Addition Of ICI Research Articles

Exposure to nitrate and nitrite disrupts cardiovascular development through estrogen receptor in zebrafish embryos and larvae.

Increasing nitrate concentration on surface and groundwater due to anthropogenic activities is an environmental concern. In this study, Tg(fli1: EGFP) zebrafish embryos were exposed to nitrate (NO3-) and nitrite (NO2-), and their cardiovascular development were investigated. Exposure to 10mg/L NO3-N and 1 and 10mg/L NO2-N decreased heart rate at 48-96-h post-fertilization (hpf), ventricular volume, and red blood cell flow rate at 96 hpf. Similar concentrations increased the number of embryos and larvae with pericardial edema and missing intersegmental and parachordal vessels in the caudal region at 48-96 hpf. Addition of ICI 182,720 (ICI) reversed the effects of nitrate and nitrite, suggesting estrogen receptors (ER) are involved. 10mg/L NO3-N and 1mg/L NO2-N decreased cardiovascular-related genes, gata4,5,6, hand2, nkx2.5, nkx2.7, tbx2a, tbx2b, and fgf1a. Gene expressions of ovarian aromatase and brain aromatase (cyp19a1a and cyp19a1b, respectively) decreased in the exposed groups, whereas ERs (esr1, esr2a, and esr2b) and nitric oxide synthase 2a (nos2a) increased. The effects on gene expression were also reversed by addition of ICI. Taken together, nitrate and nitrite disrupt cardiovascular system through ER in developing zebrafish, implying that environmental nitrate and nitrite contamination may be harmful to aquatic organisms.

Real-world (rw) clinical outcomes for advanced/metastatic non-small cell lung cancer (aNSCLC) patients (pts) treated with second line (2L) ramucirumab plus docetaxel (R+D) post frontline (1L) platinum based chemotherapy plus immune checkpoint inhibitors (Pt + ICI).

e20727 Background: R+D is approved for use in pts with aNSCLC after Pt chemotherapy. With recent approvals, ICI can now be added to Pt chemotherapy (Pt + ICI) in 1L. This retrospective observational study provides an exploratory view of baseline characteristics and rw clinical effectiveness outcomes for pts receiving 2L R+D post 1L Pt + ICI. Methods: All adult pts treated with 2L R+D after 1L Pt + ICI therapy between 03/01/2015 and 06/30/2018, with ≥ 3 months follow up, were selected from the Flatiron Health EHR-derived de-identified database (n = 15). Rw clinical endpoints during R+D therapy included rw objective response rate (rwORR), rw disease control rate (rwDCR), rw best response, as well as Kaplan-Meier estimates of rw time to first response & rw duration of response. Results: Median age was 62 years, 10 pts (66.6%) were aged < 65 years, 11 (73.3%) were men, 3 (20.0%) had no history of smoking, 14 (93.3%) had non-squamous histology, 4 (26.7%) were EGFR positive, 3 (20.0%) were KRAS positive and 6 (85.7%) were PD-L1 negative. Of the 8 pts with a documented rw tumor response assessment, 3 (37.5%) had partial response (PR), 3 (37.5%) had stable disease (SD), & 2 (25.0%) had progressive disease as their rw best response. The rwORR (PR or complete response [CR]) & rwDCR (PR, CR, or SD) were 37.5% and 75.0%, respectively. Among responding pts, median time to first response was 2.2 months (95% CI, 1.3 - not reached [NR]) & median duration of response was 2.3 months (95% CI, 1.5 - NR). Patient numbers were too small (n = 15) and duration of follow-up was too short (3.4 months [IQR, 0.7 - 5.4]) to make robust estimation of overall survival or rw progression free survival. Conclusions: Data from this small patient cohort in US community practice are not conclusive and should be considered exploratory, but do show high rates of rw objective response and rw disease control rates during 2L R+D following 1L Pt + ICI. Data with larger sample sizes and additional follow-up are needed to better understand outcomes of R+D following the addition of ICI to 1L Pt chemotherapy regimens.

Editor's Highlight: Transcriptome Profiling Reveals Bisphenol A Alternatives Activate Estrogen Receptor Alpha in Human Breast Cancer Cells.

