Microbial Transformations of Halolactones and Evaluation of Their Antiproliferative Activity.

The microbial transformations of lactones with a halogenoethylocyclohexane moiety were performed in a filamentous fungi culture. The selected, effective biocatalyst for this process was the Absidia glauca AM177 strain. The lactones were transformed into the hydroxy derivative, regardless of the type of halogen atom in the substrate structure. For all lactones, the antiproliferative activity was determined toward several cancer cell lines. The antiproliferative potential of halolactones was much broader than that observed for the hydroxyderivative. According to the presented results, the most potent was chlorolactone, which exhibited significant activity toward the T-cell lymphoma line (CL-1) cell line. The hydroxyderivative obtained through biotransformation was not previously described in the literature.

Yolkin, a Polypeptide Complex from Egg Yolk, Affects Cytokine Levels and Leukocyte Populations in Broiler Chicken Blood and Lymphoid Organs after In Ovo Administration.

Yolkin is a polypeptide complex isolated from hen egg yolk that exhibits immunomodulating properties. The aim of the present study was to determine whether in-ovo-delivered yolkin affects leukocyte populations and cytokine levels in broiler chickens. The experiment was carried out on eggs from Ross 308 broiler breeder birds. Yolkin was administered in ovo on the 18th day of incubation, once, at the following three doses: 1, 10, or 100 µg/egg. The immunological parameters were assessed in 1-, 7-, 14-, 21-, 28-, 35-, and 42-day-old birds kept under farming conditions and routinely vaccinated. The leukocyte populations were determined in the thymus, spleen, and blood. The cytokine (IL-1ß, IL-2, IL-6, and IL-10) levels were determined in the plasma of the broiler chickens. Each experimental group included eight birds. The most pronounced effect of yolkin was an increase in the population of T cells, both CD4+ and CD8+, mainly in the blood. This effect on the lymphocyte subsets may be valuable regarding chicken immune responses, mainly against T-dependent antigens, during infection or after vaccination.

Regiospecific Hydrogenation of Bromochalcone by Unconventional Yeast Strains.

This research aimed to select yeast strains capable of the biotransformation of selected 2'-hydroxybromochalcones. Small-scale biotransformations were carried out using four substrates obtained by chemical synthesis (2'-hydroxy-2″-bromochalcone, 2'-hydroxy-3″-bromochalcone, 2'-hydroxy-4″-bromochalcone and 2'-hydroxy-5'-bromochalcone) and eight strains of non-conventional yeasts. Screening allowed for the determination of the substrate specificity of selected microorganisms and the selection of biocatalysts that carried out the hydrogenation of tested compounds in the most effective way. It was found that the position of the bromine atom has a crucial influence on the degree of substrate conversion by the tested yeast strains. As a result of the biotransformation of the 2'-hydroxybromochalcones, the corresponding 2'-hydroxybromodihydrochalcones were obtained. The products obtained belong to the group of compounds with high potential as precursors of sweet substances.

Benzyl Isothiocyanate, a Vegetable-Derived Compound, Induces Apoptosis via ROS Accumulation and DNA Damage in Canine Lymphoma and Leukemia Cells.

Treatment of neoplastic diseases in companion animals is one of the most important problems of modern veterinary medicine. Given the growing interest in substances of natural origin as potential anti-cancer drugs, our goal was to examine the effectiveness of benzyl isothiocyanate (BITC), a compound found in cruciferous vegetables, against canine lymphoma and leukemia. These are the one of the most common canine cancer types, and chemotherapy is the only treatment option. The study involved established cell lines originating from various hematopoietic malignancies: CLBL-1, GL-1, CLB70 and CNK-89, immortalized noncancerous cell lines: MDCK and NIH-3T3 and canine peripheral blood mononuclear cells (PBMCs). The cytotoxic activity of BITC, apoptosis induction, caspase activity and ROS generation were evaluated by flow cytometry. H2AX phosphorylation was assessed by western blot. The study showed that the compound was especially active against B lymphocyte-derived malignant cells. Their death resulted from caspase-dependent apoptosis. BITC induced ROS accumulation, and glutathione precursor N-acetyl-l-cysteine reversed the effect of the compound, thus proving the role of oxidative stress in BITC activity. In addition, exposure to the compound induced DNA damage in the tested cells. This is the first study that provides information on the activity of BITC in canine hematopoietic malignancies and suggests that the compound may be particularly useful in B-cell neoplasms treatment.

