Andrographolide protects against doxorubicin-and arsenic trioxide-induced toxicity in cardiomyocytes

Andrographolide (AG) is a lactone diterpene with valuable biological activities. This in vitro study evaluated whether AG can protect cardiomyocytes under toxicities triggered with anti-cancer chemotherapeutic agents, doxorubicin (DOX) and arsenic trioxide (ATO). H9C2 cells were pretreated with AG (0.5–10 µM) for 24 h and then exposed to DOX (1 μM) or ATO (35 μM) for another 24 h period. For determination of cell viability or cytotoxicity, MTT and lactate dehydrogenase (LDH) assay were used. Total oxidant and antioxidant capacities were estimated by determining hydroperoxides and ferric reducing antioxidant power (FRAP) levels. Real time-polymerase chain reaction was also used for quantitative evaluation of TLR4 gene expression. AG inhibited cardiomyocytes proliferation at the concentrations of more than 20 μM. However, it considerably enhanced cell viability and decreased cytotoxicity of DOX and ATO at the concentration range of 2.5–10 μM in MTT and LDH assays. AG significantly declined hydroperoxides concentration in ATO-treated cardiomyocytes and raised FRAP value in DOX- and ATO-treated cells. Furthermore, AG notably lessened TLR4 expression in H9C2 cells after exposure to DOX- and ATO. In conclusion, these data presented that AG was able to reverse DOX- and ATO-induced cardiotoxicity in vitro. The cardiomyocyte protective activities of AG may be due to the decrease in TLR4 expression and total oxidant capacity and increase in total antioxidant capacity.


Introduction
Recent advances in cardio-oncology with development of some medicines and therapeutic approaches have partially prevented cardiotoxicity in patients treating with antitumor drugs. In spite of many advantages were reported for the use of antineoplastic agents in reducing cancer-caused mortality, cardiomyopathy is considered a progressive myocardial complication linked with chemotherapy in patients with cancer [1,2].
Anthracycline drugs such as doxorubicin (DOX), are employed to treat many types of cancers in breast, thyroid, stomach, bladder, ovarian, and other organs and have positive impacts on preventing metastasis [3]. The proposed action mechanisms of anthracyclines include the ability to inhibit topoisomerase I and II, target DNA, inhibit macromolecule synthesis, produce free radicals/DNA-damaging oxidants, induce lipid peroxidation, and increase endoplasmic reticulum stress resulting in long-term apoptosis induction [4]. Although anthracyclines play a critical role in the treatment of several neoplastic disorders, their longterm administration can lead to the cardiomyopathy and heart failure [5]. Increasing oxidative stress and producing different types of reactive oxygen species (ROS), altering energy reserves via changing phosphate concentrations and impairing synthesis of creatine kinase, inducing apoptosis, elevating expression of toll-like receptors (TLRs) including TLR2 and TLR4 and activation of nuclear factor kappa-B (NF-κB), increasing inflammatory conditions and impairing intracellular Ca 2+ are some mechanisms involved in DOX-induced cardiotoxicity [5][6][7][8].
Arsenic trioxide (ATO) is an effective antineoplastic agent for healing patients with acute promyelocytic leukemia (APL), both newly diagnosed and relapsed conditions [9]. ATO can lead to antiproliferative activities through growth inhibition of the G1 phase cell cycle, stimulating cellular differentiation by inducing the expression of cell surface maturation markers and inhibition of angiogenesis [10]. However, it has also been reported that this compound may enhance levels of ROS and inhibit antioxidant enzymes, induce stress in endoplasmic reticulum, disturb calcium balance, promote expression of apoptotic factors and ultimately increase the risk of cardiomyocyte apoptosis [11,12].
Currently, natural phytochemicals are being investigated for their therapeutic potential in cardio-oncology. Andrographolide (AG) is a lactone diterpene derived from Andrographis paniculata with a very bitter taste. Various biological activities including modulation of immune system, inhibition of oxidative stress and inflammation, heparoprotection and neuroprotection have been reported for this natural compound [13]. Evidence suggest that AG may act as both chemo-preventive and chemotherapeutic agent time-and concentration-dependently [14]. AG has also 1 3 shown cardioprotective effects in hyperlipidemia, atherosclerosis, myocardial infarction and ischemia/reperfusion via inhibiting oxidative, inflammatory and apoptotic pathways [15][16][17][18]. Furthermore, it has been reported that AG displays inhibitory action on TLR4/NF-κB signals [19].
Concerning the valuable cardiovascular activities of AG, this study was designed to inspect the possible protective effect of AG on toxicity caused by doxorubicin or arsenic trioxide in cardiomyocytes in vitro.

