Significant antitumor activity of cationic multilamellar liposomes containing human interferon-beta gene in combination with 5-fluorouracil against human renal cell carcinoma

Immunotherapy is one of the most effective treatments against metastatic renal cell carcinoma (RCC). However, the response rate is not high. Therefore, more effective therapies are necessary for patients with metastatic RCC. We previously reported on the significant antitumor activity of cationic multilamellar liposome containing human interferon-beta (huIFN-beta) gene (IAB-1) against RCC. We then examined the antitumor effect of IAB-1 in combination with anticancer drugs against RCC. The cytotoxicity of IAB-1 alone, and in combination with anticancer drugs, cisplatin, adriamycin, 5-fluorouracil, gemcitabine, paclitaxel and irinotecan hydrochloride against the human RCC cell line NC65 was examined by the colorimetric method using tetrazolium salt. For the in vivo study, we used NC65 cells inoculated into the severe combined immunodeficiency mouse. The results showed that the in vitro combination therapy with IAB-1 and 5-FU was more cytotoxic than IAB-1 alone. However, synergistic cytotoxicity was not observed when combined with IAB-1 and other anticancer drugs. NC65 tumors transfected with IAB-1 in mice were smaller than those receiving an injection of empty liposome or the recombinant huIFN-beta protein. Treatment with IAB-1 in combination with 5-FU resulted in significant anticancer activity. IAB-1 enhanced the activity of thymidine phosphorylase (TP), which converts 5-FU to the active metabolite, FdUMP. In contrast, IAB-1 decreased the activity of thymidylate synthase (TS), which is a target enzyme of 5-FU. In conclusion, these findings indicate that a combination of IAB-1 and 5-FU may have enhanced antitumor activity against human RCC, suggesting its potential clinical application. The mechanism of enhanced cytotoxicity by combination therapy with IAB-1 and 5-FU may up-regulate TP activity and down-regulate TS activity.     

Significant induction of apoptosis in renal cell carcinoma cells transfected with cationic multilamellar liposomes containing the human interferon-β gene through activation of the intracellular type 1 interferon signal pathway

We previously reported that cationic multilamellar liposome containing the human interferon-β (huIFN-β) gene (IAB-1) demonstrated significant cytotoxic effect in the NC65 human renal cell carcinoma (RCC) cell line. In this study, we investigated the molecular mechanisms of IAB-1-induced apoptosis and cytotoxicity in RCC cells. Remarkable in vitro cytotoxic and apoptosis-inducing effects of IAB-1 against NC65 cells were observed by a colorimetric method and TUNEL staining, respectively. In contrast, treatment of NC65 cells with exogenously added huIFN-β protein induced low-level cytotoxicity without apoptosis. Neutralizing antibodies against huIFN-β significantly suppressed the cytotoxic effect of huIFN-β protein, but they were unable to block the effect of IAB-1. Cytotoxicity assays using transwell plates revealed that NC65 cells treated with IAB-1 did not secrete cytotoxic soluble factors other than IFN-β. Substantial enhancement of interferon-stimulated response element (ISRE) activity of NC65 cells by IAB-1 was demonstrated by promoter reporter assays. In addition, immunofluorescence using confocal microscopy revealed the intracellular expression of IFN-β and its receptor induced by IAB-1. The induction of c-Myc by IAB-1 was suggested by a cDNA macroarray and was confirmed by western blot analysis. These findings indicate that IAB-1 induces significant cytotoxicity and apoptosis in NC65 cells, possibly through enhanced ISRE activity, that is associated with increased intracellular localization of huIFN-β and IFN-receptor. Our data support the potential clinical application of IAB-1 gene therapy for RCC resistant to IFN.     

Low concentrations of doxorubicin sensitizes human solid cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor (R) 2-mediated apoptosis by inducing TRAIL-R2 expression

