Doxorubicin-induced cardiotoxicity is suppressed by estrous-staged treatment and exogenous 17β-estradiol in female tumor-bearing spontaneously hypertensive rats

Abstract

Background: Doxorubicin (DOX), an anthracycline therapeutic, is widely used to treat a variety of cancer types and known to induce cardiomyopathy in a time and dose-dependent manner. Postmenopausal and hypertensive females are two high-risk groups for developing adverse effects following DOX treatment. This may suggest that endogenous reproductive hormones can in part suppress DOX-induced cardiotoxicity. Here, we investigated if the endogenous fluctuations in 17β-estradiol (E2) and progesterone (P4) can in part suppress DOX-induced cardiomyopathy in SST-2 tumor-bearing spontaneously hypersensitive rats (SHRs) and evaluate if exogenous administration of E2 and P4 can suppress DOX-induced cardiotoxicity in tumor-bearing ovariectomized SHRs (ovaSHRs).

Methods: Vaginal cytology was performed on all animals to identify the stage of the estrous cycle. Estrous-staged SHRs received a single injection of saline, DOX, dexrazoxane (DRZ), or DOX combined with DRZ. OvaSHRs were implanted with time-releasing pellets that contained a carrier matrix (control), E2, P4, Tamoxifen (Tam), and combinations of E2 with P4 and Tam. Hormone pellet-implanted ovaSHRs received a single injection of saline or DOX. Cardiac troponin I (cTnI), E2, and P4 serum concentrations were measured before and after treatment in all animals. Cardiac damage and function were further assessed by echocardiography and histopathology. Weight, tumor size, and uterine width were measured for all animals.

Results: In SHRs, estrous-staged DOX treatment altered acute estrous cycling that ultimately resulted in prolonged diestrus. Twelve days after DOX administration, all SHRs had comparable endogenous circulating E2. Thirteen days after DOX treatment, SHRs treated during proestrus had decreased cardiac output and increased cTnI as compared to animals treated during estrus and diestrus. DOX-induced tumor reduction was not affected by estrous-staged treatments. In ovaSHRs, exogenous administration of E2 suppressed DOX-induced cardiotoxicity, while P4-implanted ovaSHRs were partly resistant. However, ovaSHRs treated with E2 and P4 did not have cardioprotection against DOX-induced damage.

Conclusions: This study demonstrates that estrous-staged treatments can alter the extent of cardiac damage caused by DOX in female SHRs. The study also supports that exogenous E2 can suppress DOX-induced myocardial damage in ovaSHRs.

Keywords: Adriamycin; Cardiomyopathy; Cardioprotection; Doxorubicin; Estradiol; Progesterone.

Fig. 1

Estrous stage-specific DOX treatment causes cycle irregularity in tumor-bearing SHRs. a Representative vaginal cytologic images of nucleated, cornified, and neutrophil cells collected from SHRs during each stage of the estrous cycle: proestrus (PRO), estrus (EST), metestrus (MET), and diestrus (DIE). b Representative line graph depiction of the estrous cycle for 13 days following a single injection of saline, DOX, and DOX + DRZ during proestrus, estrus, metestrus, or diestrus. The estrous stages were determined by vaginal cytology in five animals per stage per treatment. Arrow indicates the onset of a prolonged diestrus phase. c SST-2 tumor volume in SHRs following a single treatment of saline, DOX, and DOX + DRZ during proestrus, estrus, metestrus, or diestrus at the indicated times. The data points represent the mean ± SEM. (two-way ANOVA performed on day 13, n = 4–5 animals per stage per treatment, *p < 0.05 according to Tukey’s multiple comparison between treatments within the stage.) d The SHRs weight change at day 13 after a single treatment during a specific estrous stage. Percentage of weight change was calculated by a day 13 to day 0 (no treatments) weight ratio. Bars represent the mean ± SEM. (Two-way ANOVA, n = 4–5 animals per treatment per stage, *p < 0.05 according to Tukey’s multiple comparison between stages and treatments.) e Weight in grams of SHR following DOX treatment during each estrous stage. (Repeated measure two-way ANOVA per stage, n = 4–5 animals per group per treatment, *p < 0.05 according to Tukey’s multiple comparison between treatments, while #p < 0.05 between the indicated days of the DOX and DOX + DRZ-treated groups.) Results of analyses are listed in Additional file 2: Table S3 and S4

