单链抗体与lidamycin辅基蛋白融合蛋白工程菌的高密度发酵及其免疫活性

目的实现抗Ⅳ型胶原酶单链抗体(Fv)与lidamycin辅基蛋白(LDP)结合构成的融合蛋白Fv-LDP产生菌的高密度发酵,并测定融合蛋白Fv-LDP免疫活性。方法先在5L发酵罐中进行工程菌分批发酵和补料分批发酵,再用固定化金属亲和层析从菌体中纯化融合蛋白Fv-LDP,分步透析使之复性,酶联免疫吸附试验和免疫荧光细胞化学染色检测其免疫活性。结果确定分阶段恒速补料分批发酵为工程菌最佳高密度发酵模式,融合蛋白Fv-LDP最高表达量为4g/L,A600为55左右,培养时间约14h。融合蛋白Fv-LDP对Ⅳ型胶原酶呈阳性免疫反应,能与PG细胞特异结合。结论确定了工程菌高密度发酵工艺,融合蛋白Fv-LDP免疫活性良好,为融合蛋白Fv-LDP与lidamycin烯二炔发色团(AE)构成的小型高效化抗体药物Fv-LDP-AE的研究奠定良好基础。

Optimization of the Assembly Efficiency for Lidamycin Chromophore Bound to its Apoprotein:A Case Study using Orthogonal Array

Objective Lidamycin （LDM） can be dissociated to an apoprotein （LDP） and an active enediyne chromophore （AE）. The detached AE can reassemble with its LDP-containing fusion protein to endow the latter with potent antitumor activity. However, the reassembly of AE with LDP is affected by several factors. Our aim was to optimize the assembly efficiency of the AE with a LDP-containing fusion protein and investigate the influence of several factors on the assembly efficacy. Methods A method based on RP-HPLC was developed to analyze the assembly rate, and an orthogonal experimental design L9 （3^4） was used to investigate the effects of temperature, assembly time, pH and molecular ratio of LDP-containing fusion protein to AE on the assembly rate. Furthermore, the determined optimum conditions for the assembly rate of the LDP-containing fusion protein with AE were applied and evaluated. Results A calibration curve based on the LDM micromolar concentration against the peak-area of AE by HPLC was obtained. The order in which individual factors in the orthogonal experiment affected the assembly rate were temperature〉time〉pH〉molar ratio of AE to protein and all were statistically significant （P〈0.01）. The optimal assembly conditions were temperature at 10 ℃, time of 12 h, pH 7.0, and the molar ratio of AE： protein of 5：1. The assembly rate of AE with a LDP-containing fusion protein was improved by 23% after condition optimization. Conclusion The assembly rate of chromophore of lidamycin with its LDP-containing fusion protein was improved after condition optimization by orthogonal design, and the optimal conditions described herein should prove useful for the development of this type of LDP-containing fusion protein.

Lidamycin inhibits proliferation and induces apoptosis in endot helial cells

目的 研究力达霉素 (lidamycin,L DM)对内皮细胞的增殖抑制作用和诱导细胞凋亡作用。方法 用 MTT测定法和[3H]胸苷掺入测定法观察 L DM对内皮细胞的增殖抑制 ;用流式细胞分析、形态观察、蛋白质印迹分析等方法研究 L DM诱导内皮细胞凋亡及对相关调节蛋白的影响。结果 L DM呈浓度依赖性抑制内皮细胞增殖和诱导内皮细胞凋亡。L DM浓度 1～ 10 nmol/L可将内皮细胞阻断在 G2 /M期。L DM可导致内皮细胞中的游离钙增高 ,可使 Bc1-2和 PCNA蛋白的表达下调 ,但对 Bax蛋白的表达无影响。结论 力达霉素抑制内皮细胞增殖与诱导内皮细胞凋亡 。

