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Realgar transforming solution as a novel arsenic agent with a lower risk of cardiotoxicity
Journal of Pharmacological Sciences 140 (2019) 162e170
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Realgar transforming solution as a novel arsenic agent with a lower risk of cardiotoxicity Yang Hai a, Peng Song b, Xin Wang a, Longhe Zhao a, Qinjian Xie c, Jianyin Li a, Yang Li a, Hongyu Li a, b, * a b c
School of Pharmacy, Lanzhou University, Lanzhou, 730020, China Institute of Microbiology, School of Life Sciences, Lanzhou University, Lanzhou, 730000, China Gansu Corps Hospital of CAPF, Gansu, 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2019 Received in revised form 2 June 2019 Accepted 4 June 2019 Available online 15 June 2019
QTc prolongation has been observed during arsenic trioxide and realgar's clinical use, and become a huge obstacle for the application. Our lab has obtained the soluble arsenic from realgar named realgar transforming solution or RTS. In this study, we first evaluated the cytotoxicity on NB4 cell and found that RTS could remarkably inhibit proliferation of NB4 than arsenic trioxide. Then we figured out the QTc prolongation of RTS treatment contrasted with arsenic trioxide; results revealed that arsenic trioxide prolonged corrected QTc of mice by 20.1% and showed 1.9-fold higher cytotoxicity on H9c2 cell than RTS. On the contrary, there could not find any QTc prolongation of mice in RTS treatment. Also, arsenic trioxide elevated the intercellular calcium accumulation of H9c2 cell by 2.02-fold v.s control and RTS. HE staining and Masson's trichrome staining had shown that there was no injured section after RTS treatments. IK1 currents of rat ventricular cardiomyocytes were diminished by 45.0% after treating with arsenic trioxide while RTS showed no significance than the control group. The results above indicated that RTS could serve as an alternative arsenic agent on leukemia and had a lower risk of cardiotoxicity. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Arsenic Cardiotoxicity RTS ATO Realgar
1. Introduction Nowadays, arsenic trioxide (As2O3, ATO) as a first-line drug not only approves for the treatment of acute promyeloid leukemia (APL) but also shows excellent activity in other malignancies, such as lung and breast cancers.1e3 Unfortunately, severe cardiac side effect occurred with ATO treatment. The main clinical manifestations were palpitation, QT interval (QTc) prolongation, S-T section down, PR interval prolonged, complete atrioventricular block, torsade de points (TdP),4e6 even sudden death has been observed.7,8 Nowadays, with the developing feedbacks of ATO clinical research, more and more data show the cardiac side effects of ATO.9e11 Specifically, 63% of APL patients under ATO treatment has found QTc prolongation.12 Chinese researcher Wu observed 95 patients who were treated with ATO for the first time acquired
* Corresponding author. School of Pharmacy, Lanzhou University, Lanzhou, 730020, China. E-mail addresses: [email protected] (Y. Hai), [email protected] (H. Li). Peer review under responsibility of Japanese Pharmacological Society.
cardiac symptoms with the incidence of 51.6%.13 In this context, it is urgent to improve the existing arsenic agents or find new ones which can not only guarantee the efficacy but reduce the cardiac side effects. Realgar is one of Chinese traditional medicine (TCM). In recent years, studies have shown that realgar has an anti-tumor effect and has been successfully used in the treatment of acute and chronic myelogenous leukemia,14 which makes realgar become one of the research hotspots,15e17 and realgar began to be re-recognize the medicinal value for its pharmaceutical value.18e20 However, insolubility is an obstacle of realgar application, and clinical studies have found that realgar also presents that 35% patients had achieved QTc prolongation in the treatment of APL.21 It can be seen that realgar has the same problem with ATO. Therefore, although both ATO and realgar can treat leukemia effectively, it is still compelled us to find new arsenic agents with the high anti-cancer ability and negligible cardiotoxic effect. In the past few years, our laboratory has conducted crossdisciplinary integration of biological metallurgy technology and mineral medicine processing and invented a new mild realgar
https://doi.org/10.1016/j.jphs.2019.06.003 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Hai et al. / Journal of Pharmacological Sciences 140 (2019) 162e170
biological method for toxicity reduction, which successfully achieved realgar leaching and dissolution under moderate conditions. This processing technology is utilized the catalytic effect of certain microorganisms to dissolve the metal sulfides in acid water. We successfully obtain arsenic extraction, realgar transforming solution (RTS), from realgar by using Acidithiobacillus ferrooxidans BY-3 (At. f BY-3).22 Previous studies in our lab showed RTS displayed more significant arsenic accumulation at the tumor site of mice which were transplanted by H22 cells than in healthy tissues22 and significant shrunken the volume in S180 solid tumor-burdened mice.23 Also, RTS showed slight cardiac toxicity in wild type Caenorhabditis elegans.24 These results hinted us that RTS might have higher anti-tumor activity and maybe has a low risk of toxicity. It is reported that the underlying mechanisms of QTc prolongation caused by ATO mainly due to myocardial apoptosis, oxidative stress, calcium overload, and changes of ion channel permeability.25e28In this study, we aim to evaluate whether RTS will induce the QTc prolongation, and the underlying mechanisms of toxicity reduction by detecting ECG recording, cell apoptosis, or ion channels balance.
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sulfuric acid. Biotransforming realgar was carried out in 250 mL flasks, each with 100 mL of iron-free 9 K medium (contained 1.0g Fe2þ/L) containing 1.0 g of realgar powder with an initial pH of 1.8. Each flask was inoculated at 150 rpm, 30 C with A. ferrooxidans suspension at 10% (v/v) for the pure culture. After 25 days of incubation, 10,000 rpm for 10 min harvested the original RTS supernatant and adjusted to pH 7.0 with NaOH and Na2EDTA. After filtered by a 0.22 mm membrane, and the arsenic concentration of RTS was determined by ICP-AES as 708.85 mg/mL. 2.4. Electrocardiogram (ECG) recordings Three-channel ECG was collected on anesthetized mice treated with sodium pentobarbitone. Before and after injecting intravenously with RTS and ATO at 1.6 mg/kg and the same volume of saline was given under the same experimental condition, the ECG of 10 mice per group were recorded during drug infusion for 2 h, and the heart-rate QTc was calculated with the Bazett formula: QTc ¼ QT/(RR)1/2. 2.5. Cell culture and MTT assay
2. Material and methods 2.1. Materials Realgar was obtained from Shimen County, Hunan Province, China, and purified through the traditional mean. Thiazolyl tetrazolium (MTT) and Hoechst 33258 staining fluid were purchased from Solarbio Science & Technology. FITC-Annexin V/PI was purchased from BD Biosciences. RPMI 1640 and DMEM culture medium was purchased from Limited Liability Corporation of Sijiqing Bioengineering Material in Hangzhou. ATO for injection was obtained from the Yida Medicinal Ltd. (Harbin, China). GSH, T-SOD, MDA, LDH, CK, CRP assay kits were purchased from Nanjing Jianheng Bioengineering Institute. Inward rectifier potassium current (IK1) were detected by patch clamp amplifier (Multi Clamp 700B, Axon CNS). Fluo-3 AM probe (5 mM) was purchased from Cayman Chemical (USA). Amplite™ Fluorometric Calcium Quantitation Kit was obtained from AAT Bioquest (CA). ATO for injection was purchased from the Yida Medicinal Ltd. (Harbin, China), and the arsenic concentration was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES; Jobin-Yvon Ultimate 2R).
