Hormone-dependent Cell Lines Research Articles

Flavonoid Calycopterin Induces Apoptosis in Human Prostate Cancer Cells In-vitro.

Prostate cancer is enumerated as one of the most prevalent cancers in men, with a mortality rate of 18%. Chemotherapy is considered as a common strategy for cancer treatment; however, toxic side effects and drug resistance associated with chemotherapy are major drawbacks with this approach. It is well known that a diet rich in flavonoids can reduce the incidence of many types of cancer in a significant manner, and it was proved that methoxy flavones have greater bioavailability compared to the nonmethylated ones. Calycopterin is a tetramethoxy flavone which was demonstrated to have anti-proliferative effects on colon, gastric, and osteosarcoma cancer cells. Therefore, in the current study, we have evaluated the apoptotic effects of this flavonoid on two prostate cancer cell lines in-vitro. The MTT assay revealed that after 48 h treatment with this flavonoid, cell viability reduced to 50% compared to the control group. However, calycopterin treatment of healthy HUVEC did not cause any significant reduction in cell viability. Moreover, the clonogenic assay demonstrated that after 14 days, colony size and numbers reduced significantly in calycopterin treated cells. In addition, the percentage of the sub-G1 population in calycopterin-treated cells augmented significantly compared to untreated group. Also, calycopterin-treated cells demonstrated shiny condensed nuclei with fragmented DNA indicative of apoptosis. Finally, a significant reduction in the migration ability was observed in both lines subjected to calycopterin after 48 h. To conclude, our results demonstrated the apoptotic and anti-metastatic effects of calycopterin in both hormone-dependent and independent prostate cancer cell lines.

Differential expression of microRNA-218 in hormone-dependent and hormone-independent prostate cancer cell lines

Objective To verify and analyze the expression of microRNA-218 in hormone-dependent and hormone-independent prostate cancer cell lines. Methods Specific SYBR Green quantitative real-time polymerase chain reaction(RT-PCR) was used to detect the relative expression of microRNA-218 in hormone-dependent prostate cancer LNCaP cell line and hormone-independent prostate cancer C4-2, PC-3 and DU-145 cell lines, and the differences were verified and analyzed. Results The expression of MicroRNA-218 was not different in C4-type, PC-3 and DU-145 three hormone-independent prostate cancer cell lines(P>0.05), but was significantly higher than that in hormone-dependent prostate cancer cell line LNCaP(P<0.05). Conclusions MicroRNA-218 is involved in the progression from steroid-dependent to hormone-independent prostate cancer and is expected to become a biomarker for predicting the progression of prostate cancer. Key words: Prostatic Neoplasms; Cell Line; MicroRNAs

Abstract 2580: Investigation of plasma exosomal miRNA as a biomarker and its potential function in prostate cancer castration resistant development

