YAP干扰腺病毒载体对宫颈癌增殖、迁移及侵袭的影响

目的 探索YAP蛋白对宫颈癌细胞的恶性增殖、迁移及侵袭性的作用.方法 分别以腺病毒靶向YAP干扰载体(pAd-si-YAP)、重组空载体腺病毒(pAd-mock)或磷酸缓冲盐溶液(PBS)感染CaSki细胞.采用定量聚合酶链反应(qPCR)、蛋白质印迹法(Western blotting)、四甲基偶氮唑盐微量酶反应比色法(MTT法)、流式细胞法、划痕试验以及细胞侵袭试验分别分析感染后CaSki细胞的YAP mRNA表达、YAP蛋白表达、细胞增殖活力、细胞周期及凋亡、细胞迁移能力以及细胞侵袭能力.结果 qPCR结果显示Ad-si-YAP感染CaSki细胞的YAP mRNA表达明显低于pAd-mock感染的CaSki细胞及PBS处理的CaSki细胞[(22.01±0.24)比(80.12±0.31)、(84.18±0.22),P<0.05],表明Ad-si-YAP可以抑制YAP基因转录.蛋白质印迹法结果显示Ad-si-YAP感染CaSki细胞的YAP蛋白表达低于pAd-mock感染的CaSki细胞及PBS处理的CaSki细胞[(0.6±0.018)比(1.5±0.031)、(1.8±0.27),P<0.05].MTT结果显示24 h后pAd-mock感染的CaSki细胞及PBS处理的CaSki细胞增殖明显快于Ad-si-YAP感染CaSki细胞(P<0.05).流式细胞结果显示通过沉默YAP后,CaSki细胞周期停滞于G0/G1期明显增加(P<0.05),同时CaSki细胞增多(P<0.01).划痕试验发现沉默YAP后CaSki细胞的迁移能力下降[(40.01±0.16)比(81.02±0.22)、(86.04±0.31),P<0.05],说明YAP可以促进CaSki细胞迁移.细胞侵袭试验发现沉默YAP后CaSki细胞的侵袭能力下降[(120±4)比(640±3)、(680±4),P<0.05].结论 YAP蛋白的表达可导致宫颈癌细胞的恶性增殖、迁移及侵袭,是应用于宫颈癌综合治疗的一个潜在靶点.     

Ribonucleotide reductase and thymidine phosphorylation: two potential targets of azodicarbonamide

Azodicarbonamide tested as an anti-HIV agent was reported to expulse zinc from viral zinc-cysteine factors and to inhibit calcium mobilization machinery. It has structural analogy with hydroxyurea that inhibits ribonucleotide reductase and could also act on this target. Azodicarbonamide was therefore tested for its capacity to modulate deoxyribonucleotides triphosphate pools alone or in combination with other agents in the lymphoblastic SUP-T1 cell line susceptible to HIV infection. The deoxyribonucleotides triphosphate were evaluated by an enzymatic assay using sequenase. Two hours exposure of SUP-T1 cells to 100 microM azodicarbonamide induced a 50% reduction of each deoxyribonucleotide triphosphate. Among other inhibitors of nucleotide metabolism (hydroxyurea, methotrexate and thymidine), hydroxyurea only reproduces the effect of azodicarbonamide. This suggests, but does not demonstrate directly, that azodicarbonamide inhibits ribonucleotide reductase activity. The combination of azodicarbonamide with each of these inhibitors affected particularly the dCTP pool. During this study it was also suggested that azodicarbonamide could interfere with thymidine phosphorylation. Thymidine phosphorylating activity was measured with 3H-thymidine as substrate. In acellular preparations, azodicarbonamide also non-competitively inhibits thymidine phosphorylating activity. This effect was not reproduced by hydroxyurea. Thus, in vitro azodicarbonamide decreases the intracellular pool of deoxyribonucleotide and thymidine phosphorylation.     

Correlativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells

DNA double-strand break (DSB) is generally regarded as the most lethal of all DNA lesions after radiation. Ku80, DNA-PK catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) proteins are major DSB repair proteins. In this study, survival fraction at 2Gy (SF2) values of eight human tumor cell lines (including four human cervical carcinoma cell lines HeLa, SiHa, C33A, Caski, three human breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453, and one human lung carcinoma cell line A549) were acquired by clone formation assay, and western blot was applied to detect the expressions of Ku80, DNA-PKcs and ATM protein. The correlativity of protein expression with SF2 value was analyzed by Pearson linear correlation analysis. We found that the expression of the same protein in different cell lines and the expression of three proteins in the same cell line had a significant difference. The SF2 values were also different in eight tumor cell lines and there was a positive correlativity between the expression of DNA-PKcs and SF2 (r =0.723, P = 0.043), but Ku80 and ATM expression had no correlation with SF2 (P > 0.05). These findings suggest that the expression level of DNAPKcs protein can be an indicator for predicting the radiosensitivity of tumor cells.     

Comparison of Cell Proliferation and Toxicity Assays Using Two Cationic Liposomes

The present study compares different cytotoxicity and cell proliferation assays including cell morphology, mitochondrial activity, DNA synthesis, and cell viability and toxicity assays. CaSki cells were exposed to two cationic liposomal preparations containing dimethyldioctadecyl-ammonium bromide (DDAB), dioleoylphosphatidylethanolamine (DOPE) and a commercial transfection-reagent DOTAP(N[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium-methylsulfate). The results provided by these assays were similar. However, the lactate dehydrogenase assay was more sensitive in measuring early damages of cell membranes than the Trypan blue assay. Also, cell morphology showed early toxic changes, such as cytoplasmic vacuolization and cell shrinking, and it should be included with such toxicity evaluations. DDAB:DOPE was more toxic than DOTAP. The cells treated with DOTAP at 10 µM were surviving as well as the control cells, while DOTAP at 40 µM and DDAB: DOPE at 10 µM had slight toxic effects on CaSki cells. The most toxic effects were seen in CaSki cells after treatment with DDAB: DOPE at 40 µM.     

Paraquat research: do recent advances in limiting its toxicity make its use safer?

The use of the herbicide paraquat (1,1′-dimethyl-4,4′-bipyridylium dichloride; PQ) has been fiercely challenged due to its severe acute toxicity, putative neurotoxicity after long-term exposure and lack of antidotes. Breakthrough research on PQ is therefore required for an effective risk control and to allow a safer use of PQ in the future. The silencing or inhibition of quinone oxidoreductase 2, a NAD(P)H-independent flavoenzyme, was shown to significantly attenuate PQ toxicity in vitro, in primary pneumocytes and astroglial U373 cells, and to strongly antagonize PQ-induced systemic toxicity and animal mortality. The novel results reported in this issue of BJP, added to recent findings using sodium salicylate and lysine acetylsalicylate, in which full survival of PQ-intoxicated rats was also achieved, open the door for new preventative and therapeutic strategies that may lead to safer use of this effective pesticide.     

Immortalized hepatocytes as in vitro model systems for toxicity testing: the comparative toxicity of menadione in immortalized cells, primary cultures of hepatocytes and HTC hepatoma cells

Immortalized differentiated liver cell lines capable of continuous proliferation, and expressing stable liver-specific functions, would be valuable for in vitro toxicity testing in the pharmaceutical, chemical, food and cosmetics industries. Immortalized rat hepatocyte cell lines have been produced by transfection of SV40 DNA by electroporation or calcium phosphate precipitation. Their utility has been assessed by studying the toxicity of a model compound, menadione, and by measuring the activities of DT-diaphorase and NADPH cytochrome c reductase. Enzyme activities and toxicity were compared in freshly isolated hepatocytes, the immortalized cell lines and dedifferentiated HTC hepatoma cells. In HTC cells DT-diaphorase activity was 100-fold elevated compared with freshly isolated hepatocytes. In only one cell line, C2.1.2. (produced by calcium phosphate precipitation), was DT-diaphorase activity increased (twofold) compared with freshly isolated hepatocytes. Menadione caused loss of viability at similar concentrations (40–80 μ ) in the immortalized cell lines and 24-hr primary cultures of hepatocytes, whereas HTC cells showed loss of viability only with menadione concentrations above 200 μ . The immortalized lines therefore appear to have potential for predicting toxicity and for menadione this can be correlated with the expression of DT-diaphorase.     