Plasticizers with estrogenic activity, such as bisphenol A (BPA), have potential adverse health effects in humans. Due to mounting evidence of these health effects, BPA is being phased out and replaced by other bisphenol variants in “BPA-free” products. We have compared estrogenic activity of BPA with 6 bisphenol analogues [bisphenol S (BPS); bisphenol F (BPF); bisphenol AP (BPAP); bisphenol AF (BPAF); bisphenol Z (BPZ); bisphenol B (BPB)] in 3 human breast cancer cell lines. Estrogenicity was assessed (10−11–10−4 M) by cell growth in an estrogen receptor (ER)-mediated cell proliferation assay, and by the induction of estrogen response element-mediated transcription in a luciferase assay. BPAF was the most potent bisphenol, followed by BPB > BPZ ∼ BPA > BPF ∼ BPAP > BPS. The addition of ICI 182,780 antagonized the activation of ERs. Data mining of ToxCast high-throughput screening assays confirm our results but also show divergence in the sensitivities of the assays. Gene expression profiles were determined in MCF-7 cells by microarray analysis. The comparison of transcriptome profile alterations resulting from BPA alternatives with an ERα gene expression biomarker further indicates that all BPA alternatives act as ERα agonists in MCF-7 cells. These results were confirmed by Illumina-based RNA sequencing. In conclusion, BPA alternatives are not necessarily less estrogenic than BPA in human breast cancer cells. BPAF, BPB, and BPZ were more estrogenic than BPA. These findings point to the importance of better understanding the risk of adverse effects from exposure to BPA alternatives, including hormone-dependent breast cancer.

Treatment of BG-1 Ovarian Cancer Cells Expressing Estrogen Receptors with Lambda-cyhalothrin and Cypermethrin Caused a Partial Estrogenicity Via an Estrogen Receptor-dependent Pathway

Synthetic pyrethroids (SPs) are the most common pesticides which are recently used for indoor pest control. The widespread use of SPs has resulted in the increased exposure to wild animals and humans. Recently, some SPs are suspected as endocrine disrupting chemicals (EDCs) and have been assessed for their potential estrogenicity by adopting various analyzing assays. In this study, we examined the estrogenic effects of lambda-cyhalothrin (LC) and cypermethrin (CP), the most commonly used pesticides in Korea, using BG-1 ovarian cancer cells expressing estrogen receptors (ERs). To evaluate the estrogenic activities of two SPs, LC and CP, we employed MTT assay and reverse-transcription polymerase chain reaction (RT-PCR) in LC or CP treated BG-1 ovarian cancer cells. In MTT assay, LC (10−6 M) and CP (10−5 M) significantly induced the growth of BG-1 cancer cells. LC or CP-induced cell growth was antagonized by addition of ICI 182,720 (10−8 M), an ER antagonist, suggesting that this effect appears to be mediated by an ER-dependent manner. Moreover, RT-PCR results showed that transcriptional level of cyclin D1, a cell cycle-regulating gene, was significantly up-regulated by LC and CP, while these effects were reversed by co-treatment of ICI 182,780. However, p21, a cyclin D-ckd-4 inhibitor gene, was not altered by LC or CP. Moreover, ERα expression was not significantly changed by LC and CP, while downregulated by E2. Finally, in xenografted mouse model transplanted with human BG-1 ovarian cancer cells, E2 significantly increased the tumor volume compare to a negative control, but LC did not. Taken together, these results suggest that LC and CP may possess estrogenic potentials by stimulating the growth of BG-1 ovarian cancer cells via partially ER signaling pathway associated with cell cycle as did E2, but this estrogenic effect was not found in in vivo mouse model.

224 A GROWTH OF HUMAN BG-1 CANCER CELLS EXPRESSING ESTROGEN RECEPTORS WAS ENHANCED BY SYNTHETIC PYRETHROIDS, LAMBDA-CYHALOTHRIN AND CYPERMETHRIN, VIA AN ESTROGEN RECEPTOR-DEPENDENT SIGNALING PATHWAY

