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The synthesis and structural characterization of the monohydrated 1:2 cocrystal salt of acriflavine with 3,5-dinitrobenzoic acid [systematic name: 3,6-diamino-10-methylacridin-10-ium 3,5-dinitrobenzoate–3,5-dinitrobenzoic acid–water (1/1/1), C14H14N3 +·C7H3N2O6 −·C7H4N2O6·H2O] are reported. Single-crystal X-ray diffraction measurements show that the title solvated monohydrate salt crystalizes in the monoclinic space group P21 with one acriflavine cation, a 3,5-dinitrobenzoate anion, a 3,5-dinitrobenzoic acid molecule and a water molecule in the asymmetric unit. The neutral and anionic forms of 3,5-dinitrobenzoic acid are linked via O—H...O hydrogen bonds to form a monoanionic dimer. Neighbouring monoanionic dimers of 3,5-dinitrobenzoic acid are linked by nitro–nitro N—O...N and nitro–acid N—O...π intermolecular interactions to produce a porous organic framework. The acriflavine cations are linked with carboxylic acid molecules directly via amine–carboxy N—H...O, amine–nitro N—H...O and acriflavine–carboxy C—H...O hydrogen bonds, and carboxy–acriflavine C—O...π, nitro–acriflavine N—O...π and acriflavine–nitro π–π interactions, or through the water molecule by amino–water N—H...O and water–carboxy O—H...O hydrogen bonds, and are located in the voids of the porous organic framework. The intermolecular interactions were studied using the CrystalExplorer program to provide information about the interaction energies and the dispersion, electrostatic, polarization and repulsion contributions to the lattice energy.

The nine derivatives of acridine-9-carboxylic acid (CMADs) capable for chemiluminescence (CL), representing various classes of compounds were isolated in a chemically pure state (assessed by RP-HPLC) and identified using high resolution mass spectrometry (ESI-QTOF) and magnetic resonance ( 1 H NMR) techniques. Among them are aryl acridinium esters, containing certainly selected and located substituents in both aromatic systems, an acridinium perfluorinated aliphatic ester and an acridinium sulfonamide. The main aim of the study was to compare, under the conditions of their optimal performance, the luminogenic properties of the above molecular systems, such as efficiency and kinetics of emission, limits of detection/quantification as well as stability in aqueous solutions. All of the investigated acridinium salts can serve as CL indicators or fragments of CL labels in modern medical diagnostics (e.g. chemiluminescence and electrochemiluminescence immunoassays (CLIA, ECLIA)), hybridization protection assays of nucleic acids (HPA) as well as other ultra-sensitive luminescence tests. Taking advantage of the flow injection mass analysis, the appearance of unstable cationic intermediates produced upon chemiluminogenic oxidation of selected acridinium salts in an alkaline environment was confirmed for the first time. The observed entities include hydroxyl and hydroperoxide adducts (as radical cations) as well as the acridan spirodioxetanone derivative, making a high energetic and direct precursor of the target emitters, an electronically excited 10-methylacridan-9-ones. The results are supported with quantum chemistry calculations at the DFT level of theory. Theoretical studies have helped to determine the unstable intermediates appearing during in CL in ESI-QTOF experiments. In the group of acridinium ester they allowed to observe the positive linear trend among between the kinetics of CL decay and calculated coefficients of p z LUMO orbitals on the carbon atoms being the reaction centers responsible for generation of light.

Antibody-dependent cellular cytotoxicity (ADCC) by cells from peripheral blood was studied in patients with; chronic lymphocytic leukaemia (CLL), chronic granulocytic leukaemia (CGL), acute lymphoblastic leukaemia (ALL), acute non-lymphoid leukaemia (ANLL) and compared with that of healthy donors. The ADCC activity in all the types of leukaemia was on the average lower as compared with the control values, being especially low in both CLL and CGL. Further, it has also been shown that the ADCC activity of non-leukaemic blood cells in ANLL was lower as compared with control values. The latter observation indicates that, at least in ANLL, the reduced ADCC potential of peripheral blood cells is not due to dilution of effector cells by poorly- or non-active leukaemic cells.