There is accumulating evidence suggesting that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor (R) 2 is a promising molecular target for cancer therapy. Therefore, we investigated the effect of chemotherapeutic agents on TRAIL-R2-mediated apoptosis and cytotoxicity in various human solid cancer cells. Treatment of the ACHN human renal cell carcinoma (RCC) cell line with agonistic TRAIL-R2 antibody (lexatumumab) in combination with 5-fluorouracil, vinblastine, paclitaxel, or docetaxel did not overcome resistance to these agents. However, treatment with lexatumumab in combination with doxorubicin had a synergistic cytotoxicity. Synergy was also achieved in two other human RCC cell lines, Caki-1 and Caki-2, and in eight primary RCC cell cultures. Sequential treatment with doxorubicin followed by lexatumumab induced significantly more cytotoxicity than reverse treatment or simultaneous treatment. Low concentrations of doxorubicin (0.1 and 1 µg/mL) significantly increased TRAIL-R2 expression at both the mRNA and protein levels. Furthermore, the combination of doxorubicin and lexatumumab significantly enhanced caspase 8 activity, Bid cleavage, Bcl-xL decrease, release of cytochrome c , and caspase 9 and caspase 3 activity, and induced synergistic apoptosis. The activation of caspases and apoptosis induced with lexatumumab and doxorubicin was blocked by the human recombinant DR5:Fc chimeric protein. In addition, synergistic cytotoxicity was also observed in human prostate, bladder, and lung cancer cells, but was inhibited by the DR5:Fc chimeric protein. These findings suggest that doxorubicin sensitizes solid cancer cells to TRAIL-R2-mediated apoptosis by inducing TRAIL-R2 expression, and that the combination treatment with lexatumumab and doxorubicin might be a promising targeted therapy for cancers, including RCC, prostate, bladder, and lung cancers. ( Cancer Sci 2007; 98: 1969–1976)     

Enhancement of Ad5-TRAIL cytotoxicity against renal cell carcinoma with histone deacetylase inhibitors

Renal cell carcinoma (RCC) will cause greater than 12,000 deaths in the United States this year. The lack of effective therapy for disseminated RCC has stimulated the search for novel treatments including immunotherapeutic strategies, but poor therapeutic responses and marked toxicity have limited their use. The tumor necrosis factor (TNF) family member TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L induces apoptosis in various tumor cell types, while having little cytotoxicity against normal cells. In this study, we investigated the tumoricidal potential of a recombinant adenovirus encoding human TNFSF10 (Ad5-TRAIL), alone and in combination with a panel of histone deacetylase inhibitors (HDACi), against the TRAIL/Apo-2L-resistant RCC line 786-O and normal human renal proximal tubule epithelial cells (RPTEC). Ad5-TRAIL was unable to induce apoptosis in either 786-O or RPTEC alone; however, tumor cell apoptosis occurred when Ad5-TRAIL was combined with HDAC inhibition. Except when combined with trichostatin A, RPTEC were not sensitized to Ad5-TRAIL by HDACi. In 786-O, HDAC inhibition induced CAR expression, permitting increased adenoviral infection and transgene expression. It also induced TRAIL-R2 expression, accelerated the death-inducing signaling complex formation and enhanced caspase-8 activation. Our results demonstrate the utility of combining Ad5-TRAIL with HDACi against RCC, and mechanistically define how this combination modulates RCC sensitivity to TRAIL/Apo-2L and adenoviral infection.     

Significance of cytotoxic activity of peripheral blood lymphocytes against autologous tumor cells in patients with bladder cancer

Cell-mediated immunity is an important and central mechanism of host resistance to cancer. Most reported studies have used cultured tumor cell lines as targets to assess antitumor cell-mediated cytotoxicity. However, it is difficult to translate the data generated from the cytotoxic activity against cultured tumor cell lines to cytotoxicity against autologous tumors. In a recent study, we have reported on the prognostic significance of circulating cytotoxic lymphocytes against autologous tumor cells in patients with bladder cancer. In this study, we examined whether established bladder cancer cell line like T24 or NK-sensitive K562 target cells can be substituted for autologous bladder cancer cells. The cytotoxic activity of peripheral blood lymphocytes (PBL) against freshly isolated autologous tumor cells, the T24 human bladder cancer cell line and the NK-sensitive K562 human myelogenous leukemia cell line was studied in 63 patients with primary initial bladder cancer by a 12-h 51Cr release assay. The mean percent cytotoxic activity of PBL directed against autologous tumor cells, T24 cells and K562 cells were 11.3%, 18.2% and 29.4%, respectively, using an E:T of 40:1. The cytotoxic activity against T24 cells in patients with bladder cancer was higher than that in normal individuals. The anti-K562 and the anti-T24 cytotoxic activities in patients with low-stage or low-grade bladder cancer were relatively higher than those in patients with high-stage or high-grade cancer, but not statistically significant. There was no correlation between the anti-autologous tumor cytotoxic activity and either the histologic grade or stage in patients with bladder cancer. The extent of the anti-autologous tumor cytotoxic activity was not paralleled with that of either the anti-K562 or the anti-T24 cytotoxic activity. In contrast, the anti-K562 cytotoxic activity correlated positively with the anti-T24 cytotoxic activity. Separation of PBL revealed that the anti-K562 and the anti-T24 cytotoxic activities were mediated mainly by the NK cells, whereas the anti-autologous tumor cytotoxic activity was mediated by both the NK cells and the T lymphocytes. These findings demonstrate that cytotoxicity against T24 or K562 cells is not of prognostic value. The magnitude of the anti-autologous tumor cytotoxic activity of PBL derived from bladder cancer patients might represent an independent and important immunological parameter to monitor disease progression.     

