Doxorubicin (DOX) is one of the most effective chemotherapeutic agents in the treatment of a variety of tumors. However, its clinical use has been compromised by the risk of cardiotoxicity. Thus, many efforts have been focused on exploring new strategies to prevent or reverse DOX-induced cardiotoxicity. Recently, deep sea water (DSW) has drawn much scientific interest for therapeutic intervention due to its enrichment in nutrients and minerals. In this study, we investigated whether DSW has protective effects against DOX-induced cardiotoxicity. Pre-treatment with DSW significantly increased the viability of DOX-treated rat H9c2 cardiac muscle cells. This protective effect of DSW appears to be mediated through the inhibition of DNA damage rather than suppression of reactive oxygen species (ROS) production in DOX-treated H9c2 cardiac muscle cells. The inhibitory effect of DSW on DOX-induced DNA damage subsequently attenuated apoptotic signaling such as activation of cysteine-aspartic acid protease-3 (caspase-3) and fragmentation of poly(ADP-ribose) polymerase (PARP), whereas the expression of anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL) was increased. Moreover, DSW treatment rescued the activation of protein kinase B (Akt) to protect cells from DOX-triggered apoptosis. Taken together, our data showed that DSW has protective effects against DOX-induced cardiotoxicity, suggesting that DSW has some promise as a novel protective supplement for promoting the successful use of DOX in clinical regimen.
The anthracycline antibiotic doxorubicin (DOX) is one of the most effective chemotherapeutic agents in the treatment of numerous solid tumors and hematological malignancies (
The precise molecular mechanisms underlying anthracycline-induced cardiotoxicity are not fully understood because the cause of cardiotoxicity is complex and multifactorial. The most common hypothesis is that the formation of reactive oxygen species (ROS) such as superoxide anion (•O2−) and hydrogen peroxide (H2O2) cause oxidative damage to the cellular components and membranes in heart tissue and reduction of energy in cardiomyocytes, which ultimately lead to cardiomyopathy and congestive heart failure (
Since DOX-induced cardiotoxicity is a major limiting factor in the use of DOX, new strategies to prevent or reverse the cardiotoxic side-effects of DOX have been explored (
Recently, deep sea water (DSW) has gained much scientific interest for therapeutic intervention due to its enrichment in nutrients and minerals. DSW is obtained from a clean area at a depth of >200 m and is rich in minerals such as calcium (Ca), magnesium (Mg), potassium (K), sodium (Na), zinc (Zn), etc. (
DSW was supplied by the Marine Deep Ocean Water Application Research Center in the Korea Institute of Ocean Science and Technology (Goseong, Gyeongsangnam-do, Korea). DSW was taken from the sea in Goseong at a depth of 500 m and subjected to a process of filtrations, reverse osmosis, and concentration by electrolysis to achieve desalinated water and 4,000 hardness DSW. Mg and Ca within DSW were present in the ratio of 3:1 and the hardness of DSW was determined from the concentration of Ca and Mg ions. The following equation was used to calculate the hardness of DSW in this study: Hardness of DSW (mg/l) = Mg (mg/l) × 4.1 + Ca (mg/l) × 2.5. DSW of hardness 1,500 was prepared by diluting hardness 4,000 DSW with desalinated DSW (hardness 0) and dissolved Dulbecco’s modified Eagle’s medium (DMEM) powder with 1% antibiotic-antimycotic solution. Further serial dilutions were performed to achieve hardness 200–800 DSW media from 1,500 hardness DSW with desalinated media (hardness 0).
H9c2 rat cardiomyocytes were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in DMEM (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA, USA) and 1% antibiotic-antimycotic solution (WelGENE). MCF-7 and MDA-MB-231 human breast cancer cell lines were purchased from the Korean Cell Line Bank. MCF-7 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 10 μg/ml insulin, while MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum without insulin.
After H9c2 cells were pre-treated with DSW of various hardness for 24 h, 0.25 μM DOX (Sigma, St. Louis, MO, USA) was added to the cells. Cells were further incubated for 24 h and harvested for RNA isolation or preparation of protein lysates. For inhibition of the PI3K/Akt-signaling pathway, cells were treated with 10 μM LY294002 (LC Laboratories, Woburn, MA, USA) with 0.25 μM DOX and cultured for 24 h before cell harvest.
Cells were seeded in 96-well plates and incubated at 37°C for 24 h. The cells were treated with conditioned media containing DSW of various hardness (200, 400, 800, 1,500) for 24 h prior to adding 0.25 μM DOX. Cells were further incubated for 24 or 48 h and their cell viabilities were measured by MTT assay (Sigma). Absorbance at 570 nm was measured in a Multi-Detection Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
After cells were treated as indicated above, cells were trypsinized and incubated with 20 μM 2′,7′-dichlorofluorescin diacetate (DCF-DA) (Sigma) for 1 h at 37°C in the dark. After incubation, cells were immediately washed and resuspended in PBS. Intracellular ROS production was detected on a FACSCalibur (BD Biosciences, San Jose, CA, USA) by the fluorescent intensity of DCF measured at 525 nm.
