Rosiglitazone metformin adduct inhibits hepatocellular carcinoma proliferation via activation of AMPK/p21 pathway

Abstract

Background: Rosiglitazone metformin adduct (RZM) is a novel compound, synthesized from rosiglitazone (Ros) and metformin (Met) combined at a molar mass ratio of 1:1. Met and Ros are widely used together for treating type 2 diabetes to improve drug effectiveness and reduce adverse drug reactions. Recent studies reported that both Met and Ros may possess antineoplastic properties in several cancers, including hepatocellular carcinoma (HCC). However, the effects of RZM in HCC and its underlying mechanisms remain unknown.

Methods: RZM was synthesized from Ros and Met at an equal molar ratio and identified by infrared spectroscopy. MTS and colony formation assays were performed to detect proliferative repression of RZM, the mixture, Met and Ros, respectively. Tumorigenesis assay in vivo was used to confirm the anti-tumorigenesis potential of RZM and Met. Moreover, cellular apoptosis caused by RZM was analyzed by hoechst staining assay and flow cytometry. RT-qPCR and western blotting were performed to reveal mechanisms for the function of RZM.

Results: Both in vitro and in vivo data showed that low doses of RZM enhanced inhibitory effect on HCC cells growth compared with Met. Flow cytometry analysis confirmed that treatment with RZM at 1 mM for 48 h triggered HCC cells apoptosis. RT-qPCR and western blotting analyses showed that p21 was upregulated in response to 1 mM RZM treatment. Furthermore, RZM could increase AMPK activation compared with Met. The increased p21 expression induced by RZM treatment was attenuated by an AMPK inhibitor compound C.

Conclusions: All these observations demonstrate that RZM increases the antiproliferative effect of Met in HCC via upregulating p21 expression in an AMPK-dependent manner. Our results suggest that RZM has the potential to be an adjuvant for HCC therapy.

Keywords: AMPK/p21 pathway; Hepatocellular carcinoma; Metformin; Proliferation; Rosiglitazone metformin adduct.

Fig. 1

Synthesis of rosiglitazone metformin adduct (RZM). a–d The infrared spectroscopy analysis results of Ros, Met, the mixture of Ros and Met, and RZM

Fig. 2

Effects of RZM, Ros and/or Met on cellular proliferation. a–c The antiproliferative effects induced by RZM, the mixture of Ros and Met, Ros and Met in HepG2, SK-Hep1 and L02 cells were detected by MTS assay (*P < 0.05 and **P < 0.01 versus the control group). d Representative images of colony formation of differently treated HCC cells were shown. e Quantitative results of colony formation assays were displayed (*P < 0.05 and **P < 0.01 versus the control group). All values were expressed as the means mean ± SD from at least three independent experiments. f HepG2 cells were treated with 1 mM RZM or Met for 48 h, flow cytometric analysis was used to determine the effects of the two compounds on cellular apoptosis. The apoptotic rates were represented by the ratios of early and late apoptotic cells (P2-Q3% + P2-Q2%). g Quantitative results of flow cytometric analysis were displayed (*P < 0.05 versus the control group)

Fig. 3

RZM upregulated the expression of p21 through the activation of AMPK. a RT-qPCR analysis of relative gene mRNA expressions in RZM-treated HepG2 cells. Transcript levels were normalized to GAPDH expressions (*P < 0.05 and **P < 0.01 versus the control group). b, c sh-p21 or/and RZM regulated the expression of p21 were assayed by RT-qPCR and western blotting assays (^##P < 0.01 and *P < 0.05 versus the control group). d MTS assay conducted in HepG2 cells under the corresponding conditions. Values shown are mean ± SD of triplicate measurements (**P < 0.01 and ^#P < 0.05 versus the control group). e Western blotting analysis of the activation of AMPK in HepG2 and SK-hep1 cells, using GAPDH as an endogenous control. f Relative protein expression of AMPK, p-AMPK and p21 in cells treated with RZM or/and compound C. GAPDH was used as an internal control

Fig. 4

Effects of RZM and Met on tumor growth in vivo. a Tumor growth curves were analyzed for RZM-treated, Met-treated and the control group. (*P < 0.05, **P < 0.01 versus the control group). b, c Nude mice were sacrificed after 4 weeks and the tumors in each group were exhibited. d, e The mean volume and weight of xenograft tumors from indicated treatment were analyzed (*P < 0.05, **P < 0.01 versus the control group). f Relative protein expressions of AMPK, p-AMPK and p21 in RZM-treated, Met-treated and the control group (n = 3, each group)