Caryophyllane sesquiterpenes reverse sorafenib-chemoresistance through Pgp and STAT3 pathways in liver cancer cells.

Introduction. Research of innovative and alternative therapeutic approaches is an important acquisition in the battle against hepatocellular carcinoma (HCC), a highly resistant tumour. In the last years, it has been highlighted the possibility to affect tumour growth with multi-target therapies, acting in synergy or on different targets but with additive effects. This approach could allow to increase the effectiveness of anticancer drug even at low-doses and to limit the systemic toxicity associated with standard chemotherapy. In this context, the aim of the present work was to investigate the ability of three structurally related natural compounds, namely ß- caryophyllene (CRY), ß-caryophyllene oxide (CRYO), and α-humulene (α-caryophyllene, HUM), to enhance the response of HCC cells to sorafenib (SOR) treatment by blocking its Pgp-mediated export or by inhibiting the sorafenib-mediated STAT3 (Ser727) phosphorylation, both mechanism being involved in sorafenib acquired resistance. Material and methods. Cytotoxicity of test compounds alone or in combination with sorafenib was evaluated by MTT assay in human hepatoma cells HepG2. The nature of interaction (synergistic, additive or antagonistic effect) was evaluated by reversal ratio value (RR) and combination index (CI). Moreover, the ability of CRY, CRYO and HUM to inhibit Pgp functional activity was measured by the efflux assay. Particularly, rhodamine 123 and verapamil were used as fluorescent probe and typical inhibitor of this pump, respectively. At last, the effect of both substances alone or in combination with sorafenib on Pgp and STAT3 (Ser 727) protein expression was evaluated by western blot analysis. Results. All compounds showed a low cytotoxicity in our cellular model (IC50 > 240 µM). The anticancer drug sorafenib was the most effective with an IC50 value of approximately 105 µM. In the combination experiments, all compounds were able to increase the sensitivity of HepG2 cell to sorafenib with a higher effect respect to the known Pgp inhibitor verapamil. In particular, CRY, CRYO, and HUM were able to increase the sorafenib cytotoxicity of about 6-, 26-, and 9-folds, while verapamil of about 2.5-fold. The CI highlighted a synergistic effect of sesquiterpenes towards sorafenib. In the efflux experiments, all substances showed to inhibit the Pgp functional activity in a similar way than the positive control verapamil. Particularly, the rhodamine 123 efflux was reduced of about 3-, 2.8-, 2.6-, 3.1-folds by verapamil, CRY, CRYO, and HUM, respectively. As regards Pgp protein expression, sorafenib determined a slight but statistically significant increase of its expression. The combination of the antitumor drug with verapamil and HUM further increased it. By contrast, both CRY and CRYO were able to reduce the Pgp expression to basal level, being CRYO the most effective. As regards STAT3 (Ser 727) phosphorilated expression, sorafenib was able to increase it of about 3-fold respect to the control and its combination with verapamil and HUM showed similar results. Conversely, both CRY and CRYO were able to reduce the STAT3 (Ser 727) phosphorilated expression of about 1.3- and 2- fold, respectively. Discussion and conclusions. Under our experimental conditions, in spite of a slight effect of HUM, CRY and CRYO showed to synergistically increase the sorafenib cytotoxicity by affecting Pgp pump at both functional and protein expression. Also, they reduced STAT3 (Ser727) phosphorylation, known to be involved in MDR1-transcription upregulation. Altogether these results highlight the possible role of CRY and CRYO as chemosensitizing agents in combination with sorafenib and support their potential usefulness to reverse the sorafenib-induced multidrug resistance by targeting Pgp and/or STAT3 pathways.