Abstract Prostate cancer (PCa) ranks as the second most frequent cancer and the fifth leading cause of cancer death in men worldwide. Androgen-deprivation therapy is the principal treatment for locally advanced and metastatic disease. Although a majority of patients initially respond well to ADT, most will progress to castration-resistant prostate cancer (CRPC), which contributes to the majority of PCa deaths. Exosomes are small vesicles that contain numerous molecular constituents, including lipids, proteins, RNAs and DNAs, and can mediate cell-cell communications. The aim of this study is to identify plasma exosomal miRNAs correlated with CRPC development, which may serve as a biomarker to monitor disease status and may reveal new role of exosome in CRPC development. Next-generation sequencing (NGS) of plasma exosomal miRNAs was performed in 24 treatment-naive PCa patients and 24 CPRC patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate candidate miRNAs in 108 treatment-naive PCa patients and 42 CRPC patients and a group of 38 non-CRPC patients under hormone therapy (treated non-CRPC). To study the miRNA function, hormone-dependent PCa cell line LNCaP was cultured in hormone-depleted media. MiRNA was overexpressed by transient transfection of miRNA mimic. Post transfection, transwell assays and cell viability assays were performed to determine the effect of miRNA on cell migration and proliferation. RNA NGS generated an average of approximately 5-million reads per sample and identified differentially expressed miRNAs. QRT-PCR validation showed six miRNAs were significantly differentially expressed between treatment-naïve PCa and CRPC (p-value&amp;lt;0.05). It also showed that five of these miRNAs were differently expressed in CRPC compared treated non-CRPC patients (p-value&amp;lt;0.001). When receiver operating characteristic curve was applied, one of the exosomal miRNAs, miR-423-3p, achieved area under the curve (AUC) = 0.784 when predicting CRPC from treatment-naïve PCa and AUC = 0.883 when predicting CRPC from treated non-CRPC. Overexpression of miR-423-3p had no effect on LNCaP proliferation, but significantly increased cell migration. This study demonstrated that plasma exosomal miRNAs play important roles in CRPC development and may serve as a biomarker for CRPC occurrence prediction. Citation Format: Tianyu Guo, Yang Wang, Xueying Mao, Lei Xu, Jacek Marzec, Edwina Burke, Glenda Scandura, Elzbieta Stankiewicz, Caitlin R. Davies, John Hines, Greg Shaw, Jonathan Shamash, Daniel Berney, Prabhakar Rajan, Karen Tipples, Alistair Grey, Yong-jie Lu. Investigation of plasma exosomal miRNA as a biomarker and its potential function in prostate cancer castration resistant development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2580.

Co-targeting the HER and IGF/insulin receptor axis in breast cancer, with triple targeting with endocrine therapy for hormone-sensitive disease

Interactions between HER2, estrogen receptor (ER), and insulin-like growth factor I receptor (IGF1R) are implicated in resistance to monotherapies targeting these receptors. We have previously shown in pre-clinical studies synergistic anti-tumor effects for co-targeting each pairwise combination of HER2, IGF1R, and ER. Strikingly, synergy for HER2/IGF1R targeting occurred not only in a HER2+model, but also in a HER2-normal model. The purpose of the current study was therefore to determine the generalizability of synergistic anti-tumor effects of co-targeting HER2/IGF1R, the anti-tumor activity of triple-targeting HER2/IGF1R/ER in hormone-dependent cell lines, and the effect of using the multi-targeting drugs neratinib (pan-HER) and BMS-754807 (dual IGF1R/insulin receptor). Proliferation and apoptosis assays were performed in a large panel of cell lines representing varying receptor expression levels. Mechanistic effects were studied using phospho-protein immunoblotting. Analyses of drug interaction effects were performed using linear mixed-effects regression models. Enhanced anti-proliferative effects of HER/IGF-insulin co-targeting were seen in most, though not all, cell lines, including HER2-normal lines. For ER+lines, triple targeting with inclusion of anti-estrogen generally resulted in the greatest anti-tumor effects. Double or triple targeting generally resulted in marked increases in apoptosis in the sensitive lines. Mechanistic studies demonstrated that the synergy between drugs was correlated with maximal inhibition of Akt and ERK pathway signaling. Dual HER/IGF-insulin targeting, and triple targeting with inclusion of anti-estrogen drugs, shows striking anti-tumor activity across breast cancer types, and drugs with broader receptor specificity may be more effective than single receptor selective drugs, particularly for ER- cells.

Restoration of the cellular secretory milieu overrides androgen dependence of in vivo generated castration resistant prostate cancer cells overexpressing the androgen receptor

