In vitro effects of epidermal growth factor (EGF) on growth of urological malignant tumor cells

It is well known that Epidermal Growth Factor (EGF) is a cell-regulating factor for variety of tissues in vitro including normal and malignant cells. Furthermore, Takano et al reported that a decreased expression of EGF receptor in clones of human cancer KB cell line might be one of the pleiotropic properties of multidrug-resistant cells. However, both the influence of EGF on human urological cancer cell lines and the relation between EGF receptors and sensitivities of antitumor drugs on these cell lines have not been fully described. We have studied the effects of EGF on growth of 4 transitional carcinoma cell lines of bladder (TCCaB), 1 squamous cell carcinoma cell line of bladder (SCCaB), 5 renal cell carcinoma cell lines (RCC) and 3 prostatic carcinoma cell lines (CaP), as well as the relationship between the number of EGF receptors and drug sensitivities of these cell lines in vitro against methotrexate, vinblastine, adriamycin, cisplatin and etoposide (VP16). The present results determined by the in vitro colony forming efficiency method showed that exogenous addition of EGF to cell cultures at 0.1 ng/ml stimulated the growth of SCCaB by 169.0%, and at 1 ng/ml inhibited that of RCC by 2.9%-79.0%, relative to control. The more EGF receptors by 125I-EGF binding assay, the higher inhibition of VP16 on the growth of these cell lines. These results suggested that EGF stimulated the growth of SCCaB and inhibited the growth of RCC in vitro, and we found that these phenomena were correlated with neither the number of EGF receptors nor affinities of that receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of keratinocyte growth factor—2 on proliferation of human adult keratinocytes

OBJECTIVE: To investigate the proliferative effect of keratinocyte growth factor (KGF-2) on human adult keratinocytes. METHODS: The standard medium was keratinocyte growth medium without bovine pituitary extract (BPE), hydrocortisone or epidermal growth factor (EGF). Keratinocytes from a 48-year-old subject were cultured and seeded on dishes with standard medium of EGF in cell density of 2 x 10(4)/32 mm(2). After 24 hours, the medium was replaced by the standard medium with 0, 4, 16, 125 and 500 ng/ml KGF-2, respectively. The standard medium with EGF was used as the positive control and the standard medium without EGF or KGF-2 was used as the negative controls. The growth of keratinocytes was monitored by 3-(4,5-dimethythiazol-2-yl)-2,5 dipheyl tetrazolium bromide (MTT) assay and by photographs on days 3, 5 and 7, respectively. RESULTS: KGF-2 in concentrations of 4-500 ng/ml showed a significant proliferative effect on days 5 and 7 as compared with that of the negative controls (P < 0.01). On day 3 the cells were proliferated to 1.5-2.5-fold, on day 5 to 3-5-fold and on day 7 to 3-12-fold in KGF-2 medium as that of the negative controls. The optimal response occurred when the concentration of KGF-2 was 125 ng/ml on day 7. Cell proliferation was also consistently higher in all KGF-2 concentrations as compared with that of the positive controls. CONCLUSIONS: KGF-2 has significant effects on the proliferation of adult keratinocytes, which are more effective than that of EGF. This study supports KGF-2 can improve the healing of chronic wounds in adults in clinic.     

前列腺癌及前列腺增生组织EGF,EGFr表达的差异及意义

对前列腺增生症及前列腺癌中ＥＧＦ、ＥＧＦｒ表达进行了观察。７５％ＢＰＨ表达ＥＧＦ，５８％ＢＰＨ有ＥＧＦｒ阳性染色。２７例前列腺癌中１３例（４８％）ＥＧＦ阳性，仅４例（１５％）表达ＥＧＦｒ。ＥＧＦｒ在前列腺癌中低水平表达可能与原癌基因Ｃ－ｅｒｂＢ－１表达调控有关。本研究未能发现ＥＧＦｒ表达与肿瘤组织学类型及预后的关系。因此认为对ＥＧＦｒ在肿瘤组织中表达的临床意义的解释应慎重。     

Inhibition of head and neck squamous cell carcinoma cell lines by transforming growth factor-β

