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Time course of TGF1 mRNA induction by DHT (10 9 mol/L) in NCI-H295 cells. Results are expressed as the mean SE number of TGF1 cDNA molecules per g total RNA from DHT-treated and nontreated NCI-H295 cells in at least seven independent experiments . *, P 0.01; **, P 0.05 (vs. control untreated cells).
Source publication
Sex steroid hormones have been shown to affect adrenocortical function and trophism, yet little is known about androgen action in human adrenocortical gland. In this study we examined the effects of androgens on transforming growth factor-beta1 (TGF/beta1) production by the human adrenocortical cell line, NCI-H295, which we recently demonstrated to...
Contexts in source publication
Context 1
... 24 h of treatment, DHT significantly (P 0.001) induced TGF1 mRNA expression in the NCI-H295 adre- nocortical cancer cell line at concentrations ranging from 10 12 -10 8 mol/L, with a 12-to 14-fold increase compared with the control value and in a dose-dependent fashion (Fig. 2). The time course (Fig. 3) of TGF1 gene induction by DHT (10 9 mol/L) showed that TGF1 mRNA levels increased time-dependently. DHT caused a significant increase in TGF1 mRNA expression after 4 and 12 h (P 0.05) and after 24 h (P ...
Context 2
... from seven individual experiments are expressed as the percentage over the value in untreated control cells. As The TGF1 log ratio of target to com- petitor band intensity was plotted against the log amount of target ini- tially added to each PCR. M, 100-bp DNA marker; , negative control; C, competitor only; T, target only. indicated in Fig. 5, [ 3 H]thy incorporation in NCI-H295 cells was reduced by TGF1 treatment after 24-h incubation, reaching statistical significance at 10 7 (P 0.01) and 10 6 (P 0.05) mol/L (mean inhibition, 29.3 ...
Citations
... To study the role androgen plays relative to TGF-β signaling in SMCs, we performed in vitro studies using ASC aortic SMCs derived from Fbn1 C1039G/+ mice. Because androgens increase TGF-β ligand gene expression, [24][25][26] we postulated that androgens potentiate TGF-β-induced p-Erk1/2 and p-Smad2 signaling in Fbn1 C1039G/+ males. TGF-β treatment ( Figure S1B). ...
Background
Male patients with Marfan syndrome have a higher risk of aortic events and root dilatation compared with females. The role androgens play during Marfan syndrome aneurysm development in males remains unknown. We hypothesized that androgens potentiate transforming growth factor beta induced Erk (extracellular‐signal‐regulated kinase)/Smad activation, contributing to aneurysm progression in males.
Methods and Results
Aortic diameters in Fbn1 C1039G/+ and littermate wild‐type controls were measured at ages 6, 8, 12, and 16 weeks. Fbn1 C1039G/+ males were treated with (1) flutamide (androgen receptor blocker) or (2) vehicle control from age 6 to 16 weeks and then euthanized. p‐Erk1/2, p‐Smad2, and matrix metalloproteinase (MMP) activity were measured in ascending/aortic root and descending aorta specimens. Fbn1 C1039G/+ male and female ascending/aortic root‐derived smooth muscle cells were utilized in vitro to measure Erk/Smad activation and MMP‐2 activity following dihydrotestosterone, flutamide or transforming growth factor beta 1 treatment. Fbn1 C1039G/+ males have increased aneurysm growth. p‐Erk1/2 and p‐Smad2 were elevated in ascending/aortic root specimens at age 16 weeks. Corresponding with enhanced Erk/Smad signaling, MMP‐2 activity was higher in Fbn1 C1039G/+ males. In vitro smooth muscle cell studies revealed that dihydrotestosterone potentiates transforming growth factor beta‐induced Erk/Smad activation and MMP‐2 activity, which is reversed by flutamide treatment. Finally, in vivo flutamide treatment reduced aneurysm growth via p‐Erk1/2 and p‐Smad2 reduction in Fbn1 C1039G/+ males.
Conclusions
Fbn1 C1039G/+ males have enhanced aneurysm growth compared with females associated with enhanced p‐Erk1/2 and p‐Smad2 activation. Mechanistically, in vitro smooth muscle cell studies suggested that dihydrotestosterone potentiates transforming growth factor beta induced Erk/Smad activation. As biological proof of concept, flutamide treatment attenuated aneurysm growth and p‐Erk1/2 and p‐Smad2 signaling in Fbn1 C1039G/+ males.
