Type 2 Diabetes in Neuroendocrine Tumors

Are Biguanides and Statins Part of the Solution?

Aura D. Herrera-Martínez; Sergio Pedraza-Arevalo; Fernando L-López; Manuel D. Gahete; María A. Gálvez-Moreno; Justo P. Castaño; Raúl M. Luque


J Clin Endocrinol Metab. 2019;104(1):57-73. 

In This Article

Materials and Methods

Patients and Tissue Samples

This study was approved by the ethics committee of the Reina Sofia University Hospital (Córdoba, Spain), and was conducted in accordance with the Declaration of Helsinki and with national and international guidelines. A written informed consent was signed by every individual before inclusion in the study. A total of 181 patients (n = 81 with LCs; n = 100 with GEP-NETs) who underwent surgery at the Reina Sofia University Hospital from 2005 to 2015 were included in the study. Clinical records were used to collect patients' full medical history. Endocrine-associated syndromes were excluded. Patients with T2DM before the diagnosis of NET (n = 31 patients) were analyzed separately; these included 14 patients in the LCs group, 6 of whom were treated with metformin, and 17 patients in the GEP-NETs group, 9 of whom were treated with metformin. A similar analysis was performed in those patients treated with statins: four in the LCs group and six in the GEP-NETs group. Demographic and clinical characteristics of both cohorts are summarized in Table 1, Table 2, Table 3 and Table 4.

Patients were treated following the available guidelines and recommendations. After surgery, if residual or relapsed disease was observed, adjuvant treatment with or without surgery was prescribed. To confirm the neuroendocrine nature of all tumors, chromogranin A, synaptophysin, cytokeratin 7, cytokeratin 20, CD56, and neuronal-specific enolase levels were determined by immunohistochemistry, which was performed following the standardized diagnosis protocol of our hospital and evaluated by two experienced pathologists. Formalin-fixed paraffin-embedded (FFPE) samples were available for 46 LCs and 55 GEP-NETs cases; total RNA was isolated from these. Tumor samples were reevaluated by two experienced pathologists before RNA isolation. Only primary tumor samples were included. Samples were analyzed individually and mRNA expression levels were correlated with the clinical and histological characteristics of the corresponding patient.

Culture of Cell Lines

We used two human pancreatic NET (PNET) cell lines: BON-1 and QGP-1.[36–39] BON-1 cells were cultured in DMEM-F12 (Life Technologies, Barcelona, Spain) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, Madrid, Spain), and 0.2% antibiotic (gentamicin/amphotericin B; Life Technologies). QGP-1cells were cultured in RPMI 1640 (Lonza, Basel, Switzerland), supplemented with 10% FBS, 1% glutamine, and 0.2% antibiotic. Cells were harvested with trypsin (0.05%) and EDTA (0.53 mM) (Sigma-Aldrich) and resuspended in culture medium. Cell viability always exceeded 85%. Both cell lines were cultured in 75 cm2 flasks at 37 °C in a 5% CO2 incubator. All experimental procedures in both cell lines were performed at least three times.

Drugs and Reagents

Metformin, phenformin, simvastatin, atorvastatin, lovastatin, and rosuvastatin were purchased from Sigma-Aldrich. Buformin was purchased from Santa Cruz Biotechnology (Dallas, TX). All treatments were dissolved in the respective FBS-free medium and diluted until final concentrations were obtained before use (metformin, 10−2 M; phenformin and buformin, 5 × 10−3 M; statins, 10−5 M). IGF1 and paclitaxel were purchased from Sigma-Aldrich. Drug doses were selected on the basis of in vitrodose-response curves (unpublished results) or in previous studies.[40,41]

Cell Viability Assay

Cells were plated in 100 μL of medium in 96-well plates at the density necessary to obtain a 65% to 70% cell confluence in the control groups at the end of the experiment. Twenty-four hours later, serum-free medium was added over 24 hours. After this, biguanides and statins were added into wells in medium with 5% serum. Cell viability was measured using the alamarBlue assay (Thermo Fisher Scientific, Waltham, MA) at basal, and 24, 48, and 72 hours of incubation by measuring the fluorescent signal exciting at 560 nm and reading at 590 nm (Flex Station 3; Molecular Devices, San Jose, CA). On the day of each measurement, cells were incubated for 3 hours in 10% alamarBlue/serum-free medium and then alamarBlue reduction was measured. After each measurement, medium was replaced immediately by fresh medium. In all cases, cells were seeded per quadruplicate and all assays were repeated a minimum of four times. IGF1 and paclitaxel treatment were used as positive and negative controls, respectively.

Migration Capacity Assay

The ability of BON-1 cells to migrate after 24 hours of treatment with biguanides and statins was evaluated by a wound-healing technique. Briefly, cells were plated at subconfluence in 12-well plates. Confluent cells were serum starved for 24 hours; after synchronization, the wound was made using a 100-μL sterile pipette tip. Cells were incubated for 24 hours in FBS-free medium. Wound healing was calculated as the area of a rectangle centered in the picture 24 hours after the wound vs the area of the rectangle just after wounding, as previously reported.[42] At least three experiments per cell line were performed on independent days, in which three random images along the wound were acquired per well.

