Potential effect of chloroquine and propranolol combination to treat colorectal and triple-negative breast cancers.

Drug repositioning explores the reuse of non-cancer drugs to treat tumors. In this work, we evaluated the effect of the combination of chloroquine and propranolol on colorectal and triple-negative breast cancers. Using as in vitro models the colorectal cancer cell lines HCT116, HT29, and CT26, and as triple-negative breast cancer models the 4T1, M-406, and MDA-MB-231 cell lines, we evaluated the effect of the drugs combination on the viability, apoptosis, clonogenicity, and cellular migratory capacity. To explore the in vivo effects of the combination on tumor growth and metastasis development we employed graft models in BALB/c, nude, and CBi mice. In vitro studies showed that combined treatment decreased cell viability in a dose-dependent manner and increased apoptosis. Also, we demonstrated that these drugs act synergically and that it affects clonogenicity and migration. In vivo studies indicated that this drug combination was effective on colorectal models but only partially on breast cancer. These results contributed to the search for new and safe treatments for colorectal and triple-negative carcinomas.

Repositioning metformin and propranolol for colorectal and triple negative breast cancers treatment.

Drug repositioning refers to new uses for existing drugs outside the scope of the original medical indications. This approach fastens the process of drug development allowing finding effective drugs with reduced side effects and lower costs. Colorectal cancer (CRC) is often diagnosed at advanced stages, when the probability of chemotherapy resistance is higher. Triple negative breast cancer (TNBC) is the most aggressive type of breast cancer, highly metastatic and difficult to treat. For both tumor types, available treatments are generally associated to severe side effects. In our work, we explored the effect of combining metformin and propranolol, two repositioned drugs, in both tumor types. We demonstrate that treatment affects viability, epithelial-mesenchymal transition and migratory potential of CRC cells as we described before for TNBC. We show that combined treatment affects different steps leading to metastasis in TNBC. Moreover, combined treatment is also effective preventing the development of 5-FU resistant CRC. Our data suggest that combination of metformin and propranolol could be useful as a putative adjuvant treatment for both TNBC and CRC and an alternative for chemo-resistant CRC, providing a low-cost alternative therapy without associated toxicity.

Comparative effectiveness of two metronomic chemotherapy schedules-our experience in the preclinical field.

Metronomic chemotherapy refers to the chronic, equally spaced, delivery of low doses of chemotherapeutic drugs, without extended interruptions. Previously, we developed two combined metronomic schemes for the treatment of murine mammary tumors. The aim of this study was to compare their effects on tumor and metastasis growth, survival, and toxicity. Metronomic chemotherapy with Cyclophosphamide + Celecoxib (Cy + Cel) showed higher antimetastatic power than Cyclophosphamide + Doxorubicin (Cy + Dox), while being similar in other aspects. That difference, plus the advantage that represents its oral administration, suggests that the Cy + Cel combination is more suitable than Cy + Dox for metronomic chemotherapy of mammary tumors and could be proposed to the translation to the clinic.

Association between baseline VEGF/sVEGFR-2 and VEGF/TSP-1 ratios and response to metronomic chemotherapy using cyclophosphamide and celecoxib in patients with advanced breast cancer.

