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Background: Staphylococcal enterotoxin C2 (SEC2) is used as an immunotherapeutic drug in China. However, SEC2 are limited due to its immunosuppressive and toxic effects. A SEC2 2M-118 (H118A/T20L/G22E) mutant generated by site-directed mutagenesis was studied to elucidate the underlying antitumor mechanism., Methods: The effects of 2M-118 on mouse fibrosarcoma (Meth-A) cells and cytokine responses were tested in vitro using a transwell assay and ELISA, respectively. 2M-118 effect on immune function in tumor-bearing mice was tested. Cytokine levels and antitumor responses were measured using ELISA and flow cytometry, respectively. TUNEL staining and immunohistochemistry were employed to detect the tumor apoptosis and CD4 + and CD8 + tumor infiltrating lymphocytes (TILs) in tumor tissue., Results: 2M-118 demonstrated the growth inhibition on tumor cells, increase of cytokines production (IL-2, IFN-γ, and TNF-α) and splenocyte proliferation in vitro. 2M-118 effectively inhibited tumor development and increased lymphocytes and cytokines in a tumor-bearing mouse model. Additionally, 2M-118 regulated the tumormicroenvironment by reducing the number of myeloid-derived suppressor cells (MDSCs), increasing the number of TILs, and inducing tumorcell apoptosis., Conclusion: 2M-118 promotes immune function and enhances antitumor response. This indicates that 2M-118 could potentially be developed as a novel anti-tumor drug with-highefficiencyandlowtoxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Flavonoids possess a broad spectrum of pharmacological properties, including anti-cancer, anti-oxidant and immunomodulatory activities. The current study explored the potential of some less-studied flavonoids in inhibiting Matrix Metalloproteinase-9 (MMP-9), a prominent biomarker, upregulated in a variety of cancers and known to promote migration and invasion of cancer cells. Amongst these, Tamarixetin, a naturally occurring flavonoid derivative of Quercetin, demonstrated significant dose-dependent inhibition of MMP-9 expression. Furthermore, a substantial inhibition of migration, invasion and clonogenic potential of HT1080 cells was also observed in the presence of Tamarixetin, which further suggests its role as a potential anti-cancer agent. It is noteworthy that Tamarixetin inhibits nuclear translocation as well the activity of nuclear factor kappa B (NFκB), both of which are functions essential for the activation of MMP-9 in promoting tumorigenesis. Additionally, the endogenous regulators of MMP-9 that tightly control its activity were also modulated by Tamarixetin, as evident from the 1.9 fold increase in the expression of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), with a concomitant 2.2 fold decrease in Matrix Metalloproteinase-14 (MMP-14) expression. The results obtained were further corroborated in three dimensional (3D) tumor models, which showed significant inhibition of MMP-9 activity as well as reduced invasive potential in the presence of Tamarixetin. Taken together, our observations demonstrate for the first time, the anti-invasive potential of Tamarixetin in cancer cells, indicating its possible use as a template for novel therapeutic applications., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Fulvic acid (FA) is known to promote electrochemical balance as a donor or a receptor possessing many biomedical functions. Nevertheless, the effect of FA on the anti-cancer activity has not been elucidated. In the current study, we first isolated FA from humus and investigated whether FA regulates immune-stimulating functions, such as production of nitric oxide (NO), in RAW 264.7 cells. Our data showed that FA slightly enhances cell viability in a dose-dependent manner and secretion of NO from RAW 264.7 cells. It upregulated the protein and mRNA expression of inducible NO synthesis (iNOS). In addition, FA enhanced the DNA-binding activity of nuclear factor-κB (NF-κB) in RAW 264.7 cells; the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) effectively attenuated the expression of FA-stimulated iNOS, suggesting that FA stimulates NF-κB to promote iNOS and NO production. Finally, FA-stimulated culture media (FA-CM) from RAW 264.7 cells were collected and MCA-102 fibrosarcoma cells were cultured in this media. The FA-CM augmented MCA-102 fibrosarcoma cell apoptosis; however, an NO inhibitor N(G)-monomethyl-l-arginine (NMMA) slightly inhibited the FA-CM-mediated MCA-102 fibrosarcoma cell apoptosis, which was accompanied by low levels of NO. In the present study, we found that FA induces the generation of NO and iNOS in RAW 264.7 cells by inducing NF-κB activation; however, NO did not significantly stimulate MCA-102 fibrosarcoma cell apoptosis in the current study. In addition, FA-CM enhanced cell death in various human cancer cells such as Hep3B, LNCaP, and HL60. Taken together, FA most likely stimulates immune-modulating molecules such as NO and induces cancer cell apoptosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Recently, our group established a relationship between tumor-induced spinal cord astrocyte activation and aromatase expression and the development of bone tumor nociception in male mice. As an extension of this work, we now report on the association of tumor-induced mechanical hyperalgesia and cold hypersensitivity to changes in spinal cord dorsal horn GFAP and aromatase expression in intact (INT) female mice and the effect of ovariectomy on these parameters. Implantation of fibrosarcoma cells produced robust mechanical hyperalgesia in INT animals, while ovariectomized (OVX) females had significantly less mechanical hyperalgesia. Cold hypersensitivity was apparent by post-implantation day 7 in INT and OVX females compared to their saline-injected controls and increased throughout the experiment. The decrease in mechanical hyperalgesia in OVX females was mirrored by significant decreases in spinal astrocyte activity in laminae I-II, III-IV, V-VI and X and aromatase expression in laminae V-VI and X in the dorsal horn of tumor-bearing animals. Administration of the aromatase inhibitor letrozole reduced tumor-induced hyperalgesia in INT females only suggesting that the tumor-induced increase in aromatase expression and its associated increase in spinal estrogen play a role in the development of bone tumor-induced hyperalgesia. Finally, intrathecal (i.t.) administration of 17β-estradiol caused a significant increase in tumor-induced hyperalgesia in INT tumor-bearing females. Since i.t. 17β-estradiol increases tumor pain and ovariectomy significantly decreases tumor pain, as well as spinal aromatase, estrogen may play a critical role in the spinal cord response to the changing tumor environment and the development of tumor-induced nociception., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

