Flaxseed Lignan Supplementation as Possible Adjuvant Therapy for Prostate and Breast Cancer
Dietary flaxseed lignans may have a chemopreventive and chemotherapeutic role against prostate and breast cancers. Flaxseed lignans, though, undergo an extensive first-pass effect and exist systemically primarily as glucuronide conjugates that are believed to be inactive. Their poor oral bioavailability (FO) likely explains the very modest benefit observed in human clinical studies. To fully realize the potential for lignan oral supplementation in prostate and breast cancer, pharmaceutical strategies are necessary to overcome the limitations imposed by poor FO. This dissertation involved proof-of-principle studies designed to first provide in vitro experimental evidence of flaxseed lignans on enhancing cytotoxicity of typical chemotherapeutic agents when used in combination, followed by the development of a pharmaceutical strategy to potentially exploit a role for lignans as adjuvant therapy against prostate cancer, and a human clinical trial to show oral safety and tolerability. Combination studies of lignans with chemotherapeutic agents were first conducted in prostate (PC3 and LNCap) and breast (SKBR3 and MDA-MB-231) cancer cell lines. SECO, the aglycone metabolite of the plant lignan, secoisolariciresinol diglucoside (SDG), was tested as it exhibits good FO but has had little investigation. As a major mammalian lignan converted from SECO, studies focused on enterolactone (ENL) and its glucuronic acid conjugate (ENL-Gluc). Typical chemotherapeutic agents with different mechanisms of action, including docetaxel, doxorubicin, cabazitaxel, MDV3100, and carboplatin were selected for combination experiments with lignans. A randomized, double-blind, placebo-controlled clinical trial in healthy older adults was conducted with oral supplementation of a standard flaxseed lignan-enriched complex (~38% SDG) equivalent to 600 mg SDG for 6-months, to assess efficacy, safety, and tolerability of flaxseed lignans. Finally, antibody directed enzyme prodrug therapy (ADEPT) system was generated by using anti-prostate specific membrane antigen (PSMA) antibody, D7 scFv as the carrier, and human β-glucuronidase (hβG) as the drug-converting enzyme. The binding affinity of fusion protein was assessed in purified PSMA as well as LNCap cells expressing cell surface PSMA. The enzymatic activity of D7-hβG fusion protein was determined using probe, 4-methylumberlliferone glucuronide, and prodrug, ENL-Gluc. C4-2 cells, expressing PSMA, were chosen to measure the conversion of ENL-Gluc into ENL by fusion protein in combination with docetaxel. In the in vitro combination studies, SECO and ENL enhanced sensitivity of cancer cells against therapeutic agents, in particular the ENL and docetaxel combination, while no obvious cytotoxicity was observed from ENL-Gluc. The in vivo assessment of flaxseed lignan-enriched product indicated no adverse side effects suggesting the safety and tolerability of flaxseed lignans for long-term oral exposure at a low pharmacological dose. Furthermore, plasma parent and total flaxseed lignans were significantly elevated in healthy older adults receiving lignan supplementation (n=19) compared with placebo (n=13), with large interindividual variation in systemic lignan levels observed. Interestingly, a significant reduction in systolic blood pressure (SBP) (from 155 mmHg to 140 mmHg) was observed in participants receiving treatment under the subcategory of SBP ³140 mmHg, while no change was observed in placebo group with SBP ³140 mmHg (n=6). Positive outcomes from in vitro and human clinical trial data supported investigations into an ADEPT strategy from which the fusion protein D7-hβG was successfully generated. The fusion protein displayed excellent binding against cell surface or purified PSMA, and favorable activity in production of active mammalian lignan, ENL, from ENL-Gluc. A slight decrease in the IC50 value was observed in the treatment group of D7-hβG with 100 µM ENL-Gluc plus docetaxel compared with docetaxel alone group in C4-2 cells. This proof-of-principle therapeutic strategy is the first attempt to expand the utility of flaxseed lignans as adjuvant therapy against prostate cancer. With the advantage of the safety and tolerability profile following convenient oral lignan consumption, this dissertation research warrants further evaluations of the ADEPT strategy as adjuvant therapy against prostate as well as breast cancer both in vitro and in vivo. In general, this dissertation provides science-based evidence to support the health benefits of flaxseed lignan-enriched product following oral consumption as a natural health product (NHP) which is required by Health Canada.
Flaxseed lignans, Adjuvant therapy, ADEPT
Doctor of Philosophy (Ph.D.)
Pharmacy and Nutrition