Substantially improved in vivo radiosensitization of rat glioma with mutant HSV-TK and acyclovir

We recently demonstrated in vitro that a mutant HSV-TK (mutant 75) expressed from an adenovirus (AdCMV-TK75) radiosensitized rat RT2 glioma cells significantly better than wild type HSV-TK (AdCMV-TK) in combination with acyclovir (ACV). To examine whether a similar improvement could also be observed in vivo, we tested these viruses in a syngeneic rat glioma tumor model (RT2/Fischer 344). First, we demonstrate that treatment with AdCMV-TK and ACV significantly radiosensitizes implanted gliomas and roughly doubles the mean survival time to 37 days, compared to 20 days for control animals implanted with Adbetagal-transduced cells (P<.02). Second, it was important to first examine the effect of AdCMV-TK75 and ACV on survival without any irradiation. We found that AdCMV-TK75 appeared to sensitize gliomas more efficiently than AdCMV-TK, although this difference was not significant ( P= .19 ). Third, and most importantly, in combined HSV-TK, ACV and irradiation experiments, we demonstrate that AdCMV-TK75 is superior over AdCMV-TK and significantly (P<.005) prolonged the survival of treated animals. Our results suggest that AdCMV-TK75 is far more efficient than AdCMV-TK in radiosensitizing rat glioma when administered in combination with ACV.     

Connexin 43-mediated bystander effect in two rat glioma cell models

In tumor models, the killing by ganciclovir of a fraction of tumor cells transfected with the thymidine kinase (TK) gene has been shown to induce complete regression of the tumor. The mechanism responsible for this bystander effect is thought to be the diffusion of toxic metabolites or apoptotic signals across gap junctions. Connexin 43 (Cx43) is the major component of astrocyte gap junctions. We investigated the susceptibility of two rat glioma cell lines (CNS1 and C6) to thymidine kinase/ganciclovir, before and after transfection with the Cx43 gene. We report a close correlation between the level of Cx43 expression, the extent of gap junctional communication and the amplitude of the bystander effect. Transfection of C6 cells (which display a weak bystander effect and low levels of connexin) with a Cx43 construct induced a strong bystander effect. Inhibition of gap junction activity by 18-alpha-glycyrrhetinic acid abolished the metabolic interaction between TK(+) and TK(-) cells. This metabolic interaction was also abolished if TK(+) and TK(-) cells were separated by a semipermeable membrane. Surprisingly, the transfection of only one of these two interacting cell types with the Cx43 gene was sufficient to induce a bystander effect, although this effect was weaker than that observed if both TK(+) and TK(-) cells expressed Cx43. Finally, Cx43 expression increased sensitivity to contact inhibition. Overall, our data provide evidence that the restoration of gap junctional communication may potentiate HSV/tk-based cancer treatment and suggest that this strategy may have wider application in cancer therapy.     

Stimulation of intercellular communication of poor-communicating cells by gap-junction-competent cells enhances the HSV-TK/GCV bystander effect in vitro

We have previously shown that gap-junctional intercellular communication (GJIC) appears to play a role in the bystander effect that is observed in anticancer suicide gene therapy mediated by herpes simplex virus (HSV) thymidine kinase (tk) and ganciclovir (GCV). We now report that when connexin-expressing (Cx+) cells are present within a noncommunicating population of cells (Cx-), there is GJIC between the Cx+ and Cx- cells and that due to this stimulation of GJIC, the bystander effect also occurs when the 2 cell types are mixed. We transfected HeLa cells, which do not express any detectable level of connexin, with Cx43. The Cx+ and Cx- HeLa cells were further transfected with the tk gene, giving 4 phenotypes: Cx+tk-, Cx+tk+, Cx-tk+ and Cx-tk-. We observed GJIC between Cx+ and Cx- cells, but not between Cx- and Cx- cells, regardless of the tk genotype. Similarly, we observed the HSV-tk/GCV bystander effect in Cx+tk-/Cx-tk+ and Cx+tk+/Cx-tk- cocultures. The extent of the bystander effect in cocultures of Cx+tk- and Cx-tk+ cells was stronger than in cocultures of Cx+tk+ and Cx-tk- cells when each mixture had the same ratio of Cx+ and tk+ cells. These results suggest that Cx-expressing HeLa cells stimulate GJIC capacity between them and non-Cx-expressing HeLa cells, which mediates the bystander effect in mixtures of Cx+ cells and Cx- cells in vitro. Thus, Cx expression even in only a limited fraction of tumor cells may enhance the efficacy of the HSV-tk/GCV strategy by inducing a bystander effect.     