Antitumor efficacy of lidamycin on hepatoma and active moiety of its molecule

AIM: To study the in vitro and in vivo antitumor effect of lidamycin (LDM) on hepatoma and the active moiety of its molecule.METHODS: MTT assay was used to determine the growth inhibition of human hepatoma BEL-7402 cells, SMMC-7721cells and mouse hepatoma H22 cells. The in vivo therapeutic effects of lidamycin and mitomycin C were determined by transplantable hepatoma 22 (H22) in mice and human hepatoma BEL-7402 xenografts in athymic mice.RESULTS: In terms of IC50values, the cytotoxicity of LDM was 10 000-fold more potent than that of mitomycin C (MMC)and adriamycin (ADM) in human hepatoma BEL-7402 cells and SMMC-7721 cells. LDM molecule consists of two moieties,an aproprotein (LDP) and an enediyne chromophore (LDC). In terms of IC50 values, the potency of LDC was similar to LDM. However, LDP was 105-fold less potent than LDM and LDC to hepatoma cells. For mouse hepatoma H22 cells, the IC50value of LDM was 0.025 nmol/L. Given by single intravenous injection at doses of 0.1, 0.05 and 0.025 mg/kg, LDM markedly suppressed the growth of hepatoma 22 in mice by 84.7%, 71.6% and 61.8%,respectively. The therapeutic indexes (TI) of LDM and MMC were 15 and 2.5, respectively. By 2 iv. Injections in two experiments, the growth inhibition rates by LDM at doses of 0.1, 0.05, 0.025, 0.00625 and 0.0125 mg/kg were 88.8-89.5%, 81.1-82.5%, 71.2-74.9%, 52.3-59.575%,and 33.3-48.3%, respectively. In comparison, MMC at doses of 5, 2.5, and 1.25 mg/kg inhibited tumor growth by 69.7-73.6%, 54.0-56.5%, and 31.5-52.2%,respectively. Moreover, in human hepatoma BEL-7402 xenografts, the growth inhibition rates by LDM at doses of 0.05 mg/kg ×2 and 0.025 mg/kg ×2 were 68.7%and 27.2%, respectively. However, MMC at the dose of 1.25 mg/kg ×2 showed an inhibition rate of 34.5%. The inhibition rate of tumor growth by LDM was higher than that by MMC at the tolerated dose.CONCLUSION: Both LDM and its chromophore LDC display extremely potent cytotoxicity to hepatoma cells. LDM shows a remarkable therapeutic efficacy against murine and human hepatomas in vivo.

Tissue distribution and excretion of ^(125)I-lidamycin in mice and rats

AIM: To investigate the tissue distribution, urinary and fecal excretions of 125I-lidamycin (125I-C-1027) in mice and its biliary excretion in rats. METHODS:The total radioactivity assay (RA method) and the radioactivity assay after precipitation with 200 mL/L trichloroacetic add (TCA-RA method) were used to dete-rmine the tissue distribution,and the urinary and fecal excretions of 125I-C-1027 in mice and its biliary excretion in rats. RESULTS:Tissue concentrations reached the peak at the fifth minute after administration of 125I-C-1027 to mice. The highest concentration was in kidney, and the lowest in brain at all test-time points. The organs of the concentrations of 125I-C-1027 from high to low were kidney, lung, liver, stomach, spleen, uterus, ovary, intestine, muscle, heart, testis, fat, and brain in mice. The accumulative excretion amounts of 0-24 h, and 0-96 h after administration of 125I-C-1027 were 68.36 and 71.64% in urine, and 2.60 and 3.21% in feces of mice, respectively, and the accumulative excretion amount of 0-24 h was 3.57% in bile in rats. CONCLUSION: Our results reflect the characteristics of the tissue distribution, urinary and fecal excretions of 125I-C-1027 in mice and the biliary excretion of 125I-C-1027 and its metabolites in rats, and indicate that 125I-C-1027 and its metabolites are mainly distributed in kidney, and excreted in urine.