While spinal cord astrocytes play a key role in the generation of cancer pain, there have been no studies that have examined the relationship of tumor-induced astrocyte activation and aromatase expression during the development of cancer pain. Here, we examined tumor-induced mechanical hyperalgesia and cold allodynia, and changes in Glial fibrillary acid protein (GFAP) and aromatase expression in murine models of painful and non-painful bone cancer. We demonstrate that implantation of fibrosarcoma cells, but not melanoma cells, produces robust mechanical hyperalgesia and cold allodynia in tumor-bearing mice compared to saline-injected controls. Secondly, this increase in mechanical hyperalgesia and cold allodynia is mirrored by significant increases in both spinal astrocyte activity and aromatase expression in the dorsal horn of fibrosarcoma-bearing mice. Importantly, we show that aromatase is only found within a subset of astrocytes and not in neurons in the lumbar spinal cord. Finally, administration of an aromatase inhibitor reduced tumor-induced hyperalgesia in fibrosarcoma-bearing animals. We conclude that a painful fibrosarcoma tumor induces a significant increase in spinal astrocyte activation and aromatase expression and that the up-regulation of aromatase plays a role in the development of bone tumor-induced hyperalgesia. Since spinal aromatase is also upregulated, but to a lesser extent, in non-painful melanoma bone tumors, it may also be neuroprotective and responsive to the changing tumor environment., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cancer is a major cause of death, and the development of new anticancer drugs is urgently needed. Invasion and metastasis are the primary causes of death due to cancer rather than growth of the primary tumor. In the current study, we examined the anti-invasive effects of p-dodecylaminophenol (1), which was developed based on N-(4-hydroxyphenyl)retinamide (2), a synthetic amide of all-trans-retinoic acid (3). In HT1080 cells 1 inhibited growth, induced apoptosis and arrested the cell cycle in S phase in a dose-dependent manner. In addition, 1 significantly suppressed cell invasion, and the activity and mRNA expression of matrix metalloproteinase-9 (MMP-9). Furthermore, the expression of the reversion-inducing cysteine-rich protein with Kazal motifs (RECK), which is a negative regulator of MMP-9, was increased by treatment with 1. These results suggest that 1 could be an effective anti-cancer agent that suppresses cell growth through apoptosis induction and cell cycle arrest, which also inhibits cell invasion by decreasing MMP-9 expression due to an increase in RECK. Compound 1 might be useful clinically as a new and potent anticancer agent that could overcome adverse side effects of the retinoids., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Fibrosarcoma is one of the fatal cancer types and there is still not satisfactory success in its treatment despite new drugs. Therefore, the search for a new compound has been going on. It is currently known that some palladium-based anti-cancer compounds seem to have powerful apoptosis-inducing effects in cancer cells. For this purpose, a palladium(II)-saccharinate complex containing terpyridine which was synthesized by our research group was investigated in terms of its anti-tumor effects against mouse embryonic fibroblast NIH/3T3 (normal cell line) and rat embryonic fibroblast 5RP7 (H-ras transformed cell line) in vitro. The MTT and ATP viability assays were used to determine anti-growth/cytotoxic effects. Cytotoxic activity was confirmed by real time cytotoxicity analysis system. Flow cytometry analysis was further used to determine the mode of cell death (apoptosis/necrosis). Apoptosis was confirmed by triple-staining the cells with Hoechst 33342/PI/Calcein-AM triple and evaluated with fluorescence microscopy. It was found that the compound showed significant anti-growth activity by inducing apoptosis in a dose dependent manner. In conclusion, taking into account the cytotoxic activity of the compound at even relatively lower doses, in vivo experiments to elucidate its potential use for the treatment of fibrosarcoma are warranted., (Copyright © 2013. Published by Elsevier Ltd.)

