Search

Background: The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is hyperactivated in lung cancer and regulates a broad range of cellular processes, including proliferation, survival, angiogenesis, and metastasis. Thus PI3K is considered a promising target for therapy. To date, PI3K inhibitors have not been approved for lung cancer. Recent studies showed that the antipsychotic agent flupentixol induced apoptosis of lung cancer cell, however the anti-tumor mechanism of flupentixol remains unclear. Methods: (1) The idock software simulated the molecular docking between the PI3Kα protein and flupentixol. (2) Inhibition of PI3Kα by the flupentixol was examined by in vitro kinase assays. (3) The cytotoxicity of flupentixol on the NSCLC cell lines was tested by MTT assays. (4) We treated A549 and H661 cells with flupentixol and then measured the percentage of apoptotic cells by the Annexin V/PI analysis. (5) We investigated the effect of flupentixol on the expression of critical PI3K/AKT signaling pathway proteins, further analyzed on the cleavage of PARP and caspase-3 by Western blotting. (6) BALB/C nude mice were subcutaneously injected with A549 cells to evaluate the effect of flupentixol on the growth of lung carcinoma. Results: Structural analysis of the predicted binding conformation suggested that flupentixol docks to the ATP binding pocket of PI3Kα. Kinase assays demonstrate that flupentixol indeed inhibited the PI3Kα kinase activity. Flupentixol exhibited cytotoxicity in lung cancer cell lines A549 and H661 in a dose- and time-dependent manner. Furthermore, flupentixol more strongly inhibited the phosphorylation of AKT (T308 and S473) and the expression of its downstream target gene Bcl-2 than two known PI3K inhibitors (BYL719 and BKM120). Flupentixol induced apoptosis as measured by PARP and caspase-3 cleavage. Finally, flupentixol significantly suppressed A549 xenograft growth in BALB/C nude mice. Conclusions: Flupentixol could be docked to the PI3Kα protein and specifically inhibit the PI3K/AKT pathway and survival of lung cancer cells in vitro and in vivo. As an old drug, flupentixol is a new PI3K inhibitor that may be used for the treatment of lung cancers.

Background: Cyclin-dependent kinases 2/4/6 (CDK2/4/6) play critical roles in cell cycle progression, and their deregulations are hallmarks of hepatocellular carcinoma (HCC). Methods: We used the combination of computational and experimental approaches to discover a CDK2/4/6 triple-inhibitor from FDA approved small-molecule drugs for the treatment of HCC.Results: We identified vanoxerine dihydrochloride as a new CDK2/4/6 inhibitor, and a strong cytotoxic drug in human HCC QGY7703 and Huh7 cells (IC50: 3.79μM for QGY7703and 4.04μM for Huh7 cells). In QGY7703 and Huh7 cells, vanoxerine dihydrochloride treatment caused G1‑arrest, induced apoptosis, and reduced the expressions of CDK2/4/6, cyclin D/E, retinoblastoma protein (Rb), as well as the phosphorylation of CDK2/4/6 and Rb. Drug combination study indicated that vanoxerine dihydrochloride and 5-Fu produced synergistic cytotoxicity in vitro in Huh7 cells. Finally, in vivo study in BALB/C nude mice subcutaneously xenografted with Huh7 cells, vanoxerine dihydrochloride (40mg/kg, i.p.) injection for 21 days produced significant anti‑tumor activity (pConclusions: The present study is the first report identifying a new CDK2/4/6 triple inhibitor vanoxerine dihydrochloride, and demonstrated that this drug represents a novel therapeutic strategy for HCC treatment.

Background Cyclin-dependent kinases 2/4/6 (CDK2/4/6) play critical roles in cell cycle progression, and their deregulations are hallmarks of hepatocellular carcinoma (HCC). Methods We used the combination of computational and experimental approaches to discover a CDK2/4/6 triple-inhibitor from FDA approved small-molecule drugs for the treatment of HCC. Results We identified vanoxerine dihydrochloride as a new CDK2/4/6 inhibitor, and a strong cytotoxicdrugin human HCC QGY7703 and Huh7 cells (IC50: 3.79 μM for QGY7703and 4.04 μM for Huh7 cells). In QGY7703 and Huh7 cells, vanoxerine dihydrochloride treatment caused G1-arrest, induced apoptosis, and reduced the expressions of CDK2/4/6, cyclin D/E, retinoblastoma protein (Rb), as well as the phosphorylation of CDK2/4/6 and Rb. Drug combination study indicated that vanoxerine dihydrochloride and 5-Fu produced synergistic cytotoxicity in vitro in Huh7 cells. Finally, in vivo study in BALB/C nude mice subcutaneously xenografted with Huh7 cells, vanoxerine dihydrochloride (40 mg/kg, i.p.) injection for 21 days produced significant anti-tumor activity (p Conclusions The present study isthe first report identifying a new CDK2/4/6 triple inhibitor vanoxerine dihydrochloride, and demonstrated that this drug represents a novel therapeutic strategy for HCC treatment.

The phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway plays a pivotal role in many cellular processes, including the proliferation, survival and differentiation of lung cancer cells. Thus, PI3K is a promising therapeutic target for lung cancer treatment. In this study, we applied free and open-source protein-ligand docking software, screened 3167 FDA-approved small molecules, and identified putative PI3Kα inhibitors. Among them, econazole nitrate, an antifungal agent, exhibited the highest activity in decreasing cell viability in pathological types of NSCLC cell lines, including H661 (large cell lung cancer) and A549 (adenocarcinoma). Econazole decreased the protein levels of p-AKT and Bcl-2, but had no effect on the phosphorylation level of ERK. It inhibited cell growth and promote apoptosis in a dose-dependent manner. Furthermore, the combination of econazole and cisplatin exhibited additive and synergistic effects in the H661 and A549 lung cancer cell lines, respectively. Finally, we demonstrated that econazole significantly suppressed A549 tumor growth in nude mice. Our findings suggest that econazole is a new PI3K inhibitor and a potential drug that can be used in lung cancer treatment alone or in combination with cisplatin.

The clinical application of RNA interference (RNAi)-based cancer gene therapy has been hampered by the lack of efficient delivery of short interfering RNA (siRNA). In this context, the use of biodegradable charged polyester-based vectors (BCPVs) for delivering mutated K-Ras-targeting siRNA in a pancreatic xenograft model was investigated in vivo. Using mice bearing pancreatic xenografts as an animal model, results show that fluorescently labeled TRAMA (carboxytetramethylrhodamine) K-Ras siRNA continuously accumulated in the xenograft via BCPVs for at least 72 h. After the treatment, the level of the targeted mRNA and protein reduced to 50% of their original level. As a consequence, significant suppression in tumor growth, decreased tumor local infiltration, and increased cell apoptosis were observed in the xenograft model after the siRNA treatment. More importantly, physiological analysis results reveal that an excessive amount of BCPV (10 times higher than the commonly treated amount) will not have a significant influence on the status of the blood stream, blood stream components, and organ tissue, suggesting that BCPVs have very low in vivo toxicity. Our results indicate that the delivery of K-Ras-targeting siRNA via BCPV nanoparticles may be a promising strategy for pancreatic cancer therapy.

Esophagus cancer is the seventh cause of cancer-related deaths globally. In this study, we analyzed interleukin 6 (IL-6) gene expression in human esophagus cancer patients and showed that IL-6 mRNA levels are significantly higher in tumor tissues and negatively correlated with overall survival, suggesting that IL-6 is a potential therapeutic target for esophagus cancer. We further demonstrated that apigenin, a nature flavone product of green plants, inhibited IL-6 transcription and gene expression in human esophagus cancer Eca-109 and Kyse-30 cells. Apigenin significantly and dose-dependently inhibited cell proliferation and promoted apoptosis while stimulating the cleaved PARP (poly ADP-ribose polymerase) (C-PARP) and caspase-8 expression. It suppressed VEGF (Vascular endothelial growth Factor) expression and tumor-induced angiogenesis. Pretreatment of cells with IL-6 could completely reverse apigenin-induced cellular changes. Finally, using a preclinical nude mice model subcutaneously xenografted with Eca-109 cells, we demonstrated the in vivo antitumor activity and mechanisms of apigenin. Taken together, this study revealed for the first time that apigenin is a new IL-6 transcription inhibitor and that inhibiting IL-6 transcription is one of the mechanisms by which apigenin exhibits its anticancer effects. The potential clinical applications of apigenin in treating esophagus cancer warrant further investigations.

Bladder carcinoma (BC) is the ninth most common cause of cancer worldwide. Surgical resection and conventional chemotherapy and radiotherapy will ultimately fail due to tumor recurrence and resistance. Thus, the development of novel treatment is urgently needed. Fibroblast growth factor receptor 3 (FGFR3) is an important and well-established target for BC treatment. In this study, we utilized the free and open-source protein-ligand docking software idock to prospectively identify potential inhibitors of FGFR3 from 3,167 worldwide approved small-molecule drugs using a repositioning strategy. Six high-scoring compounds were purchased and tested in vitro. Among them, the acaricide drug fluazuron exhibited the highest antiproliferative effect in human BC cell lines RT112 and RT4. We further demonstrated that fluazuron treatment significantly increased the percentage of apoptosis cells, and decreased the phosphorylation level of FGFR3 and its downstream proteins FRS2-α, AKT, and ERK. We also investigated the anticancer effect of fluazuron in vivo in BALB/C nude mice subcutaneously xenografted with RT112 cells. Our results showed that oral treatment with fluazuron (80 mg/kg) significantly inhibited tumor growth. These results suggested for the first time that fluazuron is a potential inhibitor of FGFR3 and a candidate anticancer drug for the treatment of BC.

Magnetic field exposure is an accepted safe and effective modality for nerve injury. However, it is clinically used only as a supplement or salvage therapy at the later stage of treatment. Here, we used a planarian Girardia sinensis decapitated model to investigate beneficial effects of early rotary non-uniform magnetic fields (RMFs) exposure on central nervous regeneration. Our results clearly indicated that magnetic stimulation induced from early RMFs exposure significantly promoted neural regeneration of planarians. This stimulating effect is frequency and intensity dependent. Optimum effects were obtained when decapitated planarians were cultured at 20 °C, starved for 3 days before head-cutting, and treated with 6 Hz 0.02 T RMFs. At early regeneration stage, RMFs exposure eliminated edema around the wound and facilitated subsequent formation of blastema. It also accelerated cell proliferation and recovery of neuron functionality. Early RMFs exposure up-regulated expression of neural regeneration related proteins, EGR4 and Netrin 2, and mature nerve cell marker proteins, NSE and NPY. These results suggest that RMFs therapy produced early and significant benefit in central nervous regeneration, and should be clinically used at the early stage of neural regeneration, with appropriate optimal frequency and intensity. Bioelectromagnetics. Bioelectromagnetics. © 2016 Wiley Periodicals, Inc.