Role of Prosurvival Molecules in the Action of Lidamycin toward Human Tumor Cells

Objective Lidamycin, an enediyne antibiotic, leads to apoptosis and mitotic cell death of human tumor cells at high and low concentrations. The reason why tumor cells have distinct responses to lidamycin remains elusive. This study was to elucidate if cellular prosurvival molecules are involved in these responses. Methods Cleavage of chromatin and DNA was observed by chromatin condensation and agarose gel electrophoresis. Accumulation of rhodamine 123 in lidamycin-treated cells was assayed by flow cytometry. Cell multinucleation was detected by staining with Hoechst 33342. Western blot and senescence-associated β-galactosidase （SA-β-gal） staining were used to analyze protein expression and senescence-like phenotype, respectively. Results SIRT1 deacetylase remained unchanged in 0.5 nmol/L lidamycin whereas cleavage occurred when apoptosis was induced by lidamycin. Increased FOXO3a, SOD-1 and SOD-2 expression and transient phosphorylation of ERK were detected after exposure of human hepatoma BEL-7402 cells to 0.5 nmol/L lidamycin. High expressions of SIRT1 and Akt were found in colon carcinoma HCT116 p53 knock-out cells exposed to lidamycin. Degradation of PARP and p53 by lidamycin as a substitute for SIRT1 and Akt was confirmed with caspase inhibitor Q-VD-OPh and proteasome inhibitor MG132. Resistance to lidamycin-induced DNA cleavage was observed in breast cancer doxorubicin-resistant MCF-7 cells. This was not induced by P-glycoprotein as no accumulation of rhodamine 123 was detected in the resistant cells following exposure to lidamycin. In contrast to sensitive MCF-7 cells, a lower multinucleation rate for the resistant cells was measured following exposure to equal concentrations of lidamycin. Conclusions Cellular prosurvival molecules, such as SIRTI, Akt, SOD-1, SOD-2 and other unknown factors can influence the action of lidamycin on human tumor cells.

Lidamycin Induces Apoptosis of B-Cell Lymphoma Cells and Inhibits Xenograft Growth in Nude Mice

OBJECTIVE To study the cytotoxicity of Lidamycin （LDM） and its induction of apoptosis in Raji and Daudi cells of B-cell lymphoma, and the inhibition of growth of the lymphoma Raji xenograft in nude mice. METHODS MTT assay was used to observe the inhibition by LDM on the proliferation of the Raji and Daudi ceils. Annexin V-FITC/PI double-stain, in combination with flow cytometry （FCM）, was used to determine the induction of apoptosis by LDM in Raji cells. The B-cell lymphoma Raji xenograft model in nude mice was set up to detect the in vivo antitumor activity of LDM. RESULTS LDM markedly inhibited the proliferation of the Raji and Daudi cells in vitro, with IC50 values of 7.13×10^-11 mol/L and 2.91×10^-10 mol/L, respectively. The apoptotic rates of Raji cells were respectively 77.98% and 67.63% at 0.5 nmol/L and 0.25 nmol/L of LDM, indicating an obvious induction of apoptosis in Raji cells. LDM inhibited the formation and growth of human B-cell lymphoma Raji xenograft in nude mice. The inhibition rates of tumor growth were respectively 74.9% and 65.2% in LDM at dosage group of 0.05 mg/kg and 0.025 mg/kg, suggesting an apparent prolongation of survival time in the nude mouse bearing lymphoma. CONCLUSION LDM can effectively induce apoptosis of the B-cell lymphoma cells and inhibit the xenograft growth in nude mice.

Antitumor effects of the molecule-downsized immunocon-jugate composed of lidamycin and Fab' fragment of monoclonal antibody directed against type IV collagenase