树突状细胞与肾癌融合细胞疫苗诱导抗肿瘤免疫

目的探讨自体树突状细胞(DC)与肾癌融合细胞疫苗诱导特异性抗肿瘤免疫应答。方法利用肿瘤细胞纯化技术,从手术切除的肾癌组织中分离出高纯度的肾癌细胞,用10%胎牛血清(FCS)的RPMI-1640进行原代培养。分离外周血单核细胞(PBMCs),在含重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素4(rhIL-4)的培养条件下,诱导分化为DC。用细胞融合技术将DC与肾癌细胞融合制备DC与肾癌融合细胞疫苗。用流式细胞仪检测肾癌细胞、DC和融合细胞的表型;用噻唑盐(MTT)比色法检测融合细胞刺激自体T细胞的增殖能力,乳酸脱氢酶(LDH)释放的细胞毒实验检测融合细胞刺激的细胞毒性T淋巴细胞(CTLs)的杀瘤活性。结果DC高表达主要组织相融性复合体(MHC)Ⅰ类分子、MHCⅡ类分子和共刺激分子(CD86,CD80);原代肾癌细胞高表达一种高分子量的糖蛋白(MUC-1);自体DC与肾癌融合细胞疫苗同时表达肾癌细胞和DC细胞的表面抗原,并能有效刺激自体T细胞增殖,诱导产生的CTLs对自体肾癌细胞具有显著的杀伤活性。结论自体DC与肾癌融合细胞疫苗能诱导特异性抗自体肾癌细胞的免疫应答,为自体DC与肾癌融合细胞疫苗在治疗肾癌的临床应用中提供了实验依据。     

Relationship between expression of drug-resistance factors and drug sensitivity in normal human renal proximal tubular epithelial cells in comparison with renal cell carcinoma

The relationship between the expression level of putative drug resistance factors and sensitivity to anticancer drugs in human normal renal proximal tubule epithelial cells (RPTEC) and 3 kinds of renal cell carcinoma (RCC) cells, VMRC-RCW (RCW), OS-RC-2 (OS2), TUHR14TKB (14TKB), was examined. RPTEC exhibited high expression of P-glycoprotein (Pgp), gamma-glutamyl cysteine synthetase (gammaGCS) and cis-diamminedichloroplatinum (II) (CDDP) resistance-related gene 9 (CRR9), low expression of vacuolar ATPase (V-ATPase) and no expression of multidrug resistance-associated protein 1 (MRP1). 14TKB exhibited high expression of gammaGCS and CRR9, low expression of Pgp and V-ATPase, and no expression of MRP1. OS2 showed high expression of CRR9, low expression of Pgp, gammaGCS and MRP1, and no expression of V-ATPase. RCW exhibited high expression of Pgp, MRP1 and CRR9 and low expression of gammaGCS and V-ATPase. The level of expression of the resistance factors varied among the cells. GST activity and GST-pi expression level of each cell were correlated, and there were high levels in OS2 and RPTEC. When the cytotoxicity of anticancer drugs against each cell was measured at 96 h, the sensitivity to CDDP and Doxorubicin (DXR) in RPTEC and RCW was lower than that in the other cells. Sensitivity to DXR was enhanced by treatment with the Pgp inhibitor, Verapamil, in proportion to the Pgp expression level, and the sensitivity to CDDP was increased by the gammaGCS inhibitor, Buthionine sulfoximine, in proportion to the gammaGCS expression level (corresponding to GSH content). Although a significant increase in sensitivity to CDDP was not observed by treatment of RCC with the V-ATPase inhibitor, Bafilomycin, the sensitivity to DXR in Bafilomycin-treated cells increased about 2-fold. However, no relation between drug sensitivity and V-ATPase expression was observed. The features (such as degree of resistance) varied among the RCC cell lines manifesting many resistance factors or to the contrary, lacking or having lowered resistance factors in comparison with normal cells. Therefore, it is necessary in clinical cancer chemotherapy to determine and measure the level of expression of each resistance factor in respective tumor tissue.     

