Yona Kalechman

Anti-IL-10 Therapeutic Strategy Using the Immunomodulator AS101 in Protecting Mice from Sepsis-Induced Death: Dependence on Timing of Immunomodulating Intervention

The role of IL-10 in experimental sepsis is controversial. The nontoxic immunomodulator, ammonium trichloro(dioxoethylene-o,o′)tellurate (AS101) has been previously shown to inhibit IL-10 expression at the transcriptional level. In this study, we show that in mice subjected to cecal ligation and puncture (CLP), treatment with AS101 12 h after, but not before, CLP significantly increased survival of septic mice. This was associated with a significant decrease in serum IL-10 and in IL-10 secretion by peritoneal macrophages 24–48 h after CLP. At that time, the ability of these cells to secrete TNF-α and IL-1β was restored in AS101-treated mice. The increased survival of AS101-treated mice was due to the inhibition of IL-10, since cotreatment with murine rIL-10 abolished the protective activity of AS101. AS101 increased class II Ag expression on peritoneal macrophages, severely depressed in control mice, while it did not affect the expression of class I Ags. This was accompanied by a significant elevation in the level of IFN-γ secreted by splenocytes. Moreover, AS101 ameliorated bacterial clearance in the peritoneum and blood and decreased severe multiple organ damage, as indicated by clinical chemistry. Furthermore, myeloperoxidase levels in the liver and lung of AS101-treated mice, an indirect means of determining the recruitment of neutrophils, were significantly decreased. We suggest that nontoxic agents such as AS101, with the capacity to inhibit IL-10 and stimulate macrophage functions, may have clinical potential in the treatment of sepsis, provided they are administered during the phase of sepsis characterized by immune suppression.

The protective role of the immunomodulator AS101 against chemotherapy‐induced alopecia studies on human and animal models

The immunomodulator AS101 has been demonstrated to exhibit radioprotective and chemoprotective effects in mice. Following phase‐I studies, preliminary results from phase‐II clinical trials on non‐small‐cell‐lung‐cancer patients showed a reduction in the severity of alopecia in patients treated with AS101 in combination with chemotherapy. To further substantiate these findings, the present study was extended to include 58 patients treated either with the optimal dose of 3 mg/m2 AS101 combined with carboplatin and VP‐16, or with chemotherapy alone. As compared with patients treated with chemotherapy alone, there was a significant decrease in the level of alopecia in patients receiving the combined therapy. The newly developed rat model was used to elucidate the protective mechanism involved in this effect. We show that significant prevention of chemotherapy‐induced alopecia is obtained in rats treated with Ara‐C combined with AS101, administered i.p. or s.c. or applied topically to the dorsal skin. We show that this protection by AS101 is mediated by macrophage‐derived factors induced by AS101. Protection by AS101 can be ascribed, at least in part, to IL‐1, since treatment of rats with IL‐1RA largely abrogated the protective effect of AS101. Moreover, we demonstrate that in humans there is an inverse correlation between the grade of alopecia and the increase in IL‐1α. In addition, protection by AS101 could be related to PGE2 secretion, since injection of indomethacin before treatment with AS101 and Ara‐C partly abrogated the protective effect of AS101. To assess the ability of AS101 to protect against chemotherapy‐induced alopecia, phase‐II clinical trials have been initiated with cancer patients suffering from various malignancies.

Bone marrow-sparing and prevention of alopecia by AS101 in non-small-cell lung cancer patients treated with carboplatin and etoposide.

