Synthesis and characterization of a biocompatible chitosan-based hydrogel cross-linked via 'click' chemistry for controlled drug release.

A chemically cross-linked chitosan-based hydrogel was successfully synthesized through Diels-Alder (DA) reaction and characterized. The final product was obtained after different steps; on the one hand, furan-modified chitosan (Cs-Fu) was synthesized by the reaction of furfural with the free amino groups of chitosan. On the other hand, highlighting the novelty of the present research, maleimide-functionalized chitosan (Cs-AMI) was prepared by the reaction of a maleimide-modified aminoacid with the amino groups of chitosan through amide coupling. The two complementary chitosan derivatives were cross-linked to the final hydrogel network. Both modification reactions were confirmed by FTIR and 1H NMR, obtaining a degree of substitution (DS) of 31% and 26% for Cs-Fu and Cs-AMI, respectively. The as-designed hydrogel was analyzed in terms of microstructure, swelling capacity and rheological behaviour. The hydrogel showed pH-sensitivity, biocompatibility and inhibitory bacterial activity, promising features for biomedical applications, particularly for targeted-drug delivery.

HR007: a family of biomaterials based on glycosaminoglycans for tissue repair.

Most new advances in tissue engineering (TE) focus on the creation of adequate microenvironments that may accelerate the repair processes of damaged tissues. Extracellular matrix (ECM) of Wharton's jelly (WJ) from umbilical cords is very rich in sulphated GAGs (sGAGs) and hyaluronic acid (HA), components which have special properties that could positively influence the regeneration of several types of tissue. Previously, we described the methodology for the extraction and purification of GAGs from WJ and, importantly, the separation of sGAGs and HA to develop various scaffolds for regenerative medicine. In this new study we hypothesized that the biomaterials obtained, called HR007s, would be excellent candidates for two different applications, chondral and dermal repair. First, we have confirmed that the GAGs obtained are biocompatible, as they do not cause cytotoxicity, haemolysis or an inflammatory response. Second, we have developed three-dimensional (3D) structures through the combination of different ratios of GAGs and their subsequent stabilization, which can be properly adapted to target tissues, cartilage or skin. Finally, we have combined these scaffolds with adipose mesenchymal stem cells (ASCs) or fibroblasts for application to chondral or dermal defects, respectively, with the goal of promoting fast reparative processes. The results show that HR007 scaffolds induce cell proliferation, enhance the expression of specific gene markers, increase the production of tissue ECM proteins and have chemotactic effects over the studied cells. In summary, the bioactive properties of HR007 scaffolds make them promising candidates for use in regenerative medicine, at least for chondral and dermal repair. Copyright © 2015 John Wiley & Sons, Ltd.

Indoleamine 2,3 dioxygenase as a prognostic and follow-up marker in melanoma. A comparative study with LDH and S100B.

BACKGROUND: To date, lactate dehydrogenase (LDH) and S100B remain the most useful biomarkers for follow-up of melanoma patients. In recent years, indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme, has been proposed as a new potential tumour biomarker for melanoma. However, further studies are needed to confirm the usefulness of IDO expression as an independent prognostic factor. OBJECTIVE: To explore the potential association between serum IDO levels and melanoma stage at diagnosis and recurrence, and to compare the results to those obtained with LDH and S100B. In addition, we also investigated a possible cut off for IDO level as a prognostic factor for overall survival. METHODS: IDO, LDH and S100B levels were measured in serum samples of 186 patients in all melanoma stages at diagnosis and twice a year thereafter. A cut-off point for IDO levels was calculated using receiver operating characteristic curves to explore the association between these levels and the likelihood of lymphatic spread. Survival curves were estimated for patient groups stratified by IDO level (higher or lower than the cut off), using the Kaplan-Meier method. RESULTS: At diagnosis, serum IDO levels were significantly higher in stages IB, II, III and IV, whereas S100B levels were significantly higher in stages III and IV, and LDH levels were only higher in stage IV. In relapsed patients, significant increases were found in levels of all three markers. Finally, overall survival was significantly longer in patients with IDO levels below a cut off of 1.65 µM at diagnosis than in those with higher levels (91.3 vs. 71.0% at 36 months). CONCLUSION: In melanoma patients, serum IDO levels are significantly associated with disease stage, relapses and overall survival. These results indicate IDO could be a useful serum prognostic marker for melanoma.

