Study of tea polyphenol as a reversal agent for carcinoma cell lines' multidrug resistance (study of TP as a MDR reversal agent)

The aim of this study was to examine MDR1 expression product P-glycoprotein (Pgp) and study the effect and mechanism of tea polyphenol (TP) in reversion of multidrug resistance (MDR) in carcinoma cell lines. Immunocytochemical method was used for qualitative detection of Pgp. A comparative study of cytotoxicity and multidrug resistance reversion effect was made by MTT assay for tea polyphenol and quinidine in MCF-7 and MCF-7/Adr cell lines. The multidrug resistance reversion effect and mechanism were studied by measuring the uptake of 99mTc-tetrofosmin in the carcinoma cell lines. (1) The Pgp overexpression in MCF-7/Adr cells was found to be strong positive, while the Pgp expression of MCF-7 was negative. (2) Although both tea polyphenol and quinidine could not remarkably change the toxicity of adriamycin to MCF-7, they could improve the sensitivity of MCF-7/Adr to adriamycin. The reversion index of tea polyphenol and quinidine was 3 and 10 respectively. (3) The cellular uptake of 99mTc-tetrofosmin was remarkably lower in MCF-7/Adr than in MCF-7. The uptake of 99mTc-tetrofosmin in MCF-7/Adr exhibited a 4, 13, 16 fold increase in the presence of 200, 400 and 500 microg/ml of tea polyphenol respectively. The uptake of 99mTc-tetrofosmin in MCF-7/Adr exhibited only a 4-fold increase in the presence of 200 microM of quinidine. Immunocytochemistry can detect P-glycoprotein expression level qualitatively. Tea polyphenol is not only an anti-tumor agent, but also a multidrug resistant modulator similar to quinidine. The multidrug resistance reversion mechanism of tea polyphenol seems to be its inhibition of the activity of P-glycoprotein. Tea polyphenol has the advantage of very low toxicity in tumor treatment.     

Studies of the UVC-sensitivity of non-tumorigenic and tumorigenic human cell hybrids (HeLa x skin fibroblasts)

The UVC-sensitivities of a non-tumorigenic and a tumorigenic human cell hybrid (HeLa x skin fibroblasts) are compared and contrasted. The tumorigenic cells differ from the non-tumorigenic cells in that they have lost one copy each of chromosomes 11 and 14. For exponentially growing cultures, the tumorigenic cells are considerably more resistant than the non-tumorigenic cells. For confluent cultures, the differential in photosensitivity is much less. Flow cytometric studies of cell cycle distributions of both exponentially growing and confluent cultures of these cells indicate that the differences in photosensitivity cannot be explained by differences in cell cycle distribution. Studies of the kinetics of potentially lethal damage repair (PLDR) in confluent cultures of both cell lines indicate little or no recovery over the first 6 h followed by an increase in survival over the next 12-24 h. These data are consistent with previously published observations in human skin fibroblasts where the kinetics of PLDR reflected the kinetics of thymine dimer loss. The data are not consistent with 6-4 photoproducts being a potentially lethal lesion since such damage is rapidly repaired in human cells.     

Magnetic resonance imaging of nude mice with heterotransplanted high-grade squamous cell carcinomas: use of a low-loaded, covalently bound Gd-Hsa conjugate as contrast agent with high tumor affinity

RATIONALE: Malignant tumors often show an increased uptake and metabolism of plasma proteins, especially albumin. OBJECTIVES: Determine whether the accumulation of low loaded Gd-albumin improves visualization of malignant tumors by MRI. METHODS: Twelve nude mice with heterotransplanted squamous cell carcinomas were studied. The signal intensity of tumor, blood, liver, kidney and muscle tissue was studied in MR images after application of Gd-albumin during a period of 144 hours. MRI results were histologically correlated after simultaneously injection of Gd- and fluorescein-labeled albumins in 9 nude mice. RESULTS: Although liver and kidney had a maximum increase in signal intensity within 30 minutes, tumors showed a delayed 51% increase in the 24 hours after application. Histologic and fluorescence evaluation demonstrated albumin localization in tumors predominantly in stroma and necroses. CONCLUSIONS: Gd-albumin is efficiently accumulated in SCC transplants. MRI with low loaded Gd-albumin may offer relevant opportunities for recognizing tumors sensitive to a therapy with cyostic drug-labeled albumins.     

