小鼠B16黑色素瘤细胞HACs的提纯及其抑瘤作用和机理的探讨

目的：从小鼠Ｂ１６黑色素瘤细胞胞浆中提纯热休克蛋白抗原肽复合物（ｈｅａｔ ｓｈｏｃｋ ｐｒｏｔｅｉｎ－ａｎｔｉｇｅｎ ｐｅｐｔｉｄｅ ｃｏｍｐｌｅｓｅｓ．ＨＡＣＣｓ）,并观察其抑瘤作用及探索其机制。方法：ＣＮＢｒ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析法纯纯Ｂ１６ ＨＡＣｓ,应用体内免疫法检测ＨＡＣ的抑瘤作用,结晶紫染色法检测γ－ＩＦＮ分泌活性,ＣｏｎＡ诱导的淋巴母细胞增殖法检测ＩＬ－２分泌活性,＾３Ｈ－     

黑色素瘤B16细胞热休克蛋白 抗原肽复合物的体内外抑瘤效应 *

黑色素瘤B16细胞胞浆HSP-抗原肽复合物（HAC）的制备、免疫原性的诱导及其抑瘤作用的研究。方法:采用Tris-HCl提取和Sephacryl S-200凝胶过滤制备B16细胞HAC,通过C57BL/6J小鼠体内诱导特异性CTL,再经体内、体外实验检测其抑瘤作用。结果:凝胶过滤获得的含60～97 kD蛋白的41,47和53管的HAC可以降低肿瘤发生率、延长肿瘤出现时间及降低小鼠死亡率。结论: B16 细胞胞浆中的60～97 kD的HAC具有免疫原性及抑瘤作用,为制备肿瘤疫苗提供了实验依据。     

mB7－1基因转染小鼠黑色素瘤细胞诱导的抗瘤效应研究

目的：研究共刺激Ｂ７－１（ＣＤ６０）在诱导有效的抗肿瘤免疫反应反应中所起的作用。方法：在体外,三种肿瘤细胞与同上鼠脾淋巴细胞混合培养（ＭＬＴＣＳ）后,测定淋巴细胞增殖指数（ＭＴＴ法）和特异杀伤活性（ＬＤＨ释放改良法）;ＭＴＴＩ地测定肿瘤细胞体外增殖能力后,将Ｂ１６－ｎｅｏ和Ｂ１６－ｍＢ７－１妆种于Ｃ５７ＢＬ／６小鼠皮下后观察成瘤期、荷瘤小鼠存活以及肿瘤结节生长速度。结果：与野生型Ｂ１６细胞和模拟转     

HER2/neu肽诱导BALB/c小鼠产生特异性CTL及其抑瘤作用的研究

目的：探讨来源于ＨＥＲ２／ｎｅｕ原癌蛋白的多肽诱导特异性细胞免疫应答及其在体内的抗癌效应。方法：利用合成的２个具有小鼠Ｈ－２Ｋ＾ｄ分子结合基序的ＨＥＲ２／ｎｅｕ肽作为肿瘤排斥抗原,在体外刺激小鼠淋巴细胞以及皮下免疫小鼠,观察淋巴细胞的增殖能力、ＣＴＬ的杀伤活性以及ＨＥＲ２／ｎｅｕ肽的抑瘤作用。结果：ＨＥＲ２／ｎｅｕ肽在体内和体外对ＢＡＬＢ／ｃ小鼠淋巴细胞的增殖都有显显的促进作用。ＨＥＲ２／ｎｅｕ肽免疫ＢＡＬＢ／ｃ小鼠后,可以从小鼠淋巴细胞中诱导出肽特异性ＣＴＬ,这些ＣＴＬ可以特异地杀伤ＨＥＲ２／ｎｅｕ阳性ＳＰ２／０细胞,而对ＨＥＲ２／ｎｅｕ阴性ＳＰ２／０细胞却无明显的杀伤活性。ＳＰ２／０ＨＥＲ２细胞在ＨＥＲ２／ｎｅｕ肽免疫小鼠体内的生长受到抑制。结论：研究结果表明ＨＥＲ２／ｎｅｕ肽可以起到一定的抑瘤保护作用,提     

