Biomedical subjects

A Alonso-Varona

Publications and source records attributed to A Alonso-Varona.

14 recordsLinked to original sources

PubMed2005

Effects of all-trans retinoic acid on tumor recurrence and metastasis.

OBJECTIVE: All-trans-retinoic acid (ATRA) promotes cell differentiation. We have studied its effect on the local recurrence and metastatic spreading of an experimental rhabdomyosarcoma in rats. DESIGN: syngenic rhabdomyosarcoma cells (S4MH) were inoculated s.c. in male WAG/RijCrl rats. After 25 days tumors were excised and a 40% hepatectomy was performed for all animals. Ten days later the rats were sacrificed and a thorough necropsy was performed. The animals were randomly allocated to receive daily doses of ATRA (5 mg/kg, i.p.) or its solvent (Clinoleic/ethanol 90/10), starting three days before surgery until the end of the experiment. RESULTS: ATRA reduced the incidence of local recurrence from 70 to 33% (p < 0.05), but the tumor size was not altered (1.8 vs. 2.0 cc). Regarding inguinal metastasis, there was a six-fold decrease (0.2 vs. 1.2 cc; p < 0.05) in mean tumor volume, although the rate of this proliferation increased sharply (86 vs. 29%; p < 0.05) for treated animals. The volume of the retroperitoneal tumor masses also decreased with ATRA (0.7 vs. 5.1 cc; p < 0.05), but the difference in rate was not significant (71 vs. 67%). Lung metastases, which were present in 100% of control animals, were found in only 33% of treated rats, while the mean number of metastatic foci dropped from 26.7 to 5.7 (p < 0.05). CONCLUSION: Protocols including retinoid administration prior to and following primary tumor excision could help in controlling both recurrence and metastatic progression in surgically treated rhabdomyosarcoma.

Animals↗

PubMed2002

L-2-Oxothiazolidine-4-carboxylate reverses the tumour growth-promoting effect of interleukin-2 and improves the anti-tumour efficacy of biochemotherapy in mice bearing B16 melanoma liver metastases.

The efficacy of sequential chemoimmunotherapy involving interleukin-2 (IL2) in metastatic melanoma is limited, in part, by the severe toxicity associated with most therapeutic regimens. Glutathione (GSH), the most prevalent intracellular non-protein thiol, plays an important role in protecting against cellular injury caused by various anticancer agents. GSH is also involved in the IL2-induced proliferative activity of immune system cells and some melanoma cells expressing IL2 receptors, such as B16 melanoma cells. The present study investigated the effect of selective manipulation of GSH using the cysteine prodrug l-2-oxothiazolidine-4-carboxylate (OTZ) on the response of B16 melanoma to sequential biochemotherapy with cyclophosphamide (CY) and IL2. We found that OTZ, by depressing GSH levels, abrogates the in vitro growth-promoting effects of IL2 on B16 melanoma cells. The combination of OTZ plus IL2 in vivo also showed antitumour activity in mice bearing B16 melanoma liver metastases, significantly increasing their life span. Schedule dependency between both compounds was found; OTZ given intermittently in combination with daily IL2 administration was found to be the best therapeutic schedule. We also observed that whereas IL2 or OTZ alone added to CY resulted in a lower or non-significant improvement in the life span of the mice compared with tolerated doses of CY alone, the addition of both OTZ and IL2 to CY produced a significantly greater increase in survival than CY alone, and markedly protected mice against CY-induced toxicity, which allowed the administration of otherwise lethal doses of CY, with the CY activity/toxicity ratio being increased by four-fold.

Animals↗

PubMed2000

Removal of N-glycans from cell surface proteins induces apoptosis by reducing intracellular glutathione levels in the rhabdomyosarcoma cell line S4MH.

Expression of determined Asn-bound glycans (N-glycans) in cell surface glycoproteins regulates different processes in tumour cell biology. Specific patterns of N-glycosylation are displayed by highly metastatic cells and it has been shown that inhibition of N-glycan processing restrains cell proliferation and induces cell death via apoptosis. However, the mechanisms by which different N-glycosylation states may regulate cell viability and growth are not understood. Since malignant cells express high levels of intracellular glutathione (GSH) and a reduction of intracellular GSH induces cell death via apoptosis, we investigated whether GSH was involved in the induction of apoptosis by removal of cell surface N-glycans. We found that removal of N-glycans from cell surface proteins by treating the rhabdomyosarcoma cell line S4MH with tunicamycin or N-glycosidase resulted in a reduction in intracellular GSH content and cell death via apoptosis. Moreover, GSH depletion caused by the specific inhibitor of GSH synthesis BSO induced apoptosis in S4MH cells. This data indicates that adequate N-glycosylation of cell surface glycoproteins is required for maintenance of intracellular GSH levels that are necessary for cell survival and proliferation.

