Huanhai Liu

Reactive oxygen species‐mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to polydatin‐induced apoptosis in human nasopharyngeal carcinoma CNE cells

Previous studies revealed that polydatin, a natural small compound, possessed protective effect against ischemia/reperfusion injury and inflammation. However, the action and molecular mechanism of its potent anti‐cancer activity remain poorly understood. In the present study, polydatin significantly killed several human tumor cell lines in a dose‐ and time‐dependent manner. The compound also dose‐dependently caused mitochondrial apoptosis in human nasopharyngeal carcinoma CNE cells. In addition, polydatin triggered endoplasmic reticulum (ER) stress and down‐regulated the phosphorylation of Akt in CNE cells, while knock‐down of CCAAT/enhancer‐binding protein homologous protein (CHOP) dramatically abrogated the inactivation of Akt and reversed the pro‐apoptotic effect of polydatin. Furthermore, polydatin provoked the generation of reactive oxygen species in CNE cells, while the antioxidant N‐acetyl cysteine almost completely blocked the activation of ER stress and apoptosis, suggesting polydatin‐induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways in CNE cells. Taken together, these findings strongly suggest that polydatin might be a promising anti‐tumor drug and our data provide the molecular theoretical basis for clinical application of polydatin. J. Cell. Biochem. 112: 3695–3703, 2011.

Reactive oxygen species-mediated mitochondrial dysfunction is involved in apoptosis in human nasopharyngeal carcinoma CNE cells induced by Selaginella doederleinii extract.

ETHNOPHARMACOLOGICAL RELEVANCE A traditional Chinese medicine Selaginella doederleinii Hieron has been combined with radiotherapy for the treatment of human nasopharyngeal carcinoma in clinic in China. However, the detailed mechanism of anti-tumor effect of Selaginella doederleinii remains elusive. AIM OF THE STUDY This study was designed to investigate the anti-tumor effect of ethanol extract of Selaginella doederleinii (SDE) on human nasopharyngeal carcinoma and its possible mechanisms. MATERIALS AND METHODS Viability, apoptosis and protein expression of tumor cells were analyzed by MTT, Annexin V staining and Western blot, respectively. Formation of intracellular reactive oxygen species was determined using dichlorofluorescin fluorescence. The in vivo anti-tumor effect was evaluated by measuring tumor volume changes and TUNEL staining in nude mice. RESULTS SDE significantly inhibited the growth and induced apoptosis in human nasopharyngeal carcinoma CNE cells. In addition, SDE triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade. Moreover, SDE provoked the generation of reactive oxygen species in CNE cells, while the antioxidant N-acetyl cysteine almost completely blocked SDE-induced disruption of mitochondrial membrane potential, caspases activation and apoptosis. Furthermore, a transplantable nude mice model was utilized to estimate the effectiveness of SDE in vivo. The treated mice displayed decreased tumor size, which was associated with enhanced apoptotic cell death. CONCLUSIONS These results, offering solid evidence of the induction of mitochondria-related apoptosis in tumor cells, provide the molecular theoretical basis of clinical application of Selaginella doederleinii for the treatment of human nasopharyngeal carcinoma.

Silencing of RASSF3 by DNA Hypermethylation Is Associated with Tumorigenesis in Somatotroph Adenomas

The pathogenic mechanisms underlying pituitary somatotroph adenoma formation, progression are poorly understood. To identify candidate tumor suppressor genes involved in pituitary somatotroph adenoma tumorigenesis, we used HG18 CpG plus Promoter Microarray in 27 human somatotroph adenomas and 4 normal human adenohypophyses. RASSF3 was found with frequent methylation of CpG island in its promoter region in somatotroph adenomas but rarely in adenohypophyses. This result was confirmed by pyrosequencing analysis. We also found that RASSF3 mRNA level correlated negatively to its gene promoter methylation level. RASSF3 hypermethylation and downregulation was also observed in rat GH3 and mouse GT1.1 somatotroph adenoma cell lines. 5-Aza-2′ deoxycytidine and trichostatin-A treatment induced RASSF3 promoter demethylation, and restored its expression in GH3 and GT1.1 cell lines. RASSF3 overexpression in GH3 and GT1.1 cells inhibited proliferation, induced apoptosis accompanied by increased Bax, p53, and caspase-3 protein and decreased Bcl-2 protein expression. We also found that the antitumor effect of RASSF3 was p53 dependent, and p53 knockdown blocked RASSF3-induced apoptosis and growth inhibition. Taken together, our results suggest that hypermethylation-induced RASSF3 silencing plays an important role in the tumorigenesis of pituitary somatotroph adenomas.

