Viviana R. Rozados

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.

A Novel Synergistic Combination of Cyclophosphamide and Gene Transfer of Interleukin-12 Eradicates Colorectal Carcinoma in Mice

Purpose: Interleukin-12 (IL-12) is an immunostimulatory cytokine with potent antitumor effects in several animal models. However, serious toxicity has been associated with its systemic application in humans. Gene transfer has emerged as a tool to specifically express therapeutic genes into the tumor/peritumoral milieu, thus avoiding systemic toxicity. The aim of this study was to analyze whether subtherapeutic doses of an adenovirus encoding IL-12 (AdIL-12) might synergize with low immunopotentiating doses of cyclophosphamide in the treatment of colorectal carcinoma. Experimental Design: The antitumor effect of combining a single low dose of cyclophosphamide with an intratumoral injection of AdIL-12 was evaluated in an in vivo murine colorectal carcinoma model. The immune responses achieved with different treatments were monitored, comparing the effect of combining both therapies with individual treatments. Results: The combined therapy induced a complete tumor regression in >50% of mice in a synergistic fashion, and it significantly prolonged their survival. This strategy was superior to each single treatment in reducing both peripheral and splenic CD4+CD25+Foxp3+ regulatory T cells, increasing the number of activated dendritic cells, and inducing IFN-γ-secreting CD4-positive T lymphocytes. Importantly, the combined treatment generated a powerful tumor-specific CTL response. Consistently, a significant reduction in IL-10 levels was found. Our data suggest that the combination of nontoxic doses of cyclophosphamide with AdIL-12 allows the generation of good antitumoral responses, thus avoiding undesired side effects of both agents. Conclusions: Our data strongly support the use of a combination of cyclophosphamide and AdIL-12 as a novel therapeutic strategy against colorectal carcinoma. (Clin Cancer Res 2009;15(23):7256–65)

The Immune Response and the Therapeutic Effect of Metronomic Chemotherapy With Cyclophosphamide

Metronomic chemotherapy (MCT) is a novel therapeutic strategy for cancer treatment endowed with an antiangiogenic effect. It refers to regular administration of low doses of cytotoxic drugs, with minimal or no drug-free breaks. Previously, we demonstrated the immunomodulating activity of a single low-dose of cyclophosphamide (Cy) and the antitumor effect of MCT with Cy on established rat lymphomas and sarcomas. Here, we examined whether the immune response is responsible for the antitumor effect of MCT with Cy on L-TACB lymphoma. Inbred e rats and nude mice were subcutaneously challenged with L-TACB. After 7 days, they were distributed into two experimental groups: 1) treated animals, which were injected IP with Cy (10 mg/kg body weight) three times per week, and 2) control animals, which received IP saline injections. Exponential growth and decay and tumor doubling time were calculated. Also, serum IL-10 levels were measured. One hundred percent of treated rats showed tumor regression versus 0% of control rats. The increase of tumor-induced IL-10 levels was reverted by the treatment with Cy. On the other hand, there were no tumor regressions, in treated or control nude mice. However, the tumor doubling times of treated nude mice were significantly higher than those of control mice, implying that other antitumor mechanism(s), independent of the adaptive immune response, might be taking place. Our present results indicate that modulation of the immune response would be involved in the antitumor effect of MCT with Cy, because the absence of the specific immune response impairs, at least in part, its therapeutic effect in a lymphoma tumor model.

Immunotherapy for liver tumors: present status and future prospects

Increasing evidence suggests that immune responses are involved in the control of cancer and that the immune system can be manipulated in different ways to recognize and attack tumors. Progress in immune-based strategies has opened new therapeutic avenues using a number of techniques destined to eliminate malignant cells. In the present review, we overview current knowledge on the importance, successes and difficulties of immunotherapy in liver tumors, including preclinical data available in animal models and information from clinical trials carried out during the lasts years. This review shows that new options for the treatment of advanced liver tumors are urgently needed and that there is a ground for future advances in the field.