... The modulation of TGF-ˇ1 gene expression by androgenic substances has already been reported in the literature. In mammals, the androgen dihydrotestosterone increased the mRNA expression of the gene encoding TGF-1 (Zatelli et al., 1999). In male gilthead seabream, T implants increased the mRNA expression level of the gene encoding this cytokine in gonad of fish implanted for 7 and 28 days and in head-kidney of fish implanted for 21 days (Sánchez-Hernández et al., 2013). ...
... The NCI-H295 and the SW13 human ACC cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). NCI-H295 cells were cultured as previously described [17,18]. SW13 cells were maintained in D-MEM High Glucose, with 10 % FBS. ...
Mitotane is currently employed as adjuvant therapy as well as in the medical treatment of adrenocor-tical carcinoma (ACC), alone or in combination with chemotherapeutic agents. It was previously demonstrated that mitotane potentiates chemotherapeutic drugs cytotox-icity in cancer cells displaying chemoresistance due to P-glycoprotein (P-gp), an efflux pump involved in cancer multidrug resistance. The majority of ACC expresses high levels of P-gp and is highly chemoresistent. The aim of our study was to explore in vitro whether mitotane, at con-centrations lower than those currently reached in vivo, may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp. NCI-H295 and SW13 cell lines as well as 4 adrenocortical neoplasia primary cultures were treated with mitotane and doxorubicin, and cell viability was measured by MTT assay. P-gp activity was measured by calcein and P-gp-Glo assays. P-gp expression was evaluated by Western blot. We found that very low mitotane concentrations sensitize ACC cells to the cytotoxic effects of doxorubicin, depending on P-gp expression. In addition, mitotane directly inhibits P-gp detoxifying function, allowing doxorubicin cytotoxic activity. These data provide the basis for the greater effi-cacy of combination therapy (mitotane plus chemothera-peutic drugs) on ACC patients. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.
... Total RNA was reverse transcribed with random hexamers using the SuperScript Preamplification System for First Strand cDNA Synthesis (Life Technologies, Inc., Monza, Italy). Reverse transcription (RT) reaction was carried out in the Minicycler (MJ Research, Inc., Watertown, MA) as described previously [24]. ...
... Interaction of AR protein is known to be dependent on tissue and promoter context and a decreased amount of AR protein (long CAGn) with low transcriptional activity in the cell would increase the breast cancer risk. Several studies have observed an association between increasing AR CAG repeat length and a linear decrease in AR transactivation activity (Choong et al.1996) [6,262728293031. Shorter alleles of the AR gene would be associated with a better response to circulating androgens, possibly resulting in better " repression " of breast cancer development and/or progression. ...
Considerably little is known about the biological role and clinical significance of androgen receptor expression in breast cancer. The objectives of this study were to characterize AR-CAG repeat genotypes in a cohort of women with breast cancer and to determine the influence of AR on response to neoadjuvant chemotherapy and clinical outcome.
Genotyping of the AR CAG repeat region was done on 70 patients and 80 healthy aged- matched female controls. To assess response to NACT, tissue samples from 30 LABC cases were evaluated quantitatively by real time for AR mRNA expression. The clinical response was correlated with both the pre and post chemotherapy AR expression. The CAG alleles did not show differences between cases and controls when the mean of short, long and average length of both CAG alleles was considered. However, analysis when done defining short allele as CAGn < 20 (AR1) and the long as CAGn ≥ 20 (AR2), risk was found associated with AR2 allele with marginal significance (P = 0.09). Stratification by age of onset, FH, stage, grade ER and AR status failed to reveal any association with breast cancer risk. Genotype carriers with ≥ 20 CAGn showed decrease of AR mRNA expression although significance could not be established (P = 0.47). Tumours in responders had the higher AR mRNA expression levels in pre neo-adjuvant chemotherapy condition (p < 0.02) which got reduced after neoadjuvant chemotherapy and the difference was found to be significant (P = 0.014).
Although, expansion of the CAGn in the AR gene doesn't show any major effect on breast cancer risk, patients with positive AR expression, pre neoadjuvant chemotherapy, were found to be good responders and a decrease in mRNA level of AR gene related to the chemotherapy-induced apoptosis could serve as an important independent predictor of response to NACT.
... TGFh can act either as a tumor suppressor or promote cancer progression. In human adrenocortical cells, TGFh treatment decreases cell proliferation (45). Here, TGFh altered the cell cycle and induced apoptosis in adrenocortical cells (this article and ref. 30). ...