树突状细胞诱导抗人肝癌细胞系的肿瘤免疫

目的 以树突状细胞（ＤＣ）在体外诱导抗肝瘤免疫。方法 自肝癌患者外周血中分离ＤＣ;以粒／巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）及白介素－４（ＩＬ－４）联合刺激ＤＣ;以人肝癌细胞系ＨｅｐＧ２细胞和ＢＥＬ－７４０２细胞的肿瘤相关抗原（ＴＡＡ）激活ＤＣ;ＤＣ诱导自体Ｔ淋巴细胞增殖、分化为细胞毒素性Ｔ细胞（ＣＴＬ）;检测ＣＴＬ对ｅｐＧ２细胞,ＢＥＬ－７４０２细胞、ＳＧＣ－７９０１细胞、ＬＯＶＯ细胞及ＨＯＳ－     

腺病毒介导的胞嘧啶脱氨酶自杀基因联合IL-2基因疗法的肿瘤治疗作用及免疫机理分析

目的以腺病毒作为载体，将大肠杆菌胞嘧啶脱氨酶（ＣＤ）基因与小鼠ＩＬ－２基因联合转移，研究其体内抗肿瘤作用及免疫机理。方法小鼠皮下接种黑色素瘤Ｂ１６Ｆ１０细胞后３天，肿瘤局部注射表达ＩＬ－２的重组腺病毒ＡｄＩＬ－２和表达ＣＤ的重组腺病毒ＡｄＣＤ，然后连续１０天给予５－氟胞嘧啶（５－Ｆｃ）３００ｍｇ／ｋｇ进行治疗。结果联合治疗组荷瘤小鼠皮下肿瘤结节的生长明显受到抑制，小鼠存活期明显长于ＡｄＩＬ－２、ＡｄＣＤ／５－Ｆｃ、ＡｄｌａｃＺ／５－Ｆｃ或ＰＢＳ组。经联合治疗后，小鼠脾细胞的ＮＫ活性和ＣＴＬ杀伤活性明显增强；肿瘤瘤体内ＣＤ４、ＣＤ８细胞浸润增加；肿瘤细胞表达Ｈ－２Ｋｂ和Ｂ７－１分子明显增加。结论联合应用自杀基因和ＩＬ－２基因治疗，一方面可以明显抑制荷瘤小鼠肿瘤生长，另一方面可以提高机体对肿瘤细胞免疫应答，增加机体的抗肿瘤作用，是肿瘤基因治疗中一条行之有效的途径。     

树突状细胞局部免疫抗瘤作用的初步研究

目的：探讨树突状细胞（ＤＣ）瘤内注射的局部免疫方式对小鼠Ｈ２２肿瘤的治疗效果。方法：体外诱导生成树突状细胞,经Ｈ２２肿瘤裂解抗原致敏,实验分为对照组、ＤＣ组和ＤＣ＋ＣｐＧ-ＯＤＮ组,分别与Ｔ细胞共培养,收获的Ｔ细胞与肿瘤细胞共培养,观察其对肿瘤细胞的杀伤。体内试验：ＢＡＬＢ／ｃ小鼠皮下接种Ｈ２２肿瘤细胞制作成荷瘤鼠,第４天时进行局部瘤内免疫治疗,分为３组：生理盐水组、ＤＣ组和ＤＣ＋ＣｐＧ-ＯＤＮ组,免疫治疗１０天后处死小鼠,观察其治疗小鼠Ｈ２２肿瘤的效果。结果：体外实验中,对照组、ＤＣ组和ＤＣ＋ＣｐＧ-ＯＤＮ组的肿瘤杀伤率分别为（１０.８０±３.２７）％、（３８.２６±５.６０）％和（４２.６６±９.００）％,后两组杀伤率均高于对照组（Ｐ＜０.０１）;体内实验中,生理盐水组、ＤＣ组和ＤＣ＋ＣｐＧ-ＯＤＮ组的平均瘤重（ｇ）分别为１.８０４±０.４２２、１.２１６±０.３３５和０.７３３±０.１９１（Ｐ＜０.０１）。结论：ＤＣ瘤苗局部瘤内注射可抑制小鼠Ｈ２２肿瘤的生长,联合应用非甲基化ＣｐＧ-ＯＤＮ可以明显提高抑瘤效果。     

