Oral delivery of tumor-targeting Salmonella for cancer therapy in murine tumor models

Tumor-targeting bacteria have been investigated intensively in recent years as anticancer agents. To ensure the reliability of infection, bacteria have conventionally been injected intravenously or intraperitoneally into animals or humans. However, systemic infection of bacteria is rather inconvenient and carries the risk of obvious toxicity. Here we tested whether Salmonella typhimurium VNP20009, a tumor-targeting strain, could be administrated orally for tumor therapy. Tumor-targeting potential, antitumor effects, as well as toxicity of orally administrated VNP20009 were investigated in this study. Oral delivery of VNP20009 not only exhibited high tumor-targeting potential, but also led to a significant anticancer effect by delaying tumor growth and prolonging survival in murine tumor models. As part of combination therapy, orally administrated bacteria notably improved the antitumor effect of cyclophosphamide. In vitro and in vivo studies showed that VNP20009 significantly induced tumor cell apoptosis. No obvious toxicity was observed during the treatments with oral inoculation of VNP20009. Comparative analysis of toxicity in tumor-bearing and tumor-free mice further revealed that orally administrated Salmonella had high safety compared to conventional systemic infection of bacteria. The findings indicated that oral administration of tumor-targeting bacteria is effective and safe. This approach provides a novel avenue in the application of bacteria as a potential antitumor agent.     

Oral delivery of tumor-targeting Salmonella exhibits promising therapeutic efficacy and low toxicity

Tumor‐targeting bacteria have been developed as powerful anticancer agents. Salmonella typhimurium VNP20009, a representative tumor‐targeting strain, has been systemically administered as a single‐agent therapy at doses of 1 × 10⁶ to 3 × 10⁶ colony‐forming unit (cfu)/mouse, or in combination with other antitumor agents at doses of 1 × 10⁴ to 2 × 10⁵ cfu/mouse. Recently, we reported that oral delivery of VNP20009 at the dose of 1 × 10⁹ cfu/mouse induced significant anticancer effects comparable to that induced by systemic administration of this strain at 1 × 10⁴ cfu/mouse. To further address the efficacy and safety of oral administration of bacteria, here we performed a systemically comparative analysis of anticancer efficacy and toxicity of VNP20009 administered: (i) orally at a dose of 1 × 10⁹ cfu/mouse (VNP9‐oral); (ii) intraperitoneally at a dose of 1 × 10⁴ cfu/mouse (VNP4‐i.p.); or (iii) intraperitoneally at a dose of 1 × 10⁶ cfu/mouse in tumor‐free and tumor‐bearing murine models. The results showed that VNP9‐oral, similar to VNP4‐i.p., induced significant tumor growth inhibition whereas VNP6‐i.p. induced better anticancer effect in the B16F10 melanoma model. Among three treatments, VNP9‐oral induced the mildest and reversible toxicity whereas VNP6‐i.p. resulted in the most serious and irreversible toxicities when compared to other two treatments. Moreover, the combination of VNP9‐oral with a low dose of chemotherapeutics produced comparable antitumor effects but displayed significantly reduced toxicity when compared to VNP6‐i.p. The findings demonstrated that oral administration, as a novel avenue in the application of bacteria, is highly safe and effective. Moreover, the present preclinical study should facilitate the optimization of bacterial therapies with improved anticancer efficacy and reduced adverse effects in future clinical trials. (Cancer Sci 2009; 100: 2437–2443)     

Enhanced therapeutic efficacy of cisplatin by combination with diethyldithiocarbamate and hyperthermia in a mouse model