Since cyclin‑dependent kinases 4/6 (CDK4/6) play pivotal roles in cell cycle regulation and are overexpressed in human skin cancers, CDK4/6 inhibitors are potentially effective drugs for skin cancer. In the present study, we present a mixed computational and experimental study attempting to repurpose approved small‑molecule drugs as dual CDK4/6 inhibitors for skin cancer treatment. We performed structure‑based virtual screening using the docking software idock, targeting an ensemble of CDK4/6 structures. We identified and selected nine compounds with significant predicted scores, and evaluated their cytotoxic effects in vitro in A375 and A431 human skin cancer cell lines. Rafoxanide was found to exhibit the highest cytotoxic effects (IC50: 1.09 µM for A375 and 1.31 µM for A431 cells). Consistent with the expected properties of CDK4/6 inhibitors, rafoxanide significantly increased the G1 phase population. Notably, we revealed that rafoxanide specifically decreased the expression of CDK4/6, cyclin D, retinoblastoma protein (Rb) and the phosphorylation of CDK4/6 and Rb. Furthermore, the anticancer effect of rafoxanide was demonstrated in vivo in BALB/C nude mice subcutaneously xenografted with human skin cancer A375 cells. Rafoxanide (40 mg/kg, i.p.) exhibited significant antitumor activity, comparable to that of oxaliplatin (5 mg/kg, i.p.). The combined administration of rafoxanide and oxaliplatin produced a synergistic therapeutic effect. To the best of our knowledge, the present study is the first to indicate that rafoxanide inhibits CDK4/6 activity and is a potential candidate drug for the treatment of human skin cancer.

Cyclin-dependent kinase 2 (CDK2) has been reported to be overexpressed in human colorectal cancer; it is responsible for the G1-to-S-phase transition in the cell cycle and its deregulation is a hallmark of cancer. The present study was the first to use idock, a free and open-source protein-ligand docking software developed by our group, to identify potential CDK2 inhibitors from 4,311 US Food and Drug Administration-approved small molecular drugs with a re-purposing strategy. Among the top compounds identified by idock score, nine were selected for further study. Among them, adapalene (ADA; CD271,6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphtoic acid) exhibited the highest anti-proliferative effects in LoVo and DLD1 human colon cancer cell lines. Consistent with the expected properties of CDK2 inhibitors, the present study demonstrated that ADA significantly increased the G1-phase population and decreased the expression of CDK2, cyclin E and retinoblastoma protein (Rb), as well as the phosphorylation of CDK2 (on Thr-160) and Rb (on Ser-795). Furthermore, the anti-cancer effects of ADA were examined in vivo on xenograft tumors derived from DLD1 human colorectal cancer cells subcutaneously inoculated in BALB/C nude mice. ADA (20 mg/kg orally) exhibited marked anti-tumor activity, comparable to that of oxaliplatin (40 mg/kg), and dose-dependently inhibited tumor growth (P

RNA interfering (RNAi) using short interfering RNA (siRNA) is becoming a promising approach for cancer gene therapy. However, owing to the lack of safe and efficient carriers, the application of RNAi for clinical use is still very limited. In this study, we have developed cadmium sulphoselenide/Zinc sulfide quantum dots (CdSSe/ZnS QDs)-based nanocarriers for in vitro gene delivery. These CdSSe/ZnS QDs are functionalized with polyethyleneimine (PEI) to form stable nanoplex (QD-PEI) and subsequently they are used for siRNA loading which specially targets human telomerase reverse transcriptase (TERT). High gene transfection efficiency (>80%) was achieved on two glioblastoma cell lines, U87 and U251. The gene expression level (49.99 ± 10.23% for U87, 43.28 ± 9.66% for U251) and protein expression level (51.58 ± 7.88% for U87, 50.69 ± 7.59% for U251) of TERT is observed to decrease substantially after transfecting the tumor cells for 48 h. More importantly, the silencing of TERT gene expression significantly suppressed the proliferation of glioblastoma cells. No obvious cytotoxicity from these QD-PEI nanoplexes were observed over at 10 times of the transfected doses. Based on these results, we envision that QDs engineered here can be used as a safe and efficient gene nanocarrier for siRNA delivery and a promising tool for future cancer gene therapy applications. ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version