A phytochemical analysis of the bulbs of Allium vavilovii M. Pop. & Vved. was attained for the first time extensively, affording to the isolation of four new furostanol saponins, named vavilosides A1/A2-B1/B2 (1a/b-2a/2b), as two couple of isomers in equilibrium, together with ascalonicoside A1/A2 (3a/3b) and 22-O-methyl ascalonicoside A1/A2 (4a/4b), previously isolated from shallot, Allium ascalonicum. High concentrations of kaempferol, kaempferide, and kaempferol 4(I)-glucoside were also isolated. The chemical structures of the new compounds, established through a combination of extensive nuclear magnetic resonance, mass spectrometry and chemical analyses, were identified as (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-galactopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside A1), (25R)-furost-5(6)-en-1β,3β,22β,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-galactopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside A2), (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-xylopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside B1), (25R)-furost-5(6)-en-1β,3β,22β,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-d-xylopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside B2). The isolated saponins showed cytotoxic activity on J-774, murine monocyte/macrophage, and WEHI-164, murine fibrosarcoma, cell lines with the following rank: vaviloside B1/B2>ascalonicoside A1/A2>vaviloside A1/A2., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the present study, we prepared NGR-modified sterically stabilized liposomes containing paclitaxel (NGR-SSL-PTX) in order to evaluate their potential targeting to aminopeptidase N receptors expressed on tumor endothelial cells and the tumor cell surface and its anti-angiogenic activity following metronomic administration. NGR-SSL-PTX was prepared by a thin-film hydration method. The in vitro targeting characteristics of NGR-modified liposomes on HUVEC (human umbilical vein endothelial cells), HT1080 (human fibrosarcoma cells) and MCF-7 (human breast adenocarcinoma cells) were then investigated. The effect of NGR-SSL-PTX on HUVEC proliferation and migration was also tested. The pharmacokinetics of NGR-SSL-PTX was studied in rats. The in vivo targeting activity of NGR-modified liposomes was investigated in HT1080 tumor-bearing mice. The anti-tumor activity of NGR-SSL-PTX following metronomic administration was evaluated in HT1080 tumor-bearing mice in vivo. The targeting activity of the NGR-modified liposomes was demonstrated by in vitro flow cytometry and confocal microscopy as well as in vivo confocal immunofluorescence microscopy and bio-distribution experiments. The results of endothelial cell proliferation and migration and microvessel density (MVD) confirmed the anti-angiogenic activity of NGR-SSL-PTX in vitro and in vivo. The sustained circulation of NGR-SSL-PTX was shown in the pharmacokinetic study. NGR-SSL-PTX is able to improve treatment efficacy producing the most significant anti-tumor activity and anti-angiogenic following metronomic administration., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Fibrosarcoma is rare in the pediatric age group. It generally involves the extremities and the trunk but rarely involves the genital area. We report a case of a large fungating infantile fibrosarcoma of the penis in a 2-year-old Malay boy. Partial recovery of the penile structure was achieved after chemotherapy. The difficulty in managing the social and surgical aspect of this case is discussed in our report. To the best of our knowledge, this is the first case report of infantile fibrosarcoma involving the penis in an Asian region., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pseudolaric acid B (PAB) is the primary biologically active compound isolated from the root bark of P. kaempferi Gordon. Previous studies have demonstrated that PAB arrests cells in G2/M phase in several cancer cell lines without significantly perturbing the G2/M transition-associated proteins. CylinB1, a marker for mitotic phase arrest, was up-regulated in cells treated with PAB. Therefore, we investigated whether PAB affects cell cycle progression at the mitotic phase. The mitotic index increased during a 24h treatment with PAB, suggesting that PAB arrested cell cycle progression at mitosis. In addition, after a prolonged mitotic arrest, the cells underwent mitotic catastrophe. After an extended treatment with PAB (longer than 24h), the protein levels of cylinB1 and cdc2 significantly decreased in both nuclear and cytosolic extracts. According to these results, we concluded that mitotic slippage could be due to the inactivation of the cylinB1-cdc2 complex resulting from prolonged treatment with PAB. The cells undergoing mitotic catastrophe died via apoptosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A phytochemical analysis of Aesculus pavia has led to the isolation of eight novel triterpenoid saponins, based on oleane type skeleton and named paviosides A-H (1a, 1b-4a, 4b). On the basis of chemical, and 2D NMR and mass spectrometry data, the structures of the new compounds were elucidated as 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-d-glucopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl barringtogenol C (1a), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-glucopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-angeloyl-22-acetyl barringtogenol C (1b), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-galactopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl barringtogenol C (2a), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-galactopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-angeloyl-22-acetyl barringtogenol C (2b), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl barringtogenol C (3a), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-d-glucopyranosiduronic acid 21-angeloyl-22-acetyl barringtogenol C (3b), 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-tigloyl-22-acetyl protoaescigenin (4a), and 3-O-[β-D-xylopyranosyl (1 → 2)] [-β-D-xylopyranosyl (1 → 4)]-β-D-glucopyranosiduronic acid 21-angeloyl-22-acetyl protoaescigenin (4b). The compounds showed cytotoxic activity on J-774, murine monocyte/macrophage, and WEHI-164, murine fibrosarcoma, cell lines. Among them, paviosides E-H (3a, 3b and 4a, 4b) showed higher activity with values ranging from 2.1 to 3.6 μg/mL. Structure-activity relationship studies indicated the positive effect on the activity of xylose unit in the place of glucose, while a little detrimental effect is observed when glucose is substituted by galactose. The aglycone structure and the presence of a tigloyl or an angeloyl group at C-21 do not affect significantly the inhibitory activity on both tested cell lines., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinases are implicated in a wide range of pathophysiological processes and potent selective inhibitors for these enzymes continue to be eagerly sought. 5,5-Disubstituted barbiturates hold promise as inhibitor types being stable in vivo and relatively selective for the gelatinases (MMP-2 and MMP-9). In this paper we describe the synthesis of 5-piperazine and -homopiperazine substituted barbiturates. The activity of these compounds as gelatinase inhibitors was evaluated using supernatants from 12-O-tetradecanoylphorbol-13-acetate (PMA)-stimulated HT-1080 cells as well as using recombinant human MMPs. N-Acyl homopiperazine compounds were found to be potent inhibitors of the gelatinases (range in nM) and generally more potent than the corresponding piperazine analogues. The panel of N-acyl homopiperazines was enlarged in order to exploit differences between the gelatinases at the S2' site in order to design MMP-2- or MMP-9-selective inhibitors. Compounds in this group exhibited single digit nano-molar potency and some selectivity between the two enzymes. Representative potent compounds were effective inhibitors of cancer cell migration., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

