The Effect of Xanthohumol Derivatives on Apoptosis Induction in Canine Lymphoma and Leukemia Cell Lines.

Xanthohumol is a cancer chemopreventive agent that can interfere with the initiation, promotion, and progression phase of carcinogenesis via a variety of inhibitory mechanisms. Xanthohumol was reported as an effective agent against leukemia/lymphoma cells. In the present study, we investigated the effect of xanthohumol and its natural and semisynthetic derivatives against various canine leukemia/lymphoma cell lines. Xanthohumol, three hops minor prenylflavonoids (xanthohumol C, xanthohumol D, α,ß-dihydroxanthohumol) and four derivatives obtained by biotransformation (xanthohumol 4'-O-ß-D-(4‴-O-methyl)-glucopyranoside) as well as by chemical modification (1″,2″-dihydroxanthohumol K, 2,3-dehydroisoxanthohumol, (Z)-6,4'-dihydroxy-4-methoxy-7-prenylaurone) were tested for their antiproliferative and pro-apoptotic activities against the following canine leukemia/lymphoma cell lines: CLBL-1 (B-cell lymphoma), CLB70 (B-cell leukemia), and GL-1 (B-cell leukemia). The compounds were tested at a final concentration range of 0.1-30 µM for 48 h. All eight of the tested flavonoids exerted concentration-dependent cytotoxicity in the selected canine lymphoma/leukemia cell lines. Three compounds markedly decreased the viability of all cell lines with IC50 in the range of 0.5 to 8 µM. Double-staining of the treated cells with AnnexinV and propidium iodide revealed that the dying cells were mostly in the late apoptosis stage. ROS production and changes in mitochondrial potential were detected. Western blot analysis showed a decreased expression of Bcl-2. Canine lymphoma and leukemia cell lines are sensitive to xanthohumol derivatives, and the compounds acted through an apoptotic cell-death mechanism. These compounds, either used alone or in combination with other therapies, may be useful for the treatment of canine leukemia/lymphoma.

Nerve-independent formation of membrane infoldings at topologically complex postsynaptic apparatus by caveolin-3.

Junctional folds are unique membrane specializations developed progressively during the postnatal maturation of vertebrate neuromuscular junctions (NMJs), but how they are formed remains elusive. Previous studies suggested that topologically complex acetylcholine receptor (AChR) clusters in muscle cultures undergo a series of transformations, resembling the postnatal maturation of NMJs in vivo. We first demonstrated the presence of membrane infoldings at AChR clusters in cultured muscles. Live-cell super-resolution imaging further revealed that AChRs are gradually redistributed to the crest regions and spatially segregated from acetylcholinesterase along the elongating membrane infoldings over time. Mechanistically, lipid raft disruption or caveolin-3 knockdown not only inhibits membrane infolding formation at aneural AChR clusters and delays agrin-induced AChR clustering in vitro but also affects junctional fold development at NMJs in vivo. Collectively, this study demonstrated the progressive development of membrane infoldings via nerve-independent, caveolin-3-dependent mechanisms and identified their roles in AChR trafficking and redistribution during the structural maturation of NMJs.

Ubiquitin-specific protease 7 as a potential therapeutic target in dogs with hematopoietic malignancies.

BACKGROUND: Ubiquitin-specific protease 7 (USP7) belongs to the group of deubiquitinating enzymes (DUBs), which remove ubiquitin which controls various cellular processes such as chromosome segregation, DNA repair, gene expression, protein localization, kinase activity, protein degradation, cell cycle progression, and apoptosis. It is critical for several important functions in the cell, and therefore dysregulation of USP7 can contribute to tumorigenesis. OBJECTIVES: Alterations in the USP7 protein have been identified in various malignancies of humans. Our aim was to examine whether USP7 could be a potential therapeutic target in hematopoietic cancers of dogs. METHODS: The expression level of USP7 in lymphocytes from healthy dogs and canine lymphoma cells was determined, and the effect of USP7 inhibition on the vital functions of canine cancer cells was examined. RESULTS: We showed that USP7 was overexpressed in lymphomas in dogs. The USP7 inhibitor P5091 has selective cytotoxic activity in canine lymphoma and leukemia cell lines. Our results indicate that inhibition of USP7 leads to a disruption of cell cycle progression, and triggers DNA damage and apoptosis. The observed proapoptotic effect of the USP7 inhibitor most likely is not dependent on the p53 pathway. CONCLUSIONS AND CLINICAL IMPORTANCE: Our results suggest that USP7 could be explored as a potential therapeutic target in dogs with lymphoma. The effectiveness of USP7 inhibition in malignant cells is predicted to be independent of their p53 status.

