Curcumin implants, not curcumin diet, inhibit estrogen-induced mammary carcinogenesis in ACI rats

Abstract

Curcumin is widely known for its antioxidant, anti-inflammatory, and antiproliferative activities in cell-culture studies. However, poor oral bioavailability limited its efficacy in animal and clinical studies. Recently, we developed polymeric curcumin implants that circumvent oral bioavailability issues, and tested their potential against 17β-estradiol (E2)-mediated mammary tumorigenesis. Female Augustus Copenhagen Irish (ACI) rats were administered curcumin either via diet (1,000 ppm) or via polymeric curcumin implants (two 2 cm; 200 mg each; 20% drug load) 4 days before grafting a subcutaneous E2 silastic implant (1.2 cm, 9 mg E2). Curcumin implants were changed after 4.5 months to provide higher curcumin dose at the appearance of palpable tumors. The animals were euthanized after 3 weeks, 3 months, and after the tumor incidence reached >80% (~6 months) in control animals. The curcumin administered via implants resulted in significant reduction in both the tumor multiplicity (2 ± 1 vs. 5 ± 3; P = 0.001) and tumor volume (184 ± 198 mm(3) vs. 280 ± 141 mm(3); P = 0.0283); the dietary curcumin, however, was ineffective. Dietary curcumin increased hepatic CYP1A and CYP1B1 activities without any effect on CYP3A4 activity, whereas curcumin implants increased both CYP1A and CYP3A4 activities but decreased CYP1B1 activity in the presence of E2. Because CYP1A and CYP3A4 metabolize most of the E2 to its noncarcinogenic 2-OH metabolite, and CYP1B1 produces potentially carcinogenic 4-OH metabolite, favorable modulation of these CYPs via systemically delivered curcumin could be one of the potential mechanisms. The analysis of plasma and liver by high-performance liquid chromatography showed substantially higher curcumin levels via implants versus the dietary route despite substantially higher dose administered.

Fig. 1

(A) Curcumin implants placed subcutaneously were removed when the rats were anaesthetized at various time points (25, 89, 138 and 184 days). In vivo release of curcumin from a 2-cm implant (200 mg, 20% load) were plotted as cumulative curcumin released at various time points and the cumulative release were divided by the number of days they remained in the rats to derive the daily average release; data represent an average of three animals (±SD). (B) The tumor-free survival (TFS) is estimated by the Kaplan-Meier method. Differences in the survival curves are evaluated through the estimated hazard rates using the un-weighted log-rank tests. The TFS time is be determined as the time from on study until the first occurrence of tumor. The four groups represent female ACI rats treated with E₂ implant (n=25) along with either two sham polymeric implants (2 cm), or two curcumin implants (2 cm, 20% load) or curcumin diet (1,000 ppm) (n = 20 each group). All 4 groups are significantly different (p=0.0328); these are mainly due to significant difference between the curcumin implant and curcumin diet groups (p=0.0012).The arrows in panels A and B indicate the time at which new curcumin implants were grafted.

Fig. 2

Plasma prolactin levels of female ACI rats treated with or without a silastic E₂ implant along with either two sham polymeric implants (2 cm), or two curcumin implants (2 cm, 20% load) or curcumin diet (1,000 ppm) for 3 weeks (n=6) (A), 3 months (n=6) (B) and 6 months (n=6–8) (C). Plasma prolactin was measured by using an EIA kit following manufacturer’s protocol and a p value of <0.05 was considered significant. Groups compared are denominated with the same alphabets (* p<0.05, ** p<0.01 and *** p<0.001).

Fig. 3

Mammary tumor volume (A) and tumor multiplicity (B) in female ACI rats treated with a silastic E₂ implant along with either two sham polymeric implants (2 cm), or two curcumin implants (2 cm, 20% load) or curcumin diet (1,000 ppm) for over a period of 6 months (n=20). The rats were palpated for tumor occurrence every week and when the E2-treated group achieved > 90% tumor incidence, the study was terminated. Mammary tumors were harvested, measured for tumor volume and counted for tumor multiplicity. A significant difference was observed between sham implant and curcumin implant group as indicated.

Fig. 4

CYP 1B1 (A), 1A (1A1 and 1A2) (B) and 3A4(C) activities of hepatic microsomes isolated from female ACI rats treated with or without a silastic E₂ implant along with either two sham polymeric implants (2 cm), or two curcumin implants (2 cm, 20% load) or curcumin diet (1,000 ppm) for over a period of 3 weeks, 3 months and 6 months. CYP1A and 1B1 activities were determined by EROD assay with and without a selective CYP1B1 inhibitor (pyrene). CYP3A4 activity was measured using P450-Glo™ CYP3A4 assay with Luciferin-IPA following manufacturer’s protocol by replacing NADPH regenerating system with NADPH (5 mM). Groups compared are denominated with the same alphabets (* p<0.05, ** p<0.01 and *** p<0.001).

Fig. 5

Serum estradiol (E₂) levels in female ACI rats treated with sham or E₂ implant along with either two sham polymeric implants (2 cm), or two curcumin implants (2 cm, 20% load) or curcumin diet (1,000 ppm) for over a period of 3 weeks (A) and 3 months (B) (n=6 for each group). Serum E₂ was measured using Estradiol II reagent kit as per manufacturer’s instructions. Groups compared are denominated with the same alphabets (* p<0.05, ** p<0.01 and *** p<0.001).

Fig. 6

Plasma (A) and liver (B) curcumin concentrations in female ACI rats treated with sham or E₂ implant along with either two sham polymeric implants (2 cm), or two curcumin implants (2 cm, 20% load) or curcumin diet (1,000 ppm) for over a period of 3 weeks, 3 months and 6 months. Plasma was pooled from all the animals (n=6) in each group during 3 week and 3 month time points, and from every 3^rd animal from 6 month time point, acidified and extracted with ethyl acetate. The dried residue was reconstituted in acetonitrile and analyzed by HPLC.