沉默hnRNP A2/B1基因后通过Bcl-2/Bax影响宫颈癌CaSki细胞的凋亡

目的 探索沉默核内不均一核糖核蛋白A2/B1（hnRNP A2/B1）基因后对宫颈癌CaSki细胞的增殖、凋亡的 影响及其相关作用机制。方法 建立稳定沉默 hnRNP A2/B1 的 CaSki 细胞株,分为未沉默 hnRNP A2/B1 的空白组 （CaSki 组）、转染阴性序列的阴性对照组（CaSki-NC 组）、转染阳性 hnRNP A2/B1 干扰序列的实验组（CaSki-shRNA 组）。运用实时荧光定量PCR（qRT-PCR）及蛋白印迹法（Western blot）对各组细胞hnRNP A2/B1的mRNA及蛋白表达 水平进行验证,四甲基偶氮唑蓝（MTT）法检测各组细胞增殖能力,细胞克隆实验检测各组细胞克隆形成能力,流式细 胞术检测各组细胞凋亡情况,Western blot检测各组细胞凋亡相关蛋白Bcl-2、Bax的表达变化。结果 与CaSki组、 CaSki-NC相比较,CaSki-shRNA组细胞hnRNP A2/B1的mRNA及蛋白表达水平均明显减低,细胞的增殖能力在48 h 降低,克隆形成能力下降,凋亡率增加,Bcl-2蛋白的表达减低,Bax表达升高（P＜0.01）,而CaSki组与CaSki-NC组之 间差异无统计学意义（P＞0.05）。结论 沉默hnRNP A2/B1基因后能抑制宫颈癌CaSki细胞的增殖能力,且可能通过 Bcl-2/Bax影响宫颈癌CaSki细胞的凋亡。     

Synthesis of berberine-piperazine conjugates as potential antioxidant and cytotoxic agents

Piperazine derivatives bearing different electron-withdrawing and electron-donating functional groups were linked to the well-known isoquinoline alkaloid derivative, berberine via efficient organic transformations. The entire target berberine-based analogues were examined for their in vitro antioxidant potency using 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid bioassays, and anticancer activities using sulforhodamine B assay against HeLa and CaSki cervical cancer cell lines in addition to the cytotoxicity using Madin-Darby canine kidney non-cancer cell lines and, ascorbic acid and berberine used as a control for antioxidant and anticancer activities, respectively. Bioassay results revealed that newer compounds were more active against CaSki and HeLa cell lines with therapeutic indices better than that of parent berberine and showed tolerable cytotoxicity to the normal cells. A final analogue 5a with 4-methylpiperazine substituent indicated most significant anticancer potency with a therapeutic index of 58.53 (HeLa) and 48.76 (CaSki), followed by those bearing meta-chloropiperazine rings with a therapeutic index of 41.83 (HeLa) and 47.35 (CaSki), respectively.In addition, newly synthesized analogues exerted a significant radical scavenging activity against 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid cation with IC₅₀ values of 8.917?µg/mL, and were good to moderate scavengers of 2,2-diphenyl-1-picrylhydrazyl radical with IC₅₀ values of 25.40?µg/mL. Synthesized compound was characterized using several techniques, fourier transform infrared spectroscopy, ¹H nuclear magnetic resonance, ¹³C nuclear magnetic resonance, mass spectroscopy and elemental (CHN) analyses.     