Stroke causes death or long-term disabilities and threatens the general health of the population worldwide. Recent studies have suggested that miRNAs are dysregulated and can be used as biomarkers for diagnosis and prognosis in stroke. The intracerebral hemorrhage (ICH) accounts for 15% of all the stroke cases. However, at present, little is known regarding the functions and clinical implications of miRNAs in ICH. In the present study, we established the collagenase-induced rat ICH model to mimic human ICH syndrome. We profiled the expression of 728 rat miRNAs at different time points in rat brain tissues and plasma post-ICH and identified a set human brain-enriched miRNAs that had changed expression level in the plasma of rat ICH. Among them, the expression levels of miR-124 displayed significantly synchronous alterations in rat plasma and brain tissue during ICH progression. They were significantly elevated at the acute injury phase (day 1 and 2), gradually decreased during the delayed recovery phase (day 7, 14 and 30), and finally restored to normal levels at late recovery phase (day 60). We further determined the plasma expression profile of miR-124 from human ICH patients. Similar to the pattern observed in rat ICH model, our results indicated that immediately after patients reached the hospital, the average plasma concentrations of miR-124 increased more than 100-fold in 24 h, then decreased gradually on day 2, 7, 14 and to near normal level on day 30. Taken together, these results strongly suggested that plasma concentration of miR-124 is a promising candidate biomarker for the early detection and predictive prognosis of human ICH.

MicroRNAs (miRNAs) are members of non-coding RNAs. They are involved in diverse biological functions. MiRNAs are precisely regulated in a tissue- and developmental-specific manner, but dysregulated in many human diseases, in particular cancers. Transcriptional regulation, post-transcriptional regulation, as well as genetic alterations, are the three major mechanisms controlling the spatial and temporal expression of miRNAs. Emerging evidence now indicates that transcriptional and epigenetic regulations play major roles in miRNA expression. This review summarizes the current knowledge and discusses the future challenges.

Background Antiangiogenic therapies are considered promising for the treatment of glioblastoma (GB). The non-collagenous C-terminal globular NC1 domain of type VIII collagen a1 chain, Vastatin, is an endogenous antiangiogenic polypeptide. Sustained enhanced expression of Vastatin was shown to inhibit tumour growth and metastasis in murine hepatocellular carcinoma models. In this study, we further explored the efficacy of Vastatin in the treatment of GB xenografts. Method Treatment of Vastatin was carried out using a nanopolymer gene vector PEI600-CyD-Folate (H1). Antiangiogenic effect of Vastatin was tested in vitro by using co-culture system and conditioned medium. An orthotopic GB murine model was established to examine the in vivo therapeutic effect of Vastatin alone treatment and its combination with temozolomide. Results Vastatin gene transfection mediated by H1 could target tumour cells specifically and suppress the proliferation of microvessel endothelial cells (MECs) through a paracrine inhibition manner. Enhancing Vastatin expression by intracerebral injection of H1-Vastatin significantly prolonged animal survival from 48 to 75 days in GB murine model, which was comparable to the effect of Endostatin, the most studied endogenous antiangiogenic polypeptide. The diminished presence of CD34 positive cells in the GB xenografts suggested that Vastatin induced significant antiangiogenesis. Moreover, a synergistic effect in extending survival was detected when H1-Vastatin was administered with temozolomide (TMZ) in GB chemoresistant murine models. Conclusion Our results suggest, for the first time, that Vastatin is an antiangiogenic polypeptide with significant potential therapeutic benefit for GB. H1-Vastatin gene therapy may have important implications in re-sensitizing recurrent GB to standard chemotherapeutic agents.

Hepatocellular carcinoma (HCC) is a hypervascular cancer without effective treatment. Here we report that polypeptide of NC1 domain of type VIII collagen (Vastatin) is an endogenous polypeptide expressed in normal liver tissue but lost in the liver of most HCC patients (73.1%). Its expression level is negatively associated with tumor size (P = 0.035) and metastasis (P = 0.016) in HCC patients. To evaluate its potential use as a therapeutic, we constructed a recombinant adeno-associated virus carrying Vastatin (rAAV-Vastatin) to treat HCC in an orthotopic Buffalo rat model. rAAV-Vastatin treatment significantly prolonged the median survival, inhibited tumor growth, and completely prevented metastasis in HCC-bearing rats by decreasing microvessel density and increasing tumor necrosis. No detectable toxicity in nontumor-bearing mice was observed. To investigate its molecular mechanisms, we performed DNA microarray, western blotting assays, and bioinformatic analysis to determine its effect on global gene expression patterns and signal transduction pathways. Our results indicated that rAAV-Vastatin significantly reduced the expressions of Pck1, JAG2, and c-Fos, thus inhibiting the cellular metabolism, Notch and AP-1 signaling pathways, respectively. Hence, we demonstrated for the first time that Vastatin is a novel, safe, and effective antiangiogenic therapeutic and a potential biomarker for HCC.

Chemokine CC-motif receptor-like 2 (CCRL2) is a 7-transmembrane G protein-coupled receptor which plays a key role in lung dendritic cell trafficking to peripheral lymph nodes. The function and expression of CCRL2 in cancer is not understood at present. Here we report that CCRL2 expression level is elevated in human glioma patient samples and cell lines. The magnitude of increase is positively associated with increasing tumor grade, with the highest level observed in grade IV glioblastoma. By gain-of-function and loss-of-function studies, we further showed that CCRL2 did not regulate the growth of human glioblatoma U87 and U373 cells. Importantly, we demonstrated that over-expression of CCRL2 significantly enhanced the migration rate and invasiveness of the glioblastoma cells. Taken together, these results suggest for the first time that elevated CCRL2 in glioma promotes cell migration and invasion. The potential roles of CCRL2 as a novel therapeutic target and biomarker warrant further investigations.