A newly established canine NK-type cell line and its cytotoxic properties.

We established a canine natural killer (NK)-type cell line called CNK-89 derived from a dog with NK cell neoplasia. Immunophenotyping analysis showed positive staining for CD5, CD8, CD45, CD56, CD79a and NKp46, while negative for CD3, CD4, CD14, CD20, CD21, CD34, Thy1, IgG, IgM and MHCII. Polymerase chain reaction analysis revealed the presence of CD56, NKG2D, NKp30, NKp44, NKp46 and perforin, but the absence of CD16, Ly49 and granzyme B mRNA. Treating CNK-89 cells with IL-2 did not change the expression of activating receptors, TNFα and IFNγ secretion and cytotoxic activity, however, treatment with IL-12 alone or in combinations with IL-15, IL-18 and IL-21 caused an increase in granzyme B and CD16 mRNA, IFNγ secretion and cytotoxic properties of the CNK-89 cell line.

Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer.

ALK gene rearrangement was observed in 3%-5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI-resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.

Hypoxia increases the apoptotic response to betulinic acid and betulin in human non-small cell lung cancer cells.

Betulinic acid and betulin show promising activity against cancers cells, but the mechanism of their action is still unclear. In this study, non-small cell lung cancer (NSCLC) cell lines: A549, H358 and NCI-H1703 were treated with betulinic acid and betulin under normoxic and hypoxic conditions as hypoxia is critically involved in the response of solid tumors to chemotherapy. The treatment inhibits viability and proliferation of NSCLC cells. The anti-proliferative effect was induced by G1 cell cycle arrest with increased p21 expression and decreased cyclin D1 and cyclin B1 expression. Additionally, downregulation of p-GSK3ß activity and the Wnt/ß-catenin pathway were also observed under hypoxia. We found that hypoxia increased apoptosis in NSCLC cells. Cell death was associated with changes in the expression of proteins involved in the mitochondrial apoptosis pathway and induction of apoptotic death by caspase activation. Additionally, hypoxia exposure deregulated HIF-1α and p53 expression levels. Importantly, treatment with betulinic acid and betulin reduced colony-forming ability under normoxia, however, only betulinic acid reduced clonogenic activity under hypoxia. Our findings that betulinic acid increases apoptotic cell death and clonogenic activity under hypoxic conditions reveal new attractive strategies for treating hypoxic cancer tumors, such as NSCLC.

An Antibody Specific for the Dog Leukocyte Antigen DR (DLA-DR) and Its Novel Methotrexate Conjugate Inhibit the Growth of Canine B Cell Lymphoma.

Canine B-cell lymphoma (CBL) is an incurable, spontaneous lymphoid malignancy constituting an accurate animal model for testing novel therapeutic strategies in human medicine. Resources of available species-specific therapeutic monoclonal antibodies (mAbs) targeting CBL are scarce. The aim of the present study was to evaluate the therapeutic potential of mAb B5, specific for the dog leukocyte antigen DR (DLA-DR) and its antibody-drug conjugate with methotrexate (B5-MTX). B5 induced caspase-dependent apoptosis of DLA-DR-expressing canine B cell lymphoma/CLBL1 and CLB70 leukemia lines, but not the GL-1 line not expressing DLA-DR. The cytotoxicity of B5-MTX to sensitive cells was further potentiated by a payload of MTX, but without any substantial off-target effects. The infusion of B5 and B5-MTX in a murine model of disseminated, advanced canine lymphoma, mediated >80% and >90% improvement in survival, respectively, and was well tolerated by the animals. Interestingly, the concentrations of soluble DLA-DR (sDLA-DR) antigens present in the blood serum of tumor-bearing mice were found proportional to the tumor burden. On this basis, sDLA-DR levels were evaluated as a potential biomarker using samples from canine lymphoma patients. In summary, the action of B5 and B5-MTX holds promise for further development as an alternative/complementary option for the diagnosis and treatment of canine lymphoma.

