Potentiation of Low-Dose Doxorubicin Cytotoxicity by Affecting P-Glycoprotein through Caryophyllane Sesquiterpenes in HepG2 Cells: an in Vitro and in Silico Study

Abstract

Doxorubicin represents a valuable choice for different cancers, although the severe side effects occurring at the high effective dose limits its clinical use. In the present study, potential strategies to potentiate low-dose doxorubicin efficacy, including a metronomic schedule, characterized by a short and repeated exposure to the anticancer drug, and the combination with the natural chemosensitizing sesquiterpenes β-caryophyllene and β-caryophyllene oxide, were assessed in human hepatoma HepG2 cells. The involvement of P-glycoprotein (P-gp) in the HepG2-chemosensitization to doxorubicin was evaluated. Also, the direct interaction of caryophyllene sesquiterpenes with P-gp was characterized by molecular docking and dynamic simulation studies. A metronomic schedule allowed us to enhance the low-dose doxorubicin cytotoxicity and the combination with caryophyllane sesquiterpenes further potentiated this effect. Also, an increased intracellular accumulation of doxorubicin and rhodamine 123 induced by caryophyllane sesquiterpenes was found, thus suggesting their interference with P-gp function. A lowered expression of P-gp induced by the combinations, with respect to doxorubicin alone, was observed too. Docking studies found that the binding site of caryophyllane sesquiterpene was next to the ATP binding domain of P-gp and that β-caryophyllene possessed the stronger binding affinity and higher inhibition potential calculated by MM-PBSA. Present findings strengthen our hypothesis about the potential chemosensitizing power of caryophyllane sesquiterpenes and suggest that combining a chemosensitizer and a metronomic schedule can represent a suitable strategy to overcome drawbacks of doxorubicin chemotherapy while exploiting its powerful activity.Int. J. Mol. Sci. 2020, 21, 633 2 of 28 Int. J. Mol. Sci. 2020, 21, 633 5 of 28 oxide were selected for the combination experiments. Under our experimental conditions, both the sesquiterpenes were able to potentiate the effect of doxorubicin in different experimental conditions, although with different profiles. Particularly, after 24 h exposure, the lower chemosensitizing concentration (50 µM) of β-caryophyllene significantly enhanced the doxorubicin-cytotoxicity starting from the concentration of 10 µM, achieving almost the maximum potentiation of about 32% at concentration of 20 µM (Figure 2a).

Chemopreventive Potential of Caryophyllane Sesquiterpenes: An Overview of Preliminary Evidence

“…Indeed, it has been characterized by a plethora of biological activities, among which analgesic, anti-inflammatory, antioxidant, neuroprotective, and antiproliferative were the most investigated; moreover, it has been reported to affect phospholipid cooperativity and membrane permeability ( Table 3 ). These properties have provided benefits in several experimental models of disease, such as neurodegeneration, inflammation, pain, anxiety, depression, autoimmune diseases, metabolic ailments, osteoarthritis and some cancer models [ 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 ,…”

“…Interestingly, a peripheral release of endogenous opioids seems to mediate the antinociceptive effects of β-caryophyllene, although nonpsychoactive responses have been also described [ 124 ]. Among further targets, β-caryophyllene has shown to inhibit the expression of pro-inflammatory factors and to potentiate the antioxidant cell defenses in different in vitro and in vivo models [ 123 , 124 , 144 , 155 , 162 , 189 , 193 ].…”

“…In HepG2 cells, β-caryophyllene and β-caryophyllene oxide produced similar cytotoxic effects in all the experimental conditions, being more toxic after long-term exposures than metronomic schedules. β-Caryophyllene was slightly more potent than the epoxide metabolite after long-term exposures of 48 and 72 h [ 162 ].…”

“…Particularly, Legault and Pichette [ 168 ] showed that β-caryophyllene was able to potentiate its cytotoxicity of paclitaxel in different in vitro models, including L-929 fibroblasts, DLD-1 colon adenocarcinoma, and MCF-7 breast cancer cells, by increasing its intracellular accumulation through an enhancement of membrane permeability. Conversely, the paclitaxel cytotoxicity was not increased by β-caryophyllene in HepG2 cells [ 162 ].…”

Chemico-Biological Characterization of Torpedino Di Fondi® Tomato Fruits: A Comparison with San Marzano Cultivar at Two Ripeness Stages

“…Cytoprotective activity of the tested extracts was evaluated towards the oxidative damage induced by tert-butyl hydroperoxide solution (tBuOOH) in HepG2 liver cancer cells (American Type Culture Collection, Milan, Italy). The cells were grown at 37 °C in 5% CO 2 in Dulbecco’s modified Eagle’s medium, supplemented with fetal bovine serum (10% v/v ), glutamine (2 mM), streptomycin (100 µg mL −1 ), and penicillin (100 U mL −1 ) [ 38 ]. All experiments were performed when cells reached the logarithmic growth phase.…”