The cyclic AMP signaling pathway can be altered at multiple levels in endocrine tumors. Its central component is the protein kinase A (PKA). Carney complex (CNC) is a hereditary multiple neoplasia syndrome resulting from inactivating mutations of the gene encoding the PKA type I alpha regulatory subunit (PRKAR1A). Primary pigmented nodular adrenocortical disease is the most frequent endocrine tumor of CNC. Transforming growth factor beta (TGFbeta) regulates adrenal cortex physiology and signals through SMAD2/3. We used an interference approach to test the effects of PRKAR1A inactivation on PKA and TGFbeta pathways and on apoptosis in adrenocortical cells. PRKAR1A silencing stimulates PKA activity and increases transcriptional activity of a PKA reporter construct and expression of the endogenous PKA target, NR4A2, under basal conditions or after forskolin stimulation. PRKAR1A inactivation also decreased SMAD3 mRNA and protein levels via PKA, altering the cellular response to TGFbeta. SMAD3 expression was also inhibited by adrenocorticorticotropic hormone in the mouse adrenal gland and by forskolin in H295R cells. TGFbeta stimulates apoptosis in H295R cells, and this effect was counteracted by PRKAR1A inactivation. PRKAR1A silencing decreased the percentage of apoptotic cells and the cleavage of apoptosis mediators [caspase-3, poly(ADP-ribose) polymerase, and lamin A/C]. Inactivating mutations of PRKAR1A observed in adrenocortical tumors alter SMAD3, leading to resistance to TGFbeta-induced apoptosis. This cross-talk between the PKA and the TGFbeta signaling pathways reveals a new mechanism of endocrine tumorigenesis.
... The medium was aspirated and replaced with medium without Nu-serum (BD Biosciences, San Jose, CA) and ITS+ Premix (BD Biosciences), and the various treatments stated below. The cells were then incubated for 24 hours at which time the medium was aspirated from the wells, and both the cells and medium were frozen at −80° C. Our time course experiments (4,8,24, 48, 72 hr) revealed near maximal steroidogenesis and mRNA at 24 hours (data not shown) as noted by other investigators (29)(30). ...
To determine changes in adrenal androgen (AA) production, and transcription of dehydroepiandrosterone (DHEA) sulfotransferase (SULT2A1) in the NCI-H295R human adrenocortical cell line in response to insulin and testosterone, an environment mimicking the polycystic ovary syndrome state.
In vitro experiment using NCI-H295R adrenocortical cell lines.
Academic medical center.
NCI-H295R human adrenocortical cell line.
The transcriptional activity of SULT2A1 and adrenal steroid production was quantified after exposure to various treatments (e.g., forskolin, insulin, testosterone, and combinations thereof).
Quantification of mRNA for DHEA sulfotransferase (SULT2A1) by real-time reverse transcription-polymerase chain reaction and measurement of steroid production by radioimmunoassay.
Testosterone decreased DHEAS and cortisol, and increased DHEA secretion by H295R cells; the inhibitory effects of testosterone on DHEAS and cortisol production were augmented by insulin. There was a trend toward an increase in the transcription of SULT2A1 by insulin and testosterone.
Testosterone and insulin appear to be modulators of adrenal androgen production in this human adrenocortical cell model. These results suggest that testosterone may augment DHEA secretion in the human adrenal, although they do not support the role of this sex steroid or insulin on the elevated DHEAS levels frequently observed in polycystic ovary syndrome.
... Upregulation of prostate-specific antigen (PSA), often a hallmark of prostate cancer development, also inhibits the apoptotic ability of TGF-b (see Kang et al., 2001). Interestingly, androgens negatively regulate the expression of both TGF-b and its receptors, thus providing a molecular basis for the marked enhancement of TGF-b-induced prostate epithelial apoptosis following androgen ablation (see Wikstrom et al., 1999;Zatelli et al., 2000;Zhu & Kyprianou, 2005). There appears to be a considerably active crosstalk between the TGF-b signalling pathway and the androgen signalling axis, the degradation of which may functionally contribute to tumorigenesis (see Guo & Kyprianou, 1999;Gerdes et al., 2004;Zhu & Kyprianou, 2005). ...