Transforming growth factor-β is known to be a potent autocrine growth inhibitor produced by a wide variety of cells, including cells of the immune system. Other investigators have noted that the growth of nontransformed keratinocytes is inhibited by transforming growth factor-β, whereas various carcinoma cell lines are resistant to these effects. Head and neck squamous cell carcinoma cells are known to have surface receptors for this cytokine. We thus assessed the effect of transforming growth factor-β on the growth of head and neck squamous cell carcinoma cell lines. Four head and neck squamous cell carcinoma cell lines were incubated with varying concentrations of transforming growth factor-β, and cytotoxicity was evaluated with a methylene blue colorimetric assay. After culturing in transforming growth factor-β for 4 days, inhibition of growth was detected in CAL-27 (maximal inhibition at 5.0 ng/ml), UMSCC-1, and UMSCC-19 (maximal inhibition at 50 ng/ml) cell lines. One other cell line, UMSCC-8 was found resistant to the inhibitory effects of transforming growth factor-β. Kinetics analysis experiments revealed minimal inhibition before day 2 of incubation, at which time inhibition increased linearly to day 4. Assessment of double-stranded DNA fragmentation suggested that DNA fragmentation occurs before significant cytotoxicity. Electron microscopic analysis and gel electrophoresis of extracted DNA revealed morphologic features consistent with apoptotic cell death. Our findings indicate that transforming growth factor-β significantly inhibits the growth of head and neck squamous cell carcinoma cell lines by inducing apoptotic cell death. (OTOLARYNGOL HEAD NECK SURG 1995;112:728-34.)     

Plasma levels of CEA as a prognostic marker in carcinoma of urinary bladder.

Plasma carcinoembryonic antigen (CEA) levels were performed preoperatively by radioimmunoassay in 124 patients with histologically proved bladder carcinoma. The level of CEA was used to determine its prognostic value in patients with bladder cancer. The correlation of CEA levels with the stage of the disease, histology, and resectability was also studied. Values above 2.5 ng/ml were taken as abnormal. Active disease was associated with high CEA levels. All patients with CEA levels greater than 10 ng/ml died in less than 1 1/2 years, while all patients who survived 1 1/2-3 years had preoperative CEA levels less than 10 ng/ml. There was a prognostic significance for patients with transitional cell or squamous cell carcinoma. All patients with squamous cell carcinoma had CEA levels less than or equal to 10 ng/ml, and all patients with transitional carcinoma had preoperative CEA values greater than 10 ng/ml. A correlation between CEA levels and resectability of the primary tumor was found. This study indicates that, in bladder carcinoma patients, preoperative CEA levels greater than 10 ng/ml are of prognostic value, since all of these patients have died and all of the long-term survivors had levels of less than or equal to 10 ng/ml.     

The effect of keratinocyte growth factor-2 (KGF-2) on the proliferation of human keratinocytes

Objective:In vitro studies have shown that KGF-2 has a proliferative effect on neonatal foreskin keratinocytes. Cells from adult donors have been shown to respond to KGF-1 to a lesser degree than neonatal keratinocytes. The purpose of the study was to investigate the proliferative effect of KGF-2 on keratinocytes from an adult subject. Methods: Standard medium was Keratinocyte Growth Medium without BPE, hydrocortisone and EGF. Keratinocytes cultured from a 48-year-old subject were seeded at 2 104 in 32 mm dishes in standard medium with EGF.After 24 hours, medium was changed to standard medium with KGF-2 at 4, 16, 125 and 500 ng/ml or without KGF-2. Standard medium with EGF was used as a positive control. The growth of keratinocytes was monitored by the MTT assay and by photographs at day 3, 5 and 7. Each treatment was performed in triplicate. Results: KGF-2concentrations from 4 to 500 ng/ml all showed a significant proliferative effect on days 5 to 7 compared to the negative control (P＜ 0.01). By day 3 the response of keratinocytes in KGF-2 was 1.5 to 2.5-fold, by day 5 was 3 to 5-fold and by day 7 was 3 to 12-fold relative to the negative control. The optimal response was in 125 ng/ml KGF-2 at day 7. Cell proliferation was also consistently higher in all KGF-2 concentrations compared to the positive control.Conclusion: KGF-2 exerted a significant effect on the proliferation of adult keratinocytes, which was higher than that of the EGF concentration in the growth medium. This supports the clinical work where KGF-2 has been shown to improve the healing in chronic wounds in adults.     

