Effects of gossypol on rat Sertoli and Leydig cells in primary culture and established cell lines

In order to evaluate the direct effect of gossypol on testicular cells, we used primary cultures of rat Leydig and Sertoli cells. No alteration in Leydig cell survival, morphology, or testosterone production was seen during three days of culture with up to 3 microgram/ml gossypol. With higher concentrations (3 to 7 microgram/ml) of gossypol, there was a reduction in cell survival but no change in androgen secretion. In contrast, there was a marked change in Sertoli cell morphology after five days of gossypol treatment. Large vacuoles and electron dense granules appeared in the cytoplasm, but these effects were reversed within six days of removing gossypol from the medium. There was a significant decrease in androgen binding protein (ABP) secretion by Sertoli cells in the presence of gossypol. We also tested the effect of gossypol on the growth of three established cell lines. Two Sertoli-derived cell lines, TM4 and TR-ST, were more sensitive than a Leydig-derived cell line (TM3). These results suggest that, of the somatic cell types in the testis, the Sertoli cells are most sensitive to gossypol.     

4‐Methylcatechol inhibits cell growth and testosterone production in TM3 Leydig cells by reducing mitochondrial activity

4‐Methylcatechol (4‐MC) is a potential neuroprotective drug because it stimulates the synthesis of brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in neurons. The present study explored the effect of 4‐MC on cell growth and testosterone synthesis in the TM3 Leydig cells of mice. 4‐MC did not enhance expression of both BDNF and NGF in these cells. However, this compound significantly inhibited cell proliferation and increased the number of apoptotic cells in a dose‐dependent manner. The expression profile of Bax/Bcl‐2 gene was altered considerably, and mitochondrial activity was significantly decreased in cells. 4‐Methylcatechol also inhibited testosterone synthesis in TM3 Leydig cells. The inhibitory roles of this compound in relation to growth and testosterone synthesis in TM3 Leydig cells maybe associated with increased Bax gene expression and decreased mitochondrial activity. As a result, caspase cascade is activated.     

Effects of combined valproic acid and the epidermal growth factor/vascular endothelial growth factor receptor tyrosine kinase inhibitor AEE788 on renal cell carcinoma cell lines in vitro

OBJECTIVE

To evaluate adhesion and growth inhibiting effects of the multiple receptor tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) on renal cell carcinoma (RCC) cells.

MATERIALS AND METHODS

Caki‐1 cells were treated with AEE788 and VPA, either alone or in combination, to investigate RCC cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins. Tumour cell proliferation was examined by MTT dye reduction assay. Effects of drug treatment on cell signalling pathways were determined by Western blotting. The expression levels of integrin α and β subtypes were evaluated by flow cytometry (surface expression) and Western blotting (intracellular protein expression).

RESULTS

RCC cell treatment with AEE788 and VPA in combination resulted in a stronger inhibition of tumour cell proliferation than that caused by either drug alone. There were also additive effects of the combined treatment on tumour cell adhesion to endothelial cells and to immobilized laminin (but not to immobilized fibronectin and collagen). AEE788 alone or combined with VPA reduced Akt expression and histone H3 acetylation. Both compounds altered integrin α and β subtype expression, in particular α1, α3 and β4, and blocked integrin‐dependent integrin‐linked kinase and focal‐adhesion kinase (total and phosphorylated) signalling.

CONCLUSIONS

Both AEE788 and VPA profoundly block the interaction of RCC cells with endothelium and extracellular matrix and reduce tumour growth in vitro. Therefore, this combined regimen warrants further preclinical and possible clinical study for treating advanced RCC.     