The mRNA expression of multi-drug resistance protein 1 (MDR1) was determined by quantitative real-time PCR. Cells were grown and treated in 6-well plates as indicated above. Total RNA was extracted with easy-BLUE™ Total RNA Extraction kit (Intron Biotechnology, Inc., Gyeonggi, Korea) and cDNA was synthesized with reverse transcriptase (Takara Bio, Inc., Shiga, Japan). The real-time PCR reactions were performed using QuantiMix SYBR-Green kit (Philekorea, Daejeon, Korea) in Eco Real-Time PCR (Illumina, San Diego, CA, USA). mRNA expression level of MDR1 was calculated after normalizing with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The utilized primer sequences were as follows: MDR1 forward, 5′-GATGGAATTGATAATGTGGACA-3′ and MDR1 reverse, 5′-GTACGTCGTCATCCAGAGC-3′; GAPDH forward, 5′-AACTTTGGCATCGTGGAAGG-3′ and GAPDH reverse, 5′-TACATTGGGGGTAGGAACAC-3′.
Cells were grown and treated in 6-well plates as indicated above. Cells were lysed with RIPA buffer (50 mM NaCl, 1% Triton X-100, 1% Na deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.5 and 2 mM EDTA). Phosphatase and protease inhibitor cocktail (GenDEPOT, Barker, TX, USA) were added immediately before use. Lysates were cleared of debris at 13,000 rpm for 10 min, and protein concentrations were determined using bicinchoninic acid reagent (Sigma). Proteins were separated by SDS-PAGE (8–15% gels) and transferred onto polyvinylidene difluoride (PVDF) membranes at 100 V for 45 min. Membranes were blocked in 5% milk in TBS-Tween (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature. The following primary antibodies were incubated with blots overnight at 4°C: Anti-rabbit phospho-H2A histone family member X (H2AX), phospho-p38, total-p38, phospho-protein kinase B (Akt), total-Akt, phospho-extracellular signal-regulated kinase 1/2 (ERK1/2), total-ERK1/2, B-cell lymphoma-extra large (Bcl-xL), cleaved cysteine-aspartic acid protease-3 (caspase-3), and poly(ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Inc., Beverly, MA, USA). HRP-conjugated secondary anti-rabbit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted 1:5,000 was incubated with blots for 1 h at room temperature. Blots were developed using Luminescent Image Analyzer LAS-4000 (Fujifilm, Tokyo, Japan).
The Student’s t-test was used for statistical analysis of the data. P<0.05 was considered significant.
To evaluate whether DSW itself has harmful effects on normal cardiomyocyte cells, we first measured viabilities of H9c2 rat cardiomyocytes after treatment with DSW of different hardness for 24 or 48 h. As shown in
Since DSW exhibited cardioprotection by inhibiting DOX-induced cell death, we tested possible interference of DSW with antitumor effects of DOX in the MCF-7 and MDA-MB-231 human breast cancer cells. Interestingly, mildly enhanced antitumor effects of DOX were observed in both MCF-7 and MDA-MB-231 cells in a dose-dependent manner, exhibiting ~10% decrease of cell viability at DSW of 1,500 hardness (
To elucidate the molecular mechanisms involved in the cardioprotective effects of DSW, we first monitored the generation of ROS in H9c2 cells treated with DOX and DSW since a number of recent reviews described the involvement of ROS in the mechanism of DOX-induced cardiotoxicity. In flow cytometry analysis using DCF-DA reagent, which can be converted to fluorescent DCF in a reaction with intracellular ROS, we observed that DOX caused a right-shift of the fluorescence intensity of DCF signal compared to untreated cells, confirming the generation of ROS by DOX. However, this DOX-induced ROS generation was not diminished by pre-treatment with DSW of 1,500 hardness, suggesting that the cardioprotective effects of DSW are independent of ROS generation (
Several studies suggested that induction of DNA damage is an early event in DOX-induced lethal cardiomyocyte injury (
As DSW suppressed DOX-induced DNA damage, which triggers the cell death program, we assessed the effects of DSW on apoptosis signaling. We first analyzed the expression of Bcl-xL, which is an anti-apoptotic protein that inhibits the release of mitochondrial cytochrome
Several studies have shown that the PI3K/Akt- and MAP kinase-signaling pathways are involved in DOX-induced apoptosis (
In the present study, we demonstrated that DSW provides a cardioprotective effect against DOX-induced cardiotoxicity in rat H9c2 cardiac muscle cells without interfering with the antitumor activities of DOX. This protective effect of DSW appears to be mediated through the inhibition of DNA damage rather than suppression of ROS, resulting in subsequent inhibition of DOX-induced apoptotic signaling. Moreover, DSW rescues the Akt-signaling pathway to protect cells from DOX-induced cell death.
