Combination of cytosine deaminase suicide gene expression with DR5 antibody treatment increases cancer cell cytotoxicity

Combined treatment using adenoviral-directed enzyme/prodrug therapy and immunotherapy has the potential to become a powerful alternative method of cancer therapy. We have developed adenoviral vectors encoding the cytosine deaminase gene (Ad-CD) and cytosine deaminase:uracil phosphoribosyltransferase fusion gene (Ad-CD:UPRT). A monoclonal antibody, TRA-8, specifically binds to death receptor 5, one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of Ad-CD:UPRT and TRA-8 against human pancreatic cancer and glioma cell lines. The present study demonstrates that Ad-CD:UPRT infection resulted in increased 5-FC-mediated cell killing, compared with Ad-CD. Furthermore, a significant increase of cytotoxicity following Ad-CD:UPRT/5-FC and TRA-8 treatment of cancer cells in vitro was demonstrated. Animal studies showed significant inhibition of tumor growth of MIA PaCa-2 pancreatic and D54MG glioma xenografts by the combination of Ad-CD:UPRT/5-FC plus TRA-8 as compared with either agent alone or no treatment. The results suggest that the combination of Ad-CD:UPRT/5-FC with TRA-8 produces an additive cytotoxic effect in cancer cells in vitro and in vivo. These data indicate that combined treatment with enzyme/prodrug therapy and TRAIL immunotherapy provides a promising approach for cancer therapy.     

Treatment of human colon cancer xenografts with TRA-8 anti-death receptor 5 antibody alone or in combination with CPT-11.

PURPOSE: This study was designed to evaluate the in vitro cytotoxicity and in vivo efficacy of TRA-8, a mouse monoclonal antibody that binds to the DR5 death receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called Apo2L), alone and in combination with CPT-11, against human colon cancer cells and xenografts. EXPERIMENTAL DESIGN: DR5 expression was assessed on human colon cancer cell lines using flow cytometry, and cellular cytotoxicity after TRA-8 treatment, alone and in combination with SN-38, was determined by measuring cellular ATP levels. Tumor growth inhibition and regression rates of well-established subcutaneous COLO 205, SW948, HCT116, and HT-29 colon cancer xenografts in athymic nude mice treated with TRA-8 or CPT-11 alone and in combination were determined. (99m)Tc-TRA-8 was used to examine tumor localization of TRA-8 in animals bearing each of the four xenografts. In addition, whole-body biodistribution and imaging was carried out in COLO 205-bearing animals using in vivo single-photon emission computed tomography imaging and tissue counting. RESULTS: DR5 expression was highest on HCT116, intermediate on SW948 and COLO 205 cells, and lowest on HT-29 cells. COLO 205 cells were the most sensitive to TRA-8-induced cytotoxicity in vitro, SW948 and HCT116 cell lines were moderately sensitive, and HT-29 cells were resistant. Combination treatment with TRA-8 and SN-38 produced additive to synergistic cytotoxicity against all cell lines compared with either single agent. The levels of apoptosis in all cell lines, including HT-29, were increased by combination treatment with SN-38. In vivo, combination therapy with TRA-8 and CPT-11 was superior to either single-agent regimen for three of the xenografts: COLO 205, SW948, and HCT116. COLO 205 tumors were most responsive to therapy with 73% complete regressions after combination therapy. HT-29 cells derived no antitumor efficacy from TRA-8 therapy. Tumor xenografts established from the four colon cancer cell lines had comparable specific localization of (99m)Tc-TRA-8. CONCLUSIONS: In vitro and in vivo effects of TRA-8 anti-DR5 monoclonal antibody on four different colon cancer cell lines and xenografts were quite variable. The HT-29 cell line had low surface DR5 expression and was resistant to TRA-8 both in vitro and in vivo. Three cell lines (COLO 205, SW948, and HCT116) exhibited moderate to high sensitivity to TRA-8-mediated cytotoxicity which was further enhanced by the addition of SN-38, the active metabolite of CPT-11. In vivo, the combination of TRA-8 and CPT-11 treatment produced the highest antitumor efficacy against xenografts established from the three TRA-8-sensitive tumor cell lines. All four colon cancer xenografts had comparable localization of (99m)Tc-TRA-8. These studies support the strategy of TRA-8/CPT-11 combined treatment in human colon cancer clinical trials.     

