Lymphokine-activated Killer Cell Cytotoxicity Research Articles

Oxidized-Desialylated Low-Density Lipoprotein Inhibits the Antitumor Functions of Lymphokine Activated Killer Cells.

Elevated concentrations of circulating low density lipoprotein (LDL) that is abnormally oxidized and desialylated is both a precursor to and a hallmark of atherosclerosis. Peripheral blood mononuclear cells (PBMCs) treated in vitro with interleukin-2 (IL-2) become lymphokine activated killer (LAK) cells, the primary effectors of which are NK cells and NKT cells. LAK cells display antitumor functions such as increased cytotoxicity and IFN-γ production, and they have been evaluated as a potential cancer therapeutic. Atherosclerotic processes may influence innate immunity against cancer. Because prior studies have shown that low density lipoprotein (LDL) reduces T-cell and NK cell antitumor functions, we asked whether oxidized-desialylated LDL affects the functionality of LAK cells in vitro. We show here that LAK cells take up oxidized-desialylated LDL to a significantly greater extent than native LDL over a period of 72 hours. This resulted in a significant downregulation of LAK cell cytotoxicity against K562 cells. In particular, the expression of IFN-γ, CD56, and NKG2D were reduced upon oxidized-desialylated LDL treatment of LAK cells and, conversely, their expression was enhanced with native LDL. It was also observed that as the number of CD56 and NKG2D positive cells decreased upon treatment with oxidized-desialylated LDL, the number of CD3 positive cells increased in proportion. Additionally, only a slight inhibition of LAK cell cytotoxicity was observed with desialylation alone of LDL, and no significant inhibition was observed with oxidation alone of LDL. Thus, this study describes a new role of oxidized-desialylated LDL as an inhibitor of the antitumor functions of LAK cells. These observations have implications for how atherosclerosis processes, namely oxidation and desialylation of LDL, may influence LAK cell antitumor activity.

117. Development of Novel Cancer Therapy Combination of Ad-SOCS Gene Therapy and LAK Cell Immunotherapy for the Treatment of Prostate Cancer

Background Suppressor of cytokine signaling 3 (SOCS3) is a promising molecule for cancer gene therapy. SOCS3 suppresses tumor growth through inhibition of mutiple signaling pathways including Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase (ERK), and focal adhesion kinase (FAK). The constitutive activation of JAK/STAT3 pathway is known to be involved in oncogenesis of various types of cancers including prostate cancer. We investigated the anti-tumor activity of Ad-SOCS, which is the recombinant adenovirus vectors carrying the SOCS3 gene, against prostate cancer cell line in vitro. Furthermore, to improve the antitumor effect against prostate cancer, we combined LAK (Lymphokine activated killer) cell immunotherapy with Ad-SOCS gene therapy. Materials and Methods LNCaP, human prostate cancer cell line was used in this study. The cytotoxicity of Ad-SOCS against LNCaP was measured by XTT assay. LAK cells were generated from PBMCs isolated from fresh blood by Ficoll-paque (GE Healthcare, Munich, Germany) density gradient centrifugation. PBMC were incubated in medium with interleukin (IL)-2 and anti CD3 antibody (OKT3) to generate LAK cells. The cytotoxicity of LAK cells against LNCaP cells infected with Ad-SOCS was investigated by the in vitro cytotoxicity assay. Expression level of SOCS3, STAT3 and ULBPs were measured by real-time PCR. Results Ad-SOCS showed the significantly higher cell growth inhibitory effect compared to Ad-LacZ and PBS controls in LNCaP cells. In real-time PCR, Ad-SOCS significantly increased the level of SOCS3 mRNA expression. Sequentially, the expression of STAT3 mRNA was decreased by Ad-SOCS infection. Upregulation of SOCS3 mRNA and downregulation of STAT3 mRNA were not observed in both Ad-LacZ and PBS infected LNCaP cells. In cytotoxicity assay, LAK cells exhibited the significantly higher cytotoxicity against LNCaP cells infected with Ad-SOCS at all effector: target cell ratios, 1:1,5:1, 10:1, compared with that of non-infected or Ad-LacZ infected cells. The mRNA expression levels of ULBP 1-5, which are NKG2D ligands to stimulate LAK cell activities, were measured and the mRNA expressions of ULBP 1, 3 and 4, in LNCaP infected with Ad-SOCS were significantly increased compared to the treatments. Conclusion In the present study, we demonstrated that Ad-SOCS could inhibit the cell growth of LNCaP cells and increased the sensitivity of LAK cell in vitro. These findings suggested that the Ad-SOCS and LAK cell combination therapy warranted the further development of novel therapeutic approach for advanced prostate cancer.

