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1.  Platycodin D from Platycodonis Radix enhances the anti-proliferative effects of doxorubicin on breast cancer MCF-7 and MDA-MB-231 cells 
Chinese Medicine  2014;9:16.
Background
It has been demonstrated that platycodin D (PD) exhibits anti-cancer activities. This study aims to investigate the anti-proliferative effects of the combination of PD and doxorubicin (DOX) on human breast cancer cells (MCF-7 and MDA-MB-231 cells).
Methods
The anti-proliferative effects of different dosages of PD, DOX, and PD + DOX on MCF-7 and MDA-MB-231 cells were determined by the MTT assay. The 10 μM PD, 5 μM DOX, and 10 μM PD + 5 μM DOX induced-protein expression of apoptosis-related molecules on MCF-7 and MDA-MB-231 cells were detected by western blot. The 10 μM PD, 5 μM DOX and 10 μM PD + 5 μM DOX-induced mitochondrial membrane potential changes on MCF-7 and MDA-MB-231 cells were stained with JC-1 before visual determination. The intracellular accumulations of DOX, induced by 10 μM PD, 5 μM DOX and 10 μM PD + 5 μM DOX, were detected by flow cytometry.
Results
PD enhanced anti-cancer activities of DOX were observed in both MCF-7 and MDA-MB-231 cell lines. Compared with mono treatment, the combined treatment increased the protein expression of cleaved poly (ADP-ribose) polymerase and decreased the mitochondrial membrane potential. The combined treatment with PD did not obviously increase the accumulation of DOX in MCF-7 cells (1.66 ± 0.13 in DOX-treated group, and 1.69 ± 0.06 in PD + DOX-treated group, P = 0.76), but it significantly increased the accumulation of DOX in MDA-MB-231 cells (1.76 ± 0.17 in DOX-treated group, 2.09 ± 0.02 in PD + DOX-treated group, P = 0.027).
Conclusion
The combined treatment of DOX and PD exhibited stronger anti-proliferative effects on MCF-7 and MDA-MB-231 cells than DOX and PD treatment did.
doi:10.1186/1749-8546-9-16
PMCID: PMC4075934  PMID: 24982689
2.  Quercetin Potentiates Doxorubicin Mediated Antitumor Effects against Liver Cancer through p53/Bcl-xl 
PLoS ONE  2012;7(12):e51764.
Background
The dose-dependent toxicities of doxorubicin (DOX) limit its clinical applications, particularly in drug-resistant cancers, such as liver cancer. In this study, we investigated the role of quercetin on the antitumor effects of DOX on liver cancer cells and its ability to provide protection against DOX-mediated liver damage in mice.
Methodology and Results
The MTT and Annexin V/PI staining assay demonstrated that quercetin selectively sensitized DOX-induced cytotoxicity against liver cancer cells while protecting normal liver cells. The increase in DOX-mediated apoptosis in hepatoma cells by quercetin was p53-dependent and occurred by downregulating Bcl-xl expression. Z-VAD-fmk (caspase inhibitor), pifithrin-α (p53 inhibitor), or overexpressed Bcl-xl decreased the effects of quercetin on DOX-mediated apoptosis. The combined treatment of quercetin and DOX significantly reduced the growth of liver cancer xenografts in mice. Moreover, quercetin decreased the serum levels of alanine aminotransferase and aspartate aminotransferase that were increased in DOX-treated mice. Quercetin also reversed the DOX-induced pathological changes in mice livers.
Conclusion and Significance
These results indicate that quercetin potentiated the antitumor effects of DOX on liver cancer cells while protecting normal liver cells. Therefore, the development of quercetin may be beneficial in a combined treatment with DOX for increased therapeutic efficacy against liver cancer.
doi:10.1371/journal.pone.0051764
PMCID: PMC3519886  PMID: 23240061
3.  Disparate Impact of Butyroyloxymethyl Diethylphosphate (AN-7), a Histone Deacetylase Inhibitor, and Doxorubicin in Mice Bearing a Mammary Tumor 
PLoS ONE  2012;7(2):e31393.
The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) synergizes the cytotoxic effect of doxorubicin (Dox) and anti-HER2 on mammary carcinoma cells while protecting normal cells against their insults. This study investigated the concomitant changes occurring in heart tissue and tumors of mice bearing a subcutaneous 4T1 mammary tumor following treatment with AN-7, Dox, or their combination. Dox or AN-7 alone led to inhibition of both tumor growth and lung metastases, whereas their combination significantly increased their anticancer efficacy and attenuated Dox- toxicity. Molecular analysis revealed that treatment with Dox, AN-7, and to a greater degree, AN-7 together with Dox increased tumor levels of γH2AX, the marker for DNA double-strand breaks and decreased the expression of Rad51, a protein needed for DNA repair. These events culminated in increased apoptosis, manifested by the appearance of cytochrome-c in the cytosol. In the myocardium, Dox-induced cardiomyopathy was associated with an increase in γH2AX expression and a reduction in Rad51 and MRE11 expression and increased apoptosis. The addition of AN-7 to the Dox treatment protected the heart from Dox insults as was manifested by a decrease in γH2AX levels, an increase in Rad51 and MRE11 expression, and a diminution of cytochrome-c release. Tumor fibrosis was high in untreated mice but diminished in Dox- and AN-7-treated mice and was almost abrogated in AN-7+Dox-treated mice. By contrast, in the myocardium, Dox alone induced a dramatic increase in fibrosis, and AN7+Dox attenuated it. The high expression levels of c-Kit, Ki-67, c-Myc, lo-FGF, and VEGF in 4T1 tumors were significantly reduced by Dox or AN-7 and further attenuated by AN-7+Dox. In the myocardium, Dox suppressed these markers, whereas AN-7+Dox restored their expression. In conclusion, the combination of AN-7 and Dox results in two beneficial effects, improved anticancer efficacy and cardioprotection.
doi:10.1371/journal.pone.0031393
PMCID: PMC3285631  PMID: 22384017
4.  Hsp20 Interacting with Phosphorylated Akt Reduces Doxorubicin-Triggered Oxidative Stress and Cardiotoxicity 
Circulation research  2008;103(11):1270-1279.
