Molecular determinants of susceptibility to oncolytic vesicular stomatitis virus in pancreatic adenocarcinoma

Background

M protein mutant vesicular stomatitis virus (M51R-VSV) has oncolytic properties against many cancers. However, some cancer cells are resistant to M51R-VSV. Herein, we evaluate the molecular determinants of vesicular stomatitis virus (VSV) resistance in pancreatic adenocarcinoma cells.

Methods

Cell viability and the effect of β-interferon (IFN) were analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Gene expression was evaluated via microarray analysis. Cell infectability was measured by flow cytometry. Xenografts were established in athymic nude mice and treated with intratumoral M51R-VSV.

Results

Four of five pancreatic cancer cell lines were sensitive to M51R-VSV, whereas Panc 03.27 cells remained resistant (81 ± 3% viability 72 h after single-cycle infection). Comparing sensitive MiaPaCa2 cells with resistant Panc 03.27 cells, significant differences in gene expression were found relating to IFN signaling (P = 2 × 10⁻⁵), viral entry (P = 3 × 10⁻⁴), and endocytosis (P = 7 × 10⁻⁴). MiaPaCa2 cells permitted high levels of VSV infection, whereas Panc 03.27 cells were capable of resisting VSV cell entry even at high multiplicities of infection. Extrinsic β-IFN overcame apparent defects in IFN-mediated pathways in MiaPaCa2 cells conferring VSV resistance. In contrast, β-IFN decreased cell viability in Panc 3.27 cells, suggesting intact antiviral mechanisms. VSV-treated xenografts exhibited reduced tumor growth relative to controls in both MiaPaCa2 (1423 ± 345% versus 164 ± 136%; P < 0.001) and Panc 3.27 (979 ± 153% versus 50 ± 56%; P = 0.002) tumors. Significant lymphocytic infiltration was seen in M51R-VSV–treated Panc 03.27 xenografts.

Conclusions

Inhibition of VSV endocytosis and intact IFN-mediated defenses are responsible for M51R-VSV resistance in pancreatic adenocarcinoma cells. M51R-VSV treatment appears to induce antitumor cellular immunity in vivo, which may expand its clinical efficacy.     

靶向性自杀基因治疗系统联合放射线治疗前列腺癌移植瘤的实验研究

目的初步判断针对整合素αv的靶向性自杀基因治疗系统(RGD4C AAVP HSV-TK/ GCV)是否能提高前列腺癌细胞株DU145移植瘤的放射效应。方法建立移植性前列腺癌细胞株DU145的裸鼠肿瘤模型48只。当位于每只裸鼠右大腿的肿瘤直径达6.0mm(5.8～6.3 mm)时,开始实验。实验共分成6组(每组8只),分别为对照组、单纯放射组、单纯RGD-4C AAVP HSV-TK/GCV组(RGD-4C组)、单纯AAVP HSV-TK/GCV组(无RGD-4C组)、RGD-4C AAVP HSV-TK/GCV联合放射组(XRT RGD4C组)和AAVP HSV-TK/GCV联合放射组(XRT 无RGD-4C组)。观察指标为肿瘤生长延迟时问(肿瘤从6.0 mm生长至12.0 mm所需时间)和肿瘤治愈情况。结果除5只在实验过程中死亡外,单纯放射组、RGD-4C组、无RGD-4C组各有1只和XRT RGD-4C组3只肿瘤被治愈。统计分析余下37只肿瘤生长情况发现,RGD-4C组、无RGD-4C组和单纯放射组的绝对延迟时间分别为(24.4±9.0)、(22.6±11.3)和(28.3±5.5)d;当RGD4C AAVP HSV-TK/GCV和AAVP HSV-TK/ GCV分别联合放射时,其绝对延迟时间分别为(64.7±23.8)和(35.4±9.6)d,而标准化的延迟时间则分别为(40.3±23.8)和(12.8±9.6)d,它们对放射的增益因子分别为1.42和0.45。结论针对整合素αv的靶向性自杀基因治疗系统RGD-4C AAVP HSV-TK/GCV能显著提高前列腺癌细胞株DU145移植瘤的放射效应,值得进一步研究。     