It is believed that growth of castration resistant prostate cancer (CRPC) cells is enabled by sensitization to minimal residual post-castrate androgen due to overexpression of the androgen receptor (AR). Evidence is derived from androgen-induced colony formation in the absence of cell-secreted factors or from studies involving forced AR overexpression in hormone-dependent cells. On the other hand, standard cell line models established from CRPC patient tumors (e.g., LNCaP and VCaP) are hormone-dependent and require selection pressure in castrated mice to re-emerge as CRPC cells and the resulting tumors then tend to be insensitive to the androgen antagonist enzalutamide. Therefore, we examined established CRPC model cells produced by castration of mice bearing hormone-dependent cell line xenografts including CRPC cells overexpressing full-length AR (C4-2) or co-expressing wtAR and splice-variant AR-V7 that is incapable of ligand binding (22Rv1). In standard colony formation assays, C4-2 cells were shown to be androgen-dependent and sensitive to enzalutamide whereas 22Rv1 cells were incapable of colony formation under identical conditions. However, both C4-2 and 22Rv1 cells formed colonies in conditioned media derived from the same cells or from HEK293 fibroblasts that were proven to lack androgenic activity. This effect was (i) not enhanced by androgen, (ii) insensitive to enzalutamide, (iii) dependent on AR (in C4-2) and on AR-V7 and wtAR (in 22Rv1) and (iv) sensitive to inhibitors of several signaling pathways, similar to androgen-stimulation. Therefore, during progression to CRPC in vivo, coordinate cellular changes accompanying overexpression of AR may enable cooperation between hormone-independent activity of AR and actions of cellular secretory factors to completely override androgen-dependence and sensitivity to drugs targeting hormonal factors.

Abstract 3576: USP22 modulates AR activity to critically regulate prostate cancer progression.

Abstract Recently, the deubiquitylase Ubiquitin-specific protease 22 (USP22) was identified as a member of the 11 gene ‘death-from-cancer’ signature, which can predict poor response to therapy and/or propensity toward metastasis of multiple tumor types, including breast and prostate. However, the mechanism for how USP22 impacts cancer is currently unknown. Our new data implicate a specific role for USP22 in hormone-dependent cancers, especially prostate cancer (PCa) where USP22 expression is elevated in primary and metastatic PCa. USP22 has an established role in regulating oncogenic c-Myc activity as it is recruited by c-Myc for transcriptional activation through deubiquitylation of histones, and is necessary for c-Myc-mediated cell transformation. Similarly, our data in PCa cells demonstrates that USP22 controls c-Myc activity but does not affect expression levels. Additional data has also identified USP22 as a critical effector of androgen receptor (AR) levels and output. This is of great clinical relevance since PCa is intricately dependent on AR signaling for disease initiation and progression to castrate resistant disease (CRPC). First, USP22 overexpression in hormone-dependent and CRPC cell lines increased AR protein expression and activity, but did not perturb transcript. Second, in the absence of ligand, USP22 upregulation resulted in enhanced AR recruitment to target genes, as determined by chromatin immunoprecipitation, which corresponded with significant enhancement of cell proliferation and BrdU incorporation. Third, USP22 depletion resulted in attenuated AR activity and caused a significant reduction in AR protein levels that could be attributed to protein stability. Importantly, in the CRPC setting, USP22 depletion dramatically inhibited in vivo tumor growth while expression correlated with decreased time for tumor regression. In sum, the data suggest that USP22 upregulation is sufficient for aberrant c-Myc activity, altered AR expression and activity, and establishment of a CRPC phenotype. We propose that modulation of UPS22 expression and/or activity may present a novel platform to achieve combinatorial suppression of AR and c-Myc function, and therefore could be potentially developed as a novel approach for treatment of PCa. Citation Format: Randy Schrecengost, Jeffry Dean, Tim Stanek, Ruth Birbe, Jessica Hicks, Tapio Visakorpi, Angelo DeMarzo, Steve McMahon, Karen Knudsen. USP22 modulates AR activity to critically regulate prostate cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3576. doi:10.1158/1538-7445.AM2013-3576

Correlation between erythropoietin receptor(s) and estrogen and progesterone receptor expression in different breast cancer cell lines