Doxorubicin (DOX) is a widely used antitumor drug, but its application is limited due to its cardiotoxic side effects. Hsp20 has been recently shown to protect cardiomyocytes against apoptosis, induced by ischemia/reperfusion injury or by prolonged β-agonist stimulation. However, it is not clear whether Hsp20 would exert similar protective effects against DOX-induced cardiac injury. Actually, DOX-treatment was associated with down-regulation of Hsp20 in the heart. To elucidate the role of Hsp20 in DOX-triggered cardiac toxicity, Hsp20 was first overexpressed ex vivo by adenovirus-mediated gene delivery. Increased Hsp20 levels conferred higher resistance to DOX-induced cell death, compared to GFP-control. Furthermore, cardiac-specific overexpression of Hsp20 in vivo significantly ameliorated acute DOX-triggered cardiomyocyte apoptosis and animal mortality. Hsp20-transgenic mice also showed improved cardiac function and prolonged survival after chronic administration of DOX. The mechanisms underlying these beneficial effects were associated with preserved Akt phosphorylation/activity and attenuation of DOX-induced oxidative stress. Co-immunoprecipitation studies revealed an interaction between Hsp20 and phosphorylated Akt. Accordingly, BAD phosphorylation was preserved and cleaved caspase-3 was decreased in DOX-treated Hsp20-TG hearts, consistent with the Hsp20's anti-apoptotic effects. Parallel ex vivo experiments showed that either infection with a dominant-negative Akt adenovirus or pre-incubation of cardiomyocytes with the PI3-kinase inhibitors significantly attenuated the protective effects of Hsp20. Taken together, our findings indicate that overexpression of Hsp20 inhibits DOX-triggered cardiac injury, and these beneficial effects appear to be dependent on Akt activation. Thus, Hsp20 may constitute a new therapeutic target in ameliorating the cardiotoxic effects of DOX-treatment in cancer patients.
doi:10.1161/CIRCRESAHA.108.182832
PMCID: PMC2763388  PMID: 18948619
apoptosis; cardiomyopathy; doxorubicin; heat-shock protein; Akt
5.  Protective effect of tetrahydroxystilbene glucoside on cardiotoxicity induced by doxorubicin in vitro and in vivo 
Acta Pharmacologica Sinica  2009;30(11):1479-1487.
Aim:
To test the effect of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) on doxorubicin (DOX)-induced cardiotoxicity.
Methods:
We used neonate rat cardiomyocytes and an acute mouse model of DOX-induced cardiotoxicity to examine the protective effect of THSG.
Results:
In the mouse model, administration of THSG significantly reduced DOX-induced cardiotoxicity, including animal mortality, histopathological changes, and levels of serum creatine kinase (CK) and lactate dehydrogenase (LDH). Moreover, THSG was able to attenuate the increased malondialdehyde (MDA) and decreased reduced glutathione (GSH) caused by DOX. In in vitro studies, THSG 10−300 μmol/L ameliorated DOX-induced cardiomyocyte apoptosis in a concentration-dependent manner. Further studies showed that THSG inhibited reactive oxygen species (ROS) generation and prevented DOX-induced loss of mitochondrial membrane potential, caspase-3 activation and upregulation of Bax protein expression. We observed a protective response against damage after DOX treatment. The level of Bcl-2 protein was increased. Additionally, THSG inhibited a DOX-induced [Ca2+] increase.
Conclusion:
These results showed that THSG protected against DOX-induced cardiotoxicity by decreasing ROS generation and intracellular [Ca2+] and by inhibiting apoptotic signaling pathways.
doi:10.1038/aps.2009.144
PMCID: PMC4003005  PMID: 19890356
2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside; doxorubicin; apoptosis; reactive oxygen species
6.  A Human Anti-c-Met Fab Fragment Conjugated with Doxorubicin as Targeted Chemotherapy for Hepatocellular Carcinoma 
PLoS ONE  2013;8(5):e63093.
c-Met is over-expressed in hepatocellular carcinoma(HCC) but is absent or expressed at low levels in normal tissues. Therefore we generated a novel conjugate of a human anti-c-Met Fab fragment (MetFab) with doxorubicin (DOX) and assessed whether it had targeted antitumor activity against HCC and reduced the side-effects of DOX. The MetFab was screened from human phage library, conjugated with DOX via chemical synthesis, and the conjugation MetFab-DOX was confirmed by HPLC. The drug release patterns, the binding efficacy, and cellular distribution of MetFab-DOX were assessed. MetFab-DOX was stable at pH7.2 PBS while release doxorubicin quickly at pH4.0, the binding efficacy of MetFab-DOX was similarly as MetFab, and the cellular distribution of the MetFab-DOX is distinct from free DOX. The cytotoxicity of MetFab-DOX was analyzed by the MTT method and the nude mouse HCC model. The MetFab-DOX demonstrated cytotoxic effects on c-Met expressing-tumor cells, but not on the cells without c-Met expression. MetFab-DOX exerted anti-tumor effect and significantly reduced the side effect of free DOX in mice model. Furthermore, the localization of conjugate was confirmed by immunofluorescence staining of tumor tissue sections and optical tumor imaging, respectively, and the tissue-distribution of drug was compared between free DOX and MetFab-DOX treatment by spectrofluorometer. MetFab-DOX can localize to the tumor tissue, and the concentration of doxorubicin in the tumor was higher after MetFab-DOX administration than after DOX administration. In summary, MetFab-DOX can target c-Met expressing HCC cells effectively and have obvious antitumor activity with decreased side-effects in preclinical models of HCC.
