In vitro exposure to human immunodeficiency virus type 1 induces apoptotic cell death of the factor-dependent TF-1 hematopoietic cell line

In this study, we evaluated the effect of a short-term exposure (2 hours) to two different lymphocytotropic strains of human immunodeficiency virus type 1 (HIV-1; HIVIIIB and ICR-3) on the survival of a factor-dependent CD34+ hematopoietic progenitor cell line (TF-1). At flow cytometry analysis, a significant (P < .05) increase in the frequency of apoptotic cell death was observed in HIV-1-treated TF- 1 cells, supplemented with low doses of either interleukin-3 (IL-3; 0.02 to 1 ng/mL) or granulocyte-macrophage colony-stimulating factor (GM-CSF; 0.02 to 0.2 ng/mL) with respect to mock-treated cells. On the other hand, higher doses of both cytokines or combinations of suboptimal concentrations of IL-3 plus GM-CSF (eg, 0.2, plus 0.2 ng/mL) completely reversed the HIV-1-induced increase of apoptosis. Remarkably, no signs of productive or latent virus replication were ever observed in HIV-1-treated TF-1 cells up to 16 days of liquid culture. In parallel experiments, the in vitro exposure to HIVIIIB induced a significant and progressive increase of apoptotic death in purified bone marrow CD34+ cells, seeded in liquid cultures in the presence of 1 ng/mL IL-3. The HIV-1-induced apoptosis of TF-1 cells was likely triggered by the simple interaction of HIV-1 envelope glycoprotein gp120 with CD4 receptor, which was expressed at a low level on the surface of TF-1 cells. In fact, treatment of TF-1 cells with recombinant gp120 plus a polyclonal anti-gp120 antibody or with anti-CD4 monoclonal antibody plus rabbit antimouse IgG significantly increased the percentage of apoptotic death. These data suggest that HIV-1, and perhaps also free gp120 in the presence of anti-gp120 antibody; could play a direct role in the pathogenesis of peripheral blood cytopenias in acquired immunodeficiency syndrome patients by inducing apoptotic death of hematopoietic progenitor cells without the need of a direct infection.     

Sorafenib inhibits MAPK‐mediated proliferation in a Barrett's esophageal adenocarcinoma cell line

SUMMARY. Esophageal adenocarcinoma continues to rise in incidence. Despite recognition of Barrett's metaplasia as the histological precursor, prognosis remains poor. The mitogen‐activated protein kinases (MAPK) pathway is activated in Barrett's‐associated dysplasia and adenocarcinoma and this activation is, in part, due to acid and bile acid reflux. We investigated the effects of sorafenib, an orally active Raf‐inhibitor, on acid and bile acid‐stimulated growth and signaling in SEG‐1 cells, derived from a Barrett's esophageal cancer. SEG‐1 cells were pretreated with sorafenib or vehicle and subsequently stimulated with acid or bile acid. MAPK signals, including phospho‐ERK and phospho‐p38, as well as cyclin D1 expression were assessed by Western blotting. Cell proliferation was measured by WST‐1 colorimetric assay. Acid (pH 3.0–4.0) and bile acid (taurocholate 50–100 µmol/L) activated ERK and p38. Acid and bile acid exposure also increased levels of cyclin D1, a G1 to S cell cycle regulator. Furthermore, acid and taurocholate exposure increased cell proliferation. Sorafenib abrogated MAPK activation and cyclin D1 up‐regulation and significantly inhibited cell growth. In summary, sorafenib inhibits acid or bile acid‐stimulated Barrett's esophageal cancer cell proliferation by a mechanism involving the MAPK pathway. Our results suggest that sorafenib might be useful in the management of Barrett's‐associated dysplasia and adenocarcinoma. These findings provide a foundation for in vivo studies to assess the efficacy of sorafenib in Barrett's‐related neoplasia.     

