洛贝林逆转肿瘤细胞多药耐药机制新进展

洛贝林,是从半边莲的品种中提取的一种哌啶生物碱。近期发现洛贝林可以逆转肿瘤细胞的多药耐药,可能作为新型、低毒、有效的耐药逆转剂。研究其作用机理、体内实验等将有助于探讨其临床实用性。     

参麦注射液对肺腺癌耐药细胞株的逆转作用

本研究探讨了参麦注射液对人肺腺癌耐药细胞株A549/DTX(多西他赛)的耐药逆转作用,并初步分析其作用机制。以MTT法检测参麦注射液、多西他赛及联合应用对耐药细胞株的增殖抑制率,并计算耐药逆转倍数。用流式细胞术测定参麦注射液联合多西他赛对A549/DTX细胞株的促凋亡作用;用Western blotting分析参麦注射液对A549/DTX细胞株的凋亡相关蛋白Bax和Bcl-2表达水平的影响。研究表明,参麦注射液增加耐药细胞株对DTX的敏感性,耐药逆转倍数为3.92,并明显增强DTX对A549/DTX细胞株的促凋亡作用。同时抑制了Bcl-2和P-gp蛋白表达,与对照组相比差异显著(p0.05)。故参麦注射液能部分逆转A549/DTX细胞株耐药性,其作用通过诱导细胞凋亡及降低P-gp表达实现。     

骨肉瘤化疗多药耐药的分子机制及其逆转

目的：总结国内外对骨肉瘤化疗多药耐药的分子机制及相应逆转措施的研究进展;方法：应用PubMed及CNKI期刊全文数据库检索系统,以＂骨肉瘤、化疗多药耐药、机制、逆转＂等为关键词,检索2000-2011年的相关文献。纳入标准：1）骨肉瘤耐药相关的蛋白质、酶类及相关基因;2）各因素发挥作用的机制;3）相关的逆转措施。结果：骨肉瘤多药耐药是当今骨肉瘤化疗治疗的一大难题,多药耐药性即肿瘤细胞对一种抗肿瘤药物产生耐药性的同时,对其他结构和作用机制不同的多种药物产生交叉耐药性。其作用的发生是多种因素共同作用的结果。结论：与骨肉瘤化疗多药耐药相关的主要有P-糖蛋白,多药耐药相关蛋白,肺耐药蛋白,谷胱甘肽,拓扑异构酶,蛋白激酶C以及DNA损伤修复和细胞凋亡抑制等相关机制。而逆转措施主要通过抑制剂和基因疗法。     

顺铂诱导人鼻咽癌细胞CNE1、CNE2后的辐射敏感性

为研究鼻咽癌细胞CNE1、CNE2多药耐药与辐射敏感性的关系,通过体外逐渐增加顺铂浓度的方法诱导鼻咽癌细胞CNE1、CNE2,MTT法分析环孢霉素A、IFN单药及联合应用对多药耐药的逆转倍数,克隆形成实验分析顺铂诱导鼻咽癌细胞CNE1、CNE2多药耐药前后辐射敏感性的改变,同时研究环孢霉素A、IFN逆转耐药处理对辐射敏感性的影响。结果显示,环孢霉素A、IFN联合应用逆转倍数明显高于单药应用,鼻咽癌CNE1细胞经顺铂诱导后辐射敏感性无明显改变,鼻咽癌CNE2细胞经顺铂诱导后辐射敏感性下调,环孢霉素A、IFN逆转耐药处理可以部分恢复其辐射敏感性。     

肿瘤多药耐药:机制及逆转剂

肿瘤细胞多药耐药性(multidrug resistance,MDR)的产生是临床上导致肿瘤化疗失败的主要原因之一,因此寻找高效低毒的MDR逆转剂已成为肿瘤药物开发领域的热点。MDR的作用机制主要包括P-糖蛋白、多药耐药相关蛋白、乳腺癌耐药蛋白、肺耐药相关蛋白等等。多药耐药逆转剂包括钙离子通道阻滞剂、维拉帕米及其衍生物等等。本文主要介绍了MDR的作用机制以及肿瘤多药耐药逆转剂的研究进展。     

