Silvia I. Gervasoni

Th2/Th1 switch induced by a single low dose of cyclophosphamide in a rat metastatic lymphoma model.

Abstract. Cyclophosphamide (Cy) is an alkylating agent widely used in cancer chemotherapy. It has a bimodal effect on the immune system, depending on the dose and schedule of administration. We have previously demonstrated that a single low dose of Cy has an antimetastatic effect, achieved through immunomodulation, in lymphoma bearing rats. Such a treatment reduced the splenic production of IL-10, TGF-β, and NO, restoring the lymphoproliferative capacity. A shift from immunosuppression to immunopotentiation induced by low-dose Cy treatment was mainly mediated by a decrease in IL-10 production. The present study focused on the analysis of the modulation of type-1 cytokine levels by treatment with a single low dose of Cy and the effect these cytokines (IL-2 and IFN-γ) and IL-10 have on primary tumor and metastatic cell growth. Our results suggest that a single low dose of Cy induces a Th2/Th1 shift in the cytokine profile of lymphoma-bearing rats, which may be responsible for its antimetastatic effect. A direct action of IL-10 as a growth factor and IFN-γ as a cytotoxic factor on metastatic cells is also shown.

Down regulation of T-cell-derived IL-10 production by low-dose cyclophosphamide treatment in tumor-bearing rats restores in vitro normal lymphoproliferative response.

In previous reports, we demonstrated an inhibitory effect of a single low-dose of cyclophosphamide (Cy) on spontaneous and experimental metastasis of a rat lymphoma (L-TACB). This antimetastatic effect could be adoptively transferred by immune spleen cells from Cy-treated tumor-bearing rats and it was abrogated by the use of immunosuppressed hosts, suggesting an immunomodulatory effect. Subsequently, we found that increased levels of TGF-beta, IL-10 and NO were involved in tumor-induced immunosuppression by inhibiting lymphocyte proliferation. The treatment of tumor-bearing rats with low-dose Cy reduced the splenic production of these suppressive cytokines, restoring the lymphoproliferative capacity otherwise diminished during tumor growth. Here, we investigated the changes of the cytokines modulated by the Cy therapy that are responsible for the restoration of the lymphoproliferative response and determined the spleen cell type producing TGF-beta, IL-10 and NO in our experimental model. Our current results show that IL-10 and NO are produced exclusively by T lymphocytes and macrophages, respectively, whereas TGF-beta is produced by both cell types. The high level of IL-10 produced by T-cells from tumor-bearing rats is responsible for the inhibition of lymphocyte proliferation. Moreover, our results suggest that the shift from immunosuppression to immunopotentiation induced by treatment of tumor-bearing rats with a single low-dose of Cy is mediated by a reduction in T-cell derived IL-10 production, which would account, to some extent, for the antimetastatic effect of Cy treatment.

Hsp25 and Hsp70 in rodent tumors treated with doxorubicin and lovastatin

Abstract Heat shock protein 27 (Hsp27) and Hsp70 have been involved in resistance to anticancer drugs in human breast cancer cells growing in vitro and in vivo. In this study, we examined the expression of Hsp25 (the rodent homologue to human Hsp27) and Hsp70 in 3 different rodent tumors (a mouse breast carcinoma, a rat sarcoma, and a rat lymphoma maintained by subcutaneous passages) treated in vivo with doxorubicin (DOX) and lovastatin (LOV). All tumors showed massive cell death under control untreated conditions, and this massive death increased after cytotoxic drug administration. In this study, we show that this death was due to classic apoptosis. The tumors also showed isolated apoptotic cells between viable tumor cells, and this occurred more significantly in the lymphoma. The tumor type that was more resistant to cell death was the sarcoma, and this was found in sarcomas growing both under control conditions and after cytotoxic drug administration. Moreover, sarcomas showed the highest expression levels of Hsp25 in the viable tumor cells growing under untreated conditions, and these levels increased after DOX and LOV administration. After drug treatment, only sarcoma tumor cells showed a significant increase in Hsp70. In other words, sarcomas were the tumors with lower cell death, displayed a competent Hsp70 and Hsp25 response with nuclear translocation, and had the highest levels of Hsp25. In sarcomas, Hsp25 and Hsp70 were found in viable tumor cells located around the blood vessels, and these areas showed the most resistant tumor cell phenotype after chemotherapy. In addition, Hsp25 expression was found in endothelial cells as unique feature revealed only in lymphomas. In conclusion, our study shows that each tumor type has unique features regarding the expression of Hsp25 and Hsp70 and that these proteins seem to be implicated in drug resistance mainly in sarcomas, making these model systems important to perform more mechanistic studies on the role of Hsps in resistance to certain cytotoxic drugs.

Inhibition ofras oncogene: A novel approach to antineoplastic therapy

The most frequently detected oncogene alterations, both in animal and human cancers, are the mutations in the ras oncogene family. These oncogenes are mutated or overexpressed in many human tumors, with a high incidence in tumors of the pancreas, thyroid, colon, lung and certain types of leukemia. Ras is a small guanine nucleotide binding protein that transduces biological information from the cell surface to cytoplasmic components within cells. The signal is transduced to the cell nucleus through second messengers, and it ultimately induces cell division. Oncogenic forms of p21(ras) lead to unregulated, sustained signaling through downstream effectors. The ras family of oncogenes is involved in the development of both primary tumors and metastases making it a good therapeutic target. Several therapeutic approaches to cancer have been developed pointing to reducing the altered gene product or to eliminating its biological function: (1) gene therapy with ribozymes, which are able to break down specific RNA sequences, or with antisense oligonucleotides, (2) immunotherapy through passive or active immunization protocols, and (3) inhibition of p21(ras) farnesylation either by inhibition of farnesyl transferase or synthesis inhibition of farnesyl moieties.