Synthetic pyrethroids (SP) are the most common pesticides in recent use, which are used as indoor pest control. The widespread use of SPs has resulted in extensive exposure to wildlife and human. Recently some SPs are suspected as endocrine disrupting chemicals (EDC) and have been assessed for their potential estrogenicity by various assays. In this study, we examined the estrogenic effects of lambda-cyhalothrin (LCT) and cypermethrin (CP), the most commonly used pyrethroid insecticides in Korea, using BG-1 ovarian cancer cells expressing oestrogen receptors (ER). To evaluate the estrogenic activities of two SPs, LCT and CP, we employed MTT assay and reverse-transcription polymerase chain reaction (RT–PCR). In MTT assay, LCT (10–6 M) and CP (10–5 M) significantly induced the growth of BG-1 cancer cells, 1.61 ± 0.1 and 1.45 ± 0.06 times, respectively, as 17β-oestradiol (E2, 10–9 M, 2.73 ± 0.25 times) did. LCT or CP-induced cell growth was reversed to a control level (DMSO) by addition of ICI 182 720 (10–8 M), an ER antagonist, suggesting that this effect appears to be mediated by an ER-dependent manner. Moreover, RT–PCR results showed that transcriptional level of ERα expression was significantly down-regulated by LCT and CP as in case of E2. Taken together, these results indicate that LCT and CP may possess estrogenic potentials to stimulate ovarian cancer cells expressing ERs via an ER-dependent manner, and these collective results confirm the carcinogenicity of these SP, LCT and CP, in ER-positive cells or tissues.

Effects of naringin on proliferation, differentiation and maturation of rat calvarial osteoblasts in vitro

To investigate the effects of naringin on the proliferation, differention and maturaion of rat calvarial osteoblasts (ROB). Segregated neonatal SD rat skull, enzyme digestion to obtain ROB. The culture medium was replaced every three days. Serial subcultivation proceeded when cells covered with 80% culture dish. Naringin supplemented into the culture at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) respectively. MTT method was adopted in proliferation analysis and the activity of ALP was examined after induced 9 days. Search the best concentration and supplemented into the medium, then the osteogenic differentiation markers including the secretion amount of osteocalcin, osteopontin and bone morphogenetic protein-2 were compared between the naringin-supplemented group and the control. Total RNA was isolated and the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERa and ERbeta was investigated by Real time RT-PCR. Total protein also was isolated and the expression ERa, ERbeta and collagen I was examined by Western blot. After the addition of ICI 182.780, an inhibitor of the estrogen signal pathway, these index also was examined and the changes were compared. The ROB proliferation was motivated by naringin dose-dependently. And it evidently leads to osteogenic process and maturation. 1 x 10(-5) mol x L(-1) is the best concentration. Naringin improved the secretion of osteocalcin, osteopontin, bone morphogenetic protein-2 and collagen I significantly. Besides, it can also enhanced the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERalpha and ERbeta. While all these effects can be restrained by ICI 182.780. The naringin with final concentration of 1 x 10(-5) mol x L(-1) enhances the osteogenic differentiation and maturation of ROB significantly, while the promoting effects vanished after the addition of ICI 182.780. These results suggesting that naringin is one of the phytoestrogens and have the activity of bone formation may via estrogen signal pathway, it can be developed into a new drug for osteoporosis therapy.

Effects of neuroactive steroids on cochlear hair cell death induced by gentamicin

As neuroactive steroids, sex steroid hormones have non-reproductive effects. We previously reported that 17β-estradiol (βE2) had protective effects against gentamicin (GM) ototoxicity in the cochlea. In the present study, we examined whether the protective action of βE2 on GM ototoxicity is mediated by the estrogen receptor (ER) and whether other estrogens (17α-estradiol (αE2), estrone (E1), and estriol (E3)) and other neuroactive steroids, dehydroepiandrosterone (DHEA) and progesterone (P), have similar protective effects. The basal turn of the organ of Corti was dissected from Sprague–Dawley rats and cultured in a medium containing 100 μM GM for 48 h. The effects of βE2 and ICI 182,780, a selective ER antagonist, were examined. In addition, the effects of other estrogens, DHEA and P were tested using this culture system. Loss of outer hair cells induced by GM exposure was compared among groups. βE2 exhibited a protective effect against GM ototoxicity, but its protective effect was antagonized by ICI 182,780. αE2, E1, and E3 also protected hair cells against gentamicin ototoxicity. DHEA showed a protective effect; however, the addition of ICI 182,780 did not affect hair cell loss. P did not have any effect on GM-induced outer hair cell death. The present findings suggest that estrogens and DHEA are protective agents against GM ototoxicity. The results of the ER antagonist study also suggest that the protective action of βE2 is mediated via ER but that of DHEA is not related to its conversion to estrogen and binding to ER. Further studies on neuroactive steroids may lead to new insights regarding cochlear protection.

Studies in the guinea-pig with ICI 185,282: a thromboxane A2 receptor antagonist.