表皮生长因子和干细胞因子对小鼠生精细胞增殖与分化的作用

目的:观察表皮生长因子(EGF)和干细胞因子(SCF)体外促小鼠生精细胞增殖、分化的效应。方法:对7~8日龄雄性昆明小鼠生精细胞进行混合细胞体外培养,在培养液中分别添加不同浓度(5、10、20、40、100 ng/ml)的EGF和SCF,并进行EGF和SCF的交互实验,观察生精细胞的存活率和形态学变化,并对粗线期精母细胞特异性磷蛋白基因(P19)、单倍体精子细胞特异性转化蛋白基因(TP1)及染色体倍性进行检测。结果:加入EGF或SCF 2~4 d,各组均可见不同程度的细胞增殖,细胞呈团或族状,以20 ng/ml EGF组和40 ng/ml SCF组最为明显;培养第7天,单一添加20 ng/ml EGF或40 ng/ml SCF组生精细胞数和存活率显著高于与其它各组(P0.05),且40 ng/ml SCF组P19/TP1基因表达显著低于其它各组,单倍体细胞率显著高于其它各组(P0.05)。EGF与SCF配伍时,可显著增加体外培养后生精细胞数(P0.05)。结论:在混合生精细胞体外培养体系中,添加一定浓度的EGF和SCF可显著提高生精细胞数和存活率,而且SCF可提高单倍体精子的形成率;两者交互实验时,对细胞增殖有一定的叠加效应。     

Inhibition of chemomigration of a human prostatic carcinoma cell (TSU-pr1) line by inhibition of epidermal growth factor receptor function

Chemoattractants expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of human prostate tumor cells to these organs. Epidermal growth factor (EGF) is a potent chemoattractant for several human metastatic prostate tumor cell lines, including the TSU-pr1 cell line, and EGF has been localized to the stroma of both bony sites and pelvic lymph nodes in humans. Hence, we investigated whether the TSU-pr1 cell line expresses a functional EGF receptor (EGFR), which when antagonized reduces EGF-mediated chemomigration of this cell line. In this context, the EGFR immunoprecipitated from cell lysates of TSU-pr1 cells comigrated with the EGFR from A431 cells at a molecular weight of 170 kD. Addition of human EGF (hEGF) to the TSU-pr1 cells for 5 min stimulated the dose-dependent biphasic phosphorylation of the EGFR, with maximal stimulation of EGFR phosphorylation occurring at 2 ng/ml hEGF. In addition, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with 0.5 μg/ml anti-hEGFR monoclonal antibody or 100 nM staurosporine inhibited EGFR phosphorylation. Conversely, as negative controls, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with K252a or dimethyl sulfoxide (DMSO) vehicle did not inhibit EGFR phosphorylation. TSU-pr1 cells were stimulated to migration in 4 hr across Boyden chambers in response to 10 ng/ml hEGF. Treatment of the TSU-pr1 cells with anti-hEGFR monoclonal antibody inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. Similarly, treatment of the TSU-pr1 cells with staurosporine inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. These results demonstrate that antagonists of hEGF-mediated hEGFR phosphorylation also antagonize chemomigration of the TSU-pr1 cells across Boyden chambers, suggesting that antagonists of the EGFR in prostate cancer may be useful in the treatment of metastatic disease. © 1996 Wiley-Liss, Inc.     

