Apigenin Reverses Drug-resistance In Hepatocellular Carcinoma Cells By Inhibiting The Nrf2-ARE Pathway

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers. Chemotherapy is an important therapeutic strategy for HCC. Unfortunately, cancer cells often develop chemoresistance, which presents a major obstacle contributing to the failure of cancer therapy in HCC.Nuclear factor erythroid-2-related factor2(Nrf2) is a redox-sensitive transcription factor regulating expression of a number of cytoprotective genes. Recently, Nrf2is now viewed as a pharmacological target to overcome chemoresistance. Therefore, identification of small molecules inhibitors that potently inhibit the Nrf2-dependent response is desirable, and such compounds may be used as an adjuvant sensitizer to combat chemoresistance.Several flavonoid compounds have been reported to be Nrf2inhibitor that can reverse drug resistance effectively, such as epigallocatechin3-gallate (EGCG), luteolin, brusatol. Similar to luteolin, brusatol, and EGCG, apigenin (APG) is a natural compound with anticancer potential. So APG structurally related to them whether sensitizing cancer cells to anticancer drugs through antagonizing the Nrf2signaling pathway is not clear. We aim to study the effect of APG on reversing drug resistance in HCC cells, as well as to explore the molecular mechanism through the Nrf2pathway.Part I Study of the correlation between Nrf2and doxorubicin-resistance in hepatocellar carcinoma cellsObjects:To explore the correlation of doxorubicin-resistance with Nrf2expression in HCC.Methods:The expression of Nrf2, aldo-keto reductase1B10(AKR1B10), multidrug resistance-associated protein5(MRP5) protein in HCC were analyzed by immunostaining. And the relationship of Nrf2expression in these tissues with clinicopathologic characteristics and correlation of AKR1B10, MRP5expression with Nrf2expression in HCC were analyzed. Using Western blot, expressions of Nrf2in BEL-7402 cells and doxorubicin resistant BEL-7402(BEL-7402/ADM) cells were compared. In addition, using Nrf2siRNA was created to inhibit Nrf2expression in BEL-7402/ADM cells, and cell viabilities in response to ADM treatment were measured by MTT analysis.Results:The expression of Nrf2, AKR1B10, and MRP5in HCC were higher than that in the corresponding normal tissue (P<0.01; P<0.01; P<0.01). The Nrf2protein level was positively correlated with AKR1B10/MRP5protein in the HCC tissues (P=0.009; P=0.016), as assessed by Spearman’s rank correlation coefficient test. Additionally, there was a significant correlations between Nrf2expression and tumor degree of differentiation (P=0.017), and TNM stage (P=0.028). Patients with liver cirrhosis and early recurrence had higher Nrf2expression (P=0.017;P=0.016). The Nrf2protein level in BEL-7402/ADM cells was higher than that in BEL-7402cells. And suppression of Nrf2expression sensitized BEL-7402/ADM cells to ADM.Conlusion:These results suggest that the high expression level of Nrf2is probably correlated with doxorubicin-resistance in HCC.Part II Reversal of apigenin on chemoresistance in BEL-7402/ADM cells via inhibiting PI3K/Akt/Nrf2pathwayObjects:Nrf2is a redox-sensitive transcription factor regulating expression of a number of cytoprotective genes. Recently, Nrf2has emerged as an important contributor to chemoresistance in cancer therapy. Here, we investigated whether APG could reverse drug resistance in BEL-7402/ADM cells via inhibiting Nrf2pathway.Methods:Reverse of drug resistance was assayed by MTT analysis. Intracellular ADM concentration was measured by Fluorometric and FCM analyses. Inhibition of Nrf2signaling pathway by APG was detected through real-time quantitative PCR and western blot analysis.Results:In the present study, we found that non-toxic dose of APG significantly sensitizes BEL-7402/ADM cells to anticancer drugs and increases intracellular concentration of ADM. Mechanistically, APG dramatically reduced Nrf2expression at both the mRNA and protein levels through down-regulation of PI3K/Akt pathway, leading to a reduction of Nrf2-downstream genes HO-1, AKR1B10, and MRP5. Conlusion:These results clearly demonstrate that APG can be used as an-effective adjuvant sensitizer to prevent chemoresistance by down-regulating PI3K/Akt/Nrf2signaling pathway.PartⅢ Synergisyic inhibitory effects by the combination of apigenin and doxorubicin on human hepatocellar carcinoma xenografts in nude miceObjects:In this study, we investigated the inhibitory effect of APG and ADM on hepatocellar carcinoma in vivo experiments using human hepatocellar carcinoma cell lines, and we aimed to elucidate the possible molecular mechanism of their anticancer effect.Methods:The animal model of BEL-7402transplantation tumor in nude mice was constructed.32nude mice were randomly assigned into four groups and treated i.p. with vehicle, ADM (3mg/kg), APG (50mg/kg), or in combination every three days for a total of seven times. The inhibitory effects of the drugs in each group were observed. The mice were sacrificed and their tumors were excised for further analysis, Tumor cell proliferation was analyzed by Ki-67immunostaining. Apoptosis was determined by TUNEL assay. Nrf2protein levels were measured by Western blot.Results:In vivo, combination treatment with APG and ADM resulted in enhanced tumor growth inhibition, apoptosis induction, inhibition of tumor cell proliferation and inhibition of Nrf2expression in tumor tissues.Conlusion:These results suggest that the combination of APG and ADM would be more efficacious for the treatment of HCC than either treatment alone. And inhibition of Ki-67expression, induction of tumor cell apoptosis and down-regulation of Nrf2expression might be one of its possible mechanisms.