGO5转染,用FCM和Western blot方法对ER的表达情况进行检测。结果：乳腺癌细胞株MM-231、MM330、MMl34Ⅵ、MM175Ⅶ、MM157、MM361、MM436、MM453、UaCC812、UACC893、BT-549、BT-20、HBL-100、Hs578t、MCF-7、T-47d、ZR-75-1的转染效率存在很大差异。细胞株HBL-100重复性转染实验的变异系数为5．1。几种转染效率较高的细胞株MCF-7、HBL-100、Hs578t、MM436、MM453和BT-20用HEG05转染后,ER阳性的表达率介于12．9％～54．8％之间,与pEGFP—N1的转染效率相当;不同细胞ER的FCM和Western blot检测结果一致。此外,ER的表达具有一定的细胞周期特异性。结论：FuGENE6试剂介导乳腺癌细胞进行转染时重复性好、结果稳定。HEG05转染后ER的表达情况可分别用Western blot和FCM进行定性、定量检测。     

Baicalein and genistein display differential actions on estrogen receptor (ER) transactivation and apoptosis in MCF-7 cells

Genistein is a soy isoflavone that has attracted much attention for its chemopreventive properties. Baicalein is a flavone found in Scutellaria species, and its structure is analogous to that of genistein. We examined the two flavones' antiestrogenicity by using a transient transfection model. Our results indicated that baicalein could suppress 17beta-estradiol-induced transactivation in cells expressing estrogen receptor alpha, whereas no antagonistic activity was seen in genistein. Cell death assay and flow cytometry analysis demonstrated that baicalein was a stronger apoptosis inducing agent than genistein. Considering all the criteria tested, baicalein could be a better chemopreventive agent than genistein.     

Anticarcinogenic effects of isoflavones may be mediated by genistein in mouse mammary tumor virus-induced breast cancer.

Isoflavones are known to exert anticancer effects. These effects were examined using two isoflavones, biochanin A and daidzein, in a mouse mammary tumor virus (MMTV)-induced spontaneous breast cancer model. Emphasis was placed on isoflavone metabolism by the intestinal microflora and changes in estrogen levels. Germ-free (Gf) mice and their conventionalized (Cv) counterparts were assigned to three diet groups: (1) control diet, (2) biochanin A and (3) daidzein. In all groups, urine was collected from virgin female mice to analyze isoflavone metabolism by high performance liquid chromatography. These studies revealed changes of biochanin A into genistein, and of daidzein into equol, which were accelerated in the Cv animals. However, the Gf mice could not transform biochanin A into genistein, or daidzein into equol. Estrogen levels in the control and daidzein diet groups were lower in the Gf mice than in the Cv mice. The biochanin A group showed no differences in estrogen levels between the Cv and Gf animals. Four-week-old male and female animals were paired in the Gf and Cv groups. The female animals delivered and lactated repeatedly and were observed for the development of mammary cancer by palpation, twice weekly, until 15 months of age. The Cv mice showed a significantly lower incidence of breast cancer in the biochanin A diet group than in the control or daidzein groups (p < 0.05). These results suggest that the anticarcinogenic effects in this system might be produced not by daidzein or equol, but by biochanin A and/or genistein. In the Gf animals, the incidence of breast cancer was significantly higher in the biochanin A group than in the control group (p < 0.05), probably due to the increased level of estradiol in the former group. The biochanin A group tended to have a higher incidence of breast cancer than the daidzein group in the Gf group, although no significant differences were noted. Thus, no anticarcinogenic effect was produced by biochanin A alone in the Gf mice. In view of the results presented, genistein derived from biochanin A following metabolic processes in the intestinal microflora most likely acts as an inhibitor in breast carcinogenesis; biochanin A is most likely a precursor of genistein.     

Serum estrogen receptor beta mediated bioactivity correlates with poor outcome in lung cancer patients

Objectives

The role of estrogen signaling in lung cancer remains unresolved. We investigate the influence of serum estrogenic compounds and estrogen receptor (ERα and ERβ) mediated bioactivity on lung cancer outcomes.

Materials and methods

Serum samples were collected from 222 postmenopausal Chinese patients diagnosed with lung cancer in five Singapore hospitals. Levels of the estrogenic compounds estradiol and estrone were measured using liquid chromatography tandem mass spectrometry. Free estradiol levels were calculated based on sex hormone binding globulin levels. ERα- and ERβ-mediated bioactivity in serum samples were analyzed using reporter gene bioassays in human cells.

