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Synergistic effect of metformin on sorafenib in in vitro study using hepatocellular carcinoma cell lines

Backgrounds/Aims Hepatocellular carcinoma (HCC) recurrence remains a great concern following hepatic resection and liver transplantation. We investigated the metformin-induced cytotoxic effects on sorafenib in an in vitro study using HCC cell lines. Methods This research was conducted through an in vitro study using one HepG2.2.15 liver tumor and two patient-derived graft HCC cell lines. Results An in vitro study revealed noticeable cytotoxic effects of metformin as well as noticeable synergistic cytotoxic effects of metformin and sorafenib on cell viability. Assays for the mechanisms of action of antitumor effects revealed that alpha-fetoprotein expression was suppressed by both metformin and sorafenib, but no synergistic effect was observed. LC3-I and LC3-II assays revealed the synergistic upregulation of autophagy and assays for IL-1β, IL-6, p53, and TNF-α revealed the synergistic upregulation of cell damage and apoptosis. In contrast, metformin did not affect HBx expression, thus no noticeable synergistic effect was considered to be present. Conclusions Our in vitro study demonstrated cytotoxic effects of metformin and synergistic antitumor effects of sorafenib. These results should be verified in further clinical studies with patients of advanced HCC.


INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most common malignancies as well as a leading cause of cancer-related death. HCC is initially treated with various locoregional therapies including resection, but tumor recurrence or progression can result in such treatments no longer being applicable. Thus, a considerable number of patients with advanced HCC undergo systemic chemotherapy including sorafenib. Sorafenib is currently regarded to be the first-line chemotherapeutic agent for treating advanced HCC following reports of prolonged survival periods in two randomized controlled trials. 1,2 However, the therapeutic effect of sorafenib is often suboptimal, and most patients still experience tumor progression. 3 Thus, there is an essential need to discover add-on agents that can enhance the therapeutic effects of sorafenib.
Metformin is a biguanide used in the treatment of type We recently reported that metformin administration was associated with reduced tumor recurrence and helped induce significant improvements in overall patient survival in patients who underwent hepatic resection for HCC. 11 It has also been reported that combined metformin and sorafenib can suppress proliferation and induce autophagy of HCC in vitro and in vivo through targeting the mammalian target of rapamycin (mTOR) pathway. 12 Therefore, the present study intended to assess whether the add-on use of metformin to sorafenib could induce synergistic antitumor effects on in vitro study with HCC cell lines. In vitro study using liver tumor cell lines The cytotoxic effects of metformin and sorafenib were evaluated using the three liver tumor cell lines mentioned above. In line with the therapeutic range in patients with type II diabetes, the in vitro drug concentrations were determined as 5-10 mmol/mL for metformin and 5-20 mol/mL for sorafenib. 14 The duration of drug administration was 20 hours.
Cell survival was expressed as the percentage of absorb-

Assessment of in vitro cell viability
The MTT assay for cell survival assessment showed a Fluorescence microscopy with 4′,6-diamidino-2-phenylindole-Hoechst staining showed noticeable apoptosis following metformin administration in all three cell lines, which was comparable to that of sorafenib administration.
The highest levels of apoptosis were observed after combination therapy (Fig. 2).
Assessment of the mechanisms of cytotoxicity RT-PCR assays to assess the mechanisms of action of antitumor effects revealed that AFP expression was suppressed by both metformin and sorafenib, but no synergistic effect was observed (Fig. 3A). LC3-I and LC3-II  assays revealed the synergistic upregulation of autophagy ( Fig. 3B).
In contrast, RT-PCR revealed that metformin did not affect HBx expression; thus, no noticeable synergistic ef-fect was present (Fig. 5).

DISCUSSION
Sorafenib is a multikinase inhibitor with therapeutic efficacy against HCC. 16 Although sorafenib markedly prolongs the survival of patients with advanced HCC, 4-6 its  potential mechanisms of action that induce clinical bene- fits are yet to be properly elucidated. Metformin is a biguanide that inhibits cancer cell growth by inducing cell cycle arrest and enhancing apoptosis, and is also reportedly associated with a reduced risk of HCC.

4-10
The mechanism of action of metformin is mainly associated with inhibition of the mTOR pathway, which plays an important role in the metabolism, growth, and proliferation of cancer cells. 17 The inhibition of mTOR pathway is exerted by metformin via AMP-activated protein kinase (AMPK) activation, which is crucial for energy homeostasis. 18 Metformin also inhibits the mTOR pathway in an AMPK-independent manner. 14 AMPK-dependent and independent pathways are thought to be associated with the antitumor effects of metformin. Sorafenib can activate AMPK. A clinical study showed that patients with KRAS-mutant advanced non-small cell lung cancer receiv-ing a combination of sorafenib and metformin had better outcomes than those receiving sorafenib alone. The study also showed that sorafenib and metformin act synergistically through inhibiting cellular proliferation in non-small cell lung cancer in vitro and in vivo and by phosphorylating the AMPK  activation site. 19 Our in vitro study also demonstrated synergistic antitumor effects of both drugs.
Thus, both sorafenib and metformin seem to activate the AMPK pathway, thus inducing synergistic antitumor effects.
In our in vitro study, we compared the potencies of the antitumor effects of metformin and sorafenib, in which the metformin-associated anti-tumor effect was variably comparable to that of sorafenib; however, sorafenib appeared to have a more powerful antitumor effect than metformin.
The variability of response to the addition of metformin indicates that the combination of sorafenib and metformin treatment may lead to different therapeutic effects in a clinical setting.
In a clinical study with 93 patients treated with sorafenib, 31 has metformin administered concurrently due to diabetes mellitus. The concomitant use of sorafenib and metformin was associated with a median progression-free survival of 2.6 months, compared to 5.0 months for patients receiving sorafenib alone (p=0.029). The median overall survival of patients treated with the combination was 10.4 months, compared to 15.1 months for those who were not given metformin (p=0.014). 20 These findings indicated increased tumor aggressiveness and resistance to sorafenib in metformin-treated patients, which is in direct contrast to the results of our in vitro study.
In conclusion, our in vitro study demonstrated the cytotoxic effects of metformin and synergistic antitumor effects with sorafenib. These results should be further verified in clinical studies with patients having advanced HCC.