Gene expression changes associated with Barrett's esophagus and Barrett's-associated adenocarcinoma cell lines after acid or bile salt exposure

Background  

Esophageal reflux and Barrett's esophagus represent two major risk factors for the development of esophageal adenocarcinoma. Previous studies have shown that brief exposure of the Barrett's-associated adenocarcinoma cell line, SEG-1, or primary cultures of Barrett's esophageal tissues to acid or bile results in changes consistent with cell proliferation. In this study, we determined whether similar exposure to acid or bile salts results in gene expression changes that provide insights into malignant transformation.     

s-thalidomide has a greater effect on apoptosis than angiogenesis in a multiple myeloma cell line

s-Thalidomide has proven efficacy in multiple myeloma. Although it has both antiangiogenic and pro-apoptotic effects, its primary mode of therapeutic action remains unclear. We have investigated the changes to the expression of genes involved with these cellular processes following culture with s-thalidomide in the U266 MM cell line. Cells were cultured with s-thalidomide (0-1000 microM), and cell parameters, including apoptosis, were assessed on day 3. RNA was extracted from cells cultured for 24 h at the IC(50) concentration of s-thalidomide, and changes to gene expression were investigated by microarray methodologies. A reduction in cell viability was observed in U266 cells cultured with s-thalidomide (IC(50): 362 microM), which were mirrored by significant increases in apoptosis (for example, 200 microM on day 3: 40.3+/-3.1% vs. 3.2+/-0.4% on day 0; P<0.001). There were changes in the expression profile of genes involved in angiogenesis and apoptosis, but the changes were most dramatic in the apoptotic genes. In particular, the expression of I-kappaB kinase was decreased by two-fold, which was associated with a four-fold decrease in NF-kappaB expression. These data correlated with immunoblotting analyses, which showed significant increases in I-kappaB protein levels and decreased NF-kappaB activity. Additionally, the Bax : Bcl-2 ratio was significantly increased. Our data suggest that both angiogenic and apoptotic genes and proteins are affected by s-thalidomide. Additionally, a dramatic decrease in Bcl-2 expression with s-thalidomide suggests a possible enhancement of cytotoxic effect if combined with other cytotoxic agents.     

去甲斑蝥酸钠对耐顺铂肺腺癌A549/DDP细胞的逆转作用及机制

Objective To investigate the reversal effect and the mechanism of sodium norcantharidate(SNCTD)on human lung adenocarcinoma cell line A549/DDP. Methods CKK assay was used to screen out non-toxic concentration (less than 10 percent of cell inhibition ratio) of SNCTD, and to measure the IC50 of cisplatin and IC50 of innoxious concentration SNCTD plus cisplatin in drug-resistant cell line. The accumulation effect of Rh123 was assayed by flow cytometry after treatment with non-toxic concentration of SNCTD. PT-PCR was used to detect the expression of mdr1, MRP1 gene for the drug-resistant cell line treated with non-toxic concentration of SNCTD for 48h. Results (1)The non-toxic concentration of SNCTD was 5μg/ml. SNCTD could decrease drug resistance to cisplatin. The reversal fold was 1.97. (2)The fluorescence intensity of Rh123 in the cells treated with 5μg/ml SNCTD was obviously increased (F=36.99, P＜0.05). (3)The expressions of mdr1, MRP1 gene decreased significantly in a concentration-dependent manner.Conclusions SNCTD could reverse the resistance to cisplatin in A549/DDP cell line. It possibly downregulates the expression of mdr1, MRP1 gene, and inhibits the function of efflux pump of membrance protein.     

