Autophagy Blockade by Ai Du Qing Formula Promotes Chemosensitivity of Breast Cancer Stem Cells Via GRP78/ß-Catenin/ABCG2 Axis.

Accumulating evidence suggests that the root of drug chemoresistance in breast cancer is tightly associated with subpopulations of cancer stem cells (CSCs), whose activation is largely dependent on taxol-promoting autophagy. Our pilot study identified GRP78 as a specific marker for chemoresistance potential of breast CSCs by regulating Wnt/ß-catenin signaling. Ai Du Qing (ADQ) is a traditional Chinese medicine formula that has been utilized in the treatment cancer, particularly during the consolidation phase. In the present study, we investigated the regulatory effects and molecular mechanisms of ADQ in promoting autophagy-related breast cancer chemosensitivity. ADQ with taxol decreasing the cell proliferation and colony formation of breast cancer cells, which was accompanied by suppressed breast CSC ratio, limited self-renewal capability, as well as attenuated multi-differentiation. Furthermore, autophagy in ADQ-treated breast CSCs was blocked by taxol via regulation of ß-catenin/ABCG2 signaling. We also validated that autophagy suppression and chemosensitizing activity of this formula was GRP78-dependent. In addition, GRP78 overexpression promoted autophagy-inducing chemoresistance in breast cancer cells by stabilizing ß-catenin, while ADQ treatment downregulated GRP78, activated the Akt/GSK3ß-mediated proteasome degradation of ß-catenin via ubiquitination activation, and consequently attenuated the chemoresistance-promoted effect of GRP78. In addition, both mouse breast cancer xenograft and zebrafish xenotransplantation models demonstrated that ADQ inhibited mammary tumor growth, and the breast CSC subpopulation showed obscure adverse effects. Collectively, this study not only reveals the chemosensitizating mechanism of ADQ in breast CSCs, but also highlights the importance of GRP78 in mediating autophagy-promoting drug resistance via ß-catenin/ABCG2 signaling.

Agkistrodon ameliorates pain response and prevents cartilage degradation in monosodium iodoacetate-induced osteoarthritic rats by inhibiting chondrocyte hypertrophy and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteoarthritis (OA), characterized by joint pain and cartilage degradation, is the most common form of joint disease worldwide but with no satisfactory therapy available. The ethanol extract of Agkistrodon acutus (EAA) has been widely used as a traditional Chinese medicine (TCM) for the treatment of arthralgia and inflammatory diseases, but there is no report regarding its efficacy on OA to date. Here, we determined the effects of EAA on the pain behavior and cartilage degradation in vivo and clarified its target genes and proteins associated with chondrocyte hypertrophy and apoptosis in vitro. MATERIALS AND METHODS: In vivo OA model was established by intra-articular injection (1.5 mg) of monosodium iodoacetate (MIA) into rats and weekly treated by intra-articular administration of EAA at a dose range from 0.3 to 0.9 g/kg for four weeks. The pain behavior parameters, thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT) were tested before and after the treatment. Then histopathologic, immunohistochemical and TUNEL analyses of the articular cartilage were conducted, followed by Mankin's scoring. In vitro, the effects of EAA on chondrocytes were evaluated via assays of cell viability, immunofluorescence, real time PCR, and Western blot. UPLC-MS was applied to determine the chemical composition of EAA. RESULTS: The animal data showed that EEA not only attenuated the pain hypersensitivity but also blocked the cartilage degeneration by improving chondrocyte survival and suppressing chondrocyte apoptosis at a dose-dependent manner in OA rats. Furthermore, EAA remarkably restored the abnormal expression of collagen type II (Col2) and matrix metalloproteinase-13 (MMP13) in cartilage of OA rats. The cellular data showed that EAA significantly increased the cell viability of chondrocytes against OA-like damage and restored the abnormal expressions of Col2 and MMP13 in damaged chondrocytes. The molecular data showed that EAA significantly restored the abnormal mRNA expressions of Col2, Col10, MMP2 and MMP13 as well as the abnormal protein expressions of MMP13, PARP (total and cleaved) in chondrocytes under pathological condition. UPLC-MS analysis showed the known main components of EAA, including amino acides (glycine, L-aspartic acid, L-glutamic acid, and L-hydroxyproline), nucleoside (uridine), purines (xanthine and hypoxanthine), and pyrimidine (uracil). CONCLUSIONS: Our data demonstrate that EAA exerts antinociceptive and chondroprotective effects on OA through suppressing chondrocyte hypertrophy and apoptosis with restoration of the molecular expressions of anabolism and catabolism in chondrocytes. It provides a promising TCM candidate of novel agent for OA therapy.

Intra-Articular Injection of Fructus Ligustri Lucidi Extract Attenuates Pain Behavior and Cartilage Degeneration in Mono-Iodoacetate Induced Osteoarthritic Rats.

Fructus Ligustri Lucidi (FLL) has been widely used as a traditional Chinese medicine (TCM) for treating soreness and weakness of waist and knees. It has potential for treating OA owing to its kidney-tonifying activity with bone-strengthening effects, but there is so far no report of its anti-OA effect. This study established a rat OA model by intra-articular (IA) injection of mono-iodoacetate (1.5 mg) and weekly treated by IA administration of FLL at 100 µg/mL for 4 weeks. Thermal withdrawal latency, mechanical withdrawal threshold, and spontaneous activity were tested for evaluation of pain behavior, and histopathological (HE, SO, and ABH staining) and immunohistochemical (Col2, Col10, and MMP13) analyses were conducted for observation of cartilage degradation. In vitro effect of FLL on chondrocytes was evaluated by MTT assay and qPCR analysis. Moreover, HPLC analysis was performed to determine its chemoprofile. The pain behavioral data showed that FLL attenuated joint pain hypersensitivity by increasing thresholds of mechanical allodynia and thermal hyperalgesia as well as spontaneous activity. The histopathological result showed that FLL reversed OA cartilage degradation by protecting chondrocytes and extracellular matrix in cartilage, and the immunohistochemical analysis revealed its molecular actions on protein expressions of MMP13, Col2, and Col10 in cartilage. The MTT assay showed its proliferative effects on chondrocytes, and qPCR assay clarified its mechanism associated with gene expressions of Mmp13, Col2, Col10, Adamts5, Aggrecan, and Runx2 in TNF-α treated chondrocytes. Our results revealed an anti-OA effect of FLL on pain behavior and cartilage degradation in OA rats and clarified a molecular mechanism in association with the suppression of chondrocyte hypertrophy and catabolism. IA FLL can be regarded as novel and promising option for OA therapy.