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ABSTRACT: In total, 16 patients with cytologically proven malignant effusion from colorectal cancer were treated by locoregional administration of the streptococcal preparation OK-432 alone or OK-432 plus the T-cell growth factor interleukin (IL)-2, and the action mechanism of the treatment was studied. A positive clinical response, showing a cytologic disappearance of cancer cells and decrease of effusion, was observed in nine of 11 (82%) patients treated with OK-432 alone and in all five patients treated with OK-432 plus IL-2. Flow cytometric analysis revealed that OK-432 plus IL-2 locally induced acute inflammation-like responses, including serial cellular infiltrations of granulocyte migration within a matter of hours, and activation of macrophages and T lymphocyte involvement within the following days, and that a predominant expansion of CD3+CD4+ lymphocytes (CD: cluster of differentiation) was induced by in vitro stimulation with IL-2 of locoregional cells after the OK-432 administration (OK/IL-2AK cells). The OK/IL-2AK cells produced tumour necrosis factor-alpha and interferon-gamma, but these cells did not produce IL-4 and IL-6. The OK/IL-2AK cells expressed potent killing activity against autologous tumour cells. This activity was abrogated by treatment of the lymphocytes with anti-CD3, -CD4, -TCRalphabeta antibody, and by the treatment of target cells with anti-human leukocyte antigen (HLA)-DR antibody. The OK/IL-2AK cells expressed Fas-L gene, and flow cytometric analysis demonstrated HLA-DR expression in approximately 75% of CEA+ or cytokeratin+ effusion cells. TCRVbeta gene analysis of the OK/IL-2AK cells showed an oligoclonal usage of TCRbeta20, which was also involved in the cytotoxic mechanism of the OK/IL-2AK cells. Single-strand conformational polymorphism analysis demonstrated the clonotypes for the TCRVbeta20 gene, and the CDR3s of the gene were sequenced. The clonotypic PCR using the TCRVbeta20-CDR3 sequences could detect the CDR3-identical TCRs in effusion lymphocytes from the other patients. Taken together, it is suggested that locoregional administration of OK-432 plus IL-2 is highly effective for the management of malignant effusion from colorectal cancer. OK-432 plus IL-2 induces autologous tumour-reactive CD4+ Th1 killer lymphocytes, which recognise tumour antigen(s) presented with HLA class II molecules on effusion tumour cells by means of preferential usage of TCRVbeta20. The clonotypic PCR using the TCRVbeta20-CDR3 sequences may be informative for treating malignant effusion from colorectal cancer using OK-432 plus IL-2.
British Journal of Cancer 12/2003; 89(10):1876-84. · 5.04 Impact Factor
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ABSTRACT: Proapototic gene is an important target to enhance the chemotherapeutic effect in cancer cells. Based on in vitro study showing that the introduction of bax gene enhanced the sensitivity to anticancer drugs, we examined whether the intratumoral administration of bax gene could enhance the anti-tumor effect in combination with anticancer drugs in gastric cancer. The human gastric cancer cell line, MKN45 was transplanted into nude mice, and the intratumoral administration of bax gene was performed using the bax cDNA plasmid complexed with a cationic lipopolyamine. The enhancement of antitumor effect was examined in the combination with 5-fluorouracil (5-FU) and cisplatin (CDDP). The anticancer drugs were administered intraperitoneally with one third of LD50 four times at 4-day intervals, and the antitumor effect was assessed by the NCI protocol. The expression of bax gene was analyzed by RT-PCR and the apoptotic cell death was assessed by TUNEL method. The intratumoral administration of bax gene alone showed slight anti-tumor effect as compared to that of control tumor injected with vector alone. The antitumor effect of 5-FU and CDDP was significantly enhanced in the combination with intra-tumoral administration of bax gene as compared to that of CDDP and 5-FU alone (p<0.05, Student's t-test). The enhancement of antitumor effect was associated with the constitutive overexpression of bax gene and with the induction of apoptosis in the tumor treated with anticancer drug and bax gene. These results indicate that the combination therapy of intratumoral administration of bax gene complexed with a cationic lipopolyamine and anticancer drugs may provide us a new strategy for cancer chemotherapy to enhance its therapeutic efficacy in gastric cancer as termed with gene-chemotherapy.