doi:10.1371/journal.pone.0063093
PMCID: PMC3652865  PMID: 23675455
7.  PEG-oligocholic acid telodendrimer micelles for the targeted delivery of doxorubicin to B-cell lymphoma 
Doxorubicin (DOX) is one of most common anti-cancer chemotherapeutic drugs, but its clinical use is associated with dose-limiting cardiotoxicity. We have recently developed a series of PEG-oligocholic acid based telodendrimers, which can efficiently encapsulate hydrophobic drugs and self-assemble to form stable micelles in aqueous condition. In the present study, two representative telodendrimers (PEG5k-CA8 and PEG2k-CA4) have been applied to prepare DOX micellar formulations for the targeted delivery of DOX to lymphoma. PEG2k-CA4 micelles, compared to PEG5k-CA8 micelles, were found to have higher DOX loading capacity (14.8% vs. 8.2%, w/w), superior stability in physiological condition, and more sustained release profile. Both of these DOX-loaded micelles can be efficiently internalized and release the drug in Raji lymphoma cells. DOX-loaded micelles were found to exhibit similar in vitro cytotoxic activities against both T- and B- lymphoma cells as the free DOX. The maximum tolerated dose (MTD) of DOX-loaded PEG2k-CA4 micelles in mice was approximately 15 mg/kg, which was 1.5-fold higher of the MTD of free DOX. Pharmacokinetics and biodistribution studies demonstrated both DOX-loaded micelles were able to prolong the blood retention time, preferentially accumulate and penetrate in B-cell lymphomas via the enhanced permeability and retention (EPR) effect. Finally, DOX-PEG2k-CA4 micelles achieved enhanced anti-cancer efficacy and prolonged survival in Raji lymphoma bearing mice, compared to free DOX and PEGylated liposomal DOX (Doxil®) at the equivalent dose. In addition, the analysis of creatine kinase (CK) and lactate dehydrogenase (LDH) serum enzymes level indicated that DOX micellar formulations significantly reduced the cardiotoxicity associated with free DOX.
doi:10.1016/j.jconrel.2011.07.018
PMCID: PMC3196055  PMID: 21787818
doxorubicin; polymeric micelles; drug delivery; biodistribution; cardiotoxicity; cancer therapy
8.  Modulation of doxorubicin cytotoxicity by resveratrol in a human breast cancer cell line 
Background
Breast cancer is the most common cancer in the Arab world and it ranked first among Saudi females. Doxorubicin (DOX), an anthracycline antibiotic is one of the most effective anticancer agents used to treat breast cancer. chronic cardiotoxicity is a major limiting factor of the use of doxorubicin. Therefore, our study was designed to assess the role of a natural product resveratrol (RSVL) on sensitization of human breast cancer cells (MCF-7) to the action of DOX in an attempt to minimize doxorubicin effective dose and thereby its side effects.
Methods
Human breast cancer cell line MCF-7, was used in this study. Cytotoxic activity of DOX was determined using (sulforhodamine) SRB method. Apoptotic cells were quantified after treatment by annexin V-FITC- propidium iodide (PI) double staining using flow-cytometer. Cell cycle disturbance and doxorubicin uptake were determined after RSVL or DOX treatment.
Results
Treatment of MCF-7 cells with 15 μg/ml RSVL either simultaneously or 24 h before DOX increased the cytotoxicity of DOX, with IC50 were 0.056 and 0.035 μg/ml, respectively compared to DOX alone IC50 (0.417 μg/ml). Moreover, flow cytometric analysis of the MCF-7 cells treated simultaneously with DOX (0.5 μg/ml) and RSVL showed enhanced arrest of the cells in G0 (80%). On the other hand, when RSVL is given 24 h before DOX although there was more increased in the cytotoxic effect of DOX against the growth of the cells, however, there was decreased in percentage arrest of cells in G0, less inhibition of DOX-induced apoptosis and reduced DOX cellular uptake into the cells.
Conclusion
RSVL treatment increased the cytotoxic activity of DOX against the growth of human breast cancer cells when given either simultaneously or 24 h before DOX.
doi:10.1186/1475-2867-12-47
PMCID: PMC3537590  PMID: 23153194
Doxorubicin; Resveratrol; Breast cancer cell line
9.  Chemosensetizing and cardioprotective effects of resveratrol in doxorubicin- treated animals 
Background
Doxorubicin (DOX), an anthracycline antibiotic is one of the most effective anticancer drug used in the treatment of variety of cancers .Its use is limited by its cardiotoxicity. The present study was designed to assess the role of a natural product resveratrol (RSVL) on sensitization of mammary carcinoma (Ehrlich ascites carcinoma) to the action of DOX and at the same time its protective effect against DOX-induced cardiotoxicity in rats.
Methods
Ehrlich ascites carcinoma bearing mice were used in this study. Percent survival of tumor bearing mice was used for determination of the Cytotoxic activity of DOX in presence and absence of RSVL. Uptake and cell cycle effect of DOX in tumor cells in the presence of RSVL was also determined. Histopatholgical examination of heart tissues after DOX and/or RSVL therapy was also investigated.
Results
DOX at a dose level of 15 mg/kg increased the mean survival time of tumor bearing mice to 21 days compared with 15 days for non tumor-bearing control mice. Administration of RSVL at a dose level of 10 mg/kg simultaneously with DOX increased the mean survival time to 30 days with 70% survival of the tumor-bearing animals. RSVL increased the intracellular level of DOX and there was a strong correlation between the high cellular level of DOX and its cytotoxic activity. Moreover, RSVL treatment showed 4.8 fold inhibition in proliferation index of cells treated with DOX. Histopathological analysis of rat heart tissue after a single dose of DOX (20 mg/kg) showed myocytolysis with congestion of blood vessels, cytoplasmic vacuolization and fragmentation. Concomitant treatment with RSVL, fragmentation of the muscle fiber revealed normal muscle fiber.
Conclusion
This study suggests that RSVL could increase the cytotoxic activity of DOX and at the same time protect against its cardiotoxicity.
doi:10.1186/1475-2867-13-52
PMCID: PMC3680308  PMID: 23714221
Doxorubicin; Resveratrol; Potentiation; Cardioprotection; Cell cycle disturbance
10.  Berberine Inhibits Doxorubicin-Triggered Cardiomyocyte Apoptosis via Attenuating Mitochondrial Dysfunction and Increasing Bcl-2 Expression 
PLoS ONE  2012;7(10):e47351.