In vitro effects of the activity of novel platinum (II) complex in canine and human cell lines.

The anticancer activity of novel platinum derivative, a complex of platinum with tris(2-carboxyethyl)phosphine (Pt-TCEP), has been evaluated in canine (D-17) and human osteosarcoma (U2-OS) cell lines. Viability of cells after incubation for 24 or 72 hours with increasing concentrations (0.625, 1.25, 2.50, 5, 10 and 20 µM) of Pt-TCEP was tested in an MTT assay and compared to effect of cisplatin. Longer-term effect of Pt-TCEP was evaluated in the colony-forming unit assay after 24 hours exposure to the Pt-TCEP (2 and 3 µM) and subsequent incubation for 2 weeks. The influence of the compound on the cell cycle was measured after 24 hours treatment with Pt-TCEP (3 µM). Its pro-apoptotic activity was examined after 24 hours treatment with Pt-TCEP (1.25, 2.50, 5, 10 and 20 µM) using flow cytometry. Expression of main proteins involved in apoptosis was measured after exposure for 24 hours to 3 or 5 µM Pt-TCEP in Western Blot. The compound much more effectively decreased cell viability than cisplatin in case of both cell lines. IC50 of Pt-TCEP was 5.93 ± 0.12 in D-17 and 3.45 ± 0.14 in U2-OS cell lines after 24 hours, and 1.77 ± 0.14 in D-17 and 1.53 ± 0.11 in U2-OS after 72 hours (P < .05). The compound arrested cells in the G2/M phase and inhibited the ability of cells to form colonies. Pt-TCEP induced caspase-dependent apoptosis. The expression of the anti-apoptotic Bcl-XL protein was decreased after Pt-TCEP treatment in both cell lines. The results confirmed anti-cancer activity of Pt-TCEP against canine and human osteosarcoma cell lines.

Effect of electrochemotherapy with betulinic acid or cisplatin on regulation of heat shock proteins in metastatic human carcinoma cells in vitro.

Betulinic acid (BTA) is naturally occurring triterpene that has received interest as a novel therapeutic substance with cytotoxicity towards a number of cancer cell lines. Despite the wide spectrum of biological and pharmacological effects, its effect may be limited its lipophobic properties. Therefore, strategies to improve the access of BTA to the cells are required to enhance the anticancer effects. Electroporation (EP) enables increased inflow of drugs into cancer cells, even at low doses, which may reduce the side effects caused by high doses of chemotherapy. The potential application of BTA in electrochemotherapy (ECT) in metastatic type of cancers was investigated in the present study. The efficacy of BTA with EP was estimated using a cell survival assay (MTT assay), microscopical morphology analysis and the immunocytochemical expression of heat shock proteins (HSPs). HSPs are molecules that protect the cell from harmful environmental, chemical and physical stresses, and ensure cell survival, recovery and proper functioning. HSP expression is induced various stress factors. Therefore, the expression of HSP27 and HSP70 was evaluated after cells were exposed to an external pulsed electric field and anticancer drugs. Facilitated drug delivery and the anticancer effect on metastatic tumor cells were evaluated in vitro. The effect of BTA was compared with cisplatin (CP), a standard cytostatic agent. Two different metastatic cancer cell lines were used, an ovary adenocarcinoma cell line (SW626) and melanoma cell line (Me45). BTA combined with EP exhibited similar efficacy to CP with EP after 24 and 48 h in SW626 and Me45 cancer cells. Me45 cells also had high HSP27 and low HSP70 immunosignals post­ECT treatment. ECT caused increased expression of HSP27 and HSP70 proteins in SW626 cells, which were less sensitive to ECT than the Me45 melanoma cell line. The results indicate that BTA may be efficiently applied instead of CP in ECT approaches, but its activity differs between tumor cell lines.