H9c2 cell is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue. The NB-4 cell was derived from the marrow of a patient with APL in the second relapse. H9c2 and NB4 cell lines were purchased from the Institute of Cancer Research of Gansu province in China. H9c2 cells were cultured in DMEM medium, and NB4 cells were cultured in RPMI-1640. All the cells were supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin-streptomycin in an incubator with humidified 5% CO2 atmosphere at 37 C. The two cell lines were grown for 15e18 days then seeded into 96 well dishes at 5 104/mL cells per well and incubated with a range of arsenic concentration of RTS in 24 h. 20 mL MTT was added into each well to make the final MTT concentration of 0.5 mg/mL. After 4 h incubation, 150 mL DMSO was added and absorbance measured by the microplate reader at OD 570 nm. IC50 was calculated by SPSS v.22.0 with a significance level of 0.05 (P ¼ 0.05). The significance levels were determined by oneway ANOVA. Multiple comparisons were carried using Turkey's post-hoc test. 2.6. Apoptosis analysis of H9c2 cell
All the animal experiments were approved by the Animal Care and Use Committee of Lanzhou University. Adult male and female KM mice were obtained from the Laboratory Animal Center of Lanzhou University (Lanzhou, China). Adult male Wistar rats were obtained from Henan University (Kaifeng, China). The mice were housed ten per cage, the rats were housed two to three per cage, and acclimatized to the testing environment for at least 3 days before experiments. Arsenic concentration: L-RTS (0.8 mg/kg), MRTS (1.6 mg/kg), H-RTS (3.2 mg/kg), ATO (1.6 mg kg1).
For the Hoechst 33258 staining, the cells were seeded on coverslips then incubated in the RTS under the same conditions described above, and also treated with 1 mg/mL ATO. Cells were harvested after 24 h, then fixed cells in 4% paraformaldehyde for 30 min, PBS washed 3 times and stained 3e5 min with Hoechst 33258 and washed 2e3 times by PBS. The cells were visualized and photographed under a fluorescence microscope. For FCM examination, cells were harvested after treating with RTS or ATO for 24 h and washed with ice-cold PBS and resuspended in the 1 binding buffer containing AV/PI. After incubated for 15 min at room temperature in the dark, the samples were analyzed by flow cytometer FACS Verse.
2.3. Preparation for RTS
2.7. Western blotting analysis
RTS was obtained following the process described previously.22,23,28 In brief, realgar was ground into powder in water first to remove the impurity and avoid oxidation. Then precipitate for 24 h and transferred the powder into a plate to dry at room temperature. A. ferrooxidans BY3 (CCTCC-M 204057) cultured in 9 K medium: 3 g of (NH4)2SO4, 0.1 g of KCl, 0.5 g of K2HPO4, 0.5 g of MgSO4$7 H2O, 0.01 g of Ca(NO3)2 per liter; pH 1.8, adjusted with
Cells were lysed on ice in RIPA buffer and 1 mM PMSF for 30 min. The protein concentration of each sample was measured by a BCA kit and adjusted to the lysis buffer and 5 sample buffer. Samples were boiled at 95 C for 10 min and kept at 37 C, and loaded equally in SDS-PAGE. The protein was electrophoretically transferred to a PVDF membrane (0.22 mm) at 4 C for 1 h. The membrane was blocked with 5% low-fat milk for 2 h, incubated
2.2. Animals
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overnight with rabbit anti-caspase-3 antibody and b-actin at 4 C, after that, washed with TBST three times. The anti-rabbit secondary antibody was added and probed at room temperature for 2 h, then washed with TBST. Immunoblotting was evaluated after chemiluminescence development. The band intensity was quantified with Image J software. 2.8. Plasma and cardiac tissue index detection Mice were treated with RTS and ATO in 1.6 mg/kg arsenic concentration by oral administration consecutively for 4 weeks, CRP, CK-MB, and LDH of mice plasma were detected followed with assay kits procedure, and oxidative stress index includes T-SOD, GSH-PX, and MDA in cardiac tissue was also tested followed with assay kits procedure. 2.9. Histological examination Mice were treated with RTS and ATO by oral administration consecutively for 4 weeks. Cardio tissues were quickly dissected and immersed fixed in 10% neutral buffered formalin, and processed for embedding in paraffin after fixing for 24 h. Sections of 4e5 mm were stained with hematoxylin and eosin or Masson's trichrome staining to detect the histopathological changes and fibrotic areas under the Olympus inverted fluorescence microscope BX 53 (Olympus; USA). The fibrous quantification was quantified with Image J software. 2.10. Intracellular calcium accumulation detection H9c2 cell was planted in 6-well plate with coverslips. Treated with 1 mg/mL RTS and ATO for 24 h, then loaded 5 mM Fluo-3 AM to detect intracellular Ca2þ imaging. It was performed by a twophoton laser confocal scanning microscope with 488 nm beam for excitation from an argon ion laser and 530 nm beam for emission. Calcium concentration also detected by Amplite™ Fluorimetric Calcium Quantitation Kit. The fluorescence intensity was quantified with Image J software.