BACKGROUND: Metronomic chemotherapy (MCT) with cyclophosphamide (Cy) and celecoxib (Cel) has therapeutic efficacy and low toxicity profile in advanced breast cancer patients (ABCP), but no reliable biomarkers of response have been found yet that allow patient selection for treatment. AIM: To investigate the potential role as biomarkers of pro- and antiangiogenic parameters and evaluate their response in ABCP receiving metronomic Cy 50 mg p.o./day + Cel 400 mg p.o./day. MATERIALS AND METHODS: Serum levels of vascular endothelial growth factor-C (VEGF-C), soluble VEGF receptors 2 and 3 (sVEGFR-2, sVEGFR-3), were measured at different time points in 13/15 patients included in a phase II trial of MCT with Cy+Cel. RESULTS: Serum levels of sVEGFR-2 and sVEGFR-3 increased significantly during treatment (P = 0.0392; P = 0.0066, respectively). VEGF-C showed no significant modifications. Previous determinations of VEGF and TSP-1 in the same patients were utilized. VEGF/sVEGFR-2, VEGF/TSP-1, and VEGF-C/sVEGFR-3 ratios decreased significantly along the treatment (P = 0.0092; P = 0.0072; P = 0.0141, respectively). Nonsignificant variations were observed for VEGF-C/sVEGFR-2 ratio. Baseline values of VEGF/sVEGFR-2 and VEGF/TSP-1 ratios were associated with time to progression (TTP) (P = 0.0407; P = 0.0394, respectively) meanwhile baseline VEGF was marginally significant (P = 0.0716). Patients with values lower than the 50th percentile for both ratios showed longer TTP. CONCLUSIONS: We have identified the baseline VEGF/sVEGFR-2 and VEGF/TSP-1 ratios as potential biomarkers of response in ABCP treated metronomically with Cy+Cel. This finding warrants its confirmation in a higher number of patients.

Therapeutic efficacy of metronomic chemotherapy with cyclophosphamide and doxorubicin on murine mammary adenocarcinomas.

BACKGROUND: Metronomic chemotherapy (MCT) refers to the chronic and equally spaced administration of low doses of different chemotherapy drugs, without extended rest periods. Herein, we investigated the therapeutic efficacy of metronomic cyclophosphamide (Cy) combined with doxorubicin (Dox) in two mouse mammary adenocarcinoma models. MATERIALS AND METHODS: Mice were s.c. challenged with M-234p or M-406 mammary tumors, and when the tumors reached ∼150 mm(3), they were treated with: (I) no treatment (controls); (II) Cy in the drinking water (30 mg/kg body weight/day); (III) Dox (0.5 mg/kg body weight i.p. three times/week); (IV) treated as (II) + (III). Mice challenged i.v. with M-234p or M-406 tumor cells received, on day 3, the same treatments. RESULTS: We found that MCT with Cy plus Dox inhibited tumor growth, decreased lung metastases, and increased the median survival time, while having low toxic effect. Combined MCT was more effective than each monotherapy causing decrease in VEGF serum concentration and tumor proliferation rate plus increase in tumor apoptosis. CONCLUSION(S): The therapeutic benefits of combined MCT with Cy and Dox on mammary adenocarcinomas together with its low toxic effect profile suggest the possibility of future translation into the clinic.

Regulatory T cells but not NKT I cells are modulated by a single low-dose cyclophosphamide in a B cell lymphoma tumor-model.

AIM: Experimental and clinical studies showed that the administration of cyclophosphamide (Cy) in low doses leads to an enhancement of the antitumor immune response. Our objective was to study the modulation, if any, by low dose Cy, of T regulatory (Treg) and natural killer T (NKT) I cells, two cell populations of the innate immune response with opposing effects on the tumors, in a rat B cell lymphoma model. METHODS: Inbred e rats were challenged s.c. with L-TACB lymphoma and on day 14 animals were distributed in two groups. Treated: injected i.p. with cyclophosphamide (10mg/kg of body weight) and CONTROL: injected i.p. with saline. Blood samples were taken from days 0 to 21 and the percentage of T regulatory and natural killer T I cells were determined by flow cytometry. RESULTS: We found that the increase of natural and inducible T regulatory cells of peripheral blood achieved during tumor growth was significantly downregulated by cyclophosphamide. On the contrary, natural killer T I cells were not modulated by the treatment. CONCLUSION: The antimetastatic effect of a single low dose of Cy would be due, at least in part, to downregulation of natural and inducible T regulatory cells.

Metronomic chemotherapy: changing the paradigm that more is better.

The introduction of the "maximum tolerated dose" in usual treatment protocols (and its concomitant overt toxicity) made necessary the imposition of rest periods between cycles of therapy-a practice that not only involves re-growth of tumour cells, but also growth of selected clones resistant to the therapy. To avoid the problems caused by traditional chemotherapeutic regimens, a new modality of drug administration called "metronomic chemotherapy" has been proposed. This name makes reference to the chronic, equally spaced administration of (generally) low doses of various chemotherapeutic drugs without extended rest periods. The novelty of this treatment modality lies not only in its antitumour efficacy with very low toxicity, but also in a cell target switch, now aiming at tumour endothelial cells. The knowledge acquired in the experimental field of metronomic chemotherapy, plus the increasing experience that is being obtained in the clinical setting, will help to lead a change in the design of therapeutic protocols against cancer.