Regulation of osteosarcoma EGF receptor affinity by phorbol ester and cyclic AMP

We studied the binding and degradation of 125I-labeled epidermal growth factor (EGF) by UMR-106 osteosarcoma cells and the regulation of EGF receptor affinity for EGF by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by treatments that raise intracellular levels of cyclic AMP. Cell surface binding of [125I]EGF to A431 cells reached a plateau after a 30 minute incubation at 37 degrees C but was undetectable in UMR-106 cells. Degradation of [125I]EGF proceeded at a 50-fold higher rate in A431 cells on a per cell basis, but receptor-bound [125I]EGF was internalized and degraded at a 3.5-fold higher rate by UMR-106 cells on a per receptor basis. At 4 degrees C, [125I]EGF labeled a single class of surface binding sites in the UMR-106 cell. Treatment with TPA at 37 degrees C reduced subsequent cell surface binding of [125I]EGF at 4 degrees C a maximum of 80% with an IC50 of 1.25 ng/ml. Maximal TPA reduction of [125I]EGF binding was observed within 5-15 minutes and was due to a reduction in the affinity of cell surface receptors of [125I]EGF without a change in receptor density. Pretreatment of the cells for 4 h with 30 microM forskolin, 1 mM isobutylmethylxanthine (IBMX) plus 30 microM forskolin, or 1 mM IBMX plus 100 ng/ml parathyroid hormone (PTH) attenuated the loss in [125I]EGF binding caused by a subsequent dose of 10 ng/ml of TPA by 17% (p less than 0.0005), 39% (p less than 0.0002), and 35% (p less than 0.002), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Augmentation of 5-fluorouracil cytotoxicity by epidermal growth factor in a newly established human signet-ring cell carcinoma of the stomach in culture

A cell line designated TSG6 was established from a signet-ring cell gastric carcinoma developed in a 57-year-old female patient. The TSG6 cells had well preserved the features of signet-ring cell carcinoma based on morphology. The cells exhibited both epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) immunoreactivities, and also secreted EGF. Moreover, the growth of TSG6 cells was stimulated in the presence of exogenous EGF. These results suggest that the possible presence of an EGF/EGFR autocrine growth mechanism is expressed in the TSG6 cells. The simultaneous treatment with EGF and 5-fluorouracil (5-FU) produced a nearly 2.4-fold enhancement of 5-FU cytotoxicity against TSG6 cells. A bromodeoxyuridine/DNA How cytometry analysis revealed that EGF augmented 5-FU cytotoxicity by inducing the accumulation of S phase cells which might be more susceptible to 5-FU. Moreover, we found that the incorporation of 5-FU into the TSG6 cells was increased with the addition of EGF. These data indicate that EGF may be a potent agent as a biological response modifier for 5-FU against the tumors which express the EGF/EGFR autocrine mechanism, and that the TSG6 cell line is useful in furthering our understanding of the interaction between anticancer drugs and EGF.     

Overexpression and regulation of expression of scatter factor/hepatocyte growth factor in prostatic carcinoma

Objectives. Scatter factor (hepatocyte growth factor) (SF/HGF) is a multifunctional polypeptide growth factor that has been implicated in tumor proliferation, angiogenesis, invasiveness, and metastasis. Little is known of the expression of SF/HGF in human prostatic carcinoma. The aims of this investigation were to quantitate the level of SF/HGF expression in benign versus malignant human prostatic tissues and to assess regulation of SF/HGF expression by human prostatic stromal myofibroblasts.Methods. We determined the level of SF/HGF expression in 10 human prostatic tissue samples (5 benign, 5 carcinoma) by Western blot analysis. Five purified growth factors—basic fibroblast growth factor (bFGF), interleukin-1beta (IL-1β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and endothelial growth factor (EGF)—were tested for their capacity to induce SF/HGF expression by a human prostatic stromal myofibroblastic cell line, as assessed by enzyme-linked immunosorbent assay. Supernatant from the normal PrEC prostatic epithelial cell line and the DU 145 carcinoma cell line were assayed for SF/HGF-inducing activity.Results. SF/HGF exhibited a mean fourfold overexpression in carcinoma tissues compared with benign prostatic tissue. Significant stimulation of SF/HGF expression by prostatic stromal myofibroblasts was detected for IL-1β (8.1-fold), PDGF (6.2-fold), bFGF (4.0-fold), VEGF (3.7-fold), and EGF (2.9-fold). DU 145-conditioned media, but not the PrEC-conditioned media, contained SF/HGF-inducing activity, which was determined to include IL-1β, bFGF, and PDGF by antibody-blocking experiments.Conclusions. SF/HGF is overexpressed in human prostatic carcinoma tissues. Prostatic carcinoma cell stimulation of SF/HGF expression by adjacent benign myofibroblastic cells as a type of epithelial–stromal paracrine interaction could potentially influence prostatic carcinoma cell behaviors.     

Role of membrane type 1-matrix metalloproteinase and gelatinase A in head and neck squamous cell carcinoma invasion in vitro.

