Tokio Osaki

Mn-SOD antisense upregulates in vivo apoptosis of squamous cell carcinoma cells by anticancer drugs and γ-rays regulating expression of the BCL-2 family proteins, COX-2 and p21

ROIs and their scavengers are associated with apoptosis induction by anticancer drugs and γ‐rays, but the details have not been clarified. We examined the effect of transfection of Mn‐SOD antisense on apoptosis by 5‐FU, PLM, CDDP and γ‐rays using nu/nu mice. After inoculation of Mn‐SOD antisense–transfected SCC cells into the subcutis of each mouses back, they slowly multiplied to form tumors sized 1,460 ± 70 mm3 at day 60, while control vector‐transfected SCC cells rapidly multiplied, with a mean tumor size of 2,330 ± 220 mm3. Inversely, mice in the Mn‐SOD antisense group survived longer (mean survival duration 94.4 ± 12.7 days) compared to those in the empty vector group (67.3 ± 6.8 days). After treatment with 5‐FU (5 μg/day), PLM (50 μg/day), CDDP (10 μg/day) and γ‐rays (2 Gy/day), mean survival times were largely prolonged, to 126.3 ± 22.7, 123.0 ± 22.1, 136.3 ± 24.0 and 143.0 ± 20.8 days, respectively, while mean survival times in the empty vector group were 91.7 ± 14.8, 85.7 ± 13.3, 97.5 ± 16.0 and 100.7 ± 17.1 days, respectively. Immunohistologically, tumors in the Mn‐SOD antisense group revealed additional nick end‐labeled cells compared to those in the empty vector group. In comparison, strong expression of Bax, Bak and p21waf1/cip1 and suppressed expression of Bcl‐2, Bcl‐XL and COX‐2 were observed in the Mn‐SOD antisense group and the expression pattern of these proteins was the inverse in the empty vector group. The increased expression of these proapoptotic proteins appeared to be p53‐independent because p53 protein expression was not increased in the antisense group. These immunohistologic results were supported by Western blotting of each protein. In conclusion, Mn‐SOD antisense transfection is advantageous for apoptosis induction of SCC cells by anticancer drugs and γ‐rays through induction of proapoptotic Bcl‐2 family proteins and suppression of antiapoptotic protein expression.

Enhanced apoptosis of squamous cell carcinoma cells by interleukin-2-activated cytotoxic lymphocytes combined with radiation and anticancer drugs

Induction of potent apoptosis is required in cancer therapy. We examined the combination effect of interleukin-2-activated lymphocytes (LAK cells) and anticancer drugs or gamma (gamma)-rays on the induction of apoptosis in an established oral squamous cell carcinoma cell line (OSC-3 cells). By pretreatment of OSC-3 cells with (137)Cs (5 Gy), 5-fluorouracil (5-FU) (0.5 microg/ml) or cis-dichlorodiammine-platinum (CDDP) (5 microg/ml), the activation of bid and caspase-3 by LAK cells was strongly increased and associated with an enhanced degradation of poly-(ADP-ribose) polymerase (PARP) and/or nuclear mitotic apparatus protein (NuMA) and the increased fragmentation of DNA. The LAK cell-enhanced caspase-3 activity in the pretreated OSC-3 cells was decreased to approximately 70% and 40% of the control by the addition of Z-AAD-CMK (a granzyme B inhibitor) and neutralising monoclonal antibody to Fas antigen (alphaFas-IgG), respectively. The combined treatment-induced DNA fragmentation was suppressed by approximately 20% and 30% of the control by the addition of Z-AAD-CMK and alpha Fas-IgG, respectively, in the co-culture system. While Ac-DEVD-CHO (a caspase-3 inhibitor) suppressed the DNA fragmentation levels to approximately half and this was similar to the amount of suppression that was obtained by the addition of both alpha Fas-IgG and Z-AAD-CMK. In addition, LAK cell-activated bid may have increased the intracellular reactive oxygen intermediates (ROI) level and induced a decrease of mitochondrial membrane potential. These influences by LAK cells were enhanced when OSC-3 cells were pretreated with each anticancer drug or (137)Cs. Furthermore, the increase of ROI by LAK cells was suppressed by alpha Fas-IgG and Z-AAD-CMK to approximately half the level of the control. These results indicate that anticancer drugs and gamma-rays prime squamous cell carcinoma cells to be susceptible to apoptosis by LAK cells, that LAK cell-induced apoptosis largely depends on the activation of caspase-3 by the Fas/Fas-ligand signal and granzyme B, and that LAK cells induce ROI in the target cells, which is largely mediated by Fas and granzyme B.