Cardiomyocyte apoptosis is an important event in doxorubicin (DOX)-induced cardiac injury. The aim of the present study was to investigate the protection of berberine (Ber) against DOX- triggered cardiomyocyte apoptosis in neonatal rat cardiomyocytes and rats. In neonatal rat cardiomyocytes, Ber attenuated DOX-induced cellular injury and apoptosis in a dose-dependent manner. However, Ber has no significant effect on viability of MCF-7 breast cancer cells treated with DOX. Ber reduced caspase-3 and caspase-9, but not caspase-8 activity in DOX-treated cardiomyocytes. Furthermore, Ber decreased adenosine monophosphate-activated protein kinase α (AMPKα) and p53 phosphorylation at 2 h, cytosolic cytochrome c and mitochondrial Bax levels and increased Bcl-2 level at 6 h in DOX-stimulated cardiomyocytes. Pretreatment with compound C, an AMPK inhibitor, also suppressed p53 phosphorylation and apoptosis in DOX-treated cardiomyocytes. DOX stimulation for 30 min led to a loss of mitochondrial membrane potential and a rise in the AMP/ATP ratio. Ber markedly reduced DOX-induced mitochondrial membrane potential loss and an increase in the AMP/ATP ratio at 1 h and 2 h post DOX exposure. In in vivo experiments, Ber significantly improved survival, increased stroke volume and attenuated myocardial injury in DOX-challenged rats. TUNEL and Western blot assays showed that Ber not only decreased myocardial apoptosis, caspase-3 activation, AMPKα and p53 phosphorylation, but also increased Bcl-2 expression in myocardium of rats exposed to DOX for 84 h. These findings indicate that Ber attenuates DOX-induced cardiomyocyte apoptosis via protecting mitochondria, inhibiting an increase in the AMP/ATP ratio and AMPKα phosphorylation as well as elevating Bcl-2 expression, which offer a novel mechanism responsible for protection of Ber against DOX-induced cardiomyopathy.
doi:10.1371/journal.pone.0047351
PMCID: PMC3471849  PMID: 23077597
11.  Overexpression of angiopoietin-1 reduces doxorubicin-induced apoptosis in cardiomyocytes 
Journal of Biomedical Research  2012;26(6):432-438.
Doxorubicin (Dox) is a major anticancer chemotherapeutic agent. However, it causes cardiomyopathy due to the side effect of cardiomyocyte apoptosis. We have previously reported that angiopoietin-1 significantly reduced myocardial infarction after ischemic injury and protected cardiomyocytes from oxidative stress-induced apoptosis. It is hypothesized that angiopoietin-1 may protect cardiomyocytes from Dox-induced apoptosis. Cardiomyocytes H9C2 were transfected with adenovirus expressing angiopoietin-1 (Ad5-Ang-1) 24 h before the cells were challenged with Dox at a concentration of 2 µmol/L. Ad5-GFP served as the vector control. Cardiomyocyte apoptosis was evaluated using Annexin V-FITC staining and caspase-3 and caspase-8 activity was determined by Western blotting. The results showed that Dox treatment significantly induced cardiomyocyte apoptosis as evidenced by the greater number of Annexin V-FITC stained cells and increases in caspase-3 and caspase-8 activity. In contrast, overexpression of angiopoietin-1 significantly prevented Dox-induced cardiomyocyte apoptosis. To elucidate the mechanisms by which angiopoietin-1 protected cells from Dox-induced apoptosis, we analyzed both extrinsic and intrinsic apoptotic signaling pathways. We observed that angiopoietin-1 prevented Dox-induced activation of both extrinsic and intrinsic apoptotic signaling pathways. Specifically, angiopoietin-1 prevented DOX-induced increases in FasL and Bax levels and cleaved caspase-3 and caspase-8 levels in H9C2 cells. In addition, overexpression of angiopoietin-1 also activated the pro-survival phosphoinositide-3 kinase (PI3K)/Akt signaling pathway and decreased Dox-induced nuclear factor-kappaB (NF-κB) activation. Our data suggest that promoting the expression of angiopoietin-1 could be a potential approach for reducing Dox-induced cardiomyocyte cytoxicity.
doi:10.7555/JBR.26.20120006
PMCID: PMC3597044  PMID: 23554782
cardiomyocyte; doxorubicin; apoptosis; angiopoietin-1; phosphoinositide-3 kinase (PI3K); nuclear factor-kappaB (NF-κB)
12.  Preparation, characterization and application of star-shaped PCL/PEG micelles for the delivery of doxorubicin in the treatment of colon cancer 
Star-shaped polymer micelles have good stability against dilution with water, showing promising application in drug delivery. In this work, biodegradable micelles made from star-shaped poly(å-caprolactone)/poly(ethylene glycol) (PCL/PEG) copolymer were prepared and used to deliver doxorubicin (Dox) in vitro and in vivo. First, an acrylated monomethoxy poly (ethylene glycol)-poly(å-caprolactone) (MPEG-PCL) diblock copolymer was synthesized, which then self-assembled into micelles, with a core-shell structure, in water. Then, the double bonds at the end of the PCL blocks were conjugated together by radical polymerization, forming star-shaped MPEG-PCL (SSMPEG-PCL) micelles. These SSMPEG-PCL micelles were monodispersed (polydispersity index = 0.11), with mean diameter of ≈25 nm, in water. Blank SSMPEG-PCL micelles had little cytotoxicity and did not induce obvious hemolysis in vitro. The critical micelle concentration of the SSMPEG-PCL micelles was five times lower than that of the MPEG-PCL micelles. Dox was directly loaded into SSMPEG-PCL micelles by a pH-induced self-assembly method. Dox loading did not significantly affect the particle size of SSMPEG-PCL micelles. Dox-loaded SSMPEG-PCL (Dox/SSMPEG-PCL) micelles slowly released Dox in vitro, and the Dox release at pH 5.5 was faster than that at pH 7.0. Also, encapsulation of Dox in SSMPEG-PCL micelles enhanced the anticancer activity of Dox in vitro. Furthermore, the therapeutic efficiency of Dox/SSMPEG-PCL on colon cancer mouse model was evaluated. Dox/SSMPEG-PCL caused a more significant inhibitory effect on tumor growth than did free Dox or controls (P < 0.05), which indicated that Dox/SSMPEG-PCL had enhanced anticolon cancer activity in vivo. Analysis with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) showed that Dox/SSMPEG-PCL induced more tumor cell apoptosis than free Dox or controls. These results suggested that SSMPEG-PCL micelles have promising application in doxorubicin delivery for the enhancement of anticancer effect.