A spontaneously metastasizing solid tumor model derived by transplanting the TA3Ha murine mammary carcinoma into the s.c. tail tissue of mice was used to develop a treatment strategy for enhancing the therapeutic efficacy of cisplatin (CDDP). This strategy was based on the findings that diethyldithiocarbamate (DDTC) reduces the toxicity of CDDP, and that localized hyperthermia (HT) augments the antitumor efficacy of CDDP. DDTC (500 mg/kg) reduced the CDDP-induced nephrotoxicity and gastrointestinal toxicity as well as increased the CDDP LD10 from 8 to 20 mg/kg in strain A mice. When CDDP and DDTC were used in multiple treatment schedules at 5-day intervals, DDTC protected the hosts but not the tumors against the toxicity of CDDP. HT administered locally to the tumor 1 h after the injection of CDDP (8 mg/kg) in 1 ml Hanks' balanced salt solution increased the antitumor effect but not the host toxicity. While administration of 8 mg/kg CDDP alone or with HT three times at 5-day intervals caused 100% host mortality, this dose of CDDP could be used with no mortality by combining it with DDTC. A combination of 8 mg/kg CDDP with DDTC (750 mg/kg) and HT (43 degree C for 60 min), administered three times at 5-day intervals, retarded the local tumor growth significantly compared to the untreated, CDDP plus DDTC plus HT control groups of mice. The frequency of lung metastasis in these groups on day 30 of tumor inoculation were 0, 90, 90, and 80%, respectively. The mean survival days of the mice treated with CDDP plus DDTC plus HT was 61 +/- 6 compared to 34 +/- 5 in the controls. The results presented here demonstrate that by combining CDDP with DDTC, high doses of CDDP can be safely administered. When localized HT is combined with high dose CDDP and DDTC, the tumor growth retardation and the host survival prolongation are significantly better than those obtained with the highest tolerable dose of CDDP alone or CDDP plus HT.     

Salmonella-mediated tumor-targeting TRAIL gene therapy significantly suppresses melanoma growth in mouse model

Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) under the control of a hypoxia-induced nirB promoter and examined the efficacy of Salmonella-mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL-resistant RM-1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM-1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing melanoma apoptosis. Our findings indicate that Salmonella-mediated tumor-targeted therapy with TRAIL could reduce tumor growth and extend host survival.     

Regression of melanoma in a murine model by RLIP76 depletion

RLIP76/RALBP1 is a stress-responsive membrane protein implicated in the regulation of multiple cellular signaling pathways. It represents the predominant glutathione-conjugate transporter in cells, and our previous studies have shown that its inhibition by antibodies or depletion by short interfering RNA (siRNA) causes apoptosis in a number of cancer cell types. The present studies were done to explore the potential clinical applicability of our previous observations by comparing the relative expression of RLIP76 in cancer versus normal cell lines and to determine whether depletion of RLIP76 activity can exert cancer-specific apoptosis. RLIP76 expression was found to be significantly greater in malignant cells compared to nonmalignant cells. Inhibition of RLIP76, using antibodies towards a cell surface epitope, or depletion of RLIP76 using either siRNA or antisense phosphorothioate oligonucleotides preferentially caused apoptosis in malignant cells. More importantly, in vivo studies showed that administration of RLIP76 antibodies, siRNA, or antisense oligonucleotides to mice bearing syngeneic B16 mouse melanoma cells caused complete tumor regression within 10 days. These findings strongly suggest that RLIP76 depletion by genetic approaches or inhibition by antibodies may be a clinically viable antineoplastic therapy, particularly for melanoma.     

Antitumor activity of tributyrin in murine melanoma model

Butyric acid has been known to inhibit growth and to induce differentiation of a variety of tumor cells. Butyrate-treated tumor cells have also been observed to undergo apoptosis. Although butyrate compounds have demonstrated antitumor activity in murine tumor models and have already been admitted to clinical trials in tumor patients, the exact mechanism of their antitumor effects has not been elucidated. The results of our study showed antitumor activity of tributyrin, a butyric acid prodrug, in murine melanoma model and are strongly suggestive that antiangiogenic effects could participate in antitumor effects of butyrate compounds in vivo.     

Enhanced therapeutic effect of amsalog (CI-921) in combination with cisplatin in vitro and in vivo

