The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer

Abstract

Background: Cancer is a significant health challenge and the leading cause of mortality globally. Tumor cells use multiple mechanisms to acquire their distinctive capacity for uncontrolled proliferation, one of which is the evasion of apoptosis. It has been shown that in breast, colon, and liver cancer, evasion of apoptosis is associated with the overexpression of enzymes that metabolize arachidonic acid (AA) because free AA is a strong inducer of apoptosis. Glycerol-3-phosphate acyltransferase 2 (GPAT2) is a key enzyme in AA metabolism and is highly expressed in breast and colon cancer, where it promotes the development of essential tumor features.

Methods: In this work, a model of GPAT2 silencing in the human breast cancer-derived cell line MDA-MB-231 was used, and the cells were exposed to exogenous AA. The role of GPAT2 in AA-induced cell death was studied using MTT and TUNEL assays and measurements of caspase activity. The underlying molecular mechanism of cell death was assessed by qRT‒PCR.

Results: The results showed that AA reduced cell viability only in GPAT2-silenced cells, and that this cell death was a consequence of an apoptotic process involving BNIP3 overexpression. Additionally, it was demonstrated that GPAT2 silencing triggered a compensatory mechanism by overexpressing other genes involved in AA utilization for eicosanoid biosynthesis.

Conclusions: We concluded that GPAT2 expression is necessary to prevent AA-induced apoptotic cell death in MDA-MB-231 cells and that the overexpression of other AA-metabolizing genes is not sufficient to compensate for the lack of GPAT2 and prevent apoptosis.

Keywords: Apoptosis; Arachidonic acid; BNIP3; Breast cancer; Cell viability; GPAT2.

Fig. 1

Concentration-dependent induction of AA-related cell death in GPAT2-silenced cells. scr-MDA and sh-MDA cells were grown in 10% FBS DMEM supplemented with 50 or 100 µM AA for 24–48 h before the cell proliferation rate was measured via an MTT proliferation assay. The values represent the means ± SDs of 3 independent experiments (***P< 0.001, **P< 0.01)

Fig. 2

AA induces apoptosis only in GPAT2-silenced cells. scr-MDA and sh-MDA cells were treated with 100 µM AA for 48 h, and the percentage of apoptotic cells was determined by counting the number of apoptotic and nonapoptotic cells via a TUNEL assay and hematoxylin staining (***P< 0.001)

Fig. 3

AA increases the activity of several caspases in GPAT2-silenced cells. sh-MDA cells were treated with 100 µM AA for 48 h, and the activity of caspases 1, 2, 3, 5, 6, 8 and 9 was quantified using the colorimetric assay kit described in the Materials and Methods section. Staurosporine (ST) was used as a positive control for caspase-3 activation. The results are expressed as the mean ± SD of 3 independent experiments (***P< 0.001)

Fig. 4

Arachidonic acid increases the mRNA expression of the proapoptotic gene BNIP3. sh-MDA cells were treated with 100 µM AA for 48 h, after which BNIP3, BNIP3L and TNFRSF21 mRNA levels were analyzed via qRT‒PCR. The values represent the means ± SDs of 3 independent experiments (***P< 0.001)

Fig. 5

GPAT2 silencing alters the mRNA expression of genes involved in arachidonic acid metabolism. Total RNA was extracted from scr-MDA and sh-MDA cells, subjected to cDNA synthesis, and amplified via qRT‒PCR with primers for human PTGS2, ALOX5, AKR1C3 and EPHX2. The values represent the means ± SDs of 3 independent experiments (***P< 0.001, **P< 0.01)

Fig. 6

Schematic model of arachidonic acid-induced apoptosis in GPAT2-silenced cells. The cellular level of unesterified arachidonic acid is a general mechanism by which apoptosis is regulated, and GPAT2 promotes carcinogenesis by decreasing this level. In scr-MDA cells, GPAT2 esterifies arachidonoyl-CoA to glycerol−3-phosphate, allowing these cells to survive AA treatment. Instead, sh-MDA cells overexpress eicosanoid-producing enzymes, and AA increases BNIP3 expression, which leads to apoptosis in GPAT2-silenced cells. (cyt. C: cytochrome C, AA: arachidonic acid)