Follicular lymphoma: in vitro effects of combining lymphokine-activated killer (LAK) cell-induced cytotoxicity and rituximab- and obinutuzumab-dependent cellular cytotoxicity (ADCC) activity.

Follicular lymphoma (FL) is a disease of paradoxes-incurable but with a long natural history. We hypothesized that a combination of lymphokine-activated killer (LAK) cells and monoclonal antibodies might provide a robust synergistic treatment and tested this hypothesis in a phase II clinical trial (NCT01329354). In this trial, in addition to R-CHOP, we alternated the administration of only rituximab with rituximab and autologous LAK cells that were expanded ex vivo. Our objective was to determine the in vitro capability of LAK cells generated from FL patients to produce cytotoxicity against tumor cell lines and to determine rituximab- and obinutuzumab-induced cytotoxicity via antibody-dependent cellular cytotoxicity (ADCC) activity. We analyzed the LAK cell-induced cytotoxicity and rituximab (R)- and obinutuzumab (GA101)-induced ADCC activity. We show that LAK cells generated from FL patients induce cytotoxicity against tumor cell lines. R and GA101 enhance cytolysis through ADCC activity of LAK cells. Impaired LAK cell cytotoxicity and ADCC activity were detected in 50 % of patients. Percentage of NK cells in LAK infusions were correlated with the R- and GA101-induced ADCC. Our results indicate that the combination of R or GA101 and LAK cells should be an option as frontline maintenance therapy in patients with FL.

Therapeutic effect of lymphokine-activated killer cells treated with low-dose ionizing radiation on osteosarcoma

The aim of the present study was to investigate the effect of lymphokine-activated killer (LAK) cells, which received low-dose ionizing radiation, on the treatment of osteosarcoma in rats. The cultured UMR-106 cells were inoculated under the anterior chest skin of 24 rats to establish an osteosarcoma model. In addition, the LAK cells from 24 mice were exposed to doses of 0 (control group), 0.65 or 3.25 mGy X-ray radiation. The tritiated thymidine (3H-TdR) release method and Winn assay were performed to determine the antitumor effects of the LAK cells. The proliferation of the mouse LAK cells treated with 3.25 mGy radiation was significantly higher than that for those treated with 0 or 0.65 mGy radiation, which suggested that low-dose ionizing radiation stimulates the proliferation of LAK cells. The tumor-bearing rats were divided into three groups and injected with LAK cells that had already received 0, 0.65 or 3.25 mGy radiation. The mean survival time of the 3.25-mGy group was longer than that of the 0- and 0.65-mGy groups. After 30 days, tumors with weights of ~6.25 and 2.0 g were identified in the rats of the 0- and 0.65-mGy groups, respectively. However, tumor proliferation was not detectable in the rats of the 3.25-mGy radiation group. Therefore, low-dose ionizing radiation effectively kills osteosarcoma cells in rats by stimulating the proliferation and enhancing the cytotoxicity of LAK cells.

Gemcitabine sensitizes lung cancer cells to Fas/FasL system‐mediated killing

Gemcitabine is a chemotherapy agent commonly used in the treatment of non-small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine-induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real-time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL-expressing cells was evaluated by flow cytometry, and caspase-8 and caspase-3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine-activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti-Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane-bound FasL (mFasL) and of mFasL-positive apoptotic H292 cells, as well as caspase-8 and caspase-3 cleavage. Moreover, gemcitabine increased CH11-induced caspase-8 and caspase-3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine-treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up-regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas-dependent apoptosis mediated by caspase-8 and caspase-3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine-induced lung cancer cell killing.