3. Results 3.1. RTS inhibited proliferation of leukemia cells NB4 is a typical t (15; 17) chromosomal translocation acute promyeloid leukemia cells.30 We incubated NB4 cell in the different arsenic concentration of RTS for 6 h, 12 h, 24 h, and the results demonstrated that RTS inhibited the proliferation of NB4 cell in a dose and time-dependent manner. As shown in Fig. 1, RTS caused 50% cell death in NB4 cells only in 0.46 mg/mL, which was much lower than ATO after treatment for 24 h. 3.2. ECG recording ECG recording showed the representative traces of ECG of the three groups were presented in Fig. 2, the data clearly showed that ATO prolonged the QTc from 67.02 ± 8.99 ms to 80.46 ± 16.01 ms (P < 0.05), while it was 62.907 ± 5.05 ms and 62.904 ± 6.49 ms before and after treating with RTS (Fig. 2). 3.3. Physiological, biochemical indexes and histological alterations It was confirmed that ATO treatment could induce cardiac peroxidative alterations and histopathological alterations.27,31 In the present study, after consecutively treating with RTS for 4 weeks, mice weight and organ coefficient maintained at a constant level. Data showed that ATO elevated the level of CRP and CK in plasma than RTS, as well as the LDH in plasma and T-SOD in cardiac tissue (Fig. 3A, C). There were no significant pathological alterations observed in the cardiac tissue of the control group or RTS treatments (Fig. 3D). In ATO treatment, the myocardial fibers showed mild interstitial oedema. Besides, Masson's trichrome staining analysis demonstrated a collagenous fibrous scar, which can lead to myocardial fibrosis treated with ATO contrast to control and RTS animals (Fig. 3E). We also found that collagenous structures were
2.11. Patch-clamp recording Isolation of adult rat ventricular cardiomyocytes according to the study procedure.29 According to the method described previously,29 the rat ventricular cardiomyocytes were laid in a recording chamber placed on an inverted microscope. Borosilicate glass electrodes were filled with pipette solution to obtain the tip resistance of 2e4 MU. Membrane currents were recorded in the voltage clamp mode by an Axopatch-200 B amplifier. The signals were low-pass filtered at 1 kHz; command pulses were controlled by the pCLAMP 10.0 software. Adjusting the sophisticated electricity, and the whole-cell was established when ruptured the membrane by gentle suction after giga-seal. Stimulated the cells and recorded under current clamp modes. The measured currents were normalized by whole-cell capacitance and presented as the current density (pA/pF). 2.12. Statistical analysis All the tests were performed with the SPSS v. 22.0 with a significance level of 0.05 (P ¼ 0.05). The significance levels were determined by one-way ANOVA. Multiple comparisons were carried using Turkey's post-hoc test.
Fig. 1. Cytotoxicity of RTS on NB4. Cells were treated with RTS on various arsenic concentrations for 24 h. ATO on indicated arsenic concentrations was also analyzed. The results were expressed as mean ± S.D. (n ¼ 3).
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Fig. 2. ECG recording of KM mice under different arsenic treatments. (A) ECG determination of control, RTS and ATO treatment after 10, 20, 30, 60, 90,120 min of KM mice (iv.). (B) Relative levels of corrected QTc after 2 h, QT ¼ QT/(RR) 1/2. (C) ECG recording data of RTS and ATO treatment. Data are expressed as mean ± S.D. (n ¼ 10) and analyzed by one-way ANOVA with SPSS version 22. *P < 0.05, **P < 0.01 v.s control; #P < 0.05, ##P < 0.01 vs ATO.
consisted of haphazardly arranged, and there were a lot of fine collagen fibers in ATO treatment, whereas the normal had a parallel arrangement of collagen fibers. This result was consistent with the report of ATO cardiotoxicity.32
treatment (Fig. 5C), consistently with the result from assay kit (Fig. 5D). On the contrary, RTS did not affect calcium dyshomeostasis. 4. Discussion
3.4. Apoptosis of RTS on H9c2 H9c2 cell is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue. In this experiment, both RTS and ATO demonstrated an arsenic-dose-dependent increase in cytotoxicity. However, ATO had shown higher toxicity as compared to RTS (P < 0.05) (Fig. 4A) in the same arsenic concentration range from 0.25 mg/mL to 2 mg/mL. As shown in Fig. 4B, C, RTS (1 mg/mL) did not significantly induce early apoptotic H9c2 cells after 24 h by the observation of Hoechst 33258 staining and FCM, and the cleaved-caspase-3 activity was not activated considerably under the RTS treatment by the western blot analysis. However, ATO induced apoptosis remarkably increase the cleaved-caspase-3 expression (Fig. 4C). 3.5. Effects of RTS on IK1 currents and intracellular calcium accumulation IK1 current plays a vital role in the maintenance of cardiac resting membrane potential (RMP), and the current density of IK1 could be down-regulated in the presence of ATO.29 Ca2þ dyshomeostasis resulting from ROS-induced myocardial injury by ATO.27 According to Fig. 5, ATO could significantly decrease the current density of IK1 by 45.0% as compared to the control group (Fig. 5A, P < 0.05). There is no difference in IK1 density between RTS treatment and control group (Fig. 5B, left panel). Data indicated that there was a significant increase of Ca2þ fluorescence intensity after treated with ATO treatment than control (P < 0.05) and RTS
Arsenic agents, widely known as ATO and realgar, have been clinically used as anticarcinogens. Treatment of APL with ATO represents the first model of targeted therapy.33 However, the clinical use of either ATO or realgar has been restricted by its cardiac toxicity such as tachycardia, especially the QTc prolongation.21,34 These disadvantages triggered us to explore other novel arsenic agents, which have better anti-cancer efficacy and negligible cardiotoxic effect. Previous studies had already demonstrated that RTS had a remarkable anti-tumor ability.35e39 In the present study, we proved the more compelling inhibition ratio of NB4 than ATO (Fig. 1). Besides, RTS had shown a relative safety than ATO in wild type C. elegans.24 Here, MTT assay showed that ATO demonstrated a higher inhibition ratio than RTS in H9c2 cell, which hinted that ATO had cytotoxicity on cardiomyocytes (Fig. 4A). It was documented that ATO could induce the QTc prolongation during clinical application leading to severe cardiac side effect.29 In the present experiments, ECG recording showed that RTS did not prolong the QTc while QTc was prolonged for 20.1% by ATO (Fig. 2). Apoptosis-induction of H9c2 has been suggested as one of the major mechanisms involved in the cardiotoxicity of ATO26,38 and the activation of caspase-3 alone is sufficient to cause apoptosis in cardiomyocytes.39 Results showed that ATO could up-regulate the caspase-3 expression (Fig. 4D). Also, Hoechst 33258 staining showed ATO resulted in cell membrane shrinkage, apoptotic body's formation, and cell necrosis (Fig. 4B, C). However, RTS did not elevate caspase-3 expression, and neither could not result in cell membrane shrinkage, and apoptotic
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Fig. 3. Physiological biochemical indexes of KM mice treated with RTS and ATO. (A) Mice weight and organ coefficient. (B) Mice plasma CRP, CK, and LDH alterations treated. (C) Oxidative stress indexes' alterations of T-SOD, GSH-PX, and MDA in cardiac tissue of mice. (D) HE staining of cardiac tissues treated with a different arsenic concentration of RTS. (E) Masson's trichrome staining of cardiac tissues treated with a different arsenic concentration of RTS. Data are expressed as mean ± S.D. (n ¼ 10) and analyzed by one-way ANOVA with SPSS version 22. *P < 0.05, **P < 0.01 v.s control; #P < 0.05, ##P < 0.01 v.s ATO.