Methoxy-Substituted γ-Oxa-ε-Lactones Derived from Flavanones-Comparison of Their Anti-Tumor Activity In Vitro.

BACKGROUND: The study investigated four flavanone-derived γ-oxa-ε-lactones: a parent unsubstituted compound and its three derivatives with the methoxy group in positions 2', 4' and 8. Our objective was to find out if the introduction of the methoxy group into the aromatic ring affects in vitro anti-tumor potency of the investigated lactones. METHODS: Cytotoxic and pro-apoptotic effects were assessed with cytometric tests with propidium iodide, annexin V, and Western blot techniques. We also investigated potential synergistic potency of the tested lactones and glucocorticoids in canine lymphoma/leukemia cell lines. RESULTS: The tested flavanone-derived lactones showed anti-cancer activity in vitro. Depending on its location, the methoxy group either increased or decreased cytotoxicity of the derivatives as compared with the parent compound. The most potent lactone was the one with the methoxy group at position 4' of the B ring (compound 3), and the weakest activity was observed when the group was located at C-8 in the A ring. A combination of the lactones with glucocorticoids confirmed their synergy in anti-tumor activity in vitro. CONCLUSIONS: Methoxy-substituted flavanone-derived lactones effectively kill canine lymphoma/leukemia cells in vitro and, thanks to their synergistic action with glucocorticoids, may potentially be applied in the treatment of hematopoietic cancers.

Hawthorn (Crataegus monogyna) Phenolic Extract Modulates Lymphocyte Subsets and Humoral Immune Response in Mice.

This study investigated the effect of hawthorn (Crataegus monogyna) phenolic extract on lymphocyte subsets in the lymphoid organs in nonimmunized mice and on humoral immune response in sheep red blood cell-immunized mice. Hawthorn phenolic extract (50, 100, 200 mg/kg) was administered orally five or ten times. Sheep red blood cells were injected 24 h after administration of the last extract dose. The lymphocyte subsets were assessed 24 and 72 h after the last dose. Humoral immune response was determined 4 and 7 days after immunization. Five doses of the extract decreased the percentage of CD4-CD8- and CD4+ thymocytes but elevated the percentage of CD4+CD8+ and CD8+ thymic cells. The extract increased the total number, percentage, and absolute count of T and B splenocytes. When administered five times, it lowered the percentage of T lymphocytes, but boosted the population of B lymphocytes of mesenteric lymph nodes (after 24 h). However, a rise in the population of T lymphocytes was observed 72 h after five and ten doses. The extract administered ten times elevated the number of plaque-forming cells and total anti-sheep red blood cell hemagglutinin titer but reduced the 2-ME-resistant antibody titer (day 7). At the same time, five doses of the extract increased antibody titers. Considering its impact on lymphocyte subsets and humoral immune response, hawthorn extract may be used as an immunomodulator.

Chalcone Methoxy Derivatives Exhibit Antiproliferative and Proapoptotic Activity on Canine Lymphoma and Leukemia Cells.

Chalcones are interesting candidates for anti-cancer drugs due to the ease of their synthesis and their extensive biological activity. The study presents antitumor activity of newly synthesized chalcone analogues with a methoxy group on a panel of canine lymphoma and leukemia cell lines. The antiproliferative effect of the 2'-hydroxychalcone and its methoxylated derivatives was evaluated in MTT assay after 48 h of treatment in different concentrations. The proapoptotic activity was studied by cytometric analysis of cells stained with Annexin V/FITC and propidium iodide and by measure caspases 3/7 and 8 activation. The DNA damage was evaluated by Western blot analysis of phosphorylated histone H2AX. The new compounds had selective antiproliferative activity against the studied cell lines, the most effective were the 2'-hydroxy-2″,5″-dimethoxychalcone and 2'-hydroxy-4',6'-dimethoxychalcone. 2'-Hydroxychalcone and the two most active derivatives induced apoptosis and caspases participation, but some percentage of necrotic cells was also observed. Comparing phosphatidylserine externalization after treatment with the different compounds it was noted that the addition of two methoxy groups increased the proapoptotic potential. The most active compounds triggered DNA damage even in the cell lines resistant to chalcone-induced apoptosis. The results confirmed that the analogues could have anticancer potential in the treatment of canine lymphoma or leukemia.