Characteristic cytokine generation patterns in cancer cells and infiltrating lymphocytes in oral squamous cell carcinomas and the influence of chemoradiation combined with immunotherapy on these patterns

Objectives: Cytokines produced by tumor cells and tumor-infiltrating lymphocytes (TIL) appear to regulate tumor cell growth and the cytotoxic activity of TIL. The objectives of the present study were to investigate cytokine generation patterns in tumor cells and TIL and to examine the influence of cancer therapy on this cytokine production and the cytotoxic activity of TIL. Methods: We determined the levels of cytokines produced by tumor cells and TIL in vitro and measured the cytotoxic activity of TIL against Daudi cells in patients with oral squamous cell carcinoma (OSC) before and 1 week after the start of concomitant chemo-radio-immunotherapy. Results: Before the therapy, OSC cells generated higher levels of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) than did oral keratinocytes isolated from the noninflamed gingivae of healthy individuals, but both kinds of cells generated similar levels of interleukin (IL)-1β and IL-6. Compared with peripheral blood mononuclear cells (PBMC) of the patients, TIL produced higher levels of IL-1β, IL-6, IL-10, TNF-α and TGF-β, whereas their production of IL-12 and interferon-γ (IFN-γ) was only slightly higher than that in PBMC. After 1 week of therapy, the cytokine production by OSC cells had largely decreased, while the production of TNF-α, IFN-γ, TGF-β and IL-12 by TIL had increased greatly, although other cytokine levels were almost constant during the investigations. The cytotoxic activity of TIL was higher than that of PBMC before the therapy, and this activity was strongly increased by 1 week of therapy. Conclusions: These results suggest that the cytokine productivities of TIL and tumor cells differ from those of PBMC and normal keratinocytes, respectively, and that chemo-radio-immunotherapy modulates in situ cytokine generation, which is advantageous for inhibition of tumor cell growth and activation of TIL.

Induction of differentiation in maxillary adenoid cystic carcinomas by adoptive immunotherapy in combination with chemoradiotherapy

The successful treatment results of a case of maxillary adenoid cystic carcinoma (ACC) and the possibility of differentiation of ACC cells with chemoradioimmunotherapy are described. Combined therapy was applied to two maxillary ACCs. Adoptive immunotherapy consisted of intra-arterial injection of lymphokine-activated killer cells (total 9.0 x 10(8) cells) and recombinant interleukin-2 (1.8 x 10(5) U) and interferon-gamma (1.8 x 10(5) U) was combined with 60Co radiation (50 Gy), 5-fluorouracil (4000 mg) and peplomycin (10 mg). Immunohistochemical staining of the biopsy material obtained during the therapy revealed a marked decrease of proliferating cell nuclear antigen-positive cells and a prominent increase of Lewis Y antigen- and bone morphogenetic protein-2-positive cells. The disappearance of tumour cells and the remodeling of the sinus wall with calcification in the sinus cavity, which had been occupied by the tumour, were observed after therapy in both patients. Adoptive immunotherapy in combination with chemoradiotherapy is useful for the treatment of ACC.