Generation of tunable glycosaminoglycan hydrogels to mimic extracellular matrices.

Biomaterials and, especially, scaffolds may function as temporary extracellular matrix (ECM), mimicking in vivo environmental structures and facilitating cell growth and tissue regeneration. ECM is composed mostly of glycosaminoglycans (GAGs) and proteins, the ratio of GAGs, hyaluronic acid (HA):sulphated GAGs (sGAGs) being characteristic of each type of tissue. Umbilical cord (UC) and particularly Wharton's jelly (WJ) have been proposed as good sources for obtaining GAGs. In this work, we present a novel methodology for the extraction, purification and separation of GAGs from UC obtained from two different species, human and pig. The new methodology is based on enzymatic digestion of WJ, precipitation of GAGs with organic solvents, purification steps and chromatographic separation of GAGs using ion exchange columns. This novel process allows highly purified HA and sGAGs to be obtained from human and pig WJ. The composition of sGAGs and molecular weight of HA were very similar in the two species and GAGs are haemocompatible and non-cytotoxic. Finally, these new biomaterials have significant bioactive properties, increasing proliferation rates of two cell lines, human adipose mesenchymal stem cells (ASCs) and fibroblasts. In summary, the separation of HA and sGAGs, linked to the improvement in the GAG quantification method described in this paper, opens new avenues for the formulation of natural biomaterials with various ratios of GAGs, mimicking tissue matrix for different tissue-engineering applications. Copyright © 2014 John Wiley & Sons, Ltd.

A study of the mechanical properties and cytocompatibility of lactide and caprolactone based scaffolds filled with inorganic bioactive particles.

The mechanical properties of highly porous (90% porosity) poly(l-lactide) (PLLA), poly(ε-caprolactone) (PCL) and poly(l-lactide/ε-caprolactone) (PLCL) were investigated. Young's modulus of non-porous PLLA, PCL and PLCL dropped from 2263.4, 183.7 and 5.7 MPa to 16.8, 1.0 and 1.0 MPa, respectively, for their ~90% porous counterparts. Elongation at break of PCL decreased noticeably with porosity fraction while PLCL maintained a highly elastomeric character and strain recovery capacity even in the presence of pores. Inorganic bioactive particles (hydroxyapatite or bioglass) were added to confer bioactivity to the aforementioned synthetic bioresorbable polymers, and their effect on the mechanical properties was also investigated. Addition of 15 vol.% of inorganic bioactive particles increased the Young's modulus of highly porous PLLA from 16.2 to ~30 MPa. On the contrary, the difference between Young's modulus of filled and unfilled PCL and PLCL scaffolds was not statistically significant. Finally, an in vitro study of the cytocompatibility and adhesion of adipose derived stem cells (ADSCs) was conducted. The observed viability and excellent adhesion of these cells to both porous and non-porous templates indicate that the employed materials can be good candidates for application in tissue engineering.

Crystallinity assessment and in vitro cytotoxicity of polylactide scaffolds for biomedical applications.

Bioresorbable polylactides are one of the most important materials for tissue engineering applications. In this work we have prepared scaffolds based on the two optically pure stereoisomers: poly(L: -lactide) (PLLA) and poly(D: -lactide) (PDLA). The crystalline structure and morphology were evaluated by DSC, AFM and X-ray diffraction. PLLA and PDLA crystallized in the α form and the equimolar PLLA/PDLA blend, crystallized in the stereocomplex form, were analyzed by a proliferation assay in contact with mouse L-929 and human fibroblasts and neonatal keratinocytes for in vitro cytotoxicity evaluation. SEM analysis was conducted to determine the cell morphology, spreading and adhesion when in contact with the different polymer surfaces. The preserved proliferation rate showed in MTT tests and the high colonization on the surface of polylactides observed by SEM denote that PLLA, PDLA and the equimolar PLLA/PDLA are useful biodegradable materials in which the crystalline characteristics can be tuned for specific biomedical applications.

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.