International Journal of Oncology 02/2001; 18(2):363-7. · 2.40 Impact Factor
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ABSTRACT: Locoregional administration using OK-432 was evaluated in treating malignant effusion. Positive clinical responses were seen in 19 (52%) of 36 gastric cancer patients, and in 9 (90%) of 10 colon cancer patients (p < 0.05), indicating its clinical benefit in treating malignant effusion of colon cancer. Fever elevation was observed in 43 (93%) patients and local pain occurred with 9 (20%) of 46 administrations. Immunological analysis for responder patients with rectal cancer revealed that OK-432 induced autologous tumor-reactive CD 3+ CD 4+ TCRV beta 20+ killer lymphocytes. The TCR gene analysis permitted us to clone a V beta 20 CDR 3 sequence, by which positive bands were shown in 3 (75%) of 4 responders and negative bands in 3 (100%) of 3 non-responders. It is suggested that cross-antigenicity exists between OK-432 and colon cancer, and that genetic analysis using the TCRCDR 3 sequence makes it possible to predict responder patients to OK-432 immunotherapy.
Gan to kagaku ryoho. Cancer & chemotherapy 10/2000; 27(12):1870-3.
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ABSTRACT: We studied the thymidine kinase (TK) gene and the interleukin (IL)-2 gene co-transduction into tumor cells for a possible strategy of cancer gene therapy. A murine ovarian cancer cell line, OVHM, was retrovirally transduced with the TK (OVHM/TK) or the IL-2 gene (OVHM/IL-2). The TK or IL-2 expression was permanent in OVHM/TK or OVHM/IL-2. OVHM/TK cells were susceptible to gancyclovir (GCV) in vitro, and their intraperitoneal growth was completely regulated with GCV administration. The bystander effect was not observed in vitro and in vivo in this model, and only the marginal emergence of immune involvement was observed in the OVHM/TK-cured mice with GCV. OVHM/IL-2 cells produced IL-2 biologically active to be immunogenic, but still tumorigenic to kill the mice when inoculated intraperitoneally. Then, OVHM/TK cells were co-transduced with the IL-2 gene to establish OVHM/TK/IL-2 cells. OVHM/TK/IL-2 cells were also susceptible to GCV and transiently produced active IL-2. A significant resistance against the challenge of parental tumor cells was observed in the mice that were inoculated with OVHM/TK/IL-2 cells and cured with GCV administration. It is suggested that tumor cells transduced with both TK and IL-2 genes could be regressed with GCV administration with subsequent generation of immune activation in the host. Since the bystander effect may not always be a common phenomenon in gene therapy using the TK gene, this type of combination may be advantageous in the clinical application of gene therapy for human cancers.
International Journal of Molecular Medicine 09/2000; 6(2):185-90. · 1.98 Impact Factor
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ABSTRACT: The possible use of percutaneous transhepatic low output microwave tissue coagulation therapy (PMCT) using ultra-sonography under local anesthesia for solitary liver cancer was studied. The subjects were 13 patients having primary or metastatic liver cancer with solitary liver tumor less than 3 cm in diameter, including 7 hepatocellular carcinomas and 6 metastatic liver cancers. PMCT was performed continuously 3 times at an output of 30 watts for 30 seconds at a time. Tumors less than 3 cm in diameter were completely coagulated by irradiation from 2 to 6 times, judging by enhanced CT. No tumor recurrence was recognized in the coagulation area. However, in two cases of metastasis from pancreatic carcinoma, multiple metastases were found at another site in the liver by 2 months after PMCT. Thus, the results suggest that PMCT is a useful therapy for small liver tumor as a local control.
Gan to kagaku ryoho. Cancer & chemotherapy 08/1998; 25(9):1366-9.
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ABSTRACT: The possible use of percutaneous transhepatic microwave tissue coagulation therapy (PMCT) using ultra-sonography under local anesthesia for solitary liver cancer was studied. The subjects were 8 patients having primary or metastatic liver cancer with solitary liver tumor less than 4 cm in size, consisting of 2 hepatocellular carcinomas, and 6 metastatic carcinomas. PMCT was performed continuously 3 times at the output of 30 watts for 30 seconds at a time. Tumors less than 3 cm in size were completely coagulated by irradiation from 2 to 5 times judged by enhanced CT. No recurrence of tumor was recognized in the coagulation area. But in some cases, multiple metastases were found at another site in the liver by 3 months after PMCT. Thus, the results suggest that PMCT is a useful therapy for small liver tumor as a local control.
Gan to kagaku ryoho. Cancer & chemotherapy 10/1997; 24(12):1643-6.
