Abstract
Background/Aim: We investigated metformin-induced cytotoxic effects in vitro and assessed the chemopreventive effects of metformin in patients undergoing hepatic resection (HR) for hepatocellular carcinoma (HCC). Materials and Methods: This study consisted of laboratory and clinical studies. Results: In vitro study using HCC cell lines revealed noticeable cytotoxic effects of metformin, that were largely weaker than those of sorafenib. In the clinical study, no statistical differences were found in tumor recurrence or overall survival between metformin and control groups. In contrast, there was a non-significant difference in tumor recurrence between metformin and propensity score-matched control groups, but there was a significant difference in overall patient survival. Metformin administration was an independent risk factor for patient survival. Conclusion: In conclusion, our in vitro laboratory study demonstrated the presence of cytotoxic effects of metformin. Metformin administration was associated with reduced tumor recurrence and helped induce significant improvements in overall patient survival in patients who underwent HR for HCC.
Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related death (1, 2). Hepatic resection (HR) is considered a first-line treatment in patients with preserved hepatic function, but the incidence of tumor recurrence is high after curative HR (3, 4). Several studies have reported the antitumor effects of new agents as postoperative adjuvant therapy following HR, but the clinical impacts of these studies have been limited (5-8). To date, no well-established strategy for lowering the risk for HCC recurrence after HR is accepted.
Metformin is a biguanide agent used to treat type 2 diabetes mellitus (DM). It regulates the blood sugar by improving insulin sensitivity and reducing hepatic glucose output through inhibition of gluconeogenesis and glycogenolysis. Recently, metformin has proven capable of inhibiting cancer cell growth by inducing cell cycle arrest and enhancing apoptosis (9-12). A considerable number of studies found that metformin plays a chemopreventive role in other cancers and is associated with reduced risk for HCC (13-15). Although a few high-volume population-based retrospective studies have suggested the possibility of chemopreventive activity in metformin, the effects of metformin on post-resection HCC recurrence remain unclear.
Therefore, in our current study we investigated whether metformin has cytotoxic effects on liver tumor cell lines in vitro and further assessed the chemopreventive effects of metformin on HCC recurrence following HR through a propensity score-matched clinical study.
Patients and Methods
Study design. This study consisted of two independent parts: a laboratory research and a clinical study to assess the antitumor effects of metformin. The laboratory research assessed whether exposure to metformin has any cytotoxic effect on liver tumor cell lines. In the clinical study, the rate of tumor recurrence and overall patient survival after HR of HCC were investigated to assess whether long-term exposure to metformin has any chemopreventive effects. These study protocols were approved by the Ethics Committee for Animal Study at the Asan Institute of Life Sciences and the institutional review board of the Asan Medical Center (AMC-IRB 2017-0576).
Liver tumor cell lines. We used three liver tumor cell lines, one established cell line and two patient-derived xenograft (PDX) tumor cell lines. First, because a majority of HCC patients in Korea have hepatitis B virus (HBV) infection, we chose the HepG2.2.15 cell line (Korean Advanced Institute of Science and Technology), which is derived from the human hepatoblastoma cell line HepG2 with HBV transfection. This liver tumor cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, both purchased from Gibco-BRL (Grand Island, NY, USA). Second, we established two PDX tumor cell lines. Small pieces of human HCC tissue were obtained during hepatic resection for HCC in HBV-associated patients who had not undergone any preoperative HCC treatment (n=2). A small tumor fragment of 0.3 g was implanted subcutaneously in the bilateral hind flanks in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse. After tumor growth for 3 months was confirmed, the tumor was harvested and implanted into a NOD-SCID mouse. After stable tumor growth was confirmed, the established first-generation xenograft tumor was serially implanted in an SCID mouse to expand the xenograft tumors, which were also implanted subcutaneously into the nude mouse for further tumor expansion. These tumors were harvested to establish new PDX tumor cell lines.