Lovastatin enhances in vitro radiation-induced apoptosis of rat B-cell lymphoma cells.

Our previous demonstration of an antimetastatic effect of lovastatin, both in rat sarcoma and lymphoma tumor-models, as well as the fact that lovastatin and radiation are able to stop the cell cycle in different phases, suggested the feasibility of a combined treatment. We studied the effect of the in vitro combined treatment of a B-cell rat lymphoma (L-TACB) with lovastatin and irradiation. The results herein obtained provide new information about the role of statins as radiosensitizers. The antitumor effect of the combined treatment was higher than that elicited by either treatment alone. This effect could be a consequence, at least in part, of an enhanced apoptosis.

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.

Inhibition of ras 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.

Mechanism of antimetastatic immunopotentiation by low-dose cyclophosphamide.

We have previously reported the antimetastatic effect of a single low-dose of cyclophosphamide (Cy) on L-TACB rat lymphoma. The phenomenon could be adoptively transferred in immunocompetent rats and is abolished in nude mice, facts for which an immunomodulatory explanation was proposed. The aim of this paper was to identify the mechanism(s) by which spleen cells from Cy-treated tumour-bearing rats could exert this antimetastatic activity. Conditioned media obtained by incubation of spleen cells from Cy-treated and non-treated tumour-bearing rats, under specific or non-specific stimulation, were assayed to evaluate their effect on lymphocyte proliferation. The production of transforming growth factor beta (TGF-beta), interleukin-10 (IL-10) and nitric oxide (NO) by conditioned media was also studied. The restoration of spleen lymphoproliferative responses to normal levels when exposed to media conditioned by splenocytes from Cy-treated tumour-bearing rats, together with a decreased production of suppressive cytokines TGF-beta, IL-10 and NO, suggest an enhancement of host antimetastatic immunity triggered by single low-dose Cy treatment.

Inhibitory effect of Lovastatin on spontaneous metastases derived from a rat lymphoma.

The HMGCoA reductase inhibitor Lovastatin (LOV) has previously shown to abrogate p21ras farnesylation, which is associated with invasive and metastatic abilities in many tumor models. Considering the scarcity of therapeutic resources against metastasis, our objective was to study LOV as an antimetastatic agent on L-TACB rat lymphoma, which as a syngeneic tumor model resembles more closely the situation in human cancer. We also aimed to analyze the effect of LOV on chemoinvasion, motility, metalloproteases secretion, angiogenic capacity, and adhesion to the reconstituted basement membrane Matrigel. Our results showed that LOV caused no effect on cell motility, metalloprotease secretion and neovascularization. Conversely, LOV produced a significant inhibition of invasiveness, which could be a consequence of an impaired cell adhesion to the basement membrane observed. These effects could explain, at least in part, the inhibitory action of LOV on L-TACB rat lymphoma metastases.

Modulation of the antimetastatic effect of a single low dose of cyclophosphamide on rat lymphoma.

The aim of this paper was to identify the mechanism/s responsible of the antimetastatic effect of a single low dose of cyclophosphamide (Cy), previously demonstrated by us in the rat lymphoma LTACB. No direct cytotoxic antimetastatic activity of Cy could be proved. In vitro treatment of L-TACB cells with mafosfamide did not alter their invasiveness or their motility. The adoptive transfer of splenocytes from Cy-treated tumor-bearing rats, together with L-TACB cells inhibited their metastatic growth. The single low dose Cy treatment of T-immunodeficient nude mice did not show the antimetastatic effect on L-TACB observed in immunocompetent mice. An inhibition of the metastatic ability due to immunomodulation by Cy is proposed.

Lovastatin inhibits tumor growth and metastasis development of a rat fibrosarcoma.

HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase, the rate limiting enzyme in cholesterol synthesis, catalyses mevalonate production and, hence, influence the synthesis of isoprenoid metabolites. It has already been demonstrated that products of the mevalonate pathway play an important role in the progress of the cell cycle and cell survival. Lovastatin (LOV) competitively inhibits HMG-CoA reductase, blocking the synthesis of mevalonic acid and the generation of non-sterol isoprenoids, such as farnesyl residues. The posttranslational farnesylation of p21ras protein is essential for its binding to the membrane and, therefore, for its transforming activity. Considering that p21ras protein was reported to have a significant rol in metastatic behavior of tumor cells, we decided to study LOV as an antimetastatic agent on a rat fibrosarcoma. We demonstrated that a short treatment with LOV diminished primary tumor growth and the number and size of lung experimental metastasis.

Angiogenesis induced by tumor-host interaction in the rat.

There is a clear-cut cause-effect relationship between vascularization of a given tumor and its capacity to disseminate and metastasize. Hence, it is very important to study the contribution of the two main factors determining the generation of new blood vessels namely the tumor and the host where it grows. The influence of the tumor-host interaction on the induction of angiogenesis was studied in a tumor model-system composed by two lines of rats selected for resistance or susceptibility to a sarcoma (S-E100) and two transplantable tumors: Sarcoma E 100 and Lymphoma TACB. One line is resistant to the sarcoma but susceptible to the lymphoma while the other line behaves in the opposite way. Studying the angiogenesis generated by the s.c. implantation of each tumor in two different hosts we found that the same tumor induced different angiogenic responses according to the host in which it is growing and that the same host shows a different response according to the tumor with which it is challenged, suggesting that tumor-host interactions determine the intensity of the neovascularization process.

[Influence of host genotype in the development of metastasis in a transplantable lymphoma in rats]. / Influencia del genotipo del huésped en la formación de las metástasis de un linfoma transplantable de rata.

For the purpose of investigating the influence of the host genotype in the metastatic process, the frequency of lymph nodes metastases of a transplantable rat lymphoma (L-TACB) in relation to tumor growth and to capacity of rejection was studied in the inbred lines m, eR and eS and in the F1 hybrids m x eR, m x eS and eR x eS. L-TACB is a spontaneous poorly differentiated lymphoma that appeared in the peritoneal cavity of a rat of the inbred line e; since 1969 it has been maintained by subcutaneous grafting in e rats. The tumor was inoculated by trocar and measured with a caliper every 3 to 4 days, the size expressed in mm2 (large diameter x minor diameter). Percentages of takes, regression, lethality and axillary and/or inguinal lymph nodes metastases were registered. The maximum size (TMx) reached by the primary tumor, in animals with (TMx-CM) and without metastasis (TMx-CM) were recorded. Tumor size (TMx) in animals of line m was greater (p less than 0.01) than in rats of subline eR while tumor regression was significantly less in eR (29.7%) than in m (72.7%) and eS (100%) (p less than 0.001), suggesting that tumor growth rate was independent of the capacity of tumor rejection. Rats of genotypes m and eS had a great capacity of tumor rejection; however, the hybrid m x eS showed a low percentage of regression (17.2%) (p less than 0.001). This result reveals an opposite effect produced by the genetic interaction of the m and eS parental lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Influencia del genotipo del huésped en la formación de las metástasis de un linfoma transplantable de rata. / [Influence of host genotype in the development of metastasis in a transplantable lymphoma in rats]

For the purpose of investigating the influence of the host genotype in the metastatic process, the frequency of lymph nodes metastases of a transplantable rat lymphoma (L-TACB) in relation to tumor growth and to capacity of rejection was studied in the inbred lines m, eR and eS and in the F1 hybrids m x eR, m x eS and eR x eS. L-TACB is a spontaneous poorly differentiated lymphoma that appeared in the peritoneal cavity of a rat of the inbred line e; since 1969 it has been maintained by subcutaneous grafting in e rats. The tumor was inoculated by trocar and measured with a caliper every 3 to 4 days, the size expressed in mm2 (large diameter x minor diameter). Percentages of takes, regression, lethality and axillary and/or inguinal lymph nodes metastases were registered. The maximum size (TMx) reached by the primary tumor, in animals with (TMx-CM) and without metastasis (TMx-CM) were recorded. Tumor size (TMx) in animals of line m was greater (p less than 0.01) than in rats of subline eR while tumor regression was significantly less in eR (29.7