Modulating effect of a new ester, 28-O-phosphatidylbetulin (DAPB), obtained from hen egg yolk lecithin and betulin on lymphocyte subsets and humoral immune response in mice.

Context: Leaf extracts of plants of the genus Betula have traditionally been used as diuretic, anti-rheumatic and diaphoretic preparations. One of the main active ingredients of Betula bark is betulin, lupane-type triterpene alcohol, with multiple biological activities. Objectives: The aim of this study was to investigate in vitro and in vivo immunomodulatory effects of a newly synthesized ester of betulin: 28-O-phosphatidylbetulin [28-O-(1,2-diacyl-sn-glycero-3-phospho)-betulin, DAPB] in comparison with betulin in mice. Materials and methods: Cytotoxic activity of DAPB or betulin was tested against non-cancer (D10.G4.1 and J774E.1) and cancer (GL-1; CL-1 and Jurkat) cell lines. The in vivo part assessed total lymphocyte count, weight ratio and subsets of lymphocytes in the lymphatic organs, and humoral immune response to sheep erythrocytes (SRBC). Results: In vitro assay showed that DAPB, contrary to betulin, had no antiproliferative activity. Exposure to four doses of DAPB increased the absolute count of immature CD4+CD8+ thymic cells as well as the percentage and absolute count of mature CD4+ and CD8+ thymocytes. DAPB enhanced the percentage or absolute count of CD3+ cells in spleen and lymph nodes with corresponding decrease in the percentage and/or absolute count of CD19+ cells. Both DAPB and betulin enhanced the percentage and absolute count of CD8+ lymphocytes in lymph nodes. In SRBC-immunized mice, betulin contrary to DAPB enhanced the number of splenocytes producing anti-SRBC antibodies (PFC). Both DAPB and betulin increased the level of total (IgM + IgG) and IgG titers. Conclusion: Despite the lack of cytotoxic activity, DAPB shows valuable immunomodulatory properties.

Microbial Asymmetric Functionalization of ß-Cyclocitral-Derived Tetramethyl-Substituted γ-Lactone.

Searching for the new anticancer compounds we prepared three new ß-cyclocitral-derived hydroxyl-γ-lactones by microbial hydroxylation of tetramethyl-substituted bicyclic γ-lactone. The substrate was transformed by the enzymatic system of filamentous fungi. Three out of fifteen strains were selected as effective biocatalysts (Fusarium culmorum AM10, Armillaria mellea AM296, Trametes versicolor AM536). The hydroxylation processes were not only regioselective but also stereoselective. The hydroxylation products of each secondary carbon atom in the cyclohexane ring were obtained by the application of the selected fungal strains. The Fusarium culmorum AM10 introduced the hydroxy function at C-3 and C-4, Armillaria mellea AM296 incorporated the hydroxy function at C-3 and C-5 and Trametes versicolor AM536 transformed the substrate to the mixture of C-3, C-4 and C-5 hydroxylactones. The hydroxylactones obtained were enantiomericaly enriched (ee values in the range 17⁻99%). The in vitro antiproliferative activities of the functionalization products were also evaluated. Regardless of the hydroxy substituent location all tested lactones exhibited similar, significant activity towards selected cancer cell lines (IC50 in the range 22.8⁻33.9 µg/mL).

Enantiomeric trans ß-aryl-δ-iodo-γ-lactones derived from 2,5-dimethylbenzaldehyde induce apoptosis in canine lymphoma cell lines by downregulation of anti-apoptotic Bcl-2 family members Bcl-xL and Bcl-2.

