Patient Samples from Taipei Veteran General Hospital Medical Center
A total of 797 patients were diagnosed with primary ovarian cancer in the Department of Gynecology and Obstetrics, Taipei Veteran General Hospital, from 1995 to 2012. After a review of the patients' clinical and drug histories, 737 patients who underwent complete surgery and were treated with platinum-based therapy plus paclitaxel were included in the analysis. Of these patients, 32 were identified as having taken metformin, either during admission or in the outpatient clinic. The overall survival (OS) was measured from the date of diagnosis to death or was censored at the date of the last follow-up. All documents were collected under protocols approved by the institutional review board of the hospital.
Patient Samples from the National Health Insurance Taiwanese Dataset
The reimbursement data of Taiwanese female patients with a new diagnosis of type 2 DM between 2000 and 2010 (n=38,886) were retrieved from the National Health Insurance database. Among these patients, none used only insulin or only metformin. Therefore, we compared two groups: (1) those who received metformin and insulin (n=24,033) and (2) those who received neither metformin nor insulin (n=14,853). Then, we followed the two groups for newly diagnosed ovarian cancer from 2000 to 2011. Thirty-seven patients across the two groups were diagnosed with ovarian cancer.
Microarray Analysis
The microarray experiments were conducted following the L1000 Operating Procedure (L1000 SOP) [36]. Briefly, the human ovarian cancer cell line ES-2 was left untreated (control) or treated with micromolar concentrations of metformin (0.5 mM), 50 μM carboplatin, or a combination in a microplate. After 6 hours of drug treatment, the medium was removed, and lysis buffer was added (included in the L1000 kit) to the wells for 30 minutes. After cell lysis, the lysate was stored at -80ºC for at least one night before being transferred to a 384-well plate, which was performed using the protocol available at http://s3.amazonaws.com/support.lincscloud.org/protocols/data_generation/L1000_SOP.pdf. Gene expression profiles were detected by L1000 array technology. Up and down probesets were selected by performing two-sample t-tests; genes with expression differences significant at a p value <0.01 and with fold changes >1.5-fold were included. The up and down probesets were input into GSEA software for analysis and to interpret the transcriptional profile data of the four groups by GSEA methods [37, 38].
Analysis of Ovarian Cancer using the Cancer Genome Atlas (TCGA) Genomics Data
Clinical data and protein expression data of ovarian cancer from TCGA were downloaded from the cBioPortal website (http://www.cbioportal.org/) [39, 40]. Patients in the ovarian cancer (cBioPortal TCGA, provisional, ovarian cancer genomics, n=606) dataset were categorized into low and high protein expression groups by a half-division approach. These two groups of patients were input as “User-defined Case List” to assess the total and phospho-protein levels, as evaluated by the RPPA z-score, of ± 0, including those of key proteins involved in the AKT/mTOR pathway and AMPK. Kaplan-Meier analyses were performed to assess the correlations among the indicated proteins (AKT [total and pSer473], mTOR [total and pSer2448], and AMPK [total and pThr172]).
Cell Lines, Cell Culture, Chemicals, and Antibodies
The MOSEC line was a kind gift from Dr. Honami Naora (The University of Texas MD Anderson Cancer Center). Stable MOSEC lines were generated as previously described [41]. The MOSEC lines were cultured in DMEM medium [42]. The human ovarian cancer cell line SKOV3 and ovarian clear cell carcinoma cell line ES-2 were provided by Dr. Gordon Mills (The University of Texas MD Anderson Cancer Center) and Dr. Patrice Morin (National Institute on Aging, Baltimore, Maryland, USA), respectively and were cultured in McCoy’s 5A medium. All cell culture reagents used were obtained from Invitrogen (Thermo Fisher Scientific Inc., Waltham, MA, USA). Metformin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMEM containing 10% fetal bovine serum (FBS) at the indicated concentration. Carboplatin (Sigma-Aldrich) was dissolved in water and diluted in DMEM to various concentrations. Primary antibodies against AMPKα, phospho-AMPKα (Thr172), AKT, phospho-AKT (Ser473), mTOR, phospho-mTOR (Ser2448) S6, phospho-S6 (Ser235-236), 4EBP1, phospho-4EBP1 (Thr37/46) and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were purchased from Sigma-Aldrich.
