Elsevier

Toxicology Letters

Volume 336, 1 January 2021, Pages 39-49
Toxicology Letters

Extended regorafenib treatment can be linked with mitochondrial damage leading to cardiotoxicity

https://doi.org/10.1016/j.toxlet.2020.11.003Get rights and content

Highlights

  • Regorafenib (RGF) inhibited cell proliferation and reduced ATP content of cells.

  • RGF disrupted mitochondrial membrane potential and mitochondrial structure.

  • High concentration of RGF up-regulated fission and fusion gene expressions.

  • RGF reduced mitochondrial complex I and V expressions and decreased mtDNA content.

  • HSP70 protein expression increased following high doses of RGF treatment.

Abstract

Regorafenib (RGF) has a great success in the treatment of colorectal cancer, gastrointestinal stromal tumours and hepatocellular carcinoma by inhibiting angiogenic, stromal and oncogenic kinases. However, RGF can induce life-threatening cardiotoxicity including hypertension and cardiac ischemia/infarction. The molecular mechanism of the adverse effects has not been elucidated. Mitochondrial dysfunction is one of the major causes of cardiac diseases since cardiac cells highly need ATP for their contractility. Therefore, we aimed to investigate molecular mechanisms of RGF-induced cardiac adverse effects using H9c2 cell model by focusing on mitochondria. Cells were treated with 0−20 μM RGF for 48 and 72 h. According to our results, RGF inhibited cell proliferation and decreased the ATP content of the cells depending on the exposure time and concentration. Loss of mitochondrial membrane potential was also observed at high dose. Mitochondrial fusion/fission genes and antioxidant SOD2 (superoxide dismutase) gene expression levels increased at high doses in both treatments. Mitochondrial DNA content decreased as exposure time and concentration increased. Also, protein expression levels of mitochondrial complex I and V have reduced and stress protein HSP70 level has increased following RGF treatment. Structural abnormalities in mitochondria was seen with transmission electron microscopy at the applied higher doses. Our findings suggest that RGF-induced cardiotoxicity may be associated with mitochondrial damage in cardiac cells.

Introduction

Tyrosine kinases are important enzymes that are responsible for initiation and progression of signaling cascades. The new generation anticancer drugs tyrosine kinase inhibitors (TKIs) have achieved great succes in the treatment of various types of cancer such as chronic myeloid leukemia, breast cancer, renal cell carcinoma, colon cancer (Chen et al., 2008; Cheng and Force, 2010; Paul and Mukhopadhyay, 2012). However, these therapies can induce life threatening cardiovascular adverse effects and this may lead to termination or discontinuation of the treatment (Cheng and Force, 2010; Mellor et al., 2011; Shah et al., 2013). TKI-induced cardiovascular adverse effects are hypertension, pulmoner hypertension, venous and arterial trombosis, pulmonary embolism, alteration of QT interval, left ventricular dysfunction (Mellor et al., 2011; Shah et al., 2013). The adverse effects can be drug specific and/or dependent of drug targets (Chaar et al., 2018; Lamore et al., 2019).

Regorafenib (RGF) which is a TKI drug, is approved by the FDA for the treatment of colorectal cancer, gastrointestinal stromal tumours, and hepatocellular carcinoma; inhibiting oncogenic, angiogenic, and stromal tyrosine kinases (Wilhelm et al., 2011). RGF has been shown to inhibit drug transporter proteins, therefore, it has been thought that the combination of RGF and conventional chemotherapy may overcome chemotherapy drug resistance (Chen et al., 2019; Wang et al., 2017). Clinical trials are still continuing for the use of RGF in the treatment of solid tumours such as; ovary and thyroid cancer (https://clinicaltrials.gov/ct2/results?cond=&term=Regorafenib&cntry=&state=&city=&dist=).

One of the most common cardiac adverse events after RGF treatment is hypertension. Regorefenib-induced hypertension can be life-threatening and it can be an underlying cause of caridovascular toxicity (De Wit et al., 2014; FDA, 2017; Shenasa and Shenasa, 2017). Also, it has been reported that RGF leads to cardiac ischemia and infarction (Eisen et al., 2012; FDA, 2017) and induces dyspne which is one of the indicators of cardiovascular diseases (Groarke et al., 2014). Literature involves case reports about RGF induced myocardial damage (Hsiao et al., 2016) and hypertensive crisis (Yilmaz et al., 2014). However, the molecular mechanisms of RGF-induced cardiac events have not been elucidated.

As mitochondria are the main source of ATP production in eukaryotic cells, the cardiomyoblastic cells are highly ATP dependent for cardiac muscle contractility. Mitochondria constitute 22–37 % of the total cardiomyocyte volume in mammals to meet the excessive ATP demand (Barth et al., 1992; Lehman et al., 2000). Any factor that affects ATP production of the mitochondria can distrupt cardiomyocyte function.

In the present study it was aimed to clarify the molecular mechanism of RGF-induced cardiotoxicity using an in vitro cardiomyoblastic model (H9c2 cells) by mostly focusing on the factors that affect mitochondrial function, mitochondrial dynamics, and the pathways that are related to mitochondrial and cellular stress.

Section snippets

Cell culture

H9c2 cells, rat cardiomyoblastic cell line, were taken from the American Type Culture Collection (ATCC, VA, USA) and were cultured in DMEM-F12 medium containing 10 % FBS (fetal bovine serum), 1 % penicillin- streptomycin-amphotericin. Subculturing was done 60–70 % confluence (every 2–3 days) using trypsinization in order to maintain the myoblastic population of the cells. All experiments were performed with cells under the passage number 25 and myoblastic cellular morphology was verified

Effects of RGF on cellular toxicity

According to trypan blue and LDH assay results, RGF did not induce cell death but reduced cell proliferation in a dose dependent manner as shown in Fig. 1 (≤14.68 %, p<0.05). Depending on the concentration and treatment time increasements cell proliferation decreased (p<0.05).

Effects of regoragenib on intracellular ATP content

RGF highly affected intracellular ATP content depending on concentration and exposure time (Fig. 2). For the 72 h treatment groups, ATP content reduced in a concentration dependent manner compared to control group and a

Discussion

The TKI drug RGF used for the treatment of colorectal cancer, gastrointestinal stromal tumours and hepatocellular cancer shows anticancer effects by inhibiting multi-tyrosine kinases. RGF induced cardiovascular toxicities are recorded as hypertension, cardiac ischemia and infarction (Agarwal et al., 2018; FDA, 2017). Although it has been suggested that VEGF-targeted therapies may be associated with these cardiovascular adverse effects, the molecular mechanisms have not been clarified clearly.

Conclusion

In conclusion RGF-induced cardiotoxicity can be associated with mitochondrial damage. RGF treatment in cardiomyoblasts causes alteration in the mitochondrial dynamics and mitochondrial structure, disturbing mitochondrial energetic pathways and eventually resulting in cellular stress. When all results of the current study are evaluated collectively, it can be seen at the mitochondrial level that after drug treatment the cardiomyoblasts try to cope with stress and when the treatment time gets

Declaration of Competing Interest

There are no conflicts of interest to declare.

Acknowledgement

This study was supported by Scientific Research Project Coordination Unit of Istanbul University. Project number: TDK-2017-25176

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