The mitochondrial uncoupler dicumarol disrupts the MTT assay

Dicumarol is routinely added to the 3-[4,4-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay to study the role of NAD(P)H:quinone oxido-reductase in drug activation and detoxification. We assessed the direct impact of dicumarol (a mitochondrial uncoupler) on the MTT assay. Mouse mammary tumor (EMT6) and Chinese hamster ovary (CHO) cells were treated with media containing either 10 or 1% fetal bovine serum and dicumarol (0-1000 microM) mimicking standard assay conditions. MTT, clonogenic, total reactive oxygen species (ROS), and oxygen consumption assays were performed. Significant increases in the apparent viability of EMT6 and CHO cells were observed with MTT assays after short time periods with maximum effects at 2 hr. Reduced serum concentrations intensified this effect. Conversely, significant decreases in viability for both cell lines occurred after longer incubations and serum withdrawal enhanced this effect in both cell lines. Clonogenic assays provided contrasting results where viability increased significantly only in EMT6 cells (not CHO) and was smaller than that reported by MTT. Furthermore, greater dicumarol toxicity was observed in clonogenic assays. Significant toxicity compared to control occurred after 4-hr treatment (vs. 12 hr MTT) and serum withdrawal also increased the toxicity of dicumarol with extended culture. ROS production in EMT6 and CHO cells increased in a concentration-dependent manner with 20-min dicumarol administration and thereafter declined. The EC(50) for dicumarol-induced oxygen consumption was 0.84 microM in CHO compared to 1.18 microM in EMT6 cells. Cell lines are differentially sensitive to the toxicity of dicumarol and cell survival data may be skewed by its inclusion, probably due to ROS production and mitochondrial uncoupling. Dicumarol is not recommended for inclusion in the MTT assay.     

A retrospective TDM database analysis of interpatient variability in the pharmacokinetics of lopinavir in HIV-infected adults

Lopinavir is one of the most-widely used protease inhibitors in the treatment of HIV-1 infected patients. Concentration-effect relationships have been described for both antiviral activity and toxicity. Less is known about patient characteristics that may determine interpatient variability in lopinavir plasma concentrations. A database was created containing all Therapeutic Drug Monitoring (TDM) results collected at our Department. Patients were included if they were using lopinavir twice daily for at least two weeks; subjects who were known to be nonadherent (based on either a lopinavir concentration <0.2 mg/L or suspected by the physician) were excluded. Demographic data were collected from TDM application forms and patient charts. Patients attending one of the 22 HIV treatment centers in The Netherlands. The Department of Clinical Pharmacy is a national referral center for TDM of antiretroviral agents. Lopinavir concentration ratios (CRs) were calculated for each patient by dividing the individual plasma concentration by the time-adjusted population value. Relationships with lopinavir CRs were tested using regression analysis and analysis of variance. A total of 802 patients were included (607 males; 150 females; 45 unknown). The age and body weight of the patients ranged from 18 to 74 years (mean 42) and 42 to 121 kg (mean 72), respectively. Race was known for 756 persons: Caucasian 76%, African 18% and Asian 6%. The median (+ interquartile range, IQR) lopinavir CR was 0.98 (IQR: 0.67-1.31). Body weight showed an inverse relationship with lopinavir CR (F = 23.1; P < 0.001). Age was not related with lopinavir CR (P = 0.99). Female patients had a significantly higher lopinavir CR than males: 1.18 vs. 1.03 (P = 0.005); race was not associated with differences in lopinavir CR. In a multivariate regression analysis body weight, but not gender, remained significantly related to lopinavir CR. Body weight is the only demographic factor that could be related to lopinavir exposure; clinicians should be alert for an increased risk of suboptimal antiviral efficacy in patients with high body weight, and for an increased risk of toxicity in patients with a low body weight.     