去甲斑蝥酸钠对耐顺铂肺腺癌A549/DDP细胞的逆转作用及机制

Objective To investigate the reversal effect and the mechanism of sodium norcantharidate(SNCTD)on human lung adenocarcinoma cell line A549/DDP. Methods CKK assay was used to screen out non-toxic concentration (less than 10 percent of cell inhibition ratio) of SNCTD, and to measure the IC50 of cisplatin and IC50 of innoxious concentration SNCTD plus cisplatin in drug-resistant cell line. The accumulation effect of Rh123 was assayed by flow cytometry after treatment with non-toxic concentration of SNCTD. PT-PCR was used to detect the expression of mdr1, MRP1 gene for the drug-resistant cell line treated with non-toxic concentration of SNCTD for 48h. Results (1)The non-toxic concentration of SNCTD was 5μg/ml. SNCTD could decrease drug resistance to cisplatin. The reversal fold was 1.97. (2)The fluorescence intensity of Rh123 in the cells treated with 5μg/ml SNCTD was obviously increased (F=36.99, P＜0.05). (3)The expressions of mdr1, MRP1 gene decreased significantly in a concentration-dependent manner.Conclusions SNCTD could reverse the resistance to cisplatin in A549/DDP cell line. It possibly downregulates the expression of mdr1, MRP1 gene, and inhibits the function of efflux pump of membrance protein.     

去甲斑蝥酸钠对耐顺铂肺腺癌A549/DDP细胞的逆转作用及机制

目的 探讨去甲斑蝥酸钠(SNCTD)对耐顺铂人肺腺癌细胞系A549/DDP的逆转作用及可能分子机制.方法 (1)采用CCK法筛选出SNCTD对A549/DDP的无毒浓度(即对细胞抑制率＜10%的药物浓度),并检测出顺铂及与无毒浓度SNCTD联合对耐药细胞株的IC50;(2)采用流式细胞仪检测无毒浓度SNCTD对细胞内罗丹明123的蓄积情况;(3)采用反转录-聚合酶链反应(RT-PCR)检测1、2倍无毒浓度SNCTD处理细胞48h后耐药相关基因mdrlmRNA,MRP1 mRNA的表达.结果 (1)SNCTD对A549/DDP的无毒浓度为5μg/ml,与顺铂联合用药降低A549/DDP的耐药性,逆转倍数为1.97;(2)无毒浓度SNCTD处理耐药细胞48h后细胞内罗丹明123荧光强度明显增强(F=36.99,P＜0.05);(3)1、2倍无毒浓度SNCTD处理耐药细胞后mdr1,MRP1mRNA表达明显减低,并具有浓度依赖性.结论 SNCTD对A549/DDP具有耐药逆转作用,其作用机制可能与下调耐药相关基因mdr1,MRP1的表达,影响膜蛋白外排泵功能有关.     

In vivo and in vitro antitumor effect of a unique nutrient mixture on lung cancer cell line A-549

The high incidence of lung cancer and ineffective toxic action of current mono and doublet chemotherapy approaches result in poor patient survival. Further, matrix metalloproteinases (MMPs) are implicated in neoplastic invasion and metastasis. Based on this, the authors investigated the effect of a dietary micronutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on the tumor growth of human lung carcinoma cell A-549 xenografts in athymic nude mice. Additionally, the authors tested the in vitro antitumor effect of NM on lung carcinoma A-549 cells by measuring cell proliferation by MTT assay, MMP-2 and -9 secretion by gelatinase zymography, and cell invasion through Matrigel. Nutrient supplementation strongly suppressed the growth of tumors without adverse effects in nude mice; tumor weight was reduced by 44% (P = .0001) and tumor burden was reduced by 47% (P < .0001) with supplementation. Zymography demonstrated in vitro secretion of MMP-2 by uninduced human lung carcinoma cells and both MMP-2 and -9 by phorbol 12-mysristate 13-acetate (PMA) (200 ng/mL)-treated cells. NM inhibited the secretion of both MMPs in a dose-dependent fashion, with virtual total inhibition at 500 microg/mL concentration. The invasion of human lung carcinoma cells through Matrigel was significantly reduced at 100 microg/mL (64%) and totally inhibited at 500 microg/mL concentration of NM (P = .01). Suppression of lung tumor growth in nude mice and inhibition of MMP secretion and Matrigel invasion suggest NM may act as an anticancer agent and as such warrants further investigation.     