Downregulation of the H-2Kd gene by siRNA affects the cytotoxicity of murine LAK cells

To investigate the effect of the H-2Kd gene on the lymphocyte membrane, we constructed a small interfering RNA (siRNA) that targets the H-2Kd gene and compared the cytotoxicity of mouse lymphokine-activated killer (LAK) cells with different H-2Kd expression states. H-2Kd-targeting siRNA was transfected into spleen lymphocytes of BALB/C mice. Flow cytometry (FCM) was then performed to examine the expression of the H-2Kd gene in the transfected and control cells. Additionally, the cytotoxicity of the transfected cells toward the H22 and K562 cell lines was evaluated in vitro using the LDH release assay. H-2Kd-targeting siRNA significantly reduced the expression levels of the target protein, whereas pure transMessenger and non-silencing siRNA did not inhibit H-2Kd expression at the concentrations tested. The cytotoxicity of siRNA-treated LAK cells toward H22 and K562 cells was reduced significantly. The knockdown of H-2Kd gene expression by siRNA may be associated with LAK cell cytotoxicity toward neoplasm cell lines.

Inducing anti-tumor cytokines and an immune response in melanoma by inhibition of MIA using the peptide AR71

Malignant melanoma is known for its aggressive metastatic spread and suppression of the host immune system. Immunosuppression in melanoma is mediated in part by the protein melanoma inhibitory activity (MIA). In this study, we assessed the in vitro and in vivo efficacy of the MIA-inhibitory peptide AR71 in the inhibition of MIA-induced immunosuppression. This follows a previous study that revealed an increase in CD3-positive cells and cleaved caspase-3 in an in vivo model of hepatic metastasis after MIA inhibition. We used Multiplex-ELISAs and qRT-PCR for determining changes in cytokine expression in vitro and in vivo and calcein release assays for determining immune cell response in vitro. By evaluating the serum levels of tumor-associated cytokines of the melanoma-bearing mice, we found beneficial decreases of several cytokines, including TNF-alpha, after AR71 treatment. Additionally, we demonstrated an increase of anti-tumor lymphokine-activated killer (LAK) cell cytotoxicity in the presence of the MIA inhibitor AR71. Stimulation of anti-tumor immune responses by AR71 could be observed via increased numbers of NK cells in the metastases-bearing murine livers in vivo. In summary, inhibition of MIA activity results in reduced immunosuppression in vitro and in vivo.

A possible mechanism of antibody-dependent cellular cytotoxicity (ADCC) of lymphokine-activated killer cells (LAK) with cetuximab for the treatment of mutated KRAS or BRAF metastatic colorectal cancer (mCRC).

e14084 Background: Cetuximab, a chimeric IgG1 antibody, recognizing the extracellular domain of epidermal growth factor (EGFR) to inhibit the activation signaling of proliferation, angiogenesis and anti-apoptosis is used in first line mCRC treatment. In addition to this mechanism, ADCC activity has been suggested. In this study, we investigated the antitumor activity of cetuximab in combination with activated immune cells against CRC cell lines. Methods: Peripheral blood mononuclear cells (PBMCs) were activated to LAK and CD3-LAK by incubation for 4 days with IL-2 (500 U/mL) or both IL-2 and immobilized anti-CD3 antibody, respectively. LAK and CD3-LAK were co-cultured with CRC cell lines, OMUS-23 (wild-type: wt), OUMSmt (BRAFmutant: mt) and HCC-56 (KRASmt), with or without cetuximab. The antitumor activity was evaluated by adherent target detachment (ATD) assay. Results: The inductions of Fcg IIIa (CD16)+ NK cells were significantly higher in LAK than CD3-LAK (P<0.05). The ATD activity of LAK was significantly higher (P<0.05) than that of CD3-LAK. Moreover, the ATD activities of LAK in combination with cetuximab were strongly enhanced especially against mutated KRAS or BRAF tumor cells. Conclusions: Our results suggest that cetuximab in combination with Fcg IIIa+ NK lich effecter cells induced by the addition of IL-2 exerts effective anti-tumor activity. Cetuximab may therapeutic not only mCRC patients with wild-type KRAS and BRAF but also patients with mutant KRAS or BRAF by its ADCC activity.