小鼠红白血病细胞来源的树突状细胞对特异性CTL的体内外诱 …

目的 探讨白细胞介素－１２（ＩＬ－１２）诱导小鼠红白血病细胞产生的树突状细胞,对白血病特异性ＣＴＬ细胞的诱导作用,及体内免疫后对抗肿瘤免疫应答的诱导效果。方法：采用４小时＾５１Ｃｒ释放法,检测ＣＴＬ杀伤活性;观察小鼠红白细胞病细胞来源的树突状细胞体内免疫治疗后对肿瘤的抑制作用,采用了ＨＥ染色和电镜等技术,分析肿瘤组织的病理学变化。结果：ＩＬ－１２诱导ＦＢＬ－３细胞产生的ＤＣ,在体外可诱导出ＦＢＬ－     

HSV-tk/GCV协同放射治疗黑色素瘤的实验研究 *

目的：了解ＨＳＶ－ｔｋ／ＧＣＶ系统协同放射治疗对小鼠黑色素瘤的联合杀伤作用。方法：通过逆转录病毒介导,将潮霉素磷酸转移酶和ＨＳＶ－ｔｋ的融合基因（ｈｙｔｋ）导入黑色素瘤细胞中并获得表达,通过体外和Ｃ５７ＢＬ／６小鼠动物实验,检测 ＧＣＶ对转基因细胞的体内外杀伤作用及联合应用放射治疗对黑色素瘤的治疗作用。结果：ＰＣＲ、ＲＮＡ Ｄｏｔ ｂｌｏｔｔｉｎｇ、免疫组化检测证实ＨｙＴＫ在转基因细胞的表达,体内外实验示ＧＣＶ对Ｂ１６／ＨｙＴＫ细胞有明显的杀伤作用,而对野生型Ｂ１６细胞无明显杀伤作用（Ｐ＜０．０５）;联合应用低剂量ＧＣＶ可使转入ｈｙｔｋ基因的黑色素瘤细胞对放疗的敏感性明显增高（ＳＥＲ＝１．６２）,体内实验也证实协同疗法可延缓荷瘤小鼠的肿瘤生长。结论：ＨＳＶ－ｔｋ／ＧＣＶ系统协同放射治疗有望成为黑色素瘤基因治疗的一种有效方法。     

GM-CSF及B7-1基因修饰的肿瘤细胞疫苗抗肿瘤的研究

目的：研究ＧＭ－ＣＳＦ及Ｂ７－１基因修饰的肿瘤细胞作为瘤苗的有效性。方法：我们将小鼠ＧＭ－ＣＳＦ及ＣＤ８０基因通过逆转录病毒载体分别导入ＥＬ－４淋巴瘤细胞中,进而研究了ＥＬ－４／ＧＭ－ＣＳＦ及ＥＬ－４／Ｂ７－１在同系Ｃ５７Ｂ习中的成瘤性及其诱导抗肿瘤免疫的效果。结果：转Ｂ７－１基因的肿瘤细胞诱发了ＣＤ８０的高表达。转基因的肿瘤细胞在同源小鼠中的成瘤性下降,免疫原笥增强。在肿瘤生长的早期对实验性荷瘤     

Phenothiazines induce apoptosis in a B16 mouse melanoma cell line and attenuate in vivo melanoma tumor growth