Effect of anti-DR5 and chemotherapy on basal-like breast cancer

The purpose is to evaluate sensitivity of basal-like breast cancer to treatment with anti-DR5 alone and in combination with chemotherapy. Cytotoxicity of TRA-8 anti-DR5 alone and in combination with doxorubicin or paclitaxel was examined. The role of a DR5-associated molecule (DDX3) in the regulation of apoptosis by recruitment of cIAP1 to the DR5/DDX3 complex was studied. SUM159 and 2LMP orthotopic xenografts were treated with TRA-8 alone and in combination with Abraxane or doxorubicin, and tumor growth inhibition determined. Diffusion-weighted magnetic resonance imaging was used to monitor early tumor response. The majority (12/15) of basal-like cell lines were very sensitive to TRA-8-induced cytotoxicity (IC(50) values of 1.0-49 ng/ml). In contrast, 8/11 luminal or HER2-positive cell lines were resistant (IC(50) > 1,000 ng/ml). Enhanced killing of basal-like cell lines was produced by combination treatment with TRA-8 and doxorubicin. Majority of basal cell lines expressed lower levels of DR5-associated DDX3 and cIAP1 than luminal and HER2-positive cell lines. TRA-8 inhibited growth of basal xenografts and produced 20% complete 2LMP tumor regressions. TRA-8 and chemotherapy produced greater 2LMP growth inhibition than either alone. An increase in apparent diffusion coefficient in 2LMP tumors was measured in a week of therapy with TRA-8 and Abraxane. Basal-like cell lines were more sensitive to TRA-8-mediated cytotoxicity than HER2-over-expressing and luminal cell lines, and chemotherapy enhanced cytotoxicity. High sensitivity of basal cells to TRA-8 correlated with low expression of DR5/DDX3/cIAP1 complex. Treatment with TRA-8 and chemotherapy may be an effective therapy for basal-like breast cancer.     

Radiation-inducible caspase-8 gene therapy for malignant brain tumors

PURPOSE: Patients with malignant gliomas have a poor prognosis. To explore a novel and more effective approach for the treatment of patients with malignant gliomas, we designed a strategy that combines caspase-8 (CSP8) gene therapy and radiation treatment (RT). In addition, the specificity of the combined therapy was investigated to decrease the unpleasant effects experienced by the surrounding normal tissue. METHODS AND MATERIALS: We constructed the plasmid pEGR-green fluorescence protein that included the radiation-inducible early growth response gene-1 (Egr-1) promoter and evaluated its characteristics. The pEGR-CSP8 was constructed and included the Egr-1 promoter and CSP8 complementary DNA. Assays that evaluated the apoptosis inducibility and cytotoxicity caused by CSP8 gene therapy combined with RT were performed using U251 and U87 glioma cells. The pEGR-CSP8 was transfected into the subcutaneous U251 glioma cells of nude mice by means of in vivo electroporation. The in vivo effects of CSP8 gene therapy combined with RT were evaluated. RESULTS: The Egr-1 promoter yielded a better response with fractionated RT than with single-dose RT. In the assay of apoptosis inducibility and cytotoxicity, pEGR-CSP8 showed response for RT. The pEGR-CSP8 combined with RT is capable of inducing cell death effectively. In mice treated with pEGR-CSP8 and RT, apoptotic cells were detected in pathologic sections, and a significant difference was observed in tumor volumes. CONCLUSIONS: Our results indicate that radiation-inducible gene therapy may have great potential because this can be spatially or temporally controlled by exogenous RT and is safe and specific.     

Treatment of small cell lung cancer with TRA-8 in combination with cisplatin and radiation

Background

Limited stage small cell lung cancer (SCLC) represents a minority of SCLC. Despite extensive clinical trials, standard treatment remains cisplatin-based chemotherapy and thoracic irradiation (TI). This study focused on the interaction of cisplatin/radiation with the anti-human DR5 monoclonal antibody TRA-8 in SCLC cells. TRA-8 binds specifically to DR5 and has been shown to activate apoptosis.