The human microRNA cluster MC-let-7a-1∼let-7d, with three members let-7a-1, let-7f-1, and let-7d, is an important cluster of the let-7 family. These microRNAs play critical roles in regulating development and carcinogenesis. Therefore, precise control of MC-let-7a-1∼let-7d level is critical for cellular functions. In this study, we first showed that the expression of these three members was significantly reduced in human hepatocellular carcinoma HepG2 cells as compared with the immortalized human liver L02 cells. We demonstrated that the MC-let-7a-1∼let-7d cluster was encoded by a single polycistronic transcript driven by a 10-kb upstream promoter, with two MYC-binding sites. Importantly, MYC inhibited MC-let-7a-1∼let-7d promoter activity via binding to the noncanonical E-box 3 downstream of the transcription start sites, whereas it enhanced promoter activity by binding to the canonical E-box 2 upstream of the transcription start sites. We found that although the binding affinity of MYC to E-box 2 was stronger than E-box 3, the binding quantum of MYC to E-box 3 was significantly higher in cancerous HepG2 cells as compared with the noncancerous L02 cells. In addition, forced expression of let-7 could reverse the MYC-mediated cell proliferation. These findings suggested that in L02 cells with a low level of MYC, MYC binds mainly to E-box 2 to enhance MC-let-7a-1∼let-7d expression. However, in HepG2 cells with an elevated MYC, the extra MYC could bind to E-box 3 to suppress the transcription of MC-let-7a-1∼let-7d and thus enable HepG2 cells to maintain a high level of MYC and a low level of let-7 microRNAs simultaneously.

Telomere maintenance is essential for cancer growth. Induction of telomere dysfunction, for example, by inhibition of telomeric proteins or telomerase, has been shown to strongly enhance cancer cells' sensitivity to chemotherapies. However, it is not clear whether modulations of telomere maintenance constitute cancer cellular responses to chemotherapies. Furthermore, the manner in which anti-cancer drugs affect telomere function remains unknown. In this study, we show that anthracyclines, a class of anti-cancer drugs widely used in clinical cancer treatments, have an active role in triggering telomere dysfunction specifically in telomerase-positive cancer cells. Anthracyclines interrupt telomere maintenance by telomerase through the downregulation of PinX1, a protein factor responsible for targeting telomerase onto telomeres, thereby inhibiting telomerase association with telomeres. We further demonstrate that anthracyclines downregulate PinX1 by inducing this protein degradation through the ubiquitin-proteasome-dependent pathway. Our data not only reveal a novel action for anthracyclines as telomerase functional inhibitors but also provide a clue for the development of novel anti-cancer drugs based on telomerase/telomere targeting, which is actively investigated by many current studies.

OBJECTIVE CD4 CD25 T regulatory (Treg) cells are a population of T cells which suppress an overactive immune system. CCR4 is a chemokine receptor involved in the recruitment of lymphocytes. Nasopharyngeal carcinoma (NPC) is resistant to immunosurveillance, owing to the increased number of tumor-infiltrating Treg cells which are recruited to the tumor by CCR4. METHODS The percentage of CD4+CD25+ Treg cells and CCR4+ cells in tissue or peripheral blood (PB) lymphocytes of patients with untreated NPC or normal subjects was analysed by flow cytometry. RESULTS In both tissue and PB lymphocytes, the percentage of CD4+CD25+ Treg cells and CCR4+cells was significantly elevated in patients with NPC in comparison with that in the normal tissue of controls. Furthermore, in the patients with NPC, a higher percentage of CD4+CD25+ Treg cells was found in the tumor-infiltrating (TI) lymphocyte population than in the PB population. In the NPC patient group, a general trend towards an increased percentage of TI Treg cells was found in the patients with advanced stage NPC. The number of CD4+CD25+ Treg cells was positively related to the number of CCR4+ cells in the tumor and in the PB of the patients with NPC, while the number of CD4+CD25+ Treg cells was negatively related to the number of CD4+CD25 － T cells. CONCLUSION Immunosuppression was observed in NPC, especially at the tumor sites. CD4+CD25+ Treg cells may suppress CD4+CD25 － T cells. CCR4 may have an important role in the recruitment of CD4+CD25+ Treg cells to tumor sites, thus causing resistance to immunosurveillance.