Effects of ectopic decorin in modulating intracranial glioma progression in vivo, in a rat syngeneic model

Given the failure of conventional treatments for glioblastoma, gene therapy has gained interest considerable in recent years. Gliomas are associated with a state of immunosuppression, which appears to be partially mediated by an increase in secretion of transforming growth factor-beta (TGF-beta) from glioma cells. Decorin, a small proteoglycan which can bind to and inactivate TGF-beta, has been successfully used as an antitumor strategy on stably transfected tumor cells and has been shown to cause growth suppression in neoplastic cells of various histological origins. In this paper, we investigated the use of gene therapy to deliver the decorin transgene in a site-specific manner in an experimental model of intracranial gliomas. Our aim was to inhibit the glioma-associated immunosuppressive state, and prolong the survival of tumor-bearing rats. We studied the effects of decorin gene transfer in the rat CNS-1 glioma model. To assess the effect of ectopic expression of decorin on glioma progression in vivo, stably transfected CNS-1 cells expressing decorin were implanted into the brain parenchyma of syngeneic Lewis rats. The rats implanted with CNS-1 cells expressing decorin survived significantly longer than those in the control groups which received CNS-1 cells that did not express decorin (P < .0001). We then investigated whether the survival observed with decorin expressing cells could be mimicked in vivo, using recombinant adenoviruses (RAds) expressing the decorin gene under the control of two different promoters: the human immediate-early cytomegalovirus (h-IE-CMV) and the glial fibrillary acidic protein (GFAP). In vivo results showed that administration of RAd expressing the human decorin under the control of h-IE-CMV promoter has a small, but significant effect in prolonging the survival of experimental tumor bearing rats (P < .0001). Our data indicate that ectopic decorin expression has the potential to slow glioma progression in vivo. Our results also indicate that expression of decorin has to be present in all cells which constitute the intracranial tumor mass for the inhibition of tumor growth and prolongation of the life expectancy of tumor-bearing rats to be effective.     

Radiosensitization of human glioma cells in vitro and in vivo with acyclovir and mutant HSV-TK75 expressed from adenovirus

PURPOSE: We recently reported that an adenovirus-expressing mutant HSV-TK75 (AdCMV-TK75) radiosensitized rat syngeneic gliomas in combination with low concentrations of acyclovir (ACV) much more effectively than a virus expressing wild-type herpes simplex virus thymidine kinase (HSV-TK). In this report we have examined whether similar radiosensitizing effects are also seen with human glioma cells in vitro and in vivo. METHODS AND MATERIALS: Human U87 MG glioma cells were transduced with AdCMV-TK75 and exposed to ACV followed by single-dose irradiation and colony-forming survival assays. Similarly, U87 MG xenografts were infused with AdCMV-TK75 or Adbetagal control virus, followed by ACV administration and fractionated irradiation. Therapeutic efficacy was monitored by tumor growth. RESULTS: U87 MG cells transduced with AdCMV-TK75 were significantly more sensitive to ACV (3 microM) than cells transduced with either wild-type HSV-TK or control virus. To determine whether human cells also demonstrate improved radiosensitization similar to that seen with rat glioma cells and tumors, we transduced U87 MG cells with either AdCMV-TK75, AdCMV-TK, expressing wild-type HSV-TK, or Adbetagal and then treated the cells with 3 microM of ACV. Cells transduced with AdCMV-TK75 were significantly more radiosensitive (dose enhancement ratio [D(37)]: 2.6) by colony-forming survival assay than cells transduced with either AdCMV-TK or Adbetagal. Furthermore, we found that U87 MG xenografts infused with AdCMV-TK75 by slow positive pressure infusion were more radiosensitive after administration of ACV than tumors infused with Adbetagal. A more dramatic result was achieved when fractionated irradiation was carried out concurrently with ACV administration, in which case AdCMV-TK75-treated tumors did not grow at all. CONCLUSIONS: These results demonstrate that transduction of human glioma cells in vitro and infusion of xenografts in vivo with AdCMV-TK75 and treatment with concentrations of ACV that can be achieved in vivo produce similar radiosensitization, as previously reported with rat glioma cells and intracerebral syngeneic tumors. In addition, concurrent treatment with ACV and radiation therapy is more efficient than when ACV is administered before radiation therapy.     