Type IV collagenase plays an important role in tumor invasion and metastasis through cleaving type IV collagen in the basement membrane and extracellular matrix. In this study a molecule-downsized immunoconjugate (Fab′-LDM) was constructed by linking lidamycin (LDM), a highly potent antitumor antibiotic, to the Fab′ fragment of a monoclonal antibody directed against type IV collagenase and its antitumor effect was investigated. As assayed in 10% SDS-PAGE gel, the molecular weight of Fab′-LDM conjugate was 65 kD with a 1:1 molecular ratio of Fab′ and LDM. The Fab′-LDM conjugate maintained most part of the immunoreactivity of Fab′ fragment to both type IV collagense and mouse hepatoma 22 cells by ELISA. By MTT assay, Fab′-LDM conjugate showed more potent cytotoxicity to hepatoma 22 cells than that of LDM. Administered intravenously, Fab′-LDM conjugate proved to be more effective against the growth of subcutaneously transplanted hepatoma 22 in mice than free LDM in two experiment settings. In Experiment I, the drugs were given intravenously on day 1 and day 8. Fab′-LDM at the doses of 0.025 mg/kg, 0.05 mg/kg and 0.1 mg/kg inhibited tumor growth by 76.7%, 93.3% and 94.8%, while free LDM at 0.05 mg/kg inhibited tumor growth by 76.1%, respectively. In experiment II, the drugs were given intravenously on day 4 and day 11, Fab′-LDM at the doses of 0.025 mg/kg and 0.05 mg/kg inhibited tumor growth by 74.2%, 80.9%, while free LDM at 0.05 mg/kg inhibited tumor growth by 60.5%, respectively. In terms of survival time, Fab′-LDM was more effective than free LDM. The results suggest that the molecule-downsized immunoconjugate directed against type IV collagenase is of high efficacy in experimental cancer therapy.

Generation and antitumor effects of an engineered and energized fusion protein VL-LDP-AE composed of single-domain antibody and lidamycin

Type IV collagenase plays a pivotal role in invasion, metastasis and angiogenesis of tumor. Single domain antibodies are attractive as tumor-targeting vehicle because of their much smaller size com-pared with antibody molecules produced by conventional methods. Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic. In this study an engineered and energized fusion protein VL-LDP-AE composed of lidamycin and VL domain of mAb 3G11 directed against type IV collagenase was prepared using a novel two-step method. First a VL-LDP fusion protein was constructed by DNA recombination. Secondly VL-LDP-AE was obtained by molecular reconstitution. In MTT assay, VL-LDP-AE showed potent cytotoxicity to HT-1080 cells and KB cells with IC50 values of 8.55×10-12 and 1.70×10-11 mol/L, respectively. VL-LDP-AE showed antiangiogenic activity in chick chrorioallantoic membrane (CAM) assay and tube formation assay. In in vivo experiments, VL-LDP-AE was proved to be more effective than free LDM against the growth of subcutaneously transplanted hepatoma 22 in mice. Drugs were given intravenously on day 3 and 10 after tumor transplantation. Compared in terms of maximal tolerated doses, VL-LDP-AE at 0.25 mg/kg suppressed the tumor growth by 89.5%, LDM at 0.05 mg/kg by 69.9%, and mitomycin at 1 mg/kg by 35%. Having a molecular weight of 25.2 kDa, VL-LDP-AE was much smaller than other reported antibody-based drugs. The results suggested that VL-LDP-AE would be a promising candidate for tumor targeting therapy. And the 2-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.

Antitumor effects of an engineered and energized fusion protein consisting of an anti-CD20 scFv fragment and lidamycin

Antibody-based fusion proteins are the next generation of antibody therapies for cancer and other diseases.CD20 antigen,which is overexpressed on cell membranes in nearly 95% of cases of B-cell Non-Hodgkin's Lymphoma,is an attractive target for the therapy of B-lymphoid malignancies.Lidamycin (LDM) is a potent enediyne-containing antitumor antibiotic that now has entered phase II clinical trials.In this study,we prepared an engineered fusion protein,scFv-LDP,consisting of an anti-CD20 scFv fragment and the apoprotein LDP of LDM using DNA recombination.After purification and refolding,scFv-LDP was found to bind specifically to CD20-positive lymphoma cells using ELISA and indirect immunofluorescent cytochemical staining assays.The energized fusion protein scFv-LDP-AE was obtained using molecular reconstitution of the active chromophore AE of LDM and scFv-LDP.MTT assay revealed potent cytotoxicity of scFv-LDP-AE to CD20-positive Raji and Daudi cells,with IC 50 values of 1.21×10-11 and 6.24×10-11 mol L-1,respectively.An in vivo subcutaneous xenograft model of CD20-positive B cell lymphoma in BALB/c (nu/nu) mice was also utilized.Drugs were given intravenously on day 14 and 21 after tumor transplantation.In terms of maximal tolerated doses,scFv-LDP-AE at 0.3 mg kg-1 suppressed tumor growth by 79.3%,and LDM at 0.05 mg kg-1 by 68.6% (P<0.05).Results suggested scFv-LDP-AE could be a potential candidate for tumor-targeting therapy.

