Absolute configuration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol formed metabolically from 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK). Using the chiral derivatizing agent, (R)- (+)-alpha-methylbenzyl isocyanate [(R)-(+)-MBIC], previous work has shown that the enantiomeric ratio of metabolically formed NNAL and its glucuronide derivative may be species dependent. However, the absolute configuration of such NNAL has not been previously reported. Synthetically prepared racemic NNAL was converted to diastereomeric esters by reaction with (R)-(+)- and (S)-(-)-alpha-methoxy-alpha- (trifluoromethyl)phenylacetic acid (MTPA) chloride (Mosher's reagent) and the products were characterized by 1H-NMR. Based on chemical shift data, the absolute configuration of NNAL in each diastereomeric ester was assigned. Hydrolysis of (R)-NNAL-(R)-MTPA gave (R)-NNAL. This was converted to the corresponding carbamate by reaction with (R)-(+)-alpha- MBIC and the absolute configurations of the diastereomeric carbamates formed by reaction of (R)- and (S)-NNAL with (R)-(+)-MBIC were thereby assigned. Conversion of metabolically produced NNAL to the same carbamates allowed us to assign the NNAL formed from NNK by rat liver microsomes as (R)-NNAL. The major and minor NNAL-glucuronide diastereomers found in the urine of patas monkeys and humans exposed to NNK were similarly assigned; they were formed from (R)-NNAL and (S)- NNAL, respectively.      

5，4’-二-正辛烷氧基-7-二氟亚甲基异黄酮对人乳腺癌细胞增殖和调亡的影响

目的：探讨金雀异黄素（gen）衍生物5,4＇-二-正辛烷氧基-7-二氟亚甲基异黄酮（5,4＇-Di-noctoxyl-7-gem-difluoromethylenegenisteinDOdFMG）体外选择性抑制人乳腺癌细胞增殖和诱导凋亡作用.方法：MTT法检测DOdFMG对MCF-7和HBL-100细胞生长的影响;流式细胞术和AO/EB荧光双染法测定DOdFMG诱导MCF-7细胞凋亡作用及对细胞周期的影响.结果：DOdFMG抑制MCF-7细胞生长,呈时间剂量依赖,比gen具有更强选择性和抗肿瘤活性;DOdFMG能诱导MCF-7细胞凋亡,DOdFMG40.0μM组比gen100μM组诱导凋亡的活性更强,DOdFMGMCF-7将MCF-7细胞阻滞于S期和G2期,呈浓度依赖.结论：DOdFMG能抑制人乳腺癌细胞系（MCF-7）细胞生长和诱导凋亡,是一种高效、毒副作用小的新型治疗乳腺癌候选药物.     

草分枝杆菌对SPCA/Ⅰ人肺腺癌细胞生长周期的影响以及促凋亡作用的研究

探讨草分子杆菌（Mycobacterium Phlei）对SPCA /Ⅰ人肺腺癌细胞系的调节细胞增殖周期及诱导细胞凋亡的作用。将SPCA /Ⅰ人肺腺癌细胞株培养传代,分别加入不同浓度的草分子杆菌,作用36小后,用倒置显微镜观察细胞形态,并以annexin V:FITC Apoptosis detection kit和cycletest plus DNA reagent kit标记,分别经FACSAria流式细胞仪检测细胞凋亡率和细胞周期改变。不同浓度的草分子杆菌体外对人肺腺癌细胞株SPCA /Ⅰ均有直接的抑制作用,其抑制率与所用药物浓度呈正相关;倒置显微镜下可见细胞由原来贴壁生长而逐渐脱落、外形变圆、体积缩小、折光性增强,核染色质凝集,细胞裂解。草分子杆菌体外能改变SPCA /Ⅰ人肺腺癌细胞株的细胞增殖周期,使细胞周期阻滞在G2-M期,对人肺腺癌细胞株SPCA /Ⅰ有诱导凋亡的作用,细胞凋亡率明显增加。草分子杆菌能通过调节细胞增殖周期和诱导细胞凋亡,直接抑制SPCA /Ⅰ人肺腺癌细胞株的增殖。     