doi:10.2147/IJN.S39532
PMCID: PMC3593767  PMID: 23493403
drug delivery; star-shaped polymer; MPEG-PCL; CMC
13.  Ocotillol Enhanced the Antitumor Activity of Doxorubicin via p53-Dependent Apoptosis 
The use of doxorubicin (Dox) was severely constrained by dose-dependent side effects, which might be attenuated by combining a “sensitizer” to decrease its cumulative dosage. In this study, it was investigated whether ocotillol could enhance the antiproliferation activity of Dox. MTT assays and xenograft tumor model were firstly conducted to evaluate the effect of ocotillol on the antitumor activity of Dox. Flow cytometry and Hoechst staining assays were then performed to assess cell apoptosis. Western blot and real-time PCR were finally used to detect the expression of p53 and its target genes. Our results showed ocotillol to enhance Dox-induced cell death in p53 wild-type cancer cells. Compared with Dox alone, Dox with ocotillol (Dox-O) could induce much more cell apoptosis and activate p53 to a much greater degree, which in turn markedly increased expression of proapoptosis genes. The enhanced cytotoxic activity was partially blocked by pifithrin-α, which might be through attenuating the increased apoptosis. Furthermore, ocotillol significantly increased the antitumor activity of Dox in A549 xenograft tumor in nude mice. These findings indicated that ocotillol could potentiate the cytotoxic effect of Dox through p53-dependent apoptosis and suggested that coadministration of ocotillol with Dox might be a potential therapeutic strategy.
doi:10.1155/2013/468537
PMCID: PMC3727205  PMID: 23956772
14.  Urotensin II Inhibits Doxorubicin-Induced Human Umbilical Vein Endothelial Cell Death by Modulating ATF Expression and via the ERK and Akt Pathway 
PLoS ONE  2014;9(9):e106812.
Background and Purpose
Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Urotensin II (U-II), a potent vasoactive peptide, participates in vascular and myocardial remodeling after injury. We investigated the protective effect of U-II on doxorubicin (DOX)-induced apoptosis in cultured human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved in this process.
Experimental Approach
Cultured HUVECs were treated with vehicle, DOX (1 µM), U-II, or U-II plus DOX. Apoptosis was evaluated by DNA strand break level with TdT-mediated dUTP nick-end labeling (TUNEL) staining. Western blot analysis was employed to determine the related protein expression and flow cytometry assay was used to determine the TUNEL positive cells.
Key Results
U-II reduced the quantity of cleaved caspase-3 and cytosol cytochrome c and increased Bcl-2 expression, which results in protecting HUVECs from DOX-induced apoptosis. U-II induced Activating transcription factor 3 (ATF3) at both mRNA and protein levels in U-II-treated cells. Knockdown of ATF3 with ATF3 siRNA significantly reduced ATF3 protein levels and U-II protective effect under DOX-treated condition. U-II downregulated p53 expression in DOX-induced HUVECs apoptosis, and it rapidly activated extracellular signal-regulated protein kinase (ERK) and Akt. The DOX induced change of p53 was not affected by U-II antagonist (urantide) under ATF-3 knockdown. The inhibitory effect of U-II on DOX-increased apoptosis was attenuated by inhibitors of ERK (U0126) and PI3K/Akt (LY294002).
Conclusion and Implications
Our observations provide evidence that U-II protects HUVECs from DOX-induced apoptosis. ERK-Akt phosphorylation, ATF3 activation, and p53 downregulation may play a signal-transduction role in this process.
doi:10.1371/journal.pone.0106812
PMCID: PMC4182104  PMID: 25268131
15.  Reversal of multidrug resistance by 5,5’-dimethoxylariciresinol-4-O-β-D-glucoside in doxorubicin-resistant human leukemia K562/DOX 
Indian Journal of Pharmacology  2013;45(6):597-602.
Objective:
The objective of this study was to investigate the reversal effects of 5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG) extracted from traditional Chinese medicines Mahonia on multidrug resistance (MDR) of human leukemia cells to chemotherapeutic agents.
Materials and Methods:
MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine the effect of DMAG on doxorubicin sensitivity to K562/DOX cells. Propidium iodide /Hoechst 33342 double staining assay was used to investigate the effect of DMAG on doxorubicin-induced cellular apoptosis. Intracellular accumulation of doxorubicin and rhodamine 123 assay were performed to evaluate the effect of DMAG on drugs efflux activity of P-glycoprotein.
Results:
DMAG significantly enhanced the doxorubicin cytotoxicity to K562/DOX cells. In the presence of 1.0 μM of DMAG, the IC50 of doxorubicin decreased from 34.93 ± 1.37 μM to 12.51 ± 1.28 μM. DMAG of 1.0 μM significantly enhanced doxorubicin-induced cell apoptosis in K562/DOX cells and the enhancement was time-dependent. A significant increase in accumulation of doxorubicin in the presence of DMAG was observed. After treatment of the K562/DOX cells for 1 h with 15.0 μM doxorubicin alone, the fluorescence intensity was 33093.12. With the addition of 1.0 μM of DMAG, the fluorescence intensity of doxorubicin was 2.3-fold higher. A significant increase of accumulation of rhodamine 123 in the presence of DMAG was also observed. With the addition of 1.0 μM of DMAG, the fluorescence intensity was increased by 49.11% compared with rhodamine 123 alone.
Conclusion:
DMAG was shown to effectively enhance chemosensitivity of resistant cells, which makes it might be a suitable candidate for potential MDR-reversing agents.
doi:10.4103/0253-7613.121371
PMCID: PMC3847250  PMID: 24347768
5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside; doxorubicin; leukemia; multidrug resistance
16.  The synergistic effect and mechanism of doxorubicin-ZnO nanocomplexes as a multimodal agent integrating diverse anticancer therapeutics 
Background
Nanomaterials have emerged as ideal multimodal nanomedicine platforms, each one combining different designs and therapeutic approaches in a single system against cancer. The aim of the current study was to explore the synergistic effect and mechanism of a doxorubicin (Dox)-ZnO nanocomplex as a multimodal drug delivery system, integrating Dox chemotherapy and ZnO-mediated photodynamic therapy, in anticancer therapeutics.