body's formation showed no significant effect of apoptosis in H9c2 cell. Researchers also had found that ATO could increase LDH release, CK and CRP concentration in plasma, and elevated CRP and CK level, which predicted future adverse cardiac events induced by ATO.40,41 Increased activation of the CRP, CK, and LDH by ATO in this study corroborated these earlier findings. However, RTS did not increase
CRP, CK, or LDH levels (Fig. 3B). These results implied that RTS might have the ability to restrict the extent of the injuries of cardiomyocytes, thus reducing the release of these cardiac markers from the cardiomyocytes. Also, RTS hardly induced oxidative damage under these doses (0.8, 1.6, 3.2 mg/kg) during 4 weeks (Fig. 3C) nor the histopathological changes (Fig. 3D). Masson's trichrome staining demonstrated that there produced a collagenous
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Fig. 4. Cytotoxicity effect of RTS on H9c2 cell. (A) MTT assay on H9c2 cell growth at various concentrations of RTS or ATO. (B) Fluorescent microscopic images of H9c2 cell stained with Hoechst 33258 blue indicator (400 ). (C) Dot plot of H9c2 cell stained with FITC-Annexin V/PI. (D) Caspase-3 protein expressions on H9c2 cell treated with different arsenic concentration RTS and ATO, full images are in supplement Fig. 1. Data are expressed as mean ± S.D. (n ¼ 3) and analyzed by one-way ANOVA with SPSS version 22. *P < 0.05, **P < 0.01 vs control, #P < 0.05, ##P < 0.01 v.s ATO.
Fig. 5. Effects of RTS on IK1 and intercellular calcium concentration. (A) Representative current traces recorded from cardiomyocytes isolated from rats and treated with RTS or ATO. (B) IeV curves of IK1, obtained the current densities (pA/pF) at amplitude of 285 ms for each voltage (n ¼ 3, **P < 0.01). (C) Intercellular calcium concentration of H9c2-treated with RTS or ATO by loading the Fluo-3 AM probe observed under laser confocal microscope. The data were quantified by Image J software (bottom panel) (D) Intercellular calcium concentration of H9c2-treated with RTS or ATO by calcium quantitation kit to quantify the intercellular calcium concentration. Data are expressed as mean ± S.D. and analyzed by one-way ANOVA with SPSS version 22. *P < 0.05, **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs ATO.
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fibrous scar which can lead to myocardial fibrosis in ATO treatment, and collagenous structures were consisted of haphazardly arranged, and there were a lot of fine collagen fibers (Fig. 3E). These results corroborate the findings of the previous work in ATO effect on cardiotoxicity.32 Calcium is an essential second messenger whose destruction activates pathways leading to apoptosis.42 ATO-induced
accumulation of intercellular calcium in H9c2 cell by 2.02-fold vs. control was found in the present study (Fig. 5C), this result was consistent with the findings of the previous work.26 On the contrary, RTS showed a slightly increasing tendency (Fig. 5C, P > 0.05). Meanwhile, assay kit was used to detect quantification of calcium in H9c2 cell, and the results were in constant with fluorescence intensity, as shown in Fig. 5D.
Fig. 6. Schemative representation of a potential mechanism of RTS.
Y. Hai et al. / Journal of Pharmacological Sciences 140 (2019) 162e170
Kþ current (IK) plays an important role in controlling the repolarization velocity. Function and density of the cardiac Kþ channels may be responsible for the pathophysiological outcomes of various cardiac diseases.43 IK1 is a crucial regulator for the repolarization and also the maintenance of RMP. It has been reported that downregulation of IK1 provokes membrane depolarization.29 ATO resulting in the QTc prolongation of ventricular cardiomyocytes of rats is due to intracellular calcium overloaded27,44 and IK1 downregulation.29 To this regard, this study examined the whole-cell patch clamp of selecting ventricular cardiomyocytes in the present study. IK1 currents treated with RTS decreased a little but not significantly (P > 0.05), inversely, it was reduced by 45.0% v.s control after treated with ATO (Fig. 5A, B, P < 0.05). IK1 currents maintaining might be one of the critical mechanisms remaining QTc interval stable of RTS. Taken all data together, we suspected that the underlying mechanism of low cardiotoxicity of RTS might relate to low cytotoxicity or maintain iron homeostasis. The results above inspired that RTS could be a potential arsenic agent with mild cardiotoxicity. Therefore, we will further investigate the effect of RTS on potassium and calcium channels on ventricular myocytes in subsequent experiments. The findings of this study, along with the probable mode of action of RTS, have been illustrated in Fig. 6. In conclusion, RTS maybe can serve as an alternative arsenic agent on leukemia with mild cardiac toxicity.
Conflict of interest No potential conflict of interest was reported by the authors.
Acknowledgments Thanks are due to Dr. Zhizeng Wang of Henan University for assistance with the experiments. This work was supported by the National Natural Science Foundation of China [No. 81403145, 81560715, 51501080, 81803779]; The Sub-Project of National Science and Technology Major Projects for “Major New Drugs Innovation and Development” [No. 2015ZX09501-004-003-008]; The Fundamental Research Funds for the Central Universities in China [No. lzujbky-2018-136, lzujbky-2018-40, lzujbky-2017-206].
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NB4, a maturation inducible cell line with t(15;17) marker isolated from a human acute promyelocytic leukemia (M3). Blood. 1991;77(5):1080e1086. 31. Mathews VV, Paul MV, Abhilash M, et al. Myocardial toxicity of acute promyelocytic leukaemia drug-arsenic trioxide. Eur Rev Med Pharmacol Sci. 2013;17(suppl. 1):34e38. 32. Chu W, Li C, Qu X, et al. Arsenic-induced interstitial myocardial fibrosis reveals a new insight into drug-induced long QT syndrome. Cardiovasc Res. 2012;96(1):90e98. 33. Nitto T, Sawaki K. Molecular mechanisms of the antileukemia activities of retinoid and arsenic. J Pharmacol Sci. 2014;126(3):179e185. 34. Chen Y, Xiang Y, Chang X, et al. Electrocardiogram (ecg) QTC interphase and ST -t of Compound Huang Dai tablets in patients with acute early young granulocyte leukemia. J Clin Hematol China. 2012;25(9). 35. Song P, Chen P, Wang D, et al. Realgar transforming solution displays anticancer potential against human hepatocellular carcinoma HepG2 cells by inducing ROS. Int J Oncol. 2017;50(1e2):660. 36. Wang X, Chen B, Zhao L, et al. Autophagy enhanced antitumor effect in K562 and K562/ADM cells using realgar transforming solution. Biomed Pharmacother. 2017;98(99):252. 37. Hai Y, Li J, Wang X, et al. Apoptosis mechanism of Realgar bioleaching solution on HL-60 cell line via mitochondrial membrane potential. Pharmaceut Biotechnol. 2018;25(04):283e287. 38. Raghu KG, Cherian OL. Characterization of cytotoxicity induced by arsenic trioxide (a potent anti-APL drug) in rat cardiac myocytes. J Trace Elem Med Biol. 2009;23(1):61e68.