Site-specific PEGylation of lidamycin and its antitumor activity

In this study,N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein(r LDP) of lidamycin(LDM) was prepared using a polyethyleneglycol(PEG) derivative(Mw20 k Da) through a reactive terminal aldehyde group under weak acidic conditions(p H 5.5).The biochemical properties of m PEG-r LDP-AE,an enediyne-integrated conjugate,were analyzed by SDSPAGE,RP-HPLC,SEC-HPLC and MALDI-TOF.Meanwhile,in vitro and in vivo antitumor activity of m PEG-r LDP-AE was evaluated by MTT assays and in xenograft model.The results indicated that m PEGr LDP-AE showed significant antitumor activity both in vitro and in vivo.After PEGylation,m PEG-r LDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP.It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM,implying the possibility that this derivative may function as a payload to deliver novel tumortargeted drugs.

Cloning,expression and characterization of gene sgcD involved in the biosynthesis of novel antitumor lidamycin

Lidamycin with high antitumor activity is a novel enediyne antitumor antibiotic produced by Streptomyces globisporus C1027. The 75 kb biosynthesis gene cluster of lidamycin containing 33 open reading frames has been cloned from S. globisporus C1027. In this paper, the function of sgcD (ORF24) is investigated. Gene disruption experiment proved that sgcD is involved in lidamy- cin biosynthesis. With homologous comparing analysis, we deduce that sgcD codes aminomutase catalyzing a-tyrosine to b-tyrosine which is one motif for lidamycin. To identify the function of en- zyme coded by sgcD, sgcD is cloned into vector pET30a for inducing expression and the activity of expression product is analyzed. The result showed that the expression product of sgcD has the activity of aminomutase. Aminomutase coded by sgcD is the first characterized enzyme involved in the biosynthesis of enediyne antitumor antibiotics. Our research will be helpful to clarifying the biosynthesis mechanism of such kind of antibiotic and to producing new antitumor compounds.

Lidamycin inhibits tumor growth and pulmonary metastasis in murine breast carcinoma and shows synergy with paclitaxel

The main goal of this study was to investigate whether lidamycin (LDM) could enhance the efficacy of paclitaxel (TAX) against breast cancer. In the MTT assay, LDM showed much more potent cytotoxicity than paclitaxel, and there was a synergy on the 4T1/luc breast cancer cells treated with a combination of paclitaxel and LDM. Western blot analysis showed that paclitaxel and LDM synergistically downregulated MMP9, MMP2, VEGF, and upregulated the cleaved PARP proteins. By wound closure cell migration assay, paclitaxel combined LDM obviously inhibited the migration of 4T1/luc cells. At therapeutic dosage level, LDM, paclitaxel, and the combination suppressed the pulmonary metastases by 70.2%, 53.8%, and 88.7%, respectively, and the CDI value was 0.82, indicating synergism. The results show that LDM enhances the antitumor effect of paclitaxel on 4T1/luc breast cancer, in particular, the antimetastatic effects on pulmonary metastasis.

