Neuropeptides B and W enhance the growth of human adrenocortical carcinoma-derived NCI-H295 cells by exerting MAPK p42/p44-mediated proliferogenic and antiapoptotic effects

Neuropeptides B and W (NPB and NPW) are endogenous ligands of two G protein-coupled receptors, named GPR7 and GPR8. GPR7 and GPR8 are expressed in the adrenal cortex, and there is evidence that NPB and NPW stimulate glucocorticoid secretion from human adrenocortical cells by activating protein kinase (PK) A and PKC signaling. To gain insight into the role of NPB and NPW in human adrenal functional regulation, we have investigated their effects on the secretion and growth of the human adrenocortical carcinoma-derived NCI-H295 cell line. NCI-H295 cells were found to express both GPR7 and GPR8 mRNAs, but neither NPB nor NPW (up to 10(-6) M) affected their secretory activity. In contrast, both peptides (from 10(-10) to 10(-6) M) enhanced the growth of NCI-H295 cells, by raising their proliferative activity and lowering their apoptotic deletion rate. NPB and NPW (10(-6) M) stimulated tyrosine kinase (TK) and mitogen-activated PK (MAPK) p42/p44 activities in NCI-H295 cells. Both these effects were blocked by the TK inhibitor tyrphostin-23, while the MAPK p42/p44 inhibitor PD-98059 annulled only MAPK p42/p44 activation. The growth-stimulating effect of 10(-6) M NPB and NPW were not affected by either the PKA and PKC inhibitors H-89 and calphostin-C or the MAPK p38 antagonist SB-293580, but were abolished by both tyrphostin-23 and PD-98059. Taken together, our findings allow us to conclude that GPR7 and GPR8 expressed in NCI-H295 cells: i) are, at variance with those present in normal human adrenocortical cells, uncoupled to PKA- and PKC-dependent cascades, thereby explaining the absence of any secretory response to NPB and NPW; and ii) are coupled to the TK-dependent MAPK p42/p44 signaling, whose activation mediates the proliferogenic and antiapoptotic effect of NPB and NPW.     

Msi1siRNA对人结肠癌SW-480细胞增殖的影响

目的 探讨siRNA沉默Msi1基因表达对人结肠癌SW-480细胞增殖的影响.方法 针对Msi1 mRNA序列设计合成siRNA,转染SW-480细胞;采用RT-PCR法检测Msi1基因表达;MTT比色实验、群体倍增时间及平皿克隆形成实验分析Msi1 siRNA对人结肠癌SW-480细胞的生长抑制效应,流式细胞术检测Msi1 siRNA 对其细胞周期的影响.结果 Msi1 siRNA能有效抑制人结肠癌SW-480细胞中Msi1基因的表达;Msi1 siRNA转染SW-480细胞24、48、72 h后,与其它各组比较,其吸光度值明显降低,Msi1 siRNA 组SW-480细胞生长、增殖速度明显减缓(P<0.05);空白对照组的SW-480细胞群体倍增时间为22.15 h,而Msi1 siRNA处理后的细胞,群体倍增时间延长至37.76 h(P<0.05);平皿克隆形成实验结果发现,Msi1 siRNA处理后的细胞与其它各组相比,细胞生长明显减慢.流式细胞术表明Msi1 siRNA可导致SW-480细胞G0/G1期阻滞,S期细胞减少.结论 沉默Msi1基因对人结肠癌SW-480细胞增殖有负性调节作用.     

Utilization of rat lymphocytes for the in vitro chromosomal aberration assay

In vitro cytogenetic assays are widely conducted to assess the mutagenic potential of chemicals. Chinese hamster ovary (CHO) cells or human lymphocytes are often used for these assays; however, these cell types have certain limitations. In order to evaluate an alternate cell system, cultured rat lymphocytes were treated for 4 h at 48 h of incubation with a variety of direct- and indirect-acting clastogens in the presence or absence of an exogenous mammalian activation system. Cytogenetic effects of in vitro physiological alterations such as medium hypertonicity or pH changes were also evaluated. The background aberration rate of rat lymphocytes is approximately 2%, and they respond positively to both direct- and indirect-acting clastogens. In contrast to CHO cells, however, neither the hyperosmolality nor pH changes in the treatment media have significant effect on background aberrations. Unlike samples of human blood, rat blood can be collected under well-controlled environmental conditions. Because of the easy access to rat blood samples, the simplicity of culture, the reproducible nature of its in vitro growth, the positive response to known clastogens and negative response to media pH changes or hyperosmolalities, the rat lymphocyte in vitro chromosomal assay presented is an optimal system to assess the mutagenic potential of chemicals.     

