Regarding the background where the α and β adrenoceptor blocking drugs were used in liver fibrosis, morphological changes and alteration in liver proliferation markers have been observed. Based on these results, the present study was developed to observe these main findings: cytotoxicity and morphological changes in hepatocytes. These morphological alterations and cytotoxicity have been regulated by pretreatment with curcumin.
In relation to the cytotoxicity of alpha adrenoblockers, investigations have been carried out on the mode of induction of apoptosis and its relationship with the cytotoxicity of different alpha adrenoblockers such as doxazosin, prazosin, 5-methylirapidil, and teratozine in HL-1 cells (derived from cardiomyocytes) as an in vitro model because they maintain the phenotype and intracellular and cell surface elements. It is reported that alpha adrenoblockers doxazosin and prazosin induced apoptosis in a dose-dependent manner, and doxazosin shows significant changes in viability decrease with the 0.1 µM/L dose [10]. These results were confirmed with Hoechst staining, and the studies were made of 24, 48, and 72 h. In our results found that doxazosin has the same behavior at the dose of 0.1 µM and time-dependent in HepG2 cells. It is important to remember that in patients, serum doxazosin concentration reaches 0.122 µmol/L with an 8 mg dose and 0.244 µmol/L with a 16 mg dose) [10]. The viability was lowed from the 0.1 µM dose at 24 hours. However, carvedilol (β adrenoblocker) with antioxidant activity and selective antagonist tamsulosin of α1A and α1B adrenoceptors showed a significant increase in dose-dependent cell viability at 24 h, and after 72 h, the cell proliferation mechanism was normalized.
Doxazosin is the only adrenoblocker used in the study with high levels of cytotoxicity in the HepG2 cell line. This adverse characteristic of doxazosin has also been reported by different authors, who describe doxazosin as an agent cytotoxic at doses of 25 µM in prostate epithelial cell lines [12]. For that reason, we used 25 µM as a marker of damage; the viability decreased to 39.07 ± 8.75% after 24 h (Fig. 1). Likewise, under this doxazosin concentration, we observed morphological changes in the hepG2 cells, such as ballooning, vesicle formation, and positive orange coloration through acridine orange, changes that are related to apoptosis. These morphological changes found in HepG2 cells are similar to those found in prostate cells (LNCaP). The proposed mechanism of damage in an epithelial cell line is through increased activation of caspase 8 by the formation of the death-inducing signaling complex (DISC), caspase 8 can stop the cell cycle in the G2-M phase and thus activate caspase 3 as the tBid responsible for the release of cytochrome c in the BAX/Bak receptor [13]. This results in the release of pro-apoptotic inducing factors related to mitochondrial stress: cytochrome c Smac/DIABLO, AMID, and AIF [14].
Forbes A. et al., 2016 reported that the cytotoxicity of doxazosin and tamsulosin were compared and was demonstrated a dose-dependent increase in the activation of caspase 3 followed by the apoptotic response when the quinazoline structure is present, to confirm this was demonstrated a decrease in HIF-1, a resistance mediator in the LNCaP line after they were exposed to quinazoline [15]. In our study, both α-adrenoblockers were also compared at the cytotoxic level, and doxazosin shows the highest decrease in cell viability even in 24 h treatments. It is proposed because doxazosin has a structure based on quinazoline and tamsulosin in sulfonamide.
It was demonstrated that doxazosin causes an increase in DNA fragmentation, cell cycle arrest in the G2 phase, and apoptosis due to the inactivation of CDK1. Also, it is suggested that doxazosin interrupts the cell cycle by quinazolines thanks to the competitive inhibition of ATP tyrosine kinase and the inhibition of PI3K phosphorylation of EGF and VEGF receptors [16].