Methods
Dox was loaded onto ZnO nanomaterials, forming complexes with the transition metal Zn to yield the Dox-ZnO nanocomplexes. After culture with SMMC-7721 hepatocarcinoma cells, the cellular uptake was quantitatively detected by flow cytometry and visualized by fluorescence microscopy. The synergistic effects of the different anticancer therapeutic modalities on the proliferation of SMMC-7721 hepatocarcinoma cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of B-cell lymphoma 2 protein (Bcl-2), Bcl-2 associated X protein (Bax), caspase 9, and caspase 3 were examined by Western blot, to elucidate the possible molecular mechanisms involved.
Results
Our observations demonstrated that Dox-ZnO nanocomplexes could act as an efficient drug delivery system for importing Dox into SMMC-7721 cells, enhancing its potential chemotherapy efficiency by increasing the intracellular concentration of Dox. With the addition of ultraviolet (UV) illumination, the ZnO nanomaterials showed excellent photodynamic therapeutic properties, attacking the cancer cells further. Thus the caspase-dependent apoptosis was synergistically induced, resulting in distinct improvement in anticancer activity.
Conclusion
The Dox-ZnO nanocomplex presents a promising multimodal agent for comprehensive cancer treatment.
doi:10.2147/IJN.S43657
PMCID: PMC3652513  PMID: 23674895
nanomedicine; nanocomplexes; drug delivery system; doxorubicin; photodynamic therapy
17.  Schisandrin B Prevents Doxorubicin-Induced Chronic Cardiotoxicity and Enhances Its Anticancer Activity In Vivo 
PLoS ONE  2011;6(12):e28335.
Background
To mitigate the cardiotoxicity of anthracycline antibiotics without compromising their anticancer activities is still an issue to be solved. We previously demonstrated that schisandrin B (Sch B) could protect against doxorubicin (Dox)-induced acute cardiotoxicity via enhancing cardiomyocytic glutathione redox cycling that could attenuate oxidative stress generated from Dox. In this study, we attempted to prove if Sch B could also protect against Dox-induced chronic cardiotoxicity, a more clinically relevant issue, without compromising its anticancer activity.
Methodology
Rat was given intragastrically either vehicle or Sch B (50 mg/kg) two hours prior to i.p. Dox (2.5 mg/kg) weekly over a 5-week period with a cumulative dose of Dox 12.5 mg/kg. At the 6th and 12th week after last dosing, rats were subjected to cardiac function measurement, and left ventricles were processed for histological and ultrastructural examination. Dox anticancer activity enhanced by Sch B was evaluated by growth inhibition of 4T1, a breast cancer cell line, and S180, a sarcoma cell line, in vitro and in vivo.
Principal Findings
Pretreatment with Sch B significantly attenuated Dox-induced loss of cardiac function and damage of cardiomyocytic structure. Sch B substantially enhanced Dox cytotoxicities toward S180 in vitro and in vivo in mice, and increased Dox cytotoxcity against 4T1 in vitro. Although we did not observe this enhancement against the implanted 4T1 primary tumor, the spontaneous metastasis to lung was significantly reduced in combined treatment group than Dox alone group.
Conclusion
Sch B is capable of protecting Dox-induced chronic cardiotoxicity and enhancing its anticancer activity. To the best of our knowledge, Sch B is the only molecule ever proved to function as a cardioprotective agent as well as a chemotherapeutic sensitizer, which is potentially applicable for cancer treatment.
doi:10.1371/journal.pone.0028335
PMCID: PMC3229562  PMID: 22164272
18.  Isorhamnetin Protects against Doxorubicin-Induced Cardiotoxicity In Vivo and In Vitro 
PLoS ONE  2013;8(5):e64526.
Doxorubicin (Dox) is an anthracycline antibiotic for cancer therapy with limited usage due to cardiotoxicity. Isorhamnetin is a nature antioxidant with obvious cardiac protective effect. The aim of this study is going to investigate the possible protective effect of isorhamnetin against Dox-induced cardiotoxicity and its underlying mechanisms. In an in vivo investigation, rats were intraperitoneally (i.p.) administered with Dox to duplicate the model of Dox-induced chronic cardiotoxicity. Daily pretreatment with isorhamnetin (5 mg/kg, i.p.) for 7 days was found to reduce Dox-induced myocardial damage significantly, including the decline of cardiac index, decrease in the release of serum cardiac enzymes and amelioration of heart vacuolation. In vitro studies on H9c2 cardiomyocytes, isorhamnetin was effective to reduce Dox-induced cell toxicity. A further mechanism study indicated that isorhamnetin pretreatment can counteract Dox-induced oxidative stress and suppress the activation of mitochondrion apoptotic pathway and mitogen-activated protein kinase pathway. Isorhamnetin also potentiated the anti-cancer activity of Dox in MCF-7, HepG2 and Hep2 cells. These findings indicated that isorhamnetin can be used as an adjuvant therapy for the long-term clinical use of Dox.
doi:10.1371/journal.pone.0064526
PMCID: PMC3665796  PMID: 23724057
19.  A Novel Doxorubicin Prodrug with Controllable Photolysis Activation for Cancer Chemotherapy 
Pharmaceutical Research  2010;27(9):1848-1860.
ABSTRACT
Purpose
Doxorubicin (DOX) is a very effective anticancer agent. However, in its pure form, its application is limited by significant cardiotoxic side effects. The purpose of this study was to develop a controllably activatable chemotherapy prodrug of DOX created by blocking its free amine group with a biotinylated photocleavable blocking group (PCB).
Methods
An n-hydroxy succunamide protecting group on the PCB allowed selective binding at the DOX active amine group. The PCB included an ortho-nitrophenyl group for photo cleavability and a water-soluble glycol spacer arm ending in a biotin group for enhanced membrane interaction.
Results
This novel DOX-PCB prodrug had a 200-fold decrease in cytotoxicity compared to free DOX and could release active DOX upon exposure to UV light at 350 nm. Unlike DOX, DOX-PCB stayed in the cell cytoplasm, did not enter the nucleus, and did not stain the exposed DNA during mitosis. Human liver microsome incubation with DOX-PCB indicated stability against liver metabolic breakdown.