Phenothiazines and related antipsychotics were reported to have an antiproliferative effect in several tissue cultures. The aims of this study were: a) to screen in vitro, the potential anti-cancer activity of phenothiazines in wild-type and multi-drug resistant (MDR) B16 mouse melanoma cell lines; and b) to determine the in vivo anti-tumor effect of an in vitro selected highly potent phenothiazine (thioridazine) in a murine melanoma model. The following phenothiazines were evaluated: perphenazine, fluphenazine, thioridazine trifluoperazine and chlorpromazine. All agents induced a dose-dependent decrease in cell viability in wild-type and in MDR B16 melanoma cells. Thioridazine displayed the highest antiproliferative activity. Flow cytometric analyses of 24-h treated B16 melanoma cells revealed an increase in fragmented DNA (16.3 vs 71.3% and 87.2% in controls, 25 microM and 50 microM thioridazine-treated, respectively). Apoptosis was confirmed by co-staining of thioridazine-treated B16 cells (12.5 microM) with propidium iodide and Hoechst 33342 reagents. Caspase-3 expression, a typical mediator of apoptosis, was markedly increased following a 4-h exposure of B16 cells to thioridazine (25 microM and 50 microM). This increase could be blocked by a specific caspase-3 inhibitor. In vivo studies were performed using female C57/Bl mice. Animals were inoculated with wild-type B16 cells by i.v. injection into the tail vein. Mice were treated with thioridazine (10 and 15 mg/kg x3/week i.p. or 15, and 25 mg/kg/day p.o.) and control animals received saline. Mice were monitored for 21-30 days. Body weight was recorded. After autopsy, the lung weight and number of pulmonary melanoma colonies were determined. Thioridazine administration (i.p. or p.o.) resulted in the reduction of lung tumor burden and an increase in mice survival. In conclusion, several phenothiazines, and particularly thioridazine, induced apoptosis of B16 melanoma cells and demonstrated in vivo anti-tumor activity.     

Melanoma B16-F1 cells coated with fusion protein of mouse calreticulin and virus G-protein coupled receptor induced the antitumor immune response in Balb/C mice

In apoptotic progress of tumor cells stimulated by special agents, the calreticulin (CRT) was relocated from endoplasmic reticulum onto the cell surface. When used as cellular antigen to immunize experimental animals, these CRT-coated apoptotic tumor cells could initiate effective anti-tumor immunoresponse against homologous tumor cells, indicating the value of CRT in anti-tumor immunotherapy. In order to develop an universal technique that could make CRT-coating more efficiently in the tumor cells, in this study, a mouse CRT recombinant gene with virus G-protein coupled receptor (vGPCR) was constructed and cloned into vector pcDNA3.1(+). When resulted plasmid pcDNA3.1(+)-mCRT/ vGPCR was stably transfected into the mouse melanoma B16-F1 cells, the mCRT-vGPCR recombinant protein was synthesized. With the membrane-locating ability of vGPCR in the recombinant protein, mCRT-vGPCR was carried onto the surface of B16-F1 cells efficiently. Overexpression of mCRT-vGPCR on the cell surface could enhance the phagocytosis of B16-F1 by macrophages in vitro. When mCRT-vGPCR coated B16-F1 cells were used as a cell-antigen to immunize mice, the specific anti-tumor immune response against the homologous tumor cells was initiated efficiently. Our data in this study may provide a new possibility for CRT-mediated tumor immune prevention and treatment.     

In vitro and in vivo correlation of the effect of granulocytemacrophage colony-stimulating factor gene transfer on the tumorigenicity and immunogenicity of B16 melanoma