Sorafenib in Combination with Betulinic Acid Synergistically Induces Cell Cycle Arrest and Inhibits Clonogenic Activity in Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers in the world due to late diagnosis and poor response to available treatments. It is important to identify treatment strategies that will increase the efficacy and reduce the toxicity of the currently used therapeutics. In this study, the PDAC cell lines AsPC-1, BxPC-3, and Capan-1 were treated with sorafenib and betulinic acid alone and in combination. We examined the effect of combined treatments on viability (MTS test), proliferation and apoptosis (annexin V staining), cell cycle arrest (PI staining), alterations in signaling pathways (Western blotting), and colony-forming ability. The combination of sorafenib with betulinic acid inhibited the viability and proliferation of PDAC cells without the induction of apoptosis. The antiproliferative effect, caused by G2 cell cycle arrest, was strongly associated with increased expression of p21 and decreased expression of c-Myc and cyclin D1, and was induced only by combined treatment. Additionally, decreased proliferation could also be associated with the inhibition of the P13K/Akt and MAPK signaling pathways. Importantly, combination treatment reduced the colony-forming ability of PDAC cells, as compared to both compounds alone. Collectively, we showed that combined treatment with low concentrations of sorafenib and betulinic acid had the capacity to inhibit proliferation and abolish clonogenic activity in PDAC cell lines.

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.

Development of novel monoclonal antibodies to dog leukocyte antigen DR displaying direct and immune-mediated cytotoxicity toward canine lymphoma cell lines.

Spontaneous canine lymphoma (CL) has become a promising, nonrodent model for advancing the therapeutic strategies of human hematological malignancies. As new resources for veterinary and comparative studies on CL-associated antigens, we developed 2 novel mouse monoclonal antibodies, denoted B5 and E11, that recognized the canine major histocompatibility Class II DR antigens (dog leukocyte antigen DR). Using flow cytometry and solid phase immunoenzymatic assays, we showed that the antigens recognized by B5 and E11 were strongly expressed in several CL cell lines and the ex vivo canine neoplastic cells of B and mixed B/T immunophenotypes. Additionally, we evaluated a minimal cross-reactivity of B5 and E11 with the human B-cell line, Raji. By the ectopic expression of the hybrid murine/canine I-E/DR dimers in the HEK293 cells, we demonstrated that the epitope of B5 was localized to the invariant DRα chain, whereas the epitope of E11 was collectively formed by the DRα and DRß chains. Both epitopes were conformational and conserved in all the tested unrelated individuals of different dog breeds. In vitro treatment of 2 CL B-cell lines (CLBL1 and CLB70) with B5 and E11 rapidly induced a direct apoptotic cell death. Similarily, both mouse monoclonal antibodies efficiently killed the above cell lines through the mechanisms of complement-dependent and antibody-mediated cellular phagocytosis. Collectively, our data support the further development of B5 and E11 as novel tools for dog leukocyte antigen DR-targeted, preclinical trials involving CL.

Flavopiridol Strongly Sensitizes Canine Lymphoma Cells to TRAIL-induced Apoptosis.

BACKGROUND/AIM: Targeting the extrinsic apoptotic pathway is an interesting option for anticancer therapy. A protein which such ability is Apo2 ligand, also known as TNF-related apoptosis-inducing ligand (TRAIL). The aim of this study was to examine the possibility of sensitizing resistant CLBL-1 canine lymphoma cells to TRAIL-induced apoptosis by using flavopiridol (FVP) a cyclin-dependent kinase inhibitor (CDKs). MATERIALS AND METHODS: The CLBL-1 (canine B-cell lymphoma cell line) was used in the study. The effect of FVP and TRAIL treatment on apoptosis induction was assessed by flow cytometry and western blot. RESULTS: Although canine lymphoma cells were resistant to TRAIL-induced apoptosis, combination of this death ligand with FVP was able to overcome TRAIL resistance of CLBL-1 lymphoma cells. CONCLUSION: Our results demonstrated that although canine lymphoma cells were resistant to TRAIL-induced apoptosis, combination of this death ligand with FVP was able to overcome TRAIL resistance of CLBL-1 lymphoma cell line. Although further investigation is required to deepen the knowledge of TRAIL as an antitumor agent in canine cancers, our results open the door to future use of TRAIL-based treatment strategies in veterinary oncology.

Methotrexate induces high level of apoptosis in canine lymphoma/leukemia cell lines.