The effect of aspirin on human's gammadeltaT cells killing digestive system tumor cell lines

AIM: To explore the effect of aspirin on human's gammadeltaT cells killing digestive system tumor cell lines. METHODS: Use the isopentenyl pyrophosphate method to amplify human peripheral blood gammadeltaT cells in vitro. Various concentrations of aspirin were used to induce gammadeltaT cells and digestive system tumor cells SGC-7901, SW-1990, SW-480, SW-1116, LOVO cells lines, LDH assays was used to measure the cytotoxic activity of gammadeltaT cells, flow cytometry analysis the apoptosis percentage of before and after induced gammadeltaT cells and SGC-7901, SW-1990, SW-480, SW-1116, LOVO cell lines. RESULTS: gammadeltaT cells were cultivated for ten days which proliferation ratio increase from 4.21% to 70.35%. When gammadeltaT cells induced via aspirin concentrations rang from 0.4 mmol/L to 0.8 mmol/L which cytotoxic activity on the five kinds of tumor cell lines was the highest, if aspirin's concentration surpassing 3.2 mmol/L, cytotoxic activity of gammadeltaT cells present decrease tendency. SGC-7901, SW-1990, SW-480, SW-1116, LOVO cells lines were induced by different concentrations of aspirin for 24 hours, just SW-480, SW-1116 and LOVO were enhanced as far as the cytotoxic activity of gammadeltaT cells on these cell lines was concerned, other groups and control group have no notable changed. The apoptosis percentage of gammadeltaT cells(52.71%) were induced by aspirin which concentration was 3.2 mmol/L, which was strikingly higher than SGC-7901, SW-1990, SW-480, SW-1116, LOVO cells lines, (respectively 7.88%, 8.89%, 6.21%, 4.47% and 3.67%). CONCLUSION: When aspirin's concentration for clinical routine used can enhance the effect of gammadeltaT cells killing the tumor cells, if surpassing this concentration can obvious inhibited the proliferation capacity and cytotoxic activity of gammadeltaT cells and augment apoptosis ratio, however, it is not obvious for digestive tract tumor lines.     

Dibutyryl cyclic AMP modulation of gap junctions in SW-13 human adrenal cortical tumor cells

Cultured human adrenal cortical adenocarcinoma cells (SW-13) form a confluent monolayer of epithelial like cells when seeded into culture flasks. Following a 24–48 hr nonmitotic period, cells begin to divide and become confluent within a week after seeding at 5×10⁴ cells/cm². The SW-13 cells were exposed to dibutyryl cyclic AMP (DbcAMP), cyclic AMP (cAMP), sodium butyrate, and adrenocorticotropin (ACTH). The rate of SW-13 cell proliferation was measured with a DNA microfluorometric assay, as well as by procedures measuring the incorporation of ³H-thymidine. In addition, following administration of ACTH and DbcAMP, the fractional area of membrane covered by gap junctions was quantitated with freeze-fracture electron microscopic techniques. Dibutyryl cyclic AMP at a concentration of 1 × 10^?3 M decreased the growth rate of the cell population. There was a corresponding increase in the fractional area of gap junctions found on the cell membrane in 96-hr DbcAMP-treated cultures. ACTH (40 mU/ml) exposure failed to produce an increase in the fractional area of gap junctions or to alter the rate of cell proliferation. From these data it can be suggested that elevations in cAMP levels within the cell can be related to both the proliferation of gap junctions and the decrease in cell proliferation in the SW-13 tumor cell.     

Ghrelin inhibits in vitro angiogenic activity of rat brain microvascular endothelial cells