Cell culture
Rat H9C2 cardiomyocytes were obtained from Iran Cell Bank and were grown in high glucose medium of Dulbecco's modified Eagle Eagle (BioIdea Co., Iran) fortified with antibiotics (penicillin-streptomycin, 1%) and fetal bovine serum (10%) (Biosera Co., France) in humidity incubator with 5% CO 2 at 37 °C.
For assessment of the protective activity of AG against DOX or ATO toxicities, cardiomyocytes were initially pretreated with AG (0.5-10 µM) or N-acetylcysteine (NAC; 1000 µM) as a reference drug for 24 h and then exposed to DOX (1 µM) or ATO (35 µM) for another 24 h period. The rest of the test was completed as above. The cells that did not receive any treatment were considered as negative control.

Lactate dehydrogenase (LDH) assay
The impact of AG on DOX-or ATO-induced cytotoxicity was estimated using the LDH release assay Kit (Kiazist, Iran) according to the manufacturer's protocol [20]. The released LDH in the collected culture media which reflects a direct measurement of the dead fraction of the cells, was quantified by microplate reader/spectrophotometer at 560 nM. For assessing the supreme LDH release (high control), some cardiomyocytes were incubated with PermiSolution reagent. To detect the unprompted LDH release (low control), cells were kept with culture media. Cytotoxicity was estimated by the following formula: Cytotoxicity (%) = (Test sample absorption−Low control absorption)/(High control absorption−Low control absorption) × 100.

Total oxidant capacity assay
Total oxidant capacity was assessed by detection of hydroperoxides concentration based on FOX-1 (ferrous ion oxidation by xylenol orange) test using a commercial kit (Hakiman Shargh Research Co., Iran) [21]. For this mean, after pretreatment of cardiomyocytes with various concentrations of AG and then incubation with DOX or ATO, the supernatant of each well was separated and mixed with FOX-1 reagent. The solution was preserved for 30 min in a dark place at room temperature and absorbance was spectrophotometerically measured at 540 nM. A standard curve was depicted with different concentrations of H 2 O 2 and used for estimation of hydroperoxides concentration.

Total antioxidant capacity assay
Total antioxidant capacity was evaluated by determination of FRAP (ferric reducing antioxidant power) value using a standard kit (Hakiman Shargh Research Co., Iran) which was designed according to the reduction of ferric-tripyridyltriazine complex to ferrous iron [22]. After pre-incubation of cardiomyocytes with various concentrations of AG and then treatment with DOX or ATO, the supernatants were isolated and mixed with FRAP reagent. The solution was kept in an incubator (40 °C) for 40 min and absorbance was spectrophotometerically read at 570 nM. A standard curve was depicted with different concentrations of FeSO 4 and FRAP assessment was performed as FeSO 4 equivalents.

TLR4 gene expression assay
After relevant treatment, extraction of total RNA was performed from H9C2 cells using BIOFACT™ Total RNA Prep kit (BioFact Ltd., Korea). A NanoDrop system spectrophotometer (260/280 nM) was used for approving the concentrations of RNA and their purity. For detecting the expression of TLR4 gene, cDNA was synthesized using a commercial kit (Yekta Tajhiz Azma Co., Iran). Then, Quantitect SYBR Green master mix kit (Qiagen, Germany) on a StepOne™ RT-PCR System (USA) was used for quantitative RT-PCR (real time-polymerase chain reaction) analyzing [23].

Statistical analysis
Data were stated as SEM (mean ± standard error of mean). SPSS 25.0 software was used for statistical evaluation. The P less than 0.05 were estimated via one-way analysis of variance (ANOVA) and Tukey post hoc test and considered as the significant level.