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39. Clerk A, Cole SM, Cullingford TE, et al. Regulation of cardiac myocyte cell death. Pharmacol Ther. 2003;97(3):223e261. 40. Dasgupta A, Wahed A. Chapter 8 - cardiac markers. In: Dasgupta A, Wahed A, eds. Clinical chemistry, immunology and laboratory quality control. San Diego: Elsevier; 2014:127e144. 41. Hemmati AA, Olapour S, Varzi HN, et al. Ellagic acid protects against arsenic trioxide-induced cardiotoxicity in rat. Hum Exp Toxicol. 2018;37(4):412e419.
42. Chen X, Zhang X, Kubo H, et al. Ca2þ influxeinduced sarcoplasmic reticulum Ca2þ overload causes mitochondrial-dependent apoptosis in ventricular myocytes. Circ Res. 2005;97(10):1009e1017. mez R, Valenzuela C, Delpo n E. Pharmacology of 43. Tamargo J, Caballero R, Go cardiac potassium channels. Cardiovasc Res. 2004;62(1):9e33. 44. Ficker E, Kuryshev YA, Dennis AT, et al. Mechanisms of arsenic-induced prolongation of cardiac repolarization. Mol Pharmacol. 2004;66(1):33e44.
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Full Paper
Realgar transforming solution as a novel arsenic agent with a lower risk of cardiotoxicity Yang Hai a, Peng Song b, Xin Wang a, Longhe Zhao a, Qinjian Xie c, Jianyin Li a, Yang Li a, Hongyu Li a, b, * a b c
School of Pharmacy, Lanzhou University, Lanzhou, 730020, China Institute of Microbiology, School of Life Sciences, Lanzhou University, Lanzhou, 730000, China Gansu Corps Hospital of CAPF, Gansu, 730000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 March 2019 Received in revised form 2 June 2019 Accepted 4 June 2019 Available online 15 June 2019
QTc prolongation has been observed during arsenic trioxide and realgar's clinical use, and become a huge obstacle for the application. Our lab has obtained the soluble arsenic from realgar named realgar transforming solution or RTS. In this study, we first evaluated the cytotoxicity on NB4 cell and found that RTS could remarkably inhibit proliferation of NB4 than arsenic trioxide. Then we figured out the QTc prolongation of RTS treatment contrasted with arsenic trioxide; results revealed that arsenic trioxide prolonged corrected QTc of mice by 20.1% and showed 1.9-fold higher cytotoxicity on H9c2 cell than RTS. On the contrary, there could not find any QTc prolongation of mice in RTS treatment. Also, arsenic trioxide elevated the intercellular calcium accumulation of H9c2 cell by 2.02-fold v.s control and RTS. HE staining and Masson's trichrome staining had shown that there was no injured section after RTS treatments. IK1 currents of rat ventricular cardiomyocytes were diminished by 45.0% after treating with arsenic trioxide while RTS showed no significance than the control group. The results above indicated that RTS could serve as an alternative arsenic agent on leukemia and had a lower risk of cardiotoxicity. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Arsenic Cardiotoxicity RTS ATO Realgar
1. Introduction Nowadays, arsenic trioxide (As2O3, ATO) as a first-line drug not only approves for the treatment of acute promyeloid leukemia (APL) but also shows excellent activity in other malignancies, such as lung and breast cancers.1e3 Unfortunately, severe cardiac side effect occurred with ATO treatment. The main clinical manifestations were palpitation, QT interval (QTc) prolongation, S-T section down, PR interval prolonged, complete atrioventricular block, torsade de points (TdP),4e6 even sudden death has been observed.7,8 Nowadays, with the developing feedbacks of ATO clinical research, more and more data show the cardiac side effects of ATO.9e11 Specifically, 63% of APL patients under ATO treatment has found QTc prolongation.12 Chinese researcher Wu observed 95 patients who were treated with ATO for the first time acquired
* Corresponding author. School of Pharmacy, Lanzhou University, Lanzhou, 730020, China. E-mail addresses: [email protected] (Y. Hai), [email protected] (H. Li). Peer review under responsibility of Japanese Pharmacological Society.
cardiac symptoms with the incidence of 51.6%.13 In this context, it is urgent to improve the existing arsenic agents or find new ones which can not only guarantee the efficacy but reduce the cardiac side effects. Realgar is one of Chinese traditional medicine (TCM). In recent years, studies have shown that realgar has an anti-tumor effect and has been successfully used in the treatment of acute and chronic myelogenous leukemia,14 which makes realgar become one of the research hotspots,15e17 and realgar began to be re-recognize the medicinal value for its pharmaceutical value.18e20 However, insolubility is an obstacle of realgar application, and clinical studies have found that realgar also presents that 35% patients had achieved QTc prolongation in the treatment of APL.21 It can be seen that realgar has the same problem with ATO. Therefore, although both ATO and realgar can treat leukemia effectively, it is still compelled us to find new arsenic agents with the high anti-cancer ability and negligible cardiotoxic effect. In the past few years, our laboratory has conducted crossdisciplinary integration of biological metallurgy technology and mineral medicine processing and invented a new mild realgar
https://doi.org/10.1016/j.jphs.2019.06.003 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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biological method for toxicity reduction, which successfully achieved realgar leaching and dissolution under moderate conditions. This processing technology is utilized the catalytic effect of certain microorganisms to dissolve the metal sulfides in acid water. We successfully obtain arsenic extraction, realgar transforming solution (RTS), from realgar by using Acidithiobacillus ferrooxidans BY-3 (At. f BY-3).22 Previous studies in our lab showed RTS displayed more significant arsenic accumulation at the tumor site of mice which were transplanted by H22 cells than in healthy tissues22 and significant shrunken the volume in S180 solid tumor-burdened mice.23 Also, RTS showed slight cardiac toxicity in wild type Caenorhabditis elegans.24 These results hinted us that RTS might have higher anti-tumor activity and maybe has a low risk of toxicity. It is reported that the underlying mechanisms of QTc prolongation caused by ATO mainly due to myocardial apoptosis, oxidative stress, calcium overload, and changes of ion channel permeability.25e28In this study, we aim to evaluate whether RTS will induce the QTc prolongation, and the underlying mechanisms of toxicity reduction by detecting ECG recording, cell apoptosis, or ion channels balance.