New strategies that stimulate cell-mediated immunity (CMI) against tumors and inhibit regulatory T cells are needed to improve the outcome of cancer immunotherapy. The aim of this study was to enhance the anti-tumor immunity of gp96 vaccine through naloxone administration. Therefore, we used BALB/c mouse model of fibrosarcoma tumor and analyzed the tumor size, splenocyte proliferation, spleen and tumor-infiltrated lymphocytes. Tumor and spleen CD4+CD25+Foxp3+ regulatory T lymphocytes, cytotoxic activity of the splenocytes, IFN-gamma and IL-4 secretion were assessed to describe the anti-tumor immune response. Our findings showed that co-administration of gp96 and naloxone has resulted in a significant reduction in CD4+CD25+Foxp3+ regulatory T cells in the spleen. The results indicated that on days 27 and 32 the tumors in the gp96+Nal group grew significantly slower. Moreover, co-administration of gp96 and naloxone significantly increased the intra-tumor CD8+ T cells and cytotoxic activity. In addition the results indicated a significant increase in the proliferation of splenocytes and IFN-gamma production. Our results demonstrate that naloxone is an effective immunoadjuvant in cancer immunotherapy.

The cytosolic members of the heat shock protein 70 (HSP-70) family have been shown to elicit protective cell mediated immunity in animal tumor models. The aim of this study was to investigate the effect of the HSP-70 enriched lysate of heated tumor cells as vaccines in cancer immunotherapy in the mouse model for WEHI-164 fibrosarcoma. Three animal bearing tumor groups were investigated: test group; vaccinated with enriched HSP-70 tumor lysate, control group I; vaccinated with tumor lysate only and control group II; received PBS. The results indicated that vaccinated mice in the test group had resulted in a significant reduction in tumor size and longer survival. To find the mechanism of these results, we measured the splenocytes proliferation, tumor infiltrated lymphocytes and cytotoxic activity of the splenocytes. The results indicated a significant increase in the proliferation of mouse splenocytes, a significant increase in the CD8+ lymphocytes as well as significant increase in the cytotoxic activity of splenocytes against the target cells in the test group. In addition, we analyzed the shifting of Th1/Th2 in all the groups. The results indicated a significant increase in the IFN-gamma production in the test group. These findings provided a useful therapeutic model for development of approaches to cancer treatments.

Principal rotenoids (deguelin, tephrosin, rotenone, and 12a-hydroxyrotenone) (3-30microM) isolated from the stems of Erycibe expansa significantly inhibited invasion of human fibrosarcoma HT1080 cells through Matrigel-coated filters and release of proMMPs-2 and 9. In addition, deguelin and tephrosin showed differentiation-inducing activity in human promyelocytic leukemia HL-60 cells. Furthermore, effects of various constituents isolated from the ethyl acetate-soluble fraction on proliferation of human leukemia U937 cells were examined. As a result, most of isoflavones and several flavans as well as rotenoids showed moderate or substantial anti-proliferative activities.