Erythropoietin (EPO) receptor (EPOR) expression in breast cancer has been shown to correlate with the expression of estrogen receptor (ESR) and progesterone receptor (PGR) and to be associated with the response to tamoxifen in ESR+/PGR+ tumors but not in ESR- tumors. In addition, the correlation between EPOR and Gprotein-coupled estrogen receptor1 [GPER; also known as Gprotein-coupled receptor30 (GPR30)] has been reported, suggesting the prognostic potential of EPOR expression. Moreover, the involvement of colony stimulating factor2 receptor, β, low‑affinity (CSF2RB) and ephrin type-B receptor4 (EPHB4) as EPOR potential receptor partners in cancer has been indicated. This study analyzed the correlation between the expression of genes for EPO, EPOR, CSF2RB, EPHB4, ESR, PGR and GPER in the MCF-7, MDA-MB-361, T-47D, MDA-MB-231, Hs578Bst, SKBR3, MCF-10A and Hs578T cell lines. The cell lines were also treated with recombinant human EPO (rHuEPO) in order to determine its ability to activate the Jak/STAT5, MAPK and PI3K signaling pathways and modify cell growth characteristics. Expression analysis stratified the cell lines in 2main clusters, hormone-dependent cell lines expressing ESR and PGR and a hormone-independent cluster. A significant correlation was observed between the expression levels of ESR and PGR and their expression was also associated with that of GPER. Furthermore, the expression of GPER was associated with that of EPOR, suggesting the connection between this orphan Gprotein and EPO signaling. A negative correlation between EPOR and CSF2RB expression was observed, questioning the involvement of these tworeceptors in the hetero-receptor formation. rHuEPO treatment only influenced the hormone-independent cell lines, since only the MDA-MB-231, SKBR3 and Hs578T cells responded to the treatment. The correlation between the expression of the analyzed receptors suggests that the receptors may interact in order to activate signaling pathways or to evade their inhibition. Therefore, breast cancer classification upon ESR, PGR and human epidermal growth factor receptor2 (HER2) may not be sufficient for the selection of suitable treatment protocol. The expression of EPOR, GPER and EPHB4 may be considered as additional classification factors.

HNF1b is involved in prostate cancer risk via modulating androgenic hormone effects and coordination with other genes

Prostate cancer is one of the most commonly diagnosed male malignancies. Genome wide association studies have revealed HNF1b to be a major risk gene for prostate cancer susceptibility. We examined the mechanisms of involvement of HNF1b in prostate cancer development. We integrated data from Gene Expression Omnibus prostate cancer genes from the Dragon Database of Genes Implicated in Prostate Cancer, and used meta-analysis data to generate a panel of HNF1b-associated prostate cancer risk genes. An RT-PCR was used to assess expression levels in DU145, PC3, LNCaP, and RWEP-1 cells. Twelve genes (BAG1, DDR1, ERBB4, ESR1, HSPD1, IGFBP2, IGFBP5, NR4A1, PAWR, PIK3CG, RAP2A, and TPD52) were found to be associated with both HNF1b and prostate cancer risk. Six of them (BAG1, ERBB4, ESR1, HSPD1, NR4A1, and PIK3CG) were mapped to the KEGG pathway, and submitted to further gene expression assessment. HNF1b, NR4A1, and HSPD1 were found to be highly expressed in the LNCaP androgenic hormone-dependent cell line. Compared to expression levels in wild-type prostate cancer cells, NR4A1, HSPD1, ERBB4, and ESR1 expression levels were also found to be significantly increased in the HNF1b-transfected cells. We conclude that the mechanism of action of HNF1b in prostate cancer involves modulation of the association between androgenic hormone and prostate cancer cells. Gene-gene interaction and coordination should be taken into account to determine relationships between specific loci and diseases.

Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q

The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10 μg/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line.

Targeting steroid hormone receptors for ubiquitination and degradation in breast and prostate cancer

