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Lung cancer is the most common leading cause of cancer-related death worldwide. Late diagnosis contributes to a high mortality rate and poor survival of this cancer. In our previous study, we found that IDH2 polymorphism rs11540478 is a risk factor for lung cancer. Here, we examined IDH2 protein expression in culture medium in which two non-small-cell lung cancer (NSCLC) cell lines, H460 and A549, were growing. We found that the IDH2 protein was elevated in the culture supernatant fraction in a time- and cell number-dependent manner. Next, we used ELISA methods to examine IDH2 protein level in serum from patients with NSCLC and healthy controls. We found that IDH2 protein levels in serum could distinguish NSCLC patients from healthy controls with an AUC (area under the curve) of 0.83 (95% confidence interval = 0.79-0.88). The IDH2 level was decreased in serum from NSCLC postsurgical patients compared with the paired presurgical serum. High serum IDH2 levels appear to correlate with poor survival in patients with NSCLC. These results suggest that IDH2 levels in the serum could be a new effective biomarker for the diagnosis and prognosis of NSCLC.

Neoalbaconol, derived from Albatrellus confluens, shows anti-cancer activities in the previously study, but its role in angiogenesis is unknown. Here, we determined whether neoalbaconol could attenuate angiogenesis and how does it occur. Data demonstrated that neoalbaconol could inhibit the proliferation of breast cancer cells and induce apoptosis. Also, neoalbaconol suppressed vascular endothelial growth factor (VEGF)-induced human umbilical vascular endothelial cells (HUVECs) proliferation, migration, invasion, and capillary-like tube formation in vitro and reduced tumor angiogenesis in vivo. VEGF receptor activation and the downstream signal transduction cascades activation were inhibited by neoalbaconol. Additionally, neoalbaconol blocked EGFR-mediated VEGF production. EGFR overexpression reversed the neoalbaconol-induced VEGF reduction, confirming the importance of the EGFR inhibition in anti-angiogenesis of neoalbaconol. Furthermore, neoalbaconol inhibited tumor growth and tumor angiogenesis in a breast cancer xenograft model in vivo. Taken together, these results indicate that neoalbaconol could inhibit tumor angiogenesis and growth through direct suppression effects on vascular endothelial cells and reduction of proangiogenic factors in cancer cells.

// Xiangjian Luo 1, 2, 3 , Namei Li 1, 2, 3 , Juanfang Zhong 1, 2, 3 , Zheqiong Tan 1, 2, 3 , Ying Liu 4 , Xin Dong 1, 2, 3 , Can Cheng 1, 2, 3 , Zhijie Xu 1, 2, 3 , Hongde Li 1, 2, 3 , Lifang Yang 1, 2, 3 , Min Tang 1, 2, 3 , Xinxian Weng 1, 2, 3 , Wei Yi 1, 2, 3 , Jikai Liu 5 , Ya Cao 1, 2, 3 1 Key Laboratory of Carcinogenesis and Invasion, Chinese Ministry of Education, Xiangya Hospital, Central South University, Changsha, Hunan 410078, China 2 Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan 410078, China 3 Key Laboratory of Carcinogenesis, Chinese Ministry of Health, Changsha, Hunan 410078, China 4 Department of Medicine, Hunan Traditional Chinese Medical College, Zhuzhou, Hunan 412000, China 5 School of Pharmacy, South-Central University For Nationalities, Wuhan, Hubei 430074, China Correspondence to: Xiangjian Luo, email: luocsu@hotmail.com Jikai Liu, email: jkliu@mail.kib.ac.cn Ya Cao, email: ycao98@vip.sina.com Keywords: grifolin, PGC1α, ROS, adhesion, migration Received: April 13, 2016 Accepted: August 29, 2016 Published: September 10, 2016 ABSTRACT Grifolin, a farnesyl phenolic compound isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens , exhibits effective antitumor bioactivity in previous study of our group and other lab. In this study, we observed that grifolin inhibited tumor cells adhesion and migration. Moreover, grifolin reduced reactive oxygen species (ROS) production and caused cellular ATP depletion in high-metastatic tumor cells. PGC1α (Peroxisome proliferator-activated receptor γ, coactivator 1α) encodes a transcriptional co-activator involved in mitochondrial biogenesis and respiration and play a critical role in the maintenance of energy homeostasis. Interestingly, grifolin suppressed the mRNA as well as protein level of PGC1α. We further identified that MMP2 and CD44 expressions were PGC1α inducible. PGC1α can bind with metastatic-associated transcription factors: Fra-1 and LSF and the protein-protein interaction was attenuated by grifolin treatment. Overall, these findings suggest that grifolin decreased ROS generation and intracellular ATP to suppress tumor cell adhesion/migration via impeding the interplay between PGC1α and Fra-1 /LSF-MMP2/CD44 axes. Grifolin may develop as a promising lead compound for antitumor therapies by targeting energy metabolism regulator PGC1α signaling.

