TheSGLT-2 inhibitor dapagliflozin ehnanced the anticancer activities and exerts cardioprotective effects during exposure to ipilimumab through NLRP3 inflammasome and pro-fibrotic cytokines
Authors: Maurea N , Quagliariello V , Bonelli A , Paccone A , Conforti G , Caronna A , Buccolo S , Iovine M , Cerrone F , Botti G 
Published: October 2021

Abstract

Background
The clinical trial “DECLARE-TIMI 58” (Dapagliflozin Effect on Cardiovascular Events-Thrombolysis in Myocardial Infarction 58), demonstrated that dapagliflozin, a Sodium glucose cotransporter 2 inhibitor, reduces the composite end point of cardiovascular death/hospitalization for heart failure in a broad population of patients with type 2 diabetes mellitus. Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, achieving unprecedented efficacy in multiple malignancies. However, ICIs are associated with immune-related adverse events involving cardiotoxicity.

Purpose
We aimed to study if dapagliflozin could affect ipilimumab-induced anticancer efficacy in human breast cancer cells and reduces its cardiotoxicity.

Methods
Co-culture of hPBMCs and human cardiomyocytes or estrogen-responsive and triple-negative breast cancer cells (MCF-7 and MDA-MB-231 cell lines) were exposed to ipilimumab (200 nM) alone or combined to SGLT-2 inhibitor (dapagliflozin) for 72h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome; expression of TLR4/MyD88; transcriptional activation of p65/NF-κB and secretion of cytokines involved in cardiotoxicity (Interleukins 1β, 8, 6).

Results
Dapagliflozin increases significantly the cardiomyocytes viability during exposure to Ipilimumab. Indeed, in human breast cancer cells, dapagloflozin showed an opposite behavior with a significant increase in cell mortality and apoptosis (p&lt;0.001 vs only ipilimumab). Cardioprotective properties of dapagliflozin are explainable by the reduction of intracellular Ca2+ overload (−56,8% vs only ipilimumab; p&lt;0,001), of the lipid peroxidation (mean reduction of 42,1–48,6% compared to cells exposed only to ipilimumab; p&lt;0,05). Moreover, cells exposed to dapagliflozin during ipilimumab reduced the protein expression of pro-inflammatory cytokines involved in cardiotoxicity and resistance to anticancer effects of ICIs (−47,2% for Interleukin-1β; −48,7 for Interleukin 6; −32,1% for Interleukin 8; p&lt;0,001 for all vs only ipilimumab groups). Notably, dapagliflozin reduces p65-NF-κB activation (−46,3 and −49,3% vs only ipilimumab, p&lt;0.05) and inhibits of 43,2–53,7% the expression of NLRP3 inflammasome, p&lt;0.05 for all). No significant effects were seen on TLR4/MYD88 expression in all groups.

Conclusion
Dapagliflozin demonstrated cardioprotective properties during Ipilimumab exposure in co-culture model of hPBMCs and cardiomyocytes. Dapagliflozin improves Ca2+ homeostasis and inhibits the pro-inflammatory “NLRP3–NF-κB–cytokines” pathways in cardiac cells.

Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ricerca corrente grant of Italian Ministry of Health