Blockade of the vascular endothelial growth factor-receptor 2 pathway inhibits the growth of human renal cell carcinoma, RBM1-IT4, in the kidney but not in the bone of nude mice

Primary and metastatic RCCs are consistently resistant to radiotherapy, chemotherapy, or immunotherapy. As recurrent or metastatic RCC after surgery is related with poor prognosis and cancer-related death, development of therapeutic modalities that can control RCC and improve patient survival is urgently needed. We determined whether blockade of the vascular endothelial growth factor-receptor 2 (VEGF-R2) signaling pathway inhibits the growth of human renal cell carcinoma cells in the kidney and bone of nude mice. Male nude mice implanted with 1x10(6) RBM1-IT4 cells in the kidney or in the tibia were treated with oral administrations of TSU-68, anti-VEGF-R2 tyrosine kinase inhibitor beginning 5 days after implantation. The tumor incidence, tumor weight and bone destruction were determined at twelve weeks after commencing the therapy. VEGF production by RCCs was determined by ELISA and alterations in VEGF production related with genetic instability were also analysed. VEGF-R expression of mouse osteoclast precursors (mOCPs) and human umbilical vascular endothelial cell (HUVEC) was determined by RT-PCR and Western immunoblotting. in vitro, the effects of TSU-68 on the cellular proliferation of HUVEC, normal human renal proximal tubule epithelial cell (RPTEC) and mOCPs were determined. RBM1-IT4 cells had loss of heterozygosity and frame shift mutation on chromosome 3p, inactivating the von Hippel-Lindau (VHL) tumor suppressor gene and resulting in the production of relatively higher levels of VEGF than the RCCs without VHL mutation. TSU-68 significantly inhibited the growth of RBM1-IT4 in the kidney (p<0.05). In contrast, TSU-68 did not inhibit the growth of RBM1-IT4 in the tibia or bone lysis. Although HUVEC, RPTEC and mOCPs expressed VEGF-R2, TSU-68 directly inhibited the VEGF-stimulated cell growth of HUVEC and RPTEC but not the mOCPs in vitro. These data indicate that the VEGF-VEGF-R2 pathway is not required for survival of the osteoclasts and anti-VEGF-R2 therapy did not contribute to the suppression of metastatic RCC growth in the bone.     

CG0070, a conditionally replicating granulocyte macrophage colony-stimulating factor--armed oncolytic adenovirus for the treatment of bladder cancer.

PURPOSE: The purpose of this study was to examine the tumor specificity, cytotoxicity, and granulocyte macrophage colony-stimulating factor expression of CG0070, a conditionally replicating oncolytic adenovirus, in human bladder transitional cell carcinoma (TCC) cell lines and determine its antitumor efficacy in bladder TCC tumor models. EXPERIMENTAL DESIGN: Virus yield and cytotoxicity assays were used to determine tumor specificity and virus replication-mediated cytotoxicity of CG0070 in a panel of human bladder TCC cell lines and primary cells in vitro. Two s.c. and one orthotopic bladder TCC xenograft tumor models were used to assess antitumor activity of CG0070. RESULTS: In a matched isogenic pair of cell lines with differing retinoblastoma (Rb) pathway status, CG0070 showed selective E1a and granulocyte macrophage colony-stimulating factor (GM-CSF) expression in Rb pathway-defective cells. CG0070 replicated in Rb-defective bladder TCC cell lines as efficiently as wild-type adenovirus but produced 100-fold less virus in normal human cells. CG0070 was up to 1,000-fold more cytotoxic in Rb pathway-defective bladder TCC cells in comparison with normal human cells. Antitumor activity of CG0070 was shown in two bladder TCC s.c. xenograft tumor models following intratumoral injections and intravesical treatment in an orthotopic xenograft tumor model when compared with PBS treatment. CONCLUSIONS: In vitro and in vivo studies showed the selective replication, cytotoxicity, GM-CSF production, and antitumor efficacy of CG0070 in several bladder TCC models, suggesting a potential utility of this oncolytic agent for the treatment of bladder cancer. Further studies are warranted to show the role of human GM-CSF in the antitumor efficacy of CG0070.     

Enhancement of arsenic trioxide-induced apoptosis in renal cell carcinoma cells by L-buthionine sulfoximine

Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemic patients and apoptosis in various tumor cells in vitro. To develop As2O3-based combination chemotherapy for renal cell carcinoma (RCC), we investigated the cytotoxic effects of As2O3 in combination with chemotherapeutic agents or L-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor. Cytotoxicity and synergy were assessed by the MTT assay and isobolographic analysis, respectively. Apoptosis was monitored by Hoechst 33342 staining, flow cytometrical analysis, and DNA fragmentation assay. Treatment of ACHN cells with As2O3 in combination with adriamycin, vinblastine, or 5-fluorouracil induced an antagonistic effect. However, combination treatment with As2O3 and BSO resulted in a synergistic cytotoxic effect. Synergy was also obtained in Caki-1, Caki-2, NC65 cells and freshly derived RCC cells from 6 patients. Simultaneous treatment of ACHN cells with As2O3 and BSO caused significantly more cytotoxicity than the As2O3 first BSO second or the reverse treatment. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of As2O3 and BSO was realized by inducing apoptosis. This combination markedly decreased intracellular GSH content and GSH-S-transferase (GST) activity. However, neither the intracellular GSH nor GST was decreased by As2O3 with adriamycin, vinblastine, or 5-fluorouracil. Furthermore, the GSH-increasing agents N-acetylcysteine and lipoic acid significantly inhibited the combined cytotoxicity of As2O3 and BSO. These findings indicate that BSO sensitizes RCC cells to As2O3-induced apoptosis through the down-regulation of the intracellular GSH redox system, suggesting the potential application of a combination of As2O3 and BSO for the treatment of RCC.     

Potent tumoricidal effects of a human cytotoxic T-cell line (TALL-104) against prostate cancer

The human TALL-104 cell line possesses major histocompatibility complex non-restricted cytotoxic activity against a large variety of tumor targets. Adequate therapies for prostate cancer that has spread outside its capsule are lacking. In order to identify effective therapies for this problem, we investigated the antiproliferative effects of TALL-104 cells against three prostate cancer cell lines (LNCaP, PC-3, DU-145). A Cr-51-release: cytotoxicity assay showed that TALL-104 cells were very cytotoxic against the prostate cancer cells. For example, at a 1:1 ratio of TALL-104 cells to prostate cancer cells, the percent release of Cr-51 at 18 h were 50, 40, and 45% for LNCaP, PC-3, and DU-145, respectively. Analysis by inhibition of clonogenic growth of prostate cancer cells also showed that TALL-104 cells were extremely effective. For instance, a short-term (4 h or 18 h) pre-incubation of TALL-104 cells with these tumor cells at the effector to target ratio of 10:1 prior to clonogenic assay resulted in a substantial reduction in clonogenic tumor growth (90%, 65%, and 50% clonal growth inhibition for LNCaP, PC-3, and DU-145, respectively). Further experiments using both Cr-51 release and clonogenic assays showed that irradiated TALL-104 cells were also effective in their anti-prostatic cancer activities. We also examined if TALL-104 cells plus a chemotherapeutic agent might complement each other in their cytotoxic effects. Preincubation of prostate cancer cell targets with etoposide (0.2-20 mu g/ml) for 18 h markedly increased their susceptibility to TALL-104 lysis. The anti-tumor efficacy of TALL-104 cells was also demonstrated in vivo utilizing the BNX murine model engrafted with subcutaneous PC-3 prostate cancer cells. A substantial reduction in PC-3 tumor cell progression was observed in mice injected with irradiated TALL-104 cells (1x10(7) cells intraperitoneally or intratumorally for 5 days beginning on days 24 and 45 after implantation) as compared to mice injected with tumors only. Taken together, these findings suggest that TALL-104 cells may be utilized as a potent anti-tumor agent, either alone or in combination with other agents (such as etoposide) in metastatic prostate cancer.     

Inhibition of murine prostate tumor growth and activation of immunoregulatory cells with recombinant canarypox viruses.

BACKGROUND: Immunization with modified tumor cells carrying recombinant immunomodulatory genes is being explored as cancer immunotherapy. In this study, we examine whether canarypox ALVAC viruses carrying immunostimulatory cytokine genes (granulocyte-macrophage colony-stimulating factor, interleukin 2, interleukin 12, and tumor necrosis factor-alpha) can induce antitumor immunity (to rechallenge) in the RM-1 model of a highly aggressive, weakly immunogenic murine prostate cancer. METHODS: For antitumor activity studies, RM-1 murine prostate cancer cells were infected with the parental ALVAC virus or one or two recombinant ALVAC-cytokine viruses and then injected into male C57BL/6 mice. For rechallenge studies, other mice were first given an injection subcutaneously with irradiated (nonproliferating) recombinant ALVAC-infected RM-1 cells and then (10 days later) with untreated RM-1 cells. For the determination of which immune cells were required for antitumor activity, mice were immunodepleted of CD4, CD8, or natural killer (NK) NK1.1 cells with the corresponding monoclonal antibodies and were then given an injection of ALVAC-cytokine-infected RM-1 cells. For all experiments, tumor outgrowth and animal survival were monitored. RESULTS: After subcutaneous injection into mice, RM-1 cells infected with one (except ALVAC-interleukin 2) or two ALVAC-cytokine recombinants had statistically significantly greater antitumor activity than RM-1 cells infected with parental ALVAC (P<.001 for all; two-sided test). The antitumor activity of RM-1 cells infected with any two ALVAC-cytokine recombinants was greater than, but not statistically significantly different from, that of RM-1 cells infected with any one ALVAC-cytokine recombinant. NK1.1 cells were necessary for antitumor activity, but tumor-specific CD4(+) regulatory T cells were also induced that inhibited CD8(+) RM-1-specific cytotoxic T cells, resulting in the lack of immunity to a rechallenge by RM-1 cells. DISCUSSION: Canarypox viruses can transfer immunostimulatory cytokine genes into RM-1 prostate cancer cells. When such cells were injected into mice, the cytokines induced an antitumor response against this highly aggressive, weakly immunogenic tumor. This response, however, did not protect the mouse against a rechallenge with RM-1 cells because suppressor CD4(+) T cells were induced that inhibited tumor-specific CD8(+) cytotoxic T cells.     