Follicle stimulating hormone, luteinizing hormone and testicular Leydig cell responses to estradiol immunization in Ile-de-France rams

Active immunization of Ile-de-France rams against estradiol (E2) resulted in the production of E2-neutralizing antibodies and an elevation in the plasma concentrations of FSH, LH, and testosterone. The presence of E2 antibodies did not affect the testosterone metabolic clearance rate, indicating that the immunization-mediated 10-fold increase in plasma testosterone was the result of a 10-fold increase in testicular testosterone production. Testis weights, as well as nuclear and cytoplasmic volumes of individual peritubular and perivascular Leydig cells, were greater in E2-immunized rams than in albumin-immunized controls. Leydig cell numbers were not affected by treatment. The E2 antibodies were capable not only of neutralizing the inhibitory effects of endogenous E2 on gonadotropin levels in intact rams, but were able to block the effects of exogenously administered E2 on their FSH and LH secretory response to castration. It is concluded that circulating E2 in the ram is involved in pituitary-testicular endocrine homeostasis and that E2 immunoneutralization can be employed to enhance testosterone secretion in this species.     

Changes in epidermal growth factor receptor expression in human bladder cancer cell lines following interferon-alpha treatment

PURPOSE: We examined the regulation of epidermal growth factor (EGF) receptor (EGFR) expression in human bladder cancer cell lines by interferon-alpha (IFN-alpha), the ability of IFN-alpha to inhibit cell proliferation and the sensitivity of IFN-alpha pretreated cells to EGF. MATERIALS AND METHODS: Cell proliferation was determined using crystal violet colorimetric and clonogenic assays. EGFR expression was measured by flow cytometry using specific antibody or ligand binding approaches. RESULTS: After IFN-alpha (100 IU/ml) treatment cell surface EGFR expression was upregulated in 6 of 11 and down-regulated in 2 of 11 bladder cancer cell lines. The over expression of cell surface EGFR peaked within 48 to 96 hours and increased by 35% to 241% in individual cell lines. High level cell surface EGFR correlated with intracellular EGFR expression. Cell growth inhibition by IFN-alpha coexisted with EGFR over expression in the 6 lines. IFN-alpha treated cells remained sensitive to EGF treatment. CONCLUSIONS: IFN-alpha transiently up-regulates EGFR expression and inhibits in vitro growth in some human bladder cancer cells. IFN-alpha does not prevent EGFR from binding EGF or signal transduction via the EGF-EGFR pathway. This may have clinical implications for improving treatment based on EGFR targeting in select patients with bladder cancer.     

Androgenic and antiandrogenic control on epidermal growth factor,epidermal growth factor receptor,and androgen receptor expression in human prostate cancer cell line LNCaP

Both androgen and antiandrogen treatments enhance the proliferation rate of the hormone-dependent prostate cancer cell line LNCaP, expressing a mutated androgen receptor (AR). We studied the modification of the expression of epidermal growth factor (EGF), of its receptor (EGF-R), and of androgen receptor (AR) in the LNCaP cell line, under basal conditions and during androgen (R1881) and antiandrogen hydroxy-flutamide (OH-FLU) treatment. After prolonged R1881 administration, a marked increase of EGF release was observed, completely blocked by the addition of OH-FLU. The Scatchard plot analysis of EGF-R binding revealed two classes of binding sites with high and low affinity. The administration of OH-FLU alone or combined with R1881 did not modify the affinity constants, while the low-affinity component disappeared after androgen administration. Both androgen and antiandrogen administration led to a significant increase of the EGF-R high-affinity component. AR mRNA and protein levels were downregulated by R1881 treatment. Following OH-FLU administration, AR mRNA was slightly downregulated, and there was not a strict parallelism between AR mRNA levels and AR binding capacity. When combined with R1881, OH-FLU partially counteracted the androgen-induced AR downregulation. Our data show that EGF-R binding capacity is the only parameter constantly raised in cell proliferation with respect to quiescent cells, and highlights the nonunivocal action of OH-FLU on androgen-induced effects.     