防己水煮液逆转MCF-7/ADM多药耐药细胞的初步研究

目的：观察中药防己水煮液对MCF-7／ADM多药耐药细胞的逆转作用,并对防己水煮液的逆转MCF-7多药耐药细胞株的有效组分作初步追踪。方法：用SRB显色法观察防己水煮液对MCF-7野生型细胞的毒性作用及对MCF-7／ADM多药耐药细胞的逆转作用,并用相同方法跟踪防己水煮液不同分离组分逆转MCF-7多药耐药细胞株的作用,确定有效组分。结果：对耐2．0μg／mLADM的MCF-7／ADM多药耐药细胞,当阿霉素浓度降到0．25μg／mL时,防己水煮液对MCF-7／ADM多药耐药细胞的逆转效果比较明显,即0．25μg／mL阿霉素为防己水煮液逆转MCF-7／ADM多药耐药细胞的敏感浓度点;对MCF-7／ADM多药耐药细胞起逆转作用的有效成分大部分集中在经正丁醇萃取所得相和水相中,而极性低的有机溶剂相中基本不合可以逆转MCF-7／ADM多药耐药细胞的有效组分。     

高温逆转人胃癌细胞SGC7901多药耐药性的初步研究

目的:观察高温对人胃癌耐药细胞多药耐药性的逆转作用.方法:对人胃癌耐药细胞株SGC7901/ADM予高温43℃处理,用MTT试验检测高温对ADM、5-FU、DDP、TAX作用下细胞生存率和IC50,并以人胃癌敏感细胞株SG-C7901为对照.结果:实验发现人胃癌耐药株sGc7901/ADM细胞除了对诱导耐药的ADM耐受,对CDDP、5-FU、TAX也有交叉耐药.加温至43℃,耐药株SGC7901/ADM细胞的耐药指数明显下降,而且对ADM组、CDDP组、TAX组人胃癌耐药株SGC7901/ADM细胞耐药逆转倍数分别为3.77、2.24、6.25,但对5-FU组SGC7901/ADM细胞的耐药逆转指数较低,为1.11.结论:高温可以增加耐药株SGC7901/ADM细胞对化疗药物ADM、DDP、TAX的敏感性,一定程度逆转细胞的多药耐药性.     

4′-甲醚-黄芩素对绒癌耐药细胞株多药耐药性的逆转作用研究

为探讨马鞭草中4′-甲醚-黄芩素对人绒癌JAR/VP16耐药细胞株的多药耐药逆转作用及可能的逆转机制,采用四甲基偶氮唑盐比色、透射电镜观察、逆转录多聚酶链反应、流式细胞术、基因芯片以及实时定量PCR等方法,检测到4'-甲醚-黄芩素对化疗药物具有协同增效作用,显著逆转耐药细胞对鬼臼乙叉甙(VP16)、甲氨蝶呤(MTX)及更生霉素(KSM)的耐药性,该药物作用后,耐药细胞超微结构呈现凋亡样改变,细胞凋亡率增高并出现明显凋亡峰.此外,耐药细胞中MDR1、MRP1、MRP2、MRP6、AHR、COMT、FGF2等耐药相关基因以及Bcl-2、BFL1、NAIP、p63等凋亡抑制基因表达降低,而Apaf-1、ASC、ATM、Bad、Bak、Bax、BimL等凋亡促进基因表达升高.上述实验结果表明:4′-甲醚-黄芩素对绒毛膜癌耐药细胞具有显著的耐药逆转作用,这种逆转作用可能是通过降低耐药基因表达、促进细胞凋亡实现的.     

4'-甲醚-黄芩素对绒癌耐药细胞株多药耐药性的逆转作用研究

为探讨马鞭草中4'-甲醚-黄芩素对人绒癌JAR/VP16耐药细胞株的多药耐药逆转作用及可能的逆转机制,采用四甲基偶氮唑盐比色、透射电镜观察、逆转录多聚酶链反应、流式细胞术、基因芯片以及实时定量PCR等方法,检测到4'-甲醚-黄芩素对化疗药物具有协同增效作用,显著逆转耐药细胞对鬼臼乙叉甙(VP16)、甲氨蝶呤(MTX)及更生霉素(KSM)的耐药性,该药物作用后,耐药细胞超微结构呈现凋亡样改变,细胞凋亡率增高并出现明显凋亡峰.此外,耐药细胞中MDR1、MRP1、MRP2、MRP6、AHR、COMT、FGF2等耐药相关基因以及Bcl-2、BFL1、NAIP、p63等凋亡抑制基因表达降低,而Apaf-1、ASC、ATM、Bad、Bak、Bax、BimL等凋亡促进基因表达升高.上述实验结果表明4'-甲醚-黄芩素对绒毛膜癌耐药细胞具有显著的耐药逆转作用,这种逆转作用可能是通过降低耐药基因表达、促进细胞凋亡实现的.     