) than in m (72.7

) and eS (100

) (p less than 0.001), suggesting that tumor growth rate was independent of the capacity of tumor rejection. Rats of genotypes m and eS had a great capacity of tumor rejection; however, the hybrid m x eS showed a low percentage of regression (17.2

) (p less than 0.001). This result reveals an opposite effect produced by the genetic interaction of the m and eS parental lines.(ABSTRACT TRUNCATED AT 250 WORDS)

[Inheritance of susceptibility to tumors and metastases in rodents]. / Herencia de la susceptibilidad a los tumores y a las metástasis en roedores.

The behavior of two spontaneous transplantable rat tumors and a mouse lymphoma was studied in different hosts. A resistant (eR) subline to Sarcoma E 100 (S-E 100) and a susceptible one (eS) were obtained by divergent selection carried out in an inbred line of rats IIM (e). The phenotypic frequencies of the resistant and susceptible individuals to S-E 100 in the base population e and eR, eS, F1 and F2 fitted with the theoretical frequencies of a model of two independent pairs of genes, one (T) with complete dominance (locus SE1) and the other (R) with partial dominance (locus SE2). Locus SE2 would regulate the antitumoral response. Resistance to S-E 100 was found to be a dominant trait. The subcutaneous challenge of eR and eS rats with Lymphoma TACB (L-TACB) showed a different behavior. Subline eR (resistant to S-E 100) was susceptible to L-TACB while subline eS (susceptible to S-E 100) was resistant. A tumor-host interaction was found, revealing that genotypes resistant to S-E 100 were susceptible to L-TACB; in addition, susceptible animals (eR) developed lymph node metastases. It was observed, using animals of different genotypes, that susceptibility to L-TACB metastases and to local tumor growth, would be conditioned by different genes. Delayed type hypersensitivity (DTH) to tumor antigens (S-E 100 and L-TACB) and humoral immune response and DTH to sheep red blood cells (SRBC) were investigated in eR and eS rats. Differences between both sublines were statistically significant suggesting that genes involved in the divergent selection would regulate the antitumoral immune response.(ABSTRACT TRUNCATED AT 250 WORDS)

Herencia de la susceptibilidad a los tumores y a las metástasis en roedores. / [Inheritance of susceptibility to tumors and metastases in rodents]

The behavior of two spontaneous transplantable rat tumors and a mouse lymphoma was studied in different hosts. A resistant (eR) subline to Sarcoma E 100 (S-E 100) and a susceptible one (eS) were obtained by divergent selection carried out in an inbred line of rats IIM (e). The phenotypic frequencies of the resistant and susceptible individuals to S-E 100 in the base population e and eR, eS, F1 and F2 fitted with the theoretical frequencies of a model of two independent pairs of genes, one (T) with complete dominance (locus SE1) and the other (R) with partial dominance (locus SE2). Locus SE2 would regulate the antitumoral response. Resistance to S-E 100 was found to be a dominant trait. The subcutaneous challenge of eR and eS rats with Lymphoma TACB (L-TACB) showed a different behavior. Subline eR (resistant to S-E 100) was susceptible to L-TACB while subline eS (susceptible to S-E 100) was resistant. A tumor-host interaction was found, revealing that genotypes resistant to S-E 100 were susceptible to L-TACB; in addition, susceptible animals (eR) developed lymph node metastases. It was observed, using animals of different genotypes, that susceptibility to L-TACB metastases and to local tumor growth, would be conditioned by different genes. Delayed type hypersensitivity (DTH) to tumor antigens (S-E 100 and L-TACB) and humoral immune response and DTH to sheep red blood cells (SRBC) were investigated in eR and eS rats. Differences between both sublines were statistically significant suggesting that genes involved in the divergent selection would regulate the antitumoral immune response.(ABSTRACT TRUNCATED AT 250 WORDS)