CI-921, the 5-methyl-4-carboxamide analog of amsacrine, in combination with cisplatin produced a 6-fold better cell kill in vitro than expected based on additive effects. The combination of CI-921 and cisplatin was subsequently evaluated in three in vivo model systems: intraperitoneally (TP) and intravenously (IV) implanted P388 leukemia, and advanced stage subcutaneously (SC) implanted LC-12 squamous cell carcinoma. All drug treatments were administered IP on an intermittent treatment schedule which was optimal for both agents. Combination therapy was superior to therapy with the best single agent alone, CI-921, in all three model systems. Against IP implanted P388 leukemia, combination therapy produced greater than 8 logs of net tumor cell kill with 60-day survivors (cures). This level of activity was 50-fold greater (1.7 log) than that obtained with CI-921 alone. An IV implant of P388 leukemia was used in a confirmatory study to provide a more rigorous evaluation against disseminated disease. Combination therapy against IV implanted P388 leukemia produced greater than 7.7 logs of net tumor cell kill, which was 630-fold greater (2.8 logs) than that obtained with CI-921 therapy alone. Against advanced stage LC-12 (200-1000 mg tumors at initial treatment), combination therapy improved tumor cell kill by 0.6 log (4-fold) over that obtained with CI-921 therapy alone and also produced greater numbers of 120-day survivors than did single agent therapy with CI-921. The combination of carboplatin and CI-921 was also evaluated against IV implanted P388 leukemia to determine if the enhanced therapeutic effect of CI-921 and cisplatin could be extended to include CI-921 and carboplatin. Combination therapy with CI-921 and carboplatin increased net log tumor cell kill by 0.8 and 1.5 log in two separate tests (6- and 32-fold, respectively) over that obtained with CI-921 therapy alone. The data indicate that combination therapy with CI-921 and platinum containing anticancer agents may have clinical application.     

Autocrine expression of both endostatin and green fluorescent protein provides a synergistic antitumor effect in a murine neuroblastoma model

Modalities that act through different mechanisms can often provide synergistic antitumor activity for the treatment of refractory tumors when used in combination. Here we report a gene therapy approach in which the genes for the angiogenesis inhibitor, endostatin, and the marker protein and potent immunogen, green fluorescent protein (GFP), were delivered to murine neuroblastoma cells prior to inoculation of the tumor cells into syngeneic immunocompetent mice. Although the effect of either angiogenesis inhibition or immunomodulation alone resulted in only a modest delay in tumor growth, when these approaches were used in combination, prevention of the formation of appreciable tumors was effected in 15 of 24 (63%) mice. The combination of endostatin and GFP expression elicited a strong immune response that was T cell-mediated and was reactive against both GFP and tumor cell line-specific antigens. This afforded treated mice protection against subsequent tumor challenge with unmodified tumor cells. These results suggest that antiangiogenic and immunotherapy strategies, when used in a gene therapy-mediated approach, can act synergistically in an effective multimodality anticancer approach.     

重组人血管内皮抑制素联合达卡巴嗪增强抗黑素瘤作用及其机制研究

目的:探讨重组人血管内皮抑制素(recombinant human endostatin, rh-endostatin)联合达卡巴嗪(Dacarbazine)能否增强抗黑素瘤作用,并初步分析其作用机制.方法:分别采用MTT法和FCM法检测重组人血管内皮抑制素单药及其联合达卡巴嗪对黑素瘤B16F10细胞增殖和凋亡的影响;Western印迹法检测重组人血管内皮抑制素对黑素瘤细胞中细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)磷酸化的影响;建立C57BL/6黑素瘤荷瘤小鼠模型,研究重组人血管内皮抑制素联合达卡巴嗪能否增强体内抗黑素瘤的作用.结果:重组人血管内皮抑制素、达卡巴嗪以及两药联合处理对黑素瘤B16F10细胞增殖的抑制率分别为(9.67±2.89)%、(22.67±3.06)%和(30.33±1.16)%,差异有统计学意义(P<0.05);重组人血管内皮抑制素组黑素瘤细胞的ERK分子磷酸化水平明显降低.接种肿瘤18 d时,重组人血管内皮抑制素组、达卡巴嗪组和两药联合组荷瘤小鼠的肿瘤体积分别为(6.45±1.24)cm3、(8.94±2.04)cm3和(4.56±0.98)cm3,两药联合组的肿瘤体积明显低于达卡巴嗪单药组(P<0.05).结论:重组人血管内皮抑制素联合达卡巴嗪体外可增强抗黑素瘤B16F10细胞增殖的作用,体内可增强达卡巴嗪对荷瘤小鼠黑素瘤生长的抑制作用,并提高荷瘤小鼠的存活率.推测抑制黑素瘤细胞中ERK分子酪氨酸磷酸化可能是重组人血管内皮抑制素发挥其抗黑素瘤作用的机制之一.     