A new class of compounds, the pyrrolo[2,3-h]quinolin-2-ones, nitrogen isosters of the angular furocoumarin Angelicin, was synthesized with the aim of obtaining new photochemotherapeutic agents with increased antiproliferative activity and lower undesired toxic effects than the lead compound. Two synthetic pathways were approached to allow the isolation both of the dihydroderivatives 10-17 and of the aromatic ring system 23. Compounds 10-17 showed a remarkable phototoxicity and a great UVA dose dependence reaching IC(50) values at submicromolar level. Intracellular localization of these compounds has been evaluated by means of fluorescence microscopy using tetramethylrhodamine methyl ester and acridine orange, which are specific fluorescent probes for mitochondria and lysosomes, respectively. A weak co-staining was observed with mitochondrial stain, whereas a specific localization in lysosomes was observed. Studies directed to elucidate the mode of action of this series of compounds revealed that they do not intercalate with DNA and do not induce photodamage to the macromolecule. On the contrary, they induce significative photodamage to lipids and proteins.

Photodynamic therapy (PDT) inflicts tumor tissue injury that is experienced by the host as a local trauma. This provokes a strong host response with pronounced neutrophilia as one of its manifestations. Mouse FsaR fibrosarcoma model was used for investigating photodynamic therapy (PDT)-induced neutrophilia and its link to the acute phase response. Compared to normal mice, the extent of neutrophilia induced following Photofrin-based tumor PDT in adrenalectomized host mice was less pronounced revealing the elicited engagement of the adrenal-pituitary axis, which is one of the principal characteristics of the acute phase response. Neutrophilia was demonstrated after tumor-localized PDT even in the host mice previously depleted of circulating neutrophils. The rise in serum levels of complement C3 protein, which is an acute phase reactant and a principal mediator of tumor PDT-induced neutrophilia, occurred at the post PDT time period when the neutrophilia was largely resolved. However, the activation of complement system (assessed by the standard erythrocyte hemolysis assay) peaked already at 6 h after PDT and correlated with the time kinetics of PDT-induced neutrophilia. The findings of this study uncover the link between tumor PDT-induced neutrophilia and key acute phase response manifestations, the activation of adrenal-pituitary axis and the expression of a complement C3 protein (major acute phase reactant).

Vascular endothelial growth factor (VEGF) and platelet-derived lipid sphingosine-1-phosphate (S1P) are two proinflammatory mediators which contribute to angiogenesis, in part through the synthesis of platelet-activating factor (PAF). The red grape skin polyphenolic extract (SGE) both prevents and inhibits angiogenesis in the Matrigel model, decreases the basal motility of endothelial and cancer cells, and reverses the chemotactic effect of S1P and VEGF on bovine aortic endothelial cells (BAECs) as well as the chemotactic effect of conditioned medium on human HT-1080 fibrosarcoma, human U-87 glioblastoma, and human DAOY medulloblastoma cells. Inhibition of VEGF- and S1P-mediated chemotaxis by SGE is associated with a down-regulation of ERK and p38/MAPK phosphorylation and a decreased in acute PAF synthesis. Notably, as do extracellular inhibitors of PAF receptor, SGE prevents S1P-induced PAF synthesis and the resulting activation of the S1P/endothelial differentiation gene-1 cascade. Given the key role of VEGF and S1P in inflammation, angiogenesis, and tumor invasion, SGE may therefore contribute to prevent (or to delay) the development of diseases associated with angiogenesis dysregulation, including cancer. The dual inhibition of S1P- and VEGF-mediated migration of endothelial cell and of serum-stimulated migration of U-87 cells suggests a usefulness of SGE against highly invasive human glioblastoma.

The influence of environmental pH on the production of tumoricidal free radical nitric oxide (NO) was investigated in mouse fibrosarcoma L929 and rat glioma C6 cell lines. A combination of IFN-gamma and IL-1 induced a significant NO release and subsequent reduction of cell viability in tumor cell lines. Acidification of cell culture medium reduced tumor cell NO production in a pH-dependent manner. While the inhibitory effect of acidosis on NO production in C6 cells was associated with a further decrease in cell viability, it completely rescued L929 cells from NO-dependent apoptotic and necrotic death. Acidic pH diminished IFN-gamma+ IL-1-induced expression of inducible NO synthase (iNOS) mRNA and protein, and abolished the activation of iNOS transcription factor IRF-1 in L929 cells. Moreover, extracellular acidosis significantly impaired cytokine-induced phosphorylation of MAP kinase p44/42 (ERK1/2) and subsequent expression of transcription factor c-Fos in L929 cells. Finally, mild acidosis (pH 6.8) augmented, while severe acidosis (pH 6.0) reduced, IFN-gamma-induced iNOS activation/NO release and NO-dependent anticancer activity of rat and mouse macrophages. Taken together, our findings indicate that modulation of macrophage and tumor cell iNOS by an acidic microenvironment might influence the progression of NO-sensitive solid tumors.