Effects of AG, DOX and ATO on H9C2 cells viability
As shown in Fig. 1A, MTT assay showed non-inhibitory effect of AG at the concentration range of 1-10 µM on H9C2 cells viability after 24 h treatment. However, AG resulted in a non-significant reduction at the concentration of 20 µM and a significant decrease at the concentration of 40 µM on H9C2 cells survival (P < 0.05).
DOX and ATO significantly inhibited the growth of cardiomyocytes after 24 h incubation and their IC 50 values were determined to be 1.07 and 35.09 μM, respectively (Fig. 1B,  C). These concentrations were used to study the possible protective activity of AG on cardiomyocytes.

Effect of AG against DOX-and ATO-induced cytotoxicity
To examine the cytoprotective effect of AG against toxicities caused by DOX and ATO in H9C2 cells, MTT and LDH assays were used. DOX induced 55% cytotoxicity in MTT and 36% cytotoxicity in LDH test. AG exhibited cytoprotective activities against DOX-induced cardiomyocyte cell death at the concentrations of 2.5-10 µM in MTT assay and at the concentrations of 10 µM in LDH assay ( Fig. 2A, B).
After exposure of H9C2 cells to ATO, cytotoxicity was observed as 46% and 42% in MTT and LDH tests, respectively. Pretreatment of cardiomyocyte with AG prominently decreased the cytotoxicity of ATO at the concentrations of 5 and 10 µM in both tests (Fig. 3A, B).
NAC significantly protected cardiomyocyte against DOX-and ATO-induced cytotoxicity in LDH assay (Figs. 2B and 3B). NAC also alone could significantly ameliorate the decline in cell viability caused by DOX and ATO in MTT assay (data are not shown). However due to the more potent effects from AG, the results of incubation with NAC were not significant in simultaneous statistical analysis of all cellular groups.

Effect of AG on total oxidant capacity
As shown in Fig. 4A, B, the hydroperoxides level as a measure of total oxidant capacity was significantly elevated after incubation of cardiomyocyte with DOX (P < 0.001) and ATO (P < 0.01) when compared to the untreated normal cells. Pretreatment of cardiomyocytes with AG only attenuated the hydroperoxides levels at the concentration of 10 µM compared to the ATO groups (P < 0.05). NAC resulted in a significant reduction in hydroperoxides levels in DOX-and ATO-treated cells (P < 0.05).

Effect of AG on total antioxidant capacity
Exposure of cardiomyocyte to DOX and ATO caused a notable decrease in total antioxidant capacity which was determined as FRAP value (P < 0.01). AG treatment prominently raised FRAP value at the concentrations of 5 and 10 µM in DOX-treated cells and at the concentration range of 2.5-10 µM in ATO-treated cells (Fig. 4C, D).

Effect of AG on TLR4 gene expression
As shown in Fig. 5, the levels of TLR4 expression were increased in cardiomyocytes after 24 h incubation with DOX or ATO (P < 0.01). Pretreatment of H9C2 cells with AG significantly lessened TLR4 expression at the concentration of 10 µM in DOX-and ATO-induced cytotoxicity (P < 0.05). NAC similarly reduced TLR4 expression (P < 0.05).

