Inactivation of NF-kappaB by 3,3'-diindolylmethane contributes to increased apoptosis induced by chemotherapeutic agent in breast cancer cells

Constitutive activation of Akt or nuclear factor-kappaB (NF-kappaB) has been reported to play a role in de novo resistance of cancer cells to chemotherapeutic agents, which is a major cause of treatment failure in cancer chemotherapy. Previous studies have shown that 3,3'-diindolylmethane (DIM), a major in vivo acid-catalyzed condensation product of indole-3-carbinol, is a potent inducer of apoptosis, inhibitor of tumor angiogenesis, and inactivator of Akt/NF-kappaB signaling in breast cancer cells. However, little is known regarding the inactivation of Akt/NF-kappaB that leads to chemosensitization of breast cancer cells to chemotherapeutic agents, such as Taxotere. Therefore, we examined whether the inactivation Akt/NF-kappaB signaling caused by B-DIM could sensitize breast cancer cells to chemotherapeutic agents both in vitro and in vivo. MDA-MB-231 cells were simultaneously treated with 15 to 45 micromol/L B-DIM and 0.5 to 1.0 nmol/L Taxotere for 24 to 72 h. Cell growth inhibition assay, apoptosis assay, electrophoretic mobility shift assay, and Western blotting were done. The combination treatment of 30 micromol/L B-DIM with 1.0 nmol/L Taxotere elicited significantly greater inhibition of cell growth compared with either agent alone. The combination treatment induced greater apoptosis in MDA-MB-231 cells compared with single agents. Moreover, we found that NF-kappaB activity was significantly decreased in cells treated with B-DIM and Taxotere. We also have tested our hypothesis using transfection studies, followed by combination treatment with B-DIM/Taxotere, and found that combination treatment significantly inhibited cell growth and induced apoptosis in MDA-MB-231 breast cancer cells mediated by the inactivation of NF-kappaB, a specific target in vitro and in vivo. These results were also supported by animal experiments, which clearly showed that B-DIM sensitized the breast tumors to Taxotere, which resulted in greater antitumor activity mediated by the inhibition of Akt and NF-kappaB. Collectively, our results clearly suggest that inhibition of Akt/NF-kappaB signaling by B-DIM leads to chemosensitization of breast cancer cells to Taxotere, which may contribute to increased growth inhibition and apoptosis in breast cancer cells. The data obtained from our studies could be a novel breakthrough in cancer therapeutics by using nontoxic agents, such as B-DIM, in combination with other conventional therapeutic agents, such as Taxotere.     

Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells

Activating epidermal growth factor receptor (EGFR) mutations have been linked with sensitivity to gefitinib and erlotinib; however, there are no established predictive markers for response to the combination of EGFR inhibitors with standard chemotherapy in non-small cell lung cancer (NSCLC) patients. In this study, we characterized a panel of human EGFR wild-type and mutant NSCLC cells for their sensitivity to gefitinib alone and in combination with cisplatin or Taxol. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and crystal violet cell viability assays. Cell cycle distribution was measured by flow cytometry. EGFR expression was measured by flow cytometry, real-time PCR, and Western blotting. EGFR/Her2/Akt and extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation were measured by Western blotting. Two of nine EGFR wild type and one of two EGFR mutant NSCLC cells were sensitive to gefitinib, and this was associated with a decrease in phospho (p)-Akt and pErk1/2 following gefitinib exposure. There was no correlation between constitutive EGFR expression or activity and sensitivity to gefitinib nor was there a correlation between Her2/Akt and Erk1/2 activity and gefitinib sensitivity. However, in cells displaying a synergistic interaction between gefitinib and chemotherapy (cisplatin or Taxol), a dose-dependent increase in pEGFR was observed following chemotherapy exposure. In contrast, in cells where no change or a decrease in pEGFR following drug treatment was observed, we found an antagonistic or (at best) an additive interaction between the two compounds. Furthermore, the nature of this interaction was not dependent on the presence of a mutant EGFR. These novel findings suggest that modulation of EGFR activity following drug treatment determines response to gefitinib in combination with chemotherapy in NSCLC cells.     