Transduction of murine B16 melanoma cells with a GM-CSF gene, the B16-MG tumor line, showed reduced tumorigenicity. In vitro studies demonstrated no remarkable difference between the parent and transduced tumor lines in their ability to induce secondary response to generate the anti-tumor killer cells (immunogenicity), or in their susceptibility to the killing by anti-tumor killer cells (immunosensitivity). Both CD4(+) and CD8(+) cells were required for the generation of the effecters. Nevertheless the effecters were determined to be Thy1.2(+), CD8(-), and NK1.1(-). At least two antigenic specificities could be defined in the cytolytic reactions. One was a broadly cross-reactive antigen shared by a variety of tumor cells, and the other apparently a tumor-specific antigen which was only present in B16 tumors. Cold target inhibition experiment confirmed these specificities. In the in vivo tumor transplantation study, the B16-MG cell line was not only more immunogenic but also was more immunosensitive than the parent line. More than 50% of the mice which were immunized with B16-MG remained tumor free after challenge with the parent tumor B16, indicating that GM-CSF gene transfer makes an effective tumor vaccine. The in vivo protective effect was specific for B16 tumor, thus only the tumor-specific antigen could function as transplantation antigen. Both CD4(+) and CD8(+) cells were required for providing the in vivo protection. Both the B16 and B16-MG tumor bearing hosts could generate anti-tumor killer cells, hence the development of progressive growth of B16 tumor was not due to the lack of anti-tumor immune response. It appears that the overall effect of in vivo tumor immunity is determined by a complex network of interactions among different compartments of host immune cells and different compartments of host immune cells and different immune-regulatory molecules derived from the host and from the tumor.     

Hsp70-肿瘤抗原肽复合物防治小鼠黑色素瘤B16肺转移的作用

目的:探讨热休克蛋白70-肿瘤抗原肽复合物(Hsp70-antigen peptide complexes)对小鼠黑色素瘤B16转移的防治作用.方法:分别从小鼠腿部接种的B16实体瘤及小鼠肺B16转移灶提取混合抗原肽,体外与Hsp70结合制得复合物,此复合物免疫小鼠后用于预防或治疗经尾静脉接种转移至肺的B16黑色素瘤,观察其对肿瘤转移的防治作用.结果:Hsp70-肿瘤抗原肽复合物免疫后肺转移灶节结数显著减少(P＜0.01),体外脾细胞表现出对B16较高的杀伤率(P＜0.01);并对肺转移灶有显著的治疗作用(P＜0.01),而从B16实体瘤提取的混合抗原肽制得的复合物比从肺转移灶提取的混合抗原肽制得复合物有更好的治疗效果(P＜0.01),表现出体外脾细胞对B16更高的杀伤率(0.01＜P＜0.05).结论:Hsp70-肿瘤抗原肽复合物对肿瘤的转移有明显的防治作用,而从实体瘤提取的混合抗原肽比从转移灶提取的混合抗原肽更为有效.     

Potentiated antitumor effects of butyrate and actinomycin D in melanoma model in mice.

Butyrate and butyric acid derivatives are well known to induce differentiation and apoptosis of tumor cells and have also recently gained acceptance as potential anticancer agents. In this study we observed an increased expression of mTNFalpha in tumor tissues in mice treated with butyrate prodrug tributyrin. Since in in vitro experiments we observed a potentiating effects of TNFalpha and actinomycin D on B16F10 cells and also a synergistic interaction was previously claimed between those agents, we investigated the anti-tumor activity of the combination therapy with butyrate and actinomycin D in the B16F10 melanoma model in mice. The combination of the drugs resulted in a strongly potentiated tumor growth retardation in melanoma bearing mice. However the B16F10 cells in vitro did not produce any detectable amounts of TNFalpha. The presented data strongly suggest that one of the mechanism of this successful drug combination could depend on the interaction of the actinomycin D with butyrate-induced TNFalpha produced by stromal or tumor infiltrating immune cells. The results illustrate also the possible application of this combination in cancer therapy.     