Tumor-targeting Salmonella typhimurium improves cyclophosphamide chemotherapy at maximum tolerated dose and low-dose metronomic regimens in a murine melanoma model

Chemotherapy for cancer is partly limited by the inability of drugs to act on poorly vascularized or avascularized areas of tumors. Tumor-targeting bacteria are capable of preferentially replicating in these poorly perfused regions. Some strains have been combined with chemotherapeutic agents and the results have been promising. However, no systematic work has been carried out to test the effect of bacteria on clinical modes of chemotherapy, such as standard maximum tolerated dose (MTD) and novel low-dose metronomic (LDM) chemotherapy. Here Salmonella typhimurium VNP20009 was combined with cyclophosphamide (CTX) at both MTD and LDM schedules in a murine melanoma model. The results showed that VNP20009 significantly improved the effects of all forms of CTX treatments. The combination of VNP20009 and CTX led to a more significant decrease in tumor microvessel density and serum vascular endothelial growth factor (VEGF) level, compared with either treatment alone. Furthermore, combination therapy remarkably increased the number of bacteria within tumors when compared with bacteria treatment alone. These findings suggest that tumor-targeting bacteria, in conjunction with CTX at standard MTD and LDM regimens, might be of clinical value for the treatment of melanoma.     

Antimetastatic efficacy of niosomal pentoxifylline and its combination with activated macrophages in murine B16F10 melanoma model

The aims of the present study were a) to enhance the effectiveness of antimetastatic agent, Pentoxifylline (PTX) by encapsulation in niosomes and b) to investigate the anticancer activity by combination therapy involving activated macrophages and PTX solution/PTX niosomes. Niosomes were prepared by lipid film hydration method. Particle size distribution revealed bimodal distribution with median vesicle size of 462 nm. The entrapment efficacy of PTX niosomes was found to be 9.64%. A cumulative release of 82.43% from niosomal suspension was observed at the end of 21 hours. Intravenous administration of niosomal PTX (6 mg/kg and 10 mg/kg) resulted in significant reduction in lung nodules in an experimental metastatic B16F10 model suggesting accumulation of PTX in a distant target organ-lung. Light microscopic observations of histologic sections showed a decrease in number of tumor islands in the lung. Macrophages activated by intraperitoneal injection of Iscove's Modified Dulbecco's Medium (IMDM) containing 20% fetal calf serum (FCS) followed by in vitro incubation with muramyl dipeptide (MDP) were more effective in controlling tumor spread than those activated by FCS alone. Combination therapy of activated macrophages and PTX solution/niosomal PTX showed no additive or synergistic effect in controlling tumor spread. Carbon clearance studies revealed that PTX inhibits the phagocytic ability of activated macrophages, thereby resulting in the failure of combination therapy.     

Enhanced therapeutic effect of multiple injections of HSV-TK + GCV gene therapy in combination with ionizing radiation in a mouse mammary tumor model.

PURPOSE: Standard therapies for breast cancer lack tumor specificity and have significant risk for recurrence and toxicities. Herpes simplex virus-thymidine kinase (HSV-tk) gene therapy combined with radiation therapy (XRT) may be effective because of complementary mechanisms and distinct toxicity profiles. HSV-tk gene therapy followed by systemic administration of ganciclovir (GCV) enhances radiation-induced DNA damage by generating high local concentrations of phosphorylated nucleotide analogs that increase radiation-induced DNA breaks and interfere with DNA repair mechanisms. In addition, radiation-induced membrane damage enhances the "bystander effect" by facilitating transfer of nucleotide analogs to neighboring nontransduced cells and by promoting local and systemic immune responses. This study assesses the effect of single and multiple courses of HSV-tk gene therapy in combination with ionizing radiation in a mouse mammary cancer model. METHODS AND MATERIALS: Mouse mammary TM40D tumors transplanted s.c. in syngeneic immunocompetent BALB-c mice were treated with either adenoviral-mediated HSV-tk gene therapy or local radiation or the combination of gene and radiation therapy. A vector consisting of a replication-deficient (E1-deleted) adenovirus type 5 was injected intratumorally to administer the HSV-tk gene, and GCV was initiated 24 h later for a total of 6 days. Radiation was given as a single dose of 5 Gy 48 h after the HSV-tk injection. A metastatic model was developed by tail vein injection of TM40D cells on the same day that the s.c. tumors were established. Systemic antitumor effect was evaluated by counting the number of lung nodules after treating only the primary tumors with gene therapy, radiation, or the combination of gene and radiation therapy. To assess the therapeutic efficacy of multiple courses of this combinatorial approach, one, two, and three courses of HSV-tk + GCV gene therapy, in combination with radiation, were compared to HSV-tk or XRT alone and to sham-treated animals. (Treatments were repeated at 7-day intervals from the HSV-tk injection.) RESULTS: Both single-therapy modalities reduced tumor growth by 11% compared to controls, while the combined therapy resulted in a decrease of 29%. Median survival was 36 days in the combined therapy group, compared to 33 days in the monotherapy groups and 26 days in the control group. In the metastatic model, the number of lung nodules was reduced by 59.5% after HSV-tk gene therapy, whereas radiotherapy had no effect on metastatic growth. Combined therapy led to an additional 66.7% reduction in lung colonization. Compared to controls, local tumor growth was maximally suppressed by three courses of combined therapy (51.5%), followed by two courses of combined therapy (37.2%), and three sessions of XRT alone (35.6%). Median survival was also significantly prolonged to 58 days with the three courses of combined therapy, followed by two courses, to 45 days. All other treatment groups demonstrated median survival times between 26 and 35 days, while controls had a median survival of 24 days. CONCLUSIONS: These results indicate that multiple courses of HSV-tk therapy in combination with radiation improve the therapeutic efficacy of this approach and may provide therapeutic implications for the treatment of human breast cancer and other solid tumors.     