子宫内膜腺癌组织中尿激酶型纤溶酶原激活系统的表达及其临床意

研究尿激酶型纤溶酶原激活系统在子宫内膜腺癌的发生、发展及浸润转移中的规律及其对临床的指导意义.应用免疫组织化学方法测定24例正常子宫内膜、30例子宫内膜增生过长及62例子宫内膜腺癌组中uPA、uPAR、PAI-1的表达,并分析与良恶性、临床分期、组织学分级、基层浸润深度及淋巴结转移的关系. uPA、uPAR、PAI-1在子宫内膜腺癌组中的表达较正常子宫内膜组和子宫内膜增生过长组中的表达显著升高（P均<0.05）,而正常子宫内膜组和子宫内膜增生过长组中的表达无显著性差异（P >0.05）.uPA、uPAR、PAI-1的高表达与肿瘤分化、临床分期、肌层浸润深度和淋巴结转移有关(P <0.05).uPA、uPAR、PAI-1的表达与子宫内膜腺癌的发生、发展及浸润转移密切相关,可作为判断预后的重要指标.     

食管癌常规分割剂量每周5天与每周7天照射的疗效观察

目的观察和比较常规分割剂量每周照射7天与每周照射5天两种方法治疗Ⅲ期食管癌的疗效及放射反应。方法对96例Ⅲ期食管癌首治病例随机分成每周照射7天组(治疗组)和每周照射5天组(对照组),治疗组48例,2.0Gy次/,1次/天,7天/周,共60~70Gy,30~35分次,30~35天完成;对照组48例,2.0 Gy次/,1次/天,5天/周,共60~70 Gy,30~35分次,40~47天完成。结果治疗组与对照组1、2、3年生存率分别为77.1%、62.5%、50%和66.7%、37.5%、22.9%,治疗组高于对照组(P<0.05)。1、2、3年局控率分别为8 1.3%、6 4.6%、5 4.1%和6 8.7%、41.6%、31.2%,治疗组高于对照组(P<0.05)。急性放射性食管炎发生率分别为10.4%和6.2%,差别无显著意义(P>0.05)。出血穿孔的发生率分别为6.2%和4.3%,差别无显著意义(P>0.05)。结论常规分割剂量7天/周照射比5天/周照射可提高疗效,未明显增加不良反应。     

DNA and hemoglobin alkylation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite 4-(methylnitros-amino)-1-(3-pyridyl)-1-butanol in F344 rats

Alkylation of DNA and hemoglobin was compared in male F344 ratsgiven a single s.c. injection of the tobacco-specific nitrosamine4-(methyInitrosamino)-1-(3-pyridyl)-1-butanone (NNK), or itsmajor metabolite formed by carbonyl reduction, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL).In hepatic DNA, levels of 7-methylguanine and O⁶-methyl-guanineformed from NNK 1-48 h after treatment were similar to thoseformed from NNAL. In nasal mucosa and lung DNA, levels of 7-methylguanineand O⁶Amethylguanine were somewhat higher after treatment withNNK than with NNAL. Acid hydrolysis of hepatric DNA, isolatedfrom rats treated with either [5-³H]NNK or [5-³H]NNAL, gave180 ± 48 or 120 ± 23 µuno/mol guanine, respectively,of 4-hydroxy-1-(3-pyridyl)-1-butanone. Basic hydrolysis of globinisolated from rats treated with either [5-³H]NNK of 5-³H]NNALgave 4.1 ± 0.7 or 2.0 ± 0.1 pmol/mg, respectivelyof 4-hydroxy-1-(3-pyridyl)-1-butanone. These results indicatethat NNAL is not a detoxification product of NNK, since treatmentof rats with NNAL results in modifications of DNA which arequalitativerly and quantitatively similar to those observedupon treatment with NNK. Alkylation of DNA and globin by NNALmay result mainly from its metabolic reconversion to NNK.     

Spontaneous and mutagen-induced transformation of primary cultures of Msh2-/- p53-/- colonocytes

Loss of function of mismatch repair (MMR) genes underlies hereditary nonpolyposis colorectal cancer (HNPCC). However, the inability to maintain primary colon epithelial cells in culture has limited the analysis of the contribution of MMR gene defects to colorectal tumorigenesis. We have now established primary cultures of epithelial cells from the colon crypts of Msh2-/- p53-/- double-knockout mice. These cells undergo spontaneous transformation (soft agar colonies and s.c. tumor formation), with a progressively shorter latency as a function of increasing passages in culture. Treatment of early passage cells with the mutagen methylmethane thiosulfonate (MMS) further decreases the transformation latency of Msh2-/- p53-/- cells. Spontaneous transformation of p53-/- colonocytes is only observed using late passage cells, and methylmethane thiosulfonate-treated early passage p53-/- colonocytes do not form tumors when injected into immunodeficient mice. Together, these findings support the pathogenic role of MMR gene inactivation in colorectal tumorigenesis and provide an experimental model for the serial assessment of the molecular phenotype associated with Msh2 deficiency.     