We conducted this research to explore the role of latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) in modulating the DNA damage response (DDR) and its regulatory mechanisms in radioresistance. Our results revealed that LMP1 repressed the repair of DNA double strand breaks (DSBs) by inhibiting DNA-dependent protein kinase (DNA-PK) phosphorylation and activity. Moreover, LMP1 reduced the phosphorylation of AMP-activated protein kinase (AMPK) and changed its subcellular location after irradiation, which appeared to occur through a disruption of the physical interaction between AMPK and DNA-PK. The decrease in AMPK activity was associated with LMP1-mediated glycolysis and resistance to apoptosis induced by irradiation. The reactivation of AMPK significantly promoted radiosensitivity both in vivo and in vitro. The AMPKα (Thr172) reduction was associated with a poorer clinical outcome of radiation therapy in NPC patients. Our data revealed a new mechanism of LMP1-mediated radioresistance and provided a mechanistic rationale in support of the use of AMPK activators for facilitating NPC radiotherapy.

Recently, mutations in isocitrate dehydrogenase 1/2 (IDH1/2) were discovered in 70% of low-grade glioma and secondary glioblastoma multiforme. The discovery of an oncogenic function and the identification of onco-metabolites of IDH1/2 support new roles for metabolism in cancer. For example, some evidence indicates that IDH2 might also exhibit oncogenic functions by promoting cellular metabolism and cancer cell growth. We examined the association between IDH2 rs11540478 and lung cancer risk in 262 lung cancer patient cases and 602 healthy control subjects and also investigated the biological function of rs11540478 in vivo. We found that a higher risk was observed in lung cancer patient carriers of rs11540478 TT and CT compared with CC carriers (OR = 1.44; 95%CI = 1.04-2.00; P = 0.03). The frequency of IDH2 rs11540478 TT and CT carriers was decreased in healthy individuals between the ages of 50-77 compared to those aged 30-49 (OR = 0.67; 95%CI = 0.47-0.96; P = 0.03). Functional analysis showed the effect of rs11540478 on IDH2 expression and lung cancer cell viability, with higher IDH2 expression and cell viability among T allele compared with C allele. IDH2 mRNA was higher in peripheral blood lymphocytes from lung cancer patients compared to healthy subjects. Herein, for the first time we identified IDH2 rs11540478 as a new susceptibility locus for lung cancer. The effect of rs11540478 on mRNA expression of IDH2 and lung cancer cell viability might provide new insight for the genetic basis of lung cancer. © 2016 Wiley Periodicals, Inc.

Grifolin, a natural product isolated from the mushroom Albatrellus confluens, has been shown to inhibit the growth of some cancer cell lines and induce significant apoptosis. However, the molecular targets and the signaling mechanism underlying the anticancer effect of this compound are not completely understood. Here, we undertook a gene expression profiling study to identify novel targets of grifolin. We found that the effect of grifolin on the human nasopharyngeal carcinoma cell line CNE1 occurs primarily via the ERK1/2 pathway. At high doses, both the ERK1/2 and the ERK5 pathways may be involved in the inhibition. Because inhibition of the ERK1/2 or the ERK5 pathway has been associated with cell-cycle arrest and growth inhibition, we evaluated the cell cycle distribution after grifolin treatment. We found that grifolin significantly caused cell-cycle arrest in G1 phase. To investigate the underlying mechanisms, G1-related proteins were assayed by Western blotting. Following grifolin treatment, a concomitant inhibition of cyclin D1, cyclin E, CDK4 expression, and subsequent reduction in pRB phosphorylation occurred. Meanwhile, grifolin treatment also resulted in a significant upregulation of CKI (p19INK4D). These results suggest that the inhibition of the ERK1/2 or the ERK5 pathway is responsible for at least part of the induction of cell-cycle arrest in G1 phase by grifolin. These results are significant in that they provide a mechanistic framework for further exploring the use of grifolin as a novel antitumor agent.

Grifolin, a secondary metabolite isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens, has been reported by us and others to display potent antitumor effects. However, the molecular target of grifolin has not been identified and the underlying mechanism of action is not fully understood. Here, we report that the ERK1/2 protein kinases are direct molecular targets of grifolin. Molecular modeling, affinity chromatography and fluorescence quenching analyses showed that grifolin directly binds to ERK1/2. And in vitro and ex vivo kinase assay data further demonstrated that grifolin inhibited the kinase activities of ERK1/2. We found that grifolin suppressed adhesion, migration and invasion of high-metastatic cancer cells. The inhibitory effect of grifolin against tumor metastasis was further confirmed in a metastatic mouse model. We found that grifolin decreased phosphorylation of Elk1 at Ser383, and the protein as well as the mRNA level of DNMT1 was also down-regulated. By luciferase reporter and ChIP assay analyses, we confirmed that grifolin inhibited the transcription activity of Elk1 as well as its binding to the dnmt1 promoter region. Moreover, we report that significant increases in the mRNA levels of Timp2 and pten were induced by grifolin. Thus, our data suggest that grifolin exerts its anti-tumor activity by epigenetic reactivation of metastasis inhibitory-related genes through ERK1/2-Elk1-DNMT1 signaling. Grifolin may represent a promising therapeutic lead compound for intervention of cancer metastasis, and it may also be useful as an ERK1/2 kinase inhibitor as well as an epigenetic agent to further our understanding of DNMT1 function.

