Flavaglines alleviate doxorubicin cardiotoxicity: implication of Hsp27

Abstract

Background: Despite its effectiveness in the treatment of various cancers, the use of doxorubicin is limited by a potentially fatal cardiomyopathy. Prevention of this cardiotoxicity remains a critical issue in clinical oncology. We hypothesized that flavaglines, a family of natural compounds that display potent neuroprotective effects, may also alleviate doxorubicin-induced cardiotoxicity.

Methodology/principal findings: Our in vitro data established that a pretreatment with flavaglines significantly increased viability of doxorubicin-injured H9c2 cardiomyocytes as demonstrated by annexin V, TUNEL and active caspase-3 assays. We demonstrated also that phosphorylation of the small heat shock protein Hsp27 is involved in the mechanism by which flavaglines display their cardioprotective effect. Furthermore, knocking-down Hsp27 in H9c2 cardiomyocytes completely reversed this cardioprotection. Administration of our lead compound (FL3) to mice attenuated cardiomyocyte apoptosis and cardiac fibrosis, as reflected by a 50% decrease of mortality.

Conclusions/significance: These results suggest a prophylactic potential of flavaglines to prevent doxorubicin-induced cardiac toxicity.

Figure 1. Flavaglines FL1–4 protect H9c2 cardiomyocytes from apoptosis induced by 1 µM doxorubicin.

H9c2 cells were pretreated with flavaglines FL1–4 or their vehicle for 10 h and then treated with doxorubicin for additional 14 h (acute model of cardiotoxicity). Survival rate was quantified by FACS analysis. Histogram shows total population of surviving cells after different concentration of flavagline and doxorubicin treatments. Remarkably, incubation of H9c2 cells with 50 or 100 nM FL3 induced a cardioprotection of 61 and 67% respectively. The structure of the tested compounds is shown in the right panel. * indicates p<0.01 (n = 4).

Figure 2. Protection of H9c2 cardiomyocytes by FL3 against doxorubicin-induced apoptosis.

A. Histogram shows % of apoptotic cells in vehicle, FL3 (100 nM), doxorubicin (1 µM) and FL3+doxorubicin treated H9c2 cells detected by FACS analyses (n = 4, P<0.01). B. Original illustration of total apoptotic cells detected by FACS analyses among the cell population pretreated with FL3 (0–100 nM) and treated with doxorubicin (1 µM). C. Representative illustration of TUNEL positive apoptotic cells (green) versus Dapi (blue) positive total cells detected by fluorescent microscopic analyses for doxorubicin (DOX) and FL3+ DOX treated cells (n = 3, P<0.001). D. Histogram shows quantification of % of apoptosis (TUNEL positive cells versus Dapi positive total number of cells) for different concentration of FL3 (10–100 nM) in a chronic model of cardiotoxicity induced by doxorubicin (1 µM) (n = 3, P<0.001). E. Representative illustration of Western blot analyses on vehicle, doxorubicin, FL3 itself and FL3+doxorubicin treated cells, using active caspase-3 antibody and GAPDH antibody to normalize the assay. Quantitative analysis of Western blots is shown in histogram as % of caspase-3 activity over the vehicle (n = 3, P<0.01).

Figure 3. FL3 protects H9c2 cardiomyocytes against serum starvation.

Histogram shows quantification of percentage of apoptotic cells in H9c2 cells that were cultured in 1% serum in presence of vehicle or FL3 (20 nM) for different time periods as detected by FACS analyses (n = 3, P<0.01).

Figure 4. FL3 protects H9c2 cardiomyocytes by inducing Hsp27 phosphorylation.

A. Quantification of the FACS analysis on H9c2 cardiomyocytes that were treated with doxorubicin (1 µM), FL3 (100 nM) in the presence or absence of Hsp27 inhibitor KRIBB3 (1 µM). B. Representative Western blot analysis on H9c2 cell lysates treated with vehicle or FL3 (100 nM) for 1 h or 4 h, utilizing antibody that recognize either phosphorylated (Ser-15 ) form of Hsp27 (upper) and total Hsp27 protein (lower bands). C. Quantitative analysis of the Western blots (% phosphorylated Hsp27 over total Hsp27 (n = 3, p<0.05). D. Histogram shows % of apoptotic cells on H9c2 control or H9c2 cells where Hsp27 expression was reduced by transfection of siRNA for Hsp27. Remarkably, in the H9c2 cells-knockdown for Hsp27, the cardioprotective effect of FL3 (100 nM) was completely reversed without modifying basal and doxorubicin-induced apoptosis as compare to H9c2 control cells (n = 3, p<0.05).

Figure 5. In vivo cardioprotective activity of FL3 against doxorubicin -induced apoptosis and fibrosis.

A. Protocol of intra peritoneal administration of compounds in mice model of doxorubicin-induced cardiotoxicity. B. Kaplan-Meier survival curves for mice (n = 45) treated with either doxorubicin (15 mg/kg i.p.) or doxorubicin +5 injections of FL3 (12.5 µg/kg i.p.). The survival rate significantly increased in the FL3+doxorubicin group (n = 45) compared with doxorubicin-treated animals (56 versus 31%, p = 0.024). There was no mortality in the control group and FL3 group. C. In vivo myocardium fibrosis. Representative illustration of Malory tetrachrome staining for doxorubicin (DOX) and FL3+DOX treated mice hearts, demonstrating different degrees of fibrosis (blue) in their hearts. The histogram shows the quantification of fibrotic areas (density/pixels²) in the cryosectioned hearts of 4 groups of mice. D. In vivo myocardial apoptosis. Representative TUNEL positive (green) apoptotic area in heart tissue sections from animals treated with DOX and FL3+DOX, original magnification is ×20. Dapi is a nucleus marker (blue). The FL3+doxorubicin groups had fewer apoptotic nuclei compared with the doxorubicin group. The plots show the quantification of TUNEL positive apoptotic area (density/pixels²) in the cryosectioned hearts of 4 groups of mice (n = 4, P<0.001). E. Quantitative RT PCR analyses of cardiomyocyte contractile genes phospholamban, ryanodin receptor and SERCA2a. † indicates p<0.05 as compare to vehicle, * indicates p<0.05 as compare to doxorubicin group.