The effects of ICI 185,282 (5(Z)-7-([ 2,4,5-cis]-4-O-hydroxyphenyl-2-trifluoromethyl-1, 3-dioxan-5-yl)heptenoic acid) have been studied on guinea-pig platelets and pulmonary smooth muscle in-vitro and in-vivo. When tested on guinea-pig lung parenchyma in-vitro. ICI 185,282 (1 x 10(-7) M) produced a significant shift in U-46619 response curves (concentration ratio of 13:3); the antagonist (1 x 10(-5) M) did not modify histamine responses. When tested on guinea-pig trachea in-vitro ICI 185,282 (1 x 10(-7) M) caused significant inhibition of U-46619 and PGD2 responses (concentration ratios of 8.3 and 14.1, respectively); the antagonist (1 x 10(-5) M proved less effective against contractions of PGF2 alpha, LTD4 and histamine (concentration ratios of 7.0, 1.5 and 1.6). When added to guinea-pig platelet rich plasma in-vitro, ICI 185,282 (x 10(-6), 1 x 10(-5) M) caused concentration-dependent parallel shifts to the right of U-46619 aggregation curves, yielding concentration ratios of 13.6 and 141.9, respectively. In-vitro, addition of ICI 185,282 (x 10(-5) M) to indomethacin-treated pulmonary smooth muscle did not modify resting tone, neither did it induce aggregation or swelling in platelet-rich plasma preparations. When administered orally to guinea-pigs ICI 185,282 (0.1, 0.5 mg kg-1) caused a significant inhibition of U-46619-induced platelet aggregation ex-vivo which persisted greater than or equal to 8 h. In-vivo, a single oral dose of ICI 185,282 (1 mg kg-1) inhibited bronchospasm induced by U-46619, PGD2, PGF2 alpha, arachidonic acid, LTD4 and PAF; responses to histamine were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological actions of ICI 180080, a novel thromboxane receptor antagonist.

ICI 180080 (5(Z)-7-[2,2-dimethyl-4-(2-hydroxyphenyl)-1,3-dioxan-cis-5-yl] heptenoic acid) potently inhibited contractions of rat and rabbit aortae and guinea-pig trachea elicited by 11,9-epoxymethano PGH2 (U-46619). This antagonism was selective because contractions of aortae to noradrenaline and 5-hydroxytryptamine and trachea to histamine were not antagonized by ICI 180080. Schild analysis of data obtained from experiments on rabbit aortae indicated that this thromboxane receptor antagonism was competitive (pA2 = 7.50, slope = 1.07). Addition of ICI 180080 to human platelet-rich plasma caused dose-related inhibition of U-46619-induced platelet aggregation. This modification of platelet aggregation was not associated with inhibition of thromboxane synthetase, cyclo-oxygenase or lipoxygenase. ICI 180080 did not modify the primary phase of ADP-induced aggregation of human platelets neither did it affect the platelet inhibitory activity of prostacyclin. When dosed orally to anaesthetized guinea-pigs, ICI 180080 (5-50 mg kg-1) caused dose-related inhibition of U-46619-evoked bronchoconstriction. We conclude that ICI 180080 is a potent, selective, competitive, orally active thromboxane antagonist.

Suppressive Effect on MDC and IP-10 Expression in Monocytes by Endocrine Disruptor Chemicals

The expression of chemokines is critical in leukocyte recruitment and inflammation, but the regulatory mechanisms involved remain incompletely defined. While endocrine disrupter chemicals (EDCs) are known to be ubiquitous in the environment and often associated with altered inflammatory response, their potential impact on chemokine expression in monocytes is at present unknown. To this end, the effects of EDCs on the expression of Th1- and Th2-related chemokines in a human monocytic cell line, THP-1, were investigated. THP-1 cells were pre-treated with varying concentrations of EDCs (nonylphenol and 4-octylphenol) with or without the addition of an estrogen receptor (ER) antagonist, ICI 182,780 and then stimulated by lipopolysaccharide (LPS). The levels of chemokines, CXCL10/ IFN-alpha-inducible protein 10 (IP-10, a Th1 chemokine) and monocyte-derived chemokine (MDC)/CCL22, a Th2 chemokine) were measured by ELISA. EDC-mediated signaling events and histone modifications were examined by the use of Western blotting and chromatin immunoprecipitation (ChIP) assay. Nonylphenol and 4-octylphenol were able to suppress LPS-induced MDC and IP-10 expression. This suppressive effect was not reversed by the addition of ICI 182,780. Nonylphenol and 4-octylphenol reduced LPS-induced activation of MAPK signaling pathway, MKK1/2 and ERK, concomitant with decreased levels of LPS-induced acetylated histone 4 (H4) at the IP-10 and MDC gene loci. Nonylphenol and 4-octylphenol suppressed LPS-induced MDC expression in monocytes via, at least in part, the MKK1/2-ERK MAPK pathway and histone H4 acetylation, but not the estrogen receptor.