Synthesis and evaluation of p-iodo-phentermine (IP) as a brain perfusion imaging agent

p-(123I and 131I)iodo alpha,alpha-dimethylphenethylamine (p-iodophentermine, IP) as the alpha-methylated analogue of iodoamphetamine has been prepared. It is hoped that this methyl substitution will increase the lipophilicity of the agent, enhance resistance to metabolism by monoamine oxidase, and will result in increased initial uptake and slower washout from the brain as compared to N-isopropyl-p-(123I)iodoamphetamine. IP was prepared by diazotization of p-aminophentermine followed by decomposition of the diazonium salt with KI. Radioiodinated IP was prepared either by the solid-phase isotopic exchange reaction or by decomposition of the piperidinotriazene derivative with a radiochemical yield of 40-60%. Biodistribution of 131I-IP in rats showed brain uptake in the range of 1.7% dose g-1 at 5, 30 and 60 min. Imaging studies with 123I-IP in dogs showed high brain extraction and slow washout of activity.     

Effect of heat on dsb repair in G1- and S-phase studied in the human HeLa S3 cell line

Purpose : The aim of this study was to investigate the relation between double-strand breaks and thermal radiosensitization in dependence on cell-cycle position. Materials and methods : The experiments were performed with the human tumour cell line HeLa S3. Cells synchronized in G1- and S-phase were exposed to X-rays alone or in combination with prior heating at 44° C for 20 min. Cell kill was determined by means of colony forming assay, double-strand breaks (dsb) using constant-field gel electrophoresis and apoptotic cell death was scored using the fraction of detached cells. Results : In both cell-cycle phases heating at 44° C for 20min prior to irradiation resulted in an increased cellular radiosensitivity, whereby the thermal enhancement ratio (TER) was significantly higher in S- than in G1-phase cells with TER=2.1 and 1.2, respectively. Prior heating at 44° C did not affect the number of radiation-induced dsb but was found to modify their repair as measured for a X-ray dose of 40Gy. In both cell cycle phases dsb repair kinetics measured after irradiation alone could be described by a fast and a slow component with the majority of dsb being repaired with fast kinetics. Prior heating at 44° C was found to have only a minor effect on these half-times but mainly to affect the number of slowly rejoined dsb. In G1-phase cells the number of slowly rejoined dsb measured 300min after irradiation was enhanced by a factor of 1.8 and in S-phase cells even by a factor of 3.2. Fraction of apoptotically dying cells was low after X-irradiation alone but was clearly enhanced after combined treatment, which was especially pronounced for S-phase cells. Conclusions : The pronounced thermal radiosensitization found for S-phase cells was attributed to the heat-mediated increase in the number of slowly rejoined dsb and partly also to the enhanced fraction of apoptotically dying cells when compared to G1-phase cells.     

Effect of heat on dsb repair in G1- and S-phase studied in the human HeLa S3 cell line