Taurolidine induces apoptosis of murine melanoma cells in vitro and in vivo by modulation of the Bcl-2 family proteins

BACKGROUND: This study evaluates whether taurolidine, a novel antibiotic agent, induces murine melanoma cell apoptosis in vitro and in vivo. METHODS: Murine melanoma cells (B16 4A5 and B16 F10) were treated with taurolidine (0-100 microM) for 12 and 24 hr. Cell viability and apoptosis were assessed by MTT assay and FACScan analysis. Expression of the Bcl-2 family proteins was detected by Western blot analysis. In vivo, taurolidine-induced anti-tumor cytotoxicity was assessed in C57BL/6 mice. Therapeutic effectiveness, by intraperitoneal injection of taurolidine (15 mg/mouse) on alternate days for 2 weeks, was evaluated in mice bearing B16 4A5 tumor xenografts. Primary and metastatic tumor growth and intra-tumor apoptotic index were measured. RESULTS: Taurolidine induced cell apoptosis and reduced cell viability in murine melanoma cells. The pro-apoptotic protein Bax was enhanced, whereas the anti-apoptotic protein Bcl-2 was inhibited by taurolidine treatment. In vivo, systemic injection of 15-mg taurolidine was identified as the maximally tolerated dose. Administration of taurolidine at 15 mg/mouse significantly inhibited primary and metastatic tumor growth, which was mirrored by a significantly increased intra-tumor apoptotic index. CONCLUSIONS: These results demonstrate that taurolidine significantly attenuated melanoma tumor growth, which may result from taurolidine-induced apoptosis by modulation of the Bcl-2 family proteins.     

人参皂甙Rg3和核糖核酸酶抑制因子转基因对小鼠黑色素瘤的抑制作用研究

目的观察人参皂甙Rg3和核糖核酸酶抑制因子(RI)转基因对小鼠B16黑色素瘤肺转移的抑制作用和影响,探讨人参皂甙Rg3和RI抗肿瘤生长和转移作用的分子机制。方法制备转RI基因的B16黑色素瘤肺转移小鼠模型,对野生型对照组(W组)、空质粒转染组(B组)和RI转基因组(RI组)以及给予Rg3的野生型对照组(Rg3/W组)、空质粒转染组(Rg3/B组)和RI转基因组(Rg3/RI组)中,荷瘤小鼠肺重量、肿瘤转移灶数目、生存期和肿瘤组织微血管密度进行检测和分析。结果Rg3和RI转基因使荷瘤小鼠肺重量降低,肿瘤转移灶数目减少,其肺重量降低和肿瘤转移灶数目减少的程度以Rg3/RI组最明显,Rg3/B组、Rg3/W组和RI组次之,与W组和B组差异有显著性(P<0.01),Rg3和RI有一定的协同性。Rg3和RI可延长荷瘤小鼠的生存期,Rg3/RI组小鼠在观察期(1.5个月)内均存活,W组和B组小鼠全部死亡(至26d),且出现死亡的时间较早。经HE染色和第Ⅷ因子相关抗原的免疫组化分析显示,Rg3和RI使肺内瘤组织的微血管密度降低,降低的程度为Rg3/RI组>Rg3/B组>Rg3/W组>RI组>B组>W组。结论人参皂甙Rg3可增强RI转基因对小鼠黑色素瘤肺转移的抑制作用,人参皂甙Rg3和RI基因在抗肿瘤生长、转移及血管生成方面有协同作用。     

Increased rejection of primary tumors in mice lacking B cells: inhibition of anti-tumor CTL and TH1 cytokine responses by B cells