Transformation of rat glioma cells with the M-CSF gene inhibits tumorigenesis in vivo

Macrophage colony-stimulating factor (M-CSF) is a potent stimulator of the effector cells such as monocytes and macrophages. To evaluate the effect of M-CSF on malignant gliomas, we transfected the rat gliosarcoma cell line (9L) with human M-CSF expression vector (pCEF-MCSF) by a liposome method. Transfectants were selected using G418-containing medium. As a control, 9L cells transfected with pRc/CMV and selected by G418 were used. The effects of M-CSF gene transfection on tumor cell proliferation in vitro and in vivo were examined. All growth rate did not change in vitro. While the control 9L cells formed progressively enlarging masses, 9L cells transfected with the M-CSF gene did not develop into tumors after the injection into rats. On the other hand, in rats receiving anti-asialo GM1 antibody, 9L cells transfected with M-CSF gene developed into tumors, though at a slower rate than control 9L cells. Histologic examination after transplantation of 9L cells transfected with M-CSF gene disclosed intense infiltration of macrophages in the tumor. Thus M-CSF gene transfection into glioma cells stimulates an antitumor effect.     

Transformation of rat glioma cells with the M-CSF gene inhibits tumorigenesis in vivo

Macrophage colony-stimulating factor (M-CSF) is a potent stimulator of the effector cells such as monocytes and macrophages. To evaluate the effect of M-CSF on malignant gliomas, we transfected the rat gliosarcoma cell line (9L) with human M-CSF expression vector (pCEF-MCSF) by a liposome method. Transfectants were selected using G418-containing medium. As a control, 9L cells transfected with pRc/CMV and selected by G418 were used. The effects of M-CSF gene transfection on tumor cell proliferation in vitro and in vivo were examined. All growth rate did not change in vitro. While the control 9L cells formed progressively enlarging masses, 9L cells transfected with the M-CSF gene did not develop into tumors after the injection into rats. On the other hand, in rats receiving anti-asialo GM1 antibody, 9L cells transfected with M-CSF gene developed into tumors, though at a slower rate than control 9L cells. Histologic examination after transplantation of 9L cells transfected with M-CSF gene disclosed intense infiltration of macrophages in the tumor. Thus M-CSF gene transfection into glioma cells stimulates an antitumor effect.     

Exogenous wt-p53 enhances the antitumor effect of HSV-TK/GCV on C6 glioma cells

Objective To study on the antitumor effect of combining wt-p53 gene with suicide gene therapy (HSV-tk+GCV) for malignant gliomas. Methods AdCMV-p53 was transfected into C6 glioma cells at MOI of (Multiplicity of infection) 0(G100), 10(TPG1), 100(TPG2), then AdCMV-tk was transducted to C6 glioma cells of G100, TPG1 and TPG2, respectively, at MOI of 100. The C6 glioma cells tranfected with both AdCMV-p53 and AdCMV-tk were exposed to various concentration of GCV. The cell survival rate was measured by MTT assay in vitro. Rat glioma model was established by injecting 5 × 10⁵ C6 glioma cells into right caudate nucleus of SD rats. AdCMV-p53 and AdCMV-tk were injected into glioma on day 5 and 6, respectively. On day 7, ganciclovir (GCV) was administrated intraperitoneally at 15 mg/kg/day for 14 days. The survival time of all rats was observed. The growth of intracerebral tumors was monitored dynamically by enhanced MRI. Cell apoptosis was evaluated by TUNEL method. Expression of HSV-tk gene was identified by in situ hybridization and expression of exogenous p53 gene was detected with Western blotting. Results In vitro, wt-p53 significantly enhanced antitumor effect of HSV-tk/GCV. The concentration of GCV for ID50 of TPG2 cells (0.001 μg/ml GCV) was 10 times lower than that for the cells of tk-GCV group (MOI = 100), while the concentration of GCV for ID100 of TPG2 (0.01 μg/ml GCV) and TPG1(0.1 μg/ml GCV) was 100 and 10 times lower than that for the cells of tk-GCV group (MOI = 100), respectively. Apoptosis of C6 glioma cells also could be induced by transfection with wt-p53 gene slightly. For in vivo study, the survival time of tumor-bearing rats treated with HSV-TK/GCV or wt-p53 combined with HSV-TK/GCV was significantly prolonged and the intracerebral tumors were regressed and disappeared earlier in the combined gene therapy group than those in the HSV-TK/GCV therapy group as shown in enhanced MRI. However, only half dose of GCV for the rats treated with both wt-p53 and HSV-TK/GCV was needed to obtain the same efficacy as those rats treated with HSV-TK/GCV alone. These results indicate that the transfection of wt-p53 potentiates the effect of HSV-TK/GCV therapy. Conclusions The combination of HSV-tk/GCV system with wt-p53 gene transduction is optimal for clinical therapeutic trials of suicide gene therapy for malignant gliomas.     