In vitro study using liver tumor cell lines. The cytotoxic effects of metformin were evaluated using the abovementioned three liver tumor cell lines. The in vitro drug concentration was determined to be 5-10 mmol/ml for metformin after repeated titration from 5 to 40 mmol/ml, with consideration of the therapeutic ranges in patients with type 2 diabetes (16). To quantitatively assess metformin-associated cytotoxicity, we used a 10 μmol/ml concentration of sorafenib as a reference control (17).
To assess cell viability, a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to quantify cell viability using 12-well plates. Optical density was assessed at 550 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell survival was expressed as the percentage of the absorbance of drug-treated cells relative to that of untreated cells. MTT was purchased from Duchefa (Haarlem, the Netherlands). The cells were also observed under fluorescence microscopy after 4’,6-diamidino-2-phenylindole (DAPI)-Hoechst staining (Sigma-Aldrich; Poole, Dorset, UK).
Propensity score-matched clinical study using a single-institution cohort. The HCC database at our institution was searched to identify patients who had undergone primary HR for HCC during the 9 years from January 2006 to December 2013. To objectively compare the study groups, patients were narrowly selected according to the following selection criteria: solitary HCC of 2-5 cm in diameter, curative surgery with anatomical HR, no macroscopic vascular invasion, no extrahepatic metastasis including lymph node metastasis, no preoperative HCC treatment, and Child–Pugh class A. Through this screening process, 939 patients were selected.
These patients were classified according to postoperative administration of metformin for control of DM. Metformin use was defined as a prescription of metformin of more than 12 months within the initial 2 years following HR for HCC. To assess long-term outcomes related to the defined metformin use, 54 patients who survived for less than 2 years after HR were excluded, leaving 885 patients who survived ≥2 years as the complete study cohort. Finally, 45 patients were grouped into the metformin group, and the 840 remaining patients were the control group. The sample number for the propensity score-matching (PSM) control group was estimated using a type I error (α) of 0.1 and a type II error (β) of 0.20, in addition to a 10% survival difference; as a result, the sample number of the PSM control group became 225. To overcome possible selection bias, PSM was conducted between the metformin study group and the control group, using multiple logistic regression and a 1:5 matching requirement via the nearest-neighbor matching method (18). We matched baseline characteristics (age, sex), background liver disease (viral hepatitis versus others), preoperative level of tumor markers (α-fetoprotein [AFP] and des-γ-carboxy prothrombin [DCP; or proteins induced by vitamin K antagonist or absence-II]), tumor characteristics (size and presence of microvascular invasion), and AFP-DCP-tumor volume (ADV) score (19, 20).
Medical records were retrospectively reviewed after approval by the institutional review board of our institution. The preoperative evaluation, follow-up, and treatment for HCC recurrence have been described previously (4, 21). Patients were followed up until June 2017 using medical record reviews and with the assistance of the National Health Insurance Service, resulting in a patient follow-up period of ≥30 months or until death. All patients were followed to identify their survival status.
Statistical analysis. Numerical data are presented as means with standard deviations or as medians with ranges. Continuous variables were compared using the Student's t-test, and incidence variables were compared using the chi-square test. Survival curves were estimated using the Kaplan–Meier method and compared with the log-rank test. Cox proportional hazards regression was used for multivariate survival analyses. p-Values <0.05 were considered statistically significant. Statistical analyses were performed using SPSS version 22 (IBM, NY).
Results
In vitro cytotoxicity in cell lines. To assess cell survival, we performed an MTT assay, which showed a concentration-dependent decrease in cell survival for 20-h treatment with 5 and 10 mmol/ml metformin in the HepG2.2.15 cell line (Figure 1A); this response was greater than treatment with 10 and 20 μmol/mL sorafenib. Cell death was lower after metformin treatment than after sorafenib treatment in PDX cell lines 1 and 2 (Figure 1B and C). Fluorescence microscopy with DAPI-Hoechst staining indicated noticeable apoptosis after exposure to metformin in all three cell lines; the results were similar after sorafenib exposure (Figure 2).
