Yong X. Xu

Immunomodulatory activity of resveratrol: Suppression of lymphocyte proliferation, development of cell-mediated cytotoxicity, and cytokine production

trans-Resveratrol, a phytoalexin found in grapes, wine, and other plant products, has been shown to have anti-inflammatory, antioxidant, and antitumor activities. Many of these beneficial effects of resveratrol require participation of the cells of the immune system; however, the effect of resveratrol on the development of immunological responses remains unknown. We have investigated the effect of resveratrol on mitogen/antigen-induced proliferation of splenic lymphocytes, induction of cytotoxic T lymphocytes (CTLs) and lymphokine activated killer (LAK) cells, and the production of the cytokines interferon (IFN)-gamma, interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, and IL-12. We found that mitogen-, IL-2-, or alloantigen-induced proliferation of splenic lymphocytes and the development of antigen-specific CTLs were suppressed significantly at 25-50 microM resveratrol. The generation of LAK cells at similar concentrations was less sensitive to the suppressive effect of resveratrol. The suppression of cell proliferation and CTL generation by resveratrol was not only reversible, but in some cases the response (mitogen/IL-2-induced proliferation and CTL generation) was actually enhanced following pretreatment of cells with resveratrol. Resveratrol also inhibited the production of IFN-gamma and IL-2 by splenic lymphocytes, and the production of TNF-alpha and IL-12 by peritoneal macrophages. The inhibition of cytokine production by resveratrol was irreversible. Further, resveratrol blocked the activation of the transcription factor NF-kappaB without affecting basal NF-kappaB activity. The latter result suggests that resveratrol inhibits cell proliferation, cell-mediated cytotoxicity, and cytokine production, at least in part through the inhibition of NF-kappaB activation.

Resveratrol selectively inhibits leukemia cells: a prospective agent for ex vivo bone marrow purging

Ex vivo purging of contaminating tumor cells may reduce the incidence of relapse in patients undergoing bone marrow transplantation. In this study we demonstrate that resveratrol, a phytoalexin with anti-oxidant and chemopreventive activity, exhibits anti-leukemic activity against mouse (32Dp210, L1210) and human (U937, HL-60) leukemic cell lines by inhibiting cell proliferation. Long-term exposure to resveratrol also inhibits the clonal growth of normal hematopoietic progenitor cells but at a higher IC50 of resveratrol than that for most of the leukemia cell lines tested. The inhibitory effect of resveratrol on hematopoietic progenitors is partially reversible, whereas the effect on leukemia cells is largely irreversible. The inhibition of leukemia cells by resveratrol involves nucleosomal DNA fragmentation (apoptosis). On the other hand, resveratrol does not induce or enhance spontaneously occurring apoptotic death in normal hematopoietic progenitor cells. In vivo experiments performed with untreated and resveratrol-treated bone marrow showed comparable hematopoietic reconstitution in lethally irradiated mice (10 Gy) as determined by survival, hematologic recovery, and the number of hematopoietic progenitor cells present in the marrow of reconstituted animals. Taken together, these results indicate the potential use of resveratrol for ex vivo pharmacological purging of leukemia cells from bone marrow autografts without significant loss in the hematopoietic activity of progenitor cells. Bone Marrow Transplantation (2000) 25, 639–645.

Interleukin-12 (IL-12) gene therapy of leukemia: Immune and anti-leukemic effects of IL-12-transduced hematopoietic progenitor cells