Proteolysis targeting chimeric molecules (Protacs) target proteins for destruction by exploiting the ubiquitin-dependent proteolytic system of eukaryotic cells. We designed two Protacs that contain the peptide 'degron' from hypoxia-inducible factor-1alpha, which binds to the Von-Hippel-Lindau (VHL) E3 ubiquitin ligase complex, linked to either dihydroxytestosterone that targets the androgen receptor (AR; Protac-A), or linked to estradiol (E2) that targets the estrogen receptor-alpha (ERalpha; Protac-B). We hypothesized that these Protacs would recruit hormone receptors to the VHL E3 ligase complex, resulting in the degradation of receptors, and decreased proliferation of hormone-dependent cell lines. Treatment of estrogen-dependent breast cancer cells with Protac-B induced the degradation of ERalpha in a proteasome-dependent manner. Protac-B inhibited the proliferation of ERalpha-dependent breast cancer cells by inducing G(1) arrest, inhibition of retinoblastoma phosphorylation and decreasing expression of cyclin D1, progesterone receptors A and B. Protac-B treatment did not affect the proliferation of estrogen-independent breast cancer cells that lacked ERalpha expression. Similarly, Protac-A treatment of androgen-dependent prostate cancer cells induced G(1) arrest but did not affect cells that do not express AR. Our results suggest that Protacs specifically inhibit the proliferation of hormone-dependent breast and prostate cancer cells through degradation of the ERalpha and AR, respectively.

The proliferative effects of 5-androstene-3β,17β-diol and 5α-dihydrotestosterone on cell cycle analysis and cell proliferation in MCF7, T47D and MDAMB231 breast cancer cell lines

Epidemiological studies suggest that precursor steroids are implicated in the aetiology of breast cancer. However, our understanding of the role of precursor steroids in breast cancer is complicated by fact that there are many precursor steroids, which are metabolically inter-related and have divergent proliferative activities on the growth of breast cancer cell lines. In this study the proliferative affects of 5α-dihydrotestosterone and 5-androstene-3β,17β-diol, which may be considered true metabolites acting at a tissue level, on MCF7, T47D and MDAMB231 breast cancer cell lines have been examined by a flow cytometric technique. DNA cell cycle analysis demonstrates that 5-androstene-3β,17β-diol stimulates the proliferation of hormone-dependent cell lines at physiological levels by an oestrogen receptor mediated mechanism whereas 5α-dihydrotestosterone does not affect the proliferation of MCF7 and T47D cell lines at physiological levels over short (48 h) incubations. Both 5α-dihydrotestosterone and 5-androstene-3β,17β-diol stimulate proliferation of hormone-dependent cell lines at pharmacological levels via and interaction with the oestrogen receptor. In long (6–9 days) incubations both 5α-dihydrotestosterone and 5-androstene-3β,17β-diol inhibit the 17β-oestradiol induced proliferation of MCF7 and T47D cell lines, however, 5α-dihydrotestosterone inhibits while 5-androstene-3β,17β-diol stimulates basal proliferation. These cell line studies suggest a model for the role of precursor steroids in pre- and postmenopausal breast cancer.

The first titanocenyl dichloride moiety vectorised by a selective estrogen receptor modulator (SERM). Synthesis and preliminary biochemical behaviour

The synthesis of 4′, a titanocene derivative of the anticancer drug tamoxifen is presented together with its biochemical properties. Compound 4′ is prepared by a McMurry coupling followed by a ligand exchange. Compound 4′ reveals an unexpected proliferative effect on the hormone-dependent cell line MCF7. Surprisingly this effect is also observed with Cp 2TiCl 2 alone.

Characterization of the role of IL-6 in the progression of prostate cancer.

We recently identified IL-6, a pleiotropic cytokine implicated in the neoplastic process of a variety of neoplasms, as a mediator of prostate cancer morbidity. In the present study, we investigated the expression of members of the IL-6 supergene family and related cytokines and the potential role of IL-6 in prostate cancer growth regulation. Five established human prostate cancer cell lines were screened by ELISA for production of granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), tumor necrosis factor (TNF), interleukin-1 (IL-1), and granulocyte macrophage colony-stimulating factor (GM-CSF). Expression of the ligand-binding component of the IL-6 receptor, IL-6Rp80, was evaluated by ELISA and RT-PCR. Sequential immunoprecipitation and immunoblotting were performed to assay for expression of the signal-transducing component of the IL-6 receptor, gp130. The effects of IL-6 on cell growth were assessed by MTT assays. The three hormone-refractory cell lines, DU-145, TSU, and PC-3, secreted distinct combinations of cytokines (DU-145: IL-6, GM-CSF; TSU: IL-6, LIF; PC-3: IL-6, G-CSF, LIF, IL-1, GM-CSF), each uniformly expressing IL-6. In contrast, neither of the two hormone-dependent cell lines, LNCaP-ATCC and LNCaP-GW, secreted significant quantities of any of the cytokines analyzed. None of the cell lines secreted detectable quantities of OSM, CNTF, or TNF. All cell lines, irrespective of hormone status, expressed both Il-6Rp80 and gp130. Addition of IL-6 in vitro inhibited growth of hormone-dependent cells, but had no effect on hormone-refractory lines. Anti-IL-6 neutralizing antibody inhibited growth of hormone-refractory cells. IL-6 appears to undergo a functional transition from paracrine growth inhibitor to autocrine growth stimulator during progression of prostate cancer to the hormone-refractory phenotype.