Cell cycle-related kinase (CCRK) is a newly identified protein kinase homologous to Cdk7. We have previously shown that CCRK is a candidate oncogene in human glioblastoma. However, whether CCRK is a bona fide oncogene remains to be tested. The aim of this study was to investigate the role of CCRK in human colorectal cancer carcinogenesis. By Western blotting, we analysed the expression profile of CCRK protein in 10 colorectal cancer tissue samples and their adjacent normal colon tissues and in seven colorectal cancer cell lines. CCRK protein expression was also investigated by immunohistochemistry in a colorectal tissue microarray, which contained 120 cases of primary colorectal cancer and adjacent normal colorectal mucosa. The effects of CCRK knock-down on cell cycle profile and proliferation of colorectal cancer cells were examined by transfecting LoVo and DLD1 human colorectal cancer cell lines by either short-hairpin RNA (shCCRK) or small interfering RNA targeting CCRK (siCCRK). We found that CCRK protein levels were elevated by more than 1.5-fold in 70% of colorectal cancer patient samples examined and CCRK was detectable in all seven colorectal cancer cell lines tested. Colorectal tissue microarray indicated that overexpression of CCRK was detected in 62/109 (56.9%) of informative colorectal cancer cases and was significantly associated with the tumour pT and pN status (p < 0.05). Suppression of CCRK by siCCRK led to G1 phase cell cycle arrest and reduced cell growth. Consistently, stable clones of LoVo and DLD1 cells expressing shCCRK exhibited decreased cell proliferation rates. Furthermore, we showed that CCRK is required for the phosphorylation of Cdk2 (on Thr-160) and Rb (on Ser-795) and the expression of cyclin E. These results suggest for the first time that CCRK is involved in colorectal cancer carcinogenesis and G1/S cell cycle transition by regulating Cdk2, cyclin E and Rb.

Herba Epimedii is one of the most commonly used Chinese herbs for treating osteoporosis. In the present study, the flavonoids of Herba Epimedii (HEF) have shown to promote the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. They were noted to enhance the mRNA expression of BMP-2, BMP-4, Runx2, beta-catenin and cyclinD1, all of which are BMP or Wnt-signaling pathway related regulators. The osteogenic effect was inhibited by the introduction of noggin and DKK-1, which is classical inhibitor of BMP and Wnt/beta-catenin signaling, respectively. These results suggest that HEF exerts promoting effect on osteogenic differentiation, which plausibly functions via the BMP and Wnt/beta-catenin signaling pathways. Considering the therapeutic efficiency and economical issues, HEF may be a potential candidate for promoting bone regeneration. On the other hand, osteogenic differentiation of MSCs may also be a promising and attractive tool to apply in bone repair.

Fructus Ligustri Lucidi (FLL) has been used in traditional Chinese medicine for over 1000 years. The ethanol extract of FLL (EFLL) has been shown to be a potential candidate in the prevention and treatment of osteoporosis. The present study aimed to determine whether EFLL carries out the effect by promoting osteogenesis in mesenchymal stem cells (MSCs). The osteogenic differentiation of MSCs was evaluated by their alkaline phosphatase (ALP) activities and mineralization. Expression of genes was detected by RT-PCR. We found that EFLL significantly stimulated the ALP activities and shortened the time needed for the mineralization of MSCs during osteogenic differentiation. The expression of several osteoblast differentiation regulators was also upregulated by EFLL during this process. Our study demonstrated that the EFLL is capable of enhancing osteogenic differentiation of MSCs. It might be useful for treating diseases with inadequate bone formation, including osteoporosis.

Chitosan is a naturally occurring cationic mucopolysaccharide. It is generally biocompatible, biodegradable, mucoadhesive, non-immunogenic and non-toxic. Although chitosan is able to condense nucleic acids (NA) (both DNA and RNA) and protect them from nuclease degradation, its poor water solubility and low transfection efficacy have impeded its use as an NA carrier. In order to overcome such limitations, a multitude of strategies for chitosan modification and formulation have been proposed. In this article, we will first give a brief overview of the physical and biological properties of chitosan. Then, with a special focus on plasmid DNA delivery, we will have a detailed discussion of the latest advances in chitosan-mediated NA transfer. For future research, the following three important areas will be discussed: chitosan-mediated therapeutic small RNA transfer, structure–activity relationships (SAR) in chitosan vector design, and chitosan-mediated oral/nasal NA therapy.

The low in vivo transduction efficiency of recombinant adeno-associated virus (rAAV) and the undesirably strong immunogenicity of adenovirus (rAdv) have limited their clinical utilization in cancer gene therapy. We have previously demonstrated that intratumoral injection of rAAV expressing a C-terminal polypeptide of human telomerase reverse transcriptase (rAAV-hTERTC27) effectively inhibits the growth of glioblastoma xenografts in nude mice. To further improve its efficacy, we combined rAAV-hTERTC27 with rAdv and investigated the efficiency of the cocktail vectors in vivo. At a nontherapeutic dose (1 x 10(8) plaque-forming units (PFUs)), rAdv-null and rAdv-hTERTC27 were equipotent in enhancing the therapeutic efficacy of rAAV-hTERTC27 (1.5 x 10(11) v.g.), and complete tumor regression was achieved in 25% of the treated animals. Importantly, the combination of rAAV-hTERTC27 and a therapeutic dose (2.5 x 10(9) PFU) of rAdv-hTERTC27 significantly augmented the therapeutic effects and led to a 38% complete tumor regression rate. In vivo optical imaging also showed that rAAV-luc/rAdv-luc cocktail vectors could synergistically enhance the early transient and latent sustained expression of luciferase, as compared to rAdv-luc and rAAV-luc alone. These findings suggest that the combination of rAAV-hTERTC27 and a therapeutic dose of rAdv-hTERTC27 is potentially a promising treatment for glioblastoma, and the rAAV/rAdv cocktail vector system warrants further development for cancer gene therapy.