Ghrelin, a 28-amino acid peptide originally isolated from rat stomach, is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R). Evidence has been provided that ghrelin and GHS-Rs are highly expressed in the cardiovascular system, including endothelial cells (ECs), of which they regulate the growth in vitro. It, therefore, seemed worthwhile to investigate the effect of ghrelin on in vitro angiogenesis, using cultures of rat ECs derived from brain microvessels (neuromicrovascular ECs, NECs). ECs, when cultured on a supportive matrix, form a network of tubule-like structures, and such process is enhanced by the classic angiogenic factors, including fibroblast growth factor-2 (FGF-2). After seeding on Matrigel-coated wells, NECs formed within 18 h a meshwork of capillary-like structures; vinblastine (2 x 10(-12) M) disrupted the meshwork, while FGF-2 (50 ng/ml) increased its density. Ghrelin (10(-8) M) exerted a vinblastine-like effect and counteracted the stimulatory action of FGF-2. Computerized image-analysis confirmed these observations. FGF-2 enhanced the proliferation rate and lowered the apoptotic rate of NECs cultured on plastic wells, and ghrelin exerted opposite effects and completely reversed the proliferogenic and antiapoptotic actions of FGF-2. In contrast to vinblastine, ghrelin did not increase lactate dehydrogenase release from cultured NECs, thereby ruling out the possibility that its effects may ensue from an aspecific cytotoxic action. FGF-2 enhanced tyrosine kinase (TK) and mitogen activated protein kinase (MAPK) p42/p44 activities of NECs. Ghrelin significantly decreased TK and MAPK p42/p44 activities and effectively counteracted the effect of FGF-2. Taken together, the present findings indicate that ghrelin exerts a marked in vitro antiangiogenic action, and that the mechanism underlying this effect involves the inhibition of TK/MAPK-dependent cascades.     

Factor VII light chain-targeted lidamycin targets tissue factor-overexpressing tumor cells for cancer therapy

The overexpression of tissue factor (TF) observed in numerous cancer cells and clinical samples of human cancers make TF an ideal target for cancer therapy. Here, we report an energized fusion protein, hlFVII-LDP-AE, which can be used for cancer therapy and is composed of a human Factor VII light chain (hlFVII) conjugated to the cytotoxic antibiotic lidamycin (LDM, LDP-AE). hlFVII-LDP-AE binds with specificity to TF expressed on tumor cells, resulting in internalization of the fusion protein and cytotoxicity induced by the LDM domain. The potential efficacy of hlFVII-LDP-AE for cancer therapy was examined in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and in vivo with a BALB/c nude mouse xenograft model of the human lung cancer line NCI-H292. hlFVII-LDP-AE caused chromatin condensation and cleavage of genomic DNA in NCI-H292 cells. In the MTT assays, the IC50 value of hlFVII- LDP-AE was 0.19 nM. In the in vivo tests, after two intravenous injections of hlFVII-LDP-AE at a dose of 0.6 mg/kg, the growth rate of the lung tumor xenograft was reduced to 15% of the control rate, and there was no excessive loss of body weight and inflammatory response in the mice. These findings suggest that hlFVII-LDP-AE is efficacious and tolerated in the mouse model of NCI-H292 human lung cancer examined and could have broad clinical applicability for treating cancer patients.     

Erlotinib inhibits osteolytic bone invasion of human non-small-cell lung cancer cell line NCI-H292

Previous preclinical and clinical findings have suggested a potential role of epidermal growth factor receptor (EGFR) in osteoclast differentiation and the pathogenesis of bone metastasis in cancer. In this study, we investigated the effect of erlotinib, an orally active EGFR tyrosine kinase inhibitor (TKI), on the bone invasion of human non-small-cell lung cancer (NSCLC) cell line NCI-H292. First, we established a novel osteolytic bone invasion model of NCI-H292 cells which was made by inoculating cancer cells into the tibia of scid mice. In this model, NCI-H292 cells markedly activated osteoclasts in tibia, which resulted in osteolytic bone destruction. Erlotinib treatment suppressed osteoclast activation to the basal level through suppressing receptor activator of NF-κB ligand (RANKL) expression in osteoblast/stromal cell at the bone metastatic sites, which leads to inhibition of osteolytic bone destruction caused by NCI-H292 cells. Erlotinib inhibited the proliferation of NCI-H292 cells in in vitro. Erlotinib suppressed the production of osteolytic factors, such as parathyroid hormone-related protein (PTHrP), IL-8, IL-11 and vascular endothelial growth factor (VEGF) in NCI-H292 cells. Furthermore, erlotinib also inhibited osteoblast/stromal cell proliferation in vitro and the development of osteoclasts induced by RANKL in vitro. In conclusion, erlotinib inhibits tumor-induced osteolytic invasion in bone metastasis by suppressing osteoclast activation through inhibiting tumor growth at the bone metastatic sites, osteolytic factor production in tumor cells, osteoblast/stromal cell proliferation and osteoclast differentiation from mouse bone marrow cells.     