Curcumin has therapeutic purposes due to its antioxidant properties [17]. It is an excellent candidate for its low toxicity. However, one of the main characteristics of curcumin is to possess antioxidant and pro-oxidant properties. We determine the concentration of curcumin that did not induce cell death in the HepG2 cell line for subsequent experiments. In vitro investigations in a model with the cell line, Huh7 exposed to curcumin in very low doses (≤ 1 µM), behaves as an antioxidant. However, the doses increased in a range of (5 to 10 µM), it behaves as an inducer of autophagy by reducing cytoplasmic proteins' acetylation and blocks the cell cycle, finally with doses greater than 25 µM, cell death is induced [18]. We used different concentrations with ranges of 0.01, 0.03, 0.05, 0.07, 0.1, 0.5, 1, 5, 10, and 25 µM during 24 h, and we observed that the doses ≤ 1 µM even increased the viability compared to control, cells treated with doses ≥ 5 µM significantly decreased viability. Therefore, it was decided to use the concentration of 1 µM (Fig. 2).
Our cell viability results with 1 µM curcumin pretreatment showed a recovery of cell viability in MTT up to 100% compared to control cells (Fig. 3). Possibly because the drugs doxazosin, carvedilol, and tamsulosin induced cell death may be due to oxidative stress, and curcumin with the defense mechanisms like the antioxidant response element (ARE) regulates the expression of genes involved in the elimination of ROS and increased the cell viability; however, these regulatory mechanisms are not entirely understood [19].
The main indicator enzymes of oxidative stress and liver damages are AST and ALT [20]. Previous studies have demonstrated in a HepG2 cell model the effect of glycyrrhizate (an antioxidant) on antioxidant defense systems by analyzing AST and ALT levels with and without H2O2, showing that H2O2 induces an increase in the levels of the enzymes. Glycyrrhizinate pretreatment effectively protected HepG2 cells from induced damage and decreased ALT and AST [21]. We reported that AST and ALT enzymes of HepG2 cells exposed to different concentrations of α and β adrenoblockers increased with the concentrations of 0.1 and 10 µM of doxazosin, 0.1 and 25 µM carvedilol, and 0.1 µM tamsulosin (Fig. 4). However, curcumin pretreatment decreased AST and ALT levels, counteracting the drugs' possible oxidative effect (Fig. 5).
The effect of cycloheximide in liver cells at the histological level with hematoxylin and eosin staining and the ultrastructural level with scanning electron microscopy described apoptosis stages in rat hepatocytes in four sequential phases: 1) cell contraction, 2) cell fragmentation, 3) release of blood cells in the sinusoids, 4) phagocytosis and digestion of blood cells by hepatocytes and Kupffer cells, we report in our study that the changes morphological of the HepG2 cells, occurred at 24 h after interaction with the alpha and beta adrenoblockers following the patterns indicated above; though after cycloheximide-induced oxidative stress, is compared to histological and ultrastructural findings thioacetamide-induced apoptosis [22]. In the evaluation of the cell monolayer by hematoxylin and eosin, we observed that α and β adrenoblockers alter the formation of the monolayer and decrease cell interaction, and there are eosinophilic aggregates or apoptotic process. However, with curcumin, the monolayer is restored and cellular interaction (Figs. 6 and 7). Another of the hepatocytes' characteristics was ultrastructures which suggest a progressive fragmentation of the cell body, and each fragment gives rise to an apoptotic body. We report the ultrastructures' with SEM that indicate that doxazosin, carvedilol, and tamsulosin induce possible pro-apoptotic processes that are time and dose-dependent. However, doxazosin shows more morphological changes such as balonization and separation of cells that result in the formation of vesicles or possible apoptotic bodies; carvedilol and tamsulosin show minor changes in cells as irregular surfaces with radiated projections. Curcumin treatment reduces the morphological variations, and the cell morphology shows normal villi and intercellular junctions without vesicles, indicating that it inhibits the apoptotic process.
In vitro studies, in HepG2 cells, cell death mechanisms were studied from the red signal of the acridine orange stain of different compounds that generate oxidative stress such as H2O2—relating a greater signal with the fragmentation of cellular structures and the beginning of the apoptotic process. The positive damage control showed high red coloration within the nucleus [23]. We observed that the three drugs induce the dose-dependent apoptotic response due to the increase in color in the cells compared to our damage control (H2O2); however, this red signal decreased when the cells were analyzed with curcumin, proving that it can have an anti-apoptotic effect.