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sulfuric acid. Biotransforming realgar was carried out in 250 mL flasks, each with 100 mL of iron-free 9 K medium (contained 1.0g Fe2þ/L) containing 1.0 g of realgar powder with an initial pH of 1.8. Each flask was inoculated at 150 rpm, 30 C with A. ferrooxidans suspension at 10% (v/v) for the pure culture. After 25 days of incubation, 10,000 rpm for 10 min harvested the original RTS supernatant and adjusted to pH 7.0 with NaOH and Na2EDTA. After filtered by a 0.22 mm membrane, and the arsenic concentration of RTS was determined by ICP-AES as 708.85 mg/mL. 2.4. Electrocardiogram (ECG) recordings Three-channel ECG was collected on anesthetized mice treated with sodium pentobarbitone. Before and after injecting intravenously with RTS and ATO at 1.6 mg/kg and the same volume of saline was given under the same experimental condition, the ECG of 10 mice per group were recorded during drug infusion for 2 h, and the heart-rate QTc was calculated with the Bazett formula: QTc ¼ QT/(RR)1/2. 2.5. Cell culture and MTT assay
2. Material and methods 2.1. Materials Realgar was obtained from Shimen County, Hunan Province, China, and purified through the traditional mean. Thiazolyl tetrazolium (MTT) and Hoechst 33258 staining fluid were purchased from Solarbio Science & Technology. FITC-Annexin V/PI was purchased from BD Biosciences. RPMI 1640 and DMEM culture medium was purchased from Limited Liability Corporation of Sijiqing Bioengineering Material in Hangzhou. ATO for injection was obtained from the Yida Medicinal Ltd. (Harbin, China). GSH, T-SOD, MDA, LDH, CK, CRP assay kits were purchased from Nanjing Jianheng Bioengineering Institute. Inward rectifier potassium current (IK1) were detected by patch clamp amplifier (Multi Clamp 700B, Axon CNS). Fluo-3 AM probe (5 mM) was purchased from Cayman Chemical (USA). Amplite™ Fluorometric Calcium Quantitation Kit was obtained from AAT Bioquest (CA). ATO for injection was purchased from the Yida Medicinal Ltd. (Harbin, China), and the arsenic concentration was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES; Jobin-Yvon Ultimate 2R).
H9c2 cell is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue. The NB-4 cell was derived from the marrow of a patient with APL in the second relapse. H9c2 and NB4 cell lines were purchased from the Institute of Cancer Research of Gansu province in China. H9c2 cells were cultured in DMEM medium, and NB4 cells were cultured in RPMI-1640. All the cells were supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin-streptomycin in an incubator with humidified 5% CO2 atmosphere at 37 C. The two cell lines were grown for 15e18 days then seeded into 96 well dishes at 5 104/mL cells per well and incubated with a range of arsenic concentration of RTS in 24 h. 20 mL MTT was added into each well to make the final MTT concentration of 0.5 mg/mL. After 4 h incubation, 150 mL DMSO was added and absorbance measured by the microplate reader at OD 570 nm. IC50 was calculated by SPSS v.22.0 with a significance level of 0.05 (P ¼ 0.05). The significance levels were determined by oneway ANOVA. Multiple comparisons were carried using Turkey's post-hoc test. 2.6. Apoptosis analysis of H9c2 cell
All the animal experiments were approved by the Animal Care and Use Committee of Lanzhou University. Adult male and female KM mice were obtained from the Laboratory Animal Center of Lanzhou University (Lanzhou, China). Adult male Wistar rats were obtained from Henan University (Kaifeng, China). The mice were housed ten per cage, the rats were housed two to three per cage, and acclimatized to the testing environment for at least 3 days before experiments. Arsenic concentration: L-RTS (0.8 mg/kg), MRTS (1.6 mg/kg), H-RTS (3.2 mg/kg), ATO (1.6 mg kg1).
For the Hoechst 33258 staining, the cells were seeded on coverslips then incubated in the RTS under the same conditions described above, and also treated with 1 mg/mL ATO. Cells were harvested after 24 h, then fixed cells in 4% paraformaldehyde for 30 min, PBS washed 3 times and stained 3e5 min with Hoechst 33258 and washed 2e3 times by PBS. The cells were visualized and photographed under a fluorescence microscope. For FCM examination, cells were harvested after treating with RTS or ATO for 24 h and washed with ice-cold PBS and resuspended in the 1 binding buffer containing AV/PI. After incubated for 15 min at room temperature in the dark, the samples were analyzed by flow cytometer FACS Verse.
2.3. Preparation for RTS
2.7. Western blotting analysis
RTS was obtained following the process described previously.22,23,28 In brief, realgar was ground into powder in water first to remove the impurity and avoid oxidation. Then precipitate for 24 h and transferred the powder into a plate to dry at room temperature. A. ferrooxidans BY3 (CCTCC-M 204057) cultured in 9 K medium: 3 g of (NH4)2SO4, 0.1 g of KCl, 0.5 g of K2HPO4, 0.5 g of MgSO4$7 H2O, 0.01 g of Ca(NO3)2 per liter; pH 1.8, adjusted with
Cells were lysed on ice in RIPA buffer and 1 mM PMSF for 30 min. The protein concentration of each sample was measured by a BCA kit and adjusted to the lysis buffer and 5 sample buffer. Samples were boiled at 95 C for 10 min and kept at 37 C, and loaded equally in SDS-PAGE. The protein was electrophoretically transferred to a PVDF membrane (0.22 mm) at 4 C for 1 h. The membrane was blocked with 5% low-fat milk for 2 h, incubated
2.2. Animals
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overnight with rabbit anti-caspase-3 antibody and b-actin at 4 C, after that, washed with TBST three times. The anti-rabbit secondary antibody was added and probed at room temperature for 2 h, then washed with TBST. Immunoblotting was evaluated after chemiluminescence development. The band intensity was quantified with Image J software. 2.8. Plasma and cardiac tissue index detection Mice were treated with RTS and ATO in 1.6 mg/kg arsenic concentration by oral administration consecutively for 4 weeks, CRP, CK-MB, and LDH of mice plasma were detected followed with assay kits procedure, and oxidative stress index includes T-SOD, GSH-PX, and MDA in cardiac tissue was also tested followed with assay kits procedure. 2.9. Histological examination Mice were treated with RTS and ATO by oral administration consecutively for 4 weeks. Cardio tissues were quickly dissected and immersed fixed in 10% neutral buffered formalin, and processed for embedding in paraffin after fixing for 24 h. Sections of 4e5 mm were stained with hematoxylin and eosin or Masson's trichrome staining to detect the histopathological changes and fibrotic areas under the Olympus inverted fluorescence microscope BX 53 (Olympus; USA). The fibrous quantification was quantified with Image J software. 2.10. Intracellular calcium accumulation detection H9c2 cell was planted in 6-well plate with coverslips. Treated with 1 mg/mL RTS and ATO for 24 h, then loaded 5 mM Fluo-3 AM to detect intracellular Ca2þ imaging. It was performed by a twophoton laser confocal scanning microscope with 488 nm beam for excitation from an argon ion laser and 530 nm beam for emission. Calcium concentration also detected by Amplite™ Fluorimetric Calcium Quantitation Kit. The fluorescence intensity was quantified with Image J software.