The intricate balance maintained between cell growth and proliferation factors and apoptosis-inducing factors is fundamental to the regulation of prostate growth. Disruptions in this homeostasis often trigger the loss of apoptosis and the over-expression of factors promoting cell survival and proliferation, inevitably leading to tumorigenesis and cancer. Deregulation of prostate growth during prostate cancer development and progression is characterized by apoptotic evasion, uncontrolled proliferation, and increased invasive potential. Thus, in advanced stages of disease progression, surviving prostate tumour cells acquire the ability to migrate and invade heterotopic tissues, with the bone and lymph nodes being the most common sites for human prostate cancer metastasis. The challenges in the implementation of effective therapeutic strategies for the treatment of advanced metastatic prostate cancer reflect the multidimensional nature and functional significance of antiapoptotic pathways in the emergence of therapeutic resistance of prostate tumours. In this chapter, we discuss the current understanding of the molecular mechanisms governing growth factor signalling pathways with often overlapping functions that contribute to loss of apoptosis control and activation of cell proliferation towards aggressive prostate tumorigenic growth and metastatic behaviour. While a full understanding of the prosurvival characteristics of these growth factor pathways is still evolving, the impact that growth factors such a epidermal growth factor and transforming growth factor-β can be recognized by the vigorous attempts at therapeutic targeting of their key signalling steps.
British Journal of Pharmacology (2006) 147, S144–S152. doi:10.1038/sj.bjp.0706635
... They were also maintained in the culture medium throughout the experiment at a lower concentration as indicated by preincubation and experiment: mouse anti-human ␣ 1  1 integrin (clone SR84, BD Biosciences Pharmingen, 10 and 2 g/ml) (Rettig et al., 1984;Setty et al., 1998), mouse anti-human ␣ 2  1 (clone BHA2.1; Chemicon; 20 and 10 g/ml) (Li et al., 2003) and rabbit anti-human TGF- 1 (Promega; 0.8 g/ml) (Zatelli et al., 2000). ...
The differentiation of fibroblasts to contractile myofibroblasts, which is characterized by de novo expression of alpha-smooth muscle actin (alpha-SMA), is crucial for wound healing and a hallmark of tissue scarring and fibrosis. These processes often follow inflammatory events, particularly in soft tissues such as skin, lung and liver. Although inflammatory cells and damaged epithelium can release transforming growth factor beta1 (TGF-beta1), which largely mediates myofibroblast differentiation, the biophysical environment of inflammation and tissue regeneration, namely increased interstitial flow owing to vessel hyperpermeability and/or angiogenesis, may also play a role. We demonstrate that low levels of interstitial (3D) flow induce fibroblast-to-myofibroblast differentiation as well as collagen alignment and fibroblast proliferation, all in the absence of exogenous mediators. These effects were associated with TGF-beta1 induction, and could be eliminated with TGF-beta1 blocking antibodies. Furthermore, alpha1beta1 integrin was seen to play an important role in the specific response to flow, as its inhibition prevented fibroblast differentiation and subsequent collagen alignment but did not block their ability to contract the gel in a separate floating gel assay. This study suggests that the biophysical environment that often precedes fibrosis, such as swelling, increased microvascular permeability and increased lymphatic drainage--all which involve interstitial fluid flow--may itself play an important role in fibrogenesis.
... To evaluate MDR1 and COX-2 expression in TT cells, RT-PCR analysis was performed. Total RNA was isolated from subconfluent TT cells and from pulverized tissues by using TRIzol reagent (Invitrogen, Milano, Italy), according to the protocol of the manufacturer, and subjected to RT with random hexamers, as described previously (10,11). To prevent DNA contamination, RNA was treated with ribonuclease-free deoxyribonuclease (Promega, Milano, Italy). ...
Medullary thyroid carcinoma (MTC) is a highly chemoresistant malignant neoplasia deriving from parafollicular C cells. Chemotherapy failure has been ascribed, at least in part, to the overexpression by MTC of the multidrug resistance 1 (MDR1) gene, encoding a transmembrane glycoprotein [permeability glycoprotein (P-gp)] that antagonizes intracellular accumulation of cytotoxic agents. P-gp expression and function in a rat model have been demonstrated to depend on cyclooxygenase (COX)-2 isoform levels, which are found elevated in many human cancers. The aim of our study was to investigate the role of the COX-2 pathway in modulating chemoresistance.
We investigated P-gp and COX-2 expression and then evaluated the sensitizing effects of COX-2 inhibitors on the cytotoxic effects of doxorubicin in the presence or in the absence of prostaglandin E2 in primary cultures and in a human MTC cell line, TT. Moreover, P-gp function has been studied. Our data show that TT cells express both MDR1 and COX-2 and that rofecoxib, a selective COX-2 inhibitor, sensitizes TT cells to the cytotoxic effects of doxorubicin, reducing P-gp expression and function.
Our data suggest that these effects are mediated by a mechanism not involving the generation of prostaglandin E2, possibly implicating the synthesis of other COX-2 products.