Amidohydrolases↗

PubMed2000

Effects of L-2-oxothiazolidine-4-carboxylate on the cytotoxic activity and toxicity of cyclophosphamide in mice bearing B16F10 melanoma liver metastases.

Glutathione (GSH) is the major non-protein thiol in cells that plays a critical role against damage from electrophilic agents such as alkylating drugs. Selective therapeutic GSH elevation in normal but not in tumour cells has been suggested as a means of protecting host tissues against more intense doses of chemotherapy. The present study investigated the response of B16 melanoma to treatment with the cysteine pro-drug L-2-oxothiazolidine-4-carboxylate (OTZ), alone and in combination with cyclophosphamide (CY). We found that OTZ decreased the GSH levels and proliferation rate of B16 melanoma cells in vitro, sensitizing them to the cytotoxic action of the activated metabolite of CY, acrolein (AC). In contrast to OTZ, the cysteine deliverer N-acetylcysteine (NAC) enhanced B16 melanoma cell proliferation by increasing GSH levels, and markedly decreased the sensitivity of these tumour cells to AC. In vivo studies showed the antitumoral activity of OTZ in B16 melanoma liver metastasis-induced mice, increasing their life span. We also observed that, whereas with CY treatment the GSH levels in peripheral blood mononuclear cells (PBMCs) were reduced and a dose-dependent leukopenia was produced, OTZ significantly increased PBMC GSH content, reducing toxicity and enhancing the survival of mice bearing established melanoma liver metastases treated with lethal dose CY. These results suggest a critical role for OTZ in protecting against alkylator agent-induced immunosuppression, which may allow the dose escalation of these cytostatic drugs to improve their therapeutic benefit in the treatment of malignant melanoma.

Acrolein↗

PubMed1999

In vitro and in vivo comparison between the effects of treatment with adenosine triphosphate and treatment with buthionine sulfoximine on chemosensitization and tumour growth of B16 melanoma.

In this study we compare the effects of treatment with external sodium adenosine 5'-triphosphate (ATP) with the effects of L-buthionine-SR-sulfoximine (BSO) on B16 melanoma growth and on the modulation of the cytotoxic antimelanoma activity of cyclophosphamide (CY). We evaluated the in vitro effects of treatment with ATP or BSO on intracellular glutathione (GSH) levels, mitochondrial membrane potential (delta psi(m)) and the proliferation rate of the B16F10 melanoma cell line. Compared with untreated cells, delta psi(m) and GSH levels were already significantly decreased (25% and 57% reduction, respectively) after the first hour of incubation in culture cells exposed to 3 mM ATP. After 24 and 48 h a major reduction was observed in delta psi(m) (nearly 30%). GSH levels were also maximally depleted at 24 h (approximately 75%) and partially recovered (up to 37% of levels of control) after ATP was removed from the medium. At 24 and 48 h, the proliferation rate was decreased 1.4- and 1.7-fold, respectively, compared with control cells. Treatment with 50 microM BSO produced a time-dependent decrease in GSH levels (0.5, 21, 48 and 97.3% reduction at 1, 4, 8 and 24 h, respectively), but up to 54% of the levels of control cells was recovered after BSO was removed from the medium. In contrast to ATP, neither delta psi(m) nor proliferation rate was significantly modified in the first 24 h with BSO treatment. At 48 h, delta psi(m) was reduced by nearly 27%, and cell proliferation decreased 1.2-fold compared with controls. When the in vitro cytotoxic effect of low dose acrolein (an active metabolite of CY) in combination with BSO or ATP was analysed, a synergistic effect was found between BSO and acrolein, with a dose modification factor (DMF) of 1.98, but the antiproliferative effects of acrolein plus ATP were only approximately additive (DMF = 1.05). In addition, in in vivo studies differential effects were found between ATP and BSO. Specifically, whereas BSO alone significantly increased the survival time of mice bearing B16 melanoma liver metastases, and enhanced the cytotoxic effect of CY on this tumour model, no therapeutic benefits could be observed with ATP treatment, either alone or in combination with diethyl maleate (a GSH-depleting agent) and CY. In conclusion, our findings show that in our experimental system, both extracellular ATP and BSO have growth-inhibitory properties against B16 melanoma in vitro. In vivo, however, only BSO produces a chemosensitizing effect, whereas ATP has not proved useful as a biological modifier of chemotherapy.