低分子量酪氨酸磷酸酶在子宫颈鳞癌中的表达及在预后判定中的应用价值

目的：分析子宫颈鳞癌患者低分子量络氨酸磷酸酶（LMW-PTP）的表达和预后价值。方法选取2008年3月至2014年8月石蜡标本160例,依照病变程度分为正常组,宫颈上皮内瘤变Ⅰ期、Ⅱ~Ⅲ期组和宫颈鳞癌组,采用 RT-PCR 及免疫组化法分析 LMW-PTP 水平。结果 C33A、SiHa 和 CaSki 细胞系中 LMW-PTPmRNA 表达明显高于 HaCaT 细胞（ P ﹤0.05）,HaCat 细胞中 LMW-PTP 表达最低,明显低于其他组（ P ﹤0.05）,C33A、SiHa 和 CaSki 细胞系中 LMW-PTP 呈高表达状态,HaCaT 细胞中 LMW-PTP 表达呈弱阳性,C33A 细胞、SiHa 细胞、CaSKi 细胞中表现均明显提高,正常宫颈上皮组织到子宫颈鳞癌进展中,LMW-PTP 表达逐渐升高,中分化子宫颈鳞癌组织中 LMW-PTP 表达明显高于高分化组织（ P ﹤0.05）,低分化子宫颈鳞癌组织中 LMW-PTP 表达明显高于中分化子宫颈鳞癌组织（ P ﹤0.05）,子宫颈鳞癌组织中 LMW-PTP 表达与患者组织学程度密切相关（ P ﹤0.05）。结论 LMW-PTP 在子宫颈鳞癌进展中起到重要作用,提示 LMW-PTP 表达有助于改善子宫颈鳞癌预后。     

Influence of the surface charge of PLGA nanoparticles on their in vitro genotoxicity,cytotoxicity, ROS production and endocytosis

With the ongoing commercialization of nanotechnology products, human exposure to nanoparticles (NPs) is set to increase dramatically and an evaluation of their potential adverse effects is essential. Surface charge, among other physico‐chemicals parameters, is a key criterion that should be considered when using a definition for nanomaterials in a regulatory context. It has recently been recognized as an important factor in determining the toxicity of NPs; however, a complete understanding of the mechanisms involved is still lacking. In this context, the aim of the present study was to investigate the influence of the surface charge modification of NPs on in vitro toxicity assays. Poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles bearing different surface charges, positive(+), neutral(n) or negative(?), were synthesized. In vitro genotoxicity assays (micronucleus and comet assays) coupled with an assessment of cytotoxicity, were performed in different cell lines (L5178Y mouse lymphoma cells, TK6 human B‐lymphoblastoid cells and 16HBE14o‐ human bronchial epithelial cells). Reactive oxygen species (ROS) production and endocytosis studies were also performed. Our results showed that PLGA(+) NPs were cytotoxic. They are endocytosed by the clathrin pathway and induced ROS in the three cell lines. They led to chromosomal aberrations without primary DNA damage in 16HBE14o‐ cells, suggesting that aneuploidy may be considered as an important biomarker when assessing the genotoxic potential of NPs. Moreover, 16HBE14o‐ cells seem to be more suitable for the in vitro screening of inhaled NPs than the regulatory L5178Y and TK6 cells. Copyright © 2015 John Wiley & Sons, Ltd.     

人尿提取物抗肿瘤活性研究

目的　研究人尿提取物的抗肿瘤活性。方法　从人尿中提取到一种小肽混合物UAP(uricantitumorpeptides) ,用细胞计数法和MTT法测定UAP对 3种人肿瘤细胞SMMC772 1,MKN4 5和U937生长的抑制作用 ,并对其进行体内实验和急性毒性实验。结果　人尿中的提取物UAP对体外培养的SMMC772 1,MKN4 5和U937细胞具有抑制作用 ,但对人正常白细胞HNL(humannormalleucocyte)无明显抑制作用 ,体内实验表明UAP对小鼠HAC肝癌 ,小鼠LEWIS肺癌和小鼠S180 肉瘤瘤体生长有明显抑制作用。急性毒性试验显示其的毒性作用很低 ,LD50 =2 137 13mg·kg-1。结论　人尿中此种小肽提取物在体外体内实验中均有明显的抑瘤作用     

The predictive value of chromosome 8p deletion for metastasis of hepatocellular carcinoma: a summary of works in 10 years

