Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. ...Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro.
The human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells.
We designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of sensitive cancer cells, including colorectal cancer (CRC). Due to its short biological half-life after ...intravenous administration and related clinical ineffectiveness, novel formulations of TRAIL need to be developed. Here we propose
bacteria as a vehicle for local delivery of human soluble TRAIL (hsTRAIL) in CRC. The use of common probiotics targeting guts as carriers for TRAIL could ensure its sustained release at the tumor site and extend the duration of its activity. We have already engineered hsTRAIL-secreting
bacteria and showed their effectiveness in elimination of human CRC cells in vitro and in vivo in a mouse subcutaneous model. Here,
(hsTRAIL+) were administered by gastric gavage to SCID mice with orthotopically developed HCT116 tumor in cecum, in monotherapy or in combination with metformin (MetF), already shown to enhance the hsTRAIL anti-tumor activity in subcutaneous CRC model. Oral administration of
(hsTRAIL+) resulted in significant progression of HCT116 tumors and shortening of the colon crypts. Secretion of hsTRAIL in the colon was accompanied by infiltration of the primary tumor with M2-macrophages, while MetF promoted transient colonization of the gut by
. Our study indicates that
bacteria after oral administration enable delivery of biologically active hsTRAIL to colon, however its potential therapeutic effect in CRC treatment is abolished by its pro-tumorigenic signalling, leading to the recruitment of M2-macrophages and tumor growth promotion.
Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) induces apoptosis of many cancer cells, including CRC cells, being non-harmful for normal ones. However, recombinant form of human ...TRAIL failed in clinical trial when administered intravenously. To assess the importance of TRAIL in CRC patients, new form of TRAIL delivery would be required. Here we used genetically modified, non-pathogenic Lactococcus lactis bacteria as a vehicle for local delivery of human soluble TRAIL (hsTRAIL) in CRC. Operating under the Nisin Controlled Gene Expression System (NICE), the modified bacteria (L. lactis(hsTRAIL+)) were able to induce cell death of HCT116 and SW480 human cancer cells and reduce the growth of HCT116-tumor spheres in vitro. This effect was cancer cell specific as the cells of normal colon epithelium (FHC cells) were not affected by hsTRAIL-producing bacteria. Metformin (MetF), 5-fluorouracil (5-FU) and irinotecan (CPT-11) enhanced the anti-tumor actions of hsTRAIL in vitro. In the NOD-SCID mouse model, treatment of subcutaneous HCT116-tumors with L. lactis(hsTRAIL+) bacteria given intratumorally, significantly reduced the tumor growth. This anti-tumor activity of hsTRAIL in vivo was further enhanced by oral administration of MetF. These findings indicate that L. lactis bacteria could be suitable for local delivery of biologically active human proteins. At the same time, we documented that anti-tumor activity of hsTRAIL in experimental therapy of CRC can be further enhanced by MetF given orally, opening a venue for alternative CRC-treatment strategies.
Although blood monocytes exhibit significant cytotoxic activity against tumor cells, the function of tumor infiltrating macrophages (TIM) is depressed in cancer patients. This study addresses the ...question of how the antitumor response of human monocytes, assessed by production of cytokines (tumor necrosis factor alpha, TNF; IL-10; IL-12p40) and cytotoxicity, is altered by exposure to cancer cells. Tumor cell--pre-exposed monocytes restimulated with tumor cells showed significantly decreased production of TNF, IL-12, increased IL-10 (mRNA and release) and inhibition of IL-1 receptor-associated kinase-1 (IRAK-1) expression. This down-regulation of cytokine production was selective, as the response of pre-exposed monocytes to lipopolysaccharide (LPS) was unaffected. Treatment of tumor cell--pre-exposed monocytes with hyaluronidase (HAase) improved their depressed production of TNF, while HAase-treated cancer cells did not cause monocyte dysfunction. The response of hyaluronan (HA)--pre-exposed monocytes to stimulation with tumor cells was also inhibited. Cytotoxic activity of monocytes pretreated with cancer cells was also decreased. This study shows that tumor cells selectively deactivate monocytes and suggests that tumor cell-derived HA by blocking CD44 on monocytes inhibits their antitumor response. These observations may provide some explanation for the depressed function of TIM in human malignancy.
This study was undertaken to determine how human pancreatic cancer (HPC-4) cells transduced with the TNF-GFP fusion gene (TLG) alter the antitumor response of human monocytes in vitro and whether ...they could act as an antitumor vaccine. In our model, HPC-4 cells were transduced with retroviral vector harboring TLG gene and designated as HPC-4(TLG). The TLG protein expression was confirmed by Western blot and flow cytometry analysis. Monocytes were co-cultured with transduced and control HPC-4 cells. The secretion of TNF, IL-10 and IL-12 was measured by ELISA. The cytotoxicity of monocytes against HPC-4 cells was determined by MTT test. The results show that the HPC-4(TLG) cells expressed membrane-bound, intracellular and secretory TLG protein. When cultured with HPC-4(TLG) cells, monocytes released a higher amount of TNF, but IL-10 and IL-12 secretion was inhibited. The pre-exposure of monocytes to HPC-4(TLG), but not to HPC-4, cells did not decrease TNF nor increase IL-10 production, thus not leading to monocyte deactivation. Also, the antitumor cytotoxicity of monocytes stimulated with HPC-4(TLG) was not downregulated, which occurred when non-transduced HPC-4 cells were used. In conclusion, compared to parental HPC-4 cells, TLG gene transduced HPC-4 cells induced stronger antitumor response of monocytes in vitro and prevented deactivation of monocytes.