Using tumor cell-restricted overexpression of glutathione peroxidase 4 (GP x 4), we investigated the contribution of tumor cell eicosanoids to solid tumor growth and malignant progression in two tumor models differing in tumorigenic potential. By lowering cellular lipid hydroperoxide levels, GP x 4 inhibits cyclooxygenase (COX) and lipoxygenase (LOX) activities. GP x 4 overexpression drastically impeded solid tumor growth of weakly tumorigenic L929 fibrosarcoma cells, whereas B16BL6 melanoma solid tumor growth was unaffected. Yet, GP x 4 overexpression did markedly increase the sensitivity of B16BL6 tumors to angio-destructive TNF-alpha therapy and abolished the metastatic lung colonizing capacity of B16BL6 cells. Furthermore, the GP x 4-mediated suppression of tumor cell prostaglandin E(2) (PGE(2)) production impeded the induction of COX-2 expression by the tumor stress conditions hypoxia and inflammation. Thus, our results reflect a PGE(2)-driven positive feedback loop for COX-2 expression in tumor cells. This was further supported by the restoration of COX-2 induction capacity of GP x 4-overexpressing L929 tumor cells when cultured in the presence of exogenous PGE(2). Thus, although COX-2 expression and eicosanoid production may be enabled by PGE(2) from the tumor microenvironment, our results demonstrate the predominant tumor cell origin of protumoral eicosanoids, promoting solid tumor growth of weakly tumorigenic tumors and malignant progression of strongly tumorigenic tumors.

The use of a novel injectable biocompatible and biodegradable camptothecin-polymer implant for sustained intra-tumoral release of high concentrations of camptothecin is described. The drug delivery vehicle is an in situ thermogelling formulation, which is based on the natural biopolymer chitosan. This formulation, containing homogeneously dispersed camptothecin, was implanted intra-tumorally into a sub-cutaneous mouse tumor model (RIF-l). The effectiveness of treatment was measured in terms of tumor growth delay (TGD). Animals treated with the polymer implants containing camptothecin had significantly longer TGDs compared to untreated animals as well as to animals treated systemically with camptothecin by intra-peritoneal injection with no evidence of toxicity in terms of loss of body weight. The results indicate that this novel biodegradable polymer implant is an effective vehicle for the sustained intra-tumoral delivery of camptothecin which might also be suitable to deliver other insoluble anti-cancer drugs such as taxol.

Given the important role of gaseous free radical nitric oxide (NO) in tumor cell biology, we investigated the ability of the anti-cancer drugs 5-Aza-2'-deoxycytidine (ADC) and paclitaxel to modulate NO production in mouse L929 fibrosarcoma cells. Both drugs reduced IFN-gamma-stimulated NO release in cultures of L929 and primary fibroblasts, but not in mouse peritoneal macrophages. The inhibitory effect was due to the reduced expression of inducible NO synthase (iNOS), the enzyme responsible for cytokine-induced intracellular NO synthesis, as both agents markedly suppressed the interferon-gamma (IFN-gamma)-triggered increase in iNOS concentration in L929 cells. In addition, ADC and paclitaxel prevented the IFN-gamma-triggered activation of p44/p42 mitogen-activated protein (MAP) kinase in L929 fibroblasts, suggesting a possible mechanism for the observed inhibition of iNOS expression. These results might have important implications for the therapeutic effect of ADC and paclitaxel, since their inhibitory action on NO release partly neutralized the NO-dependent toxicity of IFN-gamma on L929 fibrosarcoma cells.

Isothiocyanates (ITCs) have been isolated from plants. Naturally occurring and synthetic ITCs are known as effective chemopreventive agents. Ethyl 4-isothiocyanatobutanoate (E-41B) is a derivative of gamma-aminobutyric acid. Immunotoxic and canocerostatic effects of E-41B in female inbred Lewis rats implanted with experimental fibrosarcoma BP6-TU2 was evaluated in this study. On day 5 after subcutaneous application of tumor cells, animals started to be treated intraperitoneally three times a week with two different doses of E-41B: 28 and 35 mg/kg/day during 28 days. High dose of E-41B was close to maximum tolerated dose (MTD). Control groups of rats with or without tumors injected intraperitoneally only saline or 70% dimethylsulphoxide were added. Administrating of E-41B resulted in suppression of thymus, popliteal lymph node, spleen weight and spleen cellularity. Hematologic evaluation displayed decreased erythrocyte (ERY) count and level of hemoglobin (HB) in rats treated withE-41B. Immune assays--the phagocytic activity of polymorphonuclear leukocytes (PMN) and monocytes, primary antibody response and in vitro proliferative activity of spleen lymphocytes (LY) to mitogens were not significantly affected by E-41B treatment E-41B moderately decreased tumor weights, but this decrease was not statistically significant in comparison with DMSO-exposed rats with tumors. The fibrosarcoma implantation itself increased significantly spleen weight and changed hematological parameters (decreased HB, increased mean cell volume of ERY, increased leukocyte count, increased % PMN, decreased % LY, decreased % EO). Moreover, moderate decreased percentage of CD161+ positive cells (NK cells) were found in peripheral blood. Immune assays showed decline in proliferation of lymphocytes and phagocytic activity of leukocytes. Our findings indicate that administration of E-41B displayed hematoxic effect in rats implanted with fibrosarcoma. Immunotoxic effect was shown as decreased lymphoid organ weight and spleen cytotoxicity although function of immune cells was not impaired.