Effect of an epidermal growth factor receptor tyrosine kinase inhibitor on actin remodeling in an in vitro bladder cancer carcinogenesis model

Alteration of actin remodeling is a marker of malignant-associated field defect and a potential surrogate biomarker for chemoprevention trials. We tested erlotinib, a specific tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), on actin remodeling in a bladder carcinogenic model consisting of untransformed HUC-PC cells and transformed MC-T11 cells, both derived from the same normal human urothelial clone immortalized by SV40. Erlotinib had a selective growth inhibitory and actin remodeling effect on MC-T11 cells over HUC-PC cells, as examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and immunofluorescence labeling with laser scan cytometer analysis, respectively. The IC(50) of untransformed HUC-PC cells was significantly higher than that of transformed MC-T11 cells (P < 0.05, t test). The actin remodeling effect was more prominent at lower dosage levels (1/8-1/4 of IC(50)), which was accompanied by an increased cell adhesion and decreased motility. At higher dosage levels (1/2 of IC(50)), erlotinib induced a decreased adhesion and anoikis (detachment-associated apoptosis). The transformed MC-T11, but not HUC-PC, showed a weak constitutive EGFR phosphorylation activity, which was inhibited by erlotinib in a dose-response manner. However, on epidermal growth factor stimulation, both cell lines showed a similar dose-response inhibitory effect on phosphorylated EGFR and mitogen-activated protein kinase (MAPK; P44/P42) activities, and MAPK inhibitor PD98059 showed no specific effect on erlotinib-induced actin remodeling, suggesting that pathways other than MAPK (P44/P42) may be responsible for erlotinib-induced actin remodeling. The findings provide evidence to support erlotinib-based bladder cancer chemoprevention and using actin remodeling as a marker for erlotinib-based intervention trials.     

应激状态下肠上皮细胞凋亡水平的变化及其机制

目的探讨在氧化应激状态下肠上皮细胞凋亡水平的变化以及凋亡异常发生的分子机制。方法使用过氧化氢(H2O2)处理培养的HT-29细胞模拟机体活性氧(ROS)损伤肠上皮细胞的体内状况,采用四甲基偶氮唑盐(MTT)微量酶反应比色法进行细胞生存力的检测;采用流式细胞术进行细胞凋亡的检测;采用蛋白质免疫印迹法(Westernblot)检测凋亡相关蛋白的表达。结果H2O2可降低HT-29细胞生存率,且呈现剂量依赖性和时间依赖性(P均<0.05);与空白对照组相比,随着H2O2浓度的增高,细胞凋亡率增加(P均<0.05),随着作用时间的延长,细胞凋亡率也增加(P<0.05);以不同浓度H2O2刺激HT-29细胞24h后发现,与空白对照组相比,Bax的表达随着H2O2浓度的增高而增加,Bcl-2的表达随着H2O2浓度的增高而降低。以500μmol/L,浓度的H2O2刺激HT-29细胞发现,Bax表达随着H2O2作用时间延长而增加,Bcl-2表达随着H2O2作用时间延长而降低。结论应激状态下,肠上皮细胞氧化应激水平与其凋亡程度相关,凋亡调控蛋白Bcl-2/Bax比值失调可能是肠上皮细胞凋亡过度的机制之一。     

A型肉毒毒素引起人增生性瘢痕成纤维细胞的凋亡

背景：A型肉毒毒素临床上可以治疗增生性瘢痕,体外细胞培养研究发现可以抑制增生性瘢痕成纤维细胞的增殖。目的：观察A型肉毒毒素对人增生性瘢痕成纤维细胞增殖的抑制作用,以及对人增生性瘢痕成纤维细胞凋亡的影响。方法：通过消化法分离培养出人增生性瘢痕成纤维细胞,分别用不同浓度的A型肉毒毒素对细胞的生长增殖过程进行干预,通过MTT染色,于酶联免疫检测仪570nm测定吸光度来研究细胞生长增殖情况,计算抑制率。通过Hoechst33342及PI染色检测人增生性瘢痕成纤维细胞凋亡隋况,并计算凋亡率。结果与结论：人增生性瘢痕成纤维细胞生长过程中呈梭形,细胞生长旺盛,细胞融合成单层,细胞排列成高度一致性。经A型肉毒毒素处理后,细胞增殖速度明显减慢,细胞数量减少,细胞排列方向散乱。MTT染色后吸光度减弱,随A型肉毒毒素浓度增加,吸光度明显减弱,与对照细胞比较,差异有显著性意义（P〈0．05）,半数抑制率出现在0．4IU/L。在荧光显微镜下,人增生性瘢痕成纤维细胞经Hoechst33342和PI染色后细胞核呈现蓝色,细胞核为光滑的圆形或椭圆形外观。A型肉毒毒素干预后细胞核致密浓缩,染色不均匀,折光性增强,核膜皱缩,部分细胞核碎裂,出现碎块,有凋亡小体出现。随着A型肉毒毒素浓度的增加细胞凋亡率逐渐增高,与对照细胞比较差异有显著性意义（P〈0．05）,半数凋亡率在0．4IU／L。说明A型肉毒毒素可以抑制人增生性瘢痕成纤维细胞的增殖,其主要通过引起细胞凋亡的途径来抑制成纤维细胞的增殖。     