PURPOSE: The aim of this study was to investigate the relation between double-strand breaks and thermal radiosensitization in dependence on cell-cycle position. MATERIALS AND METHODS: The experiments were performed with the human tumour cell line HeLa S3. Cells synchronized in G1- and S-phase were exposed to X-rays alone or in combination with prior heating at 44 degrees C for 20 min. Cell kill was determined by means of colony forming assay, double-strand breaks (dsb) using constant-field gel electrophoresis and apoptotic cell death was scored using the fraction of detached cells. RESULTS: In both cell-cycle phases heating at 44 degrees C for 20 min prior to irradiation resulted in an increased cellular radiosensitivity, whereby the thermal enhancement ratio (TER) was significantly higher in S- than in G1-phase cells with TER=2.1 and 1.2, respectively. Prior heating at 44 degrees C did not affect the number of radiation-induced dsb but was found to modify their repair as measured for a X-ray dose of 40 Gy. In both cell cycle phases dsb repair kinetics measured after irradiation alone could be described by a fast and a slow component with the majority of dsb being repaired with fast kinetics. Prior heating at 44 degrees C was found to have only a minor effect on these half-times but mainly to affect the number of slowly rejoined dsb. In G1-phase cells the number of slowly rejoined dsb measured 300 min after irradiation was enhanced by a factor of 1.8 and in S-phase cells even by a factor of 3.2. Fraction of apoptotically dying cells was low after X-irradiation alone but was clearly enhanced after combined treatment, which was especially pronounced for S-phase cells. CONCLUSIONS: The pronounced thermal radiosensitization found for S-phase cells was attributed to the heat-mediated increase in the number of slowly rejoined dsb and partly also to the enhanced fraction of apoptotically dying cells when compared to G1-phase cells.     

5-脱氧杂氮胞苷调控p16基因表达对人宫颈癌HeLa细胞增殖及调亡的影响

目的:探讨甲基化转移酶抑制剂5-脱氧杂氮胞苷(5-Aza-CdR)对人宫颈癌HeLa细胞增殖和调亡的作用及其可能机制。方法:应用不同浓度的5-Aza-CdR处理HeLa细胞,甲基化特异PCR(MSP)法检测处理前后p16基因甲基化状态,应用RT-PCR方法检测p16基因mRNA表达,MTT法检测细胞增殖,流式细胞术检测细胞凋亡率。结果:人宫颈癌HeLa细胞p16基因启动子区呈高甲基化状态,经过5-Aza-CdR处理后,p16基因启动子区呈去甲基化状态,其mRNA重新表达,细胞生长受到抑制,细胞凋亡增加。结论:5-Aza-CdR能够逆转p16基因甲基化状态,调控p16基因表达,并有效抑制人宫颈癌HeLa细胞的增殖和诱导细胞调亡。     

Influence of Pi3-K and PKC activity on 99mTc-(V)-DMSA uptake: correlation with tumour aggressiveness in an in vitro malignant glioblastoma cell line model

Purpose Intensive proliferation and a high degree of migration and invasion are characteristic features of malignant glioblastomas, associated with a poor prognosis. Phosphatidylinositol-3-kinase (Pi3-K) and protein kinase C (PKC) are two phosphorylated proteins involved in glioblastoma cell progression. Phosphorylated focal adhesion protein kinase (FAK) has also been reported to be involved in tumour progression. In a recent study, we demonstrated a correlation between phosphorylated FAK, proliferation rate and ^99mTc-(V)-dimercaptosuccinate [(V)-DMSA] uptake. We hypothesised that ^99mTc-(V)-DMSA could be a potential imaging agent to evaluate glioblastoma aggressiveness. The aim of the present study was to assess the relationship between ^99mTc-(V)-DMSA incorporation rate and modulation of Pi3-K and PKC activity.Methods Proliferation, migration and invasion capacities in the presence of protein kinase modulators—staurosporine (PKC inhibitor), 4-phorbol 12-myristate 13-acetate (PMA; PKC activator) and LY294002 (Pi3-K inhibitor)—were correlated with ^99mTc-(V)-DMSA cell accumulation in an in vitro model of several malignant glioma cells: G111 (grade II), U-87-MG (grade III) and G152 (grade IV).Results In all cell lines tested, LY294002 and staurosporine treatment inhibited cell proliferation, migration and invasion. In contrast, treatment with PMA stimulated tumour aggressiveness. ^99mTc-(V)-DMSA uptake was strongly correlated with the % of cellular proliferation (r=0.8462) and the % of cellular migration (r=0.9081), and to a lesser extent with the % of cellular invasion (r=0.7761).Conclusion Our results clearly demonstrated that ^99mTc-(V)-DMSA reflects Pi3-K and PKC activity and is correlated with tumour aggressiveness. ^99mTc-(V)-DMSA could be a reliable in vivo marker providing additional information on the biological status of malignant glioblastoma.     