3. Results 3.1. RTS inhibited proliferation of leukemia cells NB4 is a typical t (15; 17) chromosomal translocation acute promyeloid leukemia cells.30 We incubated NB4 cell in the different arsenic concentration of RTS for 6 h, 12 h, 24 h, and the results demonstrated that RTS inhibited the proliferation of NB4 cell in a dose and time-dependent manner. As shown in Fig. 1, RTS caused 50% cell death in NB4 cells only in 0.46 mg/mL, which was much lower than ATO after treatment for 24 h. 3.2. ECG recording ECG recording showed the representative traces of ECG of the three groups were presented in Fig. 2, the data clearly showed that ATO prolonged the QTc from 67.02 ± 8.99 ms to 80.46 ± 16.01 ms (P < 0.05), while it was 62.907 ± 5.05 ms and 62.904 ± 6.49 ms before and after treating with RTS (Fig. 2). 3.3. Physiological, biochemical indexes and histological alterations It was confirmed that ATO treatment could induce cardiac peroxidative alterations and histopathological alterations.27,31 In the present study, after consecutively treating with RTS for 4 weeks, mice weight and organ coefficient maintained at a constant level. Data showed that ATO elevated the level of CRP and CK in plasma than RTS, as well as the LDH in plasma and T-SOD in cardiac tissue (Fig. 3A, C). There were no significant pathological alterations observed in the cardiac tissue of the control group or RTS treatments (Fig. 3D). In ATO treatment, the myocardial fibers showed mild interstitial oedema. Besides, Masson's trichrome staining analysis demonstrated a collagenous fibrous scar, which can lead to myocardial fibrosis treated with ATO contrast to control and RTS animals (Fig. 3E). We also found that collagenous structures were
2.11. Patch-clamp recording Isolation of adult rat ventricular cardiomyocytes according to the study procedure.29 According to the method described previously,29 the rat ventricular cardiomyocytes were laid in a recording chamber placed on an inverted microscope. Borosilicate glass electrodes were filled with pipette solution to obtain the tip resistance of 2e4 MU. Membrane currents were recorded in the voltage clamp mode by an Axopatch-200 B amplifier. The signals were low-pass filtered at 1 kHz; command pulses were controlled by the pCLAMP 10.0 software. Adjusting the sophisticated electricity, and the whole-cell was established when ruptured the membrane by gentle suction after giga-seal. Stimulated the cells and recorded under current clamp modes. The measured currents were normalized by whole-cell capacitance and presented as the current density (pA/pF). 2.12. Statistical analysis All the tests were performed with the SPSS v. 22.0 with a significance level of 0.05 (P ¼ 0.05). The significance levels were determined by one-way ANOVA. Multiple comparisons were carried using Turkey's post-hoc test.
Fig. 1. Cytotoxicity of RTS on NB4. Cells were treated with RTS on various arsenic concentrations for 24 h. ATO on indicated arsenic concentrations was also analyzed. The results were expressed as mean ± S.D. (n ¼ 3).
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Fig. 2. ECG recording of KM mice under different arsenic treatments. (A) ECG determination of control, RTS and ATO treatment after 10, 20, 30, 60, 90,120 min of KM mice (iv.). (B) Relative levels of corrected QTc after 2 h, QT ¼ QT/(RR) 1/2. (C) ECG recording data of RTS and ATO treatment. Data are expressed as mean ± S.D. (n ¼ 10) and analyzed by one-way ANOVA with SPSS version 22. *P < 0.05, **P < 0.01 v.s control; #P < 0.05, ##P < 0.01 vs ATO.
consisted of haphazardly arranged, and there were a lot of fine collagen fibers in ATO treatment, whereas the normal had a parallel arrangement of collagen fibers. This result was consistent with the report of ATO cardiotoxicity.32
treatment (Fig. 5C), consistently with the result from assay kit (Fig. 5D). On the contrary, RTS did not affect calcium dyshomeostasis. 4. Discussion
3.4. Apoptosis of RTS on H9c2 H9c2 cell is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue. In this experiment, both RTS and ATO demonstrated an arsenic-dose-dependent increase in cytotoxicity. However, ATO had shown higher toxicity as compared to RTS (P < 0.05) (Fig. 4A) in the same arsenic concentration range from 0.25 mg/mL to 2 mg/mL. As shown in Fig. 4B, C, RTS (1 mg/mL) did not significantly induce early apoptotic H9c2 cells after 24 h by the observation of Hoechst 33258 staining and FCM, and the cleaved-caspase-3 activity was not activated considerably under the RTS treatment by the western blot analysis. However, ATO induced apoptosis remarkably increase the cleaved-caspase-3 expression (Fig. 4C). 3.5. Effects of RTS on IK1 currents and intracellular calcium accumulation IK1 current plays a vital role in the maintenance of cardiac resting membrane potential (RMP), and the current density of IK1 could be down-regulated in the presence of ATO.29 Ca2þ dyshomeostasis resulting from ROS-induced myocardial injury by ATO.27 According to Fig. 5, ATO could significantly decrease the current density of IK1 by 45.0% as compared to the control group (Fig. 5A, P < 0.05). There is no difference in IK1 density between RTS treatment and control group (Fig. 5B, left panel). Data indicated that there was a significant increase of Ca2þ fluorescence intensity after treated with ATO treatment than control (P < 0.05) and RTS
Arsenic agents, widely known as ATO and realgar, have been clinically used as anticarcinogens. Treatment of APL with ATO represents the first model of targeted therapy.33 However, the clinical use of either ATO or realgar has been restricted by its cardiac toxicity such as tachycardia, especially the QTc prolongation.21,34 These disadvantages triggered us to explore other novel arsenic agents, which have better anti-cancer efficacy and negligible cardiotoxic effect. Previous studies had already demonstrated that RTS had a remarkable anti-tumor ability.35e39 In the present study, we proved the more compelling inhibition ratio of NB4 than ATO (Fig. 1). Besides, RTS had shown a relative safety than ATO in wild type C. elegans.24 Here, MTT assay showed that ATO demonstrated a higher inhibition ratio than RTS in H9c2 cell, which hinted that ATO had cytotoxicity on cardiomyocytes (Fig. 4A). It was documented that ATO could induce the QTc prolongation during clinical application leading to severe cardiac side effect.29 In the present experiments, ECG recording showed that RTS did not prolong the QTc while QTc was prolonged for 20.1% by ATO (Fig. 2). Apoptosis-induction of H9c2 has been suggested as one of the major mechanisms involved in the cardiotoxicity of ATO26,38 and the activation of caspase-3 alone is sufficient to cause apoptosis in cardiomyocytes.39 Results showed that ATO could up-regulate the caspase-3 expression (Fig. 4D). Also, Hoechst 33258 staining showed ATO resulted in cell membrane shrinkage, apoptotic body's formation, and cell necrosis (Fig. 4B, C). However, RTS did not elevate caspase-3 expression, and neither could not result in cell membrane shrinkage, and apoptotic
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Fig. 3. Physiological biochemical indexes of KM mice treated with RTS and ATO. (A) Mice weight and organ coefficient. (B) Mice plasma CRP, CK, and LDH alterations treated. (C) Oxidative stress indexes' alterations of T-SOD, GSH-PX, and MDA in cardiac tissue of mice. (D) HE staining of cardiac tissues treated with a different arsenic concentration of RTS. (E) Masson's trichrome staining of cardiac tissues treated with a different arsenic concentration of RTS. Data are expressed as mean ± S.D. (n ¼ 10) and analyzed by one-way ANOVA with SPSS version 22. *P < 0.05, **P < 0.01 v.s control; #P < 0.05, ##P < 0.01 v.s ATO.
