Alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone inhibits cell proliferation, invasion, and migration in gastric cancer in part via autophagy.

Gastric cancer is a leading cause of mortality worldwide. Alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone is a type of limonoid mainly isolated from Cedrela odorata (Meliaceae) that has been shown to suppress cell proliferation in several human carcinoma cell lines. In this study, we investigated the anti-cancer ability of alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone and its underlying mechanism in MKN45 cells. Alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone induced excess reactive oxygen species (ROS) accumulation. Transwell and wound healing assays demonstrated that alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone inhibited the invasion and migration ability of MKN45 cells. Moreover, autophagy-related proteins Beclin-1, Atg5, and Atg7 were up-regulated. Light chain 3 (LC3)-I protein was converted into LC3-II under alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone exposure. Transmission electron microscopy demonstrated that alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone treatment resulted in the formation of autophagosomes. Immunofluorescence assays suggested that alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone treatment elicited dot formation of green fluorescent protein (GFP)-LC3. 3-methyladenine (3-MA), an autophagy inhibitor, demonstrated that autophagy promoted death in MKN45 cells. Western blotting showed that ROS/mitogen activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways play crucial roles in the intrinsic mechanism of alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone's activity. The combined use of N-acetyl-L-cysteine (NAC) or U0126 validated the regulatory role of ROS/MEK/ERK signaling pathways. Alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone administration inhibited the growth of MKN45 xenograft tumors in nude mice and suppressed Ki67 expression. More importantly, a similar effect was achieved in a patient-derived xenograft (PDX) model, which is more relevant to clinical application. Taken together, alpha, 2'-dihydroxy-4,4'-dimethoxydihydrochalcone has the potential to be further developed into an anti-tumor agent for clinical treatment of gastric cancer.

PD-L1 is a critical mediator of regulatory B cells and T cells in invasive breast cancer.

Regulatory T cells (Tregs), a key mediator in regulating anti-tumor immune suppression, tumor immune escape, metastasis and relapse, are considered an important therapeutic target in immunotherapy of human cancers. In the present investigation, elevated CD19+ CD24+ CD38+ regulatory B cells (Bregs) were observed in PBMCs of invasive carcinoma of breast (IBCa) patients compared with that in patients with fibroadenoma (FIBma) or healthy individuals, and the positive correlation existed between Bregs and CD4+ CD25+ CD127- Tregs (r = 0.316, P = 0.001). We found that PD-L1 expression was higher on Bregs in IBCa patients compared with patients with FIBma or healthy individuals (P < 0.05, respectively), and that a tight correlation exists between CD19+ CD24+ CD38+ PD-L1+ Bregs and CD19+ CD24+ CD38+ Bregs (r = 0.267, P = 0.007), poor TNM phases and up-regulated expression of PD-L1 on Bregs. The pattern of PD-1 expression on CD4+ T cells indicated that high level of PD-1hi expressed on CD4+ CD25+ CD127+ effector T cells (P < 0.001). More importantly, the presence of PD-L1 on Bregs was positively correlated with Tregs (r = 0.299, P = 0.003), but negatively correlated with PD-1hi effector T cells (r = -0.22, P = 0.031). Together, results of the present study indicated that PD-L1 is an important molecule on Bregs, mediated the generation of Tregs in IBCa.

PD-L1 mediated the differentiation of tumor-infiltrating CD19+ B lymphocytes and T cells in Invasive breast cancer.