Resistance to lysis by cytotoxic T cells: a dominant effect in metastatic mouse prostate cancer cells

Better understanding of the immunology of prostate cancer is needed for the development of new therapeutic approaches that can be used in conjunction with current treatment methods. The present study was designed to compare the immunological properties of a genetically matched pair of primary tumor- and metastasis-derived prostate cancer cell lines generated from the mouse prostate reconstitution (MPR) model. Only the primary prostate cancer cells were immunogenic in that prior immunization with irradiated primary but not the metastatic prostate cancer cells delayed the growth of subsequently injected live cancer cells. The lack of immunogenicity of the metastatic cells was not attributable to their inability to induce antitumor cytotoxic T cells. Both primary and metastatic cells induced antitumor CTLs in syngeneic hosts, but unlike the primary cells, the metastatic cells were resistant to CTL lysis. Differential resistance to cytolysis in metastatic versus primary prostate cancer cells was not attributable to the differential expression of molecules such as transporter associated with antigen processing (TAP)-1, TAP-2, low molecular weight protein of the proteasome complex (LMP)-2, and LMP-7 that contribute to antigen presentation by class I MHC. IFN-gamma induced surface class I MHC expression, as well as gene expression of TAP-1, TAP-2, LMP-2, and LMP-7 in the metastatic cells, yet the cells remained resistant to cell lysis induced by CTLs. Interestingly, although in comparison to the primary cells the metastatic cells were resistant to cytolysis, both cell types were susceptible to DNA fragmentation induced by CTLs. Cell fusion between primary and metastatic cancer cells resulted in hybrids that also resisted the cytolytic activity of CTLs. Therefore, there is a dominant factor(s) in the metastatic prostate cancer cells that confers specific protection against CTL cytolysis in this model system.     

Prostate stem cell antigen is overexpressed in human transitional cell carcinoma.

Prostate stem cell antigen (PSCA), a homologue of the Ly-6/Thy-1 family of cell surface antigens, is expressed by a majority of human prostate cancers and is a promising target for prostate cancer immunotherapy. In addition to its expression in normal and malignant prostate, we recently reported that PSCA is expressed at low levels in the transitional epithelium of normal bladder. In the present study, we compared the expression of PSCA in normal and malignant urothelial tissues to assess its potential as an immunotherapeutic target in transitional cell carcinoma (TCC). Immunohistochemical analysis of PSCA protein expression was performed on tissue sections from 32 normal bladder specimens, as well as 11 cases of low-grade transitional cell dysplasia, 21 cases of carcinoma in situ (CIS), 38 superficial transitional cell tumors (STCC, stages T(a)-T(1)), 65 muscle-invasive TCCs (ITCCs, stages T(2)-T(4)), and 7 bladder cancer metastases. The level of PSCA protein expression was scored semiquantitatively by assessing both the intensity and frequency (i.e., percentage of positive tumor cells) of staining. We also examined PSCA mRNA expression in a representative sample of normal and malignant human transitional cell tissues. In normal bladder, PSCA immunostaining was weak and confined almost exclusively to the superficial umbrella cell layer. Staining in CIS and STCC was more intense and uniform than that seen in normal bladder epithelium (P < 0.001), with staining detected in 21 (100%) of 21 cases of CIS and 37 (97%) of 38 superficial tumors. PSCA protein was also detected in 42 (65%) of 65 of muscle-invasive and 4 (57%) of 7 metastatic cancers, with the highest levels of PSCA expression (i.e., moderate-strong staining in >50% of tumor cells) seen in 32% of invasive and 43% of metastatic samples. Higher levels of PSCA expression correlated with increasing tumor grade for both STCCs and ITCCs (P < 0.001). Northern blot analysis confirmed the immunohistochemical data, showing a dramatic increase in PSCA mRNA expression in two of five muscle-invasive transitional cell tumors when compared with normal samples. Confocal microscopy demonstrated that PSCA expression in TCC is confined to the cell surface. These data demonstrate that PSCA is overexpressed in a majority of human TCCs, particularly CIS and superficial tumors, and may be a useful target for bladder cancer diagnosis and therapy.     

