## 4 Discussion  
Epirubicin is an anthracycline-class antitumor agent that is used in the treatment of several types of cancer, including breast cancer, ovarian, lung cancer and oncobematologic disorders. The antitumor mechanism of epirubicin refers to its ability to intercalate, resulting in a complex formation that interferes with DNA and RNA synthesis [17]. It can also interfere with gene regulation expression by inhibiting polymerase activity [18]. However, it is a nonspecific antitumor drug, and like other chemotherapeutics, causes high DNA damage and selectively targets rapidly proliferating cells such as cancer cells and normal cells with a high self-renewal potential, including those in the gastrointestinal tract [19].  
In this respect, we aimed to assess by immunohistochemical assay the consequences of epirubicin-based therapy on the mucosal cells that undergo apoptosis and the injury of the tight junctions, immediately before and after a short time of chemotherapy administration.  
The integrity of the intestinal barrier is assured by tight junction, a complex of membrane-bound proteins, occludens and claudins and their junctional adhesion molecule, zonula occludens proteins (ZO)-1, -2 and -3 [20,21]. Recently, clinical and preclinical studies reported altered tight junction protein expression and subsequent altered intestinal permeability in the consecutive administration of Irinotecan or in combination with 5-Fluorouracil [22,23]. Studies have shown a decrease in key tight junction (TJ) proteins, such as ZO-1, in experimental models of intestinal inflammation [24]. Moreover, its depletion corresponds to an increase in intestinal permeability and a decrease in transepithelial resistance [24,25]. Our results showed that the expression of essential proteins in the tight junctions, such as Claudin-1 and ZO-1, is altered 48 h after epirubicin administration and is not completely restored even 3 weeks after administration, suggesting that the integrity of the intestinal barrier is affected. As a consequence, after chemotherapy, the passage of solutes across the epithelial barrier is impaired due to an increase in paracellular permeability [26].  
The Bcl-2 family members are key proteins that regulate the intrinsic apoptotic pathway [27]. Expression of genes that modulate the apoptosis process, such as Bcl-2 (which promotes survival) and Bax (which promote apoptosis), balance cell survival or death after damage [28]. In the present study, we investigate the susceptibility to damage of the intestinal mucosa by epirubicin administration to oncobematological patients. We showed that the expression of the Bcl-2 family (pro-apoptotic Bax and anti-apoptotic Bcl-2) was unbalanced, observing a colonic higher immunopositivity for Bax, detrimental to Bcl-2. Moreover, this finding is in accordance with our preclinical studies, where we found the same pattern in all gastrointestinal tracts of mice [16]. As well, Caspase-3 was highest expressed at 48 h after epirubicin-based chemotherapy to the oncobematological patients and remain expressed 3 weeks after cessation. Other results show alleviation of epithelial mucositis after administration of NSC321205, the protective effects being mediated by a decrease in Caspase-3 activity and the consequent inhibition of Bax expression [29], which demonstrate that apoptosis is an important event in gastrointestinal mucositis induced by chemotherapy, as well.