In vitro establishment and characterization of a human megakaryoblastic cell line

A human megakaryoblastic cell line, designated CHRF-288-11, has been established in vitro through the use of adherent stromal cells in long-term human bone marrow culture. Long-term bone marrow cultures were required for the initial adaptation of the megakaryoblastic cells to culture conditions; however, once adapted, the cells were weaned from the stromal layer until they proliferated in the complete absence of any feeder layers. The seed cells for the establishment of this line were derived from a solid tumor; the cloned cell line derived from this tumor exhibits markers characteristic of megakaryocytes and platelets. Specifically, the cells express platelet peroxidase, platelet factor 4, and platelet Ca+(+)-adenosine triphosphatase (ATPase), glycoprotein IIb-IIIa (CDw41), factor VIII antigen, and the MY7 (CD13) and MY9 (CD33) antigens. The cells do not express the erythroid markers glycophorin A and hemoglobin, the myeloid marker myeloperoxidase, nor markers specific for T and/or B cells. The established cell line produces both basic fibroblast growth factor and transforming growth factor-beta, properties demonstrated previously for the solid tumor. The clonal cell population exhibited a unique, singular karyotype, indicating cellular homogeneity. The cells display a doubling time of approximately 33 hours in either 25% horse or calf serum. Treatment of the cells with 1 X 10(-8) mol/L phorbol 12-myristate 13-acetate (PMA) leads to the induction of multi-nucleation and hyperploidy in the cells, with approximately 35% of the cells exhibiting two or more nuclei per cell, and greater than 80% of the cells enlarging in size. The establishment of this unique cell line under defined culture conditions will be beneficial for the future study of megakaryocytic properties expressed by this cell line.     

Cyclooxygenase 2 expression in Barrett's esophagus and adenocarcinoma: Ex vivo induction by bile salts and acid exposure

BACKGROUND & AIMS: Barrett's esophagus (BE) results from chronic, severe gastroesophageal reflux and predisposes to esophageal adenocarcinoma. Cyclooxygenase (COX)-2 is involved in chronic inflammation and epithelial cell growth. We investigated COX-2 expression in BE and esophageal adenocarcinoma to explore a potential relation between COX-2 expression and metaplasia or carcinogenesis. METHODS: Endoscopic mucosal biopsy specimens of Barrett's intestinal metaplasia (n = 30), Barrett's dysplasia (n = 11), and esophageal adenocarcinoma (n = 5) were compared with normal esophagus (n = 46) and duodenum (n = 46) and analyzed by Western blotting and immunohistochemistry. RESULTS: Immunoblots revealed constitutive expression of COX-2 in normal esophagus and duodenum. COX-2 protein expression was significantly higher in patients with Barrett's metaplasia, dysplasia, and adenocarcinoma compared with normal squamous esophageal or columnar duodenal epithelia and was heterogenous in different regions of the BE surface. Immunohistochemistry revealed prominent staining in the glands of BE, dysplasia, and adenocarcinoma and faint staining in the basal layers of squamous esophagus and the surface of the duodenum. In response to pulses of acid or bile salts in an ex vivo organ culture system, COX-2 expression increased significantly in BE tissues, and this effect was attenuated by the selective COX-2 inhibitor NS-398. CONCLUSIONS: The results show COX-2 expression in normal esophagus, which increases significantly in BE and esophageal adenocarcinoma. COX-2 is regulated ex vivo by exposure to acid or bile salts.     

Growth inhibitory effect of wild-type Kras2 gene on a colonic adenocarcinoma cell line