We investigated the role of B cells in tumor immunity by studying immune responses of mice genetically lacking B cells to primary tumors. IgM(-/-) B cell-deficient mice (BCDM) exhibited enhanced resistance to 3 histologically diverse syngeneic tumors as compared to the wild-type (WT) mice. EL4 thymoma and MC38 colon carcinoma grew progressively in WT mice, but regressed spontaneously in BCDM whereas growth of B16 melanoma was slowed significantly in BCDM as compared to the WT mice. BCDM exhibited increased T cell infiltration of tumors, higher T(H)1 cytokine response and, in the case of MC38, a higher anti-tumor CTL response. The increased tumor resistance of BCDM did not seem to result from intrinsic changes in their non-B immunocytes because adoptive transfer of WT splenic B cells to BCDM abrogated tumor rejection and resulted in diminished anti-tumor T(H)1 cytokine and CTL responses. Studies involving BCR-transgenic mice indicated that B cells may inhibit anti-tumor T cell responses by antigen-nonspecific mechanisms since neither tumor-specific antibodies nor cognate T:B interactions were necessary for inhibition of tumor immunity by B cells. IFN-gamma secretion in splenocyte:tumor co-cultures of tumor-challenged BCDM was inhibited by WT but not CD40(-/-) B cells indicating that B cells may inhibit anti-tumor T(H)1 cytokine responses in a CD40-dependent manner. Adoptive transfer of CD40(-/-) B cells into BCDM resulted in restored growth of MC38 suggesting additional factors other than CD40 are involved in dampening anti-tumor responses. The effects of B cells on anti-tumor response warrant further study.     

Selective expression of inhibitory Fcgamma receptor by metastatic melanoma impairs tumor susceptibility to IgG-dependent cellular response

During melanoma progression, patients develop anti-tumor immunity including the production of anti-tumor antibodies. Although the strategies developed by malignant cells to escape anti-tumor cellular immunity have been extensively investigated, little is known about tumor resistance to humoral immunity. The main effect of IgG antibodies is to activate the immune response by binding to host Fc gamma receptors (FcgammaR) expressed by immune cells. We previously reported in a limited study that some human metastatic melanoma cells ectopically express the FcgammaRIIB1, an inhibitory isoform of FcgammaR. By analyzing a large panel of different types of human primary and metastatic solid tumors, we report herein that expression of FcgammaRIIB is restricted to melanoma and is acquired during tumor progression. We show that FcgammaRIIB expression prevents the lysis of human metastatic melanoma cells by NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) in vitro, independently of the intracytoplasmic region of FcgammaRIIB. Using experimental mouse models, we demonstrate that expression of FcgammaRIIB protects B16F0 melanoma tumors from the ADCC induced by monoclonal and polyclonal anti-tumor IgG in vivo. Thus, our results identify FcgammaRIIB as a marker of human metastatic melanoma that impairs the tumor susceptibility to FcgammaR-dependent innate effector responses.     

Enhancement of anti-melanoma activity of a plasmid expressing HIV-1 Vpr delivered through in vivo electroporation

The development of novel treatment strategies for the effective delivery of new therapies directed against solid tumors, particularly metastatic melanoma, are required. A novel therapeutic property has previously been discovered for the HIV-1 accessory protein viral protein R (Vpr), based on the ability of this protein to induce G(2) cell cycle arrest as well as apoptosis in various tumor cell lines. Likewise, in vivo electroporation has been utilized as an effective delivery platform for DNA plasmids expressing potentially therapeutic proteins and has been targeted to normal tissues as well as tumors. Our previous findings demonstrated that delivery of a Vpr expression plasmid (pVpr) to established subcutaneous B16.F10 melanoma tumors by in vivo electroporation yielded long-term complete tumor regression in a small percentage of mice. In this study, we modified the electroporation regimen for pVpr with the goal of enhancing the anti-tumor activity of Vpr. pVpr was injected intratumorally, on days 0, 2 and 4, into established subcutaneous B16.F10 melanoma tumors followed by in vivo electroporation. Treatment with 100 mg of pVpr plus electroporation on the modified treatment days resulted in 50% of the mice undergoing complete tumor regressions coupled with long-term survival (i.e., greater than 100 days post treatment). Additional investigations established the intratumoral expression of Vpr and induction of apoptosis for a period of at least seven days after the modified pVpr treatment regimen. This report demonstrates that the anti-tumor activity of a pVpr plus electroporation regimen against established subcutaneous B16.F10 melanoma tumors can be significantly enhanced by a modified treatment schedule. In addition, it appeared that this enhanced anti-tumor effect was correlated with prolonged Vpr expression and the associated induction of intratumoral apoptosis.