Estrogen Receptor α Mediates Breast Cancer Cell Resistance to Paclitaxel through Inhibition of Apoptotic Cell Death

Estrogen receptors (ER) are expressed in approximately 65% of human breast cancer. Cumulative data from clinical trials and retrospective analyses suggest that some chemotherapeutic agents may be less effective in patients with ER-positive (ER+) tumors than those with ER-negative (ER-) tumors. Paclitaxel is an active agent used in breast cancer chemotherapy. To investigate the possible influence of ER on the therapeutic efficacy of paclitaxel and its underlying mechanism, we established several isogenic ER+ cell lines by stable transfection of ERalpha expression vectors into ER- breast cancer BCap37 cells. We showed that 17-beta estradiol significantly reduces the overall cytotoxicity of paclitaxel in BCap37-expressing ERalpha but has no influence on the ER- parental cells. Further analyses indicate that expression of ERalpha in BCap37 cells mainly interferes with paclitaxel-induced apoptotic cell death, without affecting paclitaxel-induced microtubule bundling and mitotic arrest. Moreover, we found that the addition of ICI 182,780 (Fulvestrant), a selective ER down-regulator, could completely reverse the resistance of ER+ BCap37 cells to paclitaxel. These findings showed that ERalpha-mediated breast tumor cell resistance to paclitaxel was through selective inhibition of paclitaxel-induced tumor cell apoptosis. Additionally, the combination of ICI 182,780 also sensitizes MCF-7 and T47D cell lines to the treatment of paclitaxel, which further confirmed the correlation between ERalpha and drug resistance in ER+ tumor cells. The results obtained from this study provide useful information for understanding ER-mediated resistance to paclitaxel and possibly other antineoplastic agents.

The estrogenic activity of synthetic progestins used in oral contraceptives enhances fatty acid synthase-dependent breast cancer cell proliferation and survival

Overexpression of the lipogenic enzyme fatty acid synthase (FAS) is a common molecular feature in subsets of sex-steroid-related tumors including breast carcinomas that is associated with poor prognosis. In this study, we explored whether breast-cancer associated FAS (oncogenic antigen-519) is regulated by the progestin component in oral contraceptives. In addition, we examined the role of FAS hyperactivity on progestin-regulated breast cancer cell proliferation, survival and metastatic properties. We found that in estrogen receptor (ER)- and progesterone receptor (PR)-positive MCF-7 human breast cancer cells, synthetic progestins used in oral contraceptives including norethynodrel (NOR) and norethindrone, induced a dose-dependent increase of FAS enzymatic activity, with a maximum response (> or = 4-fold) occurring at a concentration of 10(-8) M. FAS activity was only slightly stimulated after exposure to two other progestins, medroxy-progesterone acetate (MPA) and megestrol acetate (MGA), which are used in postmenopausal hormone replacement therapy and high-dose progestin treatment therapy. Western blot analyses showed that NOR-induced stimulation of FAS activity correlated closely with NOR-induced up-regulation of FAS protein expression. To determine the role of FAS accumulation following NOR exposure, we pharmacologically examined the requirement for FAS activity in NOR-stimulated breast cancer cell proliferation and survival. The novel small compound C75 (a slow-binding FAS inhibitor) blocked NOR-induced breast cancer cell proliferation in anchorage-dependent assays. More importantly, pharmacological inhibition of FAS activity completely abolished NOR-stimulated soft-agar colony formation of MCF-7 cells. To evaluate the involvement of PR and ER signalings in NOR-induced up-regulation of FAS expression and activity, NOR was used in combination with either the anti-progestin RU486 (mifepristone) or the pure antiestrogen ICI 182,780 (Faslodex). RU486 and ICI 182,780 similarly abolished NOR-induced FAS activation, supporting the notion that PR- and ER-mediated FAS up-regulation might play different roles in NOR-stimulated breast cancer cells. Interestingly, when we evaluated the involvement of PR and ER signalings on NOR-induced breast cancer cell proliferation, the anti-estrogen ICI 182,780, but not the anti-progestin RU486, was found to inhibit NOR-stimulated proliferation and survival of MCF-7 cells in anchorage-dependent and -independent assays. To further determine whether NOR produced their effects via the ER, we evaluated its effects on endogenous ER transcriptional activity by using transient transfection assays with an estrogen-response element reporter construct (ERE-Luciferase). In the absence of E2 stimulation, treatment with NOR dramatically increased the levels of ERE-dependent transcriptional activity. This estrogenic like-effect of NOR was blocked by the addition of ICI 182,780, whereas RU486 failed to inhibit NOR-induced ERE activity. In summary, this study provides direct evidence that: a) a number of synthetic progestins used in oral contraceptives significantly activates breast cancer-associated FAS (OA-519) activity and expression in hormone-dependent breast cancer cells; b) FAS activity is necessary for progestin-induced anchorage-independent growth and survival of human breast cancer cells, and c) activation of ER, but not PR signaling, is the stimulatory mechanism through which synthetic progestins enhance a FAS-dependent proliferative and pro-survival signaling. These findings should be helpful to explain the conflicting evidence linking oral contraceptives and breast cancer risk through the estrogenic activation of tumor-associated FAS (OA-519), a molecular marker associated with poor clinical outcome of breast cancer disease.