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ABSTRACT: To determine the significance of interleukin (IL)-10 in antitumor immune response, the effect of the down-regulation of tumor-derived IL-10 on locoregional immunotherapy was investigated. C3H/HeN mice were intraperitoneally (i.p.) inoculated with IL-10-producing murine breast cancer cell line, FM3A, and treated with locoregional administration of OK-432 with or without anti-IL-10 monoclonal antibody (mAb). Anti-IL-10 mAb did not affect the in vitro growth of FM3A cells. Administration of OK-432 plus anti-IL-10 mAb remarkably delayed the retention of malignant ascites and prolonged the survival of mice compared with the administration of OK-432 alone. Spleen cells which were collected from mice treated with OK-432 plus anti-IL-10 mAb and further stimulated in vitro with inactivated FM3A cells exhibited significantly higher cytotoxicity against FM3A cells than those from mice treated with OK-432 alone or from the control mice. The expression of major histocompatibility complex (MHC) class II molecules on spleen cells was up-regulated in vitro by the addition of OK-432 and anti-IL-10 mAb. Using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), cytokine mRNA levels of peritoneal exudate cells (PEC) and spleen cells were assessed on day 7 (before treatment) and day 14 (after treatment). In PEC, increased expression of IL-2 was observed with the administration of OK-432 plus anti-IL-10 mAb. In spleen cells, the expression of IL-2, IL-12 and IFN-gamma were strongly induced, and IL-4 expression was reduced by the administration of OK-432 plus anti-IL-10 mAb. It is suggested that down-regulation of tumor-derived IL-10 induces the up-regulation of the T helper type (Th) 1 population, resulting in an enhancement of the efficacy of locoregional immunotherapy with OK-432.
Anticancer research 19(2A):1077-84. · 1.73 Impact Factor
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ABSTRACT: Nine T-cell clones have been established from tumor-infiltrating lymphocytes (TIL) isolated from ascitic fluid of a gastric cancer patient. Five of them retained cytotoxicity against autologous tumor cells (AuTu), and were all CD4+. Each clone had different usage of T-cell receptor (TCR) V beta gene as assessed by Southern blot analysis. Using AuTu and two allogeneic gastric cancer cell lines as targets, we selected three clones with unique cytotoxic properties. Two of these clones (Clone 1 and 2) preferentially lysed AuTu, but showed no or marginal cytotoxicity against allogeneic gastric cancer cells, and one clone (Clone 7) showed appreciable cytotoxicity against AuTu and allogeneic gastric cancer cells. In the detailed analysis of TCRV beta gene usage, Clone 1, 2, and 7 expressed V beta 13.1/D beta 1/J beta 1.5/C beta 1, V beta 3/D beta 2/J beta 2.4/C beta 2, and V beta 9/D beta 1/J beta 1.4/C beta 1, respectively, and the primary structures of the three TCRVb genes did not share any common features, neither in the sizes of their complementarity determining region 3 (CDR3) nor in their amino acid compositions. Interestingly, PBL of the same patient expressed CDR3 identical to that of Clone 2 and 7, but not that of Clone 1. CDR3 identical to that of Clone 2 and 7 were also detected in TIL of other gastric cancer patients. These results show that some AuTu-specific CTL included in TIL are circulating in peripheral blood, and that the CDR3 identical to that of the CTL is expressed extensively in TIL among different gastric cancer patients. Screening of the expression of the CDR3 in other gastric cancer patients is recommended to develop an immuno-therapy of gastric cancer based on antigenic peptide.
Anticancer research 19(3A):2057-66. · 1.73 Impact Factor
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ABSTRACT: The in vitro generation of effector lymphocytes cytotoxic to cancer cells, was investigated with a mixed lymphocyte-tumor culture (MLTC) system using genetically modified human cancer cells, followed by stimulation with the interleukin (IL)-2 plus immobilized anti-CD3 antibody (IL-2/CD3) system. A gastric cancer cell line, GC022588 (HLA-A2, 24, B35, 55, C1,3), was retrovirally transduced with the human interleukin (IL)-2 gene (GC/IL-2) or the neomycin-resistance gene (GC/Neo). The secretion of biologically active IL-2 was detectable in GC/IL-2 cells but not in GC/Neo or parental GC022588 cells. The cytotoxic activity against the parental GC022588 cells of peripheral blood mononuclear cells (PBMC) was greater among PBMC activated with MLTC using GC/IL-2 than among those activated with MLTC using GC/Neo or without MLTC. The IL-2/CD3 stimulation could efficiently expand the effector lymphocytes without any reduction of the cytotoxic activity generated. The cytotoxic activity generated by this system was reproducible in several HLA-A2- or A24-positive donors. The effector lymphocytes could kill the other adenocarcinoma cells expressing HLA-A2 or A24. The phenotypes of the effector lymphocytes generated with the system were 40% CD4+ and 70% CD8+. Both phenotypes may have been responsible for the cytotoxicity. The removal of adherent cells from PBMC before the MLTC did not affect the generation of cytotoxicity, whereas neutralization of tumor-derived IL-2 with a specific antibody during the MLTC significantly inhibited the generation of cytotoxicity. These results suggest that IL-2 gene-transduction augments the immunogenicity of the tumor cells that efficiently stimulate lymphocytes to be cytotoxic, and that the IL-2/CD3 system may be practical for the expansion of effector lymphocytes for use in adoptive immunotherapy for cancer. The mechanism by which IL-2 gene-modified tumor cells stimulate immune reactivity was discussed.