body's formation showed no significant effect of apoptosis in H9c2 cell. Researchers also had found that ATO could increase LDH release, CK and CRP concentration in plasma, and elevated CRP and CK level, which predicted future adverse cardiac events induced by ATO.40,41 Increased activation of the CRP, CK, and LDH by ATO in this study corroborated these earlier findings. However, RTS did not increase
CRP, CK, or LDH levels (Fig. 3B). These results implied that RTS might have the ability to restrict the extent of the injuries of cardiomyocytes, thus reducing the release of these cardiac markers from the cardiomyocytes. Also, RTS hardly induced oxidative damage under these doses (0.8, 1.6, 3.2 mg/kg) during 4 weeks (Fig. 3C) nor the histopathological changes (Fig. 3D). Masson's trichrome staining demonstrated that there produced a collagenous
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Fig. 4. Cytotoxicity effect of RTS on H9c2 cell. (A) MTT assay on H9c2 cell growth at various concentrations of RTS or ATO. (B) Fluorescent microscopic images of H9c2 cell stained with Hoechst 33258 blue indicator (400 ). (C) Dot plot of H9c2 cell stained with FITC-Annexin V/PI. (D) Caspase-3 protein expressions on H9c2 cell treated with different arsenic concentration RTS and ATO, full images are in supplement Fig. 1. Data are expressed as mean ± S.D. (n ¼ 3) and analyzed by one-way ANOVA with SPSS version 22. *P < 0.05, **P < 0.01 vs control, #P < 0.05, ##P < 0.01 v.s ATO.
Fig. 5. Effects of RTS on IK1 and intercellular calcium concentration. (A) Representative current traces recorded from cardiomyocytes isolated from rats and treated with RTS or ATO. (B) IeV curves of IK1, obtained the current densities (pA/pF) at amplitude of 285 ms for each voltage (n ¼ 3, **P < 0.01). (C) Intercellular calcium concentration of H9c2-treated with RTS or ATO by loading the Fluo-3 AM probe observed under laser confocal microscope. The data were quantified by Image J software (bottom panel) (D) Intercellular calcium concentration of H9c2-treated with RTS or ATO by calcium quantitation kit to quantify the intercellular calcium concentration. Data are expressed as mean ± S.D. and analyzed by one-way ANOVA with SPSS version 22. *P < 0.05, **P < 0.01 vs control; #P < 0.05, ##P < 0.01 vs ATO.
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fibrous scar which can lead to myocardial fibrosis in ATO treatment, and collagenous structures were consisted of haphazardly arranged, and there were a lot of fine collagen fibers (Fig. 3E). These results corroborate the findings of the previous work in ATO effect on cardiotoxicity.32 Calcium is an essential second messenger whose destruction activates pathways leading to apoptosis.42 ATO-induced
accumulation of intercellular calcium in H9c2 cell by 2.02-fold vs. control was found in the present study (Fig. 5C), this result was consistent with the findings of the previous work.26 On the contrary, RTS showed a slightly increasing tendency (Fig. 5C, P > 0.05). Meanwhile, assay kit was used to detect quantification of calcium in H9c2 cell, and the results were in constant with fluorescence intensity, as shown in Fig. 5D.
Fig. 6. Schemative representation of a potential mechanism of RTS.
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Kþ current (IK) plays an important role in controlling the repolarization velocity. Function and density of the cardiac Kþ channels may be responsible for the pathophysiological outcomes of various cardiac diseases.43 IK1 is a crucial regulator for the repolarization and also the maintenance of RMP. It has been reported that downregulation of IK1 provokes membrane depolarization.29 ATO resulting in the QTc prolongation of ventricular cardiomyocytes of rats is due to intracellular calcium overloaded27,44 and IK1 downregulation.29 To this regard, this study examined the whole-cell patch clamp of selecting ventricular cardiomyocytes in the present study. IK1 currents treated with RTS decreased a little but not significantly (P > 0.05), inversely, it was reduced by 45.0% v.s control after treated with ATO (Fig. 5A, B, P < 0.05). IK1 currents maintaining might be one of the critical mechanisms remaining QTc interval stable of RTS. Taken all data together, we suspected that the underlying mechanism of low cardiotoxicity of RTS might relate to low cytotoxicity or maintain iron homeostasis. The results above inspired that RTS could be a potential arsenic agent with mild cardiotoxicity. Therefore, we will further investigate the effect of RTS on potassium and calcium channels on ventricular myocytes in subsequent experiments. The findings of this study, along with the probable mode of action of RTS, have been illustrated in Fig. 6. In conclusion, RTS maybe can serve as an alternative arsenic agent on leukemia with mild cardiac toxicity.
Conflict of interest No potential conflict of interest was reported by the authors.
Acknowledgments Thanks are due to Dr. Zhizeng Wang of Henan University for assistance with the experiments. This work was supported by the National Natural Science Foundation of China [No. 81403145, 81560715, 51501080, 81803779]; The Sub-Project of National Science and Technology Major Projects for “Major New Drugs Innovation and Development” [No. 2015ZX09501-004-003-008]; The Fundamental Research Funds for the Central Universities in China [No. lzujbky-2018-136, lzujbky-2018-40, lzujbky-2017-206].
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