For many years, studies focused on developing new natural or synthetic compounds with antineoplastic activity have attracted the attention of researchers. An interesting group of such compounds seem to be those with both lactone moiety and an aromatic ring which, in addition to antimicrobial or antiviral activity, also exhibit antitumor properties. The study shows antitumor activity of two enantiomeric trans isomers of 5-(1-iodoethyl)-4-(2',5'-dimethylphenyl)dihydrofuran-2-one. Our aim was to determine their antitumor activity manifested as an ability to induce apoptosis in selected canine cancer cell lines as well as to evaluate differences in their strength depending on the configuration of their stereogenic centers. The enantiomers (+)-(4R,5S,6R)-1 and (-)-(4S,5R,6S)-2 were found to induce classical caspase-dependent apoptosis through downregulation of the expression of anti-apoptotic proteins Bcl-xL and Bcl-2. Although the mechanism of apoptosis induction was the same for both enantiomers, they differed in their strength, as stronger antineoplastic activity in vitro was exhibited by isomer (+)-(4R,5S,6R)-1.

Preparation of Enantiomeric ß-(2',5'-Dimethylphenyl)Bromolactones, Their Antiproliferative Activity and Effect on Biological Membranes.

Three novel enantiomeric pairs of bromolactones possesing a 2,5-dimethylphenyl substituent at the ß-position of the lactone ring have been synthesized from corresponding enantiomeric (E)-3-(2',5'-dimethylphenyl)hex-4-enoic acids (4) by kinetically controlled bromolactonization with N-bromosuccinimide (NBS). γ-Bromo-δ-lactones (5) were isolated as the major products. Absolute configurations of stereogenic centers of γ-bromo-δ-lactones (5) were assigned based on X-ray analysis; configurations of cis δ-bromo-γ-lactones (6) and trans δ-bromo-γ-lactones (7) were determined based on mechanism of bromolactonization. Synthesized compounds exhibited significant antiproliferative activity towards the four canine cancer cell lines (D17, CLBL-1, CLB70, and GL-1) and one human cancer line (Jurkat). Classifying the compounds in terms of activity, the most active were enantiomers of trans δ-bromo-γ-lactones (7) followed by enantiomers of cis isomer (6) and enantiomeric γ-bromo-δ-lactones (5). Higher activity was observed for all stereoisomers with S configuration at C-4 in comparison with their enantiomers with 4R configuration. Synthesized compounds did not induce hemolysis of erythrocytes. The results of the interaction of bromolactones with red blood cell membranes suggest that these compounds incorporate into biological membranes, concentrating mainly in the hydrophilic part of the bilayer but have practically no influence on fluidity in the hydrophobic region. The differences in interactions with the membrane between particular enantiomers were observed only for γ-lactones: stronger interactions were found for enantiomer 4R,5R,6S of cis γ-lactone (6) and for enantiomer 4S,5R,6S of trans γ-lactone (7).

Effects of polysaccharide fractions isolated from Caltha palustris L. on the activity of phagocytic cells & humoral immune response in mice with collagen-induced arthritis: A comparison with methotrexate.

BACKGROUND & OBJECTIVES: The extracts from Caltha palustris L. have been shown to be beneficial for treating arthritis and rheumatism. In this study, the immunomodulatory effects of polysaccharide fractions B and C of C. palustris extracts were studied, using the collagen-induced arthritis (CIA) mouse arthritis experimental model. The aim was to determine the activity of blood phagocytic cells and humoral immune response in CIA mice treated with polysaccharide fractions from C. palustris. METHODS: The effects of fractions B and C of C. palustris were explored by evaluating phagocytic activity of peripheral blood granulocytes and monocytes and humoral immune response in sheep red blood cell (SRBC)-immunized mice. The results were compared with methotrexate (MTX) treatment. Following the onset of CIA, DBA/1J mice were treated for 21 days with B or C fractions (10 mg/kg; i.p.) or MTX (every 48 h, 6.6 mg/kg; i.p.). RESULTS: The results showed that fraction B reduced the level of interleukin (IL)-1ß, boosted nitric oxide synthesis in murine peritoneal macrophages stimulated in vitro with lipopolysaccharide and enhanced the monocyte phagocytic activity. Exposure of SRBC-immunized mice to fraction B and MTX during the course of CIA resulted in decreased total anti-SRBC haemagglutinin titres. INTERPRETATION & CONCLUSIONS: Fraction B of C. palustris polysaccharides modulated macrophage function and exerted beneficial effects on the clinical course of CIA in mice. The results also suggested efficacy of fraction B was comparable to that of MTX treatment for certain parameters.

The effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in SRBC-immunized mice.