Pulse Interleukin-2 with Famotidine Induces CD56+ Lymphocytes in the Peripheral Blood of Patients with Metastatic Melanoma or Kidney Cancer

Increased lymphokine-activated killer (LAK) cell numbers and cytotoxicity against tumor cell lines have been seen in patients receiving high-dose continuous and bolus infusion interleukin-2 (IL-2) regimens. LAK are CD56 positive on flow cytometry. Daily intravenous doses of IL-2 of 18-21.6 MIU/m(2) over 15-30 minutes ("pulses") have been developed to attempt to lessen the toxicity of this therapy. It has been previously shown that the patients with metastatic melanoma or kidney cancer may be treated safely with pulse IL-2 daily for 5 days preceded by intravenous famotidine. Cycles were repeated every 21 days. Because LAK numbers have not been previously described with this regimen, the present study has examined CD56 numbers via peripheral blood flow cytometry in 11 patients with samples scheduled at baseline, after two cycles, and after four cycles. Eight (8) patients had melanoma and 3 had kidney cancer. Median CD56 counts after two cycles was significantly higher than baseline (p = 0.001). Similarly, CD56 counts at 2 months later were also greater than baseline (p = 0.009). There was no difference between median values after two cycles versus after four cycles. Patients who were clinical responders had a median CD56 count of 650 after two cycles when compared with nonresponders who had a median CD56 count of 290 (p = 0.005). CD56 counts are significantly elevated in patients treated with pulse IL-2 with famotidine and clinical responders have significantly higher CD56 than nonresponders.

Alterations in the Th1/Th2 balance in breast cancer patients using reflexology and scalp massage

The diagnosis and treatment of breast cancer can adversely affect quality of life. Here the aim was to determine the effects of reflexology on host defences and endocrine function in women with early breast cancer. Six weeks after surgery for early breast cancer, 183 women were randomly assigned to self-initiated support (SIS), SIS plus foot reflexology, or SIS plus scalp massage. Peripheral blood mononuclear cells and serum were isolated at T1 (6 weeks post surgery; baseline), T2 and T3 (4 and 10 weeks post completion of intervention, respectively). Lymphocyte phenotyping found that CD25(+) cells were significantly higher in the massage group compared with the SIS group at T3. The percentage of T cells, and more specifically the T helper subset expressing IL4, decreased significantly in the massage group compared with the SIS group at T3. This change was accompanied by an increase in the percentage of CD8(+) T cytotoxic cells expressing IFNγ in the massage group. Natural killer and lymphokine activated killer cell cytotoxicity measurements, serum levels of cortisol, prolactin and growth hormone, and flow cytometric assessment of their corresponding receptors all revealed no significant differences between the three groups of patients. This study provides evidence that the immunological balance of patients can be altered in a potentially beneficial manner by massage. The original trial was registered with the International Standard Randomised Controlled Trial Registry (ISRCTN87652313).

Alterations in the Th1/Th2 balance in breast cancer patients using reflexology and scalp massage

The diagnosis and treatment of breast cancer can adversely affect quality of life. Here the aim was to determine the effects of reflexology on host defences and endocrine function in women with early breast cancer. Six weeks after surgery for early breast cancer, 183 women were randomly assigned to self-initiated support (SIS), SIS plus foot reflexology, or SIS plus scalp massage. Peripheral blood mononuclear cells and serum were isolated at T1 (6 weeks post surgery; baseline), T2 and T3 (4 and 10 weeks post completion of intervention, respectively). Lymphocyte phenotyping found that CD25+ cells were significantly higher in the massage group compared with the SIS group at T3. The percentage of T cells, and more specifically the T helper subset expressing IL4, decreased significantly in the massage group compared with the SIS group at T3. This change was accompanied by an increase in the percentage of CD8+ T cytotoxic cells expressing IFNγ in the massage group. Natural killer and lymphokine activated killer cell cytotoxicity measurements, serum levels of cortisol, prolactin and growth hormone, and flow cytometric assessment of their corresponding receptors all revealed no significant differences between the three groups of patients. This study provides evidence that the immunological balance of patients can be altered in a potentially beneficial manner by massage. The original trial was registered with the International Standard Randomised Controlled Trial Registry (ISRCTN87652313).

Chronic antidepressant treatment exerts sexually dimorphic immunomodulatory effects in an experimental model of major depression: do females lack an advantage?