Therapeutic effect of suicide gene-transferred mesenchymal stem cells in a rat model of glioma

We evaluated a new therapeutic strategy for malignant glioma, which combines intratumoral inoculation of mesenchymal stem cells (MSCs) expressing cytosine deaminase gene with 5-fluorocytosine (5-FC) administration. For in vitro and in vivo experiments, MSCs were transfected with adenovirus carrying either enhanced green fluorescent protein gene (AdexCAEGFP) or cytosine deaminase gene (AdexCACD), to establish MSC-expressing EGFP (MSC-EGFP) or CD (MSC-CD). Co-culture of 9L glioma cells with MSC-CD in a medium containing 5-FC resulted in a remarkable reduction in 9L cell viability. The migratory ability of MSC-EGFP toward 9L cells was demonstrated by double-chamber assay. For the in vivo study, rats harboring 9L brain tumors were inoculated with MSC-EGFP or MSC-CD. Immunohistochemistry of rat brain tumors inoculated with MSC-EGFP showed intratumoral distribution of MSC-EGFP. Survival analysis of rats bearing 9L gliomas treated with intratumoral MSC-CD and intraperitoneal 5-FC resulted in significant prolongation of survival compared with control animals. In conclusion, molecular therapy combining suicide gene therapy and MSCs as a targeting vehicle represents a potential new therapeutic approach for malignant glioma, both with respect to the antitumor potential of this system and its neuroprotective effect on normal brain tissue.     

Suicide gene therapy with HSV-TK in pancreatic cancer has no effect in vivo in a mouse model.

AIM: To study in vivo whether pancreatic cancer tumour growth and metastasis can be modified by a gene construct with HSV-TK suicide gene and IL2 co-expression. METHODS: Seventy-eight female SCID mice were i.p. inoculated with retrovirally transduced or control MIA PaCa 2, CAPAN-1 and PANC-1 cell lines. The animals were then randomly selected for saline or ganciclovir (GCV) treatment from the second week, for a total of two weeks. RESULTS: Most inoculated mice developed tumour nodules and spleen metastases. The liver was colonized by control CAPAN-1 and MIA PaCa 2, but not by PANC-1. Tumours in transduced MIA PaCa 2 cell injected mice were smaller, and in transduced CAPAN-1 injected mice larger, than in control-inoculated mice. There were increased pancreatic and decreased spleen metastases from transduced CAPAN-1, and diminished liver involvement from transduced MIA PaCa 2. No differences were found between mice inoculated with transduced and control PANC-1 cell lines. GCV treatment had no effect on tumour's size or metastases. CONCLUSIONS: The HSV-TK suicide gene does not confer GCV sensitivity to pancreatic cancer in this in vivo model. Different pancreatic cancer cell lines cause different growth and metastasis patterns after inoculation in SCID mice, possibly because of variations in their inherent characteristics. The different effects of our vector on cell growth and metastasis may be attributable to the effects of the immunostimulatory cytokine IL2.     