Recombinant interleukin-12 (rIL-12) is a potent immunomodulatory cytokine that has been shown to exert strong antitumoral and antimetastatic activity against several mouse tumors grown as solid lesions. The therapeutic efficacy of rIL-12 against hematological tumors and the transfer of IL-12 genes into hematopoietic progenitor cells to deliver IL-12 to the bone marrow (BM) to treat residual leukemia has not been studied adequately. We have investigated the retroviral-mediated transduction of hematopoietic progenitor cells with IL-12 genes and the in vivo anti-leukemic activity of transduced cells against the murine myeloid leukemia cell line 32Dp210. We were able to efficiently transduce the IL-3-dependent 32Dc13 myeloid progenitor cell line and primary hematopoietic progenitor cells using an MFG-based polycistronic retroviral vector containing the cDNAs of p35 and p40 murine IL-12 genes. 32Dc13 myeloid progenitor cells expressing IL-12 genes (32DIL-12 cells) have stably secreted biologically active murine IL-12 for >9 months. Mice transplanted with 32DIL-12 cells transiently express the transgene in the BM and spleen, which is associated with a rapid elevation of interferon-γ (IFN-γ) in the circulation and with secretion of IFN-γ by spleen cells in vitro. In addition, spleen and BM cells of mice injected with 32DIL-12 cells readily acquire the capacity to lyse natural killer cell-sensitive YAC-1 target cells and 32Dp210 myeloid leukemia cells. Furthermore, whereas mice challenged with leukemia cells suffered 100% mortality within 14 days, ∼40% of mice coinjected with 32Dp210 leukemia cells and 32DIL-12 progenitor cells exhibited long-term, leukemia-free survival (>60 days). This study demonstrates that IL-12 can be stably expressed in hematopoietic cells; in addition, when transplanted, transduced cells induce IFN-γ production and activation of natural killer cells, both of which may be involved in inhibiting the progression of leukemia in vivo.

TNF-alpha Gene Therapy with Myeloid Progenitor Cells Lacks the Toxicities of Systemic TNF-alpha Therapy

We examined the antileukemic activity and the toxicity of HPC transduced with human tumor necrosis factor (TNF) cDNA. Both clonal (32Dcl3) and BM-derived primary hematopoietic progenitors (BM-Prog) expressing hTNF-alpha gene (32DTNF-alpha and BMTNF-alpha cells, respectively) inhibited the development of leukemia in mice with a small dose of 32Dp210 cells, a myeloid leukemia cell line. Whether the trans-gene expressing 32DTNF-alpha cells produce toxicities commonly associated with systemic TNF-alpha therapy was determined by examining the effect of TNF-alpha-secreting progenitor cells on body weight, tissue histology, growth of HPC, and engraftment of BMT. Administration of a low or high dose of TNF-alpha-secreting 32DTNF-alpha cells to mice failed to produce loss in body weight, a measure of TNF-alpha-related cachexia. There was also no evidence of tissue necrosis or mononuclear cell (MNC) infiltration in lung, liver, kidney, or intestine of mice injected with transduced progenitor cells. Furthermore, 32DTNF-alpha cells showed no effect on the clonal growth of HPC in colony-forming assays or loss of cellularity in BM, spleen, or blood. Finally, TNF-alpha-secreting cells were found not to interfere with the engraftment of BM transplant and hematopoietic reconstitution thereafter. We conclude from these findings that unlike systemic administration of TNF-alpha, TNF-alpha gene therapy with transduced HPC is nontoxic and may have a role in eradicating residual leukemia after BMT.

Resveratrol for ex vivo purging of hematopoietic autografts

Abstract Novel purging strategies that would selectively eliminate contaminating tumor cells without affecting the hematopoietic activity of progenitor cells are needed for ex vivo purging of autografts. In this study we demonstrate that resveratrol, a chemopreventive agent naturally present in grapes, exhibits antiproliferative activity against mouse and human leukemic cell lines. Long term exposure to resveratrol also inhibited the clonal growth of normal hematopoietic progenitor cells but at a higher IC 50 than the leukemia cell lines tested. The inhibitory effect of resveratrol on hematopoietic progenitors is reversible, whereas the effect on leukemia cells is largely irreversible. Resveratrol induced apoptosis in leukemia cells, but it neither induces or enhances the spontaneously occurring DNA fragmentation in normal progenitor cells. More importantly, in vivo experiments demonstrated that treatment of bone marrow cells with resveratrol does not impair their ability to engraft in lethally irradiated mice. Overall, these data indicate that resveratrol may be used for pharmacological purging of autografts without significant decrease in the hematopoietic activity of progenitor cells.