5-Amino-3-methyl-4-isoxazolecarboxylic acid hydrazide is a non-cytotoxic synthetic isoxazole derivative with considerable immunomodulatory properties demonstrated in in vitro experiments. The aim of this study was to investigate the influence of this compound, depending on the dosage and schedule of treatment, on lymphocyte subsets in non-immunized mice and humoral immune response in SRBC (sheep red blood cells)-immunized mice. An analysis of lymphocyte subsets was carried out by flow cytometry, using specific monoclonal antibodies stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). In the SRBC-immunized mice, the influence of the compound on the humoral response was determined, depending on the time of administration relative to the antigen. The number of plaque forming cells (PFC) was determined by a local hemolysis technique in an agar gel. Total and 2-mercaptoethanol resistant serum agglutination titers were defined by active hemagglutination test carried out on microplates. The investigated hydrazide was able to modulate the percentage and absolute number of T lymphocyte subsets in the thymus, and T and B lymphocytes in the peripheral lymphatic organs. It also enhanced humoral immune response in SRBC-immunized mice by increasing the number of cells producing hemolytic anti-SRBC antibodies (PFC) and by augmenting the level of total and 2-mercaptoethanol resistant hemagglutinin. The present study showed modulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in mice. This compound could be potentially useful for the treatment of autoimmune diseases, infections or as an adjuvant for boosting the efficacy of vaccines.

In vitro immunomodulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the cellular immune response.

The aim of this study was to determine the immunomodulatory activity of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide in vitro. This compound was used for the synthesis of a series of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides with documented immunotropic activity. The performed measurements assessed the cytotoxic effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the murine macrophages (cell line J774E.1) and lymphoblasts (cell line D10.G4.1), the influence of this compound on the proliferation of murine lymphocytes isolated from peripheral lymphatic organs and murine peritoneal macrophages stimulated with mitogens (concanavalin A(ConA), lipopolysaccharide (LPS), phytohemagglutinin A (PHA)). Moreover, the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß by the murine peritoneal macrophages stimulated with LPS from Escherichia coli was assessed. It was found that 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide displayed no cytotoxic effects in the murine J774E.1 and D10.G4.1 cell lines in a wide range of concentrations (0.5-200 µg/ml). Furthermore, the compound stimulated proliferation of lymphocytes isolated from the spleen and mesenteric lymph nodes when used alone and in combination with mitogens (ConA and PHA). This effect was stronger in the nonstimulated cells, and it followed a dose-response relationship. The same phenomenon was observed for the proliferation of the murine peritoneal macrophages. The investigated hydrazide, at the highest used concentration of 150 µg/ml, increased the LPS-induced production of IL-1ß and did not affect the level of TNF-α. These results confirmed the immunomodulatory properties of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide and indicated that this compound could be useful in further research aimed at finding novel functional drugs.

The effects of bestatin on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunocompromised mice.

The effects of bestatin on humoral immune response to sheep erythrocytes (SRBC) and restoration of the response impaired by a single cyclophosphamide dose (350 mg/kg) were tested on mice. Bestatin (at doses of 10, 1, and 0.1 mg/kg) was administered intraperitoneally (i.p.) 5 or 10 times. The pharmacological immunosuppression was induced by a single i.p. injection of cyclophosphamide (350 mg/kg) administered 24 h before the first bestatin dose. The mice were immunized i.p. with SRBC 24 h after the last dose of bestatin. It was found that multiple administration of bestatin at all three doses potentiated the humoral response to SRBC in non-treated mice, resulting in an increased number of plaque-forming cells (PFC) and 2-mercaptoethanol (2-ME)-resistant anti-SRBC antibodies. However, five times administration of bestatin at the doses under investigation caused further decreases in total anti-SRBC hemagglutinins. A single injection of cyclophosphamide (350 mg/kg) suppressed humoral response of mice to the antigen. Administration of bestatin after pharmacological immunosuppression partially prevented the suppressive action of cyclophosphamide in the in vivo model of the humoral immune response to SRBC. The protective action of bestatin was both dose- and schedule-dependent. Ten times' exposure to a bestatin dose of 0.1 mg/kg after a high cyclophosphamide dose partially reduced the suppressive effect of this drug on humoral response of SRBC-immunized mice, increasing PFC on days 4 and 7 after immunization, which coincided with restored ability of the lymphocytes to produce the 2-ME-resistant hemagglutinins on day 7 and the total anti-SRBC hemagglutinins on day 14 after priming.