Phase 2 trial of talactoferrin in previously treated patients with metastatic renal cell carcinoma

BACKGROUND: Talactoferrin (TLF), a recombinant form of human lactoferrin (hLF), is an immunomodulatory iron-binding glycoprotein first identified in breast milk. Its immunomodulatory functions include activation of natural killer (NK) and lymphokine-activated killer cells and enhancement of polymorphonuclear cells and macrophage cytotoxicity. Studies in animal models have shown promising anticancer activity, and clinical antitumor activity has been observed in nonsmall cell lung cancer and other tumor types. The purpose of the current study was to evaluate the activity and safety of TLF in patients with refractory metastatic renal cell carcinoma (RCC). METHODS: Forty-four adult patients with progressive advanced or metastatic RCC who had failed prior systemic therapy received oral talactoferrin at a dose of 1.5 g twice daily on a 12-week-on 2-week-off schedule. Patients were evaluated for progression-free survival at 14 weeks, overall response rate, and progression-free and overall survival. RESULTS: TLF was well tolerated. No significant hematologic, hepatic, or renal toxicities were reported. The study met its predefined target with a 14-week progression-free survival rate of 59%. The response rate was 4.5%. The mMedian progression-free survival was 6.4 months and the median overall survival was 21.1 months. CONCLUSIONS: TLF is a well-tolerated new agent that has demonstrated preliminary signs of clinical activity. Given the lack of toxicity, the lack of rapid disease progression in this cohort, and the preclinical data on immune activation, a randomized study assessing its effects on disease progression in patients with metastatic RCC is rational.     

What are the immunologically active components of bacille Calmette-Guérin in therapy of superficial bladder cancer?

The subcomponents of bacille Calmette-Guérin (BCG) involved in the mechanism of action of intravesical BCG immunotherapy used for prophylaxis of superficial bladder cancer recurrences have been poorly investigated. We purified various BCG subcomponents and analyzed in vitro their ability to enhance a Th1 polarized immune response as well as to increase lymphocyte-mediated cytotoxicity against bladder tumors. Human peripheral blood mononuclear cells (PBMCs) from healthy purified protein derivative-positive subjects were incubated for 7 days with whole BCG and various fractions (BCG cell wall, plasma membrane, cytosol, purified polysaccharides as glucan or arabinomannan, purified native proteins from BCG culture filtrate, recombinant 22 kDa protein, phosphate transporter PstS-2 and -3 proteins). IFN-gamma, IL-12, IL-2, and IL-6 production by stimulated PBMCs was compared to unstimulated controls and the phenotype of expanded cells analyzed by flow cytometry (FACS analysis). A (51)Cr-release assay monitored the cytotoxicity of amplified effector cells against T24 bladder tumor cells. Live BCG and most of its subcomponents (with the exception of cytosol, PstS-2 and -3) significantly enhanced IFN-gamma and IL-12 secretion, expanded CD3(-)CD56(+) cells and the non-MHC-restricted cytotoxicity against bladder tumor cells compared to unstimulated controls (all P < 0.001, t-test). IL-2 receptor blockage resulted in a clear reduction in the cytotoxic activity of stimulated PBMCs. Numerous BCG subcomponents thus provide positive stimuli for Th1 cell differentiation and enhance in vitro, non-MHC-restricted cytotoxicity against bladder tumor cells. Our findings provide the basis for the therapeutic use of several of these subfractions in experimental animal models bearing bladder tumors.     

Connexin 32 potentiates vinblastine-induced cytotoxicity in renal cell carcinoma cells

We have reported that connexin (Cx) 32 gene, a member of gap junction protein family, acts as a tumor suppressor gene in human renal cell carcinoma (RCC). Of solid tumors, RCC is one of the most chemoresistant cancers, and there is no effective cancer chemotherapy against RCC at present. In this study, we examined if the combination of Cx32-dependent tumor-suppressive effect and vinblastine (VBL), a chemotherapeutic agent which has been utilized for clinical RCC treatment, could be effective in enhancing the sensitivity of RCC to VBL treatment. Cx32 expression in a human metastatic RCC cell (Caki-1 cell) significantly enhanced in vitro and in vivo VBL-induced cytotoxicity on the cell. Cx32 expression in the RCC cells potentiated VBL-induced apoptosis compared to the Cx32-negative RCC cells in vitro as well as in vivo. The enhancing apoptosis in the RCC cells by Cx32 mainly depended on the decrease of P-glycoprotein (P-gp), a multidrug resistance gene-1 (MDR-1) product responsible for reduction of VBL accumulation into the cells. We also observed that silencing of Cx32 by short interfering RNA (siRNA) treatment elevated the level of P-gp in Caki-1 cells and that inhibition of P-gp function enhanced VBL-induced apoptosis in the RCC cells. These results suggest that Cx32 is effective to enhance VBL-induced cytotoxicity in Caki-1 cells via the reduction of P-gp. Overall, it seems that the combination of Cx32-dependent tumor-suppressive effect and VBL is promising as a new cancer therapy against RCC.     