It was previously demonstrated that a single injection of OK-432 (a penicillin-treated freeze-dried Streptococcus) mixed with fibrinogen into cancer tissues induces marked infiltration by eosinophils of the tumor stroma and leads to tumor necrosis. In the present study, we examined mechanisms regulating the local accumulation of eosinophils and the role of infiltrating eosinophils in tumor regression using the OK-432/fibrinogen injected Meth-A fibrosarcoma tumor. After injection of OK-432/fibrinogen into the tumor on the left flank of the BALB/c mice, eosinophil infiltration became obvious in the tumor stroma on day 3 following the accumulation of macrophages and neutrophils, was massive on day 5 and decreased by day 10. After the decrease in the infiltration of eosinophils, the tumor injected with OK-432/fibrinogen diminished markedly in size with ulceration as compared with control. Northern blot analysis revealed that expression of IL-5 mRNA in the tumor tissue was not detected on day 0, was significantly on day 3, reached the maximum on day 5, and thereafter decreased by day 10. Although intraperitoneal injection of rat anti-IL-5 monoclonal antibody in tumor bearing mice prior to OK-432 injection inhibited the infiltration of eosinophils, the antitumor effects of OK-432 persisted. In the blood, neither eosinophilia nor IL-5 activity was recognized during the course of the experiment. These results suggest that intratumoral injection of OK-432/fibrinogen induces local production of IL-5, which in turn recruits eosinophils into the tumor tissue, however, the infiltrating eosinophils do not play an important role in tumor regression.

Inhibitory effects on some immunological responses and MethA fibrosarcoma in the double grafted tumor system in mice were compared between 6-mercaptopurine (6-MP) and its novel water-soluble derivative, gamma-(9H-purine-6-yl)thiomethyl L-glutamate (6-MPG). The dose-dependent inhibitory effects by 6-MPG on the hemagglutinin response to SRBC, DTH reaction to MBSA, contact sensitivity to oxazolone, GVH response and growth of the primary tumor were 3-10 times weaker than those by 6-MP, probably reflecting the difference in their cytotoxicities antimetabolites. However, the two drugs were nearly equipotent in reproducing inhibition of the secondary tumor growth, which is a host-mediated immunological response to tumor antigen as shown by its dependency on the primary inoculation with 1 x 10(4) or more MethA cells and by the production of anti-tumor splenocytes in tumor-bearing animals (the Winn assay). Thus, 6-MPG may point to the direction of derivatization towards anti-tumor immunopotentiators with an improved therapeutic index.

5'-Phosphatidyl-5-fluorouridines, with the same backbone structure as that of natural phospholipids, in which a polar-head group of usual phospholipids is replaced by 5-fluorouridine, were designed to be potent antitumor agents. 5'-Phosphatidyl-5-fluorouridines with a variety of diacyl or dialkyl residues in the glycerol moiety, were synthesized by phospholipase D-catalyzed transphosphatidylation from the corresponding phosphatidylcholine and 5-fluorouridine. These new compounds were evaluated in mice with experimental tumors by ip and po administration. Dipalmitoyl and distearoyl derivatives 1b and 1c had the greatest antitumor activity against both P388 leukemia and Meth A fibrosarcoma in mice.

Mixed amine platinum complexes have been identified as a new class of antitumor agents with activity in some cisplatin-resistant tumor models. cis-Ammine/cyclohexylamine-dichloroplatinum(II) is one such analog that we have evaluated in vivo and found it to have antitumor activity that was comparable to that of cisplatin in a solid murine fibrosarcoma tumor model. In contrast to the nephrotoxicity observed with cisplatin, the analog was free from inducing this side-effect. Pharmacokinetics of the two compounds administered i.v. at equitoxic dose levels to tumor-bearing mice indicated similar decay kinetics of total platinum in plasma, kidney and the tumor. Furthermore, DNA-platinum adducts of the two agents were similar in the tumor. Total adduct levels in the kidney, on the other hand, were significantly greater (P < 0.5) by up to 4-fold for cisplatin compared with the mixed amine analog. Likewise, the levels of interstrand cross-links of the two platinum complexes were comparable in the tumor, but significantly greater (P < 0.05) in the kidney for cisplatin. The data indicate that the greater renal levels of total and interstrand DNA-platinum adducts formed by cisplatin correlate with renal damage associated with this agent, and suggest that adduct levels, and not total tissue platinum levels, provide a more useful correlation with pharmacodynamic observations.