RNA Isolation and Reverse Transcription

Total RNA from FFPE samples was isolated using the RNAeasy FFPE Kit (Qiagen, Limburg, Netherlands) according to the manufacturer's instructions. Quantification of the recovered RNA was assessed using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, NC). One microgram of total RNA was retrotranscribed to cDNA with the First Strand Synthesis kit using random hexamer primers (Thermo Fisher Scientific), as previously reported.[43,44]

Quantitative Real-time PCR

cDNA was amplified with the Brilliant III SYBR Green Master Mix (Stratagene, La Jolla, CA) using the Stratagene Mx3000p system and specific primers for each transcript of interest. Specifically, expression levels (absolute mRNA copy number per 50 ng of sample) of insulin receptor (INSR) and GLUT4 genes were measured in the cells using previously validated primers,[41] and the expression level of each transcript was adjusted by the expression of β actin (BACT; used as housekeeping gene). Experiments were performed at least three times.

In human tumor samples, somatostatin system [i.e., somatostatin (SST), cortistatin (CORT), and their receptors SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, and sst5TMD4], and ghrelin system [i.e., ghrelin (GHRL), In1-ghrelin variant, ghrelin-o-acyltransferase enzyme (GOAT), and the receptors GHSR1a/GHSR1b] were evaluated using previously validated primers (41, 45–48). mRNA levels were normalized by 18S in GEP-NETs and by BACT in LCs, as previously described.[45,46] In NET cell lines, the expression of INSR and glucose transporter GLUT4 was analyzed in response to biguanides and statins treatment using previously validated primers.[49,50]

All samples were run, in the same plate, against a standard curve to estimate mRNA copy number and a no reverse transcriptase sample as a negative control. Thermal profile consisted of an initial step at 95°C for 30 seconds, followed by 50 cycles of denaturation (95°C for 20 seconds) and annealing and elongation (60°C for 20 seconds), and finally, a dissociation cycle (melting curve; 55°C to 95°C, increasing 0.5°C/30 seconds) to verify that only one product was amplified.

Serotonin Assay

BON-1 and QGP-1 cells were cultured in 12-well plates. At 70% confluence, cells were serum starved and after a 24-hour incubation period with specific treatments or with vehicle, media were collected and stored at −20°C until measurements were made. Secretion of serotonin was detected using a serotonin ELISA kit (ALPCO, Salem, NH) following the instructions of the manufacturer.

Apoptosis Assay

BON-1 and QGP-1 cells were cultured in 24-well plates. At 70% confluence, cells were serum starved and after a 48-hour incubation with specific treatments or with vehicle-treated controls in 5% FBS medium, apoptosis levels were measured using a cell-death detection ELISA kit (Sigma-Aldrich) following the manufacturer's instructions.

Measurement of ERK1/2 and AKT Signaling Pathways by Western Blotting

A total of 500,000 of each cell type (BON-1 and QGP-1) were cultured in six-well plates and incubated for 8 minutes with specific treatments and vehicle-treated controls. Briefly, after the corresponding treatment, medium was removed and cells were washed twice using PBS, detached using a scraper, and immediately lysed in prewarmed SDS-dithiotreitol sample buffer at 65°C (62.5 mM Tris-HCl, 2% SDS, 20% glycerol, 100 mM dithiotreitol, and 0.005% bromophenol blue) followed by sonication for 10 seconds and boiling for 5 minutes at 95°C, as previously described.[51,52] Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Darmstadt, Germany). Then membranes were blocked with 5% nonfat dry milk in Tris-buffered saline/0.05% Tween 20 and incubated with the primary antibodies for total-ERK1/2, p-ERK1/2, total-AKT, p-AKT (Santa Cruz, CA) and then with the appropriate secondary antibodies (anti-rabbit antibody; Cell Signaling, Danvers, MA). Protein analyses were developed using an enhanced chemiluminescence detection system (GE Healthcare, Chicago, IL) with dyed molecular-weight-markers. A densitometric analysis of the bands was carried out with ImageJ software.[53] Relative phosphorylation of ERK and AKT was obtained from normalization of p-ERK1/2 or p-AKT against the total ERK1/2 or AKT, respectively.

Statistical Analysis

In functional experiments, results are expressed as percentage vs vehicle control (nontreated cells). mRNA levels are expressed as mean ± SEM. Cell survival rate compared with control was assessed by multiple comparison tests. Mann-Whitney Utests were used to evaluate clinical relations within LC and GEP-NET samples. χ 2 test was used to compare categorical data. All statistical analyses were performed using SPSS statistical software, version 20, and GraphPad Prism, version 6. P values < 0.05 were considered statistically significant.