Genistein at a Concentration Present in Soy Infant Formula Inhibits Caco-2BBe Cell Proliferation by Causing G2/M Cell Cycle Arrest

Fifteen percent of all U.S. infants are fed soy formulas containing up to 47 mg/L of isoflavones (>65% as genistin + genistein); thus, these infants' intestines are exposed to a high dose of genistein, a phytoestrogen and tyrosine kinase inhibitor. Little attention has been focused on genistein's impact on the developing intestine. We hypothesized that a high dose of genistein would inhibit intestinal cell growth. Caco-2BBe human intestinal cells were exposed to 0, 3.7, and 111 micro mol/L (0, 1, and 30 mg/L) genistein in DMEM + 0.5% fetal bovine serum for 24-48 h. Cell number, thymidine incorporation, apoptosis, and cell cycle analyses were performed. The low genistein concentration increased intestinal cell proliferation by 28% (P = 0.001), but did not affect cell number or caspase-3 activity compared to the control. Furthermore, the addition of ICI, an estrogen receptor antagonist, negated the proliferative effect of the low genistein. In contrast, the high genistein concentration reduced cell number by 40%, proliferation by 94%, and caspase-3 activity by 50% compared to the control (P < 0.05). Cell cycle analysis after 48 h exposure to high genistein revealed 37% of cells in G0/G1 and 35% in G2/M vs. 71% in G0/G1 and 17% in G2/M for the control and low genistein groups. Thus, a biphasic effect of genistein was seen with a low dose stimulating intestinal cell proliferation through the estrogen receptor, whereas a high dose of genistein inhibited intestinal cell proliferation and altered cell cycle dynamics. A high dose of genistein may potentially compromise intestinal growth.

The neuroprotective effects of estrogen in SK-N-SH neuroblastoma cell cultures

Estrogen has been considered to be a neuroprotectant and a neuromodulator in many neuronal cell lines and tissue preparations. The protective effects of estrogen may be mediated through classical estrogen receptors (ERs), or may be due to its anti-oxidant properties which are independent of receptors. The current studies show that 17β-estradiol (E2) is neuroprotective against β-amyloid protein 25–35 (Aβ)-, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-, high density culture condition-, and serum deprivation-induced neuronal death in SK-N-SH human neuroblastoma cells. SK-N-SH cells express ERβ, but not ERα, as detected by Western blot analysis. Among all the insults, MPTP, high density culture and serum deprivation induce apoptotic cell death in this cell system as detected by ELISA determination of mono/oligonucleosomes and DNA laddering, while Aβ induces necrotic cell death. The protective effects of E2 are abolished by the addition of tamoxifen and ICI 182,780 in the MPTP treated cells, but not in the other models, suggesting that the effect of E2 in the MPTP model is probably associated with activation of ERβ. The addition of ICI 182,780 shows a mitogenic effect in SK-N-SH cells in the presence of E2 in control culture or in the Aβ treated groups. Also, ICI 182,780 induced expression of ERα. Collectively, the current studies suggest that E2 is neuroprotective in apoptotic and necrotic death induced by multiple insults in SK-N-SH human neuroblastoma cells. Involvement of ER is insult type dependent. ICI 182,780 is able to influence the expression of ERs, probably through upregulation of ERα when ERβ is totally antagonized.