Hepatocellular carcinoma (HCC) represents an extremely poor prognostic cancer, which is mainly due to the high frequency of metastasis/recurrence after surgical operation. Exploring the molecular mechanisms involved in HCC metastasis could be helpful in the prediction and early diagnosis of HCC recurrence and could also provide new therapeutic targets for HCC metastasis. In the recent decade, we analyzed the genomic aberrations of the clinical specimens, as well as the metastatic models and cell lines of human HCC to identify the genetic markers related to HCC metastasis and to verify their clinical values in the prediction and control of metastasis of HCC. Using the comparative genomic hybridization (CGH) technique, we compared the differences of chromosomal aberrations between primary HCC tumors and their matched metastatic lesions, and found that chromosome 8p deletions might contribute to HCC metastasis. This novel finding was further confirmed by comparison between nude mice models of HCC with different metastatic potentials. By the more sensitive genome-wide microsatellite analysis, 8p deletion was defined to 8p23.3 and 8p11.2, which are two likely regions harboring metastasis-related genes of HCC. Using ‘8p-specific’ microarrays, two novel metastatic suppressors (HTPAP and MRSA) were identified, and were proven to suppress in vitro invasion and in vivo metastasis of HCC. Clinical studies indicate that 8p deletion detected in HCC or circulating plasma DNA of patients is a useful predictor for metastatic recurrence and prognosis, even for patients with early stage HCC. These novel findings are regarded as important advances in the study of the molecular mechanisms of HCC metastasis, which provide not only a holistic view on the molecular cytogenetic bases of HCC metastasis, but also candidate regions for further study to identify metastatic suppressor genes.     

5'-deoxy-5'-methylthioadenosine phosphorylase--II. Role of the enzyme in the metabolism and antineoplastic action of adenine-substituted analogs of 5'-deoxy-5'-methylthioadenosine

The biological activities of several previously synthesized [J. A. Montgomery et al., J. med. Chem. 17, 1197 (1974)] adenine-substituted analogs of 5'-deoxy-5'-methylthio- or 5'-deoxy-5'-ethyl-thioadenosine, including the 2-fluoroadenine, 2-chloroadenine, 2,6-diaminopurine, 8-azaadenine, and 4-aminopyrazolo [3,4-d]pyrimidine-containing derivatives, have been reexamined. It is demonstrated that many of these analogs are cleaved to their respective free base analogs by 5'-deoxy-5'-methyl-thioadenosine phosphorylase (MTAPase), an enzyme associated with polyamine biosynthesis, and that this reaction is necessary for the cytotoxic action of these MTA analogs to be fully expressed. Evidence to support this includes: (1) the growth of two MTAPase-containing human colon carcinoma cell lines (the HCT-15 and DLD-1 lines) was inhibited by these analogs, whereas an MTAPase-deficient cell line, the CCRF-CEM human T-cell leukemia, was relatively insensitive to their cytotoxic action; (2) extracts of the MTAPase-containing colon carcinoma cell lines were able to cleave these analogs to their respective free base analogs; in contrast, extracts of MTAPase-deficient CCRF-CEM cells were unable to cleave these analogs; (3) intact colon carcinoma cells converted these MTA analogs to their corresponding 5'-phosphorylated analog nucleotides, whereas CCRF-CEM cells did not, at least to detectable levels; and (4) the MTA analog, 5'-deoxy-5'-ethylthio-4-aminopyrazolo [3,4-d]pyrimidine ribonucleoside, which is not a substrate of MTAPase, did not form analog nucleotides and was essentially noncytotoxic to all cell lines tested, whereas the corresponding adenine analog, 4-aminopyrazolo [3,4-d]pyrimidine, readily formed analog nucleotides and was highly cytotoxic to all the lines. It is postulated that the corresponding adenine analog 5'-phosphorylated nucleotides are the primary active metabolites of these MTA analogs, having been formed by the cleavage of these nucleosides to free adenine analogs by MTAPase, followed by the conversion of these base analogs to analog nucleotides by adenine phosphoribosyltransferase and the enzymes of adenine nucleotide phosphorylation. This pathway represents a novel drug-activation system for the synthesis of analog nucleotides and has the potential to be exploited chemotherapeutically.     