Major depression is a stress-related disorder that affects about 20% of the population, with women outnumbering men by 2:1. However, research focusing on stress/antidepressant-related immunomodulation overlooks sex differences, although an established sexual dimorphism also characterizes the immune system. We report for the first time that both chronic clomipramine treatment (10 mg/kg, twice daily) and chronic mild stress (CMS) application in rats, exert sexually dimorphic effects on cellular immunoreactivity (natural killer and lymphokine-activated killer cell cytotoxicity and interleukin-2-induced T-cell proliferation), with females presenting a relatively immunosuppressed phenotype compared to males. Moreover, following chronic antidepressant treatment, thymic monoamines presented sex-related alterations, as well as intriguing associations with peripheral T-cell responses. This study highlights the sex-related effects of chronic clomipramine treatment and CMS application on the cellular arm of immunity, and represents a preliminary exposé of a thymus-dependent route pertaining to the interactions between antidepressants and the immune system.

Impact of surgery and chemotherapy on cellular immunity in pancreatic carcinoma patients in view of an integration of standard cancer treatment with immunotherapy

As surgery and chemotherapy may act as adjuvants providing antitumor immunity benefits, we ran phenotypical and functional immunomonitoring in patients with resectable pancreatic adenocarcinoma and advanced metastatic disease receiving combined treatment (cisplatin, gemcitabine, 5-FU). Blood was taken before/one month after resection; before/during chemotherapy. Controls were age- and gender-matched. Circulating lymphocyte, myeloid and plasmacytoid dendritic cell (MDC and PDC) subsets were examined by flow cytometry; functional activity by mixed lymphocyte reaction (MLR) for DC allostimulation, through 4-h 51Cr-release assay for Natural Killer (NK) and lymphokine-activated-killer (LAK) cell cytotoxicity; ELISA for spontaneous/activated cytokine release by PBMC and T cells. Significant differences occurred in several parameters between pretreatment patient and control values: fewer CD8+ cells and increased apoptosis-prone CD3+/CD95+ lymphocytes, higher frequency of MDC, reduced allostimulatory activity by ex vivo-generated DC, depressed LAK activity, elevated IL-10 and IL-12p40 production; impaired IL-12p70 and IFN-gamma production by stimulated PBMC and T cells. Only IL-12p70 level was correlated with survival. One month after radical, but not palliative surgery, the percentage of T-lymphocytes coexpressing CD3/CD95 decreased significantly, the stimulatory capacity of DC increased, and LPS-induced IL-12p70 release by PBMC rose concomitantly with the anti-CD3 stimulated-IFN-gamma production by T cells. In patients with locally advanced or metastatic disease, one and/or two combined drug cycles increased percentage of CD4+ cells and LAK cell cytotoxicity and decreased PDC frequency and spontaneous/LPS-stimulated IL-10 by PBMC. Results suggest immunological changes induced by surgical resection/combined chemotherapy indicate specific precisely-timed windows of opportunity for introducing immunotherapy in pancreatic cancer, possibly improving survival in this highly lethal disease.

Activity of Continuous Infusion + Pulse Interleukin-2 with Famotidine in Metastatic Melanoma

High-dose interleukin-2 (IL-2), given via continuous intravenous (i.v.) infusion, induces lymphokine-activated killer (LAK) cell cytotoxicity against tumor cells. These LAKs exhibit enhanced cytotoxicity against tumor cells in vitro when they are subsequently pulsed with additional IL-2. Famotidine may increase LAK cytotoxicity against neoplastic cells by allowing for greater IL-2 uptake at the IL-2 receptor on lymphocytes. Twenty-three (23) patients received famotidine 20 mg i.v. twice per day and continuous-infusion IL-2 (18 MIU/m(2)/24 hours) for 72 hours, followed by a 24-hour rest, then 1-3 daily-pulse IL-2 doses of 18 MIU/m(2) over 15-30 minutes preceded by famotidine 20 mg i.v. Cycles were repeated every 3 weeks. The most common metastatic sites were lung, lymph node, and subcutaneous/soft tissue. The most common toxicities were fever, rigor, nausea/emesis, hypophosphatemia, hypotension, elevated creatinine, and pulmonary edema. There were no treatment-related deaths. One (1) complete (4%) and 9 partial responses (39%) were seen (43% total response rate; 95% confidence interval: 22%-65%). Median survival for all patients is 13 months. The combination of famotidine and high-dose continuous infusion + pulse IL-2 is active in metastatic melanoma.