Since oxidative stress is generally accepted as the major mechanism by which DOX causes toxicity to the heart, numerous antioxidants have been investigated as cardioprotective agents to prevent or reverse the cardiotoxic side-effects of DOX. However, administration of antioxidants with DOX has failed to show favorable outcomes in clinical studies, implying the involvement of additional mechanisms in the cardiotoxic action of DOX. More recent studies suggest that DNA damage plays an important role in mediating DOX-induced cardiomyocyte death through a pathway involving p53 and the mitochondria (
It is not clear which component of DSW is responsible for the protective effects against DOX-induced cardiotoxicity. However, it is assumed that the combined ionic action of several minerals such as Ca, Mg, K, and Na may play important roles in mediating diverse biological effects of DSW including its cardioprotective effect. Indeed, it is now well known that these essential metal ions are crucial to maintain cellular functions and their deficiency is considered to be a potential health hazard. In particular, Mg and Ca may be the primary minerals responsible for the protective effect of DSW against DOX-induced cardiotoxicity due to their profound existence within DSW. Mg is an essential intracellular ion necessary for normal cellular function (
This study was supported by the project entitled ‘Development of Technology for support of DSW industry (PJT200014)’ from the Ministry of Land, Transport and Maritime Affairs, Korea.
doxorubicin
deep sea water
reactive oxygen species
2′,7′-dichlorofluorescin diacetate
multi-drug resistance protein 1
H2A histone family member X
B-cell lymphoma-extra large
cysteine-aspartic acid protease-3
poly(ADPribose) polymerase
protein kinase B
extracellular signal-regulated kinase 1/2
glyceraldehyde-3-phosphate dehydrogenase
Deep sea water (DSW) protects H9c2 cells from doxorubicin (DOX)-induced cytotoxicity. (A) DSW itself did not induce cytotoxicity when cells were treated with various hardness of DSW for indicated times. (B) DOX-induced cytotoxicity of H9c2 cells exposed to various concentrations of DOX for 24 or 48 h. *P<0.005 and **p<0.001 compared to control. (C) DSW at different hardness attenuated DOX-induced cell cytotoxicity when treated with 0.25 μM DOX for 48 h. **P<0.001 compared to DOX only. The values represent the mean ± SD.
Deep sea water (DSW) enhances antitumor activity of doxorubicin (DOX) in MDA-MB-231 human breast cancer cells. DSW enhanced DOX-induced cell cytotoxicity in (A) MDA-MB-231 and (B) MCF-7 human breast cancer cells when treated with 0.3 μM DOX in combination with DSW of different hardness for 72 h. The values represent the mean ± SD.
Protective effect of deep sea water (DSW) is associated with DNA damage responses rather than reactive oxygen species (ROS) generation or expression of multi-drug resistance protein 1 (MDR1). (A) The generation of ROS was induced by 24 h treatment with 0.25 μM doxorubicin (DOX) in H9c2 cells pre-treated with DSW of 1,500 hardness. (B) MDR1 gene expression of cells pre-treated with DSW of various hardness for 24 h and co-incubated with 0.25 μM DOX for 24 h. The values represent the mean ± SD. (C) H2A histone family member X (H2AX) phosphorylation was analyzed by western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control and the relative intensity of each sample was expressed as fold change compared to control after normalization to respective internal control.
Deep sea water (DSW) inhibits doxorubicin (DOX)-induced apoptosis signal. Protein lysates were prepared from cells treated with DSW of different hardness in the presence of 0.25 μM DOX. The expression of (A) B-cell lymphoma-extra large (Bcl-xL), (B) cleaved cysteine-aspartic acid protease-3 (caspase-3), and (C) fragmented poly(ADP-ribose) polymerase (PARP) was analyzed by western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control and the relative intensity of each sample was expressed as fold change compared to control after normalization to respective internal control.
Deep sea water (DSW) rescues the protein kinase B (Akt)-signaling pathway to protect cells from doxorubicin (DOX)-induced cell death. Cells were treated with conditioned media containing DSW of different hardness for 24 h prior to addition of 0.25 μM DOX. Cells were further incubated for 24 h and harvested for protein lysates. (A) DSW did not alter the expression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). (B) Treatment with DSW significantly decreased the phosphorylation of p38. (C) DSW treatment rescued the activation of Akt from DOX-mediated Akt suppression. (D) Blocking the PI3K/Akt-signaling pathway with LY294002 significantly increased the cleavage of cysteine-aspartic acid protease-3 (caspase-3). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.