PURPOSE The aim of this study was to evaluate the ability of the immunomodulator AS101 to prevent chemotherapy-induced neutropenia and thrombocytopenia and thus allow patients to receive full-dose antineoplastic agents according to protocol design. We also aimed to determine the production level of various hematopoietic growth factors in treated patients. PATIENTS AND METHODS This study of 44 unresectable or metastatic non-small-cell lung cancer (NSCLC) patients was an open-label prospective randomized study of standard chemotherapy alone versus chemotherapy plus AS101. Each patient received carboplatin (300 mg/m2 intravenously [IV] on day 1 of a 28-day cycle, and etoposide (VP-16) (200 mg/m2 orally) on days 3, 5, and 7 of each cycle. AS101 was administered at 3 mg/m2 three times per week starting 2 weeks before chemotherapy. RESULTS AS101, which manifested no major toxicity, significantly reduced neutropenia and thrombocytopenia and thus allowed all treated patients to receive full-dose antineoplastic agents, in contrast to only 28.5% of the control group. Continuous treatment with AS101 significantly reduced the number of days per patient of thrombocytopenia and neutropenia and did not provide protection to tumor cells as reflected by the higher overall response rate compared with the chemotherapy-alone arm. Interestingly, AS101 treatment also significantly prevented chemotherapy-induced alopecia. These effects correlate with the ability of AS101-treated patients to increase significantly the production of colony-stimulating factors (CSFs) interleukin-1 alpha (IL-1 alpha) and IL-6. CONCLUSION AS101 has significant bone marrow (BM)-sparing effects and prevents hair loss in chemotherapy-treated patients, with minimal overall toxicity. These effects are probably due to increased production of IL-1 alpha, IL-6, and granulocyte-macrophage (GM)-CSF.

Ammonium Trichloro(dioxoethylene-o,o′)tellurate (AS101) Sensitizes Tumors to Chemotherapy by Inhibiting the Tumor Interleukin 10 Autocrine Loop

Cancer cells of different solid and hematopoietic tumors express growth factors in respective stages of tumor progression, which by autocrine and paracrine effects enable them to grow autonomously. Here we show that the murine B16 melanoma cell line and two human primary cultures of stomach adenocarcinoma and glioblastoma multiforme (GBM) constitutively secrete interleukin (IL)-10 in an autocrine/paracrine manner. This cytokine is essential for tumor cell proliferation because its neutralization decreases clonogenicity of malignant cells, whereas addition of recombinant IL-10 increases cell proliferation. The immunomodulator ammonium trichloro(dioxoethylene-o,o′)tellurate (AS101) decreased cell proliferation by inhibiting IL-10. This activity was abrogated by exogenous addition of recombinant IL-10. IL-10 inhibition by AS101 results in dephosphorylation of Stat3, followed by reduced expression of Bcl-2. Moreover, these activities of AS101 are associated with sensitization of tumor cells to chemotherapeutic drugs, resulting in their increased apoptosis. More importantly, AS101 sensitizes the human aggressive GBM tumor to paclitaxel both in vitro and in vivo by virtue of IL-10 inhibition. AS101 sensitizes GBM cells to paclitaxel at concentrations that do not affect tumor cells. This sensitization can also be obtained by transfection of GBM cells with IL-10 antisense oligonucleotides. Sensitization of GBM tumors to paclitaxel (Taxol) in vivo was obtained by either AS101 or by implantation of antisense IL-10-transfected cells. The results indicate that the IL-10 autocrine/paracrine loop plays an important role in the resistance of certain tumors to chemotherapeutic drugs. Therefore, anti-IL-10 treatment modalities with compounds such as AS101, combined with chemotherapy, may be effective in the treatment of certain malignancies.

The effect of a single whole-blood transfusion on cytokine secretion

The effect of a single whole-blood transfusion on the cascade of cytokine secretion was studied in patients with chronic renal failure. The results indicate that 1 week after blood transfusion, no significant changes were observed in the secretion of interleukin-2, colony-stimulating factor, tumor necrosis factor, and γ-interferon. However, 2 weeks after blood transfusion, a sharp decrease was observed in the generation of these cytokines. A decrease of about 70% was observed in interleukin-2, tumor necrosis factor, and γ-interferon secretion. The production of colony-stimulating factor 2 weeks after blood transfusion amounted to about 30% less than baseline levels. No statistical differences in interleukin-1 production were observed throughout the study. In addition, we found that the decrease in cytokine secretion was paralleled by a sharp increase in thein vitro secretion of prostaglandin E2. Thus the beneficial effect of blood transfusion on graft survival might be due in part to an immunosuppressive effect brought about by immuno-regulatory changes via the cascade of cytokine secretion.