Discussion
Several approaches have been evaluated for the management of chemotherapy-induced cardiotoxicity. Some prophylactic and chemoprotective agents such as dexrazoxane, statins, cyclooxygenase inhibitors, beta-blockers, angiotensin antagonists, diuretics, spironolactone, digoxin and erythropoietin are reported to be somewhat effective to reduce cardiotoxicity [8,24]. However, prevention of cardiotoxicity remains a major problem in cancer patients and recent investigations have focused on natural compounds.
In the present investigation, in vitro evaluation of AG as a bioactive agent revealed cytoprotective and antioxidant activities against toxicities caused by DOX and ATO in H9C2 cells through raising the cellular viability and FRAP value, and lessening the cytotoxicity, hydroperoxides concentration and TLR4 expression.
In our study, the MTT cell viability assay presented non-inhibitory effect of AG at the concentration range of 1-10 µM on H9C2 cells after 24 h treatment however there In the study of Woo et al., AG at the concentrations of 1, 3 and 10 μM had no obvious effect on the viability of neonatal rat cardiomyocyte and showed protective activity against cellular injury caused by hypoxia/reoxygenation in LDH release test [18]. Dey et al. also evaluated AG at the range of 2.5-40 µM concentrations in liver cancerous and non-cancer cell lines and reported the viability percentage as 90% after incubation with 20 µM AG and IC50 value of more than 40 µM in non-cancer cell lines [25]. In another study, AG showed anti-apoptotic effect at the concentrations of 1, 5, and 10 μM in H9C2 cells during high glucose condition through alleviating caspase-3 activity and Bax/ Bcl-2 ratio [26]. However, Wu et al. reported that AG did not disturb cardiomyocyte viability at the concentrations of 12.5, 25 or 50 μM using the cell counting kit assay-8 and it significantly prohibited the cytotoxicity induced by Ang II [27]. They reported that 7-weeks treatment with AG reduced heart dysfunction and diminished fibrosis and hypertrophy through down regulation of expression of atrial natriuretic peptide, brain natriuretic peptide, transforming growth factor β, connective tissue growth factor, collagen I and III genes in a mouse model of cardiac hypertrophy. In their study, AG also protects cardiomyocytes against hypertrophic changes induced by angiotensin II via suppressing mitogen-activated protein kinases (MAPKs) pathway [27].
The protective effects of AG against some cardiac disorders have similarly been described in several investigations. In myocardial infarction, AG has shown protective activity against heart remodeling via augmenting the Nrf2 pathway [17]. AG has dampened myocardial dysfunction caused by lipopolysaccharide in mice through suppressing phosphorylation of IκB and activation of NF-κB and hindering synthesis of NO and inflammatory markers such as TNF-α and IL-1β, and apoptosis of cardiac cells [28]. In diabetic cardiomyopathy, AG has been able to inhibit oxidative, inflammatory and apoptotic changes and consequently improved hypertrophy and fibrosis in heart tissue [26].
Furthermore, our data displayed lessening in hydroperoxides content and noteworthy raising in FRAP value in H9C2 cells after incubation with AG confirming its potent antioxidant properties. The mechanisms associated with anti-oxidative activities of AG are as follows: maintaining the integrity of mitochondrial, counteracting free radicals, reducing ROS formation via inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and iNOS and inactivation of NF-κβ signaling pathway, scavenging of several free radicals such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals, increasing NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) activity [29].
Previous studies have also shown that treatment of cardiomyocytes with AG has enhanced the cellular glutathione stores and the activities of enzymes including superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase and hence the heart antioxidant defense [18,26].
Moreover, our findings revealed that AG can attenuate the expression of TLR4 gene in cardiotoxicity caused by DOX and ATO. There are several documents for the contribution of TLR2 and TLR4 overexpression in the pathogenesis of heart toxicity in response to DOX [8,30]. Activation of TLRs triggers downstream signaling pathways including production of NF-κB-dependent inflammatory cytokines and interferons [31]. TLR4 activity is also increased in other heart pathology such as hypertension or heart failure and is linked with oxidative stress, inflammatory and fibrotic pathways [32].
Unlike DOX, little data has been described for the role of TLR overexpression in ATO-induced cardiomyopathy. In a recent article, Zheng et al. reported that ATO-induced cardiac injury was related to the stimulation of TLR4/NF-κB signaling and inflammatory reactions [33].
Instead, various analogs of AG have shown powerful anti-inflammatory properties through suppression of TLR4/ NF-κB pathways [34]. AG also has shown anticancer activity via inhibitory action on TLR4/NF-κB system in some cancerous cells and tumors [19,35]. Various studies reported the inhibitory effects of AG against NF-κB in different models of inflammation such as lipopolysaccharide-induced acute lung injury and influenza A virus-induced inflammation in mice [36,37]. A recent investigation reported the protective effect of AG against ischemic brain damages via NF-κB inhibiting and subsequently decreasing production of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and prostaglandin E2 [38]. In the study of Chen et al., AG exhibited suppressive activity against NF-κB via JNK-AKT-p65 signaling pathway in TNF-α-stimulated vascular smooth muscle cells [39]. Li et al. also showed that AG can hinder production of inflammatory cytokines such as TNF-α, IL-6, and IL-1β in lipopolysaccharide-stimulated RAW264.7 cells via preventing NF-κB/MAPK cascade [40].

Conclusion
In summary, these data demonstrated that AG effectively ameliorated the cardiotoxicity of DOX-and ATO in vitro. The cardiomyocyte protective activities of AG could be attributed mainly to the reduction in TLR4 expression and total oxidant capacity and elevation in total antioxidant capacity. Though, additional in vivo investigations are suggested to delineate the clinical value of AG in human cardiotoxicity.