Resistance to Streptozotocin-Induced Autoimmune Diabetes in Absence of Complement C3: Myeloid-Derived Suppressor Cells Play a Role

The contribution of complement to the development of autoimmune diabetes has been proposed recently. The underlying mechanisms, however, remain poorly understood. We hypothesize that myeloid-derived suppressor cells (MDSC), which act as regulators in autoimmunity, play a role in resistance to diabetes in absence of complement C3. Indeed, MDSC number was increased significantly in STZ-treated C3−/− mice. These cells highly expressed arginase I and inducible nitric oxide synthase (iNOS). Importantly, depletion of MDSC led to the occurrence of overt diabetes in C3−/− mice after STZ. Furthermore, C3−/− MDSC actively suppressed diabetogenic T cell proliferation and prevented/delayed the development of diabetes in arginase and/or iNOS-dependent manner. Both Tregs and transforming growth factor-β (TGF-β) are crucial for MDSC induction in STZ-treated C3−/− mice as depletion of Tregs or blocking TGF-β bioactivity dramatically decreased MDSC number. These findings indicate that MDSC are implicated in resistance to STZ-induced diabetes in the absence of complement C3, which may be helpful for understanding of mechanisms underlying preventive effects of complement deficiency on autoimmune diseases.

Silencing of HEPN1 is responsible for the aggressive biological behavior of pituitary somatotroph adenomas.

Background/Aims: The pathogenic mechanisms underlying pituitary adenoma formation, progression, and invasion are poorly understood. To identify candidate tumor suppressor genes, we selected somatotroph adenomas as representative of pituitary adenomas. Methods/Results: We used genome-wide differential expression analysis in 15 invasive and 12 noninvasive somatotroph adenomas. HEPN1 reduction was more frequent in the invasive group, and this result was confirmed by qRT-PCR. To understand the function of HEPN1, the pituitary adenoma cell lines, GH3 and GT1.1, were stably transfected with short hairpin RNA (shRNA) targeting HEPN1 or ectogenic HEPN1 by lentivirus-mediated transfection. We found that HEPN1 overexpression in GH3 and GT1.1 cells inhibited cell proliferation, induced apoptosis, and attenuated invasive capacity, whereas HEPN1 silencing enhanced cell proliferation and invasion accompanied by decreased apoptosis. Western blot analysis revealed that HEPN1 overexpression decreased MMP-2, MMP-9, and Bcl-2 expression, but increased BAX, p53, and caspase-3 expression. In contrast, HEPN1 silencing increased MMP-2, MMP-9, and Bcl-2 expression, but decreased BAX, p53, and caspase-3 expression. Conclusion: Taken together, our results suggest that reduction of HEPN1 may play an important role in the progression of pituitary somatotroph adenomas. HEPN1 may thus be a candidate as a prognostic predictor or an anticancer therapeutic target for patients with somatotroph adenoma.

Pectolinarigenin Suppresses the Tumor Growth in Nasopharyngeal Carcinoma

Background/Aims: Nasopharyngeal cancer (NPC) is one of the common human malignant diseases all over the world, and chemotherapy remains the main therapy for NPC. However, the survival and life quality of NPC patients are still very poor. Thus, novel and selective anti-tumor agents are pressingly needed. Our previous study identified pectolinarigenin as a novel effective anti-tumor drug candidate for NPC. In this study, we further investigated its anti-tumor activities and explored the potential molecular mechanism. Methods: NPC C666-1 cells were cultured and treated by pectolinarigenin. Cell proliferation assay, colony formation assay, Transwell assay and wound healing assay were conducted and cell apoptosis was detected by flow cytometry. Mitochondrial transmembrane potential and ROS were also observed. NPC subcutaneous xenograft mice model was established to evaluate the anti-tumor effect of pectolinarigenin in vivo. Results: We observed that treatment of pectolinarigenin inhibited cell viability and cell migration of NPC C666-1 cells in concentration- and time-dependent manner. Pectolinarigenin induced cell apoptosis in C666-1 cells detected by flow cytometry analysis, which was associated with the activation of mitochondrial-related apoptosis and the accumulation of reactive oxygen species (ROS). Pectolinarigenin also activated caspase signaling pathway. The in vivo experiment of subcutaneous xenograft mice model also indicated that the administration of pectolinarigenin could decrease the tumor growth of NPC and no severe toxicity was observed. Conclusions: Based on our findings, we conclude that pectolinarigenin could suppress the tumor growth of NPC, which verifies it as a new therapeutic agent for treating this devastating disease.

S100P is associated with proliferation and migration in nasopharyngeal carcinoma

In the present study, the function of S100 calcium binding protein P (S100P) in the C666-1 nasopharyngeal carcinoma (NPC) cell line was examined. The levels of S100P protein in NPC tissues were analyzed using immunohistochemistry, and small interfering RNA silenced S100P expression in C666-1 cells. Subsequently, cell proliferation, colony formation, migration and wound-healing assays were performed in order to assess whether the knockdown of S100P was able to influence the biological behavior of C666-1 cells. The expression levels of the receptor for advanced glycation end products (RAGE) were analyzed using a western blot following the inhibition of S100P. The immunohistochemistry results revealed that S100P was elevated in expression in 45/78 (57.7%) of patients with NPC, as compared with 5/30 (16.7%) of patients with benign inflammation. The S100P protein levels correlated with the rates of proliferation and migration in C666-1 cells. Additionally, reduced S100P expression levels altered a series of intracellular events, including the downregulation of epidermal growth factor receptor, cluster of differentiation (CD) 44, matrix metalloproteinase (MMP) 2 and MMP9 protein expression. In addition, RAGE expression was downregulated in the S100P silenced C666-1 cells, as detected by western blot analysis. These data suggest that S100P is important during the development and progression of nasopharyngeal cancer. Therefore, S100P may provide a novel treatment target for NPC.