人内皮抑素转基因治疗B16黑色素瘤的实验研究

目的　研究瘤内直接注射携带人内皮抑素 (hEN)基因逆转录病毒包装的细胞株对体内B16黑色素瘤细胞生长的抑制作用。方法　修饰、鉴定hEN基因。构建含hEN基因的逆转录病毒载体pLNC hEN ,转染包装细胞株 ,筛选稳定转染该基因之PA317 pLNChEN细胞。建立小鼠B16黑色素瘤动物模型。瘤内直接注射PA317 pLNChEN细胞 ,分时间测量肿瘤体积 ,切取肿瘤 ,免疫组化检测肿瘤组织微血管密度 (MVD) ,TUNEL染色检测肿瘤组织中的凋亡细胞。结果　细胞接种后第 7～ 9天 ,全部小鼠均长出 2～ 3mm直径肿瘤。hEN基因转染后第 3,5 ,7,9天 ,转基因组肿瘤的平均体积 (mm3 )分别为 4 .6 7± 1.10、2 2 .2 5± 13.0 6、84 .17± 4 3.5和 15 5 .0 8± 81.1;空载体对照组分别为 136 .17± 30 .6 1、390 .17± 2 2 0 .4 7、10 2 1.6 7± 5 37.4 0和 2 92 0 .2 0± 2 2 0 .0 1,两组差异有显著性。转基因组和对照组的平均MVD分别为 8.0 0± 2 .2 8和 2 8.17± 5 .31,平均凋亡细胞数分别为 2 3.33± 3.83和 2 .33± 1.2 1,差异均有显著性。结论　瘤内注射携带hEN基因逆转录病毒包装的细胞 ,可抑制小鼠种植性B16黑色素瘤的微血管数目 ,促进肿瘤细胞凋亡 ,抑制肿瘤生长     

Improved therapeutic outcome following combination immunogene vaccination therapy in murine myeloma

Increasing evidence suggests a role for immunologic vaccination and therapy in the management of minimal residual myeloma. We have previously demonstrated a synergistic effect of combining the Th1 stimulating cytokine IL-12 with the co-stimulatory molecule CD80 in murine myeloma vaccination therapy. We reasoned that the efficacy of such treatment might be further improved by incorporating additional gene products which enhance the function of antigen presenting cells. Studies were therefore conducted with murine myeloma BM1 cells expressing Flt3L (membrane bound or soluble forms) or GM-CSF and the IL-12·CD80 combination. Single agent and combined therapeutic approaches were explored. All gene-modified BM1 cells, except BM1/IL-12·CD80, developed tumors when subcutaneously injected into BALB/c mice. As prophylactic tumor vaccines, the combined use of gene-modified BM1/sFlt3L+GM-CSF+IL-12·CD80 was most effective, providing 100% protection against subsequent parental BM1 tumor challenge. By comparison, only partial protection was observed with any single gene-engineered tumor vaccine. Notably, IL-12·CD80 coexpressing BM1 cell vaccines were the most effective therapeutic vaccine in a minimal disease model. Such protective vaccination was achieved by stimulation of lymphocyte proliferation and enhancement of cytotoxic lymphocyte activity.     