The synergistic anti-tumour activity of ICI 182,780 in combination with docetaxel is mediated by P-glycoprotein inhibition

Docetaxel is a semi-synthetic drug that has been shown to be effective in refractory advanced breast cancer. Its main mechanism of action seems to be to block microtubule depolymerisation. In this study we investigated the interaction between docetaxel and the pure anti-oestrogen ICI 182,780 on different oestrogen receptor-negative cancer cells, some of which express the classical multi-drug resistance (MDR) phenotype. ICI 182,780 potentiated the anti-proliferative effect of docetaxel only in the MDRpositive cells. Isobolic analysis demonstrated that this chemosensitising effect was a synergism. In the same conditions where synergism was detected, cell cycle analysis demonstrated an augmentation of cells blocked at the G2/M phase of the cell cycle, suggesting that ICI 182,780 increases the activity of docetaxel. In order to test this hypothesis, we performed bcl-2 western blot analysis and demonstrated that the addition of ICI 182,780 to docetaxel induced bcl-2 phosphorylation only in MDR-positive cells. The functional inactivation of bcl-2 is probably responsible for the commitment to apoptosis, since the combined docetaxel/ICI 182,780 treatment was able to foster a massive apoptosis in MDR-bearing cells as demonstrated by morphological analysis. Our results suggest that the synergism between docetaxel and ICI 182,870 is due to a block in Pglycoprotein activity, thus determining cell cycle block, bcl-2 inactivation and apoptosis induced by

Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture

The effects of estrogen on bone are possibly mediated by several cell types. In the present study, the effect of 17β-estradiol (E 2) on osteoblast-like cells was investigated by using mouse bone marrow cultures. Bone marrow cells were harvested from the shafts of femurs of 10-week-old NMRI mice and cultured. On day 6, confluent primary cultures were trypsinized and subcultured. Under the conditions used (Keila, S., Pitaru, S., Grosskopf, A., and Wernreb, M. Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro. J Bone Miner Res 9:321–327; 1994), the bone marrow cultures showed differentiation towards the osteoblastic phenotype. This was demonstrated by the appearance of osteoblastic markers such as α1(I) collagen (COL1), alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OP), and transforming growth factor-β1 (TGF-β1), which were detected by using reverse transcriptase polymerase chain reaction (RT-PCR). Bone nodule formation, including deposition of collagen fibers and matrix mineralization, was also studied at several time points of the 3-week culture period. The effect of E 2 on the appearance of osteoblastic markers was studied by incubating cultures in the presence or absence of the hormone. The messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) was found to be expressed at all time points as demonstrated by RT-PCR. When grown with E 2, the rate of cell proliferation was increased in the early phase of cultures, but not after day 6. The addition of E 2 in subcultures resulted in an increase of levels of mRNA for COL1, ALP, OCN, OP, and TGF-β1. ALP activity was also increased. Bone nodule formation, as well as calcium contents, were significantly increased in the cultures grown in the presence of E 2. All E 2 concentrations used (0.01–10 nmol/L) were effective but the maximum response was obtained with 0.1 nmol/L E 2. Addition of the antiestrogen ICI 182,780 abolished the E 2-induced stimulation of proliferation and later an increase in ALP activity. Addition of ICI 182,780 without the hormone did not cause any changes when compared to control cultures. In conclusion, our results demonstrate that E 2 stimulates sequential differentiation of osteoblasts and increases deposition and mineralization of matrix in mouse bone marrow cultures in an estrogen receptor-dependent manner.

Clinical studies with the specific ‘pure’ antioestrogen ICI 182780

Abstract We have shown that ICI 182780 inhibits the growth of metastatic tumour cells derived from patients failing treatment with tamoxifen when grown in vitro. In this system tamoxifen stimulates growth of tumour cells which can be reversed by addition of ICI 182780 to tamoxifen in culture. In a phase I study we have demonstrated that treatment of patients for 7 days before surgical excision of the tumour resulted in marked down regulation of ER, inhibition of growth and oestrogen-induced gene expression. More recently we demonstrated that treatment of advanced breast cancer patients with ICI 182780, after tamoxifen failure, resulted in a high remission rate and prolonged periods of remission with very few side-effects. These data indicate that further clinical trials of ICI 182780 are warranted. We expect that it will show superiority over conventional endocrine therapy.