The combined effects of the synthetic glucosaminylmuramyl dipeptide (GMDP) on the antitumor activity of chemically synthesized lipid A analogs, compound A-103 (glucosamine-4-phosphate with (R)-3-tetradecanoyloxytetradecanoyl group at the C-2 and C-3 positions), Escherichia coli-type lipid A (506), Salmonella typhimurium LT-2 lipopolysaccharide (LPS) against Meth A fibrosarcoma in mice were examined. Meth A fibrosarcoma cells (5 x 10(5) were inoculated intradermally into BALB/c mice on day 0, and compound A-103 and/or GMDP was administered intravenously (i.v.) on days 7 and 9. Two i.v. injections of A-103 (50 micrograms) alone or GMDP (10 micrograms) alone induced 42.8 or 51.8% inhibition of the rate of tumor growth, however, A-103 (100 micrograms) with GMDP (10 micrograms) exhibited a high 68.7% inhibition rate 19 days after tumor inoculation. The inhibition of the tumor growth rate by the combination A-103 (100 micrograms) or 506 (50 micrograms) with GMDP (10 micrograms) was stronger than that of A-103 or 506 with MDP (10 micrograms). The combination of LPS (1 or 10 micrograms) with GMDP (10 micrograms) exhibited a higher inhibition rate than that of LPS with MDP, and three or four tumor-free mice out of five mice were observed, suggesting that the combined effect of GMDP is more potent than that of MDP. With the addition of GMDP, A-103 did not enhance the production of tumor necrosis factor (TNF) on the basis of L929 cell lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Combined effects of chemically synthesized lipid A analogs, the compound A-171 (acylglucosamine-4-phosphate with (R)-3-hydroxytetradecanoyl and (R)-3-hydroxytetradecanoyloxy]tetradecanoyl group at the C-2 and C-3 positions), or the compound A-172 (with (R)-3-hydroxytetradecanoyloxy]tetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl group at the C-2 and C-3 positions), and muramyl dipeptide (MDP) on antitumor activity against Meth A fibrosarcoma, were examined. Meth A fibrosarcoma cells (5 X 10(5)) were inoculated intradermally into BALB/c mice on day 0, compound A-172 and/or MDP were administered intravenously (i.v.) on day 7. Although the antitumor activity by single i.v. injection of A-172 (50 micrograms/mouse) with MDP (10 micrograms) was weaker than that of 50 micrograms of synthetic lipid A analogs (506), or 10 micrograms of bacterial lipopolysaccharide (LPS) with MDP, A-172 alone and with MDP exhibited tumor inhibition rates of 49.0 and 70.6%, respectively. When A-171 (50 micrograms) with MDP (10 micrograms) was administered i.v. twice (days 7 and 10) into mice inoculated Meth A fibrosarcoma, two of five mice caused complete tumor regression. Furthermore, L929 cell lysis by the combination of A-171, A-172 with MDP was higher than that by the analogs or MDP alone, suggesting that the lipid A analogs of monosaccharide type as well as LPS are able to enhance the production of tumor necrosis factor in the presence of MDP.

The combined effects of rIL-2 and OK-432 were investigated against a Meth-A tumor, a syngeneic tumor of inbred BALB/c mice. An analysis of the effector cells was also performed. The treatment resulted in an inhibition in vivo of tumor growth and increased survival of the Meth-A tumor-bearing mice. Splenic cells obtained from Meth-A inoculated mice which received combination therapy were not only NK-sensitive YAC-1 and LAK-sensitive EL-4 cells, but also NK-resistant Meth-A cells, as shown in a 4-h 51Cr-release assay. Syngeneic killer cell activity against Meth-A cells was abolished almost completely with anti-Thy 1.2 treatment and about 70% of the activity was abolished with anti-asialo GM1 treatment in a complement-dependent cytotoxic assay. It was not changed by the removal of macrophages and B cells from the splenic cells. Mice which survived for 60 days after the start of therapy rejected Meth-A inoculation when rechallenged, suggesting the establishment of a specific immunity. Combination therapy appeared to be beneficial against Meth-A cells and T-cells appeared to play a determining role in the treated Meth-A bearing mice. It was suggested that more than two populations of killer cells exist in the spleen treated with the combined therapy and they may have the same characteristics as activated T and NK cells with or without specific killer T-cells.