Combined immunogene therapy of IL-6 and IL-15 enhances anti-tumor activity through augmented NK cytotoxicity

Many tumors evade host immunity by lowering expression of major histocompatibility complex (MHC) molecules. Theoretically, low MHC expression should activate natural killer (NK) cells and in some cases suppress tumor growth; nevertheless, some tumors also produce high concentrations of immunosuppressive cytokines, such as transforming growth factor (TGF)-β, to inhibit the activity of NK cells. Using a canine transmissible venereal tumor (CTVT) model, we have previously demonstrated that IL-6 is a strong antagonist for TGF-β. Herein, we found that IL-6 alone was unable to significantly promote TGF-β-inhibited NK activities. Conversely, IL-15 alone strongly promoted NK activities; however, NK activities were inhibited to baseline levels following the addition of TGF-β. Therefore, a new strategy using combined immunogene therapy of both IL-6 and IL-15 mediated by electroporation was used in this study. This combined IL-6 and IL-15 treatment effectively relieved the inhibitory effect of TGF-β and activated NK cell cytotoxicity of lymphokine-activated killer (LAK) cells. Similarly, in isolated DX5 + NK cells, only IL-6 and IL-15 in combination significantly overcame the inhibitory effect of TGF-β and promoted NK cytotoxicity. The group of BALB/c mice injected with plasmids with IL-6 and IL-15 genes (pIL-6/pIL-15) had the highest percentages of DX5 + NK cells as compared with either the pIL-6 or pIL-15 groups. Further, in SCID mice inoculated with CTVT, electroporation-mediated delivery of pIL-6/pIL-15 was significantly more efficient in suppressing both tumor establishment and tumor growth as compared with pIL-6 or pIL-15 inoculation alone. In addition, the anti-asialo GM-1 antibody abolished NK activities in SCID mice and resulted in outgrowth of the tumors. Together, these results suggest that the TGF-β-associated inhibition of NK cytotoxicity cannot be adequately restored by simply antagonizing TGF-β with IL-6: the co-existence of NK activating factors such as IL-15 is also important in restoring TGF-β-inhibited cytotoxicity. This study highlights the therapeutic potential of the pIL-6/pIL-15 combination by inhibiting TGF-β activity and enhancing NK cytotoxicity.

Lymphokine-activated killer cytotoxicity against leukaemic blast cells

While the sensitivity of cell lines and solid tumours to lymphokine-activated killer (LAK) cell cytotoxicity is extensively reported, the ability of these generated cytotoxic cells to lyse leukaemic blast cells is controversial. This study reports the successful generation of LAK cells with specificity for leukaemic blast cells. Control donors, patients in remission and patients with active leukaemia were capable of lysing allogeneic leukaemic blast cells. One patient was also capable of lysing autologous leukaemic blast cells. Successful LAK generation was achieved by the use of high dose recombinant interleukin-2 (rIL-2) while combinations of lymphokines did not improve LAK specificity or efficacy. These findings suggest that LAK immunotherapy may be considered for the treatment of patients with acute leukaemia.

Lymphokine-activated killer cell susceptibility in epirubicin-resistant and parental human non-small cell lung cancer (NSCLC)

The aim of this study was to evaluate that: (i) epirubicin-HCl (EPI) and lymphokine-activated killer (LAK) cells cytotoxicity may be mediated by free radical generation; and (ii) resistant H1299 cells may be more sensitive to combined treatment of LAK cells plus EPI than the LAK or EPI treatment alone. Viability of H1299 cells treated with EPI, LAK and LAK plus EPI was measured using the MTT test. Amount of glutathione (GSH), protein content and enzymatic activity were measured by spectrophotometer. Glutathione S-transferase (GST)-pi expression in the cells was determined by western blot analysis. LAK plus EPI combined treatment increased susceptibility of H1299 WT and H1299 EPI(R) (300-fold EPI resistant) cells to LAK cell cytotoxicity. The resistance of H1299 EPI(R) cells to EPI appears to be associated with a developed tolerance to free radicals, most likely because of a 2-fold increase in NADPH-dependent-cytochrome-P450 reductase (NADPH-CYP reductase) activity, 11-fold GST activity and 11-and 7-fold augmented selenium dependent and independent glutathione peroxidase (GSH-Px) activity, respectively. Amount of GST-pi in H1299 EPI(R) cells is statistically different from negative control and H1299 WT (p < 0.01). It is proposed that production of reactive oxygen species and hydrogen peroxide by the treatment of EPI and LAK cells can cause cytotoxicity of H1299 WT and H1299 EPI(R) cells. Superoxide dismutase, catalase, GSH-Px, GST, NADPH-CYP reductase and GSH must be considered as part of the intracellular antioxidant defense mechanism of H1299 WT and H1299 EPI(R) cells against reactive oxygen species. Combined treatment of EPI plus LAK cells caused the increasing cytotoxicity on the H1299 EPI(R) cells.