Synergistic effect of metronomic dosing of cyclophosphamide combined with specific antitumor immunotherapy in a murine melanoma model

Immunotherapy could be combined with conventional chemotherapeutic modalities aimed at reducing tumor burden. Such combination therapy may be most useful when "metronomic" doses of antineoplastic drugs are used, thereby potentially avoiding some of the immunosuppressive effects of these drugs. Recent studies have shown that some conventional antineoplastic drugs can be exploited for antiangiogenic capacities, a strategy that requires drugs to be administered at regular intervals. We therefore investigated whether such metronomic therapy with the alkylating agent cyclophosphamide (CTX) could be effectively combined with immunotherapy eliciting tumor-reactive CTLs. An immunization protocol using injection of recombinant DNA followed by injection of recombinant modified vaccinia virus Ankara strain was used to initiate a specific CTL response in mice capable of providing resistance to challenge with the murine melanoma B16.F10. Combining this immunotherapeutic regime with metronomic delivery of CTX resulted in antitumor activity that was dramatically enhanced over either treatment administered alone and was also significantly greater than combining immunotherapy with CTX administered by a maximum tolerated dose regime. Whereas both metronomic and maximum tolerated dose delivery of CTX did cause deletion of proliferating tumor-specific CTLs in the blood, this deletion occurred with slower kinetics with the metronomic schedule. Further analysis showed that metronomic CTX treatment did not delete cells with low expression of CD43, a "memory" phenotype, and that these cells maintained potent restimulatory capacity. The combination of immunotherapy and metronomic CTX therapy may be well suited to clinical management of cancer.     

Human endostatin inhibits growth of human non-small-cell lung cancer in a murine xenotransplant model.

Overall prognosis in human NSCLC remains poor. Antiangiogenic treatment has become a promising concept for the treatment of solid malignancies. Our purpose was to evaluate the efficacy of recombinant HSENDO for the treatment of human NSCLC in an orthotopic murine xenotransplantation model. The efficacy of HSENDO was tested in vitro in cell-proliferation, cell-migration and tube-formation assays. In vivo, the effect of HSENDO on tumor growth was tested in s.c. xenotransplanted human NSCLC and on intrapulmonary induced human NSCLC. In vitro, HSENDO inhibited both human and rodent endothelial cell proliferation in a time- and dose-dependent fashion. Endothelial cell migration was inhibited by 97%. Tube formation of murine endothelial cells was inhibited and preexisting tubes degenerated after HSENDO exposure. In vivo, HSENDO delayed growth of s.c. xenotransplanted tumors. Immunohistochemic staining demonstrated no change in microvessel density but a significant reduction of proliferating tumor cells and an increase in bFGF and VEGF expression, reflecting the antiangiogenic effect of HSENDO. Intrapulmonary tumor induction caused death subsequent to metastatic disease. Systemic HSENDO application extended survival significantly. HSENDO was demonstrated to inhibit endothelial cell proliferation, migration and tube formation effectively. In vivo growth of s.c. transplanted tumors was delayed and survival extended by 32% and 69%, respectively, after intrapulmonary NSCLC induction.     