Methods

Four human SCLC cell lines were utilized for experimentation (SCLC-41, SCLC-58, SCLC-68, and SCLC-74). Immunoblot analysis was used to determine relative protein levels of DR5, DR4 and pro-caspase 8 for each cell line. Using a tetrazolium-based assay (XTT), the IC₅₀ values for cisplatin with or without TRA-8 were determined for the SCLC cell lines. Four SCLC lines were assayed with a combination of TRA-8 (10 μg/ml), 2 Gy radiation and various concentrations of cisplatin. Apoptosis was evaluated using Annexin V-FITC and cleaved caspase immunoblotting. Using a SCLC-58 subcutaneous xenograft model, treatment began 21 d after tumor cell injection. Treatment included weekly cisplatin (4 mg/kg) and radiation of 1 Gy (24 h after cisplatin) and TRA-8 (200 μg) was administered i.p. twice weekly for three weeks.

Results

Immunoblot analysis showed similar levels of DR5 for all cell lines with variable levels of DR4. Various concentrations of TRA-8 antibody (?10 μg/ml) induced no significant cytotoxicity in the SCLC cell lines. The in vitro combination treatment with TRA-8 (10 μg/ml), 1.25 μg/ml cisplatin and 2 Gy radiation showed increased cytotoxicity when compared to combinations without TRA-8. Furthermore, the triple combination demonstrated the greatest amount of apoptosis as measured by Annexin V staining. The in vivo studies showed the combination of 1 Gy, cisplatin and TRA-8 extended the tumor doubling time to 44 d as compared to any doublet treatment groups that ranged from 12 to 20 d. Analysis of survival data showed 100% of the combination group (RT + cisplatin + TRA-8) were alive 65d after treatment began whereas all doublet treatment groups showed 50% or less survival.

Conclusions

These studies showed increased cytotoxicity when TRA-8 was added to radiation/cisplatin in SCLC. This effect was demonstrated in vitro and in vivo. TRA-8 represents a promising new agent in the treatment of SCLC.     

Systemic delivery of neutralizing antibody targeting CCL2 for glioma therapy

Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immune responses and facilitate tumor growth. Precursors for these immune cell populations migrate to the tumor site in response to tumor secretion of chemokines, such as monocyte chemoattractant protein-1 (MCP-1/CCL2), which was originally purified and identified from human gliomas. In syngeneic mouse GL261 glioma and human U87 glioma xenograft models, we evaluated the efficacy of systemic CCL2 blockade by monoclonal antibodies (mAb) targeting mouse and/or human CCL2. Intraperitoneal (i.p.) administration of anti-mouse CCL2 mAb as monotherapy (2 mg/kg/dose, twice a week) significantly, albeit modestly, prolonged the survival of C57BL/6 mice bearing intracranial GL261 glioma (P = 0.0033), which was concomitant with a decrease in TAMs and MDSCs in the tumor microenvironment. Similarly, survival was modestly prolonged in severe combined immunodeficiency mice bearing intracranial human U87 glioma xenografts treated with both anti-human CCL2 mAb and anti-mouse CCL2 antibodies (2 mg/kg/dose for each, twice a week) compared to mice treated with control IgG (P = 0.0159). Furthermore, i.p. administration of anti-mouse CCL2 antibody in combination with temozolomide (TMZ) significantly prolonged the survival of C57BL/6 mice bearing GL261 glioma with 8 of 10 treated mice surviving longer than 70 days, while only 3 of 10 mice treated with TMZ and isotype IgG survived longer than 70 days (P = 0.0359). These observations provide support for development of mAb-based CCL2 blockade strategies in combination with the current standard TMZ-based chemotherapy for treatment of malignant gliomas.     

Effects of toxic doses of glutamate on Cu–Zn and Mn/superoxide dismutases activities in human glioma cell lines

