Background/Aim: Betulinic acid (BA) and betulin (BT) exhibit a variety of pharmacological properties including anti-cancer, anti-inflammatory and anti-oxidant ones. Canine lymphoma and osteosarcoma have a high mortality rate and need more effective therapeutic approaches. In this study, the anti-proliferative and pro-apoptotic effects of BA and BT were investigated in canine T-cell lymphoma (CL-1), canine B-cell lymphoma (CLBL-1) and canine osteosarcoma (D-17) cell lines. Materials and Methods: The cultured cells were treated with several concentrations of BA or BT for 24, 48 and 72 h, and cell proliferation was assessed by the MTT assay. Cell apoptotic rate and cell cycle were analyzed using flow cytometry. Results: Anti-proliferative effect of BT and BA was concentration- and time-dependent. Moreover, BA and BT arrested cell cycle in S phase in CL-1 and D-17 cells, and in G₀/G₁ phase in CLBL-1 cells. Conclusion: Both compounds showed an antitumor activity, and the effects of BA were stronger than that of BT.

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.

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.