Results and conclusion

High ERβ-mediated bioactivity predicted poorer lung cancer survival (p = 0.001) on multivariable Cox regression analysis with adjustment for age, stage of tumor, smoking status, body mass index and histology. In comparison, levels of estrogens and ERα-mediated bioactivity were not associated with prognosis. Compared to the lowest tertile of ERβ-mediated bioactivity, patients in the middle and highest tertiles had HR (95%CI) 1.60 (1.10–2.33) and 1.93 (1.32–2.82) (p for trend = 0.001) higher risk of death from lung cancer. Using Kaplan–Meier survival curves, patients with high ERβ-mediated bioactivity correlated with poorer overall survival (p = 0.033). ERβ-mediated bioactivity did not differ in terms of age, use of hormone replacement therapy, smoking, stage of tumor or histological subtype. High ERβ-mediated bioactivity levels in patients’ serum were associated with poorer prognosis in lung cancer patients. Our findings suggest that that compound(s) other than endogenous estrogens may be exerting this ERβ bioactivity and studies to identify these compounds or groups of compounds need to be performed. Furthermore, the measurement of ERβ activity in sera could potentially serve as a prognostic marker to predict lung cancer survival, and selective blockage of ERβ signaling may have a role in lung cancer therapy.     

Influence of estrogen receptor alpha and progesterone receptor polymorphisms on the effects of hormone therapy on mammographic density.

Postmenopausal hormone therapy increases mammographic density, a strong breast cancer risk factor, but effects vary across women. We investigated whether the effect of hormone therapy use is modified by polymorphisms in the estrogen receptor (ESR1) and progesterone receptor (PGR) genes in the Dutch Prospect-EPIC and the English EPIC-Norfolk cohorts. Information on hormone therapy use was obtained through questionnaires at recruitment and after 5 years. Blood samples were collected and consecutive mammograms were available through breast cancer screening programs. For 795 hormone therapy users, one mammogram before and a second mammogram during hormone therapy use was included. For 781 never hormone therapy users, mammograms with similar time intervals were included. Mammographic density was assessed using a computer-assisted method. Changes in density were analyzed using linear regression. A statistically significant difference in percentage density change between hormone therapy users and never users was seen in women with the ESR1 PvuII Pp or pp genotype (2.24%; P < 0.01), but not in those with the PP genotype (0.90%; P = 0.47). Similarly, effects of hormone therapy on percentage density were observed in women with the ESR1 XbaI Xx or xx genotype (2.20%; P < 0.01), but not in those with the XX genotype (-0.65%; P = 0.70). Also, effects were seen in women with the PGR +331 GG genotype (2.04%; P < 0.01), but not in those with the GA or AA genotype (0.98%; P = 0.53). The PGR PROGINS polymorphism did not seem to make women more susceptible to the effects of hormone therapy use. In conclusion, our results suggest that specific polymorphisms in the ESR1 and PGR genes may make women more susceptible to the effects of hormone therapy use on mammographic density.     

Antitumor effects of radioiodinated antisense oligonuclide mediated by VIP receptor

A 15-mer phosphorothioate antisense oligonuclide (ASON) complementary to the translation start region of the C-myc oncogene mRNA was radioiodinated to enhance its antitumor activity, and vasoactive intestinal peptide bound covalently polylysine (VIP-polylysine) was used as a carrier to deliver the oligonucleotide into VIP receptor-positive tumor cells. The antitumor activity of radioiodinated ASON conjugated to VIP-polylysine(VIP-131I-ASON) was investigated in athymic mice bearing HT29 tumor xenografts in comparison with unconjugated radioiodinated ASON(131I-ASON), unlabelled ASON (VIP-ASON) and scrambled oligonucleotide (VIP-131I-MON) conjugated to VIP-polylysine. Conjugation 125I-ASON to VIP-polylysine resulted in a 5.6-fold decrease in the plasma clearance and a 3.4-fold increase in tumor uptake of the radiopharmaceutical. Athymic mice bearing HT29 tumor xenografts were treated with 4 weekly doses of VIP-131I-ASON and the antitumor effects were assessed by use of the slope of the tumor growth curve. VIP-131I-ASON exhibited strong antitumor effects against HT29 xenografts, decreasing tumor growth rate 9.67-, 7.90-fold more effectively than 131I-ASON and VIP-ASON at equivalent doses of ASON. Conversely, 131I-ASON, VIP-ASON or VIP-131I-MON caused no significant effect compared with the normal saline. These data indicated that use of a VIP-polylysine carrier greatly increased HT29 tumor uptake of ASON and treatment with the VIP-131I-ASON complexes resulted in tumor growth delay in human colon cancer xenograft.