Efficacy of large doses of IL-2-activated human leukocyte antigen haploidentical peripheral blood stem cells on refractory metastatic renal cell carcinoma

Traditional immunotherapy for patients with refractory metastatic renal cell carcinoma (RCC) is limited because the tumors themselves induce immunosuppression. The aim of this article was to evaluate the clinical efficacy of the infusion of a high dose of interleukin (IL)-2-activated allogeneic haploidentical peripheral blood stem cells (haplo-PBSCs) in patients with advanced intractable RCC. Ten advanced RCC patients and their haploidentical relatives, who were haplo-PBSC donors, were enrolled in this study. All patients accepted one cycle of activated haplo-PBSCs. The clinical and immunologic responses were evaluated. A range from 2.3 to 5.5×10(10) of activated haplo-PBSCs were harvested after exposure to recombinant human IL-2 (rhIL-2), along with a significant increase in the proportion of natural killer cells and activated lymphocytes (CD69+ and CD25+). Enhanced cytotoxicity of haplo-PBSCs for RCC was also observed. After treatment, 2 (2/10) cases of partial remission, 6 (6/10) cases of stable disease, and 2 (2/10) cases of progressive disease were identified in these 10 patients. The median progression-free survival of the 10 patients was 5.5 months (3-14 months). The adoptive transfusion of IL-2-activated haplo-PBSCs can induce sustained antitumor effects for advanced intractable RCC patients who have had no response to conventional immunotherapy.     

Antitumor effect of the tumor necrosis factor against various types of human cancer cells

The antitumor activity of tumor necrosis factor (TNA) against various human cancer cells (32 cases) was investigated by 51Cr cytotoxic release assay and tumor stem cell assay. Over 50% sensitivity (the ratio of cytotoxicity for L929 cells) was shown by 4 of 14 cases of gastric cancer (28.6%), 7 of 9 cases of leukemic cells (77.8%), and 1 case each of pancreatic carcinoma and ovarian cancer. However, scarcely any sensitivity was shown by APL, a portion of the gastric cancer cells, normal lymphocytes or colony-forming cells tested. No correspondence was observed between the histological type of the cancer and TNF sensitivity. The above results seem to confirm the significant antitumor activity of TNF against human cancer cells.     

Antitumor therapeutic potential of activated human umbilical cord blood cells against leukemia and breast cancer.

In this study, in vitro and in vivo antitumor effects of mononuclear cells from human umbilical cord blood cells (UCBCs) and peripheral blood stem cells (PBCs) harvest obtained by leukapheresis were compared. Interleukin 2 (IL-2)-activated mononuclear cells from UCBCs showed increased cytotoxicity against K562 and Raji hematopoietic malignant cells compared with PBCs (P < 0.05). After IL-2 activation, both UCBCs and PBCs showed significant cytotoxicity against MDA-231 human breast cancer cells. The UCBC population involved in this antitumor activity appeared to be CD56+ natural killer precursors. The cytotoxicity of UCBCs was inhibited in the absence of Ca2+ (P < 0.05), supporting a perforin/granzyme-mediated target of cell lysis. In addition, antibodies to Fas ligand blocked cytotoxic activity, suggesting that some of the antitumor cytotoxicity was Fas ligand mediated. In vivo antitumor effects of UCBCs and PBCs were studied using a human leukemic cell-bearing severe combined immunodeficient mouse model. There was a significant increase in the survival of K562 leukemia-bearing mice that also received 5 million in vitro IL-2-activated UCBCs or PBCs i.v. on days 3 and day 5 after tumor transplantation compared with untreated mice (P < 0.01). Similar antitumor cytotoxicity of UCBCs and PBCs was also observed against MDA-231 human breast cancer grown in severe combined immunodeficient mice (P < 0.01). These studies suggest that IL-2-activated UCBCs may be a useful source of cellular therapy for patients with hematological malignancies and breast cancer.