Enhancer of zeste homolog 2 (EZH2) is suggested to be a potential therapeutic target and a diagnostic marker for cancer. Our previous study also showed the critical role of EZH2 in hepatocellular carcinoma (HCC) tumorigenesis. The present study is aimed at revealing the comprehensive downstream pathways of EZH2 by functional proteomic profiling. Lentivirus mediated RNA interference (RNAi) was employed to knockdown EZH2 in HCC cells. The 2-DE was employed to compare the expression profile difference between parental and EZH2-knockdown HCC cells. In total, 28 spots were differentially expressed during EZH2 inhibition. Among all, 18 proteins were identified by PMF with MALDI-TOF MS. Western blotting further validated upregulation of 60S acidic ribosomal protein P0 (L10E), and downregulation of two proteins with EZH2 inhibition: stathmin1 and probable protein disulfide isomerase (PDI) ER-60 precursor (ERp57). Moreover, L10E was downregulated with overexpression of EZH2 in hepatocytes, and L10E reversed the effect of EZH2 on cell proliferation, suggesting it a downstream target of EZH2. The comprehensive and comparative analyses of proteins associated with EZH2 could further our understanding on the downstream signal cascade of EZH2 leading to tumorigenesis.

// Zifeng Wang 1 , Sheng Lin 4 , Ji Zhang 1 , Zhenhua Xu 5 , Yu Xiang 1 , Hong Yao 6 , Lei Ge 8 , Dan Xie 1 , Hsiang-fu Kung 7 , Gang Lu 3 , Wai Sang Poon 3 , Quentin Liu 1 , Marie Chia-mi Lin 2 1 Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China; Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China 2 Shenzhen Key Laboratory of Translational Medicine of Tumor, School of Medicine, Shenzhen University, Shenzhen, China 3 Brain Tumor Centre and Division of Neurosurgery, Department of Surgery, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China 4 Laboratory of Medical Genetics, Shenzhen Research Institute of Population and Family Planning, Shenzhen, China 5 Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA 6 Jiangsu Eng. Laboratory of Cancer Biotherapy, Xuzhou Medical College, Xuzhou, China 7 School of Biomedical Science, and State Key Laboratory in Oncology in South China, Chinese University of Hong Kong, Shatin, Hong Kong 8 Department of Gastrointestinal Surgery, Tumor Hospital, Xinjiang Medical University, Urumqi, Xinjiang Uyghur Autonomous Region, China Correspondence to: Marie Chia-mi Lin, email: mcmlin@163.com Quentin Liu, email: liuq9@mail.sysu.edu.cn Keywords: MC-let-7a-1~let-7d, MYC, glioblastoma, transcriptional regulation Received: October 16, 2015 Accepted: May 20, 2016 Published: July 09, 2016 ABSTRACT Previously, we reported that MYC oncoprotein down-regulates the transcription of human MC-let-7a-1~let-7d microRNA cluster in hepatocarcinoma (HCC). Surprisingly, in silico analysis indicated that let-7 miRNA expression levels are not reduced in glioblastoma (GBM). Here we investigated the molecular basis of this differential expression. Using human GBM U87 and U251 cells, we first demonstrated that forced over-expression of MYC indeed could not down-regulate the expression of human MC-let-7a-1~let-7d microRNA cluster in GBM. Furthermore, analysis of MC-let-7a-1~let-7d promoter in GBM indicated that MYC failed to inhibit the promoter activity. Pearson's correlation and Linear Regression analysis using the expression data from GSE55092 (HCC) and GSE4290 (GBM) demonstrated a converse relationship of MC-let-7a-1~let-7d and MYC only in HCC but not in GBM. To understand the underlying mechanisms, we examined whether MYC could bind to the non-canonical E-box 3 located in the promoter of MC-let-7a-1~let-7d. Results from both chromatin immune-precipitation (ChIP) and super-shift assays clearly demonstrated the loss of MYC and E-box 3 binding in GBM, suggesting for the first time that a defective MYC and E-box3 binding in GBM is responsible for the differential MYC mediated transcriptional inhibition of MC-let-7a-1~let-7d and potentially other tumor suppressors. MYC and let-7 are key oncoprotein and tumor suppressor, respectively. Understanding the molecular mechanisms of their regulations will provide new insight and have important implications in the therapeutics of GBM as well as other cancers.

Magnetic field exposure is an accepted safe and effective modality for nerve injury. However, it is clinically used only as a supplement or salvage therapy at the later stage of treatment. Here, we used a planarian Girardia sinensis decapitated model to investigate beneficial effects of early rotary non-uniform magnetic fields (RMFs) exposure on central nervous regeneration. Our results clearly indicated that magnetic stimulation induced from early RMFs exposure significantly promoted neural regeneration of planarians. This stimulating effect is frequency and intensity dependent. Optimum effects were obtained when decapitated planarians were cultured at 20 °C, starved for 3 days before head-cutting, and treated with 6 Hz 0.02 T RMFs. At early regeneration stage, RMFs exposure eliminated edema around the wound and facilitated subsequent formation of blastema. It also accelerated cell proliferation and recovery of neuron functionality. Early RMFs exposure up-regulated expression of neural regeneration related proteins, EGR4 and Netrin 2, and mature nerve cell marker proteins, NSE and NPY. These results suggest that RMFs therapy produced early and significant benefit in central nervous regeneration, and should be clinically used at the early stage of neural regeneration, with appropriate optimal frequency and intensity.