The proteolytic activity of gelatinase A, a member of the matrix metalloproteinase (MMP) family, is considered to be a critical factor in tumor cell penetration of the extracellular matrix. To express catalytic activity, however, gelatinase A requires activation by another MMP, membrane type 1-matrix metalloproteinase (MT1-MMP). The head and neck squamous cell carcinoma cell line, UM-SCC-1, forms a quiescent monolayer atop collagen unless stimulated with epidermal growth factor (EGF; 3.5 nmol/L), which induces single cell invasion within 48 hours. To determine the role of the MT1-MMP/gelatinase A protease system in an in vitro stromal invasion model, expression vectors for MT1-MMP and gelatinase A were transfected into UM-SCC-1 (SCC-1/MT and SCC-1/gelA, respectively). SCC-1/MT tumor cells were found to invade in the absence of growth factor stimulation. Additionally, these cells displayed shorter onset to invasion and penetrated deeper into the collagen gel with EGF stimulation than did control vector transfectants. SCC-1/gelA cells similarly demonstrated invasion in the absence of EGF and a heightened invasive potential under EGF-stimulated conditions. These results suggest that the MT1-MMP/gelatinase A protease system participates in squamous cell carcinoma invasion of collagenous matrices.     

Instant hepatic differentiation of human embryonic stem cells using activin A and a deleted variant of HGF

Human embryonic stem (hES) cells have the ability to differentiate into a variety of different cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. Here we investigated an efficient method of hepatic differentiation from hES cells. A human ES cell line, KhES-1, was used and maintained by a nonfeeder method. KhES-1 cells were cultured for 5 days in the presence of human activin A (50 ng/ml) and then treated with a deleted variant of hepatocyte growth factor (dHGF) at 0, 100, or 500 ng/ml for 7 days. The resultant cells were biologically analyzed. The expression of the endodermal genes SOX17 and FOXA2 increased in KhES-1 cells after activin A treatment. In contrast, Oct4, a self-renewal undifferentiated marker, decreased in a time-dependent manner in KhES-1 cells. Following a 7-day treatment of the resultant cells with dHGF, especially at 500 ng/ml, KhES-1 cells showed an expression of the hepatic makers albumin, AFP, and CK18. Transitional electron microscopy showed well-developed glycogen rosettes and a gap junction in KhES-1 cells treated with 500 ng/ml of dHGF. We developed an efficient method to differentiate KhES-1 cells into hepatocyte-like cells in vitro using 50 ng/ml of activin A and 500 ng/ml of dHGF.     

重组人生长激素对体外培养的Bel-7402肝癌细胞生长激素受体的调控作用

目的 了解重组人生长激素(rhGH)对体外培养的Bel-7402肝癌细胞生长激素受体(GHR)的调控作用.方法 采用放射配体法了解7402肝癌细胞生长激素受体(GHR)的表达情况,检测不同浓度(0,1,10,100,1000,10 000 ng/ml)rhGH对肝癌细胞GHR表达的影响,用辣根过氧化酶法了解7402肝癌细胞培养上清液的胰岛素样生长因子-Ⅰ(IGF-Ⅰ)的变化,采用肿瘤细胞计数、噻唑蓝比色法了解肝癌细胞在不同浓度rhGH作用下药物敏感性,计算细胞生长率;用流式细胞计了解肝癌细胞在上述浓度rhGH作用下细胞周期各时相细胞比率、细胞增殖指数(PI)以及凋亡率变化.结果 放射配体法发现7402肝癌细胞表达GHR,在rhGH作用后24 h,7402肝癌细胞GHR的位点数量在10与100 ng/ml实验组较对照组增加(P<0.05),在10 000 ng/ml组较对照组降低(P<0.05);rhGH作用后的24 h与48 h,7402肝癌细胞上清液IGF-Ⅰ浓度在100 ng/ml组较对照组显著增高(P<0.05);与此同时,rhGH对体外培养的7402肝癌细胞增殖具有一定程度刺激作用,表现为rhGH在100 ng/ml浓度时促进肝癌细胞增殖较显著,而rhGH在其它浓度(1,10,1000,10 000 ng/ml)对肝癌细胞的增殖虽能表现出一定程度促进作用,但总体效果较rhGH在100 ng/ml浓度为弱.rhGH作用48 h流式细胞检测发现各实验组S期所占比率、PI均较对照组升高(P<0.05);G2-M细胞所占比率,10 ng/ml组与100 ng/ml组较对照组显著增高(P<0.05);各实验组凋亡率与对照组比较无显著差异.结论 rhGH可促进7402肝癌细胞DNA的合成,提高肿瘤细胞分裂增殖能力,原因可能与7402肝癌细胞表达GHR,rhGH可在一定浓度范围内上调7402肝癌细胞GHR的表达,促进肿瘤细胞合成IGF-Ⅰ有关,其确切途径仍有待进一步验证.     