VX2乳腺癌兔的阿霉素致心脏毒性模型的建立

目的建立VX2乳腺癌兔的阿霉素致的心脏毒性模型。方法先建立兔VX2乳腺癌模型。2周后，将接种VX2乳腺癌成功模型兔随机分为阿霉素（ADR）组和对照组，阿霉素组10只，雌雄各5只，对照组8只，雌雄各4只。ADR组，每周1次于兔耳缘静脉注射ADR（用生理盐水配制成1mg／ml）2mg／kg，共6次。对照组，每周1次于耳缘静脉推注生理盐水每次2ml／kg，共6次。比较左心室射血分数的变化，并作心脏和肿块的病理切片，观察其组织学变化，记录动物的生存时间。结果模型成功率为90％，两组左心室射血分数（LVEF）差异有统计学意义（P〈0．05），阿霉素组的心脏经病理检查证实为有心肌病变，病理学结果证实符合心肌病样改变，肿块经病理检验证实为鳞状细胞癌。结论成功建立了VX2乳腺癌兔的阿霉素致心脏毒性模型，它将为临床研究提供的理想动物模型     

Therapeutic efficacy of the topoisomerase I inhibitor 7-ethyl-10-(4-[1-piperidino]-1-piperidino)-carbonyloxy-camptothecin against pediatric and adult central nervous system tumor xenografts

 Therapy of patients with malignant central nervous system tumors is frequently unsuccessful, reflecting limitations of current surgical, radiotherapeutic, and pharmacotherapeutic treatments. The camptothecin derivative irinotecan (CPT-11) has been shown to possess antitumor activity in phase II trials for patients with carcinoma of the lung, cervix, ovary, colon, or rectum and for patients with non-Hodgkin’s lymphoma. The current study was designed to test the efficacy of the drug against a panel of human tumor xenografts derived from adult and pediatric central nervous system malignancies. Tumors included childhood high-grade gliomas (D-212 MG, D-456 MG), adult high-grade gliomas (D-54 MG, D-245 MG), medulloblastomas (D341 Med, D487 Med), ependymomas (D528 EP, D612 EP), and a rhabdomyosarcoma (TE-671), as well as sublines with demonstrated resistance to busulfan (D-456 MG (BR)), cyclophosphamide (TE-671 CR), procarbazine (D-245 MG (PR)) or melphalan (TE-671 MR), growing subcutaneously and intracranially in athymic nude mice. In replicate experiments, CPT-11 was given at a dosage of 40 mg/kg per dose via intraperitoneal injection in 10% dimethylsulfoxide on days 1–5 and 8–12, which is the dosage lethal to 10% of treated animals. CPT-11 produced statistically significant (P<0.001) growth delays in all subcutaneous xenografts tested, including those resistant to busulfan, cyclophosphamide, procarbazine, and melphalan, with growth delays ranging from 21.3 days in D487 Med to 90+ days in several tumor lines. Further, tumor regression was evident in every treated animal bearing a subcutaneous tumor, with some xenografts yielding complete tumor regression. Statistically significant (P<0.001) increases in survival were demonstrated in the two intracranial xenografts – D341 EP (73.0% increase) and D-456 MG (114.2% increase) – treated with CPT-11. These studies demonstrate that, of over 40 drugs evaluated in this laboratory, CPT-11 is the most active against central nervous system xenografts and should be advanced to clinical trial as soon as possible. Received: 4 December 1995/Accepted: 18 May 1996     