Imaging agents that enable direct detection of apoptosis are highly desirable in the field of monitoring chemotherapeutic response as well as early diagnosis and disease monitoring. Previous work demonstrated that the dansyled amino acid DNSBA is used to specifically and selectively detect apoptotic cancer cells at the both early and late stages, but the mechanism remains unclear. In this work, we evaluated DNSBA as a tool for monitoring cell apoptosis in CNE1 tumor cell models both in vitro and ex vivo after its in vivo administration, which was confirmed by other assays. The ability of DNSBA to detect multiple pathways and different stages of apoptosis leading to cell death may be advantageous in the evaluation of cancer treatment indicative of a positive therapeutic outcome. The uptake change of molecular probes DNSBA in CNE1 cells represented the changes of apoptotic rate in a caspase-dependent manner. However, the accumulation of DNSBA in apoptotic cells did not increase with the enhanced membrane permeability. Furthermore, ex vivo study demonstrated DNSBA has a similar pattern as the TUNEL-positive cells. In conclusion, DNSBA cellular imaging is useful for the early assessment of treatment-induced apoptosis, and thus may act as a substitute for Annexin V for assessing treatment response.

Grifolin is a natural biologically active substance isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens. Here, for the first time, we describe a novel activity of grifolin, namely its ability to inhibit the growth of tumor cells by the induction of apoptosis. Grifolin strongly inhibited the growth of tumor cell lines: CNE1, HeLa, MCF7, SW480, K562, Raji and B95-8. Analysis of acridine orange (AO)/ethidium bromide (EB) staining and flow cytometry showed that grifolin possessed apoptosis induction activity to CNE1, HeLa, MCF7 and SW480. Furthermore, the cytochrome c release from mitochondria was detected by confocal microscopy in CNE1 cells after a 12h treatment with grifolin. The increase of caspase-8, 9, 3 activities revealed that caspase was a key mediator of the apoptotic pathway induced by grifolin, and the underexpression of Bcl-2 and up-regulation of Bax resulted in the increase of Bax: Bcl-2 ratio, suggesting that Bcl-2 family involved in the control of apoptosis. Owing to the combination of the significant antitumor activity by inducing apoptosis and natural abundance of the compound, grifolin holds the promise of being an interesting antitumor agent that deserves further laboratory and in vivo exploration.

Recently, immunoglobulins (Igs) were unexpectedly found to be expressed in epithelial cancers. Immunoglobulin class switching or class switch recombination (CSR) is a natural biological process that alters a B cell's production of antibodies (immunoglobulins) from one class to another. However, the mechanism of CSR of Ig genes in cancer is still unknown. Here, we confirmed by detecting the hallmark of CSR that the Igα gene in cancer underwent CSR. Then we focused on activation-induced cytidine deaminase (AID), a crucial factor for initiating CSR. Further studies using tumor necrosis factor (TNF)-α stimulation and specific inhibitor of NF-κB revealed that TNF-α could increase AID expression through NF-κB signaling. Finally, we demonstrated that AID could co-localize with protein kinase A and bind to the switching (Sα) region of the Igα gene. Overexpression of AID obviously enhanced Igα heavy chain expression and its binding ability to the Sα region. These findings indicated that TNF-α-induced AID expression is involved with CSR in cancer.