Synthesis and localization of inducible nitric oxide synthase mRNA (iNOS-mRNA) and iNOS protein in the cultures of human monocytes (Mphi) and colon carcinoma cell line (DeTa) that resulted in nitric ...oxide (NO) synthesis has been studied. The iNOS-mRNA was observed around the sixth hour of culture and peaked at the twelfth hour. The iNOS-mRNA, as determined by the in situ hybridization and iNOS protein, as detected by staining with specific anti-iNOS monoclonal antibodies, were observed preferentially in the cytoplasm of some Mphi, but not in cancer cells. Mphi cultured alone did not show detectable iNOS-mRNA expression and iNOS protein. Mphi sorted out from tumor cells after 8 h of co-culture expressed iNOS protein and iNOS-mRNA, which were not detected in Mphi without previous contact with cancer cells. Prevention of NO synthesis by (L-N5-1-iminoethyl)-ornithine (L-NIO) partly inhibited Mphi cytotoxic activity against DeTa (NO-inducing cancer cell line) but not against the human pancreatic cancer (HPC-4) cell line that does not induce NO production in Mphi. This suggests that Mphi cytotoxic activity, at least in some cases, may be NO dependent. These observations provide further evidence that Mphi can be directly stimulated by cancer cells for de novo production of NO and suggest that iNOS occurring in the tumor-infiltrating macrophages may arise as a result of their interactions with tumor cells. However, because only some tumor cells are able to induce NO production in a small proportion of Mphi, its role in the anti-tumor response of the host is probably limited.
Tumour necrosis factor alpha (TNF) mRNA is detected in the macrophage infiltrate surrounding the tumour, but the cellular/molecular interactions leading to TNF gene expression in macrophages are ...unknown. The in vitro system in which human blood monocytes are stimulated with human cancer cells for TNF release was used to study such interactions. Monoclonal antibodies (MAbs) against various adhesion molecules (LFA-1, LFA-3, ICAM-1, VNR, VLA beta I chain) were unable to block TNF production in co-culture of monocytes with a human pancreatic carcinoma (HPC) cell line. However, anti-CD44 and anti-HLA-DR MAbs effectively blocked TNF release and TNF-mRNA induction in monocytes. Pre-incubation of monocytes with anti-HLA-DR and tumour cells with anti-CD44 MAbs had a similar effect. It was concluded that CD44 molecules are involved in tumour-monocyte interactions and that HLA-DR determinants of monocytes are engaged in signal transduction for TNF gene activation. These findings may suggest that certain surface determinants of tumour cells act as ligands for MHC class-II molecules and induce TNF production in monocytes.
The control of expression of MHC class II molecules on antigen-presenting cells is important for the induction of immunity, while aberrant expression of these molecules plays a role in the ...immunopathology of autoimmune diseases. This study explored the role of tumor necrosis factor alpha (TNF) in controlling the level of HLA class II mRNA in human monocytes. Exposure of monocytes to exogenous recombinant TNF (rTNF) selectively up-regulated DR alpha-mRNA but not DP or DQ alpha-mRNA. Inhibitors of TNF synthesis, pentoxifylline (PTX) and thalidomide, inhibited TNF mRNA accumulation in LPS-activated monocytes and down-regulated DR mRNA but not DP or DQ mRNA. The inhibitory effect of anti-TNF monoclonal antibody (MAb) indicated that endogenously generated TNF acted extracellularly. Anti-p75 TNF-R2 receptor and to a lesser extent anti-p55 TNF-R1 MAbs inhibited TNF-mediated up-regulation of DR mRNA and TNF mRNA. Taken together, this implies that endogenously generated TNF plays a role in controlling isotype-specific MHC class II gene expression in human monocytes/macrophages. These results may have some implications for anti-tumor response and autoimmunity.
Human peripheral blood monocytes cocultured with tumour cells were used as an in vitro model of in situ interactions between tumour-infiltrating macrophages and the tumour. Tumour cells stimulated de ...novo expression of the human tumour necrosis factor alpha (TNF) gene in monocytes and caused the release of TNF into the culture supernatant. A group of 14 patients with stage IVA gastric cancer receiving adjuvant chemotherapy (5-FU, Adriamycin, mitomycin C: FAM) or immunochemotherapy (BCG+FAM) was investigated for the ability of monocytes to produce TNF in vitro upon stimulation with tumour cells or purified protein derivative of tuberculin (PPD). Patients were followed at biweekly intervals, i.e. before each instillation of BCG epicutaneously over a period of 10 weeks. It was found that monocytes of some patients receiving BCG at the end of the observation period had an enhanced ability to produce TNF following stimulation with tumour cells. In contrast, such production was not substantially altered during the study period in patients on chemotherapy. PPD-induced TNF production was much weaker and was not significantly changed during this observation time. We infer that BCG immunotherapy may induce the subtle changes in some cancer patients that lead to an increased interaction between monocytes and tumour cells and result in enhanced production of cytokine(s) with antitumour properties.
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