内皮抑素抑制SKOV3细胞生长及其在裸鼠皮下移植瘤中的作用机制

目的:探讨内皮抑素对卵巢癌SKOV3细胞及其荷瘤鼠卵巢癌组织的生长作用及机制。方法:MTT法观测内皮抑素对体外SKOV3细胞的抑制作用,透射电镜观察细胞凋亡、免疫细胞化学检测SKOV3;细胞中bcl-2和bax蛋白表达;将SKOV3细胞移植至裸鼠皮下,观察内皮抑素对皮下移植瘤生长的影响;TUNEL和电镜法观察肿瘤细胞的凋亡,免疫组织化学和RT-PCR法检测瘤组织中bcl-2和bax的表达。结果:内皮抑素具有体外抑制SKOV3细胞增殖的作用(P<0．01);能诱导SKOV3细胞凋亡,但对bcl-2和bax的表达无明显影响。内皮以抑素能抑制裸鼠皮下移植瘤的生长(P<0．05);其中bcl-2的表达低于对照组,而bax表达无明显改变。结论:内皮抑素具有抑制SKOV3细胞及皮下移植瘤生长的作用,其机制可能与诱发细胞凋亡和调节bcl-2／bax表达相关。     

Human Skin Xenograft Rejection in CD45 Exon-6 Knockout Mice: The Implication of Involvement of a Direct Pathway

The results of previous studies indicate that only CD4⁺ T cells generated via the indirect pathway play an essential role in causing discordant skin xenograft rejection. The present study was conducted in an attempt to clarify further the roles of effector T cells generated via direct pathways on discordant xenograft rejection using CD45 exon-6 knockout (CD45−/−; C57BL/6 (B6): H-2^b) mice. It has been strongly suggested that CD45 exon-6 knockout mice have profound impairment in T-cell functions via an indirect pathway. When human skin was grafted onto untreated normal C57BL/6 (B6; H-2^b) mice, rejection occurred within 12 days; however, in the CD45 exon-6 knockout mice, the grafts lasted for slightly longer as in fully allogeneic C3H (H-2^k) skin rejection, with a mean survival time ± SD of 19.4 ± 1.5 days and median survival times of 19 days. The difference in survival periods between the human and C3H skin grafts in the CD45 knockout mice was not statistically significant. Both CD4⁺ and CD8⁺ T cells seemed activated in the spleens of these CD45 exon-6 knockout mice 10 days after the human skin grafting. These results suggest that effector T cells generated via a direct pathway can cause discordant skin xenograft rejection, and that CD45 exon-6 knockout mice can generate effector T cells via a direct pathway to reject discordant skin xenografts, similarly to fully allogeneic skin allografts. Received: June 24, 1999 / Accepted: May 30, 2000     

Necrotic cell death of ovarian adenocarcinoma caused by seminal plasma

Objective. From the knowledge of risk factors of epithelial ovarian cancer, we deduced a hypothesis that human seminal plasma (HSP) has a preventive role in the development of epithelial ovarian cancer. To examine whether HSP directly influences the growth of ovarian cancer, we have investigated the in vitro and in vivo effect of HSP on ovarian adenocarcinoma cell lines (SK-OV-3 and OVCAR-3) in comparison with its effects on normal ovarian surface epithelial cells (NOSE).Methods. Cell viability was determined by MTT assay. Cytotoxic effect was evaluated by flow cytometry analysis, by DNA laddering, and by morphological analysis. In vivo therapeutic effect of HSP was evaluated by the subcutaneous inoculation of SK-OV-3 cells in nude mice (BALB-c) model.Results. HSP at a final concentration of 1:50 induced a time- and dose-dependent inhibition of SK-OV-3 and OVCAR-3 growth, whereas NOSE was not affected. Flow cytometric analysis, DNA laddering, and morphological analysis indicated that HSP induced necrosis, rather than apoptosis, of both ovarian carcinoma cell lines. In in vivo experiment that used the nude mice (Balb-C) with tumor inoculation of SK-OV-3 cells, HSP induced necrosis of tumor with no detectable toxic effects on the major organs.Conclusion. These results show that HSP inhibits the growth and induces the necrosis of epithelial ovarian cancer cells and suggests that one or more components of HSP may provide a scientific basis for preventing epithelial ovarian cancer.     