Acrolein↗

PubMed1997

Interleukin-2 increases intracellular glutathione levels and reverses the growth inhibiting effects of cyclophosphamide on B16 melanoma cells.

Glutathione (GSH) plays an essential role in the metabolism of melanoma. As changes in intracellular GSH content can modify the processes of cell proliferation and detoxification, this could determine the therapeutic response to some cancer treatment strategies. The purpose of this study was to test the effects of treatment with interleukin-2 (IL-2), alone and in combination with cyclophosphamide (CY), on survival of mice bearing B16 melanoma liver metastases, and to determine the influence of these therapeutic agents on the GSH metabolism of B16 cells. In the in vivo test system, B16 melanoma liver metastases were induced in C57BL/6 mice which were subsequently treated with IL-2, CY and CY plus IL-2. Survival time was used to determine the response to treatment. In the in vitro system, we evaluated the effects of IL-2, acrolein (an active metabolite of CY responsible for GSH depletion) and acrolein plus IL-2 on GSH levels and proliferation of B16 melanoma cells. Results indicated that, in vivo, all treatments increased mouse survival times with respect to control mice. However, the addition of IL-2 to CY therapy decreased survival time compared with treatment with CY alone. In vitro, whereas acrolein produced a GSH depletion and inhibited B16 cell proliferation, IL-2 increased GSH content and cell proliferation rate compared with untreated cells. Moreover, addition of IL-2 to cells preincubated with acrolein increased GSH levels and proliferation with respect to acrolein alone. In summary, the data suggest that GSH plays a critical role in the growth-promoting effects of IL-2 on B16F10 melanoma cells and in the antagonistic effect of IL-2 on CY inhibitory activity on these tumor cells.

Animals↗

PubMed1996

Tumor-host interaction in non-random metastatic pattern distribution.

In the clinical evolution of malign tumors, prognosis depends on whether metastasis develops or not. Biologically speaking, the formation of metastasis implies the existence of tumor cells capable of successfully performing all the steps in the metastatic process: local invasion, lymphatic or hematogenous dissemination, arrest in the microvascular bed of an organ, extravasation and growth of a secondary colony. Clinical observations have demonstrated that for each primary tumor there is a colonization pattern determined by the characteristics of the microvascular endothelium and the functional environment of the target organ. Moreover, the formation of metastasis depends on at least two additional factors: a) tumor cell-tumor cell and tumor cell-host cell relations modulated by intercellular contact and/or soluble paracrine or autocrine growth factors; b) the antitumor efficiency of the immune system, mediated primarily by the action of NK/LAK cells, macrophages and cytolytic T-lymphocytes, whose activity is in turn regulated by a complex of cytokines, including interferons, tumor necrosis factors and interleukins. In this work, we first review certain aspects of tumor biology that are specifically involved in tumor cell-host cell interactions determining non-random metastatic pattern distribution, and then review the implication of certain cytokines in the regulation of tumor proliferation.

Animals↗

PubMed1995

The addition of interleukin-2 to cyclophosphamide therapy can facilitate tumor growth of B16 melanoma.

The role of interleukin-2 (IL-2) on tumor growth of B16F10 melanoma cells was assessed in two sets of mice with different immune status: normal (immunocompetent) mice and immunodeficient mice. The two sets of animals were treated with cyclophosphamide (CY) or IL-2 alone or with a combined therapy of CY+IL-2. On days 6 and 10 after tumor cell injection, we evaluated the incidence of hepatic B16 melanoma metastases and the percentage of hepatic volume occupied by metastatic tissue. We observed that the CY alone (300 mg/kg, days 3 and 8 post-tumoral inoculation) significantly reduced tumor growth in all treated mice; however, CY proved more effective in normal recipients than in immunodeficient hosts. On the other hand, whereas administration of IL-2 alone (10(5) IU daily, from day 3 to day 7) in immunocompetent mice significantly reduced tumor growth on days 6 and 10, in immunodeficient mice, no significant differences were observed in tumor growth either on the 6th or on the 10th day, in comparison to control groups. Finally, when the combined CY+IL-2 therapy was administered, an antisynergistic effect between these therapeutic agents was achieved both in normal and in immunodeficient mice. Thus, the addition of low-dose IL-2 (25 x 10(3) IU daily, from day 4 to day 7) to high-dose CY (300 mg/kg, days 3 and 8) significantly increased tumor growth in both the early and later periods, compared to the effect of CY alone. It is concluded that exogenous IL-2 can facilitate tumor growth of B16 melanoma cells in vivo.