AIM: To observe the growth inhibitory effect of wild-type Kras2 gene on a colonic adenocarcinoma cell line Caco-2. METHODS: Recombinant plasmid pCI-neo-Kras2 with wild type Kras2 open reading frame was constructed. The Caco-2 cells were transfected with either pCI-neo or pCI-neo-Kras2 using Lipofectamine 2000. The expression of wild type Kras 2 was examined by Northern blot analysis. And the expression of wild type Kras2 protein was examined by Western blot analysis. The effects of wild-type Kras2 on cell proliferation were analyzed by monotetrazolium (MTT) assay, meanwhile analyses of cell cycle and spontaneous apoptosis rate were carried out by flow cytometry (FCM). RESULTS: The plasmid of pCI-neo-Kras 2 was successfully established. The growth rate of cells transfected with pCI-neo-Kras2 was significantly lower than the control cells transfected with the empty pCI-neo vector (P < 0.05). Cell cycle analysis revealed arrest of the pCI-neo-Kras2 transfected cells in G0/G1 phases, decreased DNA synthesis and decreased fractions of cells in S phase. The proliferative index of cells transfected with pCI-neo-Kras2 was decreased compared with the control cells (49.78% vs 64.21%),while the apoptotic rate of Caco-2 cells with stable Kras 2 expression increased (0.30% vs 0.02%). CONCLUSION: The wild-type Kras2 gene effectively inhibits the growth of the colonic adenocarcinoma cell line Caco-2.     

Capsaicin-induced cell death in a human gastric adenocarcinoma cell line

AIM: Capsaicin, a pungent ingredient found in red pepper,has long been used in spices, food additives, and drugs.Cell death induced by the binding of capsaicin was examined in a human gastric adenocarcinoma cell line (AGS cells).METHODS: By using XTT-based cytotoxicityassay, flow cytometry using the TUNEL method, and quantitation of DNA fragmentation, both cell death and DNA fragmentation were detected in AGS cells treated with capsaicin. By using Western blotting methods, capsaicin reduced the expression of Bcl-2, the antiapoptotic protein, in AGS cells in a concentration-dependent manner.RESULTS: After incubation of AGS cells with capsaicin for 24 h, cell viability decreased significantly in a dose-dependent manner. After incubation of AGS cells with capsaicin for 24 h, apoptotic bodies also significantly increased, and were again correlated with the dose of capsaicin. When the concentration of capsaicin was 1 mmol/L, the amount of DNA fragments also increased. Similar results werealso in the lower traces.CONCLUSION: These results suggest that capsaicininduced cell death might be via a Bcl-2 sensitive apoptotic pathway. Therefore, capsaicin might induce protection from gastric cancer.     

Capsaicin-induced cell death in a human gastric adenocarcinoma cell line

AIM: Capsaicin, a pungent ingredient found in red pepper, has long been used in spices, food additives, and drugs. Cell death induced by the binding of capsaicin was examined in a human gastric adenocarcinoma cell line (AGS cells). METHODS: By using XTT-based cytotoxicity assay, flow cytometry using the TUNEL method, and quantitation of DNA fragmentation, both cell death and DNA fragmentation were detected in AGS cells treated with capsaicin. By using Western blotting methods, capsaicin reduced the expression of Bcl-2, the antiapoptotic protein, in AGS cells in a concentration-dependent manner. RESULTS: After incubation of AGS cells with capsaicin for 24 h, cell viability decreased significantly in a dose-dependent manner. After incubation of AGS cells with capsaicin for 24 h, apoptotic bodies also significantly increased, and were again correlated with the dose of capsaicin. When the concentration of capsaicin was 1 mmol/L, the amount of DNA fragments also increased. Similar results were also in the lower traces. CONCLUSION: These results suggest that capsaicin-induced cell death might be via a Bcl-2 sensitive apoptotic pathway. Therefore, capsaicin might induce protection from gastric cancer.     

Signaling pathways activated in a unique mast cell line where interleukin-3 supports survival and stem cell factor is required for a proliferative response

Stem cell factor (SCF) and interleukin-3 (IL-3) both act on several target hematopoietic populations, including mast cells. We have isolated a unique murine mast cell line, B6M, that is phenotypically similar to immature mast cells. For B6M cells, IL-3 is a survival factor and alone does not stimulate proliferation. SCF can stimulate proliferation of B6M cells, and together IL-3 and SCF synergize to stimulate optimal proliferation and long-term growth. A sustained induction of c-myc is observed only in the presence of SCF (with or without IL-3). In B6M cells, both IL-3 and SCF stimulate phosphorylation of Shc and activation of the Ras, Raf-1, MAPK pathway. Interestingly, IL-3 plus SCF synergistically activate MAPK. IL-3, but not SCF, leads to activation of Jak 2 and Stat 5 and induces pim-1 expression. From these data, we suggest that the induction of pim-1 and c-myc is independently regulated. Furthermore, IL-3-stimulated activation of the Jak 2/Stat 5 pathway, induction of pim-1, and activation of the Ras/MAPK pathway are insufficient to mediate proliferation of B6M cells. The unusual IL-3 response of B6M cells provides a useful model to dissect signals required for IL-3-stimulated survival and proliferation.     