Retinoic acid suppresses insulin-induced cell growth and cyclin D1 gene expression in human breast cancer cells.

We examined the effects of all-trans retinoic acid (RA) on the insulin-induced cell growth, cell cycle progression and cyclin D1 gene expression in breast cancer cells. RA exerted a dose-dependent growth inhibition on insulin-induced proliferation in T47D and MCF-7 hormone-dependent cell lines, whereas MDA-MB231 hormone-independent cells were not affected. The RA antagonism of insulin growth effect was associated with an inhibition of cell cycle progression and a suppression of insulin-induced cyclin D1 mRNA. The effect of RA on cyclin D1 mRNA was dose-dependent and was observed within 5 h of treatment when insulin response was maximal.

Effects of danazol and progesterone on sex hormone-binding globulin mRNA expression in human endometrial cancer cell line Ishikawa

To ascertain one of the biological effects of danazol and progesterone on the uterine endometrial cancer cell line, Ishikawa, we investigated the effects of these steroids on sex hormone-binding globulin (SHBG) mRNA expression by competitive reverse transcription-polymerase chain reaction-Southern blot analysis (RT-PCR-SBA). Estradiol-17β (E2) in any concentration given did not exert any significant effect on the expression of SHBG mRNA. Danazol and progesterone significantly ( P < 0.05) suppressed the expression of SHBG mRNA dose-dependently starting at a concentration of 10 −6 and 10 −8 M, respectively. Progesterone, in a low concentration (10 −10 M) with E2 (10 −8 M), significantly ( P < 0.05) increased the expression of SHBG mRNA, but danazol did not. In contrast, danazol and progesterone in high concentrations (10 −6 to 10 −5 M) with E2 (10 −8 M) significantly ( P < 0.05) suppressed its expression. The time course study showed the time-dependent decrease of SHBG mRNA level by danazol and progesterone (10 −6 M) with or without E2 (10 −8 M), except for a temporal increase by progesterone. These findings suggest that danazol and progesterone in a superphysiological milieu down-regulate the intracellular SHBG-related steroidal actions, and that progesterone in a physiological milieu with estrogen up-regulates it in a hormone-dependent cell line. A decrease of intracellular SHBG caused by high-dose danazol or progesterone might partly contribute to the abolition of the intracellular estrogen-dominant milieu, and be related to the inhibition of estrogen-dependent growth of some endometrial cancer cells.

Effect of sodium butyrate on human breast cancer cell lines

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 mM. The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G2M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.

Androgen-responsive expression and mitogenic activity of schwannoma-derived growth factor on an androgen-dependent Shionogi mouse mammary carcinoma cell line

We report here, using the androgen-dependent SC2G cell line derived from Shionogi mouse mammary carcinoma SC115, that cDNA encoding mouse homologue of the rat schwannoma-derived growth factor (SDGF) was isolated and its protein product was identified to be an autocrine growth factor which is expressed in response to androgen in this hormone-dependent cell line. The androgen-independent SC1G cells derived from SC115 were shown to express SDGF even in the absence of androgen, while Northern analysis probed with mouse mammary tumor virus (MMTV) DNA showed that the androgen inducible transcriptional machinery remains functional in this hormone independent cell line.