Summary Object: To investigate the effect of dendritic cells(DC) pulsed with apoptotic tumor cells for treatment of intracranial gliomas in rats. Methods: C6 glioma cells were injected into brain of Wistar rats under stereotactic monitor to establish an animal model of glioma. The precursors of DCs were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). These DCs were then pulsed ex vivo with apoptotic C6 glioma cells induced by heating and subsequently injected subcutaneously into rats harboring intracranial C6 glioma. Rats were treated with five weekly subcutaneous injections of either control media, unpulsed DCs, or DCs pulsed with apoptotic tumor cells. The animals were followed for survival, volume of tumor by MRI, CD8 + T cells, cytotoxicity assay in vitro and proliferational function of lymphocytes in peripheral blood were determined by flow cytometry(FCM). The concentration of cytokines interferon-γ (IFN-γ) and interleukin-10(IL-10) were monitored through enzyme-linked immunosorbent assay(ELISA) using ELISAkit. Results: Our results indicated that C6 glioma model rats treated with apoptotic tumor cells pulsed DCs prolonged survival, inhibited the tumor growth and increased the level of CD8 + T lymphocytes in peripheral blood comparing with control group. Cytotoxicity assay suggested that vaccination with these apoptotic cells pulsed DCs can induce cytotoxic T lymphocytes response against C6 tumor cells compared with control group. Furthermore, significantly enhanced IFN-γ and reduced IL-10(even undetectable) were observed in peripheral blood of rats treated with pulsed-DCs. No evident autoimmune response were detected. Conclusions: Our data demonstrated that systemic vaccination with DCs pulsed with apoptotic cells is a safe and effective immunotherapy for intracranial glioma.     

Tetrandrine suppresses tumor growth and angiogenesis of gliomas in rats

Tetrandrine, a bisbenzylisoquinoline alkaloid, has antitumor effects against some cancers, but its effects on gliomas are unknown. In this study, we investigated the effects of tetrandrine on the growth and angiogenesis of rat RT‐2 gliomas. We treated RT‐2 glioma cells with tetrandrine and then measured cytotoxicity, apoptosis and expression of vascular endothelial growth factor (VEGF). We also examined the cytotoxic effect of tetrandrine on the ECV304 human umbilical vein endothelial cells and the effects of tetrandrine on the in vivo angiogenesis. Tumor size and animal survival were followed in tetrandrine‐treated rats with subcutaneous or intracerebral gliomas. Expression of CD31 in tetrandrine‐treated gliomas was followed to study its effect on glioma‐induced angiogenesis. Tetrandrine had cytotoxic effects and induced apoptosis of glioma cells in a concentration‐ and time‐dependent manner. Tetrandrine also inhibited the expression of VEGF in glioma cells, induced cytotoxicity effect on the ECV304 cells and suppressed the in vivo angiogenesis. Tetrandrine (150 mg/kg/day) had significant antitumor effects on subcutaneous tumors and led to slower tumor growth rate, longer animal survival time and higher animal survival (p < 0.05). Tetrandrine also affected intracerebral tumors and prolonged animal survival (p < 0.05) without affecting survival rate. Immunohistochemical analyses showed that the subcutaneous gliomas from tetrandrine‐treated rats had fewer microvessel densities than control rats (p = 0.01). The results demonstrate that tetrandrine is cytotoxic to RT‐2 glioma cells, has antitumor effects on subcutaneous and intracerebral gliomas, and inhibits angiogenesis in subcutaneous gliomas. Tetrandrine has potential as a treatment for gliomas. © 2008 Wiley‐Liss, Inc.     

STI571 (Gleevec) improves tumor growth delay and survival in irradiated mouse models of glioblastoma

PURPOSE: Glioblastoma multiforme (GBM) is a devastating brain neoplasm that is essentially incurable. Although radiation therapy prolongs survival, GBMs progress within areas of irradiation. Recent studies in invertebrates have shown that STI571 (Gleevec; Novartis, East Hanover, NJ) enhances the cytotoxicity of ionizing radiation. In the present study, the effectiveness of STI571 in combination with radiation was studied in mouse models of GBM. METHODS AND MATERIALS: Murine GL261 and human D54 GBM cell lines formed tumors in brains and hind limbs of C57BL6 and nude mice, respectively. GL261 and D54 cells were treated with 5 micromol/L of STI571 for 1 h and/or irradiated with 3 Gy. Protein was analyzed by Western immunoblots probed with antibodies to caspase 3, cleaved caspase 3, phospho-Akt, Akt, and platelet-derived growth factor receptor (PDGFR) alpha and beta. Tumor volumes were assessed in mice bearing GL261 or D54 tumors treated with 21 Gy administered in seven fractionated doses. Histologic sections from STI571-treated mice were stained with phospho-Akt and phospho-PDGFR beta antibodies. Kaplan-Meier survival curves were used to study the response of mice bearing intracranial implants of GL261. RESULTS: STI571 penetrated the blood-brain barrier, which resulted in a reduction in phospho-PDGFR in GBM. STI571-induced apoptosis in GBM was significantly enhanced by irradiation. STI571 combined with irradiation induced caspase 3 cleavage in GBM cells. Glioblastoma multiforme response to therapy correlated with an increase in tumor growth delay and survival when STI571 was administered in conjunction with daily irradiation. CONCLUSION: These findings suggest that STI571 has the potential to augment radiotherapy and thereby improve median survival.     