Isoproterenol and 8-bromo-cyclic adenosine monophosphate stimulate the expression of the angiotensinogen gene in opossum kidney cells

To investigate whether the expression of the renal angiotensinogen (ANG) gene is regulated by beta-adrenoceptors and the cAMP-dependent protein kinase A pathway, we introduced stably the fusion gene containing the 5'-flanking regulatory sequence of the ANG gene with a human growth hormone (hGH) gene as a reporter, pOGH (ANG N-1498/+18), into opossum kidney (OK) cells. We successfully obtained several stable transformants with a high expression of the pOGH (ANG N-1498/+18) fusion gene. One stable transformant (OK 27) that is able to maintain the expression of pOGH (ANG N-1498/+18) in culture for more than a year was used in the present study. The level of expression of the pOGH (ANG N-1498/+18) in OK 27 was evaluated by the amount of immunoreactive-hGH (IR-hGH) secreted into the culture medium. The addition of isoproterenol (10(-11) M to 10(-9) M) stimulated the expression of pOGH (ANG N-1498/+18) and increased the accumulation of intracellular cAMP. Higher concentrations of isoproterenol (that is, greater than 10(-9) M) had low or minimal effect. In contrast, the addition of 8-bromo-cAMP (8-Br-cAMP) and forskolin stimulated the expression of pOGH (ANG N-1498/+18) in a dose-dependent manner. The stimulatory effect of isoproterenol was blocked by the presence of propranolol, atenolol and ICI 118,551. The addition of ICI 118,551, however, was less effective than atenolol. Furthermore, the stimulatory effect of isoproterenol and 8-Br-cAMP on the expression of the pOGH (ANG N-1498/+18) was inhibited by the presence of Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A I and II).(ABSTRACT TRUNCATED AT 250 WORDS)

Norgestrel and gestodene stimulate breast cancer cell growth through an oestrogen receptor mediated mechanism.

There is great concern over the long-term influence of oral contraceptives on the development of breast cancer in women. Oestrogens are known to stimulate the growth of human breast cancer cells, and this laboratory has previously reported (Jeng & Jordan, 1991) that the 19-norprogestin norethindrone could stimulate the proliferation of MCF-7 human breast cancer cells. We studied the influence of the 19-norprogestins norgestrel and gestodene compared to a 'non' 19-norprogestin medroxyprogesterone acetate (MPA) on MCF-7 cell proliferation. The 19-norprogestins stimulated proliferation at a concentration of 10(-8) M, while MPA could not stimulate proliferation at concentrations as great as 3 x 10(-6) M. The stimulatory activity of the 19-norprogestins could be blocked by the antioestrogen ICI 164,384, but not by the antiprogestin RU486. Transfection studies with the reporter plasmids containing an oestrogen response element or progesterone response element (vitERE-CAT, pS2ERE-CAT, and PRE15-CAT) were performed to determine the intracellular action of norgestrel and gestodene. The 19-norprogestins stimulated the vitERE-CAT activity maximally at 10(-6) M, and this stimulation was inhibited by the addition of ICI 164,384. MPA did not stimulate vitERE-CAT activity. A single base pair alteration in the palindromic sequence of vitERE (resulting in the pS2ERE) led to a dramatic decrease in CAT expression by the 19-norprogestins, suggesting that the progestin activity required specific response element base sequencing. PRE15-CAT activity was stimulated by norgestrel, gestodene and MPA at concentrations well below growth stimulatory activity. This stimulation could be blocked by RU486. These studies suggest that the 19-norprogestins norgestrel and gestodene stimulate MCF-7 breast cancer cell growth by activating the oestrogen receptor.

Mucus hypersecretion in bronchiectasis. The role of neutrophil proteases.

To investigate the role of neutrophil proteases in the pathogenesis of mucus hypersecretion in bronchiectasis, we collected sputum samples from seven patients with bronchiectasis and measured their secretagogue activity by examining secretion of radiolabeled macromolecules by bovine airway submucosal gland cells incubated with sputum supernatants. There was marked secretagogue activity in bronchiectasis sputum, reaching a maximum of 1,963 +/- 292% (mean +/- SEM) above baseline at 1:15 dilution. Addition of ICI 200,355 (10(-5) M), a selective human neutrophil elastase inhibitor, decreased the secretory response markedly (72.53 +/- 5.89% reduction). The combination of aprotinin, an inhibitor of cathepsin G, and ICI 200,355 caused significantly more reduction in the secretory response than ICI 200,355 alone (89.12 +/- 3.8 versus 72.53 +/- 5.89% reduction, p < 0.05). We conclude that bronchiectasis sputum causes a large secretory response from tracheal submucosal glands due mostly to neutrophil proteases.