Nonsteroidal anti-inflammatory drugs have been shown to reduce the incidence and mortality from colorectal cancer. It has recently been demonstrated that these drugs are capable of suppressing the production of pro-angiogenic factors from tumor cells. The mechanisms of antitumor action of interleukin 12 include the enforced secretion of anti-angiogenic factors and stimulation of antitumor immunity. Therefore, we hypothesized that the combination of a model nonsteroidal anti-inflammatory drug — indomethacin and interleukin 12 would result in enhanced angiogenesis-dependent antitumor effects against a colon-26 carcinoma cells transplanted into syngeneic mice. As expected the combined administration of both agents simultaneously resulted in a strengthened antitumor activity that was manifested as a retardation of tumor growth and prolongation of mouse survival. Importantly some mice were completely cured after the combined treatment. As administration of interleukin 12 and indomethacin resulted in enhanced inhibition of angiogenesis it seems possible that prevention of new blood vessel formation is one of the mechanisms responsible for the observed antitumor effects.

Lovastatin, a drug commonly used in the treatment of hypercholesterolemia, has previously been reported to exert potentiated antitumor activity when combined with either tumor necrosis factor-α (TNF-α), cisplatin or doxorubicin in a melanoma model in mice. Since lovastatin interferes with the function of ras oncogene-encoded (Ras) proteins, we have investigated the antitumor activity of lovastatin and TNF-α using a Ha-ras-transformed murine tumor model. In in vitro studies, lovastatin inhibited the growth of cells transformed with Ha-ras oncogene (Ras-3T3 and HBL100-ras cells) more effectively than control NIH-3T3 and HBL100-neo cells. In in vivo experiments, the Ras-3T3 tumor demonstrated significantly increased sensitivity to combined treatment with both lovastatin (50 mg/kg) and TNF-α (1 μg/day) compared with either agent alone. Combined treatment with both agents also resulted in greater inhibition of blood-vessel formation. Ras-3T3 tumor cells produced increased amounts of vascular endothelial growth factor (VEGF) and lovastatin effectively suppressed VEGF production by these cells. Our results suggest that lovastatin increases antitumor activity of TNF-α against tumor cells transformed with v-Ha-ras oncogene via inhibition of tumor-induced blood-vessel formation. Int. J. Cancer 81:560–567, 1999. © 1999 Wiley-Liss, Inc.

Retinoids combined with interferon alpha-2a (IFN alpha) or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] have shown marked synergistic inhibitory effects on angiogenesis induced by tumor cell lines harboring DNA of oncogenic human papillomaviruses (HPV) type 16 or 18. This report demonstrates comparable effects of these compounds on angiogenesis induced by non-HPV-bearing transformed cell lines, including breast carcinoma (T47D) and vulval carcinoma (A431) cell lines. Systemic treatment of mice with all-trans retinoic acid, 13-cis retinoic acid, 9-cis retinoic acid, IFN alpha or 1,25(OH)2D3 significantly decreased tumor cell-induced angiogenesis (TIA). In vitro pretreatment of T47D and A431 cells with these compounds also led to inhibition of their angiogenic capability when tested in the TIA assay. The inhibitory effects of retinoids could be counteracted by a selective antagonist of the nuclear retinoic acid receptor RAR alpha, suggesting a RAR alpha mediated mechanism of angiogenesis inhibition. The antiangiogenic effect of retinoids could be significantly enhanced by combination with IFN alpha or 1,25(OH)2D3. The results provide a further basis for the use of combinations of retinoids with IFN alpha or 1,25(OH)2D3 in the treatment of angiogenesis-dependent malignancies.

Various retinoids and interferons exert anti-tumor effects both in experimental studies and in clinical trials. Recent reports indicate that they have a synergistic antineoplastic activity. Our study aimed to determine whether these synergistic anti-tumor effects are related to inhibition of tumor-cell-induced angiogenesis. A further aim was to compare the anti-angiogenic activity of various retinoids including 9-cis retinoic acid, a ligand for nuclear retinoic acid receptor RXR, given alone and in combination with interferon alpha-2a (IFN alpha-2a). An in vivo experimental model of cutaneous angiogenesis in the mouse was used. Angiogenesis was induced by intradermal injection of HPV16- or HPV18 DNA-harboring tumor-cell lines. All-trans retinoic acid (all-trans RA), 13-cis retinoic acid (13-cis RA) and 9-cis retinoic acid (9-cis RA) as well as IFN alpha-2a applied to mice intraperitoneally for 5 consecutive days before induction of angiogenesis resulted in significant inhibition of angiogenesis. Combination of retinoids with IFN alpha-2a led to a synergistic inhibition of angiogenesis, as compared to the effects of the drugs given alone. Similar results were obtained when tumor cells were preincubated in vitro with the compounds, before injection into untreated mice. Our findings on synergistic anti-angiogenic effects of retinoids and IFN alpha-2a could explain, at least partially, the anti-tumor efficacy of combined therapy with these agents, and provide support for the role of angiogenesis in tumor growth.

