Clinical attempts to reduce the cardiotoxicity of arsenic trioxide (ATO) without compromising its anticancer activities remain to be an unresolved issue. In this study, we determined whether Sal B can protect against ATO-induced cardiac toxicity
Arsenic trioxide (ATO) was discovered more than 2000 years ago for treating diseases such as cancer, syphilis, and tuberculosis [
Salvianolic acid B (Sal B), the most abundant and bioactive member of the salvianolic acids in
In this work, we examined the effect of an ATO and Sal B combination treatment on the development of cardiotoxic side effects
Sal B (>98% purity) was purchased from Shanghai Winherb Medical S & T Development Co., Ltd., (Shanghai, China). ATO was purchased from Harbin Yi-da Pharmaceutical Ltd., (Harbin, China). HepG2 and HeLa cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V/propidium iodide (PI) apoptosis detection kit was obtained from Invitrogen (Eugene, USA). Kits for determining total creatine kinase (CK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity, and superoxide dismutase (SOD) activity were obtained from Jiancheng Bioengineering Institute (Nanjing, China). The primary antibodies against poly (ADP-ribose) polymerase (PARP), procaspase-3, Bcl-2, Bax, p-Akt, and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The
Male BALB/c mice weighing from 18 g to 20 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. The animals were housed under standard laboratory conditions (25°C ± 1°C temperature, 60% humidity, and 12 h photoperiod) and given free access to sterile food and water. All procedures were performed in accordance with the China Physiological Society “Guiding Principles in the Care and Use of Animals” and with the approval of the Laboratory Animal ethics Committee of Institute of Medicinal Plant Development, Peking Union Medical College.
A total of 60 mice were randomly assigned to four groups: control (10 mL/kg saline), ATO-treated (1 mg/kg ATO), ATO and Sal B combination-treated (2 mg/kg Sal B 1 h before ATO administration), and Sal B treated (2 mg/kg Sal B). All treatments were injected via the tail vein for 2 weeks.
Cardiac function was analyzed using echocardiography 24 h after drug treatment. The mice were anesthetized using from 1.5% to 2% isoflurane, and M-mode ultrasound images were obtained using a Vevo 770 micro-ultrasound system (VisualSonics, Toronto, ON, Canada).
After the completion of the echocardiography, blood samples were obtained from the inner canthus using a capillary tube under chloral hydrate anesthesia. The samples were centrifuged at 3000 ×g for 15 min within 1 h after collection. The activities of LDH, CK, AST, GSH-PX, CAT, and SOD in the plasma were determined using commercially available kits purchased from Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions.
The apex of the heart was fixed in 10% formalin, routinely processed, and then embedded in paraffin. Paraffin sections (3 mm) were placed on glass slides, stained with hematoxylin and eosin (HE), and then examined under a light microscope (CKX41, Olympus, Tokyo, Japan) by a pathologist blinded to the groups studied.
HepG2 and HeLa cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100
Cell viability was determined using MTT assay. Briefly, HepG2 or HeLa cells were seeded on 96-well plates at a density of 5 × 103 cells/well. The cells were treated for 48 h with ATO alone (4
The percentage of early apoptosis and necrosis was measured using an Annexin V-FITC/PI apoptosis kit for flow cytometry according to the manufacturer’s instructions (Invitrogen). After treatment, the cells were harvested, washed twice with cold phosphate-buffered saline (PBS), and then incubated with 5
After the designated treatment, heart tissue or HepG2 and HeLa cells were lysed for 30 min on ice with T-PER Tissue or Cell Protein Extraction Reagent (Pierce Chemical Co., Rockford, IL, USA) containing 1% phenylmethylsulfonylfluoride. A clear lysate was obtained through centrifugation at 12000 ×g for 15 min at 4°C. The supernate was collected. Protein concentration was determined through bicinchoninic acid assay. Equal amounts of lysates (10
Data from at least three independent experiments were expressed as means ± SD. Statistical comparisons between different groups were measured using one-way ANOVA followed by the Student-Newman-Keuls test. The level of significance was set at
We examined the effect of the combination treatment using an
Effects of Sal B and ATO on cardiac dysfunction. Mice were treated with saline or ATO with or without Sal B pretreatment. Echocardiography was performed after 2 weeks. (a) Representative M-mode echocardiograms were shown. (b) Changes in ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at diastole (LVIDd), and left ventricular internal diameter at systole (LVIDs) in the four groups. Values (
An overall view of the distribution of myocardial damage at the light microscopy level is shown in Figure
Effects of Sal B on ATO-induced myocardial injury
Next, we measured the levels of serum cardiac enzymes (CK, AST, and LDH), which are the biomarkers used for monitoring myocardial damage [
As shown in Figure
The levels of pAkt and Bcl-2 decreased in the ATO-treated group but not in the combination-treated group (Figures
Effects of Sal B treatment on protective protein expressions in heart tissue
Through MTT assay, we examined the viability of HepG2 and HeLa cell lines after treatment with ATO and Sal B alone (4 and 10
Effects of Sal B and ATO treatment on the cell viability of cancer cells. HepG2 and HeLa cells were treated with ATO (4
To analyze the effect of Sal B and ATO combination treatment on apoptosis, we performed annexin V/PI flow cytometry. The apoptotic fraction in the ATO and Sal B combination-treated group significantly increased compared with that in the ATO-group or Sal B-treated group in HepG2 and HeLa cells (Figures
Effects of Sal B on the apoptosis of cancer cells induced by ATO. Annexin V-FITC/PI flow-cytometric data from HepG2 (a) and HeLa (b) cells treated with ATO, Sal B, or both (4
We tested the involvement of caspase-3, the major effector of caspase, and PARP, the main substrate of caspases. Activation of caspase-3 was assessed by the decreased amount of procaspase-3 based on the western blot analysis [
Effects of Sal B and ATO treatment on apoptosis-related proteins
ATO attracted worldwide attention in the past decade because of its remarkable efficacy in acute APL [
Many reports revealed that Sal B is a promising compound for cardiovascular therapy and may act on multiple targets, leading to a concerted positive effect [
Another equally important issue is if Sal B could decrease ATO anticancer activity. Recent studies have demonstrated that Sal B exhibited significant growth arrest and apoptosis in many human cancer cell lines, such as head and neck as well as liver and HL-60, with no apparent toxicity to noncancerous cells [
ATO induced apoptosis in both cancer cells and cardiomyocytes, but Sal B and ATO combination could provide both cytotoxicity and cardioprotective properties, indicating that the different effects of Sal B and ATO are dependent on cell type. These results were also observed in several other antioxidant studies [
In conclusion, we confirmed the protective effects of Sal B against ATO-induced cardiotoxicity
The authors declare no conflict of interests.
The present work was supported by the Key Projects of the National Science and Technology Pillar Program (Grant no. 2008BAI51B02), the National 973 Program (no. 2009CB522805), the Major Scientific and Technological Special Project for “Significant New Drugs Formulation” (Grant nos. 2012ZX09301002001 and 2012ZX09501001004), and the Graduate Innovation Foundation of Peking Union Medical College (Grant no. 20121007025).