Reinhold Ramoner

IL-4 inhibits the TNF-α induced proliferation of renal cell carcinoma (RCC) and cooperates with TNF-α to induce apoptotic and cytokine responses by RCC: implications for antitumor immune responses

Objective: While previous reports clearly demonstrated antiproliferative effects of IL-4 on renal cell carcinoma (RCC) in vitro, the administration of IL-4 to patients with metastatic RCC in clinical trials could not recapitulate the promising preclinical results. In the present study we wanted to examine the context of IL-4 action and to establish conditions of enhanced IL-4 efficacy. Methods: Primary and permanent human RCC cells were cultured in either serum-supplemented or chemically defined, serum-free culture medium in the presence or absence of cytokines. Cell proliferation was assessed as [3H]-thymidine incorporation. Cell apoptosis was measured using the fluorescent DNA intercalator 7-aminoactinomycin D and flow cytometry. In addition, culture media conditioned by RCC were subjected to cytokine antibody array and cytokine multiplex analysis. Results: Our results indicate that the previously reported antiproliferative effects of IL-4 are serum-dependent. Under serum-free conditions, IL-4 failed to exhibit growth-inhibitory effects or was even growth-stimulatory. In a chemically defined, serum-free medium (AIM-V), however, IL-4 inhibited the TNF-α induced proliferation of RCC. IL-4 and TNF-α synergistically induced apoptosis of RCC as well as a complex cytokine response by RCC, which included the synergistic upregulation of RANTES and MCP-1. Conclusions: IL-4 alone has little effect on the spontaneous proliferation of RCC but can prevent the enhancement of proliferation induced by growth promoters like FBS and TNF-α. The concomitant growth inhibitory, apoptosis-inducing, and cytokine-enhancing effects of IL-4 in combination with TNF-α on RCC support the view that Th2 cytokines may be required for productive immune responses against RCC.

Bee venom secretory phospholipase A2 and phosphatidylinositol-homologues cooperatively disrupt membrane integrity, abrogate signal transduction and inhibit proliferation of renal cancer cells

Bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate (PtdIns(3,4)P2) act synergistically to induce cell death in tumour cells of various origins with concomitant stimulation of the immune system. Here, we investigated the mechanisms involved in such actions and examined structural requirements of PtdIns-homologues to inhibit tumour cells in combination with bv-sPLA2. Renal cancer cells were treated with bv-sPLA2 alone or in combination with PtdIns-homologues. Inhibitory effects on [3H] thymidine incorporation and intracellular signal transduction pathways were tested. Reaction products generated by bv-sPLA2 interaction with PtdIns(3,4)P2 were identified by mass spectrometry. Among the tested PtdIns-homologues those with a phosphate esterified to position 3 of the inositol head group, were most efficient in cooperating with bv-sPLA2 to block tumour cell proliferation. Growth inhibition induced by the combined action of bv-sPLA2 with either PtdIns(3,4)bisphosphate or PtdIns(3,4,5)trisphosphate were synergistic and accompanied by potent cell lysis. In contrast, PtdIns, which lacked the phosphate group at position 3, failed to promote synergistic growth inhibition. The combined administration of PtdIns(3,4)P2 and bv-sPLA2 abrogated signal transduction mediated by extracellular signal regulated kinase 1 and 2 and prevented transduction of survival signals mediated by protein kinase B. Surface expression of the epidermal growth factor (EGF)-receptor was reduced after PtdIns(3,4)P2-bv-sPLA2 administration and associated with a blockade of EGF-induced signalling. In addition, mass spectroscopy revealed that bv-sPLA2 cleaves PtdIns(3,4)P2 to generate lyso-PtdIns(3,4)P2. In conclusion, we suggest that the cytotoxic activity mediated by PtdIns(3,4)P2 and bv-sPLA2 is due to cell death that results from disruption of membrane integrity, abrogation of signal transduction and the generation of cytotoxic lyso-PtdIns(3,4)P2.

C-reactive protein is a strong predictor for anaemia in renal cell carcinoma: role of IL-6 in overall survival.

Study Type – Prognostic (case series)

Antigen-independent immune responses after dendritic cell vaccination

The ability of cultured, antigen-loaded dendritic cells (DCs) to induce antigen-specific T cell immunity in vivo has previously been demonstrated and confirmed. Immune monitoring naturally focuses on immunity against vaccine antigens and may thus ignore other effects of DC vaccination. Here we therefore focused on antigen-independent responses induced by DC vaccination of renal cell carcinoma patients.In addition to the anticipated response against the vaccine antigen KLH, vaccination with CD83+ monocyte-derived DCs resulted in a strong increase in the ex vivo proliferative and cytokine responses of PBMCs stimulated with LPS or BCG. In addition, LPS strongly enhanced the KLH-induced proliferative and cytokine response of PBMCs. Moreover, proliferative and cytokine responses of PBMCs stimulated with the homeostatic cytokines IL-7 and IL-15 were also clearly enhanced after DC vaccination. In contrast to LPS induced proliferation, which is well known to depend on monocytes, IL-7 induced proliferation was substantially enhanced after monocyte depletion indicating that monocytes limit IL-7 induced lymphocyte expansion.Our data indicate that DC vaccination leads to an increase in the ex vivo responsiveness of patient PBMCs consistent with a DC vaccination induced enhancement of T cell memory. Our findings also suggest that incorporation of bacterial components and homeostatic cytokines into immunotherapy protocols may be useful in order to enhance the efficacy of DC vaccination and that monocytes may limit DC vaccination induced immunity.

Monitoring of CD4+ and CD8+ T-Cell Responses After Dendritic Cell-Based Immunotherapy Using CFSE Dye Dilution Analysis

CFSE dye dilution analysis and [3H] thymidine incorporation were used side by side to assess proliferative responses of peripheral blood mononuclear cells (PBMCs) after vaccination of renal cell carcinoma patients (n=6) with antigen-loaded dendritic cells. Immune responses against the control antigen keyhole limpet hemocyanin (KLH) were induced in all patients. While [3H] thymidine incorporation revealed a 4 to 977-fold increase in KLH-induced proliferation (mean: 209-fold), CFSE-labeling experiments demonstrated that the KLH-responsive population of postvaccination PBMCs represented 7–53% (mean: 23%). Combining CFSE-labeling with T-cell subset analysis confirmed the presence of CD4+ KLH-reactive T cells but also revealed a substantial population of CD8+ KLH-reactive T cells in one patient as well as minor populations of CD8+ KLH-reactive T cells in three other patients. Our data indicate that CFSE dye dilution analysis is a valuable tool for immune monitoring after dendritic cell vaccination.

Attenuation of nucleoside and anti-cancer nucleoside analog drug uptake in prostate cancer cells by Cimicifuga racemosa extract BNO-1055

This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.