Rofecoxib has different effects on chemokine production in colorectal cancer cells and tumor immune splenocytes.

Cyclooxygenase-2 (COX-2) is overexpressed in colon tumors. Its main product is the immunosuppressive prostaglandin PGE2 that aids tumor immune escape. In this study, we analyzed mechanisms of action of the COX-2 inhibitor rofecoxib on the immune response to colorectal cancer in an animal model. The murine colorectal cancer cell line MC26, and splenocytes from BALB/c mice immune to irradiated MC26 cells, were incubated with rofecoxib or PGE2. In MC26 cells, 100 nM rofecoxib caused a complete abrogation of PGE2 production and inhibited cell proliferation. Splenocytes from tumor immune mice showed a 300% (P<0.01) increase in proliferation in response to irradiated MC26 cells, amplified to 450% (P<0.01) by 1 microM rofecoxib (n=3). MC26 cells incubated with 1 microM rofecoxib showed increased gene expression of CCL3, CCL5, and CCL20 (P<0.01). enzyme-linked immunosorbent assay tests also showed increased production of CCL5 and CCL20 (P<0.01). PGE2 reversed this effect causing a 40% reduction in chemokine gene expression (n=3). In contrast, splenocytes from naive BALB/c mice stimulated with irradiated MC26 cells had only a marginal chemokine response to rofecoxib. PGE2 caused a 50% down-regulation of CCL5 and CCL20 at the gene level (n=2) and 30% and 40% reduction of CCL3, CCL4, CCL5, and CCL20 at the protein level (n=2). Hence rofecoxib has a 2-fold effect upon the immune response to MC26 cells, by enhancing production of chemokines chemotactic for dendritic cells and also reducing PGE2-mediated inhibition of lymphoproliferation. Together, these may be sufficient for an effective TH1-mediated antitumor response. Rofecoxib may have potential as an addition to existing immunotherapy strategies.

Both soluble and membrane-bound forms of Flt3 ligand enhance tumor immunity following "suicide" gene therapy in a murine colon carcinoma model.

In prodrug-activated ("suicide") gene therapy, tumor cells are transfected with the gene for an enzyme that converts an inactive prodrug, such as ganciclovir (GCV), to a toxic compound. Transfected cells are killed on administration of GCV, as also are untransfected "bystander" cells. The ability of the dendritic cell stimulatory cytokine Flt3 ligand (Flt3-L) to modulate prodrug-activated gene therapy has been investigated. Transfectants of the murine colon carcinoma MC26 were generated expressing soluble (FLS) and membrane-bound forms of Flt3-L. They were inoculated together with wild-type MC26 cells and cells expressing herpes simplex virus-1 (HSV1) thymidine kinase into BALB/c mice, which were then administered GCV. Expression of Flt3-L or FLS prevented regrowth of tumor in most mice, which was comparable to the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), while tumors recurred in all mice receiving "suicide" gene therapy alone. Recurring tumor cells were resistant to direct killing by GCV but sensitive to "bystander" killing in vitro. Mice without tumor recurrence were rechallenged with unmodified MC26 cells. Of those mice given transfectants expressing GM-CSF, Flt3-L, or FLS, approximately 50% were immune to rechallenge. These mice also showed cytotoxic and proliferative responses to MC26 cells. These experiments show that both soluble and membrane-bound forms of Flt3-L were able to induce a protective immune response to colon carcinoma cells in a fashion similar to GM-CSF.