GM-CSF基因修饰肺癌细胞疫苗的抗肿瘤活性及其与化疗的协同作用

目的评价粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因修饰肿瘤疫苗的抗肿瘤活性,探讨其与化疗联合应用的抗肿瘤效果。方法以小鼠肺癌Lewis细胞系接种C57BL/6小鼠建立动物模型。利用含GM-CSF基因的重组质粒GM-CSF-pIRES2-EGFP,转染小鼠肺癌细胞系Lewis和LA795细胞,分别制备自体(Lewis细胞)和同种异体(LA795细胞)肿瘤疫苗。小鼠皮下接种Lewis细胞(1×107个)后5 d,采用GM-CSF分泌性肿瘤疫苗接种3次,同时设PBS组和单纯疫苗组作为对照,检测疫苗治疗前后脾细胞对Lewis细胞杀伤活性的变化,以及血清白介素4(IL-4)和γ干扰素(IFN-γ)的水平。观察GM-CSF分泌性肿瘤疫苗接种及联合化疗对小鼠生存期的影响。结果GM- CSF分泌性自体或同种异体肿瘤疫苗,均可诱导小鼠脾细胞对Lewis细胞杀伤活性增高,第3次接种后分别为(42.0±2.5)%和(39.6±7.3)%;同时血清中Th1类细胞因子IFN-γ的水平升高,而Th2类因子IL-4的水平无显著变化。GM-CSF分泌性肿瘤疫苗治疗组小鼠生存期与对照组比较,差异无统计学意义(P>0.05);而化疗联合疫苗组小鼠生存期较单独治疗组及对照组明显延长(P<0.05)。结论GM-CSF基因修饰的肿瘤疫苗可刺激机体产生特异性免疫反应;化疗的联合应用有助于提高肿瘤疫苗的治疗效果。     

MIP-1α Antagonizes the Effect of a GM-CSF-Enhanced Subcutaneous Vaccine in a Mouse Glioma Model

Subcutaneous vaccination using granulocyte-macrophage colony-stimulating factor (GM-CSF)-transduced glioma cells substantially prolongs survival in the mouse GL261 glioma model. To potentiate the efficacy of GM-CSF-based vaccination, syngeneic C57BL/6 mice bearing pre-implanted intracerebral GL261 gliomas were vaccinated twice subcutaneously with various combinations of glioma cells retrovirally engineered to release GM-CSF, interleukin (IL)-4 or macrophage inflammatory protein (MIP)-1alpha. More than 80% of the animals vaccinated with GM-CSF-secreting or GM-CSF- and IL-4-secreting cells were long-term survivors (> 120 days). Their survival was significantly prolonged compared with animals vaccinated with wild-type cells, which died after a median survival time of 30 days. The combination of IL-4 with GM-CSF did not provide a survival advantage over GM-CSF alone, regardless of whether the animals carried a small or large intracranial tumor load. Further, when the animals were vaccinated with a mixture of GM-CSF-, IL-4- and MIP-1alpha-secreting cells, the median survival was 37 days, and only 22% of the animals in this group were long-term survivors, similar to the vaccination effect of non-modified glioma cells. Thus, unexpectedly, the co-expression of MIP-1alpha, which was meant to attract T cells for stimulation by GM-CSF- and IL-4-stimulated dendritic cells, nullified the induction of an immune response against the GL261 glioma by a GM-CSF- and IL-4-expressing subcutaneous vaccine.     

