Site‐Specific Deglucuronidation of Turmeric‐Derived Curcuminoids in Bone

Abstract

IntroductionThe majority of women with advanced breast cancer (BCa) develop osteolytic bone metastases. Our laboratory has previously demonstrated that curcuminoids, bioactive components isolated from turmeric rhizomes, prevent the progression of osteolytic bone lesions in a murine xenograft model of human BCa bone metastasis and inhibit tumor cell secretion of TGFβ‐stimulated parathyroid‐related protein (PTHrP), a signaling pathway known to drive bone metastasis progression. In humans ingesting curcuminoid‐enriched turmeric dietary supplements, glucuronidated phase II metabolites of curcumin are the primary form detected in the circulation. This has given rise to the untested postulate that curcuminoids may be deglucuronidated at sites of action to form bioactive aglycone (free) curcumin. Studies were therefore performed to compare tumor cell (human MDA‐MB‐231 breast cancer cell [MDA])‐specific effects of glucuronidated curcumin (G‐CURC) vs. curcumin (CURC) in vitro and to assess site‐specific deglucuronidation of circulating G‐CURC in the bone microenvironment in the BCa bone metastases model.MethodsDose dependent effects of G‐CURC vs. CURC on Smad‐mediated TGFβ‐stimulated PTHrP secretion and their relative effects on constitutive Smad2 and 3 expression in MDA cells were determined by RIA and Western, respectively. G‐CURC and CURC levels were quantified directly, or after hydrolysis using β‐glucuronidase, by LC‐MS in either positive or negative modes in: 1) conditioned media of G‐CURC‐treated murine bone marrow (isolated from nude mice hind legs) or MDA BCa cells, or in 2) plasma and bone marrow specimens isolated from female nude mice treated in vivo with curcuminoids (100 mg/kg ip). Endogenous β‐glucuronidase enzyme was localized by immunohistochemical (IHC) staining in paraffin sections of decalcified metastases‐containing hind limbs of nude mice inoculated, via intracardiac injection 21 days previously, with MDA cells (1 × 105 cells).ResultsCURC dose‐dependently inhibited TGFβ stimulated PTHrP secretion from MDA cells and decreased constitutive Smad2 and 3 levels, while G‐CURC was without effect. G‐CURC levels in conditioned media after 4 hours of G‐CURC treatment of MDA cells were no different from levels detected in cell‐free media incubated under the same conditions. In contrast, G‐CURC levels were 37% lower (p < 0.05) in conditioned media obtained from bone marrow cells cultured under the same conditions. As has been reported in humans, the majority of circulating curcumin in mice was glucuronidated (94.5 ± 1.5%). Free curcumin levels were 4‐fold higher (p < 0.01) in the bone marrow (vs. plasma) of curcuminoid‐treated nude mice, representing 23.3 ± 5.2 % of total detectable curcumin (glucuronidated plus aglycone). Within the tumor/bone microenvironment, bone marrow cells expressed β‐glucuronidase, while MDA BCa tumor cells expressed little or no β‐glucuronidase.ConclusionResults from these studies suggest that G‐CURC may essentially act as a prodrug that is specifically activated (deconjugated) by murine bone marrow cells to form CURC, which can mediate anti‐tumor cell effects within bone that limit the progression of osteolytic human BCa metastases in turmeric‐treated mice. To our knowledge, these data provide the first evidence of bone‐specific targeting of active turmeric metabolites in vertebrates.Support or Funding InformationR01CA174926‐01, R01AT006896, and R03CA159382