Stathmin, a microtubule associated protein (MAP), is an important molecular target for cancer therapy. Paclitaxel is one of the primary antitumor drugs targeting microtubules (MTs). We hypothesized that decreasing the expression level of Stathmin might improve the effectiveness of paclitaxel in the treatment of nasopharyngeal carcinoma (NPC). NPC cell lines, CNE1-LMP1 and HNE2, and a CNE1-LMP1 tumor xenograft mouse model were used to test both in vitro and in vivo our siRNA-based Stathmin silencing strategy. The effects of Stathmin silencing on cell proliferation, apoptosis, and viability were investigated using MTT, AO/EB staining, TUNEL, caspase protein detection, and FCM assays. Cell migration and invasion were assayed using a Transwell assay. The combined effects of Stathmin silencing and paclitaxel were investigated using MTT, FCM, Western blot and indirect immunofluorescence assays. The effect of paclitaxel on Stathmin expression in NPC cells and, in addition, A375, MGC and HeLa cells was determined by RT-PCR and Western blotting. We found that siRNA-mediated silencing of Stathmin suppresses proliferation, induces apoptosis through the mitochondrial pathway, and causes G2/M-phase cell cycle arrest in the NPC cell lines CNE1-LMP1 and HNE2. Also, the migration and invasion of the respective NPC cells were found to be inhibited. In addition, we show that a combination of Stathmin silencing and paclitaxel is more effective than either agent alone in inhibiting proliferation and inducing apoptosis, cell cycle arrest, and MT polymerization. Furthermore, we found that Stathmin expression in the tumor cells is down-regulated by paclitaxel treatment. siRNA-mediated silencing of Stathmin suppresses the proliferation, invasion and metastasis, and induces the apoptosis of NPC cells. Paclitaxel reduces the expression of Stathmin, and combining Stathmin silencing with paclitaxel treatment enhances MT polymerization. This combined strategy may provide a new approach for clinical NPC treatment.

To identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-kappaB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC).A stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining.LMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMP1. Moreover, LMP1-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the kappaB site kappaB1 or kappaB5 was disrupted, whereas mutation of kappaB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene.LMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-kappaB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.

To clarify if Epstein-Barr virus encoded LMP1 induces matrix metalloproteinase 9 expression via NF-kappa B or AP-1 signaling pathway, which gives evidence to the elucidation of the mechanism of LMP1- mediated carcinogenesis.To determine whether LMP1 or its mutants contribute to MMP9 production via NF-kappa B or AP-1 transcription factor, MMP9-chloramphenicol acetyl transferase (CAT), NF-kappa B mut 9-CAT, AP-1 mut MMP9-CAT were transfected into human nasopharyngeal carcinoma cells stably expressing LMP1 (HNE2-LMP1) or its mutants, [HNE2-LMP1 (1-185), HNE2-LMP1 (1-231), HNE2-LMP1 delta 187-351] by electroporation technic. The difference of MMP9 reporter activity among those cell lines was detected by CAT assay and expression of MMP9 was determined in nasopharyngeal carcinoma cells stably expressing LMP1 or its mutants by zymographic analysis. In the meantime, efforts were made to demonstrate if LMP1 regulates NF-kappa B or AP-1 activation using reporter gene analysis.In contrast with vector-transfected cells, MMP9 CAT activity in HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231), HNE2-LMP1 delta 187-351 increased 7.2, 1.3, 3.3, 4.0 times respectively. Zymographic analysis demonstrated that the 92 kDa MMP9 expression was induced in HNE2-LMP1, HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells, whereas it was negative in HNE2-pSG5 and HNE2-LMP1 (1-185) cells. As compared to the HNE2 cells, NF-kappa B or AP-1 reporter activity in HNE2-LMP1 cells were increased 13.8, 8.4 fold respectively. Moreover, In contrast with MMP9 CAT-transfected cells, MMP9 CAT activity in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells were significantly decreased by 18.1% or 16.3%, 35.0% or 33.3%, 29.1% or 26.1% from the original level. However, there was no difference in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-pSG5, HNE2-LMP1 (1-185) cells.In nasophargyngeal carcinoma, Epstein-Barr virus-encoded LMP1 induces MMP9 transcription and enzymatic activity via an NF-kappa B or AP-1 signaling pathway, which may contribute to invasiveness and metastasis.

Hotspot mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) have been studied in several cancers. However, the function of wild-type IDH2 in lung cancer and the mechanism of its contribution to growth of cancer cells remain unknown. Here, we explored the role and mechanism of wild-type IDH2 in promoting growth of lung cancer. Methods: Information regarding genomic and clinical application focusing on IDH2 in cancer was examined in several databases of more than 1,000 tumor samples. IDH2 expression was assessed by immunohistochemistry in tissues from lung cancer patients. The biological functions of IDH2 were evaluated by using cell-based assays and in vivo xenograft mouse models. Results: Here we reported that wild-type IDH2 is up-regulated and is an indicator of poor survival in lung cancer and several other cancers. Targeting IDH2 with shRNA resulted in decreased HIF1α expression, leading to the attenuation of lung cancer cell proliferation and tumor growth. Treatment of lung cancer cells with AGI-6780 (a small molecule inhibitor of IDH2), PX-478 (an inhibitor of HIF1α) or incubation with octyl-α-KG inhibited lung cancer cell proliferation. Conclusion: IDH2 promotes the Warburg effect and lung cancer cell growth, which is mediated through HIF1α activation followed by decreased α-KG. Therefore, IDH2 could possibly serve as a novel therapeutic target for lung cancer.