The morphological characteristics of adrenergic and cholinergic innervation are described in the vas deferens of the domestic fowl. Adrenergic innervation was much better developed than the cholinergic. Both types of nerve fibre were found in the muscular membrane, submucosal membrane and in the mucosa. The largest number of adrenergic nerve fibres was observed in the muscular membrane. These were less numerous in the submucosa, mucosa and in the wall of small blood vessels. The largest number of cholinergic nerve fibres was noted under the mucosa. Single cholinergic nerve fibres were seen to penetrate between the epithelial cells.

Inhibition of the proteasome, a multicatalytic proteinase complex, is an attractive approach to cancer therapy. Here we report that a selective inhibitor of the chymotrypsin-like activity of the proteasome, PSI (N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal) may inhibit growth of solid tumors not only through apoptosis induction, but also indirectly--through inhibition of angiogenesis. Two murine tumors: colon adenocarcinoma (C-26) and Lewis lung carcinoma (3LL) were chosen to study the antitumor effect of PSI. In an in vivo model of local tumor growth, PSI exerted significant antitumor effects against C-26 colon carcinoma, but not against 3LL lung carcinoma. Retardation of tumor growth was observed in mice treated with both 10 nmoles and 100 nmoles doses of PSI and in the latter group prolongation of the survival time of tumor-bearing mice was observed. PSI inhibited angiogenesis in the C-26 growing tumors with no such effect in 3LL tumors. Unexpectedly, that activity was associated with upregulation of vascular endothelial growth factor (VEGF) at the level of mRNA expression and protein production in C-26 tumors treated with PSI. C-26 cells treated with PSI produced increased amounts of VEGF in vitro in a dose- and time-dependent manner. We demonstrated that in C-26 colon adenocarcionoma higher VEGF production may render endothelial cells susceptible to the proapoptotic activity of PSI and is associated with inhibition of tumor growth.

Purpose. To investigate antitumour efficacy of the combination of the antiangiogenic agent TNP-470 combined with chemoimmunotherapy in different tumour models in mice Materials. B6D2F1 mice and BALB/c mice were inoculated in the footpad of the right hind limb with B16F10 melanoma cells or colon adenocarcinoma cells C-26, respectively. Subsequently, they received therapy consisting of TNP-470 and/or IL-12 and tumour growth was observed. In the melanoma model this therapy regimen was combined with cisplatin in a subtherapeutic dose. The antiangiogenic action of the tested agents was evaluated using tumour-induced angiogenesis assay in vivo. In order to analyse interactions between TNP-470 (or cisplatin) and IFN-γ on tumour cells in vitro, the following methods were used: MTT assay, Western blot analysis, and flow cytometry analysis. Results. Administration of the combined therapy with TNP-470 and IL-12 resulted in augmented antitumour activity in colon-26 and B16F10 melanoma models. Addition of cisplatin further enhanced efficacy of this combined therapy in the melanoma model. We showed that antitumour activity of this combined therapy is mediated by multiple mechanisms: not only is enhancement of the antiangiogenic activity mediated by TNP-470 and IL-12 but also by the synergistic cytostatic/cytotoxic action of IL-12-induced IFN-γ and TNP-470 or cisplatin on tumour cells. The experiments revealed that TNP-470 together with IFN-γ leads to the increased expression of p21 protein in cancer cells, which in turn may contribute to their cytostatic/cytotoxic action in vitro. Conclusion. Our experiments show a successful TNP-470-based combination therapy and suggest that the enhancement of the antitumour activity could be explained by a concomitant effect on both endothelial and tumour cell compartments.