Hair growth induction by the Tellurium immunomodulator AS101: association with delayed terminal differentiation of follicular keratinocytes and ras-dependent up-regulation of KGF expression

The synthetic immunomodulator AS101{ammonium trichloro(dioxoethylene‐o,o’)tellurate} was previously found to protect cancer patients from chemotherapy‐induced bone marrow toxicity and alopecia. Here we show that AS101 induces hair growth in nude and normal mice. AS101 possesses the dual ability to both induce anagen and retard spontaneous catagen in the C57BL/6 mouse model. Anagen induced by AS101 is mediated by keratinocyte growth factor (KGF), as it is abrogated both in nude mice co‐treated with AS101 plus neutralizing anti KGF antibodies and in AS101‐treated transgenic mice expressing a dominant‐negative KGF receptor transgene in basal keratinocytes. AS101 up‐regulates KGF expression by activating the ras signaling pathway in cultured fibroblasts. AS101‐induced delayed catagen is associated with inhibition of terminal differentiation marker expression both in nude and C57BL/6 mice epidermal follicular keratinocytes and in cultures of primary mouse follicular keratinocytes induced to differentiate. This activity is associated with relatively sustained elevation of p21waf. Delayed expression of terminal differentiation markers was not induced by AS101 in follicular keratinocytes from p21waf knockout mice. Because similar results were obtained with cultures of primary human keratinocytes and fibroblasts, preliminary case report studies revealed substantial hair growth when AS101 was topically applied on three adolescents who had remained alopeciac 1–2 years after chemotherapy. The results emphasize the unique mode of action of AS101 and highlight its potential clinical use for treating certain types of alopecia.

Synergistic anti-tumoral effect of paclitaxel (taxol)+AS101 in a murine model of B16 melanoma: Association with ras-dependent signal-transduction pathways

Optimal doses of paclitaxel (Taxol) combined with the immunomodulator AS101, previously shown to have anti‐tumoral effects, administered to B16 melanoma‐bearing mice decreased tumor volume and resulted in over 60% cure. Paclitaxel+AS101 directly inhibited the clonogenicity of B16 melanoma cells in a synergistic, dose‐dependent manner. We suggest that this results from both reduced paclitaxel‐induced bone marrow toxicity and induction of differential signal‐transduction pathways, which lead to apoptosis of tumor cells. Paclitaxel+AS101 synergistically activated c‐raf‐1 and MAPK ERK1 and ERK2. This activation was essential for the synergistic induction of p21waf protein. Cell‐cycle analysis of B16 cells treated with both compounds revealed an increased accumulation in G2M, though AS101 alone produced significant G1 arrest. These activities were ras‐dependent. AS101+paclitaxel induced significant synergistic phosphorylation (inactivation) of the anti‐apoptotic protein Bcl‐2. Whereas phosphorylation of Bcl‐2 by paclitaxel was raf‐dependent only, the synergistic effect of both compounds together was ras‐, raf‐ and MAPK‐dependent. No effect of the combined treatment on Bax protein expression was observed. We suggest that AS101 renders more cells susceptible to Bcl‐2 phosphorylation by paclitaxel, possibly by increasing the accumulation of paclitaxel‐induced cells in G2M. Exposure of B16 cells to clinically achievable concentrations of paclitaxel+AS101 increased the rate of apoptosis of treated cells. Apoptosis induced by AS101 alone was both raf‐ and MAPK‐dependent, while that induced by paclitaxel was raf‐dependent only. Int. J. Cancer 86:281–288, 2000.