IL-1β promotes the migration of olfactory epithelium neural stem cells through activating matrix metalloproteinase expressions

BACKGROUND To investigate the effects of IL-1β on the migration of olfactory epithelium neural stem cells (OENSCs), and to assess the mechanisms. METHODS The effects of different concentrations of IL-1β on cell proliferation, apoptosis and migration were evaluated by cell counting assay, flow cytometry and transwell migration assay, respectively. Matrix metalloproteinase (MMP)-2 and MMP-9 expression in both protein and mRNA levels were detected. Small interfering RNA (siRNA) technique was employed to knockdown MMP-2 and MMP-9 expression. Additionally, c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB) inhibitors were applied to assess the potential signaling pathways involved in the effects of IL-1β on cell migration. RESULTS IL-1β promoted cell migration of OENSCs in a concentration-dependent manner at the concentration range of 0-80 ng/ml, but did not affect cell proliferation and apoptosis. Mechanically, IL-1β promoted MMP-2 and MMP-9 expressions. Knockdown of MMP-2 or MMP-9 could significantly reduce IL-1β-induced cell migration. IL-1β activated JNK, NF-κB, Extracellular Signal-Regulated Kinase (ERK) and p-65 phosphorylation. Finally, we evidenced that inhibition of JNK or NF-κB significantly inhibited cell migration. CONCLUSION Our study demonstrated that IL-1β promoted the migration of OENSCs through activating MMP expression. Moreover, JNK and NF-κB signaling pathways were involved in the regulation. This study provides important experimental evidence for the application of OENSCs in the transplantation therapy.

Electrical stimulation promotes regeneration and re-myelination of axons of injured facial nerve in rats

Abstract Objective To investigate the effects of electrical stimulation (ES) on the nerve regeneration and functional recovery of facial expression muscles in facial nerve defect rats. Methods Sixty rats were surgically introduced with a 1-cm defect on the right facial nerves and evenly divided into the Surgery group (Group A, the main trunk of the right facial nerve was surgically cut-off with a 1.0 cm at the foramina stylomastoideum) and the Surgery + ES group (Group B). Twenty normal rats were as normal control group (without receiving surgery or ES). For rats in group B, the orbicularis oris muscle of the right paralyzed face was stimulated with an electrical pulse of 3 V, 20 Hz and 0.3 mA for 1 h each day. The effects of ES on the facial muscle movement, compound muscle action potentials (CMAPs), histological structure, and the expression levels of S100B and NF200 proteins were comparatively studied. Results In group A, facial paralysis scores were slightly improved from day 1 to 28; the facial nerve trunks had swelled and malformed till day 14; and CMAPs could be induced in fewer animals and were abnormal, resulting in a slow recovery of the facial muscle movement. In group B, facial paralysis scores were improved from 4 to 2.6 during the 4 weeks; more rats showed a higher amplitude and shorter latency of CMAPs from day 14 to 28 after surgery; and increased axons and the expression of S100B and NF200 proteins and gradually decreased swelling in the injured facial nerve. Conclusion ES promotes outgrowth and myelination of axons and a partial functional recovery of facial muscles in injured facial nerve rats.

Olfactory-ensheathing cells promote physiological repair of injured recurrent laryngeal nerves and functional recovery of glottises in dogs

The aim of this study was to investigate whether the transplantation of olfactory-ensheathing cells (OECs) could physiologically repair severely injured recurrent laryngeal nerve (RLN) in dogs. Adult Beagle dogs were surgically introduced with a 10-mm defect in the left RLN and transplanted with a nerve guide (NEUROLAC) containing dog olfactory mucosa-olfactory-ensheathing cells (OM-OECs) in matrigel. The effects of OM-OECs on the morphology, histology, and electrophysiology of the injured RLNs, glottis movement, and voice acoustics were comparatively studied. Two months after transplantation, the normal dogs (group N) had intact left RLNs that contained axons well organized as bundles, transmitted action potentials of high amplitudes without latent phases, and modulated glottis movement to produce normal voices. The RLN-damaged dogs transplanted with OM-OECs (group CTT) had pieces of nerves regenerated in the place of the defects, which contained fewer axons scattered in the internal nerve membrane and wrapped peripherally by the connective tissue, prevented the distal trunk of the defected RLN from shrinking, transmitted action potentials of lower amplitudes with latent phases, and modulated a slightly impaired glottis movement to produce voices with slight differences compared to the N dogs. The RLN-damaged dogs without OM-OECs (group NC) had no nerves generated at the defective or the damaged area, leading to a shrinkage in the enervated distal nerve trunks; a blockage in nerve pulse transit; a paralysis of the left vocal cords; an impaired glottis movement; and abnormal voices. Transplantation of OM-OECs promoted nerve regeneration, and the recoveries of glottises and voices in dogs with RLN injury.