人肺癌NCI-H446细胞生成肿瘤干细胞球

目的 探索人肺癌NCI-H446细胞系干细胞球体的培养,并鉴定其生物学特性.方法 用无血清培养基培养人肺癌NCI-H446细胞得到肿瘤细胞球.将肿瘤细胞球传代扩增,并用含血清培养基培养促使其分化;四甲基偶氮唑盐(MTT)比色检测肿瘤球和普通NCI-H446细胞的增殖能力,并将肿瘤球和普通NCI-H446细胞分别植入裸鼠皮下,观察肿瘤形成;Transwell侵袭实验检测肿瘤球和普通NCI-H446细胞的侵袭能力的不同;流式细胞术检测CD133、CD44在肿瘤球和普通NCI-H446细胞中的表达,并筛选细胞表面标志物.结果 在无血清培养基中,人肺癌NCI-H446细胞可以形成少量的肿瘤细胞球,并显示很强的自我更新和增殖能力,在含血清环境中能够诱导肿瘤球分化而贴壁生长;在动物实验中,接种5×105个细胞时,肿瘤球细胞较普通NCI-H446细胞显示更强的致瘤能力;在侵袭实验中,肿瘤球细胞的侵袭能力高于普通NCI-H446细胞;干细胞标志物CD133及CD44在肿瘤球细胞的表达较普通NCI-H446细胞明显增高.结论 人肺癌细胞NCI-H446中存在癌干细胞,且可以通过无血清培养、分离和富集.     

干扰IDH-2基因对小细胞肺癌生长抑制作用的研究

 目的：研究siRNA特异干扰沉默异柠檬酸脱氢酶2（IDH-2）基因后对人小细胞肺癌细胞NCI-H446生长作用的影响。方法：用siRNA沉默NCI-H446细胞的IDH-2基因表达,用real-time PCR及Western blotting技术检测mRNA-IDH-2和蛋白的表达;CCK-8法测定细胞生长抑制,Western blotting检测MAPK p42的表达和流式细胞术检测细胞周期;用Transwell细胞迁移系统检测siRNA-IDH-2对NCI-H446细胞迁移能力的影响;将转染siRNA-IDH-2、阴性对照siRNA的细胞或未转染细胞皮下接种于BALB/c裸鼠背部,观察移植瘤的生长状况。结果：siRNA-IDH-2有效沉默NCI-H446细胞中IDH-2的表达;与对照组相比,下调IDH-2基因表达抑制NCI-H446细胞的增殖;MAPK p42蛋白表达下降且S期细胞明显增加;下调IDH-2基因显著抑制了NCI-H446细胞的迁移能力;体内实验显示siRNA-IDH-2组的肿瘤体积明显小于对照组。结论：siRNA-IDH-2能显著抑制IDH-2基因在人小细胞肺癌细胞NCI-H446中的表达,并降低细胞迁移效率,在体外和体内明显抑制NCI-H446细胞的生长。     

The nonreceptor-type tyrosine phosphatase PTPN13 is a tumor suppressor gene in non-small cell lung cancer

The aim of the present work was to identify protein tyrosine phosphatases (PTPs) as novel, candidate tumor suppressor genes in lung cancer. Among the 38 PTPs in the human genome that show specificity for phosphotyrosine, we identified six PTPs by quantitative RT-PCR whose mRNA expression levels were significantly down-regulated in lung cancer-derived cell lines (ie, PTPRE, PTPRF, PTPRU, PTPRK, PTPRD, and PTPN13). After validation in primary samples of non-small cell lung cancer (NSCLC), we selected PTPN13 for further studies. The results presented here demonstrate that PTPN13 is a candidate tumor suppressor gene that is frequently inactivated in NSCLC through the loss of either mRNA and protein expression (64/87, 73%) or somatic mutation (approximately 8%). Loss of PTPN13 expression was apparently due to the loss of one or both copies of the PTPN13 locus at 4q (approximately 26% double deletion and approximately 37% single deletion) but not to promoter methylation. Finally, the manipulation of PTPN13 expression in lung cancer cells (ie, NCI-H292, A549) demonstrated that PTPN13 negatively regulates anchorage-dependent and anchorage-independent growth in vitro and restrains tumorigenicity in vivo, possibly through the control of the tyrosine phosphorylation of both EGFR and HER2. In conclusion, the expression screening of PTPs in lung cancer reported here has identified PTPN13 as a novel candidate tumor suppressor in NSCLC whose loss increases signaling from epidermal growth factor receptor and HER2 tyrosine kinase receptors.     