Fig. 2

SHRs treated during proestrus are more sensitive to DOX-induced myocardial damage. a Echocardiograms were performed 12 days post treatment in estrous stage-specific SHRs. Cardiac output, % ejection fraction, and % fractional shortening were analyzed to assess myocardial dysfunction. b Serum concentrations of cardiac troponin I (cTnI) were measured from animals that received estrous stage specific treatments at day 13 post treatment. Bars represent the mean ± SEM. Two-way ANOVA results are displayed in Additional file 2: Table S3. n = 3 animals per treatment per stage. *p < 0.05 according to Tukey’s multiple comparison between stages and treatments, while #p < 0.05 between the indicated groups

Fig. 3

Cyclic regulation of the 17β-estradiol is lost in DOX-treated SHRs. a, c Serum concentrations of 17β-estradiol and progesterone were measured in estrous-staged SHRs at day 0 (no treatment) (ordinary ANOVA, n = 12 per stage, *p < 0.05 according to Tukey’s multiple comparison between stages). b, d 17β-estradiol and progesterone were measured in the presence of saline, DOX and DOX + DRZ at day 6 and 13 (Two-way ANOVA, n = 3–4 per treatment per stage, *p  <0.05 according to Tukey’s multiple comparison between treatments and stages). Bars represent the mean ± SEM. e, f Correlation between the levels of serum cardiac troponin I and serum 17β-estradiol or progesterone in SHRs. The data points represent the mean ± SEM

Fig. 4

DOX suppresses exogenous 17β-estradiol- and progesterone-induced estrous cycle stimulation in ovariectomized SHRs. a Representative line graph depiction of the estrous cycle before any treatment for 3 days, after implantation at days 3 to 6 prior to DOX treatment, and post DOX treatment at days 3 to 8. Animals were implanted with time-release pellets that contain a control matrix (vehicle), 17β-estradiol (E2), progesterone (P4), tamoxifen (Tam), and combination treatments of E2 + Tam, and E2+ P4. DOX was administered 6 days following implantation. The estrous stages were determined by vaginal cytology in at least three animals per stage and treatment. Red arrow indicates implantation date and DOX treatment, respectively. b, c SST-2 tumor volume and weight of ovariectomized SHRs implanted with the time-release pellets at day 12 of a single saline, or DOX treatment (n = 6–7 per implant group per treatment). The bars represent the mean ± SEM. Two-way ANOVA results are displayed in Additional file 2: Table S3. *p < 0.05 according to Tukey’s multiple comparison between implants and treatment

Fig. 5

DOX affects the serum concentrations of E2 and P4 following exogenous administration in ovariectomized SHRs. a, b Serum concentrations of E2 and P4 were measured in ovariectomized SHRs implanted with no pellet (sham) or a pellet containing a vehicle matrix (control), E2, P4, Tam, and a combination of E2 + P4 and E2 + Tam at day 5, which is before any DOX treatment (ANOVA, n = 6–8 per implant, *p  <0.05 according to Dunnett’s multiple comparison between implants). c, d E2 and P4 concentrations were measured at days 8 and 12 after DOX treatment in pellet-implanted SHRs (two-ANOVA, n = 3–4 per implant per treatment, *p  <0.05 according to Tukey’s multiple comparison between implants and treatment). The bars represent the mean ± SEM

Fig. 6

Exogenous 17β-estradiol and progesterone suppresses cardiac damage in ovariectomized SHR. a Serum concentrations of cardiac troponin I (cTnI) were measured from animals that contained pellet implants at days 8 and 12 post DOX treatment (two-way ANOVA, n = 4–6 per implant per treatment, *p  <0.05 according to Tukey’s multiple comparison between implants and treatment). Bars represent the mean ± SEM. b, c Scatter graph to evaluate the relationship between the levels of serum cardiac troponin I and serum E2 or P4 of pellet-implanted SHRs in the presence and absence of DOX treatment at day 12 (n = 3–4 per implant per treatment)