INTRODUCTION: Methotrexate is an antimetabolite used in the treatment of cancer and non-malignant diseases including rheumatoid arthritis, psoriasis and graft vs. host disease. Combination therapy with methotrexate was successful in the treatment of canine lymphoma, mammary tumor and invasive urinary bladder cancer. Lymphoma, the most common hematopoietic cancer in dogs, and leukemia are sensitive to chemotherapy, which is why methotrexate may be an important treatment option for these diseases. Although methotrexate is already used in veterinary oncology its effects on canine cancer cells has not been tested. The aim of the study was to evaluate for the first time methotrexate concentration-dependent cytotoxicity and its capability of inducing apoptosis in selected canine lymphoma/leukemia cell lines: CLBL-1, GL-1 and CL-1 as a first step before the in vitro development of new therapeutic options with the use of methotrexate. RESULTS: Methotrexate exhibited concentration-dependent inhibitory effect on proliferation of all the examined cell lines with different degree of apoptosis induction. The most methotrexate sensitive cells belonged to CL-1 cell line derived from T cell neoplasia and previously characterized by high resistance to the majority of anticancer drugs used in the therapy of lymphoma/leukemia in dogs. Canine lymphoma and leukemia cell lines are sensitive to methotrexate, and this drug may be useful in effective treatment of canine neoplasms and especially of T-type leukemia/lymphoma.

Synergistic activity of sorafenib and betulinic acid against clonogenic activity of non-small cell lung cancer cells.

The highly selective multi-targeted agent sorafenib is an inhibitor of a number of intracellular signaling kinases with anti-proliferative, anti-angiogenic and pro-apoptotic effects in various types of tumors, including human non-small cell lung cancer (NSCLC). Betulin displays a broad spectrum of biological and pharmacological properties, including anticancer and chemopreventive activity. Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key signaling pathways in NSCLC progression. NSCLC cell lines, A549, H358 and A427, with different KRAS mutations, and normal human peripheral blood lymphocyte cells, were treated with sorafenib and betulinic acid alone and in combination. We examined the effect of different combined treatments on viability (MTS test), proliferation and apoptotic susceptibility based on flow cytometry, alterations in signaling pathways by western blotting and colony-forming ability. The combination of sorafenib with betulinic acid had a strong effect on the induction of apoptosis of different NSCLC cell lines. In addition, this combination was not toxic for human peripheral blood lymphocytes. Combination treatment changed the expression of proteins involved in the mitochondrial apoptosis pathway and induced apoptotic death by caspase activation. Importantly, combination treatment with low drug concentrations tremendously reduced the colony-forming ability of A549, H358 and A427 cells, as compared to both compounds alone. In this study, we showed that combination therapy with low concentrations of sorafenib and betulinic acid had the capacity to induce high levels of cell death and abolish clonogenic activity in some NSCLC cell lines regardless of KRAS mutations.

Non-small cell lung cancer - mutations, targeted and combination therapy.

Year after year, a growing number of cases of non-small cell lung cancer (NSCLC), mostly caused by smoking, have been noted. Most patients die because of the late detection of cancer and tumor resistance to treatment with cytostatics. Treatment of patients with advanced NSCLC is impeded by the low sensitivity of the tumor to cytostatic agents and the co-existence of many diseases, which substrate is, like lung cancer, cigarette smoking. Along with the development of molecular biology, targeted therapy has started to be used, affecting specific signaling pathways involved in the processes of oncogenesis. Compounds that inhibit the activity of receptor tyrosine kinases are very well examined and already used in clinical practice. NSCLC is characterized by multiple mutations, including EGFR (epidermal growth factor receptor) and KRAS. Rarer but clinically significant is the rearrangement of the ALK gene. Currently, for NSCLC treatment a number of EGFR inhibitors such as erlotinib, gefitinib, afatinib and two compounds targeted in ALK kinase crizotinib and ceritinib are applied. Unfortunately, despite numerous studies, we are still not able to improve the treatment effectiveness of patients with KRAS mutations. The most efficient solution would be to use a combination of the compounds exhibiting synergistic effects on tumor cells. The literature data describes numerous examples of the combination treatment of NSCLC cells. Some combinations of compounds are already in clinical trials. Most attempts relate to tyrosine kinase inhibitors in combination with other types of pharmacologic inhibitor or immunotherapy. This paper describes the mutations occurring in NSCLC and drugs used in clinical practice as well as being in preclinical development.