**1. Introduction**

Doxorubicin (DOX) is a first-line anticancer agen<sup>t</sup> that is highly effective against a wide spectrum of malignancies, including breast, lung, gastric, ovarian, and thyroid ones, as well as lymphoma, myeloma, sarcoma, and some forms of pediatric neoplasms. Despite good clinical effectiveness, DOX induces cumulative, dose-dependent toxicity and adverse effects, such as cardiotoxicity, and affects the brain, kidney, and liver [1]. Currently, both cancer treatments and in vivo studies are usually based on combined therapies that include various antineoplastic agents, possibly resulting in drug–drug interactions and even an increase in toxicity [2]. Studies investigating the managemen<sup>t</sup> of DOX-induced toxicity have focused on the administration of antioxidant and/or antiapoptotic compounds in combination with DOX, the development of effective delivery systems, and the synthesis of DOX analogs [1]. One potential approach to the minimization of adverse effects is reducing

the therapeutic dose of DOX by combining its application with that of other anticancer and/or organ-protective agents [3]. However, although some of these strategies fail to decrease DOX toxicity, recent investigations have demonstrated that certain phytocompounds in combination with DOX can ultimately be more successful [4,5].

The genus *Mahonia* includes approximately 60 species, which are widely distributed throughout Asia, North America, and Europe. Species belonging to the genus *Mahonia*, including the *Mahonia aquifolium* plant, have been shown to have antibacterial, antifungal, anti-inflammatory, and antioxidant effects and have been used in traditional Chinese and North American medicine [6]. Some research has shown that several representatives of this genus, such as *Mahonia oiwakensis* and *Mahonia bealei*, which are native to China, demonstrate antiproliferative activity against human cancer cells as well [7,8]. Berberine and similar alkaloids represent a major class of secondary metabolites of the *Mahonia* genus with a wide spectrum of di fferent properties. These alkaloids have been reported to significantly inhibit growth of cancer cells and exhibit other anticancer e ffects [9–12]. Although *M. aquifolium* has been used in traditional medicine solely for treatment of inflammatory skin disorders [13], its chemical composition, as well as previously obtained results demonstrating the activity of di fferent plants belonging to this genus, sugges<sup>t</sup> that this plant possesses anticancer properties as well, as we have previously confirmed and reported [14].

Previous studies have demonstrated that the phytocompound berberine in combination with DOX can e ffectively limit the toxicity and adverse e ffects of DOX [4] and that *M. aquifolium*, whose main constituents are berberine and protoberberine alkaloids, has anticancer properties [14,15]. Therefore, we investigated the anticancer e fficacy of the combination of DOX and water or ethanol extracts of *M. aquifolium* (MAW and MAE, respectively) in vitro.

The objective of our study was to elucidate the e ffects of DOX and MAW or MAE combinations on proliferation, migratory potential, and invasiveness of malignant cells. Furthermore, we examined the influence of these extracts on cellular uptake and retention of DOX. In order to understand the mechanisms underlying the e ffects of these extracts on migration and invasiveness, we analyzed gene expression changes of matrix metalloproteinases 2 and 9 (*MMP2* and *MMP9*), occludin (*OCLN*), catenin beta-1 (*CTNNB1*), and excision repair cross-complementation group 1 (*ERCC1*) in the treated human malignant cells.
