AbstractActivation of the adenosine monophosphate protein kinase (AMPK) signalling pathway is known to result in the inhibition of ATP-consuming anabolic mechanisms in the heart and is associated with prevention of cardiac myocytes loss. The multityrosine kinase inhibitor (TKI) Sunitinib has been associated with cardiotoxicity via the inhibition of AMPK signalling. In contrast, the agent Metformin, used for treatment of type-2 diabetes mellitus, is associated with cardioprotective properties with the activation of the AMPK signalling pathway. We aimed to demonstrate the potential use of Metformin with Sunitinib in order to prevent Sunitinib-induced cardiotoxicity. Sprague-Dawley (2/3 m/o, male) rat hearts were Langendorff perfused with vehicle control, Sunitinib (1 µM) ± Metformin (50 µM) ± AMPKassociated inhibitor S-(4-Nitrobenzyl)-6-thioinosine (NBTI) (1 µM) for 155 minutes, following 20 minutes of stabilisation. Haemodynamic parameters were measured for heart rate (HR), left ventricular developed pressure (LVDP) and coronary flow (CF). Dismounted hearts were used for triphenyltetrazolium chloride (TTC) staining for infarct percentage or Western blot SDS page analysis for protein levels for phosphorylated-AMPK (Thr172), total-AMPKα and GAPDH. Isolated primary cardiac myocytes were obtained from Sprague-Dawley (2/3 m/o, male) rat hearts following Langendorff perfusion. HepG2 and HL60 cells were incubated with Sunitinib (0.1- 100 µM) ± Metformin (50 µM) ± NBTI (1 µM) for (3-(4,5-Dimethylthiazol-2-yl)-2-5- Diphenyltetrazolium Bromide (MTT) analysis. Sunitinib administration (1 µM) demonstrated a significant increase in infarct percentage of Sprague-Dawley rat hearts using the Langendorff model, compared to vehicle control. Sunitinib administration further demonstrated a significant decrease in LVDP compared to vehicle control at selected time-points. Co-administration of Sunitinib with Metformin (50 µM) attenuated the increase in infarct percentage (co-treatment 20 ± 2 % vs. Sunitinib 31 ± 2 %) and attenuated the Sunitinib-induced decrease in LVDP at selected time-points (145 minute; Sunitinib 64 ± 5 % vs. co-treatment 80 ± 6 %, 160 minute; Sunitinib 57 ± 6 % vs. co-treatment 75 ± 3 %, 175 minute; Sunitinib 58 ± 6 % vs. co-treatment 74 ± 3 %). Live cell population of cardiac myocytes was decreased during Sunitinib administration (1 µM). Co-administration of Metformin (50 µM) with Sunitinib attenuated Sunitinib-induced decrease of live cell population of isolated cardiac myocytes (co-treatment 41 ± 3 % vs. Sunitinib 12 ± 2 %).
Metformin-induced activation of AMPK and Metformin-induced cardioprotection was abolished during co-administration of Sunitinib and Metformin with NBTI (1 µM). Sunitinib demonstrated a dose-dependent decrease in cell viability in HepG2 and HL60 cells, co-administration of Sunitinib (0.1-100 µM) with Metformin (50 µM) demonstrated an increase in EC50 concentration in HepG2 and HL60 cells (HepG2 cells; co-treatment 34.7 µM vs. Sunitinib 15.4 µM., HL60 cells; co-treatment 18.2 µM vs. Sunitinib 10 µM). The addition of NBTI (1 µM) with Sunitinib (0.1-100 µM) and Metformin (50 µM) demonstrated an increase in EC50 concentration in HepG2 (43.3 µM) and HL60 cells (33.3 µM).
We demonstrated for the first time Metformin-induced cardioprotection against Sunitinib-induced cardiotoxicity in an ex-vivo Langendorff-based model. Furthermore, Western blot analysis determined that Metformin-induced cardioprotection is associated with an increase in phosphorylation of AMPK, an effect that was abolished upon AMPK inhibition with NBTI. Moreover, we demonstrated the adjunctive treatment in human cancer cell lines of HepG2 and HL60, that Metformin co-administration resulted in an increase in EC50 concentration compared to Sunitinib administration alone, however Metformin did not inhibit Sunitinib’s anti-proliferative properties.
In conclusion, this study demonstrates the cardioprotective properties of Metformin during co-administration with Sunitinib and the role of AMPK signalling. This study adds to the growing interest of potential adjunctive treatment during TKI therapy.
|Date of Award||Mar 2020|
|Supervisor||Hardip Sandhu (Supervisor), Irmgard Haussmann (Supervisor), Helen Maddock (Supervisor) & Mayel Gharanei (Supervisor)|