Effects of tumour necrosis factor-alpha (TNF-α), IL-1β and monocytes on lymphokine-activated killer (LAK) induction from natural killer (NK) cells and T lymphocytes

Roles of monocytes and cytokines were investigated on LAK induction from T and NK cells. Monocytes augmented more T‐LAK induction than did NK‐LAK. Expression of IL‐β, TNF‐α and interferon‐gamma (IFN‐γ)‐mRNA and their cytokine production were superior in NK cells compared with T cells in parallel with their LAK activities. An increase of TNF‐α, IL‐1β and IFN‐γ production was induced by co‐culturing NK or T cells with autologous monocytes. The augmentation of T cell cytokine production and T‐LAK activity by monocytes was more prominent than that of NK cells. TNF‐α and IL‐1β were generated 24 h after IL‐2 stimulation, and these cytokines were able to almost substitute for monocytes in LAK induction. Conversely, LAK induction was almost completely suppressed by both anti‐IL‐1β and anti‐TNF‐α antibodies, if they were added within 24 h after the start of the LAK induction. IFN‐γ, which was produced at a later stage, scarcely affected LAK induction in spite of the cooperation with TNF‐α. The results obtained indicate conclusively that the superiority of NK‐LAK depends on their superior productivity of both IL‐1β and TNF‐α, and that the up‐regulation of LAK induction by monocytes is largely due to the enhanced generation of both cytokines.

Induction of lymphokine-activated killer cells with low-dose interleukin 2 and interferon-γ in oral cancer patients

Lymphokine-activated killer (LAK) cells were induced with low-dose recombinant interleukin 2 (rIL-2) and recombinant interferon-γ (IFN-γ) in 28 oral carcinoma patients. The patients received daily intravenous injections of rIL-2 (1.2×105 U/m2) and rIFN-γ (7.0×104 U/m2), and both natural killer (NK) and LAK activities were periodically examined. A significant increase in CD16+CD57+ and CD16+CD57− NK subsets was observed after the induction. An increase in the T-cell population was also found, with a significant increase in CD3+HLA-DR+, CD8+Leu8−, and CD4+Leu8− cells. Significant increases in NK activity, from the original level of 32.0±13.7 to 49.9±15.2%, and LAK activity, from 4.8±3.5 to 11.0±6.1%, at Day 7 were observed. Both activities were maintained at high levels during the cytokine injections, but greater enhancement of the killing activities could not be obtained subsequently. When NK and LAK activities were investigated in each subpopulation of CD3− and CD16− cells, no remarkable cytotoxic activity could be observed before induction in any subset without NK activity in CD3− cells (31.1±14.3%). At Day 7, NK activity of CD16− cells increased up to 21.4±14.9%, accompanied by an increase in CD3−-cell activity (54.5±20.6%). LAK activities of both subsets were also enhanced, with activity at Day 7 of 6.5±5.6 and 9.4±6.6% in CD16− and CD3− cells, respectively. These increased activities were maintained at the same level during the induction. Phorbol myristate acetate-induced polymorphonuclear leukocyte (PMNL) O2− generation was significantly increased, from the original 81.1±28.1 to 95.6±34.9 pmol/min/104 cells, after 1 week of treatment. Protein kinase C activity in the cytosol decreased, and the activity in the membrane fraction conversely increased. No remarkable adverse effects except for mild fever were observed. Together with LAK induction ability and PMNL enhancement, with scarce toxicity, a combination of low-dose rIL-2 and rIFN-γ is thought to be useful in cancer treatments.

Longer local retention of adoptively transferred T-LAK cells correlates with lesser adhesion molecule expression than NK-LAK cells

The local retention of adoptively transferred lymphokine (IL‐2)‐activated killer (LAK) cells was examined in 11 patients with head and neck carcinoma. Unseparated lymphocytes, T and natural killer (NK) cells isolated from patients were cultured with IL‐2 for 7 days, labelled with 99mTc‐HMPAO, and immediately injected back into the respective donors via the superficial temporal artery or locally into the tumour tissue. The injected LAK cells were periodically traced using a gamma camera, and the LAK cell retention rate was calculated from the radioactivity. One hour after the injection, about 70% of the locally infiltrated LAK cells remained in the tumour tissue, while about half of the LAK cells transferred via the regional artery were dislodged from the tissue. LAK cells induced from T cells (T‐LAK) were retained in the tissue for a longer time than LAK cells induced from NK cells (NK‐LAK). T‐LAK were less chemotactic and less adherent to human umbilical vein endothelial cells (EC), and showed lesser migration through EC. Flow cytometric analysis revealed higher expression of CD11a, CD11b, CD18 and CD49d on NK‐LAK compared with T‐LAK. MoAbs against these adhesion molecules suppressed adhesion and migration of LAK cells. These results indicate that the rapid disappearance of NK‐LAK from the tissue is associated with their greater chemotactic and adhesive as well as migratory activities depending on differing expression of adhesion molecules.