白藜芦醇抑制胶质瘤细胞增殖的体内外实验研究

目的 探讨白藜芦醇(Res)对C6胶质瘤细胞增殖的抑制作用及其机制.方法 将不同浓度Res作用于C6大鼠胶质瘤细胞系,MTT法检测细胞增殖率,流式细胞术检测细胞周期及凋亡,蛋白印迹(Western blot)检测Res处理前后C6细胞表皮生长因子受体(EGFR)、胶质纤维酸性蛋白(GFAP)的表达;免疫组化分析基质金属蛋白酶(MMP)-9、转录核因子-κB(NF-κB)的表达.应用立体定向术建立大鼠动物模型,将20只大鼠分为对照组和治疗组,治疗组8只以8 mg·kg-1·d-1的剂量饲喂,2只以16 mg·kg-1·d-1的剂量饲喂,观察动物一般表现及生存期.MRI动态监测肿瘤大小,脑标本进行病理组织学及相关指标检测.结果 在体外Res可明显抑制C6胶质瘤细胞生长,细胞周期被阻滞在S期,诱导肿瘤细胞凋亡,Res可延长荷瘤SD大鼠生存期,免疫组化检测EGFR、MMP-9、NF-κB表达降低,而GFAP表达升高.结论 Res可抑制胶质瘤细胞增殖,延长荷瘤动物生存期,具有潜在临床应用价值.     

Therapeutic effect of genetically engineered mesenchymal stem cells in rat experimental leptomeningeal glioma model

Disseminating disease of high grade gliomas is difficult to treat. We examined the therapeutic effect of intrathecal administration of mesenchymal stem cells transduced with herpes simplex virus-thymidine kinase gene (MSCtk) followed by systemic ganciclovir (GCV) administration in rat experimental leptomeningeal glioma model. First, to examine in vivo bystander effect, rats were intrathecally co-injected with a mixture of MSCtk and C6 cells and then, intraperitoneally administered with GCV or saline for 10 days (co-injection model). Next, to examine the therapeutic effect of MSCtk/GCV therapy, MSCtk cells were intrathecally administered 1 day after C6 injection and then, GCV or saline was administered (treatment model). GCV administration significantly reduced tumor size on day 14 both in the co-injection model (0.41 ± 0.22 vs. 3.10 ± 0.97 mm², p < 0.01) and in the treatment model (0.73 ± .29 vs. 2.84 ± 0.82 mm², p < 0.01). Survival was also significantly prolonged in GCV group both in the co-injection model (29.2 ± 3.3 vs. 18.8 ± 0.8 days, p < 0.001) and in the treatment model (21.5 ± 1.5 vs. 17.2 ± 0.5 days, p < 0.001). This study provided a novel treatment strategy for leptomeningeal glioma dissemination using intrathecal MSCtk injection followed by systemic GCV administration.     

白藜芦醇抑制胶质瘤细胞增殖的体内外实验研究

目的探讨白藜芦醇(Res)对C6胶质瘤细胞增殖的抑制作用及其机制。方法将不同浓度Res作用于C6大鼠胶质瘤细胞系,MTT法检测细胞增殖率,流式细胞术检测细胞周期及凋亡,蛋白印迹(Western blot)检测Res处理前后C6细胞表皮生长因子受体(EGFR)、胶质纤维酸性蛋白(GFAP)的表达;免疫组化分析基质金属蛋白酶(MMP)-9、转录核因子-κB(NF-κB)的表达。应用立体定向术建立大鼠动物模型,将20只大鼠分为对照组和治疗组,治疗组8只以8 mg·kg-1·d-1的剂量饲喂,2只以16 mg·kg-1·d-1的剂量饲喂,观察动物一般表现及生存期。MRI动态监测肿瘤大小,脑标本进行病理组织学及相关指标检测。结果在体外Res可明显抑制C6胶质瘤细胞生长,细胞周期被阻滞在S期,诱导肿瘤细胞凋亡,Res可延长荷瘤SD大鼠生存期,免疫组化检测EGFR、MMP-9、NF-κB表达降低,而GFAP表达升高。结论Res可抑制胶质瘤细胞增殖,延长荷瘤动物生存期,具有潜在临床应用价值。     

Nitric oxide-mediated signaling in the bystander response of individually targeted glioma cells