Morphine improved the antitumor effects on MCF-7 cells in combination with 5-Fluorouracil

Background

The most frequently used opioid in cancer pain management is morphine which remains a cornerstone for the management of cancer pain, due to the largest experience existing among physicians and widely availability in a variety of formulation. Considering that analgesics on cancer pain is often under the condition of chemotherapy and 5-Fluorouracil (5-FU) is widely used today as a potent drug for the treatment of advanced cancers, whether analgesics such as morphine, interferes the chemotherapy such as 5-FU, arose as a considerable problem.

Methods

In this study, the MCF-7 breast cancer cells were used to determine the antitumor effects of the 5-FU in combination with morphine. The cell proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the apoptosis was determined by the Annexin V/PI staining and flow cytometry. The immunocytochemistry and western blot was used to determine the Bcl-2 and Bax expression.

Results

It was shown that in MCF-7 cells, the proliferation was inhibited, the apoptosis was promoted, the Bcl-2 expression was suppressed and the Bax expression was promoted by both 5-FU alone and morphine alone, while the superior effects were achieved in combination with the two drugs.

Conclusion

These results suggest that the morphine may have the beneficial effects on the antitumor chemotherapy with 5-FU, in stead of interferential effects.     

5-Methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1, exhibits activity against human pancreatic cancer in vitro and in vivo

The enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) has been found to be up-regulated in pancreatic cancer as well as many other solid tumors. A recent study showed that inhibition of NQO1 in pancreatic cancer cells using the nonselective inhibitor dicumarol suppressed the malignant phenotype. The authors suggested that inhibition of cell growth might result from an increase in intracellular superoxide production due to inhibition of NQO1. We have recently shown that NQO1 can directly scavenge superoxide and this effect may become physiologically relevant in cells containing high NQO1 levels. We therefore tested the hypothesis that 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a specific mechanism-based inhibitor of NQO1, would be an effective agent for the treatment of pancreatic tumors. The human pancreatic tumor cell lines BxPC-3 and MIA PaCa-2 contain high levels of NQO1 activity and protein as verified by immunoblot and immunocytochemical staining of human pancreatic tumor cells. ES936 treatment inhibited NQO1 activity by >98% in MIA PaCa-2 and BxPC-3 cells. In addition, ES936 treatment induced growth inhibition [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] in MIA PaCa-2 and BxPC-3 cells with an IC(50) of 108 and 365 nmol/L, respectively. Treatment of MIA PaCa-2 cells with ES936 also inhibited the ability of these cells to form colonies and grow in soft agar in a dose-dependent manner. Treatment of mice carrying MIA PaCa-2 xenograft tumors with ES936 resulted in a significant difference in growth rates in ES936-treated and DMSO-treated (control) tumors. Our data did not show an increase in either intracellular superoxide production or oxygen consumption after treatment of cells with ES936, contrary to the effects seen with dicumarol. In summary, mechanism-based inhibitors of NQO1, such as ES936, may be useful therapeutic agents for the treatment of pancreatic cancer, although the underlying mechanism seems to be independent of superoxide generation.     