Accumulating evidence suggests that B cells play important roles in inhibiting the immune response in autoimmune disorders and human tumors as well as murine tumor models. In an effort to explore the role of B cells in human breast cancer etiology, we examined the presence of CD19+ B lymphocytes in 134 cases of invasive breast carcinoma (IBCa) and 31 breast fibroadenoma, and assessed its relationship with PD-L1 (programmed death-ligand 1) expression in breast cancer. We found that the density of CD19+ B lymphocytes was higher in IBCa compared with fibroadenoma, and significantly associated with increasing tumor grade, negative estrogen status. Similar findings were observed for the expression of IL-10 in IBCa. Meanwhile, CD19+ B lymphocytes were shown to be highly coincident with PD-L1 and IL-10 in IBCa. We further demonstrated that CD19+ B cells can differentiate into CD19+CD24+CD38+ B cells when co-cultured with PD-L1hi MDA-MB231 cells. In addition, the percentage of CD19+CD24+CD38+ B cells was higher in breast tissue and peripheral blood cells of IBCa patients than that of benign tumor and health individuals. And CD19+CD24+CD38+ B cells were found to be IL-10 secreting B cells. Finally, we showed that CD19+ B cells from IBCa patients but not healthy individuals induced formation of CD4+CD25+Foxp3+ T cells when co-cultured with T cells from IBCa patients and healthy subjects (80.4% and 30.8% respectively). The induction of CD4+CD25+Foxp3+ T cells by CD19+ B cells was further shown to be mediated by PD-L1. Together, these results are suggestive of a role for CD19+ B lymphocytes in immune suppression and tumor evasion via PD-L1 in breast cancer.

Curcumin attenuated acute Propionibacterium acnes-induced liver injury through inhibition of HMGB1 expression in mice.

Curcumin is a phenolic product isolated from the rhizome of Curcuma longa and has protective effects on inflammatory diseases. Here we investigated the protective effect of curcumin in acute Propionibacterium acnes (P. acnes)-induced inflammatory liver injury. C57BL/6 mice were primed with P. acnes followed by LPS challenge to induce fulminant hepatitis. Curcumin or vehicle control was administered perorally by gavage once daily starting 2days before P. acnes priming. We found that curcumin significantly improved mouse mortality. Then, to investigate the underlying mechanisms of curcumin in this acute inflammatory liver injury model, we primed C57BL/6 mice with P. acnes only. We found that curcumin treatment attenuated P. acnes-induced liver injury as evidenced by decreased production of ALT. In addition, curcumin treatment reduced the production of proinflammatory cytokines such as TNF-α and IFN-γ, accompanied by reduced hepatocyte apoptosis. Furthermore, curcumin treatment significantly reduced HMGB1 cytoplasmic translocation and expression by down-regulating acetylation of lysine. Taken together, our results suggest that curcumin protects mice from P. acnes-induced liver injury through reduction of HMGB1 cytoplasmic translocation and expression.

Increased prediction of right nonrecurrent laryngeal nerve in thyroid surgery using preoperative computed tomography with intraoperative neuromonitoring identification.

BACKGROUND: A nonrecurrent laryngeal nerve (NRLN) is a rare but potentially serious anatomical variant. Although the incidence is reported to be 0.3% to 1.3%, it carries a much higher risk of palsy during thyroid surgery. The objective of this study is to investigate the usefulness of computed tomography (CT) for preoperative identification and intraoperative neuromonitoring identification (IONM) of NRLN in thyroid cancer patients. METHODS: The preoperative neck CT scans from 1,574 patients who needed thyroid surgery were examined. Absence of the brachiocephalic artery (BCA) and the presence of arteria lusoria were defined as positive with NRLN. Systematic intraoperative neuromonitoring (IONM) was also carried out for these 1,574 patients to localize and identify NRLN. A negative electromyography (EMG) response from lower vagal stimulation but a positive EMG response from the upper position indicated the occurrence of an NRLN. RESULTS: Nine NRLN (0.57%) were intraoperatively identified out of the 1,574 patients, and no patient with a NRLN showed preoperative clinical symptoms related to NRLN. Prior to the operation, surgeons identified only seven suspected NRLN cases based on identification of arteria lusoria. But a review of CT scans revealed that all cases could be identified by vascular anomalies. All patients were successfully detected at an early stage of operation using intraoperative neuromonitoring (IONM). Postoperative vocal cord function was normal in all patients. CONCLUSIONS: CT of the neck is a reliable method for predicting NRLN before thyroid cancer surgery. However, some image features can be easily missed. Neurophysiology helps the surgeon to identify the NRLNs more precisely. Combining the two evaluation methods may decrease the incidence of nerve palsy, especially in cases of NRLN. Considering that CT is expensive, requires an X-ray, and achieves less information than ultrasound (US) concerning thyroid nodules, we suggest that applying US and IONM is more reasonable.