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. In this study, human glioblastoma T98G cell nuclei were individually irradiated with an exact number of helium ions using a single-cell microbeam. It was found that when only 1 cell in a population of approximately 1200 cells was targeted, with one or five ions, cellular damage measured as induced micronuclei was increased by 20%. When a fraction from 1% to 20% of cells were individually targeted, the micronuclei yield in the population greatly exceeded that predicted on the basis of the micronuclei yield when all of the cells were targeted assuming no bystander effect was occurring. However when 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide (NO)-specific scavenger was present in the culture medium, the micronuclei yields reduced to the predicted values, which indicates that NO contributes to the bystander effect. By using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), NO was detected in situ, and it was found that NO-induced fluorescence intensity in the irradiated population where 1% of cell nuclei were individually targeted with a single helium ion was increased by 1.13 +/- 0.02-fold (P < 0.005) relative to control with approximately 40% of the cells showing increased NO levels. Moreover, the medium harvested from helium ion-targeted cells showed a cytotoxic effect by inducing micronuclei in unirradiated T98G cells, and this bystander response was also inhibited by c-PTIO treatment. The induction of micronuclei in the population could also be decreased by c-PTIO treatment when 100% of cells were individually targeted by one or two helium ions, indicating a complex interaction of direct irradiation and bystander signals.     

In vivo growth of C6 glioma cells transfected with connexin43 cDNA.

In order to examine the possible role of intercellular communication via gap junctions in the control of tumor growth, we have transfected C6 glioma cells with connexin43 cDNA. We obtained several clones with variable expression of connexin43. The growth rate of these clones in culture was inversely related to the degree of expression of the transfected cDNA. To examine the growth of these transfected cells in vivo, cells were grown in spinner culture flasks to form spheroids 250-300 microns in diameter. Spheroids of nontransfected C6 cells produced large gliomas. Immunohistochemical and in situ hybridization analyses revealed relatively high levels of connexin43 protein and mRNA in the host tissue, while little of this protein was detected in the glioma. In contrast, spheroids of connexin43-transfected cells grew more slowly and exhibited elevated levels of connexin43 protein and mRNA. These findings suggest that the expression of connexin43 may be associated with the control of brain tumor growth in vivo.     

Chromosomal instability in unirradiated cells induced in vivo by a bystander effect of ionizing radiation

Using a bone marrow transplantation protocol in which we transplanted a mixture of irradiated and nonirradiated bone marrow cells that were distinguishable by a cytogenetic marker, we have demonstrated chromosomal instability in the progeny of nonirradiated hemopoietic stem cells. This first demonstration of a link between a bystander effect of ionizing radiation and the induction of genomic instability in vivo clearly poses a major challenge to current views of the mechanisms of radiation-induced DNA damage with mechanistic implications for the health consequences of radiation exposure particularly in the context of the induction of malignancy.     

Intratumoral infusion of topotecan prolongs survival in the nude rat intracranial U87 human glioma model

Topotecan is a topoisomerase (topo) I inhibitor with promising activity in preclinical studies. We hypothesized that low-dose intratumoral delivery of topotecan would be highly effective for gliomas. Human glioma cell lines (U87, U138 and U373) displayed different sensitivities to topotecan (IC₅₀ range: 0.037 M to 0.280 M) in cell culture. The most resistant of the glioma cell lines (U87) was implanted stereotactically into the brains of nude rats. Twelve days later, at which time tumor diameter measured 2 to 2.5 mm, animals were randomized to three groups: group I, intratumoral topotecan infused via osmotic pump (n = 12); group II, intratumoral saline infusion (n = 7); and group III, no treatment (n = 10). Animals were sacrificed when signs of deterioration developed, or at 60 days. Animals in group I had a mean survival time (MST) of > 55 days (range=40–60); whereas, those in groups II and III had MST of 26.1 (range=21–31) and 26.5 (range = 20–30) days, respectively. The differences in survival between group I and each of the other groups were statistically significant (p < 0.0001; Logrank Mantel-Cox). None of the animals that survived 60 days had histological evidence of residual tumor at sacrifice. Measurement of topotecan levels in normal brain revealed cytotoxic concentrations up to 4.5 mm from the site of infusion. This study demonstrates that intratumoral topotecan delivered via an osmotic pump prolongs survival in the U87 human glioma model.     