Efficacy of HMAF (MGI-114) in the MV522 metastatic lung carcinoma xenograft model nonresponsive to traditional anticancer agents

Summary Illudin analogs are cytotoxic to a variety of multidrug resistant cell lines, and display an unusual toxicity towards DNA helicase-deficient cell lines. Earlier illudin analogs demonstrated efficacy in several xenograft models, including a metastatic MV522 lung cancer model, resistant to conventional anticancer agents. These illudin analogs prolonged life span as compared to conventional agents, but did not induce complete remission of primary tumors. In vitro screening studies identified a semisynthetic derivative, hydroxymethylacylfulvene (HMAF, MGI-114), with increased selective cytotoxicity towards carcinoma cells. The HMAF analog was markedly effective in the experimental MV522 metastasizing lung carcinoma xenograft system, a model refractory to treatment with existing anticancer agents. Treatment with paclitaxel, doxorubicin, or cisplatin failed to significantly inhibit primary tumor growth or prolong life span of MV522 tumor-bearing animals. Treatment with mitomycin C at the LD₂₀ increased life span in surviving animals up to 61% (p = 0.04). Treatment with HMAF induced primary tumor regression in all animals and increased life span greater than 150% (p<0.001). Thus, administration of HMAF inhibited development of lung metastasis in a model refractory to treatment with conventional anticancer agents. These results support further evaluation of HMAF as a therapeutic agent for treatment of solid tumors such as adenocarcinoma of the lung.     

基于FDA不良事件数据库对洛匹那韦/利托那韦安全信号的检测与分析

目的挖掘和评价新型冠状病毒肺炎(COVID-19)治疗方案中建议试用的抗病毒药"洛匹那韦/利托那韦"上市后的安全信号,为临床合理用药提供参考。方法检索美国FDA不良事件报告系统(FDA adverse event reporting system,FAERS)数据库2004年1月1日-2019年12月31日收录以"洛匹那韦/利托那韦"为首要怀疑对象的不良事件(Adverse drug events,ADEs)报告,采用报告比值比法和贝叶斯可信区间递进神经网络法检测该ADE信号,重点评估胃肠、肝肾、神经以及代谢等系统所涉及的安全信号。结果纳入分析的11170959份ADEs报告中,以洛匹那韦/利托那韦为首要怀疑药物的ADEs报告共10120份,发现该药不良反应信号累及多个系统,具有临床参考意义的高风险信号包括急性胰腺炎(ROR=4.32,IC-2SD=1.65)、细胞溶解性肝炎(ROR=20.90,IC-2SD=3.66)、高甘油三酯血症(ROR=27.80,IC-2SD=4.07)、脑室扩张(ROR=58.04,IC-2SD=4.26)、获得性脂肪营养不良(ROR=165.80,IC-2SD=6.10)等;另有高风险且说明书中未收录的安全信号包括呼吸急促(ROR=5.27,IC-2SD=1.79)、获得性范可尼综合征(ROR=122.34,IC-2SD=5.48)和线粒体毒性(ROR=225.12,IC-2SD=5.61),药物-不良事件组合的时间扫描图谱显示这3种安全信号与该药关联性较强。结论基于FAERS不良事件信号检测显示,COVID-19疫情中使用洛匹那韦/利托那韦除密切关注该药的急性胰腺炎、肝功能不全、高甘油三酯血症、脑室扩张、获得性脂肪营养不良等不良事件外,也应注意可能的呼吸急促、获得性范可尼综合征、线粒体毒性等风险。     

A thermally responsive Tat-elastin-like polypeptide fusion protein induces membrane leakage,apoptosis, and cell death in human breast cancer cells