Patient demographics and post-resection outcomes according to metformin administration. The process of patient selection is depicted in Figure 3. The clinicopathological features of patients belonging to the metformin study group (n=45), the non-metformin all control group (n=840), and the PSM control group (n=225) are given in Table I. The data for the metformin and PSM control groups are very similar. During follow-up, a median period of 62 months (range=24-135 months), HCC recurrence developed in 351 of 885 patients (39.7%) and all-cause death occurred in 118 of 885 patients (13.3%).
MTT assay for cell survival assessment using HepG2.2.15 (A) and patient-derived xenograft (PDX) tumor cell lines 1 (B) and 2 (C) with metformin and sorafenib treatment.
Tumor recurrence rates after HR according to metformin administration were compared: the 1-, 3- and 5-year rates were 11.1%, 32.3%, and 42.4% in the metformin study group; 13.7%, 34.3%, and 42.7% in the control group (p=0.61) (Figure 4A); and 17.7%, 42.0%, and 54.5% in the PSM control group (p=0.083), respectively (Figure 5A). For overall survival, the data were 100%, 97.8%, and 83.2% in the metformin study group; 100%, 96.4%, and 88.6% in the all control group (p=0.52) (Figure 4B); and 100%, 89.5%, and 67.8% in the PSM control group (p=0.028), at 2, 3, and 5 years, respectively (Figure 5B).
Risk factor analysis for tumor recurrence and overall survival. The results of univariate analyses for post-resection prognosis are given in Table II. Significant risk factors were tumor size >3.1 cm and microvascular invasion for tumor recurrence; and tumor size >3.1 cm and metformin administration for patient survival. Multivariate analyses revealed that independent risk factors were tumor size >3.1 cm for tumor recurrence; and tumor size >3.1 cm and metformin administration for patient survival (Table III).
Fluorescence microscopy with DAPI-Hoechst staining in the HepG2.2.15 cell line with treatment of metformin or sorafenib. Arrows indicate apoptosis.
Discussion
For patients with HCC and preserved hepatic function, HR is a first-line treatment, but tumor recurrence is high even after curative HR (3, 4). Thus, there have been many attempts to decrease the risk for tumor recurrence by postoperative adjuvant therapy such as interferon Alfa-2b, acyclic retinoid, vitamin K, and so on (5-8), but none of these has proven effective in a prospective controlled trial setting. Vitamin K administration has been reported to have antitumor effects in a few patients, but meta-analyses, including a randomized controlled study, have failed to prove its preventive and therapeutic effects (22, 23). Researchers have also found that oral administration of vitamin K2 with or without sorafenib does not show adverse side-effects and that noticeable antitumor effects occur in some patients with HCC recurrence after HR or liver transplantation (24, 25). Therefore, there is a need to discover agents that would be usable in an adjuvant chemopreventive setting.
DM is a common chronic disease that is not life threatening in the short term and is estimated to affect 4-5% of the population worldwide. Along with the increasing prevalence of the Western lifestyle and obesity in the general population, the prevalence of DM is expected to increase rapidly in Asian countries, including Korea. DM per se is not life-threatening as a disease, but severe forms can be accompanied by serious complications that lead to deterioration of quality of life and even death. Moreover, there are accumulating data showing that patients with DM are also prone to the development of cancers, including HCC (26-31). Thus, a considerable number of patients with HCC have had DM.
A nationwide Taiwanese study found that DM has an adverse effect on patients with HCC regardless of treatment modality, but the use of metformin significantly reduces the risk for HCC recurrence and improves the overall outcome of patients after HR if patients survive the first 2 years (32). In our current study, there was a noticeable reduction in the rates of post-resection tumor recurrence in the metformin study groups compared to the PSM control group; however, this was not statistically significant, probably due to the relatively small number of cases. In contrast, there was a significant improvement in overall patient survival in the metformin group.
Selection process of the study patients for metformin, control and propensity score-matching (PSM) control groups.