Recent data on estrogen sulfatases and sulfotransferases activities in human breast cancer

• Of the total number of breast cancers approx. 30–50% are hormone-dependent and estradiol is one of the main factors of cancerization. Consequently, the control of this hormone inside the cancer cell is of capital importance because it is well established that the inhibition of estradiol biosynthesis can have a positive effect on the evolution of the disease. The blockage of estradiol can be obtained by the action of anti-aromatases, anti-sulfatases, the control of the 17β-hydroxysteroid dehydrogenase activity or by the stimulation of the sulfotransferase which converted the estrogens in their sulfates. In breast cancer tissue estrone sulfate is quantitatively the most important source of estradiol. In the intact cell, estrone sulfatase activity is very intense in the hormone-dependent cell lines (e.g. MCF-7, T-47D) but very small activity is observed in the hormone-independent (e.g. MDA-MB-231, MDA-MB-436) cell lines. However, this activity became very strong after homogenization in the hormone-independent cells, suggesting the presence of repressive factor(s) for this enzyme or its sequestering in an inactive form, in the intact cells of these cell lines. • In a series of previous studies it was found that in hormone-dependent cell lines different anti-estrogens: tamoxifen and derivatives, ICI 164,384, very significantly decrease the estradiol concentration originated from estrone sulfate, and recently it was observed that Decapeptyl (D-Trp 6-gonadotropin-releasing hormone) in the presence of heparin can also decrease the conversion of estrone sulfate into estradiol. No significant effect was obtained in the presence of heparin or Decapeptyl alone. The estrone sulfatase activity can be inhibited by progesterone, the progestagen R-5020, and testosterone. In another series of recent studies the presence of very strong estrogen sulfotransferase activity has been shown in one breast cancer cell line, the MDA-MB-468. • We can conclude that: (1) the control of estradiol concentration can be carried out in the breast cancer tissue itself; (2) estrone sulfate can play an important role in the bioavailability of estradiol in the breast cancer cell; and (3) as in the case for the aromatase, the control of: the estrogen sulfatase, estrogen sulfotransferase, and 17β-hydroxysteroid dehydrogenase can be new targets for therapeutic applications in breast cancer.

Interaction between hormone-dependent and hormone-independent human breast cancer cells

We developed two different models based on in vitro co-culture of hormone-dependent and hormone-independent cell lines to simulate the cell population heterogeneity of human breast cancer tumours. Oestrogen-dependent (MCF-7, ZR 75.1) and oestrogen-independent cell lines (MDAMB-231 BT-20) were grown under serum-free conditions. Co-culture of hormone-dependent and hormone-independent cell lines resulted in an increased cell yield compared to single cell cultures carried out at the same seeding ratios. Such an increase was not affected by addition of oestradiol and single growth factors (EGF, bFGF and IGF-I). These results allow us to conclude that in a heterogeneous cell population like human breast tumours, interaction between hormone-dependent and hormone-independent cell lines may result in a complex regulation of cell growth.

Influence of culture conditions on the estrogenic cell growth stimulation of human breast cancer cells

17β-Estradiol is a potent mitogen for hormone-dependent cell lines (MCF-7, T47D and ZR 75.1). However, the degree of hormone sensitivity is very much influenced by culture conditions. In order to understand which factors modulate estrogenic effects on cell growth, four different culture conditions were used: (a) medium with dextran-coated charcoal-treated fetal calf serum (DCC-FCS); (b) medium with dextran-coated charcoal-treated growth factor-inactivated serum (DCC-FCSd); (c) serum-free medium, after a 24-h incubation with serum to allow cell attachment; and (d) serum-free medium on collagen IV-treated plates. In all cell lines the highest cell growth stimulation was achieved when estradiol was added in the presence of 5% DCC-FCS, whereas reducing or removing serum from the culture medium resulted in a decrease in cell proliferation stimulation. We postulate that serum contains some still unknown components able to modulate the degres of estrogenic action in endocrine-dependent breast cancer cell lines.