In Vitro Cell Viability Assays and Cell Proliferation Assay
To assay cell viability following 0.5 mM metformin treatment, we seeded MOSECs in 12-well plates (2x104 per well), cultured the cells for 1 to 5 days in medium containing 0.2% FBS, and collected and stained the cells with trypan blue for quantification at different time points. Day 0 represents the day of treatment. Cell proliferation was measured with the MTT assay, 2D colony-formation assay [43], or the sulforhodamine B (SRB) assay [44]. Briefly, MOSECs were seeded in 96- or 6-well plates and treated with different concentrations of metformin, carboplatin or both for the indicated times. The results were analyzed as described by Chou [45] using the CompuSyn program downloaded from http://www.combosyn.com/. The IC50 values for each drug were determined by interpolation from the dose-response curves. The resulting combination index (CI) is a quantitative measure of the degree of interaction between different drugs. CI=1 denotes additivity; CI>1 denotes antagonism; and CI<1 denotes synergism. For interpretation, the combination was plotted as the log10(CI) versus the fraction affected (Fa; defined as 1–survival fraction). On these plots, additivity was defined as log(CI)=0, synergy was defined as log10(CI)<0; and antagonism was defined as log10(CI)>0. All of the results were experimentally reproducible.
Western Blot Analysis
Cancer cells were treated with control vehicle, metformin, carboplatin, or a combination for 48 hours, and the cells were pelleted by centrifugation and rinsed with PBS. The cell pellets were then lysed in RIPA buffer followed by sonication. Lowry assays (Bio-Rad) were performed to determine the protein concentration. Equal amounts of protein were loaded in each lane and resolved by 10% to 12% gradient Bis-Tris gels. All Western blot analyses were performed using whole-cell lysates prepared as described above. SDS-PAGE and Western blotting were performed using standard methods. The protein band intensities were quantified using ImageJ analysis by determining the relative intensity for each experimental band and normalizing its absolute intensity to that of the control.
Tumor Xenografts in a Mouse Model
C57BL/6 (B6) mice (4 weeks of age) were purchased from Taiwan National Laboratory Animal Center and LASCO laboratory. The research protocol was approved, and the mice were maintained in accordance with the Institutional Guidelines of Taipei Medical Center and Taipei Veteran General Hospital. MOSECs (1x106 cells) were subcutaneously injected into the right flank of B6 mice (6 weeks of age). One week post-injection, mice were randomly divided into 4 groups: a control group, an oral metformin (150 mg/kg once per day) group, an IP carboplatin (30 mg/kg twice a week: Wednesday and Friday) group, and a combined-treatment (neoadjuvant metformin from Monday, combined with carboplatin from Wednesday) group. There were 5 mice per group (20 total). Drugs were applied one week after tumor injection, which was designated week 0 in all groups. Tumor length and width were measured using a caliper, and tumor volume was calculated using the following formula: volume=[length×width2]/2. The change in tumor size is expressed as the fold change in tumor volume. The fold change in tumor size each week was calculated as follows: fold change in tumor size=(week)n/tumor size initial (week 1). At the end of the experiment, the mice were sacrificed, and tumor samples from each group were collected for Western blot analysis.
Statistical Analysis
Statistical analysis was carried out using the PASW package (PASW Statistics V18, Chicago, IL, USA). Survival analysis was based on the Kaplan-Meier method. Comparisons of clinical characteristics between two groups were performed by Student's t-test, the chi-square test or Fisher’s exact test. Comparisons between survival curves were performed using the log-rank or Breslow test. Comparisons of relative fold-changes in tumor cell survival among different treatment groups were performed by 2-way ANOVA with Bonferroni post-tests. A value of p<0.05 was considered statistically significant.