Efecto del ácido trans-retinoico sobre la recidiva tumoral y el desarrollo metastásico / Effects of all-trans retinoic acid on tumor recurrence and metastasis

Objetivo: el ácido trans-retinoico (ATRA, "all-trans retinoic acid") es un agente inductor de la diferenciación celular. Se estudia su efecto sobre la recidiva local y la diseminación metastásica de los tumores sólidos. Diseño experimental: mediante inoculación subcutánea de células de rabdomiosarcoma (S4MH) se ha inducido un tumor en ratas WAG/RijCrl. Tras 25 días, se practicó una tumorectomía y simultáneamente hepatectomía del 40%. Los animales se sacrificaron el día 35, y fueron sometidos a estudio necrópsico. La mitad de los animales fueron tratados con ATRA (5 mg/kg, i.p.) desde el día 22 al 35, mientras que los controles recibieron el excipiente (Clinoleico®/etanol 90/10). Resultados: el tratamiento redujo la tasa de recidiva local del 70 al 33% (p < 0,05), aunque no afectó a su tamaño (1,8 vs. 2,0 cc). El volumen medio de las metástasis inguinales se redujo a la sexta parte (0,2 vs. 1,2 cc; p < 0,05), si bien su frecuencia aumentó con el ATRA (86 vs. 29%; p < 0,05). La extensión retroperitoneal del rabdomisoarcoma también se redujo (0,7 vs. 5,1 cc; p < 0,05), aunque no hubo variación en la incidencia (71 vs. 67%). La incidencia de afectación pulmonar (100% en controles) se redujo hasta el 33% (p < 0,05), a la vez que el número medio de focos en el pulmón pasó de 26,7 a 5,7 (p < 0,05). Conclusión: la estrategia terapéutica fundamentada en el tratamiento pre y postextirpación quirúrgica con retinoides podría favorecer el control local de rabdomiosarcomas sometidos a cirugía

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

[Effect of hepatic resection on development of liver metastasis]. / Efecto de la resección hepática sobre el desarrollo de metástasis en el hígado.

In the early stages of metastasis, development of the disease is dependent on growth factors produced by the host. There are clinical situations associated with an increase in these factors, such as partial resection of metastasized liver. Given the important role of hepatotrophic factors in liver regeneration, we have studied the effect of partial hepatectomy on the development of residual micrometastases in the liver, and on the neoplastic process as a whole. We used a murine model in which a rabdomiosarcoma was established by subcutaneous inoculation of syngeneic tumor cells in male Wag rats. Subsequently, the primary tumor was resected and/or a 40% hepatectomy was performed. The effect of these two surgical procedures on the tumor process was analyzed on the 25th and 35th days post-inoculation, and the percentage of regenerating hepatocytes was assessed. Both the tumorectomy and liver resection, when not combined, produced an increase in regional adenopathies without modifying the evolution of metastasis in the liver. However, when tumor excision and partial hepatectomy were performed simultaneously, there was a net increase in the metastatic process. In addition to a rapid spread of the disease (lung, mediastinum, retroperitoneum), the number of liver metastases increased by 300%. This development coincided with a steep rise in the percentage of regenerating hepatocytes, which nearly doubled that of the group subjected only to liver resection. We conclude that liver resection, alone or combined with excision of the primary tumor, may enhance tumor progression, both locally and at the metastasic level.

The emotional response of families to children with leukemia at the lower socio-economic level in central Mexico: a preliminary report.

Little research has been done in developing countries on the emotional impact experienced by families who have a child diagnosed with leukemia. This preliminary study looked at parents in Mexico who had to cope with their child's leukemia in the face of meager financial and social resources. The 51 children in the study were under 15 years and being treated for leukemia in hospitals affiliated with the Mexican Social Security Institute (IMSS) where their parents were interviewed using a questionnaire to ascertain their emotional responses to the illness. The data are analyzed and reported in five domains: perceived illness; psychological impact; coping strategies; family relationships; socio-economic impact. A strengthening of family bonds was found the most common response (82.4%). The second most common responses were concern for the expenses incurred by the illness and the time dedicated to caring for the sick child (both 78.4%). It is especially important to assess families with meager social and financial resources as to their emotional responses to life-threatening illness because these limitations impose greater burdens and make coping more difficult. Psychosocial interventions are key to ensuring adequate treatment of the child in these circumstances.

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.

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.

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.

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.

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.

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.

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.

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.