ZNRF3 acts as a tumour suppressor by the Wnt signalling pathway in human gastric adenocarcinoma.

E3 ubiquitin ligases regulate a variety of biological processes through the ubiquitin-proteasome system, together with ubiquitin activating enzyme E1 and ubiquitin-conjugating enzyme E2. Previous studies have demonstrated that zinc and ring finger 3 (ZNRF3), which belongs to the E3 ubiquitin ligases family is involved in the Wnt signalling pathway, which plays an important role in causing cancer. However, the expression and function of ZNRF3 in human gastric adenocarcinoma still remains unclear. Immunohistochemical and western blot analysis showed a significant down-regulation of ZNRF3 protein in gastric adenocarcinoma tissues compared with adjacent normal gastric tissues. In addition, there was a correlation between the down-regulation of ZNRF3 and poor tissue differentiation in gastric adenocarcinoma. To investigate the potential function of ZNRF3 in cell proliferation and apoptosis, a gastric cell line SGC7901 was employed. The over-expression of wild-type ZNRF3, which was accomplished by the transient transfection of recombinant pEGFP-ZNRF3 (or empty plasmids as control) into the cell line SGC7901, was confirmed by western blot analysis. Flow-cytometry-based and Cell Counting Kit-8 assays showed that over-expression of wt ZNRF3 induced apoptosis and suppressed proliferation. ZNRF3-overexpressing gastric cells displayed partly attenuated protein levels of beta-catenin and TCF-4 compared with those transfected with the empty plasmid. Our study demonstrates a novel gastric adenocarcinoma suppressor and reveals that ZNRF3 inhibits gastric cancer cell growth and promotes the cell apoptosis by affecting the Wnt/beta-catenin/TCF signalling pathway.

Collagen membrane alleviates peritendinous adhesion in the rat Achilles tendon injury model.

BACKGROUND: Tendon adhesion is one of the most common causes of disability following tendon surgery. Therefore, prevention of peritendinous adhesion after surgical repair of tendon is a major challenge. The aim of this study was to explore the possible application of a collagen membrane for the prevention or attenuation of peritendinous adhesions. METHODS: Sprague-Dawley (SD) rat Achilles tendon was cut and sutured by a modified Kessler's technique with or without the collagen membrane wrapped. Macroscopic, morphological and biomechanical evaluations were applied to examine the recovery of the injured tendon at 4 and 8 weeks after surgery. RESULTS: The surgery group wrapped by collagen membranes had a better outcome than the group with surgery repair only. In the collagen membrane-treated group, less adhesion appeared, stronger tensile strength was detected, and more tendon fibers and collagen I expression were observed morphologically. CONCLUSION: Wrapping the tendon with a collagen membrane may be an efficient approach for tendon repair and preventing tendon adhesion after its ruptures.

Blockade of 4-1BB/4-1BB ligand interactions prevents acute rejection in rat liver transplantation.

BACKGROUND: Blocking the 4-1BB/4-1BB ligand (4-1BBL) signal may modulate the secretion of Th1/Th2 cytokines and prolong the survival of the grafts, which play a key role in organ transplantation tolerance. The aim of this study was to investigate the role of blockade of the 4-1BB/4-1BBL co-stimulatory pathway with 4-1BBL monoclonal antibody (mAB) in acute rejection of rat orthotopic liver transplantation. METHODS: The orthotopic liver transplantation model was set up, while male Lewis rats were used as liver donors and Brown-Norway rats as recipients. The recipient rats were intravenously injected with anti 4-1BBL mAB or isotype control antibody. Groups were monitored for graft survival after transplantation. Plasma chemistry, including aspartate transaminase (AST), alanine aminotransferase (ALT), and bilirubin (BIL), was assayed. The concentrations of interleukin (IL)-2, IL-10 and interferon (IFN)-gamma in plasma were also measured by enzyme-linked immunosorbent assay. Allograft histology images were collected under light microscope and electron microscope. RESULTS: Isotype antibody treated recipients exhibited elevated plasma levels of liver injury markers including AST, ALT and BIL, progressive portal and venous inflammation and cellular infiltration of the liver allografts, and a mean graft survival time (MST) of 10.9 days. Administration of anti 4-1BBL mAB resulted in a decrease in plasma levels of liver injury markers and the concentrations of IL-2, IL-10 and IFN-gamma. The histological grade of rejection on day 7 decreased and MST (17.3 days) increased substantially. CONCLUSIONS: These results demonstrate that attenuation of acute rejection follows the blockade of the 4-1BB/4-1BBL co-stimulatory pathway with 4-1BBL monoclonal antibody and strongly suggest it is a promising strategy to prevent progression of graft rejection by suppressing T cell-mediated immunity.