A few studies have investigated the association between antidiabetic drugs and the risk for developing HCC and have reported reduced risks with metformin treatment (22-34). Metformin has also been demonstrated to inhibit cancer cell growth and proliferation through cell cycle arrest (35). Metformin-associated antitumor effects were clearly demonstrated in our present analyses, although we did not investigate the potential action mechanisms underlying these effects. Metformin can attenuate the risk of developing HCC associated with DM in terms of dosage and medication duration, inhibiting the proliferation of hepatoma cell lines in a dose-dependent manner; further, the risk of developing HCC can also be decreased by increasing the duration of metformin use (13). Our in vitro cell line study findings also indicated that the antitumor effects of metformin appear to be dose-dependent, supporting the suggestion of high-dose long-term administration. However, these antitumor effects have been demonstrated in only high-volume cohort studies or laboratory research, implying that these effects exist but their prognostic power is not great enough to be an independent prognostic factor in small- or medium-sized volume studies. In our in vitro study, we compared the potency of antitumor effects between metformin and sorafenib, and found that the metformin-associated effects were variably comparable to those of sorafenib. This implies that some certain patients may benefit more from chemoprevention with metformin. Further laboratory studies should be performed to demonstrate its cytotoxic effects and the mechanisms underlying its antitumor effects.
Comparison of clinicopathological profiles of patients in the metformin and control groups.
Univariate analyses of factors associated with tumor recurrence and patient survival in 270 patients of the metformin and propensity-score-matching control groups.
Multivariate analyses of factors independently associated with tumor recurrence and patient survival in 270 patients of the metformin and propensity-score-matching control groups.
Comparison of tumor-recurrence (A) and overall patient survival (B) curves in the metformin study group compared to the all control group.
Comparison of tumor-recurrence (A) and overall patient survival (B) curves in the metformin study group compared to the propensity score-matching (PSM) control group.
In our clinical study with the PSM control group, the independent risk factors were a tumor size >3.1 cm for tumor recurrence and a tumor size >3.1 cm and metformin administration for patient survival. These results imply that metformin may be an agent for post-resection chemoprevention. Thus, further clinical studies should also be performed to establish the guidelines for patient selection and dosage setting with the aim of achieving a wide use of metformin for chemopreventive purposes.
Recently, the antitumor effect of metformin has been testified for various malignancies other than HCC. A study on metastatic castration-resistant prostate cancer revealed that metformin-docetaxel treatment significantly reduced PC3 cell viability but it did not significantly affect cell migration or intracellular ATP levels. It was suggested that metformin may be an effective chemosensitizer for certain types of castration-resistant prostate cancer cells (36). Another study on cholangiocarcinoma presented that metformin significantly suppressed proliferation of cholangiocarcinoma cells in a dose- and time-dependent manner, that was induced by targeting signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-ĸB) (37). Another study also revealed that metformin induced a cell cycle arrest of gastric cancer stem cells and patient-derived primary tumor xenografts tumor study showed growth delay and decrease of the self-renewal ability of the gastric cancer stem cells (38). These studies suggest that the use of metformin can be associated with therapeutic strategy to treat various malignancies.
There were some limitations of this study of note. This was a retrospective single-center study, and the study population was not large; thus, our results may not be generalizable. It will also be necessary to validate the effects of metformin in other geographic regions to extend our results to HCC patients with various background liver diseases other than HBV infection.
In conclusion, our present in vitro laboratory study has demonstrated the existence of cytotoxic effects of metformin. Metformin administration showed a tendency to reduce the tumor recurrence rate and helped induce significant improvement in overall survival in patients who underwent HR for HCC. High-volume multicenter studies and refined laboratory studies are necessary to validate these effects on HCC.
Acknowledgements
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (Grant No 2015R1A2A2A04007141 to Shin Hwang and 2015K1A4A3046807 to Gi-Won Song).
Footnotes
↵* These Authors contributed equally as first co-authors.
Conflicts of Interest
No Author has any conflict of interest.
- Received December 19, 2017.
- Revision received February 4, 2018.
- Accepted February 5, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