Intratumoral injection of interleukin-2 augments the local and abscopal effects of radiotherapy in murine rectal cancer

Recent studies have suggested that tumor shrinkage in response to radiotherapy (RT) is greatly dependent on the host immune response. A Balb/c mouse model of simultaneous subcutaneous tumor and liver metastasis of Colon26 was prepared and, after irradiation of the subcutaneous tumor (2 Gy × 5 day × 2 cycles), interleukin-2 (IL-2) (2 × 10(4) U) was injected intra-tumorally, and the fate of both the subcutaneous tumor and liver metastatic lesions was evaluated. Intratumoral injection of IL-2 greatly enhanced the anti-tumor effects of RT and completely eradicated the established subcutaneous tumor. Interestingly, although RT was given locally to the subcutaneous tumor, liver metastasis formation was also inhibited in mice receiving only local RT. More impressively, the combination of RT + IL-2 completely inhibited liver metastasis formation. Splenocytes in mice receiving RT + IL-2 contained a higher percentage of CD4(+) T cells, but lower percentages of CD4(+)CD25(+) regulatory T cells and CD11b(+) Gr-1(+) myeloid-derived suppressor cells. Immunohistochemical investigation of human rectal cancer revealed that the density of CD8(+) cells infiltrating into irradiated rectal tumor was positively associated with a lower frequency of distant metastasis as well as histological response grade. Local administration of IL-2 not only enhances shrinkage of the irradiated tumor itself, but can also suppress the development of distant metastasis located outside the RT field, possibly though the induction of a systemic T cell response. Augmentation of T-cell-mediated antitumor immunity during RT might be critical for improvement of the clinical efficacy of neoadjuvant RT for the treatment of advanced rectal cancer.     

Therapeutic efficacy of OX-40 receptor antibody depends on tumor immunogenicity and anatomic site of tumor growth

The OX-40 receptor (OX-40R) is a cell surface glycoprotein of the tumor necrosis factor receptor family that is expressed primarily on activated CD4 T cells. Engagement of OX-40R by the OX-40 ligand (OX-40L) is known to costimulate the production of cytokines by activated T lymphocytes and to rescue effector T cells from activation-induced cell death. It was previously reported that in vivo ligation of OX-40R by administration of OX-40L:immunoglobulin fusion protein or OX-40R monoclonal antibody (mAb) resulted in a significant prolongation of survival of tumor-bearing mice in four histologically distinct solid tumors. In this study, we demonstrate that the therapeutic efficacy of OX-40R mAb was influenced by the tumor burden, the intrinsic immunogenicity of the tumor as well as by the histological site of tumor growth. Whereas subdermal and intracranial growth of weakly immunogenic MCA 203 and MCA 205 sarcomas and GL261 glioma were susceptible to the mAb treatment, established pulmonary MCA 205 metastases were refractory to the same regimen of treatment. Furthermore, the mAb administration had no impact on the growth of the poorly immunogenic B16/D5 mela noma. Tumor regression mediated by OX-40R mAb was dependent on the participation of both CD4 and CD8 T cells and as a result of tumor rejection, a long-term tumor-specific immunity was established. Analysis of tumor-infiltrating T cells revealed the presence of a far greater number of OX-40R+ T cells of both CD4 and CD8 phenotypes in the intracranial immunogenic GL261 glioma than that in the poorly immunogenic B16/D5 melanoma. These results suggest that ligation of OX-40R on activated T cells in situ in the tumor may provide a necessary costimulatory signal to augment immune responses leading to tumor regression and immunological memory.     

腺病毒介导的IL—2基因及B7—1基因联合转染G422细胞致瘤原性和免疫原性的变化

目的研究联合细胞因子基因转染的D422胶质母细胞瘤细胞体内致瘤原性和免疫原性的变化,为胶质瘤的免疫基因治疗打下基础.方法IL-2基因和B7-1基因转染的G422细胞1×105皮下和脑内接种,观察肿瘤生长速度和荷瘤小鼠的存活期,2周取脾脏,检测NK、LAK和CTL的杀伤活性.结果IL-2和B7-1基因联合转染的G422细胞,皮下接种后肿瘤生长明显减慢,脑内接种动物存活期明显延长,NK、LAK和CTL的杀伤活性增强.结论IL-2基因和B7-1基因联合转染的G422细胞,致瘤原性下降,免疫原性增强,能有效激活机体特异性与非特异性抗肿瘤免疫反应.     

Enhancement of antitumor activity of ionizing radiation by combined treatment with the selective epidermal growth factor receptor-tyrosine kinase inhibitor ZD1839 (Iressa).