[The dog as a model for comparative studies of lymphoma and leukemia in humans]. / Pies jako model do badan porównawczychnad ludzkimi chloniakami i bialaczkami.

Dogs have accompanied humankind for thousands of years. They share the same environment, and thus are exposed to the same environmental factors such as air pollution, tobacco smoke, and various chemicals. Recent development of veterinary care has led to a significant extension of dogs' lifespan and allowed the diagnosis and treatment of a growing number of different diseases in this species. Among all diseases in dogs, cancer is considered the main cause of mortality, with lymphoproliferative disorders accounting for up to 30% of all canine cancers. Some of them, such as non-Hodgkin lymphoma (NHL) and lymphocytic leukemia, are very similar in the etiology, pathogenesis and response to treatment to the diseases occurring in humans. Due to anatomical and physiological similarities to humans, the dog is a useful model for the study of new therapeutic strategies for humans. Studies on the canine neoplasia are currently limited by the lack of well-characterized and widely available cell lines; thus, recently obtained canine NHL cell lines may become a valuable model for such studies. Investigation of their sensitivity to the antiproliferative effects of different factors should allow the creation of a database similar to the existing classification of human leukemias and lymphomas. This should enable quick and accurate diagnosis and selection of appropriate treatment based on phenotypic analysis and histopathological examination of clinical samples. The cooperation between human and veterinary oncologists gives the opportunity to use the dog as a model for the study of certain types of cancers presenting a challenge for modern medicine.

An initial characterisation of the Unfolded Protein Response pathway in haematopoietic canine cancer cell lines - a necessary step for the future development of new therapies in dogs with neoplasia.

Introduction: New and more effective therapies for canine cancer patients are urgently required and this necessitates advanced experimental research. Dogs are good models for studies in comparative oncology; however, canine cancer cell biology research is currently limited by low availability of validated antibody reagents and techniques. This study characterises the expression of key components of the unfolded protein response (UPR) in a panel of haematopoietic canine cancer cell lines using commercially available antibodies, and validates the methods used to study this pathway. Material and Methods: The CLBL-1 canine lymphoma cell line and the GL-1 canine leukaemia cell line sourced externally and two counterparts established in house (CNK-89 and CLB70) were used as models of different lymphoma and leukaemia canine cell lines for the study. The human U2OS cell line served as the control. Antibodies were selected for identifying UPR proteins according to known canine cell reactivity and canine-murine and canine-human homology. Endoplasmic reticulum stress was induced with thapsigargin and MG132 in the cell lines. Etoposide was used to induce DNA damage in the cells. The techniques used for this validation analysis were RNA sequencing to observe the expression of UPR components in canine cell lines, Western blot to observe changes of protein expression levels after inducing ER stress in the cells, and flow cytometry in order to study cell death. Results: Substantial variations in both the basic expression and agonist-induced activation of the UPR pathway were observed in canine cancer cell lines, although the biological significance of these differences requires further investigation. Conclusion: These findings will be a starting point for future studies on cancer biology in dogs. They will also contribute to developing novel anticancer therapies for canine patients and may provide new insights into human oncology.

Studying the DNA damage response pathway in hematopoietic canine cancer cell lines, a necessary step for finding targets to generate new therapies to treat cancer in dogs.

Background: Dogs present a significant opportunity for studies in comparative oncology. However, the study of cancer biology phenomena in canine cells is currently limited by restricted availability of validated antibody reagents and techniques. Here, we provide an initial characterization of the expression and activity of key components of the DNA Damage Response (DDR) in a panel of hematopoietic canine cancer cell lines, with the use of commercially available antibody reagents. Materials and methods: The techniques used for this validation analysis were western blot, qPCR, and DNA combing assay. Results: Substantial variations in both the basal expression (ATR, Claspin, Chk1, and Rad51) and agonist-induced activation (p-Chk1) of DDR components were observed in canine cancer cell lines. The expression was stronger in the CLBL-1 (B-cell lymphoma) and CLB70 (B-cell chronic lymphocytic leukemia) cell lines than in the GL-1 (B-cell leukemia) cell line, but the biological significance of these differences requires further investigation. We also validated methodologies for quantifying DNA replication dynamics in hematopoietic canine cancer cell lines, and found that the GL-1 cell line presented a higher replication fork speed than the CLBL-1 cell line, but that both showed a tendency to replication fork asymmetry. Conclusion: These findings will inform future studies on cancer biology, which will facilitate progress in developing novel anticancer therapies for canine patients. They can also provide new knowledge in human oncology.