人食管鳞癌细胞系RJEC-2的建立及其生物学特性分析

目的:建立新的人食管鳞状细胞癌细胞系并分析其生物学特性,为食管癌分子机制和治疗干预的研究提供新的实验模型。方法:采用组织块培养法,从病人食管癌组织中分离纯化鳞状上皮癌细胞并建立细胞系。对细胞系的形态特点、细胞角蛋白表达、生长特征、细胞周期分布、细胞遗传特征和致瘤能力进行了研究分析。结果:建立了食管鳞状细胞癌细胞系RJEC-2,已在体外持续传代4个多月,传至46代,生长稳定;该细胞系具有鳞状上皮细胞的形态和特点:单层贴壁生长,免疫组化显示细胞角蛋白表达阳性,电镜下可见明显的胞质内张力纤维束和细胞间桥粒;群体倍增时间为46.5 h,细胞培养至90%汇合时,细胞周期分析显示G0/G1期平均占56.72%,S期平均占33.96%,G2/M期平均占9.32%;细胞染色体结构和数量异常,呈现肿瘤细胞特性;细胞呈克隆性生长,平板克隆平均形成率为13.93%,裸鼠移植瘤实验表明细胞具有致瘤能力,病理分析显示移植瘤与病人肿瘤病理形态特征相似,均为中、高分化鳞状细胞癌。结论:成功建立的人食管鳞癌细胞系RJEC-2,为食管癌发病机制和治疗方案的研究提供了新的研究实验模型。     

An autopsy case of primary intracranial squamous cell carcinoma

An autopsied case of primary intracranial squamous cell carcinoma (PISCC) is reported, and 25 previously reported cases of PISCC, followed by the Garcia's criteria, are reviewed. A 72-year-old female was admitted to our service with chief complaints of headache and nausea on March 30, 1988. She had no neurological deficits on admission. However, CT examination revealed a round mass lesion in the left hypothalamus with dislocation of the brain stem. The cerebrospinal fluid (CSF) examination showed squamous cell carcinoma cytologically, and slightly higher levels of beta-HCG (13.0 ng/ml) and CEA (14.2 ng/ml). Because of progressive worsening in the level of her consciousness, total removal of a suprasellar tumor was performed on April 19, 1988. Gross appearance of the tumor was yellowish, soft and encapsulated. Histologically, it was squamous cell carcinoma. She did well for several days after the operation, then deteriorated. Finally she expired because of dissemination of the carcinoma on May 14, 1988. Postmortem examination revealed a large mass of squamous cell carcinoma in her right cerebellopontine angle. Except for that in the brain, no cancer was found in her body. Immunohistological study of the tumor specimen demonstrated positive for HCG in some of the large-sized neoplastic cells. Twenty-six cases of PISCC have been reported previously, so far. However, 21 cases out of the 26 PISCC were thought to have originated from intracranial epidermoid, one from the dermoid and the other one from craniopharyngioma. In the other three cases of PISCC, including the present case, the origin of the tumor was not able to be identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