人牙髓干细胞增殖与瞬时受体电位M7通道的作用

背景：瞬时受体电位M7通道广泛存在于机体组织和细胞,对细胞存活、增殖和维持细胞镁离子平衡是必须的.目的：探讨瞬时受体电位M7通道对人牙髓干细胞增殖的作用.方法：酶消化法分离培养人牙髓干细胞,运用不同浓度（50,100μmol/L）瞬时受体电位M7通道抑制剂2-氨基乙基二苯硼酸酯作用于人牙髓干细胞72 h后,Western-blot 检测2-氨基乙基二苯硼酸酯对瞬时受体电位M7通道蛋白水平的影响,MTT法观察其对人牙髓干细胞增殖的影响;构建特异抑制瞬时受体电位M7通道表达慢病毒载体,运用慢病毒感染人牙髓干细胞.进行RT-PCR和Western-blot沉默效果分析,在1,3,5,7 d进行MTT法检测特异沉默瞬时受体电位M7通道后对细胞增殖的影响.结果与结论：50,100μmol/L 2-氨基乙基二苯硼酸酯均能抑制瞬时受体电位M7通道蛋白水平的表达并且能明显抑制人牙髓干细胞增殖（P 〈0.01）,特异性沉默瞬时受体电位M7通道表达后,在不同时间段人牙髓干细胞增殖能力均明显降低（P 〈0.01）.提示瞬时受体电位M7通道对维持人牙髓干细胞增殖能力有重要作用.     

Histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in clear cell sarcoma models

Clear cell sarcoma is an aggressive malignancy occurring most commonly in the distal extremities of young adults, characterized by t(12;22)(q13;q12) creating the chimeric fusion oncoprotein EWS-ATF1. We assessed growth inhibition and differentiation effects of histone deacetylase inhibitors MS-275 and romidepsin (depsipeptide, FK228) on clear cell sarcoma cells and evaluated drug sensitivity among related translocation-associated sarcomas and other cell models. Three clear cell sarcoma cell lines, seven other sarcomas, six nonsarcoma malignant cell lines, and two nonneoplastic mesenchymal cell models were treated with MS-275 or romidepsin. Growth inhibition was assayed by monolayer 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of cell cycle arrest and apoptosis were assessed by propidium iodide/Annexin V flow cytometry in monolayer and spheroid cultures and by immunoblotting analysis. Expression levels of key genes involved in mesenchymal differentiation and of EWS-ATF1 were measured by quantitative real-time PCR in clear cell sarcoma cells treated with histone deacetylase inhibitors. MS-275 and romidepsin inhibited growth in clear cell sarcoma cells by inducing cell cycle arrest and apoptosis in a time- and dose-dependent manner. Sarcomas showed greater sensitivity than other tumor types, with clear cell sarcomas most sensitive of all, whereas nonmalignant mesenchymal cells were highly resistant. MS-275 at 1 micromol/L and romidepsin at 1 nmol/L induced histone H3 acetylation, cell cycle arrest, apoptosis, and differentiation in clear cell sarcoma cells within 24 hours. Histone deacetylase inhibitors increased expression of SOX9, MYOD1, and PPARG and decreased EWS-ATF1 expression in clear cell sarcoma cells. Histone deacetylase inhibitors show promising preclinical activity in multiple clear cell sarcoma models.     

Antiproliferative Effects of Withanolides from Withania adpressa

Extracts of Withania adpressa Coss. (Solanaceae), a medicinal plant endemic to Moroccan Sahara, were tested for their cytotoxicity towards a panel of cancer cell lines (Hep2, HT29, RD, Vero and MDCK), using the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) [MTT assay, Sigma-Aldrich]. The bioassay-guided fractionation of this plant extracts results a novel withanolide 14α,15α,17β,20β-tetrahydroxy-1-oxo-(22R)-witha-2,5,24-trienolide and the already identified withanolides F and J extract, semi-purified fractions and pure compounds exhibits potent cytotoxicity against human cancer cell lines tested, in dose-dependant manner. Morphological features of treated Hep2 cells with the novel withanolide and characteristic DNA fragmentation revealed that the cytotoxicity was due to induction of apoptosis. Taken together, the results suggest that withanolides from W. adpressa Coss. hold potential as antiproliferative agents.     

Heat-induced superaggregation of amphotericin B reduces its in vitro toxicity: a new way to improve its therapeutic index.