PURPOSE: The epidermal growth factor receptor (EGFR) is expressed in the majority of human epithelial cancers and has been implicated in the development of cancer cell resistance to cyotoxic drugs and to ionizing radiation. Experimental Design: We used ZD1839, a selective small molecule EGFR tyrosine kinase inhibitor currently in clinical development. We tested the antiproliferative and the proapoptotic activity of ZD1839 in combination with ionizing radiation in human colon (GEO), ovarian (OVCAR-3), non-small cell lung (A549 and Calu-6), and breast (MCF-7 ADR) cancer cell lines. The antitumor activity of this combination was also tested in nude mice bearing established GEO colon cancer xenografts. RESULTS: With ionizing radiation or ZD1839, a dose-dependent growth inhibition was observed in all of the cancer cell lines growing in soft agar. A cooperative antiproliferative and proapoptotic effect was obtained when cancer cells were treated with ionizing radiation followed by ZD1839. This effect was accompanied by inhibition in the expression of the antiapoptotic proteins bcl-xL and bcl-2, and by a suppression of the activated (phosphorylated) form of akt protein. Treatment of mice bearing established human GEO colon cancer xenografts with radiotherapy (RT) resulted in a dose-dependent tumor growth inhibition that was reversible upon treatment cessation. Long term GEO tumor growth regressions were obtained after RT in combination with ZD1839. This resulted in a significant improvement in survival of these mice as compared with the control group (P < 0.001), the RT-treated group (P < 0.001), or the ZD1839-treated group (P < 0.001). The only mice alive 10 weeks after tumor cell injection were in the RT-plus-ZD1839 group. Furthermore, 10% of mice in this group were alive and tumor-free after 26 weeks. Similar results were obtained in mice bearing established human A549 lung adenocarcinoma xenografts. Finally, the combined treatment with RT plus ZD1839 was accompanied by a significant potentiation in the inhibition of transforming growth factor alpha, vascular epidermal growth factor, and basic fibroblast growth factor expression in cancer cells, which resulted in significant antiangiogenic effects as determined by immunohistochemical count of neovessels within the GEO tumors. CONCLUSION: This study provides a rationale for evaluating in cancer patients the combination of ionizing radiation and selective EGFR tyrosine kinase inhibitors such as ZD1839.     

Antitumor activity of combined treatment of human cancer cells with ionizing radiation and anti-epidermal growth factor receptor monoclonal antibody C225 plus type I protein kinase A antisense oligonucleotide.

Recent studies have suggested that selective inhibition of mitogenic pathways may improve the antitumor activity of ionizing radiation. The epidermal growth factor receptor (EGFR) is overexpressed and is involved in autocrine growth control in the majority of human carcinomas. Protein kinase A type I (PKAI) plays a key role in neoplastic transformation and is overexpressed in cancer cells in which an EGFR autocrine pathway is activated. We used two specific inhibitors of EGFR and PKAI that are under clinical evaluation in cancer patients: C225, an anti-EGFR chimeric human-mouse monoclonal antibody (MAb); and a mixed-backbone antisense oligonucleotide targeting the PKAI RIalpha subunit (PKAI AS). We tested in human colon cancer (GEO) and ovarian cancer (OVCAR-3) cell lines the antiproliferative activity of MAb C225 and/or PKAI AS in combination with ionizing radiation. In vivo antitumor activity was evaluated in nude mice bearing established GEO xenografts. Dose-dependent inhibition of soft agar growth was observed in both cancer cell lines with ionizing radiation, C225, or PKAI AS oligonucleotide. A cooperative antiproliferative effect was obtained when cancer cells were treated with ionizing radiation followed by MAb C225 or PKAI AS oligonucleotide. This effect was observed at all doses tested in both GEO and OVCAR-3 cancer cell lines. A combination of the three treatments at the lowest doses produced an even greater effect than that observed when two modalities were combined. Treatment of mice bearing established human GEO colon cancer xenografts with radiotherapy (RT), MAb C225, or PKAI AS oligonucleotide produced dose-dependent tumor growth inhibition that was reversible upon treatment cessation. A potentiation of the antitumor activity was observed in all mice treated with RT in combination with MAb C225 or PKAI AS oligonucleotide. Long-term GEO tumor growth regression was obtained following treatment with ionizing radiation in combination with MAb C225 plus PKAI AS oligonucleotide, which produced a significant improvement in survival compared with controls (P < 0.001), the RT-treated group (P < 0.001), or the group treated with MAb C225 plus PKAI AS oligonucleotide (P < 0.001). All mice of the RT + MAb C225 + PKAI AS group were alive 26 weeks after tumor cell injection. Furthermore, 50% of mice in this group were alive and tumor-free after 35 weeks. This study provides a rationale for evaluating in cancer patients the combination of ionizing radiation and selective drugs that block EGFR and PKAI pathways.     