宫颈癌细胞摄取99TcmN(NOEt)2与99Tcm-MIBI动力学对比研究

目的研究99Tcm-氮-二(N-乙基-N-乙氧基二硫代氨基甲酸盐)[N(NOEt)2]在人宫颈癌Hela细胞中的摄取动力学.方法用放射性核素示踪法研究人宫颈癌Hela细胞在37 ℃和22 ℃时对99TcmN(NOEt)2和99Tcm-甲氧基异丁基异腈(MIBI)的摄取动力学.结果 Hela细胞摄取99TcmN(NOEt)2的峰值为46.15%,99Tcm-MIBI为12.60%(P<0.01);5 min时Hela细胞摄取99TcmN(NOEt)2为峰值的65%,99Tcm-MIBI为50%(P<0.05);60 min时99TcmN(NOEt)2在Hela细胞中的滞留率为66.02%,99Tcm-MIBI为56.67%(P<0.05);Hela细胞对99TcmN(NOEt)2的摄取不依赖温度,99Tcm-MIBI摄取则呈温度依赖性.结论 99TcmN(NOEt)2可能较99Tcm-MIBI更适用于肿瘤显像,有潜在的临床应用价值.     

Preparation and in vivo evaluation of (99m)TcN-tertiary butyl xanthate as a potential myocardial agent.

(99m)Tc-tertiary butyl isonitrile (TBI), a complex structurally similar to (99m)Tc-sestamibi, has been reported to be taken up in the myocardium, albeit with significant retention in liver and lungs, thereby leading to obscure heart images when used for in vivo imaging. In the present study, tert-butyl xanthate, which is similar to TBI, but possesses two sulphur donors, has been synthesized in excellent yield by reacting tert-butanol with carbon disulphide and sodium hydroxide in dry ether. The prepared ligand was then labeled with [(99m)TcN]2+ core and the resultant complex was characterized by paper electrophoresis and HPLC. The complex was obtained in more than 95% yield and was found to be neutral. The preparation was evaluated in Swiss mice for its myocardial perfusion characteristics. The maximum heart uptake observed was 2.34%ID/g at 5 min p.i., which was cleared rapidly, with retention of 0.50%ID/g of the activity at 60 min p.i. However, owing to slow clearance from blood, liver and lungs, the heart/blood, heart/liver and heart/lung ratios at all the time points of study were not significantly high.     

辐射诱导人细胞系CEM、外周血单个核细胞DNA修复基因hHR21~(sp)的表达研究

目的　研究辐射对人T淋巴细胞白血病细胞系 (CEM)、外周血单个核细胞的hHR2 1sp基因转录表达水平的影响及意义。方法　分别对人T淋巴细胞白血病细胞系CEM和正常人外周血单核细胞在UV或γ辐射后不同时间提取细胞总RNA ,通过RT PCR与hHR2 1sp 基因特异引物杂交 ,以 β actin为内参照放射影像密度扫描检测人T淋巴细胞白血病细胞系CEM、单核细胞DNA修复基因表达。结果　在UV 辐射、γ 辐射后早期 (3～ 6h) ,人T淋巴细胞白血病细胞系CEM和淋巴细胞hHR2 1sp基因的表达水平明显增加 ,照射后 6hhHR2 1sp基因的表达水平增加最多且UV辐射更明显 ,在晚期 (9h)降低。比较人细胞系CEM和淋巴细胞两者hHR2 1sp基因的表达水平 ,γ辐射 (3Gy)后淋巴细胞对hHR2 1sp基因表达高于人细胞系CEM ,且表达增加时间较长 ,达 9h ;而人细胞系CEM受到γ辐射后早期表达增加 ,在 6～ 9h后表达降低。结论　人T淋巴细胞白血病细胞系 (CEM)和人淋巴细胞的DNA修复基因hHR2 1sp基因在一定剂量辐射 (UV、γ辐射 )范围内其表达水平随辐射剂量增加而诱导表达水平增高 ,且对UV辐射更敏感 ;提示hHR2 1sp基因在人细胞系CEM细胞和人单核细胞在照射损伤后表达增加 ,可能是促进单核细胞损伤修复的原因之一。     