Butyric acid has been known to inhibit growth and to induce differentiation of a variety of tumor cells. Butyrate-treated tumor cells have also been observed to undergo apoptosis. Although butyrate compounds have demonstrated antitumor activity in murine tumor models and have already been admitted to clinical trials in tumor patients, the exact mechanism of their antitumor effects has not been elucidated. The results of our study showed antitumor activity of tributyrin, a butyric acid prodrug, in murine melanoma model and are strongly suggestive that antiangiogenic effects could participate in antitumor effects of butyrate compounds in vivo.

Tumor cell-induced angiogenesis, i.e., new blood vessel formation within tumor tissue, is an essential requirement for the growth of solid neoplasms. Interleukin-12 (IL-12) inhibits growth of a variety of experimental tumors in vivo. We tested whether antitumor activity of IL-12 is related to the inhibition of angiogenesis induced by tumor cell lines. Angiogenesis was induced in x-ray immunosuppressed Balb/c mice by intradermal injection of the following human tumor cells: T47D, originating from mammary carcinoma; A431, derived from vulval carcinoma; and Skv, established from bowenoid papulosis, Systemic treatment of the mice with murine IL-12 significantly decreased angiogenesis induced by human tumor cells in a time-and dose-dependent manner. Preincubation of human cells in vitro with IL-12 did not inhibit tumor cell-induced angiogenesis, suggesting that the antiangiogenic capacity of IL-12 is restricted to in vivo conditions. Treatment of the mice with rat antibody against murine interferon-gamma (IFN-gamma) resulted in counteracting the antiangiogenic effect of murine IL-12. Furthermore, human IFN-gamma inhibited the angiogenic activity of human tumor cell lines. This indicates that IFN-gamma is a mediator of the antiangiogenic effect of IL-12. The results show that the mechanism of antitumor action of IL-12 may depend not only on the immunostimulatory activity of this cytokine but also on its effect on tumor cell-induced angiogenesis. IL-12 should be considered as a potential candidate for the treatment of angiogenesis-dependent malignancies.

The growth of solid tumors requires a vascular supply, formed in the process of tumor-induced angiogenesis (TIA) (Folkman, 1990). TIA is an active sprouting of capillaries and new blood vessel formation due to some tumor cell-derived angiogenic factors capable of stimulating endothelial. cell proliferation and migration (Folkman and Klagsburn, 1987). TIA was shown to precede or accompany malignancy not only in various experimental. systems (Bouck, 1990) but also in natural. human tumors of the cervix, skin, breast and bladder (Folkman, 1990). The presence of newly formed blood vessels on the surface of the cervix can indicate an underlying but invisible tumor (Sillman, et al., 1981). Quantitation, either by routine histology or immunohistochemistry, of the number of blood vessels on tissue sections of breast carcinoma was shown to be useful for estimation of relapse-free survival. rate (Weidner et al., 1992) and probability of the metastatic disease (Horak et al., 1992).

Tumor-induced angiogenesis (TIA), i.e., the ability of transformed cells to stimulate new blood vessel formation is an important factor contributing to tumor growth and invasiveness. The antiangiogenic effect of the retinoids, all-trans retinoic acid, 13-cis retinoic and 9-cis retinoic acid, of 1,25-dihydroxyvitamin D3, and of their combinations were studied using an experimental system in vivo. TIA was induced in immunosuppressed mice by intradermal injection of the two human transformed keratinocyte lines, Skv-e2, harboring DNA of human papillomavirus (HPV) type 16, and HeLa, harboring HPV18 DNA. The three retinoids and 1,25-dihydroxyvitamin D3, when administered systemically to mice, before the angiogenesis assay significantly decreased TIA. Their combination led to a synergistic inhibition of TIA. These results provide the basis for the use of combination of retinoids and 1,25-dihydroxyvitamin D3 in treatment of neoplastic diseases.

As reported in a recent article in the Journal, Joseph and Isaacs (1) tried to combine Linomide® with other antiangiogenic agents known to inhibit tumorassociated macrophages (TAMs) to evaluate their joint antitumor effects. Unfortunately, the combination of Linomide with other antiangiogenic agents did not result in potentiation of therapeutic efficacy. Instead, one of these agents, pentoxifylline, inhibited the an