An adenovirus encoding proapoptotic Bax synergistically radiosensitizes malignant glioma

PURPOSE: We explore the utility of the adenovirus-mediated delivery of proapoptotic Bax for enhancing the cytotoxicity of radiotherapy (RT) in RT-refractory glioma cells. MATERIALS AND METHODS: Cell lines D54 MG and U87 MG (p53 wild-type), and U251 MG and U373 MG (p53 mutant), and patient-derived astrocytes were evaluated. Cells were irradiated and infected with an inducible adenovirus encoding Bax. Cell proliferation, colony formation assay, quantification of early apoptotic alteration in the plasma membrane by fluorescence-activated cell sorter using annexin V, and nuclear staining with H33258 were used to evaluate apoptosis. The capacity of the combined treatment to induce regression of subcutaneous D54 MG tumors was tested in nude mice. A dose of 5 Gy was administered every other day, four times, for a total dose of 20 Gy. One day after each irradiation, tumors were injected with 1 x 10(9) plaque-forming units (PFU). RESULTS: Apoptotic death was enhanced by the combination of Ad/Bax and RT. In D54 MG, levels of apoptosis after RT alone, Ad/Bax alone, or the combination were, respectively, 12.3%, 32.1%, and 78.5%. In contrast, treatment of astrocytes did not significantly induce apoptosis. A colony-formation assay showed a 2-log inhibition with respect to controls after combined treatment, irrespective of the endogenous levels of p53. The other apoptosis assays also showed the defining characteristics of apoptosis in the combination group. Remarkably, combined treatment induced regression of tumors in mice. CONCLUSIONS: Ad/Bax synergistically radiosensitizes glioma, with a seemingly favorable therapeutic index.     

The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma

Neural stem cells (NSCs) are capable of tracking migrating glioma cells. To exploit this tropism to generate an antitumor T-cell response, particularly against disseminating tumor pockets, we inoculated intracranial glioma-bearing mice with interleukin 12 (IL-12) producing NSCs. Intratumoral therapy with IL-12-secreting NSCs prolonged survival compared to treatment with nonsecretory NSCs or saline. NSCs demonstrated strong tropism for disseminating glioma, and IL-12-secreting NSC therapy was associated with enhanced T-cell infiltration in tumor microsatellites and long-term antitumor immunity. These results indicate that the use of tumor tracking NSCs represents a potent new therapeutic modality for glioma.     

Inducible resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand receptor 2-mediated apoptosis by generation of a blockade at the death domain function

Induction of tumor cell resistance to therapeutics has been a major obstacle in cancer therapy. Targeting of the death receptors by a natural ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), or agonistic monoclonal antibodies against TRAIL receptor 1 (TRAIL-R1) or TRAIL receptor 2 (TRAIL-R2) has been thought to be a promising cancer therapy. To determine whether tumor cells are able to generate a resistance to apoptosis induced by an anti-TRAIL-R2 antibody, TRA-8, we examined the apoptotic response of human breast and ovarian cancer cell lines after treatment with TRA-8. Our results show that tumor cell resistance to TRA-8 can be induced by repeated treatment of tumor cells with low, non-apoptosis-inducing doses of TRA-8. Interestingly, the induced resistance to apoptosis was not due to a global apoptotic defect in tumor cells but rather a selective defect in the TRAIL-R2 signaling pathway. Whereas TRA-8-treated tumor cells developed a selective resistance to TRAIL-R2-mediated apoptosis, the apoptotic responses induced by TRAIL, an anti-TRAIL-R1 antibody (2E12), and other apoptotic stimuli were not impaired. The expression levels of cell surface TRAIL-R2 were not altered and mutations of TRAIL-R2 were not found in the resistant cells. The induced TRA-8 resistance was due to a selective blockade at the level of the death domain and could be reversed by a wide array of chemotherapeutic agents. Proteomic analysis of death-inducing signaling complex formation during TRA-8 treatment shows that the translocation of TRAIL-R2-associated apoptotic proteins was significantly altered. Our results suggest that the prevention of tumor cell resistance to therapeutic agents that target the death receptors must be taken into consideration.