The thermally responsive elastin-like polypeptide (ELP) has great potential as a macromolecular drug delivery vehicle due to its ability to be actively targeted to solid tumors by application of focused hyperthermia. Since, the toxicity properties of a new therapeutic delivery vehicle are crucial to its utility as an effective delivery vehicle, we evaluated the cytotoxicity of a thermally responsive Tat-ELP1 in various cell lines in response to hyperthermia. We report that Tat-ELP1 was not cytotoxic at 37°C in SK-MEL-2, SKOV-3 and WI-38 cells, and only mildly toxic in the MCF-7 breast carcinoma cell line. Application of hyperthermia (42°C) in combination with Tat-ELP1 resulted in cytotoxicity in all cell lines tested, and this toxicity was most prominent in the MCF-7 cell line, which was chosen to study the mechanism behind this increased toxicity. We found that Tat-ELP1 combined with hyperthermia caused membrane leakage and apoptosis, resulting in cell death, but no hemolytic effect was observed on murine erythrocytes.     

Defining the molecular requirements for the selective delivery of polyamine conjugates into cells containing active polyamine transporters

Several N(1)-substituted polyamines containing various spacer units between nitrogen centers were synthesized as their respective HCl salts. The N(1)-substituents included benzyl, naphthalen-1-ylmethyl, anthracen-9-ylmethyl, and pyren-1-ylmethyl. The polyamine spacer units ranged from generic (4,4-triamine, 4,3-triamine, and diaminooctane) spacers to more exotic [2-(ethoxy)ethanoxy-containing diamine, hydroxylated 4,3-triamine, and cyclohexylene-containing triamine] spacers. Two control compounds were also evaluated: N-(anthracen-9-ylmethyl)-butylamine and N-(anthracen-9-ylmethyl)-butanediamine. Biological activities in L1210 (murine leukemia), alpha-difluoromethylornithine (DFMO)-treated L1210, and Chinese hamster ovary (CHO) and its polyamine transport-deficient mutant (CHO-MG) cell lines were investigated via IC(50) cytotoxicity determinations. K(i) values for spermidine uptake were also determined in L1210 cells. Of the series studied, the N(1)-benzyl-4,4-triamine system 6 had significantly higher IC(50) values (lower cytotoxicity) in the L1210, CHO, and CHO-MG cell lines. A cellular debenzylation process was observed in L1210 cells with 6 and generated "free" homospermidine. The size of the N(1)-arylmethyl substituent had direct bearing on the observed cytotoxicity in CHO-MG cells. The N(1)-naphthalenylmethyl, N(1)-anthracenylmethyl, and N(1)-pyrenylmethyl 4,4-triamines had similar toxicity (IC(50)s: approximately 0.5 microM) in CHO cells, which have an active polyamine transporter (PAT). However, this series had IC(50) values of >100 microM, 66.7 microM, and 15.5 microM, respectively, in CHO-MG cells, which are PAT-deficient. The observed lower cytotoxicity in the PAT-deficient CHO-MG cell line supported the premise that the conjugates use PAT for cellular entry. In general, moderate affinities for the polyamine transporter were observed for the N-arylmethyl 4,4-triamine series with their L1210 K(i) values all near 3 microM. In summary, the 4,4-triamine motif was shown to facilitate entry of polyamine conjugates into cells containing active polyamine transporters.     

Species-specific toxicity of copper nanoparticles among mammalian and piscine cell lines

The four copper nanoparticles (CuNPs) with the size of 25, 50, 78 and 100 nm and one type of micron-sized particles (MPs) (～500 nm) were exposed to two mammalian (H4IIE and HepG2) and two piscine (PLHC-1 and RTH-149) cell lines to test the species-specific toxicities of CuNPs. The results showed that the morphologies, ion release and size of the particles all played an important role when investigating the toxicity. Furthermore, the authors found that the particle forms of CuNPs in suspensions highly contribute to the toxicity in all exposed cell lines whereas copper ions (Cu²⁺) only caused significant responses in mammalian cell lines, indicating the species-specific toxicity of CuNPs. This study revealed that the morphologies, ion release rate of NPs as well as the species-specific vulnerabilities of cells should all be considered when explaining and extrapolating toxicity test results among particles and among species.