Conclusions
The development of the DOX-PCB prodrug demonstrates the possibility of using light as a method of prodrug activation in deep internal tissues without relying on inherent physical or biochemical differences between the tumor and healthy tissue for use as the trigger.
doi:10.1007/s11095-010-0183-x
PMCID: PMC2916115  PMID: 20596761
prodrug; photocleavable; photoactivatable; doxorubicin; toxicity
20.  Codelivery of doxorubicin and curcumin with lipid nanoparticles results in improved efficacy of chemotherapy in liver cancer 
Liver cancer is a leading cause of cancer deaths worldwide. The combination therapy of cytotoxic and chemosensitizing agents loaded in nanoparticles has been highlighted as an effective treatment for different cancers. However, such studies in liver cancer remain very limited. In our study, we aim to develop a novel lipid nanoparticles loaded with doxorubicin (DOX) (an effective drug for liver cancer) and curcumin (Cur) (a chemosensitizer) simultaneously, and we examined the efficacy of chemotherapy in liver cancer. DOX and Cur codelivery lipid nanoparticles (DOX/Cur-NPs) were successfully prepared using a high-pressure microfluidics technique, showing a mean particle size of around 90 nm, a polydispersity index <0.3, and a zeta potential <−10 mV. The encapsulation efficacy was >90% for both DOX and Cur. The blank lipid nanoparticles were nontoxic, as determined by a cell cytotoxicity study in human normal liver cells L02 and liver cancer cells HepG2. In vitro DOX release studies revealed a sustained-release pattern until 48 hours in DOX/Cur-NPs. We found enhanced cytotoxicity and decreased inhibitory concentration (IC)50 in HepG2 cells and reduced cytotoxicity in L02 cells treated with DOX/Cur-NPs, suggesting the synergistic effects of DOX/Cur-NPs compared with free DOX and DOX nanoparticles (NPs). The optimal weight ratio of DOX and Cur was 1:1. Annexin-V-fluorescein isothiocyanate/propidium iodide double staining showed enhanced apoptosis in HepG2 cells treated with DOX/Cur-NPs compared with free DOX and DOX-NPs. An in vivo experiment showed the synergistic effect of DOX/Cur-NPs compared with DOX-NPs on liver tumor growth inhibition. Taken together, the simultaneous delivery of DOX and Cur by DOX/Cur-NPs might be a promising treatment for liver cancer.
doi:10.2147/IJN.S73322
PMCID: PMC4284012  PMID: 25565818
doxorubicin; curcumin; codelivery; liver cancer; cytotoxicity; tumor growth inhibition
21.  Cardioprotective Effects of 20(S)-Ginsenoside Rh2 against Doxorubicin-Induced Cardiotoxicity In Vitro and In Vivo 
Doxorubicin (DOX) is considered as one of the best antineoplastic agents. However, its clinical use is restricted by its associated cardiotoxicity, which is mediated by the production of reactive oxygen species. In this study, 20(S)-ginsenoside Rh2 (Rh2) was explored whether it had protective effects against DOX-induced cardiotoxicity. In vitro study on H9C2 cell line, as well as in vivo investigation in one mouse and one rat model of DOX-induced cardiomyopathy, was carried out. The results showed that pretreatment with Rh2 significantly increased the viability of DOX-injured H9C2 cells. In the mouse model, Rh2 could suppress the DOX-induced release of the cardiac enzymes into serum and improved the occurred pathological changes through ameliorating the decreased antioxidant biomolecules and the cumulated lipid peroxidation malondialdehyde in heart tissues. In the rat model, Rh2 could attenuate the change of ECG resulting from DOX administration. Furthermore, Rh2 enhanced the antitumor activity of DOX in A549 cells. Our findings thus demonstrated that Rh2 pretreatment could effectively alleviate heart injury induced by DOX, and Rh2 might act as a novel protective agent in the clinical usefulness of DOX.
doi:10.1155/2012/506214
PMCID: PMC3483725  PMID: 23125868
22.  Anti-inflammatory agents and monoHER protect against DOX-induced cardiotoxicity and accumulation of CML in mice 
British Journal of Cancer  2007;96(6):937-943.
Cardiac damage is the major limiting factor for the clinical use of doxorubicin (DOX). Preclinical studies indicate that inflammatory effects may be involved in DOX-induced cardiotoxicity. Nɛ-(carboxymethyl) lysine (CML) is suggested to be generated subsequent to oxidative stress, including inflammation. Therefore, the aim of this study was to investigate whether CML increased in the heart after DOX and whether anti-inflammatory agents reduced this effect in addition to their possible protection on DOX-induced cardiotoxicity. These effects were compared with those of the potential cardioprotector 7-monohydroxyethylrutoside (monoHER).
BALB/c mice were treated with saline, DOX alone or DOX preceded by ketoprofen (KP), dexamethasone (DEX) or monoHER. Cardiac damage was evaluated according to Billingham. Nɛ-(carboxymethyl) lysine was quantified immunohistochemically.
Compared to saline, a 21.6-fold increase of damaged cardiomyocytes was observed in mice treated with DOX (P<0.001). Addition of KP, DEX or monoHER before DOX significantly reduced the mean ratio of abnormal cardiomyocytes in comparison to mice treated with DOX alone (P⩽0.02). In addition, DOX induced a significant increase in the number of CML-stained intramyocardial vessels per mm2 (P=0.001) and also in the intensity of CML staining (P=0.001) compared with the saline-treated group. Nɛ-(carboxymethyl) lysine positivity was significantly reduced (P⩽0.01) by DOX-DEX, DOX-KP and DOX-monoHER. These results confirm that inflammation plays a role in DOX-induced cardiotoxicity, which is strengthened by the observed DOX-induced accumulation of CML, which can be reduced by anti-inflammatory agents and monoHER.
doi:10.1038/sj.bjc.6603640
PMCID: PMC2360105  PMID: 17325706
doxorubicin; cardiotoxicity; inflammation; Nɛ-(carboxymethyl)lysine; monoHER; anti-inflammatory agents
23.  Fabrication and characterization of nuclear localization signal-conjugated glycol chitosan micelles for improving the nuclear delivery of doxorubicin 
Background
Supramolecular micelles as drug-delivery vehicles are generally unable to enter the nucleus of nondividing cells. In the work reported here, nuclear localization signal (NLS)-modified polymeric micelles were studied with the aim of improving nuclear drug delivery.