Synergistic Antitumor Interaction of Risedronate Sodium and Standard Anticancer Agents in Canine (D-17) and Human Osteosarcoma (U-2 OS) Cell Lines.

The study discusses in vitro cytotoxicity of a combination of cytostatic drugs (doxorubicin, cisplatin, carboplatin, etoposide) and risedronate sodium against canine and human osteosarcoma (D-17 and U-2 OS). Standard protocols were used for the preparation of cell cultures and evaluation of their viability and apoptosis. MTT assay assessed the culture viability and EC50, while the apoptotic effect of the drugs was checked with a TUNEL assay. Doxorubicin alone showed the strongest cytotoxicity against D-17 (0.056 ± 0.019 µg/mL) and U-2 OS (0.051 ± 0.003 µg/mL), while the lowest cytotoxicity was observed for carboplatin (D-17, 6.45 ± 0.2 µg/mL and U2-OS, 27.5 ± 2.3 µg/mL). Risedronate sodium at 100, 10 and 1 µg/mL lowered viability in OS cell lines by 53.38 ± 1.46 and 49.56 ± 0.7%, 97.08 ± 3.32 and 74.92 ± 4.01%, and 102.67 ± 3.56 and 94.56 ± 3.52%, respectively. In all analyzed drug combinations, risedronate sodium significantly (* p < 0.05) increased the cytotoxicity against tested osteosarcoma cell lines. The decrease in cell viability caused by the studied compound combinations was weaker in canine than in human cell cultures. A combination of doxorubicin (all concentrations), cisplatin (1 µg/mL) and etoposide (1 µg/mL) with 100 µg/mL of risedronate sodium significantly improved the cytotoxicity of the drugs against canine and human osteosarcoma. Administration of carboplatin (1 µg/mL) and risedronate sodium (100 µg/mL), compared to carboplatin per se, produced no significant differences in cytotoxicity against the D-17 cell culture but significantly enhanced cytotoxicity in the U-2 OS line. The strongest apoptosis in both lines was detected for 0.01 µg/mL doxorubicin combined with 100 µg/mL risedronate sodium or 1 µg/mL cisplatin and 100 µg/mL risedronate sodium. In all combinations, the tested compounds revealed a synergistic mechanism of action.

In Vitro Studies on the Influence of Meloxicam on Cytotoxic Activity Induced by Risedronate Sodium in Canine (D-17) and Human (U-2 OS) Osteosarcoma Cell Lines.

The study describes the cytotoxic effect against human and canine osteosarcoma (U-2 OS and D-17) cell lines induced by risedronate sodium and meloxicam per se and in combination. Both cell lines were prepared according to standard procedures for cell cultures studies. The cell viability was estimated in both cell lines treated with chosen concentrations of risedronate sodium and meloxicam. The apoptosis assessment was carried out using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. EC50 values, computed for risedronate sodium and meloxicam cytotoxicity, showed comparable effects against the canine OS cell line in similar concentration of both drugs. In case of human OS, the stronger cytotoxic effect of risedronate sodium was proved. The EC50 values for meloxicam in both cell lines were, statistically, significantly different (* p < 0.05). Moreover, the cytotoxic effect of a combined administration of meloxicam and risedronate sodium in doses 100 µg/mL, compared with the negative control showed statistically significant differences. The human OS cell line was more resistant to both compounds than the canine OS cell line. The apoptotic effect in canine and human osteosarcoma triggered by risedronate sodium and meloxicam was statistically significant (p < 0.05). The cytotoxic effect induced with 100 µg/mL of risedronate sodium proved statistically significant differences between both tested cell lines compared to negative control. The results obtained with 10 and 100 µg/mL of meloxicam were not statistically significant. The study showed the synergic mechanism of action of risedronate sodium and meloxicam, but the concentrations used in vitro will not be possible to achieve in in vivo. Therefore, our results serve as basis only to design future studies on the tissue level.