The action of calcitonin on the TM4 Sertoli cell line and on rat Sertoli cell-enriched cultures

The effects of synthetic salmon calcitonin on primary Sertoli cell-enriched cultures and on an established cell line (TM4 cells, derived from immature mouse Sertoli cells) were studied. Synthetic salmon calcitonin stimulated the conversion of [3H]adenine to [3H]cyclic AMP in both cell systems. In addition, this peptide stimulated the secretion of rABP in primary Sertoli cell-enriched cultures prepared from rat testis. Calcitonin also increased the total concentration of both androgen and estrogen receptors in TM4 cells. Because cAMP analogs decreased androgen and estrogen receptor concentrations, the effect of calcitonin on sex steroid receptors may not be mediated by its effect on cyclic AMP in these cells. The possibility that the action of synthetic salmon calcitonin on the receptors might be mediated by a change in cellular Ca2+ was investigated. Lowering extracellular Ca2+ concentrations from 1.5 mM to less than 0.01 mM markedly reduced the concentration of androgen and estrogen receptors; restoration of Ca2+ to 1.5 mM returned receptor levels to normal. When the receptor concentrations were decreased by lowering extracellular Ca2+ concentrations to 0.5 mM, treatment with the calcium ionophore, A23187, restored receptor levels to normal. Although the calcium channel blocker, verapamil, decreased receptor levels, calcitonin partially counteracted its effect. Trifluoperazine, an inhibitor of calmodulin, also diminished androgen and estrogen receptor, levels in the cytosol of TM4 cells. It was concluded that calcitonin stimulates the formation of cyclic AMP and the secretion of rABP by Sertoli cells. This peptide also increases the concentration of androgen and estrogen receptors, possibly by a mechanism that is, in part, Ca2+ -mediated. These results, along with those on Leydig cells, suggest that calcitonin could be a regulator of testicular function.     

Response of malignant glioma cell lines to epidermal growth factor and platelet-derived growth factor in a serum-free medium

The use of a serum-free culture system for assessing the growth factor responsiveness of malignant glial cells is described. The mitogenic properties of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) were examined in three human malignant glioma cell lines (T98G, U87, and U138). Each of the three had high-affinity EGF receptors and all responded in a dose-dependent fashion to physiological concentrations of EGF. These cell lines also showed a pronounced mitogenic response to PDGF which equaled or exceeded that achieved with EGF. Simultaneous stimulation with both factors produced an additive response, which approximated that obtained in medium supplemented with 10% fetal calf serum. The authors conclude that functional EGF and PDGF receptors were present in the human malignant glial tumors studied. The response of the human glioma lines to these growth factors in many respects parallels the response seen in fetal astrocytes tested under similar conditions. In contrast, the behavior of two chemically induced rat gliomas (9L and C6) differed significantly from that seen in the human lines, suggesting that the rat lines may not be entirely acceptable as models for studying the growth characteristics of human malignant glial tumors.     

K-Ras突变对表皮生长因子受体抑制剂敏感细胞株的影响

目的 观察K-Ras突变对于携带表皮生长因子受体(EGFR)突变细胞的EGFR抑制剂敏感性的影响.方法 构建K-Ras突变真核表达质粒,采用脂质体转染技术转染肺癌细胞HCC827(EGFR突变,K-Ras野生)和H292(EGFR、K-Ras均野生),噻唑蓝(MTT)比色法测定转染K-Pas突变质粒和空白质粒后各细胞对EGFR抑制剂的半数抑制浓度(IC_(50)).结果 真核表达质粒构建成功.细胞HCC827未转染K-ras突变质粒对吉非替尼(Iressa)的IC_(50)为0.007,转染后对Iressa的IC_(50)为12.3,差异有统计学意义(P<0.01).细胞H292未转染K-ras突变质粒对Iressa的IC_(50)为0.04,转染后对Iressa的IC_(50)为12.0,差异有统计学意义(P<0.01).细胞A549(K-Ras突变)对Iressa的IC_(50)为12.8,与转染K-ras突变质粒的细胞HCC827及H292比较,差异无统计学意义(P>0.05).结论 野生型或突变型EGFR出现K-Ras突变均可引起吉非替尼耐药,其程度与K-Ras突变的细胞株相当.     