Tissue Expression and Pharmacological In Vitro Analyses of mTOR and SSTR Pathways in Adrenocortical Carcinoma

New therapies for advanced adrenocortical carcinoma (ACC) are urgently needed, as the majority of the patients experience a rapid and inexorable progression despite surgery and adjuvant mitotane. In vitro data suggest that somatostatin receptors (SSTRs) and mTOR pathway might represent reasonable targets for novel therapies, being involved in functionality and growth of ACC cells. However, in vitro analysis of combination treatments targeting both mTOR and SSTR as compared to mitotane are poorly explored in ACC. This study aimed to investigate in vitro the effects on cell growth of pasireotide, everolimus, and mitotane, alone or combined, in the two ACC cell lines H295R and SW13 (mitotane sensitive and resistant, respectively). Moreover, the tissue expression of mTOR pathway molecules and SSTR (types 1–5) was assessed in 58 ACCs. In both cell lines, only everolimus induced a significant inhibition of cell growth. Conversely, the combinations among mitotane, pasireotide, and everolimus produced antagonistic effects on mitotane-induced growth inhibition on H295R cell line. A heterogeneous profile of mTOR-related molecules and SSTR expression was observed in ACC samples, being the mTOR pathway found activated in approximately 30% of cases. In conclusion, our data suggest caution in designing combinations of mitotane with other drugs potentially active in ACC, such as mTOR inhibitors or somatostatin analogs.     

Presence of organic anion transporters 3 (OAT3) and 4 (OAT4) in human adrenocortical cells

Since the organic anion transporter-1 (OAT1) has been implicated in cortisol release from bovine and rat adrenal zona fasciculata cells, we addressed the question of whether OATs are present in human adrenal cortical cells. In the human adrenal cell line NCI-H295R, 24-h cortisol secretion increased up to 30-fold on exposure to forskolin. Incubation of forskolin-treated cells for 24 h with the OAT substrates probenecid, p-aminohippurate (PAH), glutarate or cimetidine inhibited cortisol release partly. RT-PCR did not reveal expression of human OAT1 and OAT2, but OAT3 and OAT4 mRNAs were detected in both NCI-H295R cells and human adrenal tissue. When human OAT3 (hOAT3) and hOAT4 were expressed in Xenopus laevis oocytes, only hOAT3 showed [³H]cortisol uptake in excess of that of water-injected control oocytes. Cortisol uptake via OAT3 was saturable with an apparent K_t of 2.4 µM. In NCI-H295R cells, [³H]estrone sulphate uptake was saturable, cis-inhibited by OAT substrates and trans-stimulated by preloading with glutarate or cortisol. Likewise, [³H]PAH uptake was cis-inhibited by estrone sulphate and trans-stimulated by preloading the cells with PAH, glutarate or cortisol, indicating functional expression of OATs in the plasma membrane of NCI-H295R cells.     

