**Aleksandra Pawlak 1,\*, Marta Henklewska 1, Beatriz Hernández Suárez 1, Mateusz Łu ˙zny 2, Ewa Kozłowska 2, Bo ˙zena Obmi ´nska-Mrukowicz <sup>1</sup> and Tomasz Janeczko <sup>2</sup>**


Academic Editor: H.P. Vasantha Rupasinghe Received: 4 September 2020; Accepted: 21 September 2020; Published: 23 September 2020

**Abstract:** 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.

**Keywords:** chalcones; apoptosis; DNA damage; anticancer activity; canine cancer cell lines

### **1. Introduction**

The search for novel natural or synthetic compounds with potential antitumor activity is one of the major goals of contemporary oncology. The research involves both known compounds with proven biological activity as well as newly synthesized molecules that are structural analogues of biologically active substances already used in the treatment of cancer. Interest is aroused by compounds that may constitute the basis of chemotherapy in the future, as well as compounds that may support therapy or sensitize cancer cells to classic cytostatic drugs. Because the high toxicity of many anticancer substances (particularly bone marrow toxicity) significantly reduces the effectiveness of the treatment, to maximize the efficacy and reduce adverse effects, chemotherapy is usually based on a combination of drugs with various molecular mechanisms of action and different side effects. With regard to this principle, research focusing on the antitumor activity of new natural compounds or their derivatives is highly justified. Such a potentially interesting group of compounds may be chalcones, an essential group of natural compounds classified as flavonoids [1]. Their typical structural feature is an open, α,β-unsaturated three-carbon fragment connecting two aromatic rings [2]. They are yellow [3] and commonly occur in the world of plants: in citrus fruits, vegetables and spices [4,5]. Chalcones (1-(2- -hydroxyphenyl)-3-phenylprop-2-en-1-ones) and their derivatives are often obtained from natural sources or as a result of chemical synthesis [6,7]. The most commonly used chemical synthesis method is the Claisen–Schmidt condensation of the corresponding aromatic aldehyde and 2- -hydroxyacetophenones in alkaline or acid catalysis [8–10].

The great interest in chalcones is influenced by the ease of synthesis of these compounds and the extensive biological activity characteristics they exhibit: in particular antiviral, anthelmintic, antibacterial, antiprotozoal, insecticidal, antiulcer, cytotoxic, and anticancer [11–13]. Due to the confirmed therapeutic effect of chalcones on human cancer, we decided to check their activity against canine cancer cells.

Cancer in dogs is a major challenge in modern veterinary medicine and research into the search for new cancer therapies is highly needed [14]. On the other hand, of the various animal species that develop cancer (e.g., cats, horses, rabbits, ferrets), dogs turned out to be the most appropriate model for comparative research due to their body size, life expectancy, cancer incidence and sharing of living conditions with humans. Moreover, dogs together with mice and rats, are popular laboratory animals necessary for different types of toxicological studies [15].

In conclusion, the presented study examined the antitumor activity of new chalcone analogues with the methoxy group on selected canine lymphoma and leukemia cell lines. After the hydroxyl group, the methoxy group is the second most commonly identified group in natural flavonoid compounds, and because of this, we decided to test a series of chalcones containing a different amount and places of methoxy group substitution. The cytotoxic, antiproliferative and proapoptotic effects of newly synthesized compounds were analyzed. The study showed that the obtained analogues could have anticancer potential in the treatment of canine lymphoma or leukemia.

#### **2. Results**