Characterization of tetrandrine,a potent inhibitor of P-glycoprotein-mediated multidrug resistance

Multidrug resistance (MDR) is one of the main obstacles in tumor chemotherapy. A promising approach to solving this problem is to utilize a nontoxic and potent modulator able to reverse MDR, which in combination with anticancer drugs increases the anticancer effect. Experiments were carried out to examine the potential of tetrandrine (Tet) as a MDR-reversing agent. Survival of cells incubated with Tet at 2.5 mol/l for 72 h was over 90%. Tet at 2.5 mol/l almost completely reversed resistance to vincristine (VCR) in KBv200 cells. Tet at a concentration as low as 0.625 mol/l produced a 7.6-fold reversal of MDR, but showed no effect on the sensitivity of drug-sensitive KB cells in vitro. In the KBv200 cell xenograft model in nude mice, neither Tet nor VCR inhibited tumor growth. However, VCR and Tet combined inhibited tumor growth by 45.7%, 61.2% and 55.7% in three independent experimental settings. In the KB cell xenograft model in nude mice, Tet did not inhibit tumor growth, but VCR and the combination of VCR and Tet inhibited tumor growth by 40.6% and 41.6%, respectively. Mechanism studies showed that Tet inhibited [³H]azidopine photoaffinity labeling of P-gp and increased accumulation of VCR in MDR KBv200 cells in a concentration-dependent manner. The results suggest that Tet is a potent MDR-reversing agent in vitro and in vivo. Its mechanism of action is via directly binding to P-gp and increasing intracellular VCR accumulation.Abbreviations DMSO Dimethyl sulfoxide - FBS Fetal bovine serum - MDR Multidrug resistance - MTT 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide - PBS Phosphate-buffered saline - P-gp P-glycoprotein - SDS Sodium dodecyl sulfate - Tet Tetrandrine - VCR Vincristine     

无细胞异种真皮基质降解因素的研究

目的　探讨影响异种无细胞真皮基质 (ADM )降解吸收的相关因素及其与炎症反应之间的关系。方法　猪和兔断层中厚皮片经Trypsin等脱细胞 ,分别制成异种 /异体ADM (xeno /allo ADM ) ,再按预处理方法不同分组 :戊二醛交联的xeno ADM (A)组 ;网状打孔和交联的xeno ADM (B)组 ;xeno ADM (C)组和allo ADM (D)组 ,分别埋植于兔皮下 ,术后 4～ 32周作大体观察和组织学检查。结果　埋植术后 4～ 12周期间 ,A组和B组可见较多的异物巨细胞 ,各组炎症反应和移植物被降解 /同化程度依次为 :C >B >A >D(P <0 .0 5～ 0 .0 0 1) ,二者之间有显著的正相关 (n =84,r =0 .185 ,P <0 .0 1)。结论　xeno ADM生物相容性远不及allo ADM ,其降解除受戊二醛交联作用和网状打孔因素影响外 ,还与局部血运和所受应力有关 ,其中炎症 免疫反应可能是降解最直接的原因。     

Microencapsulated bovine chromaffin cell xenografts into hemiparkinsonian rats: a drug-induced rotational behavior and histological changes analysis

Bovine chromaffin cells were microencapsulated within alginate-polylysine-alginate (APA) membranes. Microencapsulated bovine chromaffin cells as well as unencapsulated cells and empty microcapsules were grafted into the brain of hemiparkinsonian rats with 6-hydroxydopamine (6-OHDA) lesions. Apomorphine-induced rotational behavior of the host animals and the survival of the grafted chromaffin cells were examined after transplantation. The animals receiving microencapsulated bovine chromaffin cells showed a significant decrease (17.6--35.6%) in apomorphine-induced rotation 1 week postimplantation that remained stable for the 10 month test period. Fluorescent histochemistry further revealed that microencapsulation increased the chromaffin cell survival with only a minimum host reaction for up to 10 months posttransplantation while the survival of free, unencapsulated chromaffin cells was only modest and was accompanied by a large inflammatory response. The reduction of apomorphine-induced rotations was correlated with the survival of bovine chromaffin cells in the host brain. The data indicate that encapsulation of bovine chromaffin cells in APA membranes reduces the host immune response to the xenograft and prolongs the viability of the grafted cells.