Angiogenic and antiangiogenic balance regulates concomitant antitumoral resistance

Concomitant antitumoral resistance (CAR), the phenomenon by which the growth of distant secondary tumor implants or metastases in some tumor-bearing hosts is inhibited by the presence of a primary tumor, has been previously ascribed to an antiangiogenic process. Here, we investigated vascular endothelial growth factor (VEGF) and endostatin serum levels in nude or BALB/c mice bearing human lung tumors (Calu-6 and H460) or murine mammary tumors (M3MC, M-234p and M-234m), respectively. In these experimental models we previously found an association between in vivo generation of CAR and in vitro conversion of plasminogen into angiostatin. Serum endostatin level in CAR+ Calu-6-bearing mice was significantly higher than in CAR− H460 counterpart. Sera from mammary tumor-bearing mice showed similar levels of endostatin, regardless of their ability to induce CAR. Conversely, serum VEGF levels in mice bearing CAR+ tumors were lower than those found in CAR− tumor-bearing hosts. Immunostaining with an anti-CD31 antibody revealed that secondary tumors subjected to CAR were significantly less vascularized than primary tumors, while this difference was not observed in CAR− tumors. In vitro studies showed an inhibitory effect of sera from CAR-inducing tumors on endothelial cell proliferation as compared to normal sera, whereas sera from non-CAR-inducing tumors did not alter endothelial proliferation and, in some instances, even caused stimulation of endothelial proliferation. These data suggest that the antiangiogenic mechanism operating in concomitant antitumoral resistance is the result of an increase in the ratio of antiangiogenic/proangiogenic regulators. The levels of the factors involved in this phenomenon can vary in the different tumor models, but the trend favoring the inhibition of angiogenesis is always conserved.

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.

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.

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.

Metformin and propranolol combination prevents cancer progression and metastasis in different breast cancer models

Discovery of new drugs for cancer treatment is an expensive and time-consuming process and the percentage of drugs reaching the clinic remains quite low. Drug repositioning refers to the identification and development of new uses for existing drugs and represents an alternative drug development strategy. In this work, we evaluated the antitumor effect of metronomic treatment with a combination of two repositioned drugs, metformin and propranolol, in triple negative breast cancer models. By in vitro studies with five different breast cancer derived cells, we observed that combined treatment decreased proliferation (P < 0.001), mitochondrial activity (P < 0.001), migration (P < 0.001) and invasion (P < 0.001). In vivo studies in immunocompetent mice confirmed the potential of this combination in reducing tumor growth (P < 0.001) and preventing metastasis (P < 0.05). Taken together our results suggest that metformin plus propranolol combined treatment might be beneficial for triple negative breast cancer control, with no symptoms of toxicity.

Highlights from the 1st Latin American meeting on metronomic chemotherapy and drug repositioning in oncology, 27-28 May, 2016, Rosario, Argentina.

Following previous metronomic meetings in Marseille (2011), Milano (2014), and Mumbai (2016), the first Latin American metronomic meeting was held in the School of Medical Sciences, National University of Rosario, Rosario, Argentina on 27 and 28 of May, 2016. For the first time, clinicians and researchers with experience in the field of metronomics, coming from different countries in Latin America, had the opportunity of presenting and discussing their work. The talks were organised in three main sessions related to experience in the pre-clinical, and clinical (paediatric and adult) areas. The different presentations demonstrated that the fields of metronomic chemotherapy and repurposing drugs in oncology, known as metronomics, constitute a branch of cancer therapy in permanent evolution, which have strong groups working in Latin America, both in the preclinical and the clinical settings including large, adequately designed randomised studies. It was shown that metronomics offers treatments, which, whether they are combined or not with the standard therapeutic approaches, are not only effective but also minimally toxic, with the consequent improvement of the patient’s quality of life, and inexpensive, a feature very important in low resource clinical settings. The potential use of metronomic chemotherapy was proposed as a cost/effective treatment in low-/middle-income countries, for adjuvant therapy in selected tumours. The fundamental role of the governmental agencies and non-governmental alliances, as the Metronomic Global Health Initiative, in supporting this research with public interest was underlined.