低频脉冲电场逆转MCF-7/ADR对高三尖杉酯碱和长春新碱的耐药性

为了探讨低频脉冲电场对人乳腺癌多药耐药细胞系MCF-7/ADR耐药性的逆转作用及机制,采用MTT比色法检测MCF-7/ADR的耐药指数和耐药性的逆转倍数,荧光显微镜观察脉冲电场对MCF-7/ADR细胞内DiOC2(3)(P-gp的特异性荧光底物)积累和外排的影响。结果发现,在低频脉冲电场不影响MCF-7/ADR细胞生长的情况下,不同时间的电场作用均能逆转MCF-7/A的多药耐药,对高三尖杉酯碱(HHT)耐药性的逆转倍数在1.429～1.848之间,对长春新碱(VCR)耐药性的逆转倍数在1.473～2.090之间,45min电场作用的逆转效果最好,其次是30min电场作用。药物积累和外排实验结果表明,脉冲电场作用45min能使细胞内的DiOC2(3)积累明显增加,而30min电场作用能显著抑制DiOC2(3)的外排。促进药物积累和抑制其外排可能是脉冲电场逆转多药耐药的机制之一。     

Lobeline,a piperidine alkaloid from Lobelia can reverse P-gp dependent multidrug resistance in tumor cells

Multidrug resistance (MDR) can limit efficacy of chemotherapy. The best studied mechanism involves P-gp (P-glycoprotein) mediated drug efflux. This study focuses on MDR reversal agents from medicinal plants, which can interfere with P-gp. Rhodamine 123 accumulation assay and flow cytometry analysis were employed to screen for P-gp dependant efflux inhibitors. Lobeline, a piperidine alkaloid from Lobelia inflata and several other Lobelia species, inhibited P-gp activity. MDR reversal potential of lobeline could be demonstrated in cells treated with doxorubicin in that lobeline can sensitize resistant tumor cells at non-toxic concentrations. However, lobeline cannot block BCRP (Breast Cancer Resistance Protein) dependent mitoxantrone efflux. Lobeline could be a good candidate for the development of new MDR reversal agents.     

Lobeline augments and inhibits cocaine-induced hyperactivity in rats

Lobeline has high affinity for nicotinic receptors and alters presynaptic dopamine storage and release in brain. Moreover, lobeline decreases the reinforcing and locomotor-activating properties of methamphetamine, suggesting that lobeline may be a pharmacotherapy for psychostimulant abuse. This study determined if lobeline alters cocaine-induced hyperactivity and if lobeline alters the induction and/or expression of sensitization to cocaine. On Days 1-12, male rats were administered lobeline (0.3 or 1.0 mg/kg) or saline, placed in an automated activity monitor for 20 min, administered cocaine (10, 20 or 30 mg/kg) or saline and returned to the monitor for 60 min. On Day 13, the effect of lobeline on the induction and expression of sensitization to cocaine was determined. Lobeline did not alter the effect of cocaine after acute injection. However, 1.0 mg/kg lobeline attenuated cocaine (10 and 20 mg/kg)-induced hyperactivity after repeated administration and prevented the development of sensitization to these cocaine doses. Interestingly, 0.3 mg/kg lobeline augmented cocaine (10 mg/kg)-induced hyperactivity after repeated administration. Lobeline did not alter the effect of 30 mg/kg cocaine. The present results indicate a complex interaction of lobeline with cocaine and support other research indicating a role for nicotinic receptors in the development of sensitization to psychostimulants.     

