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

Anti-Gouty Arthritis and Anti-Hyperuricemia Properties of Sanghuangporus vaninii and Inonotus hispidus in Rodent Models

Acute inflammation and hyperuricemia are associated with gouty arthritis. As an edible and therapeutic mushroom, Sanghuangporus vaninii (SV) has an inhibitory effect on tumorigenesis, and Inonotus hispidus (IH) exhibits anti-tumor, anti-inflammatory, and antioxidant properties. In this study, uric acid (UA) and xanthine oxidase (XOD) levels in hyperuricemic mice were examined to determine the regulatory effects of SV and IH. SV and IH reversed the pathogenic state of elevated UA levels in the serum and reduced levels of XOD in the serum and liver of mice with hyperuricemia. SV and IH affected the inflammatory response in rats with acute gouty arthritis. Compared to vehicle-treated rats, monosodium urate crystals (MSU) increased the swelling ratio of the right ankle joints. SV and IH administration significantly reduced swelling and inflammatory cell infiltration. SV reduced the levels of interleukin-8 (IL-8) and chemokine ligand-2 (CCL-2), whereas IH reduced the levels of matrix metalloproteinase-9 (MMP-9), CCL-2, and tumor necrosis factor-α (TNF-α), which were confirmed in articular soft tissues by immunohistochemistry. In summary, our data provide experimental evidence for the applicability of SV and IH in gouty arthritis and hyperuricemia treatment

SERS determination of hydroxy-α-sanshool in spicy hotpot seasoning: The strategy to restrain the interference of capsaicin and its mechanism

Hydroxy-alpha-sanshool (alpha-SOH) is the principal ingredient responsible for the numbing sensation in spicy hotpot. However, utilizing surface-enhanced Raman scattering (SERS) to analyze the alpha-SOH in hotpot seasoning is challenging due to the significant interference of capsaicin (CAP). Therefore, two schemes were proposed to address CAP interference in hotpot seasoning, namely laccase-catalyzed conversion and metal-organic frame-work (MOF) interaction. Among them, Fe-BTC MOF exhibited significant anti-interference effect and the un-derlying mechanism is elucidated. The motion of CAP aromatic ring was constrained by steric hindrance and electrostatic interactions of Fe-BTC. Additionally, the interaction between CAP aromatic ring and conjugated triene group in alpha-SOH was quenched, enhancing the alpha-SOH SERS signal. The proposed method had a significant anti-interference effect on alpha-SOH quantification in the presence of CAP, significantly enhancing the alpha-SOH SERS signal in a range of 0.85 to 4.00 x 107. The linearity and reproducibility of the proposed hotpot seasoning testing method were also validated

SARS-CoV-2 Variant-Specific Infectivity and Immune Profiles Are Detectable in a Humanized Lung Mouse Model

Small animal models that accurately model pathogenesis of SARS-CoV-2 variants are required for ongoing research efforts. We modified our human immune system mouse model to support replication of SARS-CoV-2 by implantation of human lung tissue into the mice to create TKO-BLT-Lung (L) mice and compared infection with two different variants in a humanized lung model. Infection of TKO-BLT-L mice with SARS-CoV-2 recapitulated the higher infectivity of the B.1.1.7 variant with more animals becoming infected and higher sustained viral loads compared to mice challenged with an early B lineage (614D) virus. Viral lesions were observed in lung organoids but no differences were detected between the viral variants as expected. Partially overlapping but distinct immune profiles were also observed between the variants with a greater Th1 profile in VIDO-01 and greater Th2 profile in B.1.1.7 infection. Overall, the TKO-BLT-L mouse supported SARS-CoV-2 infection, recapitulated key known similarities and differences in infectivity and pathogenesis as well as revealing previously unreported differences in immune responses between the two viral variants. Thus, the TKO-BLT-L model may serve as a useful animal model to study the immunopathobiology of newly emerging variants in the context of genuine human lung tissue and immune cells

Snail Contributes to the Maintenance of Stem Cell-Like Phenotype Cells in Human Pancreatic Cancer

<div><p>Snail, a potent repressor of E-cadherin expression, plays a key role in epithelial-to-mesenchymal transition (EMT) in epithelial cancer. Recently, EMT and stemness programs are found linked together. In the current study, the expression of Snail and its contribution to cancer stem cell (CSC) marker expression, invasiveness, self-renewal, clonogenicity, and tumorigenicity of pancreatic cancer cells were studied. Our results showed that Snail was highly expressed in CSC^high cell line Panc-1. Stable, short hairpin RNA (shRNA)-mediated Snail knockdown decreased invasion in Panc-1 cells, in line with increased E-cadherin expression and its translocation from the nucleus to the membrane. Snail silencing in Panc-1 also inhibited CSC marker ALDH expression, together with decreased sphere and colony forming capacity, which was highly consistent with the expression of stem cell associated transcription factors like Sox2 and Oct4. In mouse xenograft models, knockdown of Snail led to a reduced number of tumor-bearing mice and a reduced average size of tumors, which had a stronger membrane staining of E-cadherin and lighter staining of Oct4. Collectively, these findings implicate Snail is required for the maintenance of stem cell-like phenotype in pancreatic cancer, and inhibition of Snail could be an efficient strategy to treat pancreatic cancer by targeting CSCs.</p></div

Snail is crucial for properties attributed to cancer stem cells.

<p>A. Snail silencing decreases ALDH^high population in Panc-1 cells. Dot plots of cells analyzed by flow cytometry for ALDH activity. The values presented are the averages of three independent experiments. B. Snail silencing significantly inhibits the ability of sphere formation with serial passaging and the capability of the clonogenicity in Panc-1 cells. Representative pictures of colony are shown above the column diagram. Similar experiments were repeated three times. ** P<0.01, compared with Panc-1/NC. C. Western blot analysis of cell extracts from Panc-1/NC and Panc-1/shSnail cells for Bmi1, Nanog, Sox2, and Oct4 expression. GAPDH was used as a loading control. D. Quantification of protein levels of Bmi1, Nanog, Sox2, and Oct4 in Panc-1/NC and Panc-1/shSnail cells. * P<0.05, **P<0.01 compared with Panc-1/NC.</p