Animals↗

PubMed1995

A simple cell labeling technique by means of lectins linked to fluorochromes for the detection of cells on tissue sections.

We designed a protocol for cell labeling with the lectin wheat germ agglutinin (WGA) linked to the fluorochrome tetramethyl-rhodamine isothiocyanate (TRITC) for effective detection of the B16F10 melanoma and Lewis lung carcinoma (LLc) cells on pulmonary histological sections from C57BL/6 mice. We have also determined a suitable concentration of WGA-TRITC (10 micrograms/ml), which leads to a very intense and homogeneous labeling of the cells, as it avoids cell clumping due to the presence of the lectin WGA. In order to determine to what extent the method affects these tumor cells, we have studied some important aspects related to their metastatic behavior, taking into account three parameters: a) viability and rate of proliferation of the cells cultured in vitro; b) percentage of animals (C57BL/6 mice) bearing metastasis 15 days after intravenous inoculation with 10(5) B16F10 or LLc cells; and c) pattern of distribution of tumor foci in lung. There were no significant differences in these three parameters between the WGA-TRITC labeled-cells compared to the cultures of non-labeled cells in either of the cell lines (B16F10, LLc). Thus, we conclude that B16F10 and LLc tumor cells can be labeled following the protocol set-up in our study, as it allows these cells to be neatly identified on tissue sections and it causes no important physiological changes in the cells, with regard to metastatic behavior. These points make this technique very suitable for the detection of B16F10 and LLc cells on histological sections in studying their behavior during the first stages of the metastatic process.

Animals↗

PubMed1990

Coincident implantation, growth and interaction sites within the liver of cancer and reactive hematopoietic cells.

We have examined the anatomical-functional sites within mouse liver where phenylhydrazine (PHZ)-induced hematopoietic foci, and M5076 reticulum cell sarcoma, B16F10 melanoma and Lewis lung-carcinoma cells specifically develop as colonies after intrasplenic injection. Cancer foci occurred predominantly in the 2.4 to 4.0 segment of the sinusoidal pathway, corresponding to hepatic acinar zone I. No significant differences were detected between different types of tumor, including their different tendencies to spontaneously metastasize liver, or as a result of the different procedures used for obtaining foci or metastases. In addition, PHZ-treatment of mice previously injected with tumor cells, resulted in double colonization of the liver tissue by both hematopoietic and cancer cells, predominantly in zone I. This spatial coincidence indicates that non-cancer-specific mechanisms operate in zone I, either promoting implantation and/or growth of cell colonies or, alternatively, inhibiting these processes in the region surrounding the central vein (Rappaport zone 3). Our observations failed to reveal mutual displacement of cancer or hematopoietic foci by potential competition for development sites in zone I. Enumeration and diameter measurements of cancer foci in PHZ-treated animals showed that the presence of hepatic hematopoietic foci coincided with a significant increase in the hepatic metastasis volume. However, the fact that no significant differences in pulmonary metastases occurred in both the PHZ-treated and control mice given tail-vein injection of cancer cells, and that PHZ reduces cancer cell proliferation in vitro, reveal evidence of local interactions with hematopoietic foci which promote growth of cancer foci in liver.

Animals↗

PubMed1990

Estimating anatomical-functional position coordinates in liver tissue.

Hepatocyte enzyme activity was demonstrated by examining adult C57BL/6 mouse liver cryostat sections under a succinate dehydrogenase (SDH) histochemical reaction, and quantified by microspectrophotometry and microdensitometry. The hepatocyte SDH activity gradient along the path between the portal veins (PV) and efferent terminal hepatic venules (THV) was analyzed by measuring the concentration of the chromophore precipitated in 10 consecutive hepatic parenchymal domains located along imaginary lines drawn across the entire PV-to-THV distance. The profiles of intensity or of normalized relative optical density obtained on a high number of lines were correlated with distance values along the PV-to-THV pathway, enabling us to establish a general mathematical function relating SDH activity (chromophore concentration) to position values on a scale of 0 to 10 corresponding to the theoretical PV-to-THV distance. The equation can be used to interpolate the SDH activity surrounding any intrahepatic object located between the PV and the THV, thus making it possible to calculate the object's anatomical-functional position coordinates in the liver acinus. To demonstrate how this method is used, we have calibrated the intrahepatic position of hemopoietic foci induced in the liver tissue of adult mice treated with phenylhydrazine (PHZ), and show that these foci are located on coordinate 3.31 (maximum range 1.25-4.86) of the sinusoidal domain-that is, on the borderline between Rappaport's acinar zones 1 and 2.

