Jingping Fan

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.

Curcumin as therapeutics for the treatment of head and neck squamous cell carcinoma by activating SIRT1.

SIRT1 is one of seven mammalian homologs of Sir2 that catalyzes NAD+-dependent protein deacetylation. The aim of the present study is to explore the effect of SIRT1 small molecule activator on the anticancer activity and the underlying mechanism. We examined the anticancer activity of a novel oral agent, curcumin, which is the principal active ingredient of the traditional Chinese herb Curcuma Longa. Treatment of FaDu and Cal27 cells with curcumin inhibited growth and induced apoptosis. Mechanistic studies showed that anticancer activity of curcumin is associated with decrease in migration of HNSCC and associated angiogenesis through activating of intrinsic apoptotic pathway (caspase-9) and extrinsic apoptotic pathway (caspase-8). Our data demonstrating that anticancer activity of curcumin is linked to the activation of the ATM/CHK2 pathway and the inhibition of nuclear factor-κB. Finally, increasing SIRT1 through small molecule activator curcumin has shown beneficial effects in xenograft mouse model, indicating that SIRT1 may represent an attractive therapeutic target. Our studies provide the preclinical rationale for novel therapeutics targeting SIRT1 in HNSCC.

Olfactory epithelium neural stem cell implantation restores noise-induced hearing loss in rats

OBJECTIVE In this study, we aimed to elucidate the restorative effects of olfactory epithelium neural stem cells (oe-NSCs) implantation on noise-induced hearing loss and establish their mechanism of action. METHODS To model hearing loss, rats were subjected to consecutive seven-day noise exposure. Then, oe-NSCs were implanted into cochlear tissue by retroauricular approach. Auditory brainstem response (ABR) method was used to evaluate the hearing function. Immunohistochemical staining was utilized to determine cell survival and migration of oe-NSCs. After IL-1β stimulation, nerve growth factor (NGF), neurotrophin-3 (NT-3), and NT-4 levels were evaluated in oe-NSCs. The protective action of oe-NSCs against hydrogen peroxide-induced cell injury was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). RESULTS oe-NSCs implantation into cochlear tissues ameliorated the noise-induced hearing impairment (p<0.05). After implantation, green fluorescent cells were observed in an even suspension in the lymph fluid of the cochlea, and 65% of the GFP(+) cells reached the cochlear duct wall three days after implantation, but did not expand to the Corti-organ. After IL-1β stimulation, olfactory epithelial stem cell increased their secretion of NGF and NT-3 (p<0.05), but not that of NT-4. TUNEL assay results revealed that oe-NSCs co-culturing with injured neurons reduced the apoptotic cell death induced by hydrogen peroxide. CONCLUSION After transplantation into the inner ear, oe-NSCs not only survived, but also migrated around the spiral ganglion neurons (SGNs) in Rosenthals canal (RC). Hearing loss induced by noise exposure was restored after oe-NSCs implantation. Mechanically, oe-NSCs secreted NGF and NT-3, which likely contributed to the prevention of neuronal injury. This study provides novel data in support of the effective action of implanted oe-NSCs in the restoration of noise-induced hearing loss in a rat model.

Identification of thyroid carcinoma related genes with mRMR and shortest path approaches.

Thyroid cancer is a malignant neoplasm originated from thyroid cells. It can be classified into papillary carcinomas (PTCs) and anaplastic carcinomas (ATCs). Although ATCs are in an very aggressive status and cause more death than PTCs, their difference is poorly understood at molecular level. In this study, we focus on the transcriptome difference among PTCs, ATCs and normal tissue from a published dataset including 45 normal tissues, 49 PTCs and 11 ATCs, by applying a machine learning method, maximum relevance minimum redundancy, and identified 9 genes (BCL2, MRPS31, ID4, RASAL2, DLG2, MY01B, ZBTB5, PRKCQ and PPP6C) and 1 miscRNA (miscellaneous RNA, LOC646736) as important candidates involved in the progression of thyroid cancer. We further identified the protein-protein interaction (PPI) sub network from the shortest paths among the 9 genes in a PPI network constructed based on STRING database. Our results may provide insights to the molecular mechanism of the progression of thyroid cancer.