Evaluation of mutagenic and genotoxic activities of lobeline and its modulation on genomic instability induced by ethanol

Aim

Lobeline is a natural alkaloid derived from Lobelia inflata that has been investigated as a clinical candidate for the treatment of alcoholism. In a pre-clinical trial, lobeline decreased the preference for and consumption of ethanol, due to the modulation of the nicotinic acetylcholine receptor. However, the interaction between lobeline and ethanol is poorly known and thus there are safety concerns.The present study was conducted to evaluate the mutagenic and genotoxic effects of lobeline and assess its modulation of ethanol-induced toxicological effects.

Main methods

CF-1 male mice were divided into five groups. Groups received an intraperitoneal injection of saline solution, lobeline (5 or 10 mg/kg), ethanol (2.5 g/kg), or lobeline plus ethanol, once a day for three consecutive days. Genotoxicity was evaluated in peripheral blood using the alkaline comet assay. The mutagenicity was evaluated using both Salmonella/microsome assay in TA1535, TA97a, TA98, TA100, and TA102 Salmonella typhimurium strains and the micronucleus test in bone marrow. Possible liver and kidney injuries were evaluated using biochemical analysis.

Key findings

Lobeline did not show genotoxic or mutagenic effects and did not increase the ethanol-induced genotoxic effects in blood. Lobeline also protected blood cells against oxidative damage induced by hydrogen peroxide. Biochemical parameters were not altered, indicating no liver or kidney injuries or alterations in lipid and carbohydrate metabolisms.

Significance

These findings suggest that lobeline does not induce gene or chromosomal mutations, and that this lack of genetic toxicity is maintained in the presence of ethanol, providing further evidence of the safety of this drug to treat alcohol dependence.     

Lobeline Displaces [3H]Dihydrotetrabenazine Binding and Releases [3H]Dopamine from Rat Striatal Synaptic Vesicles: Comparison with d-Amphetamine

Abstract: Lobeline, an alkaloid from Indian tobacco (Lobelia inflata), is classified as a nicotinic agonist and is currently used as a smoking cessation agent. However, our previous in vitro studies demonstrate that lobeline does not act as a nicotinic agonist but alters presynaptic dopamine (DA) storage by potently inhibiting DA uptake into synaptic vesicles. Recently, d-amphetamine has been reported to act at the level of the synaptic vesicle to alter presynaptic function. The present in vitro studies further elucidate the mechanism of lobeline's action and compare its effects with those of d-amphetamine. [³H]Dihydrotetrabenazine ([³H]DTBZ), used routinely to probe a high-affinity binding site on the vesicular monoamine transporter (VMAT2), bound to vesicle membranes from rat striatum with a K_D of 1.67 nM and B_max of 8.68 pmol/mg of protein. Lobeline inhibited [³H]DTBZ binding with an IC₅₀ of 0.90 µM, consistent with its previously reported IC₅₀ of 0.88 µM for inhibition of [³H]DA uptake into vesicles. These results suggest that lobeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d-amphetamine inhibited [³H]DTBZ binding to vesicle membranes with an IC₅₀ of 39.4 µM, a concentration 20 times greater than reported for inhibition of VMAT2 function, suggesting that d-amphetamine interacts with a different site than lobeline on VMAT2 to inhibit monoamine uptake. Kinetic analysis of [³H]DA release from [³H]DA-preloaded synaptic vesicles in the absence of drug revealed a t_1/2 of 2.12 min. Lobeline and d-amphetamine evoked [³H]DA release with EC₅₀ values of 25.3 and 2.22 µM, respectively. At a concentration 10 times the EC₅₀, lobeline and d-amphetamine significantly decreased the t_1/2 of [³H]DA release to 1.58 and 1.48 min, respectively. Thus, in contrast to d-amphetamine, which is equipotent in inhibiting DA uptake and promoting release from the synaptic vesicles, lobeline more potently (28-fold) inhibits DA uptake (via an interaction with the DTBZ site on VMAT2) than it evokes DA release to redistribute presynaptic DA storage.     