力达霉素经线粒体依赖通路介导细胞凋亡

力达霉素(lidamycin,LDM)是具有我国自主知识产权的烯二炔类抗生素,具有较强的抗肿瘤活性,其抗肿瘤机制有待深入阐明。本研究主要利用人肝癌BEL-7402和乳腺癌MCF-7细胞研究了力达霉素对线粒体凋亡通路相关因子的影响。通过MTT法检测不同剂量力达霉素对肿瘤细胞增殖毒性;蛋白印迹技术检测线粒体和细胞质细胞色素c、总Bax,Bcl-2及p53表达;RT-PCR检测p53和baxmRNA表达等。结果发现,LDM能迅速激活肿瘤细胞线粒体凋亡通路,在2h内就引起线粒体中细胞色素c释放至细胞质中。同时Bcl-2家族成员中促凋亡成员Bax持续增加,而抗凋亡成员Bcl-2先增加后减少。p53蛋白在6h显著增加。Bax蛋白表达升高与其mRNA转录水平升高有关,p53蛋白表达升高与转录后水平有关。Caspase抑制剂可以部分抑制LDM的增殖毒性。因此LDM可以通过激活细胞色素c启动的线粒体凋亡通路发挥抗肿瘤作用。

力达霉素效价单位及效价测定方法的确定

目的:确定力达霉素的效价单位,并建立力达霉素效价测定方法。方法:采用DNA断裂法确定力达霉素标准品引起DNA断裂的最低浓度,并据此浓度确定力达霉素标准品的效价;采用HPLC法分析力达霉素效价与其发色团峰面积的相关性,用力达霉素发色团的峰面积表征力达霉素的效价,用HPLC法测定力达霉素的效价。结果:将首批力达霉素标准品引起pBR322 DNA断裂成Ⅲ型的最低剂量规定为10-4力达霉素单位(u),即每1 mg首批力达霉素标准品的效价为896 u;力达霉素效价与其发色团峰面积呈线性关系(r=0.9995),根据力达霉素发色团的峰面积可以确定力达霉素的效价。结论:本文采用的效价单位确定方法"溯源性"明确;采用HPLC法测定力达霉素的效价可以大大提高传统生物检定的精度。

力达霉素增强顺铂诱导人肝癌BEL-7402细胞凋亡作用及机制研究

目的 研究抗肿瘤抗生素力达霉素与顺铂的体外协同抗肿瘤作用及其机制。方法 采用克隆形成法、流式细胞术、琼脂糖凝胶电泳、Hoechst3 3 3 42和PI双荧光染色法、Westernblot等方法。结果 力达霉素与顺铂联合对人肝癌BEL 740 2细胞有很强的协同杀伤作用 ;可以使BEL 740 2细胞的DNA出现明显的梯状断裂 ;可以使阻滞于S期的BEL 740 2细胞减少而使凋亡细胞比例明显增多 ;联合用药后BEL 740 2细胞核出现明显的凋亡形态变化 ;使BEL 740 2细胞内Bcl 2的表达显著降低。结论 力达霉素可以增强顺铂的抗肿瘤作用 。

力达霉素对人肝癌bel-7402细胞凋亡基因表达和细胞骨架的影响

背景与目的:力达霉素是我所分离的烯二炔类抗肿瘤抗生素,因其独特的化学结构和对体内外肿瘤的强烈抑制作用,而倍受关注。除了断裂DNA外,力达霉素对细胞凋亡基因表达和细胞骨架的影响,可能与其高活性有关。本文通过观察力达霉素对人肝癌bel-7402细胞多指标的影响,进一步阐明其分子机制。方法:用Northernblot印迹法和dot印迹法检测癌基因表达,间接免疫荧光法检测微丝和微管,线粒体特异染料MitosensorTM检测细胞凋亡。结果:低浓度的力达霉素(0.1nmol/L)处理人肝癌bel-7402细胞8h,明显促进c-myc、c-fos基因表达,而抑制N-ras表达。10nmol/L力达霉素处理细胞8h后,细胞伸展,微丝排列整齐,向非恶性细胞转变,但对微管没有影响。用线粒体特异凋亡染料MitosensorTM检测发现,力达霉素处理8h后的细胞未出现凋亡,说明力达霉素引起肿瘤细胞凋亡首先需要诱导c-myc基因表达。结论:低浓度的力达霉素明显影响人肝癌bel-7402细胞的有关凋亡基因表达和细胞骨架的分布,这些结果有助于解释力达霉素高活性地作用肿瘤细胞的分子机制。