RASSF1A expression inhibits cell growth and enhances cell chemosensitivity to mitomycin in BEL-7402 hepatocellular carcinoma cells.

BACKGROUND: The antitumor role of Ras association domain family 1A (RASSF1A) gene and its potential molecular mechanisms are not well understood. The objective of this study was to observe the antitumor ability of RASSF1A in hepatocellular carcinoma, and study the mechanisms of cell apoptosis induced by RASSF1A. METHODS: After stably transfecting a RASSF1A (wild-type or mutant) expression vector into the BEL-7402 hepatocellular carcinoma cell line, RT-PCR and Western blotting was used to detect the RASSF1A expression levels in recombinant cells. The effects of wild-type RASSF1A on cell growth were observed in vitro by analyzing cell proliferation rate, cell colony formation, and in vivo by analyzing tumorigenesis in nude mice. In addition, the effect of RASSF1A gene expression on the chemosensitivity of human hepatocellular carcinoma cells to antitumor drugs was examined by inhibition of cell proliferation and the percentage of apoptotic cells. RESULTS: Wild-type RASSF1A, not the mutant, suppressed cell growth in vitro and in vivo. Re-expression of wild-type RASSF1A could enhance the inhibition of cell proliferation and the percentage of apoptotic cells following cell treatment with mitomycin, but had no significant effect when combined with adriamycin, etoposide, 5-fluorouracil and cisplatin treatment. CONCLUSION: Wild-type RASSF1A inhibits cell growth and enhances cell chemosensitivity to mitomycin in hepatocellular carcinoma, suggesting that RASSF1A may serve as a new target for gene therapy in hepatocellular carcinoma patients.

RASSF1A expression inhibits the growth of hepatocellular carcinoma from Qidong County.

BACKGROUND AND AIM: The tumor-suppressing role of Ras-association domain family 1A (RASSF1A) has been described in several systems. In this study, we tested its tumor-suppressing ability and the potential molecular mechanisms in hepatocellular carcinoma (HCC) from Qidong County. METHODS: Reverse transcription polymerase chain reaction and Northern blotting were employed to detect the expression of RASSF1A in HCC. After establishing stable RASSF1A (wild type or mutant) expressing 'qi dong gan ai yan jiu suo' ([Qidong Institute of Liver Cancer] QGY)-7703 cell lines, we tested the effects of RASSF1A expression on cell growth by cell proliferation rate, cell colony formation, and cell cycle progression. We also tested the effects of RASSF1A expression on tumorigenesis in nude mice and on cellular sensitivity to mitomycin treatment. RESULTS: The RASSF1A transcript was not found in 75% (three of four) of HCC cell lines and 67% (32/48) of HCC primary biopsies. The stepwise regression analyses indicated that the loss of RASSF1A expression was more frequent in patients who were hepatitis B virus surface antigen positive (HBsAg+) compared to those who were HBsAg(-), both in tumor and corresponding non-cancerous tissues. The wild-type (wt)-RASSF1A expression in the QGY-7703 cell line resulted in fewer and smaller clones, decreased xenograft tumor volume and weight, and G(1)/S arrest in vitro and in vivo. The wt-RASSF1A expression also decreased the cyclin D1 protein expression, which appeared to be at the level of post-transcriptional control. In addition, the wt-RASSF1A expression increased cell growth inhibition and the percentage of cells with sub-G(1) DNA content when the cells were treated with mitomycin. CONCLUSION: RASSF1A is a tumor suppressor in HCC. The loss of RASSF1A expression may be related to HBsAg+ in hepatocarcinogenesis. Its inactivation may play an important role in the development of HCC.