Continuous Infusion Interleukin-2 and Intravenous Famotidine in Metastatic Melanoma

Lymphokine-activated killer cell (LAK) cytotoxicity against tumor cells is induced by the use of high-dose infusional interleukin-2 (IL-2). LAK cytotoxicity against neoplastic cells may be augmented by famotidine. Twelve (12) patients have been treated with continuous infusion IL-2 (18 MIU/m2/24 hours) for 72 hours and famotidine 20 mg IVPB twice per day. Cycles were repeated every 3 weeks. These patients were of median age--67 years (range, 25-79), had a median performance status of 1 (range, 0-1), and had metastatic sites, including lung, lymph node, subcutaneous/soft tissue, and liver. The most common toxicities of this regimen were fever, rigors, nausea/emesis, hypophosphatemia, and hypomagnesemia. Three (3) partial responses have been seen (25% response rate). One (1) of these responders has undergone complete surgical resection and is disease-free at 15+ months. Four (4) patients are alive at a median of > 25 months. The median survival for all patients is 13 months. This combination of infusional IL-2 with famotidine is active in metastatic melanoma.

Continuous Infusion Interleukin-2 and Famotidine in Metastatic Kidney Cancer

Infusional interleukin-2 (IL-2) is able to elicit lymphokine-activated killer cell (LAK) cytotoxicity against kidney cancer in vitro and in vivo. Famotidine may be able to augment LAK cytotoxicity against neoplastic cells. Fifteen (15) patients were treated with continuous-infusion IL-2 (9-18 MIU/m2/24 hours) for 72 hours and famotidine 20 mg intravenously twice per day. Cycles were repeated every 3 weeks. These patients had a median age of 60 years (range, 29-72), had a median performance status of 1 (range, 0-1), and had metastatic sites, including lung, bone, lymph node, and liver. The most common toxicities of this regimen were hypophosphatemia, fever, nausea/emesis, rigors, elevated creatinine, and hypomagnesemia. One (1) complete and 6 partial responses have been seen (47% response rate). The median duration of response is 9 months. The median survival for all patients is 20 months. Five (5) patients are alive at a median of 36+ months. This combination of infusional IL-2 with famotidine is active in metastatic kidney cancer.

Enhancement of Lymphokine-Activated Killer Cell Cytotoxicity by Mistletoe Plant Extracts.

Anthroposophical cancer therapies focus on the use of mistletoe (MT) preparations and extracts from European mistletoe (Viscum album l.) belong to the complementary therapeutic regimens. Although widely used in Europe as an additional therapy in the treatment of patients suffering from different malignancies, these cancer therapies have remained relatively unknown in North America. To date, mistletoe is the most commonly used complementary cancer drug in Germany, applied as monotherapy or in combination with conventional therapies. ABNOBAviscum fraxini is a standardized extract from all parts of fresh mistle, obtained from the ash tree. This study aims to investigate the effects of MT on the activity of lymphokine-activated killer (LAK) cell cytotoxicity in vivo.Here, we demonstrate that in vivo treatment of mice with a standardized MT plant extract results in activation and function of LAK cells. We show an enhanced cytotoxicity against the NK-sensitive target cell line YAC-1 by flow cytometry. Our data show that in vivo application of MT can double the cytotoxic activity against malignant disease and suggest that one mechasim of action of MT is the induction of LAK-cell mediated immune response.