Effects of the polyamine analogues BE-4-4-4-4, BE-3-7-3, and BE-3-3-3 on the proliferation of three prostate cancer cell lines

Purpose: Polyamines are biologic cations necessary for normal cell growth. Polyamine analogues have been shown to be effective inhibitors of tumor growth. We tested the effect of the polyamine analogues 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), N¹,N¹¹-bis(ethyl)norspermine (BE-3-3-3) and 1,15-bis(ethylamino)-4,12-diazapentadecane (BE-3-7-3) on the growth of the prostate cancer cell lines DU145, LNCaP and PC-3 in vitro. We also tested the effect of␣BE-4-4-4-4 on androgen-independent DU145 cells in␣vivo via a nude mouse xenograft model.Methods: In␣vitro, cell proliferation was measured using a DNA assay or a colony-formation assay. In vivo, mice were given saline or BE-4-4-4-4 3 mg/kg or 5 mg/kg intraperitoneally twice daily on days 7–10 and 14–17 (cycle 1), days 49–52 and 56–59 (cycle 2) and days 91–94 and 98–101 (cycle 3).Results: The proliferation of DU145, LNCaP and PC-3 prostate cancer cell lines was inhibited in a dose-dependent manner by BE-4-4-4-4. Intracellular putrescine, spermidine and spermine levels in all three cell lines declined after only 24 h exposure to BE-4-4-4-4 in vitro. Animals receiving BE-4-4-4-4 showed inhibition of tumor growth which continued throughout the experiment with 74% (3 mg/kg) and 81% (5 mg/kg) growth inhibition seen on day 101. No overt toxic reactions besides weight loss were observed in BE-4-4-4-4-treated animals. Tumor tissue from animals treated with BE-4-4-4-4 showed a dose-dependent decrease in spermidine and spermine levels but no decline in putrescine levels as compared with control. BE-4-4-4-4 levels were highest in tumors on day 63 with levels reaching 0.33 and 1.45 nmol/mg protein from animals treated at the 3 mg/kg and 5 mg/kg doses, respectively. Conclusion: These results show the polyamine analogues BE-4-4-4-4, BE-3-3-3 and BE-3-7-3 to be effective inhibitors of prostate cancer cell growth in vitro and BE-4-4-4-4 to be an effective inhibitor of DU145 cells in vivo with minimal toxicity. Received: 18 June 1996 / Accepted: 28 September 1996     

Effects of deuterium substitution on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in A/J mice

Bioassays and DNA-binding studies of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its analogs with deuterium substitution at the positions alpha to the nitrosamino group ([4,4-D2]NNK and [CD3]NNK) were carried out in A/J mice in order to assess the potential importance of DNA methylation or pyridyloxobutylation in lung tumor induction. The tumorigenic activities of the major NNK metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its analog with deuterium at the carbinol carbon ([1-D]NNAL) were also determined. Groups of A/J mice were given single i.p. injections of either 10 or 5 mumol of NNK, [4,4-D2]NNK, [CD3]NNK, NNAL and [1-D]NNAL, and were killed 16 weeks later. Lung tumor multiplicities were as follows in mice treated with 10 mumol: NNK, 7.3 +/- 3.5; [4,4-D2]NNK, 1.4 +/- 1.6; [CD3]NNK, 11.7 +/- 5.4; NNAL, 3.2 +/- 2.0; [1-D]NNAL, 3.2 +/- 2.0. Similar relative tumorigenic activities were observed in mice treated with 5 mumol of these compounds. These results demonstrated that [4,4-D2]NNK was less tumorigenic than NNK and [CD3]NNK was more tumorigenic than NNK. NNAL was less tumorigenic than NNK; substitution of deuterium at the carbinol carbon did not affect its activity. Levels of O6-methylguanine (O6-mG) were measured in pulmonary DNA of A/J mice treated with 10 mumol of NNK, [4,4-D2]NNK or [CD3]NNK, and killed 2 or 24 h later. O6-mG levels were lower in mice treated with [4,4-D2]NNK than in those treated with NNK; no difference in O6-mG levels was observed between those treated with NNK and [CD3]NNK. The results of this study support the hypothesis that O6-mG formation in pulmonary DNA is the key step in lung tumor induction by NNK in A/J mice.