重组人血管内皮抑素间插联合顺铂对H22小鼠腹水瘤抑制作用的研究

目的 观察重组人血管内皮抑制素（恩度）间插联合顺铂腹腔内注射治疗小鼠腹水瘤的疗效和安全性。方法 采用肝细胞癌H22腹水瘤细胞株建立小鼠腹水瘤模型。120只造模后的ICR小鼠随机分为4组：对照组（生理盐水d1～d10）、恩度组（恩度8mg／kg,d1～d5,d7～d10,生理盐水d6）、顺铂组（顺铂004mg／kg,d6,生理盐水d1～d5,d7～d10）及恩度联合顺铂组（恩度8mg／kg,d1～d5,d7～d10;顺铂0.04mg／kg,d6）,每组30只,每只小鼠腹腔注射的药物体积均为0.2ml。记录各组小鼠腹水体积、腹水中肿瘤细胞、红细胞计数以及生存期;观察各组荷瘤小鼠腹膜和腹腔脏器种植转移情况;检测各组小鼠的腹膜渗透性、血常规和肝肾功能;流式细胞仪检测各组小鼠腹腔积液中肿瘤细胞凋亡情况。结果 与对照组相比,恩度组和顺铂组和恩度联合顺铂组均能减少荷瘤小鼠腹腔积液的体积、肿瘤细胞数和红细胞数,延长荷瘤小鼠的生存时间。在上述指标中,恩度联合顺铂组与恩度组和顺铂组比较,差异有统计学意义（P＜0.05）。恩度联合顺铂组小鼠腹腔积液中肿瘤细胞的凋亡率显著高于恩度组和对照组（P＜0.05）,但与顺铂组比较差异无统计学意义（P＞0.05）;恩度联合顺铂组小鼠的腹膜渗透性明显低于顺铂组和对照组（P＜0.05）,但恩度联合顺铂组与恩度组比较差异无统计学意义（P＞0.05）。恩度联合顺铂组小鼠的体重增加及腹腔种植转移均少于其他3组。各组小鼠均未出现血常规和肝肾功能明显异常。结论 恩度间插联合顺铂腹腔内应用治疗小鼠H22腹水瘤疗效显著,安全性好。     

In vivo anti-tumour activity of recombinant human and murine TNF, alone and in combination with murine IFN-gamma, on a syngeneic murine melanoma

TNF, a protein released by induced macrophages, is believed to mediate, at least in part, the tumoritoxic effects of activated macrophages. In vitro, it has cytotoxic effects on transformed cells but not on normal cells, and in vivo it causes necrosis of tumours. Recently, both human and murine TNF became available as pure recombinant proteins. Subsequent work confirmed its in vitro cytotoxic activity, selective for transformed cells, and revealed other, non-cytotoxic effects on some normal cells. In vitro, the B16BL6 melanoma cells, syngeneic with C57BL6 mice, are resistant to the cytotoxic effects of rTNF but become sensitive when they are also treated with rIFN-gamma. We report that established, s.c. B16BL6 tumours in vivo can be induced to necrotize and regress by a combined systemic treatment with rTNF and murine rIFN-gamma. Although TNF is not species-specific in vitro, the effects of treatment with human and murine rTNF in vivo are different: with murine rTNF, the synergism with rIFN-gamma is relatively less clear, the addition of IFN-gamma is not necessary to induce regression, toxicity is more pronounced and additional mechanisms of tumoritoxicity could be involved. Relapses are frequent but complete cures have been observed. These results give further evidence in favour of a potential clinical use of TNF in combination therapy, e.g. with IFN-gamma. However, there is still a need to develop better regimens, especially for consolidation, and to continue research in order to understand and limit the toxicity, which could be mediated by the activating effects of TNF on some normal cell types.     

Enhanced anti-tumor effect of trastuzumab in combination with cisplatin.

The oncogenenic transmembrane tyrosine kinase receptor HER-2/neu is a promising target for treatment of HER-2-overexpressing cancers. The humanized anti-HER-2/neu antibody Trastuzumab is under clinical evaluation in combination with chemotherapy against breast cancer. The combination of Trastuzumab and cisplatin is expected to be active against HER-2 / neu-expressing tumors. We examined the mechanisms of this combination effect against human solid tumor cells in the presence of human peripheral blood mononuclear cells (PBMCs) using an in vitro MTT assay. The growth-inhibitory effects of cisplatin (CDDP) on the tumor cells were not significantly affected by Trastuzumab in the absence of effector cells. CDDP alone at a dose of less than 12.5 mM did not affect the viability of PBMCs, as determined by MTT assay, suggesting that PBMCs could exert antibody-dependent cell-mediated cytotoxicity (ADCC) at this CDDP concentration. The combination of Trastuzumab and CDDP showed higher cytotoxic effects against the tumor cells in the presence of PBMCs. The CDDP concentration required to inhibit tumor cell growth by 50% was reduced to approximately 20% by Trastuzumab in the presence of PBMCs at an effector/target ratio of 10. It may be important to select combined chemotherapeutic agents which do not diminish the ADCC activity of Trastuzumab via PBMCs. Both the expression of HER-2 / neu and the ADCC activity may be important determinants of the therapeutic benefit of the Trastuzumab/CDDP combination.