Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways.

Degenerative disc disease (DDD) is an important cause of low back pain. Repetitive tensile stress from the daily motion of the spine predisposes it to injury of the annulus fibrosus (AF) which causes IVD degeneration. This study aims to determine the causal relationship between mechanical stretch and osteogenesis in the AF cells of IVD as affected by bone morphogenic proteins (BMPs), specifically BMP-2/6 heterodimers. Our results found that 15% tensile stress (high cyclic stretching, HCS) may induce the expression of osteogenesis-related markers (Runx2, osterix) by upregulating BMP-2/6 heterodimeric ligands and their receptors on the human AF cell line. HCS also induced transient phosphorylation of p38 mitogen-activated protein (MAP) kinase and SMAD1/5/8. Neutralizing antibodies to the BMP-2/6 receptor (ALK3) blocked the expression of Runx2 and osterix, as well as the phosphorylation of p38 and SMAD1/5/8. In addition, treatment with a p38 MAPK inhibitor (SB203580) or siRNA to neutralize the effects of SMAD1/5/8 suppressed tensile stress-induced Runx2 and osterix expression. Mechanical stretching induces activation of p38 MAP kinase and SMAD1/5/8 signaling pathways, followed by the upregulation of BMP-2/6 heterodimer expression, thereby stimulating osteogenic Runx2 and osterix expression on AF cells. HCS may accelerate the progression of IVD degeneration by promoting an osteogenic response.

Inhibition of NLRP3 by Fermented Quercetin Decreases Resistin-Induced Chemoresistance to 5-Fluorouracil in Human Colorectal Cancer Cells.

The drug resistance of colorectal cancer (CRC) cells against 5-fluorouracil (5-FU) therapy is a major challenge to successful cancer treatment. While previous studies have proposed several 5-FU resistance mechanisms, the effects of the adipokines on cancer cells remain unclear. Thus, this study investigated the effect of resistin on 5-FU-treated CRC cell lines. The upregulation of NLRP3 can regulate the inflammatory responses in cancer cells and then enhance cancer progression. This study investigated the expression level and the function of NLRP3 on 5-FU-induced cytotoxicity in CRC cells and found that resistin-induced ERK activation and increased NLRP3 expression in CRC HCT-116 and DLD-1 cells were mediated by Toll-like receptor 4 (TLR4). The inhibition of TLR4 and ERK by pharmacological inhibitors attenuated the resistin-induced NLRP3 mRNA and protein levels. In contrast, the knockdown of NLRP3 enhanced the cytotoxic effects of 5-FU. Furthermore, quercetin is an effective chemopreventive compound. This study showed that quercetin fermented by Lactobacillus could exhibit low cytotoxicity on normal mucosa cells and improve the function of inhibiting CRC cells. The treatment of CRC cells with fermented quercetin increased the cytotoxicity and enhanced cell death in the presence of resistin. In this study, fermented quercetin induced the cytotoxicity and cell death of 5-FU in resistin-treated CRC cells, which is associated with the downregulation of NLRP3 expression and ERK phosphorylation. These results indicate the role of NLRP3 in the development of drug resistance to 5-FU in CRC cells. Elucidating the mechanism regarding the cytotoxicity effect of quercetin may provide another vision for the development of a chemotherapy strategy for CRC in the future.

6-Shogaol Antagonizes the Adipocyte-Conditioned Medium-Initiated 5-Fluorouracil Resistance in Human Colorectal Cancer Cells through Controlling the SREBP-1 Level.

The resistance of colorectal cancer (CRC) to chemotherapy, e.g., 5-fluorouracil (5-FU), is an impediment to successful cancer treatment. Although many mechanisms have been proposed to explain the occurrence of resistance, little is known concerning the role of the adipocyte-containing microenvironment of CRC. Accumulating data have proposed that the combined therapy of clinical drugs with ginger derivatives, e.g., 6-shogaol, might improve resistance development. In the present study, we examined the effect of adipocyte-conditioned medium (ACM) on 5-FU-treated CRC cells (human DLD-1 and SW480 cells) and further examined the possible antagonized role of 6-shogaol in this situation. It was shown that the level of sterol-regulatory element-binding protein-1 (SREBP-1), a critical transcription factor involved in lipid synthesis and metabolism, would be upregulated through Akt and p70S6K signaling pathways while CRC cells are cultured in ACM, which subsequently decreases the cell sensitivity to 5-FU cytotoxicity. Moreover, our results also demonstrated the antagonized role of 6-shogaol in attenuating the ACM effects on CRC cells through activating AMPK signaling. Overall, the present study elucidated the role of adipocyte-containing microenvironment in 5-FU resistance development of CRC through controlling the SREBP-1 level and further enhanced the concept of clinical application of 6-shogaol and AMPK signaling in CRC therapy.

Drug Resistance of CPT-11 in Human DLD-1 Colorectal Cancer Cells through MutS Homolog 2 Upregulation.

Colorectal cancers (CRCs) is the most commonly diagnosed and deadly cancer types in the world. Despite advances in chemotherapy for CRCs, drug resistance remains a major challenge to high incurable and eventually deadly rates for patients. CPT-11 is one of the current chemotherapy agents for CRC patients and the CPT-11 resistance development of CRCs is also inevitable. Recently, accumulating data has suggested that DNA repair system might be an inducer of chemotherapy resistance in cancer cells. Thus, this study was aimed to examine whether MutS homolog (MSH) 2, one member of DNA repair system, plays a role to affect the cytotoxicity of CPT-11 to CRCs. Human DLD-1 CRC cells were used in this study. It was shown that MSH2 gene and protein expression could be upregulated in DLD-1 cells under CPT-11 treatment and this upregulation subsequently attenuates the sensitivity of DLD-1 cells to CPT-11. Moreover, ERK1/2 and Akt signaling and AP-1 transcription factor have been found to modulate these effects. These results elucidate the drug resistance role of MSH2 upregulation in the CPT-11-treated DLD-1 CRC cells. Our findings may provide a useful thought for new adjuvant drug development by controlling the DNA repair system.

The antagonism of 6-shogaol in high-glucose-activated NLRP3 inflammasome and consequent calcification of human artery smooth muscle cells.

BACKGROUND: Vascular calcification is the major reason for high mortality of cardiovascular complications for diabetes. Interleukin (IL)-1ß has been implicated in this pathogenesis, but its precise role and clinical evidence have not been clearly identified. Hence, this study was aimed to investigate whether high concentration of glucose (HG), which mimics the hyperglycemia environment, could initiate vascular calcification through NLRP3/IL-1ß inflammasome and the underlying mechanism. Recently, 6-shogaol, a major ginger derivate, has been elucidated its pharmaceutic role for various diseases. Therefore, the aims of this study also determined 6-shogaol effect in vascular calcification of HG initiation. RESULT: Human artery smooth muscle cells (HASMCs) were used in this study. Glucose concentrations at 5 and 25 mM were defined as normal and HG status, respectively. The results showed that HG could increase the NLRP3, cleaved caspase 1, and pro/mature IL-1ß levels to induce the expressions of bone-related matrix proteins and subsequent HASMC calcification. This process was regulated by Akt activation and reactive oxygen species (ROS) production. Moreover, 6-shogaol could inhibit the Akt/ROS signaling and NLRP3/caspase 1/IL-1ß inflammasome and hence attenuated HASMC calcification. CONCLUSIONS: This study elucidates the detailed mechanism of HG-initiated HASMC calcification through NLRP3/caspase 1/IL-1ß inflammasome and indicates a potential therapeutic role of 6-shogaol in vascular calcification complication of diabetes.