部分脱抗原同种异体牙本质盖髓剂的临床应用研究

目的 :评价部分脱抗原同种异体牙本质 (HPDD)盖髓剂的疗效 ,为临床盖髓剂的选择提供依据。方法 :以HPDD、氢氧化钙 (CH)两种材料盖髓剂 ,进行临床远期疗效观察。结果 :HPDD间接盖髓成功率 10 0 % ,直接盖髓成功率 87 88% ;CH组间接盖髓成功率 94 87% ,直接盖髓成功率 80 6 5 % ,两组盖髓剂临床成功率统计学分析无显著差异性 ,P >0 0 5。结论 :两组盖髓剂临床效果良好 ,HPDD抗原性弱 ,不产生免疫反应 ,对牙本质修复有促进作用 ,是可选择的盖髓剂。     

Improved route for the visualization of stem cells labeled with a Gd-/Eu-chelate as dual (MRI and fluorescence) agent.

A simple labeling procedure of stem/progenitor cells based on the use of Gd-HPDO3A and Eu-HPDO3A, respectively, is described. The Gd-chelate acts as T(1)-agent for MRI visualization, whereas the corresponding Eu-chelate acts as reporter in fluorescence microscopy. Owing to their substantial chemical equivalence, the two chelates are equally internalized in EPCs (endothelial progenitor cells), thus allowing their visualization by both techniques. The lanthanide chelates are entrapped in endosomic vesicles and the labeled cells retain biological activity with preservation of viability and pro-angiogenesis capacity. Hyperintense spots in MR have been observed for Gd-labeled EPCs injected under mice kidney capsule or grafted on a subcutaneous Matrigel plug up to 14 days after transplantation.     

Synthesis and characterization of (99m)Tc- and (188)Re-complexes with a diamido-dihydroxymethylenephosphine-based bifunctional chelating agent (N(2)P(2)-BFCA).

A diamido-dihydroxymethylenephosphine (N(2)P(2)) bifunction chelating agent (BFCA) was shown to form well-defined (99m)Tc- and (188)Re-chelate structures. The 4, 4-bis [bis-hydroxymethyl-phosphonyl-propylcarbonmoyl]-butyric acid bifunctional chelating agent (N(2)P(2)-BFCA) formed stable complexes with (99m)Tc and (188)Re in >95% yield with high radiochemical purity (RCP). The biodistribution of the (99m)Tc- and (188)Re-N(2)P(2)-BFCAs after intravenous injection studied in normal mice showed the activity was excreted primarily via renal-urinary pathway indicating their use for labeling peptides with (99m)Tc and (188)Re.     

二氧化硒诱导肺腺癌SPCA-1细胞凋亡的研究

目的：研究二氧化硒诱导肺腺癌细胞系SPCA—l细胞凋亡。方法：采用台盼兰拒染法和四甲基偶氮唑蓝(MTT)比色法测定不同浓度二氧化硒作用不同时间对肺癌SPCA—l细胞生长的抑制作用；采用流式细胞(FCM)技术，观察二氧化硒作用后SPCA—l细胞的凋亡率及细胞周期的变化；通过HE染色和透射电镜观察SPCA—l细胞的形态变化。结果：二氧化硒可有效的地抑制SPCA—l细胞的生长，具有时间、浓度依赖性；二氧化硒诱导SPCA—l细胞的凋亡具有浓度依赖性；二氧化硒低浓度时阻滞SPCA—l细胞于S期，高浓度时选择性诱导S期细胞凋亡；二氧化硒作用后，SPCA—l细胞呈现典型的凋亡细胞形杰特征。结论：二氧化硒通过诱导S期细胞凋亡而抑制肺腺癌细胞系SPCA-1细胞的生长，具有时效、量效及周期特异性。     