基因重组人生长激素对体外培养的肝癌细胞增殖的影响及配体调控

目的 了解基因重组人生长激素(rhGH)对体外培养的Bel-7402细胞增殖的影响,并探讨rhGH对肝癌细胞生长激素受体(GHR)的调控作用.方法 分别采用肿瘤细胞计数、MTT比色法和集落形成实验等方法 了解Bel-7402细胞株在不同浓度rhGH(0、1、10、100、1000、10 000 ng/ml)作用下的药物敏感性,计算细胞生长率;用3H-TdR掺入法研究肿瘤细胞DNA代谢情况;应用放射配体法了解Bel-7402细胞GHR的表达,以及rhGH在上述浓度下对肝癌细胞GHR的影响.结果 rhGH对体外培养的Bel-7402细胞的生长有一定程度促进作用,表现为rhGH在100 ng/ml浓度下促进肝癌细胞增殖较为显著,rhGH在其他浓度(1、10、1000、10000 ng/ml)的部分时段,对肝癌细胞的增殖虽亦有一定程度的促进作用,但总体效果较rhGH在100 ng/ml浓度时弱;放射配体法发现Bel-7402细胞表达GHR,在rhGH作用后24 h,Bel-7402细胞GHR的位点数量(103个/细胞)在10、100 ng/ml组较对照组显著增加,在10000ng/ml组较对照组显著减少.结论 一定浓度范围rhGH对Bel-7402细胞的增殖有促进作用,原因可能与Bel-7402细胞表达GHR有关;同时rhGH对Bel-7402细胞的GHR有一定调控作用.     

Adenoviral-mediated gene transfer of interleukin-2 in a human breast cancer cell line and a fresh primary breast cancer culture.

Adenovirally-mediated cytokine gene transfer has proven safe in the treatment of metastatic breast cancer. Unfortunately, the optimal conditions for gene transfer in the human breast remain largely unknown. Viral-mediated gene transfer was studied in a human breast cancer cell line and a fresh primary breast cancer culture using a type five adenoviral vector (AD5) containing the human interleukin 2 (IL-2) gene driven by a cytomegalovirus (CMV) promoter (AD5.CMV-IL2). IL-2 production was measured using an enzyme-linked immunosorbent assay (ELISA). In the human breast cancer cell line (MCF-7), IL-2 production increased logarithmically with viral dose and demonstrated peak production at 2000 ng/10(6) cells/24 h using a multiplicity of infection (MOI) of 3000:1. Transduction at a higher MOI resulted in cell death. IL-2 concentration reached over 2000 ng/ml 2 days after transduction and peaked 13 days after transduction at 5700 ng/ml. IL-2 levels declined thereafter. A fresh primary breast cancer culture, transduced with Ad5.CMV-IL2 at an MOI of 1000:1, secreted IL-2 at 15 ng/24 h 1 day after transduction and peaked at 85 ng/24 h 5 days after transduction. Adenoviral-mediated gene transfer was accomplished in breast cancer cells with high efficiency across a wide range of conditions. The optimal IL-2 dose required to maximally stimulate the immune system remains unknown.     

The effect of quercetin on topotecan cytotoxicity in MCF-7 and MDA-MB 231 human breast cancer cells

BACKGROUND: Topotecan, which is a Camptothecin derivative, shows a large spectrum in anti-tumor activity. Topotecan exerts its cytotoxic effect on tumor cells mainly by inhibition of topoisomerase I activity resulting in double-strand DNA breaks. In our study, we investigated the combined cytotoxic action of Topotecan and Quercetin in MCF-7 and MDA-MB 231 human breast cancer cells. To examine the possible relation between the cytotoxic activity of Topotecan and oxidative stress, we measured ROS and nitrite levels in both human breast cell lines. MATERIALS AND METHODS: MCF-7 and MDA-MB 231 cells were exposed to Topotecan, Quercetin, or a combination of both agents for 24 h at 37 degrees C. The viability of the cells was measured using the colorimetric MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. We determined reactive oxygen species and nitrite levels as indicators of oxidative stress in both cell lines with and without Topotecan and/or Quercetin incubations using fluorometric dichlorofluorescin diacetate (DCFH-DA) and diaminonaphtalene (DAN) assay. RESULTS: The IC(50) concentration of Topotecan was 100 ng/ml in MCF-7 cell line and 160 ng/ml in MDA-MB231 cell line. Treatment with Quercetin enhanced cytotoxicity of Topotecan as 1.4-fold in MCF-7 and 1.3-fold in MDA-MB-231 cell line. A significant increment on ROS and nitrite levels was found in MCF-7 and MDA-MB-231 cells following Topotecan incubation. CONCLUSIONS: Our results suggest that Topotecan has cytotoxic activity against both of the breast cancer cell lines in vitro. A combination with Quercetin increases efficacy of Topotecan in the treatment of breast cancers. Our results indicate that increased oxidative stress plays a role in the cytotoxic action of Topotecan.