Superaggregation of amphotericin B (AmB) was previously shown to occur upon heating of solutions at 70 degrees C. In the present study, we demonstrate that heat pretreatment of Fungizone (deoxycholate salt of AmB [AmB-DOC]) solutions induces a drastic decrease in the in vitro toxicity of this antibiotic. Heated AmB-DOC colloidal solutions, which mainly contained superaggregated and monomeric forms of the antibiotic, were strongly less hemolytic than unheated solutions (aggregates and monomers). Thermal pretreatment of AmB-DOC solutions also reduced the toxicity to the cell line HT29, as deduced from two simultaneous cell viability assays (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release). These heated colloidal solutions were only slightly less efficient than the unheated ones at inhibiting the growth of Candida albicans cells in vitro. Such results suggest that mild heat treatment of AmB-DOC solutions could provide a new and simple solution for improving the therapeutic index of this antifungal agent by reducing its toxicity to mammalian cells.     

乙酰化可降低聚酰胺-胺的细胞毒性

背景：聚酰胺-胺型树枝状高分子纳米材料已被广泛应用于药物载体的研究,但由于整代聚酰胺-胺表面有大量带正电荷的氨基,具有一定的细胞毒性.目的：观察乙酰化对聚酰胺-胺细胞毒性的影响.方法：①细胞增殖检测：采用 MTT 法检测在含0,0.125,0.25,0.5,1,2,4μmol/L 乙酰化聚酰胺-胺的培养液中人胚肾293T 细胞的增殖.②细胞形态：倒置荧光显微镜观察在含4μmol/L 乙酰化聚酰胺-胺的培养液中人胚肾293T 细胞的形态.③细胞周期：流式细胞术检测在含0,5,10,15,20 mg/L 乙酰化聚酰胺-胺的培养液中人胚肾293T 细胞的细胞周期.结果与结论：聚酰胺-胺对293T 细胞具有一定的细胞毒性,在4μmol/L 浓度下48 h 的细胞存活率仅为52%,而乙酰化可显著降低聚酰胺-胺的细胞毒性（P 〈0.01）;聚酰胺-胺孵育的细胞发生团缩,伸展性变差,而乙酰化聚酰胺-胺孵育的293T 细胞与正常培养细胞基本一致,具有良好的伸展性;乙酰化聚酰胺-胺对细胞周期无影响,而聚酰胺-胺在20 mg/L 较高质量浓度时可使细胞 S 期产生阻滞.表明乙酰化可以降低聚酰胺-胺的细胞毒性.     

MDM2 inhibition sensitizes neuroblastoma to chemotherapy-induced apoptotic cell death

Novel therapeutic approaches are urgently needed for high-stage neuroblastoma, a major therapeutic challenge in pediatric oncology. The majority of neuroblastoma tumors are p53 wild type with intact downstream p53 signaling pathways. We hypothesize that stabilization of p53 would sensitize this aggressive tumor to genotoxic chemotherapy via inhibition of MDM2, the primary negative upstream regulator of p53. We used pharmacologic inhibition of the MDM2-p53 interaction with the small-molecule inhibitor Nutlin and studied the subsequent response to chemotherapy in neuroblastoma cell lines. We did 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and terminal deoxynucleotidyl transferase assays to measure proliferation and apoptosis in several cell lines (IMR32, MYCN3, and JF) treated with combinations of cisplatin, etoposide, and Nutlin. We found consistent and robust decreases in proliferation and increases in apoptosis with the addition of Nutlin 3a to etoposide or cisplatin in all cell lines tested and no response to the inactive Nutlin 3b enantiomer. We also show a rapid and robust accumulation of p53 protein by Western blot in these cells within 1 to 2 hours of treatment. We conclude that MDM2 inhibition dramatically enhances the activity of genotoxic drugs in neuroblastoma and should be considered as an adjuvant to chemotherapy for this aggressive pediatric cancer and for possibly other p53 wild-type solid tumors.     