S100A11 is a migration-related protein in laryngeal squamous cell carcinoma.

Objective: As a member of the S100 proteins family, the involvement of S100A11 has been suggested in a wide range of biological processes such as cell growth and motility, cell-cycle progression, transcription, differentiation and smooth muscle cell migration. However, the expression of S100A11 and its exact function in laryngeal squamous cell carcinoma (LSCC) have not been elucidated. Methods: The protein and mRNA expression levels of S100A11 were analyzed in primary tumors and matched tumor-adjacent tissues of LSCC by western blotting and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) or quantitative real time PCR (Q-RT-PCR), respectively. Cell proliferation, colony formation, migration and wound-healing assays were performed to assess whether the knockdown of S100A11 by small interfering RNA (siRNA) could influence the biological behavior of human laryngeal carcinoma Hep-2 cells in vitro. Results: We found that both protein and mRNA levels of S100A11 were overexpressed in laryngeal tumor tissues when compared to the corresponding noncancerous tissues. Further, it was demonstrated that the expression of S100A11 could induce migration but not proliferation of Hep-2 cells. Additionally, S100A11 altered a series of intracellular events, including the down-regulation of epidermal growth factor receptor (EGFR), CD44 and MMP2. Conclusions: These results highlight the significance of S100A11 in LSCC progression and suggest that the dysregulation of S100A11 might contribute to the metastatic progression of LSCC.

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.

Curcumin induces G2/M cell cycle arrest and apoptosis of head and neck squamous cell carcinoma in vitro and in vivo through ATM/Chk2/p53-dependent pathway

Studies have demonstrated that curcumin (CUR) exerts its tumor suppressor function in a variety of human cancers including head and neck squamous cell carcinoma (HNSCC). However, the exact underlying molecular mechanisms remain obscure. Here, we aim to test whether CUR affects ATM/Chk2/p53 signaling pathway, leading to the induction of cell cycle arrest, inhibition of angiogenesis of HNSCC in vitro and in vivo. To this end, we conducted multiple methods such as MTT assay, Invasion assay, Flow cytometry, Western blotting, RT-PCR, and transfection to explore the functions and molecular insights of CUR in HNSCC. We observed that CUR significantly induced apoptosis and cell cycle arrest, inhibited angiogenesis in HNSCC. Mechanistically, we demonstrated that CUR markedly up-regulated ATM expression and subsequently down-regulated HIF-1α expression. Blockage of ATM production totally reversed CUR induced cell cycle arrest as well as anti-angiogenesis in HNSCC. Moreover, our results demonstrated that CUR exerts its antitumor activity through targeting ATM/Chk2/p53 signal pathway. In addition, the results of xenograft experiments in mice were highly consistent with in vitro studies. Collectively, our findings suggest that targeting ATM/Chk2/p53 signal pathway by CUR could be a promising therapeutic approach for HNSCC prevention and therapy.Studies have demonstrated that curcumin (CUR) exerts its tumor suppressor function in a variety of human cancers including head and neck squamous cell carcinoma (HNSCC). However, the exact underlying molecular mechanisms remain obscure. Here, we aim to test whether CUR affects ATM/Chk2/p53 signaling pathway, leading to the induction of cell cycle arrest, inhibition of angiogenesis of HNSCC in vitro and in vivo. To this end, we conducted multiple methods such as MTT assay, Invasion assay, Flow cytometry, Western blotting, RT-PCR, and transfection to explore the functions and molecular insights of CUR in HNSCC. We observed that CUR significantly induced apoptosis and cell cycle arrest, inhibited angiogenesis in HNSCC. Mechanistically, we demonstrated that CUR markedly up-regulated ATM expression and subsequently down-regulated HIF-1α expression. Blockage of ATM production totally reversed CUR induced cell cycle arrest as well as anti-angiogenesis in HNSCC. Moreover, our results demonstrated that CUR exerts its antitumor activity through targeting ATM/Chk2/p53 signal pathway. In addition, the results of xenograft experiments in mice were highly consistent with in vitro studies. Collectively, our findings suggest that targeting ATM/Chk2/p53 signal pathway by CUR could be a promising therapeutic approach for HNSCC prevention and therapy.

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.