Vaccination with tumor cell lysate-pulsed dendritic cells augments the effect of IFN-beta gene therapy for malignant glioma in an experimental mouse intracranial glioma

Interferon-beta (IFN-beta) has been used as an antitumor drug against human glioma, melanoma and medulloblastoma since the 1980s. Recently, we developed a new gene therapy using the IFN-beta gene against malignant gliomas and then began clinical trials in 2000. Since stimulation of immune system was one mechanism of antitumor effect induced by IFN-beta gene therapy, we hypothesized that combination of IFN-beta gene therapy with immunotherapy might increase its effectiveness. In the present study, we tested whether combination therapy with IFN-beta gene therapy and immunotherapy using tumor cell lysate-pulsed dendritic cells (DCs) would increase the efficacy of IFN-beta gene therapy. In an experimental mouse intracranial glioma (GL261), which cannot be cured by either IFN-beta gene therapy or DC immunotherapy alone, IFN-beta gene therapy following DC immunotherapy resulted in a significant prolongation in survival of the mice. Moreover, when this combination was performed twice, 50% of treated mice survived longer than 100 days. Considering these results, this combination therapy may be one promising candidate for glioma therapy in the near future.     

Cytotoxic effect and uptake of estramustine in a rat glioma model

The cytotoxic effect of estramustine-phosphate (EMP) and the uptake in tumor tissue were investigated in a rat glioma model in vitro and in vivo. EMP, a combination of nornitrogen mustard and 17beta-estradiol, is a cytotoxic drug which main target is assumed to be the microtubule system. EMP and its metabolite estramustine (EaM) have a demonstrated anti-tumorous effect on human glioma cells in vitro. The drug uptake in tumor tissue and subsequently also the cytotoxic effect, is believed to depend, at least partially, on a specific estramustine-binding protein (EMBP) which is present in human glioma tissue. In this study we have examined the effects and pharmacokinetics of EaM in the nitrosourea induced BT4C rat glioma model. The tumor was characterized by infiltrative growth with a histopathological picture resembling gliosarcoma. The presence of EMBP was demonstrated by immunohistology. In vitro EMP caused a dose-related inhibition of BT4C-cell growth. In vivo, in the rat model, a significant inhibition of tumor growth was obtained after administration of EaM 20 mg/kg/d i.p. The pharmacokinetics of EaM resembled that found in the human clinical situation with EaM as the main metabolite accumulating in tumor tissue. The mean concentration ratio of EaM was 15.6 in tumor versus serum, and 1.8 in tumor versus normal brain of 1.8. The cytotoxic effect demonstrated in the rat glioma model justifies further evaluation of EMP/EaM in the treatment of malignant gliomas.     

Treatment of intracranial rat glioma model with implant of radiosensitizer and biomodulator drug combined with external beam radiotherapy

PURPOSE: To evaluate an intracranial polymer implant containing bromodeoxyuridine (BrdUrd) and N-(phosphonacetyl)-L-aspartic acid (PALA) in combination with external beam radiotherapy (EBRT) in the treatment of a rat glioma. METHODS AND MATERIALS: Combinations of the biomodulators 5-fluorouracil, methotrexate, or PALA with BrdUrd were evaluated as radiosensitizers in vitro by clonogenic assay. In in vivo experiments, BrdUrd and PALA were incorporated into a polyanhydride-based polymer, bis(p-carboxyphenoxy)propane sebacic acid, and implanted in the C6 rat glioma growing intracranially. The effectiveness of treatment was evaluated on the basis of survival. EBRT was given as 10-MV X-rays. RESULTS: In tissue culture experiments, C6 cells were refractory to radiosensitization by BrdUrd even when the thymidine analog was combined with a biomodulator intended to reduce de novo thymidine synthesis. The most effective compound in vitro was PALA. When PALA and BrdUrd in a polymer formulation were implanted intracranially and combined with 10-Gy EBRT, the treatment was highly effective, with 83% of treated rats surviving 180 days. CONCLUSION: Although the in vitro results were not encouraging, the combination of intratumoral BrdUrd and PAL with 10-Gy EBRT was highly effective in treating a rat glioma. These results indicate the clinical potential of combined and mixed modality treatments involving intratumoral sustained-release drug delivery.