不同力达霉素与抗VI型胶原酶单抗偶联物的抗肿瘤作用

研究不同偶联方法制备的力达霉素(LDM)与抗IV型胶原酶单抗3G11免疫偶联物的选择性抗肿瘤作用。采用SPDP和SMBS作为偶联剂,将LDM辅基蛋白69位赖氨酸与2-亚氨基四氢噻吩修饰的单抗3G11连接,制备免疫偶联物;ELISA法测定偶联物的免疫活性,克隆形成法及人HT-1080细胞免疫缺陷小鼠移植模型观察两种偶联物的抗肿瘤作用。偶联物保留了单抗3G11对IV型胶原酶的免疫活性,3G11-SMBS-LDM对体外培养的HT-1080细胞的杀伤作用强于LDM和3G11-SPDP-LDM。动物实验显示,LDM在等摩尔剂量条件下3G11-SMBS-LDM的抑瘤率为86.1%,游离的LDM组为71.2%,3G11-SPDP-LDM组为77.1%,3G11-SMBS-LDM的抑制作用显著增强。3G11-SMBS-LDM组动物的平均生存时间延长为163.7%,3G11-SPDP-LDM组为125.3%,LDM组为71.9%,3G11-SMBS-LDM能显著延长荷瘤鼠的生存期,两个偶联物之间有显著性差异。3G11-SMBS-LDM偶联物更具有选择性抗肿瘤作用,疗效显著提高,荷瘤动物生存期延长,毒性明显降低,可能成为新的肿瘤治疗药物。

力达霉素诱导人胃癌BGC823细胞凋亡和抑制裸鼠移植瘤生长

观察力达霉素(LDM)对人胃癌BGC823细胞的诱导凋亡作用及体内抗肿瘤活性。采用MTT法观察LDM对人胃癌BGC823细胞增殖的抑制作用。利用Annexin V-FITC/PI双染结合流式细胞仪和脱氧核糖核酸末端转移酶介导的缺口末端标记技术检测细胞凋亡的改变。采用Western blotting法检测细胞中VEGF蛋白的表达情况。建立裸鼠胃癌皮下移植瘤模型,观察LDM的体内抗肿瘤活性。LDM能够明显抑制BGC823细胞增殖,诱导细胞凋亡,降低细胞VEGF蛋白的表达,抑制胃癌裸鼠移植瘤的生长。LDM剂量0.02和0.04 mg.kg-1的抑瘤率分别为57%和72%(P<0.01)。LDM可诱导胃癌细胞凋亡并抑制裸鼠移植肿瘤的生长。

基因mdr1高表达多药耐药肿瘤细胞对力达霉素的药物敏感性

目的利用经药物诱导获得的mdr1基因高表达细胞株以及通过mdr1基因转染建立的稳定高表达细胞株,研究多药耐药肿瘤细胞对力达霉素(C-1027)的药物敏感性。方法构建mdr1重组真核表达质粒pcDNA3.1/mdr1,利用脂质体转染技术,获得mdr1高表达HepG2肝癌细胞。经RT-PCR、细胞荧光免疫化学及罗丹明外排实验,鉴定了细胞的mdr1表达水平和药物外排活性。MTT方法测定敏感细胞及相对应的多药耐药细胞对力达霉素等多种抗肿瘤药物的药物敏感性。结果mdr1稳定转染细胞株HepG2/mdr1、多药耐药KBv200细胞和MCF-7/ADR细胞对力达霉素的IC50值分别为(0.020±0.011)nmol.L-1,(0.24±0.20)nmol.L-1和(0.028±0.011)nmol.L-1。相对于各自的敏感细胞,多药耐药细胞HepG2/mdr1,KBv200和MCF-7/ADR对力达霉素的抗药倍数分别是1.3,6.8和1.6倍,对阿霉素的抗药倍数分别是8.8,37.2和181.3倍,对紫杉醇的抗药倍数分别是40.3,336.8和49.2倍。结论mdr1高表达的多药耐药肿瘤细胞对力达霉素仍高度敏感,未表现出抗药性。