Abstract
Study question
Does metformin treatment lead to temporal expression changes of specific genes and proteins in endometrial cancer cell lines?
Summary answer
The expression of three different genes and proteins was investigated, of which all displayed changes over time.
What is known already
Endometrial cancer (EC) is one of the most common malignancies among postmenopausal women. A long-term estrogen effect on the endometrium often seen in women with obesity or the polycystic ovary syndrome (PCOS), as well as type II diabetes mellitus (T2DM) are well-known risk factors for the development and progression of EC. Metformin is a biguanide used in the treatment of T2DM patients and off label in women with PCOS. Moreover, metformin displays anti-tumor and anti-proliferative effects in various cancer types, including EC. In that regards BCL2L11, CDH1 and CDKN1A play an important role in apoptotic pathways, proliferation and invasion processes.
Study design, size, duration
The EC cells were cultured in normal (5.5 mmol/L) or high (17 mmol/L) glucose medium supplemented with 10 nmol/L ß-estradiol. The cells were treated with low dose metformin (1.0 mmol/L) for 2, 6, 24, 48 and 168 h (7 d). In addition, EC cells were treated with a combination of metformin and insulin (100 ng/mL) or remained untreated. Five independent experiments were fulfilled and untreated cells served as controls.
Participants/materials, setting, methods
The study was accomplished using two different human EC cell lines. HEC–1A represents an estrogen-independent EC, whereas Ishikawa represents the more common, estrogen-dependent EC. Proteins were extracted, quantified with a BCA assay, and the protein expression of BCL2L11, CDH1 and CDKN1A was analyzed by western blots. Furthermore, total RNA was extracted, transcribed to cDNA and Taqman real-time PCR was carried out to measure the expression of the associated genes, using fold change (FC) as parameter.
Main results and the role of chance
The expression of the selected genes, analyzed by RT-PCR, changed in both cell lines over time as follows: After 6 h, metformin induced a decrease in the expression of BCL2L11 (FC = 0.7) and CDH1 (FC = 0.75), whereas the expression of CDKN1A slightly increased (FC = 0.95–1.35). After 24 h, BCL2L11 expression increased in normal glucose groups (FC = 1.3, high glucose: FC = 0.93) and CDH1 expression decreased in combination with metformin and high glucose (FC = 0.7, normal glucose: FC = 1.1). CDKN1A expression was increased by metformin in both cell lines after 24 h (FC = 1.2–1.8). After 48 h of metformin treatment, expression for all three genes was only slightly changed (FC = 0.9–1.0). After 7 d it was observed that the combination of high glucose and metformin (i.e. like obese T2DM patients) led to an increased expression of BCL2L11, CDH1 and CDKN1A (FC = 1.4–2.9) in the presence and absence of insulin, whereas metformin induced a decreased expression of CDH1 and CDKN1A (FC = 0.5–0.75) in normal glucose medium. BCL2L11, CDH1 and CDKN1A expression was investigated at the protein level as well.
Limitations, reasons for caution
The results cannot be directly transferred to metformin treatment of patients, since the study was carried out in vitro. Additionally, further studies including more timepoints would indicate a more precisely gene and protein expression over time.
Wider implications of the findings: This is the first in vitro study showing the temporal changes of BCL2L11, CDH1 and CDKN1A expression, genes related to tumorigenesis due to low dose metformin over time, suggesting differentially pathways in long term metformin treatment using physiologically achievable metformin levels.
Trial registration number
Not applicable
High Glucose Levels Interferes the Endometrial Cancer Cell Response to Metformin Treatment over Time