雷公藤甲素介导AMPK/Akt/mTOR通路对小鼠TM3睾丸间质细胞活性的影响

目的:探究雷公藤甲素(TP)对小鼠TM3睾丸间质细胞活性及AMPK/Akt/mTOR通路的影响。方法:用不同浓度(50、100、200 nmol/L)的TP处理TM3细胞,对照组为含0.1%DMSO的等体积无血清培养液,37℃恒温孵育箱培育24h后,使用乳酸脱氢酶(LDH)活性检测和凋亡检测试剂盒测定细胞膜受损程度及凋亡率,利用Western印迹检测AMPK/Akt/mTOR通路蛋白变化情况。结果:对照组LDH的活力为(157.5±20.3)%,同时其活性随TP浓度升高显著增强[50 nmol/L:(163.4±33.6)%,100 nmol/L:(346.8±148.8)%, 200 nmol/L:(422.8±113.9)%,P0.01]。流式细胞术检测显示,对照组凋亡细胞百分率为(6.27±1.41)%,TP各浓度组凋亡百分率与对照组相比均上调[50 nmol/L:(189.9±73.5)%,100 nmol/L:(284.0±103.5)%,200nmol/L:(419.2±155.7)%],其中200 nmol/L TP组与对照组比较差异显著(P0.05)。Western印迹结果显示,与对照组相比,p-AMPK/AMPK比值显著降低(P0.01),p-Akt/Akt比值升高(其中50nmol/L组,P0.05),p-mTOR磷酸化水平升高(其中200 nmol/L组P0.05)。结论:TP能破坏TM3细胞活性,诱导其凋亡,同时伴随着AMPK抑制、Akt/mTOR通路活化。     

Receptors for luteinizing hormone-releasing hormone, somatostatin, prolactin, and epidermal growth factor in rat and human prostate cancers and in benign prostate hyperplasia

Using sensitive multipoint micromethods, we estimated membrane receptors for [D-Trp6]-luteinizing hormone-releasing hormone ([ D-Trp6]-LH-RH), somatostatin (SS-14), human prolactin (hPRL), and epidermal growth factor (EGF) in experimental Dunning rat prostate cancers and in samples of normal human prostate, benign prostatic hyperplasia (BPH), and human prostate cancer (PC) obtained from biopsy, after prostatectomy, or at autopsy. In the Dunning R-3327 rat prostate adenocarcinoma specimens, the receptors were characterized in untreated animals and following in vivo treatment with microcapsules of the agonist [D-Trp6]-LH-RH and the somatostatin analog RC-160. Two populations of binding sites were found for [D-Trp6]-LH-RH, one with high affinity and low capacity and another with low affinity and high capacity. Treatment with [D-Trp6]-LH-RH and RC-160 alone or with the combination of these analogs increased the binding capacity (Bmax) of the low-affinity binding sites for [D-Trp6]-LH-RH and decreased Bmax for hPRL and EGF. Therapy with [D-Trp6]-LH-RH also reduced Bmax of SS-14 binding and dissociation binding constant of high-affinity binding sites for [D-Trp6]-LH-RH, whereas administration of RC-160 or the combination treatment with both analogs increased Bmax of SS-14 binding. These findings are compatible with the view that analogs of LH-RH and SS-14 might exert some direct inhibitory effects on the Dunning prostate cancer. Among 13 human BPH samples examined, only one had receptors for [D-Trp6]-LH-RH, and seven specimens exhibited binding for prolactin. [D-Trp6]-LH-RH receptors were found in all seven samples of human PC but not in any of the eight specimens of normal human prostate. All samples of normal human prostate, BPH, and human PC exhibited binding sites for EGF but not for SS-14. Our findings on the membrane receptors in the human and rat prostate cancers raise the intriguing possibility that LH-RH, acting as a growth factor, along with EGF and prolactin, might be involved in complex interactions that contribute to the promotion of prostate cancer in man.     