Anticancer research 21(1B):669-77. · 1.73 Impact Factor
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ABSTRACT: There exists cancer-associated immunosuppression, and the generation of lymphokine-activated killer (LAK) cells is impaired in patients with advanced cancer. Telomerase has been reported to be upregulated in the activation of lymphocytes to proliferate against immune stimulation as well as in the malignant transformation of immortal cancer cells. We attempted to clarify the involvement of telomerase in the impairment of LAK cell generation in patients with advanced cancer. LAK cells were generated by stimulation with interleukin (IL)-2 and immobilized anti-CD3 antibody (IL-2/CD3 system) from peripheral blood mononuclear cells of healthy volunteers (he-LAK) or patients with advanced cancer (ca-LAK), and proliferative potential of LAK cells was evaluated on the basis of population doubling level (PDL). Telomere length and telomerase activity of LAK cells were measured by the hybridization with oligonucleotide (TTAGGG)4 and by the telomeric repeat amplification protocol (TRAP) assay, respectively. Effects on telomerase activity in LAK cells of serum from cancer patients, transforming growth factor (TGF)-beta, and IL-10 were also examined. The lifespan of ca-LAK (15.2 +/- 5.1 PDLs) was significantly shorter than that of he-LAK (22.6 +/- 8.3 PDLs) (p = 0.0358). There were no significant differences between he- and ca-LAK in telomere length before IL-2/CD3 stimulation and maximal telomerase activity induced. The telomerase activity induced in ca-LAK failed to elongate sufficiently the telomeric ends (-35.2 +/- 46.2 bp) compared with that in he-LAK (16.8 +/- 41.5 bp) (p = 0.0448). The telomerase activity was initially detectable on day 2 in all he-LAK, whereas 8 (61.5%) of 13 ca-LAK expressed telomerase activity on day 3 or later following the stimulation, showing a significant retardation of telomerase expression (p = 0.0116). The addition to the LAK cell generation system of serum from cancer patients, as well as IL-10, but not transforming growth factor (TGF)-beta, suppressed the telomerase activity. This serum-induced suppression of telomerase activity in LAK cells was abrogated with the addition of anti-IL-10 antibody but not with anti-TGF-beta antibody. It is suggested that the dysregulation of telomerase activity and expression exists in LAK cells of cancer patients, resulting in the impairment of LAK cell generation in patients with advanced cancer. Serum IL-10 may be involved in the impairment of LAK cell generation by the suppression of telomerase activity of lymphocytes in vivo. Thus, the dysregulation mechanism of telomerase activity and expression in lymphocytes of cancer patients may be attributable, in part, to cancer-associated immunosuppression.
Oncology Reports 8(3):649-53. · 1.84 Impact Factor
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ABSTRACT: The immunological properties of interleukin-2 (IL-2) gene-transduced tumor cells were investigated in mice. A murine ovarian cancer cell line, OVHM, was retrovirally transduced with the human IL-2 gene (OVHM/IL-2) and the neomycin resistance gene (OVHM/Neo). OVHM/IL-2 cells continuously secreted IL-2 detected by ELISA using an antibody specific for human IL-2, and by a bioassay using an IL-2-reactive cell line (CTLL-2). When OVHM cells were inoculated subcutaneously into syngeneic B6C3F1 mice, OVHM/IL-2 cells but not parental (OVHM/P) or OVHM/Neo cells were regressed even though their rate of in vitro growth was comparable. This was not observed in nude mice, indicating the involvement of T lymphocytes in the regression of OVHM/IL-2 cells. The survival of mice inoculated intraperitoneally with OVHM/IL-2 cells was prolonged compared with those inoculated with OVHM/P cells. In irradiation experiments, IL-2 secretion by irradiated OVHM/IL-2 cells was retained for at least 5 days, although in vitro growth and in vivo tumorigenicity of irradiated OVHM/IL-2 cells were completely diminished. When mice were challenged with viable OVHM/P cells, survival of mice previously immunized with irradiated OVHM/IL-2 cells was prolonged compared to those immunized with irradiated OVHM/P cells, indicating a vaccine property of irradiated OVHM/IL-2 cells. Moreover, survival of mice with established ascites was improved upon injection of irradiated OVHM/IL-2 cells, indicating a therapeutic potential of OVHM/IL-2 cells. Tumor cells genetically engineered to secrete IL-2 may therefore be promising candidates for tumor vaccines and may provide a new mode of cancer immunotherapy.
Anticancer research 19(6B):5299-306. · 1.73 Impact Factor