Differential Effect of Nicotinic Agonists on the [3H]Norepinephrine Release from Rat Hippocampal Slices

The aim of this study was to investigate the mechanisms involved in the effect of nicotinic agonists on the [³H]norepinephrine ([³H]NE) release from rat hippocampal slices. The stimulatory effect of nicotine, cytisine, epibatidine and anatoxin-A was completely blocked by the nicotinic antagonist mecamylamine (10 M). In contrast, the effect of dimethylphenylpiperazinium (DMPP) was only partially inhibited by mecamylamine but was completely blocked by the NE uptake inhibitor desipramine (DMI, 10 M). Finally, the effect of lobeline was not affected by mecamylamine and was only partially blocked by DMI. Our data indicate that the majority of nicotinic agonists increase the release of [³H]NE exclusively via stimulation of nicotinic acetylcholine receptors (nAChRs). DMPP, in addition to the stimulation of nAChRs, also evokes a carrier-mediated release. Lobeline has no stimulatory effect on nAChRs, induces a carrier-mediated release and has a further action of unidentified mechanism. Our results suggest that special caution is required for the interpretation of data, when DMPP or lobeline are used as nicotinic agonists.     

Evolutionary ecology of resistance to herbivory: an investigation of potential genetic constraints in the multiple-herbivore community of Solanum carolinense

As part of a study of the ecological and evolutionary dynamics of herbivore resistance in Solanum carolinense (horsenettle), potential genetic constraints to the evolution of resistance to 11 of its most common herbivores were investigated. Leaf, flower, fruit, and stem herbivory were measured in a field experiment involving 24 ramets of each of 40 horsenettle genets. The experimental plant population contained significant genetic variation for resistance to all 11 species of herbivore. For only one species was there an indication of a genotype-by-environment interaction in the expression of resistance that might constrain its evolution. Genetic correlations in resistance to different species were common but not universal, with seven negative and 12 positive correlations out of the 55 pairwise species comparisons. Correlations were independent of plant part fed upon. The evolution of the resistance of horsenettle to most of its diverse community of herbivores does not appear to be prevented by a lack of genetic variation or by genotype-by-environment interactions in resistance. Negative genetic correlations in resistance to different herbivores may play a small role in slowing the evolution of resistance, but positive correlations may play at least as large a role in facilitating its evolution.     

Stability of resistance to neguvon in the fly Musca domestica L. after cessation of laboratory selection.

Stability of resistance to neguvon was studied in three strains of Musca domestica L. after selection using different numbers of generations and different degrees of resistance. It has been found that the stability of resistance to neguvon is not always the same after cessation of selection. The loss of resistance depends on the duration of selection, i.e., on the degree of homogenization of populations with respect to resistant specimens. The degree of resistance is the function of the duration of selection to the same extent as the loss of resistance depends on it. The period of time during which the insecticide has been used is of decisive importance for the stability of resistance. The process of loss of resistance, similarly to that of its development, takes its course according to a zigzag curve, but in the opposite direction. This variation of resistance, occurring also in its development, can be explained by the heterogenity of the strains.     

Restoration of effectiveness of R. meliloti ineffective mutants by transduction of high level streptomycin resistance.

Rhizobium meliloti lysine dependent mutant, L5-30lys, was ineffective and this mutation was not cotransducible to lys. Transduction of chloramphenicol and linked temperature sensitive mutations did not change its symbiotic properties. Subsequent transduction of high level streptomycin resistance restored its effectiveness. Streptomycin resistance marker was linked to chloramphenicol resistance and temperature sensitive markers.     

单核苷酸多态性与甜瓜抗枯萎病分子育种研究

目的:结合单核苷酸多态性标记技术,利用甜瓜本身的抗病性以解决新疆甜瓜病害问题。方法:对新疆甜瓜抗枯萎病基因Fom-2基因进行克隆分析,并根据Fom-2基因在不同抗性甜瓜亲本的单核苷酸多态性,设计检测SNP标记的PCR扩增引物,验证其多态性;并利用F2代分析该标记与筛选获得的甜瓜抗枯萎病基因连锁的SSR标记的遗传关系。结果:在抗病与感病甜瓜品种中均扩增获得PCR条带,试验中设计单核苷酸多态性分子标记在抗病品种为显性,与筛选的和抗枯萎病基因紧密连锁的共显性标记SSR430共分离。结论:不同抗性甜瓜品种均含有Fom-2基因或其高度同源序列,SNP显性标记和共显性标记SSR430均可用于甜瓜抗枯萎病分子标记辅助育种。