Regulation of testicular ornithine decarboxylase in vitro. Effect of age, follicle-stimulating hormone, and luteinizing hormone

We have examined hormonal regulation of ornithine decarboxylase (ODC) activity in decapsulated rat testis, isolated testicular interstitial cells, and purified Leydig cells under defined conditions in vitro. Both immature (15 to 26 days old) and adult (60 to 90 days old) rat testes were employed. Basal (fresh tissue) ODC activity varied widely among rats of the same age but was similar (less than 5% difference) in pairs of testes from the same animal. For this reason, pairs of testes were compared in subsequent in vitro studies. ODC activity of decapsulated testes of adult rats declined (to 25 to 30% of basal) during 4 hours of incubation in Medium 199 + 0.1% bovine serum albumin + 0.1 mM 3-isobutyl-1-methylxanthine at 34 C. The addition of FSH, LH, prolactin, prostaglandin E2, epidermal growth factor, insulin, or 10% fetal calf serum singly or in combination failed to prevent this decline in ODC activity. In contrast, ODC activity of decapsulated testes of immature rats remained stable (versus fresh tissue) during 4 hours of incubation. The addition of FSH (100 ng/ml) caused a small but statistically significant (P less than 0.005) stimulation of the enzyme activity, and 8-bromo cyclic AMP (0.5 mM) mimicked the effect of FSH. In isolated interstitial cells from adult rats, LH stimulated ODC activity in a dose- (10 pg-200 ng/ml) and time-dependent fashion. 8-Bromo cyclic AMP mimicked the effect of LH. Prolactin, FSH, estradiol, insulin, prostaglandin E2, and epidermal growth factor did not alter the enzyme activity. LH also stimulated ODC activity of purified Leydig cells. This study demonstrates for the first time direct in vitro stimulation of rodent testicular ODC activity by gonadotropins and reveals marked age-dependent differences in regulation of this enzyme in vitro.     

Effect of epidermal growth factor on prostate cancer cell line PC3 growth and invasion

Elevated levels of epidermal growth factor (EGF) and epidermal growth factor receptor (EGF-R) have been demonstrated in prostate cancer cell lines and clinical specimens suggesting a role for polypeptide growth factors in prostate tumor cell growth and invasion. To more clearly define the role of EGF in prostate cancer invasion, we undertook a series of studies utilizing the PC3 prostate cancer cell line, an aggressive, hormone-independent cell line derived from a metastatic lesion. No statistical differences were noted in the growth of PC3 cells under serum-free conditions when EGF (10^?10 M-10^?8 M) or monoclonal anti-EGF-R antibody (10^?11 M-10^?8 M) were added. Utilizing the Boyden chamber microinvasion assay, EGF supplemented cells demonstrated a statistically significant augmentation in invasion (P < 0.05) when compared to control cells at each time point in the study. With increasing length of exposure to EGF, the number of concentrations that produced significant invasion increased: day 1 (10^?8 M), day 3 (10^?9, 10^?9 M), and day 5 (10^?7, 10^?8, 10^?10 M). Northern blot analysis of EGF supplemented cells revealed an increase in expression of urokinase plasminogen activator (uPA) RNA, a serine protease involved in the regulation of pericellular proteolysis and membrane degradation. Protein analysis confirmed these findings. Statistically significant inhibition of invasion by anti-uPA antibodies was demonstrated for EGF-stimulated and PC3 control cells. Our results demonstrate that certain concentrations of EGF augment invasion in the PC3 cell line. This enhancement of invasion occurs in part by an overproduction of uPA, an extracellular protease. These findings suggest that the autocrine production of EGF may potentiate tumor cell invasion. © 1994 Wiley-Liss, Inc.     

线粒体呼吸链相关基因Cox7a2对睾丸Leydig细胞睾酮生成影响研究

目的 探讨线粒体相关基因Cox7a2编码蛋白对睾酮生成的影响.方法 构建Cox7a2-EYFP-N1荧光表达载体,鉴定后转染TM3小鼠睾丸Leydig细胞,荧光显微镜观察转染效率和蛋白表达.ELISA的方法测定上清液中睾酮生成水平,CCK-8细胞增殖和生长测定试剂盒检测Cox7a2-YFP融合蛋白对细胞增殖状态的影响.结果 Cox7a2-YFP在TM3细胞中表达后抑制LH诱导的睾酮分泌,细胞增殖试验显示,Cox7a2融合蛋白表达后能抑制TM3细胞的增殖.结论 线粒体定位蛋白Cox7a2能抑制TM3细胞睾酮合成,与Cox7a2对细胞增殖有抑制作用有关.