The transition to the metastatic phenotype of rat lymphoma cells involves up-regulation of IL-10 receptor expression and IL-10 secretion.

Interleukin 10 (IL-10) is a Th2 anti-inflammatory cytokine that participates in the regulation of the immune response at several levels. Its production has been implicated in the immunosuppression frequently observed in tumor bearing hosts. The broad spectrum of IL-10 biologic activities is mediated by its binding to its cognate receptor (IL-10R). We have already demonstrated the overproduction of IL-10 by B-cell lymphoma tumor bearing rats and, also, that IL-10 could act as a growth factor for metastatic cells. Considering the importance to unravel each feature of the complex biology of metastasis, the goal of the present study was to investigate the expression of IL-10 receptor (IL-10R), at mRNA and protein level, in primary tumor and metastatic cells from a rat B-cell lymphoma, along with the production of IL-10 by both tumor cell types. Our results indicate that IL-10, besides its immunoregulatory effect, would act as an autocrine growth factor for cells with metastatic phenotype. Also, the up-regulation of IL-10 and IL-10R expression would be part of the transition from primary tumor to the metastatic phenotype.

Differential production of angiostatin by concomitant antitumoral resistance-inducing cancer cells.

The phenomenon by which tumor‐bearing hosts are capable of inhibiting secondary tumor implants or metastases, known as concomitant antitumoral resistance (CAR), is presumably due to antiangiogenesis at places distant from the primary tumor. Although angiostatin, a potent inhibitor of angiogenesis, has been reported to be one of the factors responsible for suppressing the growth of secondary tumors in mice bearing previous tumors, it has not been definitively proven yet. With the aim of investigating whether CAR‐inducing cancer cells display a differential angiostatin production and to support the role ascribed to that molecule concerning the inhibition of secondary tumor implants, 5 tumor models with different CAR‐inducing capacities were studied herein. One of the 2 human lung cancer cell lines analyzed revealed a strong CAR against secondary s.c. tumor implants in nude mice, and 2 of 3 of the murine mammary tumors used exhibited inhibitory effect on secondary s.c. and i.v. tumor inoculations in syngeneic hosts. Since angiostatin is a proteolytic fragment from plasminogen, we examined by Western blot the ability of all conditioned media collected from the tumor cells studied to convert plasminogen to angiostatin. An association between in vivo generation of CAR and in vitro conversion of plasminogen into angiostatin was found. Since different enzymatic mechanisms were described to explain the generation of angiostatin, we also studied gelatinase and urokinase‐type plasminogen activator secretion in conditioned media by zymography. The conversion of plasminogen into angiostatin by conditioned media was mainly inhibited by broad‐spectrum serine proteinase inhibitors, suggesting a possible role for 1 or more enzymes of that group in the process. These findings suggest the existence of a differential angiostatin generation by CAR‐inducing cancer cells, providing additional support to previous data obtained by other authors.

Abstract 3655: Metronomic chemotherapy with cyclophosphamide (Cy) and celecoxib (Cel) in murine mammary adenocarcinoma tumor models: Mechanism of action

Metronomic chemotherapy (MCT) refers to the chronic, equally spaced, delivery of low doses of different chemotherapeutic drugs, without extended interruptions. CY, administered in low dose, is able to modulate the immune response. It can also reduce tumor angiogenesis, like the COX-2 inhibitor CEL does. We have already demonstrated that the administration, as an intervention strategy, of metronomic CY + CEL in M-406 and M-234p mammary adenocarcinomas-bearing mice inhibited tumor growth, increasing the survival rate. Our present aim was to study the mechanism by which that therapeutic effect was achieved. CBi and Balb/c inbred mice were s.c. challenged on day 0 with M-406 or M-234p mammary tumors, respectively, and treated from day 8 (M-406) and 5 (M-234p) on with: I) no further treatment (controls); II) CY in the drinking water (25-30 mg/kg body weight/day); III) CEL (30 mg/kg p.o.), five times/week; IV) Treated as II + III. Blood samples were taken on days 0, 20 (M-406) and 30 (M-324p) to evaluate VEGF serum level determined by ELISA and the % of circulating regulatory T cells (Tregs) by flow cytometry. The same tumor challenges and therapeutic schedules were performed in nude mice. Tumors were excised on days 20 and 30 for M-406 and M-234p, respectively, fixed and paraffin embedded for detection by immunohistochemistry of CD34 and Ki67 molecules. The VEGF serum level of M-406-bearing control animals (group I) was, on day 20, significantly higher than that measured on day 0 (p + CD25 + Foxp3 + Treg cells in serum showed no differences between days 0 and 20 in all the experimental groups. The antitumoral effect of MCT was the same in euthymic and athymic mice. Immunohistochemistry for CD34 showed slight differences between control and treated animals that were not significant. Also, the staining for the proliferation marker Ki67 did not evinced differences among the experimental groups. The results obtained with M-234p tumor model were similar to those of M-406. In conclusion, the antitumor effect of MCT with CY plus CEL in mammary tumor models would be achieved, at least in part, by the inhibition of tumor angiogenesis, without modulation of the antitumor immune response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3655.