Redox Modulation of Adjacent Thiols in VLA-4 by AS101 Converts Myeloid Leukemia Cells from a Drug-Resistant to Drug-Sensitive State

Interaction between the integrin VLA-4 on acute myelogenous leukemia (AML) cells with stromal fibronectin is a decisive factor in chemotherapeutic resistance. In this study, we provide a rationale for a drug repositioning strategy to blunt integrin activation in AML cells and restore their sensitivity to chemotherapy. Specifically, we demonstrate that the nontoxic tellurium compound AS101, currently being evaluated in clinical trials, can abrogate the acquired resistance of AML. Mechanistic investigations revealed that AS101 caused redox inactivation of adjacent thiols in the exofacial domain of VLA-4 after its ligation to stromal fibronectin. This effect triggered cytoskeletal conformational changes that decreased PI3K/Akt/Bcl2 signaling, an obligatory step in chemosensitization by AS101. In a mouse xenograft of AML derived from patient leukemic cells with high VLA-4 expression and activity, we demonstrated that AS101 abrogated drug resistance and prolonged survival in mice receiving chemotherapy. Decreased integrin activity was confirmed on AML cells in vivo. The chemosensitizing activity of AS101 persisted in hosts with defective adaptive and innate immunity, consistent with evidence that integrin deactivation was not mediated by heightening immune attack. Our findings provide a mechanistic rationale to reposition the experimental clinical agent, AS101, to degrade VLA-4-mediated chemoresistance and improve clinical responses in patients with AML.

Tellurium Compound AS101 Induces PC12 Differentiation and Rescues the Neurons from Apoptotic Death

Abstract: Parkinsons disease is characterized by the loss of dopaminergic neurons in the substantia nigra (SN). Studies show that anti‐apoptotic and neurotrophic agents are suitable candidates to prevent delayed cell death and/or restore neural function. Here we present the nontoxic immunomodulating compound AS101, which has the ability to induce neurite outgrowth and neural differentiation in PC12 cells. The present study shows that components of the ras signaling pathway are crucial for AS101‐induced PC12 differentiation. These include p21ras and its downstream effectors, c‐raf‐1 and MEK, as well as PI3K. Moreover, these components mediate AS101‐induced upregulation of p21waf, which is obligatory for AS101‐induced PC12 differentiation. Furthermore, nitric oxide plays a significant role in these AS101 activities. Finally, we show that AS101 prevents apoptosis of NGF‐differentiated PC12 cells after NGF withdrawal. Taken together, these results suggest that AS101 induces PC12 cell differentiation and survival by activating the ras‐ERK1/2 and ras‐PI3K signal transduction pathways, as well as inducing NO production. Our findings may be important in understanding the regulation of survival/apoptosis of neurons deprived of neurotropic support. Futhermore the data propose that AS101 may have clinical potential in the treatment of neurodegenerative disorders like Parkinsons disease.

Photofrin II induces cytokine secretion by mouse spleen cells and human peripheral mononuclear cells

The aim of our study was to find out if Photofrin II, a cytotoxic drug used routinely in photodynamic therapy (PDT), can induce immune responses in vitro, and to compare its effects with those of the protoporphyrin 9, hemin, which also has antitumor properties. We tested the effect of these porphyrins on lymphocyte proliferation and secretion of interleukin-2, interleukin-3, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma), by human or murine mononuclear cells (MNC) without an activating light. Both the Photofrin II- and hemin-treated cells showed a significant increase in cytokine secretion in the presence of suboptimal concentrations of mitogen. Moreover, Photofrin II and hemin significantly increased production of TNFalpha and IFNgamma even in the absence of mitogen. The cellular binding sites of Photofrin II and hemin to MNC were localized by electromicroscopy or fluorescence. Combined stimulation of cells by mitogens and porphyrins maintained optimal vital ionic balance of potassium, sodium and chlorine in the lymphocytes. In the cells thus treated there was a significant increase in intracellular calcium, a vital second messenger for lymphokine secretion. We demonstrate that the effect of Photofrin II on the immune system involves enhanced cytokine secretion which may account for the subsequent tumor eradication by PDT.