阿托伐他汀对人NK 细胞杀伤结肠癌细胞的影响及其机制研究

目的:探讨阿托伐他汀对人NK 细胞杀伤结肠癌细胞的影响及其机制。方法:不同浓度的阿托伐他汀作用于3 株结肠癌细胞(HCT-116、SW-480、Caco-2),CCK-8 法测定阿托伐他汀对结肠癌细胞生长抑制率的影响。SCGM 培养基体外扩增人NK 细胞,自动生化分析仪测定NK 细胞对3 株结肠癌细胞的杀伤活性;流式细胞仪检测结肠癌细胞MICA/ B 的表达率。结果:(1)NK 细胞的培养:培养前CD3- CD56+的NK 细胞占外周血单个核细胞的比例为4.5%,培养10 d 时NK 细胞的比例增至93.1%。(2)阿托伐他汀对3 株结肠癌细胞生长抑制率的影响:阿托伐他汀作用48 h 后,在浓度5 ~40 μmol/ L 的4 个实验组中,HCT-116 细胞的生长抑制率较对照组升高(P<0.05)。作用96 h 后,在1.25 ~ 40 μmol/ L 的所有浓度组(6 个)中,HCT-116 细胞的生长抑制率均显著高于对照组(P<0.05)。另外, HCT-116 细胞的生长抑制率随着阿托伐他汀浓度的升高而逐渐上升,相关分析显示,阿托伐他汀的浓度与HCT-116 细胞的生长抑制率呈正相关(r[48 h] = 0.13,r[96 h] = 0.22,P<0.05)。(3)NK 细胞经阿托伐他汀作用96 h 后,除浓度为20 μmol/ L 和40 μmol/ L 时抑制率高于对照组,其他各组对NK 细胞生长无明显影响。(4)阿托伐他汀对NK 细胞杀伤结肠癌细胞活性的影响:NK 细胞对HCT-116 细胞的杀伤活性在阿托伐他汀浓度2.5 ~10 μmol/ L 组均显著高于对照组,对SW-480 的杀伤活性在5 ~20 μmol/ L 组较对照组明显升高,对Caco-2 的杀伤活性在2.5 ~20 μmol/ L 组显著高于对照组(P<0.05),其中同一浓度时对HCT-116 细胞杀伤作用最强。(5)阿托伐他汀对结肠癌细胞MICA/ B 表达的影响:在阿托伐他汀浓度2.5 μmol/ L 及5 μmol/ L 组,HCT-116 细胞MICA/ B 的表达率与对照组比较显著升高(P <0.05),在10 μmol/ L 及20 μmol/ L 组,SW-480 细胞MICA/ B 表达率显著高于对照组(P <0.05),在2.5 ~40 μmol/ L 组,Caco-2 细胞MICA/ B 的表达率均较对照组显著升高(P<0.05)。结论:(1)阿托伐他汀能够呈剂量依赖性抑制结肠癌细胞HCT-116、SW-480 及Caco-2 的生长;(2)阿托伐他汀能够增强NK 细胞对结肠癌细胞的杀伤活性,可能与阿托伐他汀上调结肠癌细胞MICA/ B 的表达有关;(3)阿托伐他汀可以上调3 株结肠癌细胞MICA/ B 的表达率,提高结肠癌细胞的免疫原性。     

Overexpression of pigment epithelium-derived factor decreases angiogenesis and inhibits the growth of human malignant melanoma cells in vivo

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, and is involved in the pathogenesis of angiogenic eye disease such as proliferative diabetic retinopathy. However, a functional role for PEDF in tumor growth and angiogenesis remains to be determined. In this study, we have investigated both the in vitro and in vivo growth characteristics of human malignant melanoma G361 cell lines, stably transfected to overexpress human PEDF. Expression levels of PEDF proteins in melanoma cell lines G361 and A375 were comparable with that of human cultured melanocytes, whereas vascular endothelial growth factor levels in two tumor cell lines were much stronger than that in normal melanocytes. Overexpression of PEDF was found to significantly inhibit tumor growth and vessel formation in G361 nude mice xenografts. Furthermore, in vitro proliferation rates of G361 cells were decreased in PEDF-transfected cells. PEDF proteins showed dose-dependent induced growth retardation and apoptotic cell death in nontransfected G361 cells, which were completely prevented by treatment with antibodies against the Fas ligand. Our present study highlights two beneficial effects of PEDF treatment on melanoma growth and expansion; one is the suppression of tumor angiogenesis, and the other is induction of Fas ligand-dependent apoptosis in tumor cells. PEDF therefore might be a promising novel therapeutic agent for treatment of patients with melanoma.     

Accumulation of steroidogenic acute regulatory protein mRNA, and decrease in the secretory and proliferative activity of rat adrenocortical cells in the presence of proteasome inhibitors

Sporadic findings indicate that proteolysis may affect steroid secretion in rat ovary granulosa cells. We examined the effects of the proteasome inhibitors MG115 and MG101 on the in vitro secretion and growth of rat adrenocortical cells. MG115 and/or MG101 decreased within 120 min the aldosterone and corticosterone secretion from freshly dispersed zona glomerulosa and zona fasciculata-reticularis (ZF/R) cells. After a 24-h incubation MG115 alone or with MG101 lowered corticosterone production and enhanced proliferation rate of cultured ZF/R cells, while MG101 was per se ineffective. Real-time polymerase chain reaction demonstrated that MG101 decreased steroidogenic acute regulatory protein (StAR) mRNA in cultured cells. MG115 was per se ineffective, but when added together with MG101 evoked a marked rise in StAR mRNA content. In light of the present findings, we conclude that i) protein breakdown by proteasomes is required for the maintenance of a normal secretory and proliferative activity of freshly dispersed or cultured rat adrenocortical cells; and ii) in long-term experiments, great caution must be taken in correlating StAR mRNA content and steroidogenic capacity.