Immunoregulatory effects of Sizofiran (SPG) on lymphocytes and polymorphonuclear leukocytes

The effect of SPG on leukocytes has been studied in 20 patients with oral carcinoma and the actions have been analysed in vitro. SPG 1 mg/kg was administered intramuscularly twice weekly. Peripheral venous blood was collected before, and 1 week and 2 weeks after the initiation of SPG treatment. Both CD16+ CD57– and CD16–CD57+ cell populations were significantly increased after treatment, but no T cell subset varied. While enhancement of lymphokine‐activated killer activity could not be found, an increase in natural killer (NK) activity was observed in 15 of the subjects, and the mean NK level was significantly increased from an initial 34.7 ± 18.7% to 46.4 ± 16.5% after two weeks of injections. O2‐production by polymorphonuclear leukocytes (PMNL) was stimulated 6 h after SPG injection. When PMNL were treated in vitro with SPG 32 μg/ml. enhanced 02‐generation was induced and protein kinase C (PKC) activity in a membrane fraction increased. SPG did not directly affect non‐specific PMNL killing of K562 cells or antibody‐dependent cell mediated cytotoxicity against Raji cells, but non‐specific PMNL killing was enhanced by culture‐conditioned medium from peripheral blood mononuclear cells (PBMC) containing 10 μg/ml SPG. Interleukin‐1β, ‐3, ‐4, ‐6, tumour necrosis factor‐α, granulocyte‐macrophage colony stimulating factor and IFN‐γ levels in the conditioned medium were not increased compared with medium from PBMC not treated with SPG. No clear increase of these cytokines was found in serum from the SPG‐treated patients. From the above results, enhancement of PMNL O2‐generation by SPG seems to be a direct action of SPG, but the mechanism of elevation of the non‐specific killing activity of PMNL and NK cells is not known. Perhaps other cytokines than those assayed have participated in increasing non‐specific cytotoxicity.

IL-2 signalling in T and natural killer (NK) cells associated with their class I-non-restricted killing activity.

The signal transduction of IL‐2 in NK cells and T cells was compared. On 5u2003min incubation of these cellsu2003u2003with IL‐2, we observed tyrosine phosphorylation of 105‐kD and 110‐kD proteins in NK cells and of 95‐kD and 110‐kD proteins in T cells. The phosphorylation reached maximal levels in 15u2003min in both NK and T cells, but the levels were higher in NK cells, which showed superior killing against Daudi cells. With this phosphorylation, p52shc was also tyrosine‐phosphorylated and p21ras was activated by the short term (10u2003min) treatment of NK and T cells with IL‐2. These signals were completely suppressed by anti‐IL‐2Rβ MoAb, but only slightly suppressed by anti‐IL‐2Rα MoAb, correlated with the suppression of the class‐I‐non‐restricted cytotoxic activity of NK and T cells by these MoAbs. When tyrosine phosphorylation was inhibited by herbimycin A and genistein, the cytotoxic activities of NK and T cells were nearly completely suppressed. In addition, the tyrosine phosphorylation of JAK3 by IL‐2 was more prominent in NK cells than in T cells, but JAK1, JAK2, STAT1α, STAT2 and STAT3 were not phosphorylated. These results indicate that the IL‐2 signal flows downstream via both ras‐dependent and ras‐independent pathways and that the superior killing activity of NK cells depends on their high susceptibility to protein tyrosine phosphorylation by IL‐2.