Aminoguanidine has both an anti-inflammatory effect on experimental colitis and a proliferative effect on colonic mucosal cells

BACKGROUND: The aim of this study was to assess the effect of aminoguanidine (AG) on developed colitis and cell proliferation. METHODS: Colitis was induced by means of trinitrobenzene sulphonic acid (TNB) in male Wistar rats weighing about 250 g. Seven days after induction of TNB colitis the rats were divided into two groups at random, and one group was orally treated with 1.5 micromol/kg AG each day. We assessed the effect of AG by measuring the mucosal damage, the ulcer area, myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOs) activity, and nitrogen oxide in serum 7 days after the beginning of treatment. RESULTS: AG significantly ameliorated the macroscopic damage score (AG versus control, 5.25 +/- 0.80 versus 7.50 +/- 0.50), the microscopic damage score (5.88 +/- 1.13 versus 9.25 +/- 0.31), ulcer area (0.57 +/- 0.14 versus 1.24 +/- 0.17 cm2), decreased MPO activity (51.5 +/- 9.4 versus 192.2 +/- 60 units/g tissue), and nitrogen oxide in serum (27.2 +/- 1.4 versus 32.3 +/- 1.8 microM) but did not decrease iNOs activity (8732 +/- 435 versus 8672 +/- 357 cpm/g tissue). Moreover, AG accelerated T84 cell proliferation in a dose-dependent manner. CONCLUSIONS: These results suggest that AG ameliorates TNB colitis not only by its anti-inflammatory effect but also by accelerating the proliferation of colonic mucosal cells. AG, accordingly, might well be a useful new medicine to ameliorate inflammatory bowel disease.     

Upregulation of the oncogene c-myc in Barrett's adenocarcinoma: induction of c-myc by acidified bile acid in vitro

BACKGROUND AND AIMS: C-myc over expression is implicated in malignancy although to date this has not been studied in Barrett's metaplasia. We sought to determine c-myc expression in the malignant progression of Barrett's metaplasia and whether it may be induced by bile acids seen in gastro-oesophageal refluxate. METHODS: C-myc protein and mRNA levels were assessed in 20 Barrett's metaplasia and 20 oesophageal adenocarcinoma samples by western blotting and real time polymerase chain reaction. Levels of c-myc and proliferation were also assessed in cell lines OE21, OE33, SW-480, and TE-7 stimulated with pulses or continuous exposure to the bile acids deoxycholic acid and chenodeoxycholic acid. RESULTS: C-myc protein was upregulated in 50% of Barrett's metaplasia and 90% of oesophageal adenocarcinoma samples compared with squamous, gastric, and duodenal controls. C-myc immunolocalisation in Barrett's metaplasia revealed discrete nuclear localisation, becoming more diffuse with progression from low to high grade dysplasia to adenocarcinoma. Both continual and pulsed bile acid induced c-myc at pH 4, with no effect at pH 7 or with acidified media alone. Pulsed bile acid treatment induced proliferation (p<0.05); in contrast, continuous exposure led to suppression of proliferation (p<0.05). CONCLUSIONS: We have shown upregulation of c-myc with malignant progression of Barrett's metaplasia and suggest that acidified bile may be a novel agent responsible for induction of this oncogene.     

Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway

OBJECTIVES: Ghrelin is a brain-gut peptide with GH-releasing and appetite-inducing activities and a widespread tissue distribution. Ghrelin is the endogenous ligand of the GH secretagogue receptor type 1a (GHS-R1a), and both ghrelin and the GHS-R1a are expressed in the pituitary. There are conflicting data regarding the effects of ghrelin on cell proliferation. A positive effect on proliferation and activation of the mitogen-activated protein kinase (MAPK) pathway has been found in hepatoma, adipose, cardiomyocyte and prostate cell lines. However, ghrelin has also been shown to have anti-proliferative effects on breast, lung and thyroid cell lines. We therefore examined the effect of ghrelin on the rat pituitary cell line GH3. METHODS: RT-PCR was used for the detection of GHS-R1a and pre-proghrelin mRNA expression in GH3 cells. The effect of ghrelin on cell proliferation was studied using [(3)H]thymidine incorporation; cell counting and the activation of the MAPK pathway were studied using immunoblotting and inhibitors of the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. RESULTS: GHS-R1a and ghrelin mRNA expression were detected in GH3 cells. Ghrelin, at 10(-10) to 10(-6) M concentrations, significantly increased [(3)H]thymidine incorporation (at 10(-9) M, 183+/-13% (means+/-s.e.m.) compared with untreated controls), while 12-phorbol 13-myristate acetate (PMA) at 10(-7) M (used as a positive control) caused a 212+/-14% increase. A reproducible stimulatory effect of desoctanoyl ghrelin was also observed on [(3)H]thymidine incorporation (135+/-5%; P<0.01 at 10(-9) M compared with control), as well as on the cell count (control 6.8 x 10(4)+/-8.7 x 10(3) cells/ml vs desoctanoyl ghrelin (10(-9) M) 1.04 x 10(5)+/-7.5 x 10(3) cells/ml; P<0.01). Ghrelin caused a significant increase in phosphorylated ERK 1/2 in immunoblotting, while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The positive effect of ghrelin and desoctanoyl ghrelin on [(3)H]thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPK-dependent pathway and via a tyrosine kinase-dependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. CONCLUSION: We have shown a novel role for ghrelin in stimulating the proliferation of a somatotroph pituitary tumour cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Desoctanoyl ghrelin showed a similar effect. As ghrelin has been shown to be expressed in both normal and adenomatous pituitary tissue, locally produced ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway.     

埃罗替尼对胰腺癌细胞BxPC3生长的影响及其机制

目的 观察表皮生长因子受体(EGFR)抑制剂埃罗替尼对体外培养的胰腺痛细胞BxPC3生长的影响,并探讨其作用机制.方法 应用MTT法检测埃罗替尼作用后BxPC3细胞的增殖情况;用流式细胞分析、透射电镜和原位末端标记(TUNEL)法观察细胞凋亡和细胞周期的变化;RT-PCR法检测细胞bcl-2、bax、bel-xl、bak mRNA表达.结果 埃罗替尼呈剂量和时间依赖性抑制BxPC3细胞生长.72h后,1、100μmol/L的埃罗替尼处理组细胞存活率分别为(90.25±2.62)%和(40.75±2.98)%,两者比较具有统计学意义(P<0.01).50 μmol,/L埃罗替尼处理BxPC3后24、96 h的细胞存活率分别为(74.0±4.08)%和(49.50 ±1.29)%,两者比较也具有统计学意义(P<0.01).50μmaol/L埃罗替尼处理组的细胞凋亡率为(11.0±1.1)%,显著高于对照组的(6.2±1.1)%(P<0.01);G_0/G_1细胞占(73.4 ±1.3)%,也显著高于对照组的(63.3 ±1.0)%;透射电镜可见细胞呈现明显凋亡形态,并见凋亡小体形成.埃罗替尼处理组细胞bcl-2、bcl-xl mRNA表达下调,bax mRNA表达轻微上调,bak mRNA的表达不受影响.结论 EGFR抑制剂埃罗替尼体外可抑制胰腺癌细胞系BxPC3的生长,其机制可能与阻滞细胞周期,上调促凋亡蛋白和下调凋亡抑制蛋白有关.