Animals↗

PubMed1990

Functional variations in liver tissue during the implantation process of metastatic tumour cells.

We have examined several properties of sinusoidal cells in the unaffected tissue of micrometastasis-containing livers. Tumour cells from either B16 melanoma (B16F10) or Lewis lung carcinoma (LLC) were injected intrasplenically in syngeneic mice and sacrificed on the 7th day. Light and scanning electron microscopy (SEM) showed tumour cells in hepatic veins and sinusoids in close contact with endothelial walls and macrophages. Following quantitative analysis of SEM images from sinusoidal walls it was found that endothelial fenestrae from B16F10 or LLC-colonized livers were diffusely reduced both in size and density/microns 2 throughout the sinusoid wall, although especially affected zone 3 segments. Following the intrasplenic injection of 1 microns fluorescent latex particles 1 h prior to sacrifice of the mice a significant reduction of the latex particle uptake by sinusoidal cells was detected in B16F10-colonized livers (27% of controls) which was in contrast to the significant increase in LLC-colonized mice (180% of controls). Despite the focal character of the tumour cell implantation process, hepatic sinusoidal cells reacted diffusely to metastatic cells. However, over liver acini, endothelial cell changes were mainly expressed in zone 3 while phagocytic properties mainly varied in zone 1 and depending on the tumour type. Although the significance of these sinusoidal changes on metastatic development is unclear, data suggests that "soil" conditions in the liver are different before and after being metastasized by tumour cells.

Animals↗

PubMed1989

Selective implantation and growth in rats and mice of experimental liver metastasis in acinar zone one.

The functional zonation of liver tissue provides a framework for studying the implantation and growth of metastatic colonies within given zones of the hepatic acini. A very exact method for calibrating the position of metastatic foci in the hepatic acini was made possible by using the succinate-dehydrogenase reaction, which reveals the functional differences of the acinar zones. In mouse livers, metastasis was induced by intrasplenic injection of both high and low metastatic-capacity B16 melanoma and Lewis lung carcinoma cells. In rats, liver metastasis resulting from rhabdomyosarcoma was studied by injecting these cells into the s.c. tissue. All cases of metastasis occurred in hepatic acinar zone 1, and no significant differences were detected resulting from the type of tumor, its metastatic potential, or the procedure used for obtaining the metastasis. In subsequent experiments, metastasis was induced after first altering the zonal distribution of the hepatic extracellular matrix; distribution of the sinusoidal macrophages; and the sinusoidal diameter. However, even under these conditions, metastasis continues to occur exclusively in hepatic acinar zone 1. Thus, metastatic predilection for hepatic acinar zone 1 cannot be explained solely in terms of hemodynamic causes or the influence of extracellular matrix. Although it may still turn out that these elements play some secondary role in the phenomenon, our research points to the sinusoidal endothelial cells as a factor directly responsible for metastatic predilection for zone 1.

Animals↗

PubMedDate not supplied

Tunicamycin treatment reduces intracellular glutathione levels: effect on the metastatic potential of the rhabdomyosarcoma cell line S4MH.

Highly metastatic cells are known to overexpress certain Asn-linked oligosaccharides in the plasmatic membrane. Another phenotypic characteristic of malignant cells consists in the expression of high levels of intracellular glutathione (GSH). The aim of the present work was to demonstrate that the inhibition of N-glycosylation induces changes in intracellular GSH levels, and in turn participates in the inhibition of the metastatic potential of tumor cells by tunicamycin treatment. Firstly, we demonstrated that in comparison to the poorly metastatic cell line F21, the highly metastatic cells S4MH express a higher number of Asn-linked beta1-6 branched oligosaccharides and sialic acid (SA) and/or chitobiose oligosaccharides in glycoproteins involved in the regulation of the adhesion efficiency of tumor cells on endothelial cells and extracellular matrix. Our results showed that the decrease in S4MH cell adhesion efficiency on endothelial cells and extracellular matrix after the inhibition of N-glycan processing by tunicamycin treatment was caused by: (1) inhibition of the expression of N-glycan structures recognized by endothelial endogenous lectins, including beta1-6 branched oligosaccharides and SA and/or chitobiose oligosaccharides, and (2) redistribution of cell surface glycoproteins with beta1-6 branched oligosaccharides and/or SA and/or chitobiose oligosaccharides in their structures, caused by the depletion of intracellular GSH levels. The latter condition prevents the organization of these glycoproteins in the plasmatic membrane of S4MH cells necessary for anchoring to the substratum.

Actins↗