Methods
In this research, cholesterol-modified glycol chitosan (CHGC) was synthesized. NLS-conjugated CHGC (NCHGC) was synthesized and characterized using proton nuclear magnetic resonance spectroscopy, dynamic light scattering, and fluorescence spectroscopy. Doxorubicin (DOX), an anticancer drug with an intracellular site of action in the nucleus, was chosen as a model drug. DOX-loaded micelles were prepared by an emulsion/solvent evaporation method. The cellular uptake of different DOX formulations was analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity of blank micelles, free DOX, and DOX-loaded micelles in vitro was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HeLa and HepG2 cells.
Results
The degree of substitution was 5.9 cholesterol and 3.8 NLS groups per 100 sugar residues of the NCHGC conjugate. The critical aggregation concentration of the NCHGC micelles in aqueous solution was 0.0209 mg/mL. The DOX-loaded NCHGC (DNCHGC) micelles were observed as being almost spherical in shape under transmission electron microscopy, and the size was determined as 248 nm by dynamic light scattering. The DOX-loading content of the DNCHGC micelles was 10.1%. The DOX-loaded micelles showed slow drug-release behavior within 72 hours in vitro. The DNCHGC micelles exhibited greater cellular uptake and higher amounts of DOX in the nuclei of HeLa cells than free DOX and DOX-loaded CHGC (DCHGC) micelles. The half maximal inhibitory concentration (IC50) values of free DOX, DCHGC, and DNCHGC micelles against HepG2 cells were 4.063, 0.591, and 0.171 μg/mL, respectively. Moreover, the IC50 values of free DOX (3.210 μg/mL) and the DCHGC micelles (1.413 μg/mL) against HeLa cells were nearly 6.96- and 3.07-fold (P < 0.01), respectively, higher than the IC50 value of the DNCHGC micelles (0.461 μg/mL).
Conclusion
The results of this study suggest that novel NCHGC micelles could be a potential carrier for nucleus-targeting delivery.
doi:10.2147/IJN.S36150
PMCID: PMC3459689  PMID: 23049255
polymeric micelles; drug delivery; nucleus-targeting delivery
24.  G Protein Inactivator RGS6 Mediates Myocardial Cell Apoptosis and Cardiomyopathy Caused by Doxorubicin 
Cancer research  2013;73(6):1662-1667.
Clinical use of the widely used chemotherapeutic agent doxorubicin is limited by life-threatening cardiotoxicity. The mechanisms underlying Dox-induced cardiomyopathy and heart failure remain unclear, but are thought to involve p53-mediated myocardial cell apoptosis. The tripartite G protein inactivating protein RGS6 has been implicated in reactive oxygen species (ROS) generation, ATM/p53 activation and apoptosis in Dox-treated cells. Thus, we hypothesized that RGS6, the expression of which is enriched in cardiac tissue, might also be responsible for the pathological effects of Dox treatment in heart. In this study, we show that RGS6 expression is induced strongly by Dox in the ventricles of mice and isolated ventricular myocytes (VCM) via a post-transcriptional mechanism. While Dox-treated wild type (WT) mice manifested severe left ventricular dysfunction, loss of heart and body mass, along with decreased survival five days after Dox administration, mice lacking RGS6 were completely protected against these pathogenic responses. Activation of ATM/p53-apoptosis signaling by Dox in ventricles of WT mice was also absent in their RGS6−/− counterparts. Dox-induced ROS generation was dramatically impaired in both the ventricles and VCM isolated from RGS6−/− mice, and the apoptotic response to Dox in VCM required RGS6-dependent ROS production. These results identify RGS6 as an essential mediator of the pathogenic responses to Dox in heart, and they argue that RGS6 inhibition offers a rational means to circumvent Dox cardiotoxicity in human cancer patients.
doi:10.1158/0008-5472.CAN-12-3453
PMCID: PMC3602152  PMID: 23338613
RGS6; doxorubicin cardiotoxicity; apoptosis; p53; reactive oxygen species
25.  Overexpression of Nrdp1 in the Heart Exacerbates Doxorubicin-Induced Cardiac Dysfunction in Mice 
PLoS ONE  2011;6(6):e21104.
Background
Cardiac cell death and generation of oxidative stress contribute to doxorubicin (DOX)-induced cardiac dysfunction. E3 ligase Nrdp1 plays a critical role in the regulation of cell apoptosis, inflammation and production of reactive oxygen species (ROS), which may contribute to heart failure. However, the role of Nrdp1 in DOX-induced cardiac injury remains to be determined.
Methods and Results
We examined the effect of Nrdp1 overexpression with DOX treatment in rat neonatal cardiomyocytes and mouse heart tissue. Cardiomyocytes were infected with adenovirus containing GFP (Ad-GFP), Nrdp1 wild-type (Ad-Nrdp1) or the dominant-negative form of Nrdp1 (Ad-Dn-Nrdp1), then treated with DOX for 24 hr. DOX treatment increased cell death and apoptosis, with Ad-Nrdp1 infection enhancing these actions but Ad-Dn-Nrdp1 infection attenuating these effects. Furthermore, 5 days after a single injection of DOX (20 mg/kg, intraperitoneally), Nrdp1 transgenic mice (TG) showed decreased cardiac function and increased apoptosis, autophagy and oxidative stress as compared with wild-type (WT) mice (P<0.01). Survival rate was significantly lower in Nrdp1 TG mice than in WT mice 10 days after DOX injection (P<0.01).
Conclusions/Significance
These results were associated with decreased activation of Akt, extracellular signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) signaling pathways. Nrdp1 may be a key mediator in the development of cardiac dysfunction after DOX treatment and associated with inhibition of Akt, ERK1/2 and STAT3. Nrdp1 may be a new therapeutic target in protecting against the cardiotoxic effects of DOX.
doi:10.1371/journal.pone.0021104
PMCID: PMC3124482  PMID: 21738612

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