Preparation and preliminary biological evaluation of a (166)Ho labeled polyazamacrocycle for possible use as an intravascular brachytherapy (IVBT) agent.

(166)Ho can be considered as a potential radionuclide for intravascular brachytherapy (IVBT) using liquid-filled balloons owing to its suitable nuclear decay characteristics. The possibility of producing (166)Ho with adequate specific activity using moderate flux reactors and natural holmium target makes it an attractive alternative of (188)Re for developing IVBT agents. Keeping in mind the high thermodynamic stability of lanthanide complexes with polyazamacrocycles, (166)Ho complex of 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) was prepared and studied for its suitability as a possible agent for IVBT. (166)Ho was produced with adequate specific activity and high radionuclidic purity by irradiating natural Ho(2)O(3) powder. TETA was synthesized by a single step procedure using cyclam as the starting material. (166)Ho-TETA complex was prepared with excellent radiochemical purity and the complex was found to retain its stability for 7 days at room temperature. Biodistribution studies carried out in Wistar rats showed major renal clearance of the injected activity with almost no retention in any of the vital organ/tissue.     

Preparation of 99mTc(CO)3-Carboxymethylthioethyl iminodiacetic acid and evaluation as a potential renal imaging agent

The ligand, carboxymethylthioethyl iminodiacetic acid (CMT-IDA) has a suitable array of donor atoms for coordination with [99mTc(CO)3]+ core, wherein the resultant complex is expected to possess free carboxylic residues contributing towards hydrophilicity of the complex. The aim of the studies was to study the renal clearance of 99mTc(CO)3- labeled CMT-IDA and determine the potential of the complex towards its use as a renal tubular imaging agent. Methods: CMT-IDA was radiolabeled with the [99mTc(CO)3(H2O)3]+ precursor and was characterized by reverse phase HPLC gradient elution system. Stability, hydrophilicity and plasma protein binding studies were carried out for the complex. Biodistribution studies were carried out in normal male Swiss mice at 10 min.p.i. and 2 h.p.i. The clearance was estimated from the activity observed in the urinary bladder by tying the urethra prior to injection of the complexes under study. Imaging studies were performed with male Swiss mice administered with [99mTc(CO)3(CMT-IDA)]-2 at 30 min. p.i. and blocking studies were carried out by intraperitoneal injection of probenecid 10 min. prior to the injection of the radiotracer. Results: [99mTc(CO)3(CMT-IDA)]-2 could be obtained in > 98% radiochemical purity. The complex showed renal clearance of 71.0? 5.9% ID at 10 min.p.i. which increased to 84.1? 10.6% ID at 2 h.p.i., with no major activity in blood, liver, heart, lungs, stomach and spleen. However, the intestinal uptake was high (10.3? 2.0% ID) at 2 h.p.i. Scintigraphic image of the animal injected with probenecid showed an increase in the activity in kidneys indicating excretion of the [99mTc(CO)3(CMT-IDA)]-2 complex via tubular pathway. Conclusion: The complex, [99mTc(CO)3(CMT-IDA)]-2 has shown excellent renal clearance and thereby can be explored further for potential use as an agent towards assessing effective renal plasma flow.     

Lack of correlation between radiosensitivity and inhibition of DNA synthesis in hybrids (A-T x HeLa)

Hybrid cells obtained from A-T and D98/AH(HeLa) cells showed normal radiation sensitivity to cell killing, but retained radioresistant DNA synthesis similar to parental A-T cells.