Antitumor activity of histone deacetylase inhibitors in non-small cell lung cancer cells: development of a molecular predictive model

To ascertain the potential for histone deacetylase (HDAC) inhibitor-based treatment in non-small cell lung cancer (NSCLC), we analyzed the antitumor effects of trichostatin A (TSA) and suberoylanilide hydroxamic acid (vorinostat) in a panel of 16 NSCLC cell lines via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TSA and vorinostat both displayed strong antitumor activities in 50% of NSCLC cell lines, suggesting the need for the use of predictive markers to select patients receiving this treatment. There was a strong correlation between the responsiveness to TSA and vorinostat (P < 0.0001). To identify a molecular model of sensitivity to HDAC inhibitor treatment in NSCLC, we conducted a gene expression profiling study using cDNA arrays on the same set of cell lines and related the cytotoxic activity of TSA to corresponding gene expression pattern using a modified National Cancer Institute program. In addition, pathway analysis was done with Pathway Architect software. We used nine genes, which were identified by gene-drug sensitivity correlation and pathway analysis, to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. The prediction performance of the support vector machine model was validated by an additional nine cell lines, resulting in a prediction value of 100% with respect to determining response to TSA and vorinostat. Our results suggested that (a) HDAC inhibitors may be promising anticancer drugs to NSCLC and (b) the nine-gene classifier is useful in predicting drug sensitivity to HDAC inhibitors and may contribute to achieving individualized therapy for NSCLC patients.     

A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations

PURPOSE: Cytotoxicity assays in 96-well tissue culture plates allow rapid sample handling for multicondition experiments but have a limited dynamic range. Using DIMSCAN, a fluorescence digital image system for quantifying relative cell numbers in tissue culture plates, we have developed a 96-well cytotoxicity assay with a >4-log dynamic range. METHODS: To overcome background fluorescence that limits detection of viable cells with fluorescein diacetate, we used 2'4'5'6'-tetrabromofluorescein (eosin Y) to quench background fluorescence in the medium and in nonviable cells to enhance the reduction of background fluorescence achieved with digital image thresholding. The sensitivity and linearity of the new assay were tested with serial dilutions of neuroblastoma and leukemia cell lines. DIMSCAN was compared with other in vitro cytotoxicity assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and trypan blue dye exclusion. RESULTS: Without background fluorescence reduction, scans produced a nearly flat curve across various cell concentrations from 100 to 10(6) cells per well. Either digital image thresholding or eosin Y dramatically reduced background fluorescence, and combining them achieved a linear correlation (r > 0.9) of relative fluorescence to viable cell number over >4 logs of dynamic range, even in the presence of 4 x 10(4) nonviable cells per well. Cytotoxicity of deferoxamine for neuroblastoma cell lines measured by the DIMSCAN assay achieved dose-response curves similar to data obtained by manual trypan blue counts or colony formation in soft agar but with a wider dynamic range. Long-term cultures documented the clonogenic ability of viable cells detected by DIMSCAN over the entire dynamic range. The cytotoxicity of two drug combinations (buthionine sulfoximine + melphalan or fenretinide + safingol) was tested using both DIMSCAN and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the wider dynamic range of DIMSCAN facilitated detection of synergistic interactions. CONCLUSION: DIMSCAN offers the ability to rapidly and efficiently conduct cytotoxicity assays in 96-well plates with a dynamic range of >4 logs. This assay enables rapid testing of anticancer drug combinations in microplates.     

氧化应激诱导肺泡Ⅱ型上皮细胞凋亡及Bax和p53的表达变化

目的 探讨氧化应激状态下肺泡Ⅱ型上皮细胞(ATⅡ)凋亡的变化规律及Bax和p53表达的变化.方法 用过氧化氢(H2O2,500 μmol/L)处理不同时间模拟机体活性氧攻击损伤ATⅡ细胞的体内情况,建立氧化损伤性细胞模型;用四甲基偶氮唑盐(MTT)比色法检测细胞存活率,流式细胞仪检测细胞凋亡率,荧光显微镜及流式细胞仪检测细胞线粒体膜电位,蛋白质免疫印迹法(Western blotting)检测Bax和p53的蛋白表达变化.结果 与对照组比较,随H2O2刺激时间延长,ATⅡ细胞存活率明显下降(F=85.211,P＜0.05),线粒体膜电位也明显下降(F=72.453,P＜0.05),凋亡率却明显增加(F=54.002,P＜0.05);同时发现Bax蛋白和p53蛋白表达均明显增加(F1=28.118,F2=43.456,P均＜0.05).结论 氧化应激能损伤ATⅡ细胞,使细胞线粒体膜电位下降,细胞凋亡率增加、存活率下降,Bax和p53可能参与了ATⅡ细胞的凋亡.