Background The immune non-recognition is often the underlying cause of failure in tumor immunotherapeutic. This is because most tumor-related antigens are poorly immunogenic, and fail to arouse an efficient immune response against cancers. Here we synthesized a novel TLR7 agonist, and developed a safe and effective immunotherapeutic vaccine by conjugating this TLR7 agonist with the pluripotency antigen OCT4. Methods Purified recombinant OCT4 protein was covalently linked with a novel TLR7 agonist to form a TLR7-OCT4 conjugate (T7-OCT4). After conjugation, the in vitro release of IL-12 and IFN-γ was observed in spleen lymphocytes. Mice were immunized with TLR7-OCT4, and the release of IFN-γ, the percentages of CD3+/CD8+ T cells and the OCT4-specific cytotoxicity rates were measured. The immunized mice were challenged with mouse embryonic carcinoma (EC), and the tumor volume and tumor weight were determined. Blood routine examination was performed to evaluate the biosafety of TLR7 agonist and TLR7-OCT4 conjugate in mice. Results T7-OCT4 conjugate significantly increased the in vitro release of IL-12 and IFN-γ by mouse spleen lymphocytes. In addition, the release of IFN-γ, the percentages of CD3+/CD8+ T cells and the tumor-specific cytotoxicity rates in immunized mice were significantly higher. Importantly, in EC xenografted mice, immunization with T7-OCT4 conjugate decreased the growth of the tumor dramatically up to 90 %, as compared to mice immunized with OCT4 protein or TLR7 agonist alone. Furthermore, blood routine examination demonstrated that no abnormalities of the blood cells and components in the blood fluids were detected by T7-OCT4 and TLR7 agonist injections. Conclusions Our results showed that conjugating OCT4 protein to the novel TLR7 agonist produced a vaccine which is effective and safe in preventing tumor growth in mice. Our results suggest that this type of vaccine formulation has great potentiality in preventive vaccines against OCT4 expressing tumors. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0524-y) contains supplementary material, which is available to authorized users.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Surgical resection and conventional chemotherapy and radiotherapy ultimately fail due to tumor recurrence and HCC’s resistance. The development of novel therapies against HCC is thus urgently required. The cyclin-dependent kinase (CDK) pathways are important and well-established targets for cancer treatment. In particular, CDK2 is a key factor regulating the cell cycle G1 to S transition and a hallmark for cancers. In this study, we utilized our free and open-source protein-ligand docking software, idock, prospectively to identify potential CDK2 inhibitors from 4,311 FDA-approved small molecule drugs using a repurposing strategy and an ensemble docking methodology. Sorted by average idock score, nine compounds were purchased and tested in vitro. Among them, the anti-psychotic drug fluspirilene exhibited the highest anti-proliferative effect in human hepatocellular carcinoma HepG2 and Huh7 cells. We demonstrated for the first time that fluspirilene treatment significantly increased the percentage of cells in G1 phase, and decreased the expressions of CDK2, cyclin E and Rb, as well as the phosphorylations of CDK2 on Thr160 and Rb on Ser795. We also examined the anti-cancer effect of fluspirilene in vivo in BALB/C nude mice subcutaneously xenografted with human hepatocellular carcinoma Huh7 cells. Our results showed that oral fluspirilene treatment significantly inhibited tumor growth. Fluspirilene (15 mg/kg) exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil (10 mg/kg). Moreover, the cocktail treatment with fluspirilene and 5-fluorouracil exhibited the highest therapeutic effect. These results suggested for the first time that fluspirilene is a potential CDK2 inhibitor and a candidate anti-cancer drug for the treatment of human hepatocellular carcinoma. In view of the fact that fluspirilene has a long history of safe human use, our discovery of fluspirilene as a potential anti-HCC drug may present an immediately applicable clinical therapy.

Oxaliplatin, the first line chemotherapeutic of colon cancer, induces damage to tumors via induction of apoptosis. PUMA (p53 up-regulate modulator of apoptosis) is an important pro-apoptotic member of Bcl-2 family and regulated mainly by p53. Here we investigated the role of PUMA in oxalipaltin-induced apoptosis and the potential mechanism. We showed that oxaliplatin-induced PUMA expression in a time- and dose-dependent manner and suppression of PUMA expression by stable transfecting anti-sense PUMA plasmid decreased oxaliplatin-induced apoptosis in colon cancer cells. By abrogating the function of p53, we further demonstrated that the induction was p53-independent. We also found that oxaliplatin could inactivate ERK and suppression of ERK activity by its specific inhibitor (PD98059), and dominant negative plasmid (DN-MEK1) enhanced the oxaliplatin-induced PUMA expression and apoptosis in a p53-independent manner. Taken together, our data suggest that PUMA plays an important role in oxaliplatin-induced apoptosis and the induction could be both p53-dependent and p53-independent. Moreover, PUMA expression and apoptosis in oxaliplatin-treated colon cancer cells could be regulated partly by ERK inactivation. Identification of the molecular components involved in regulating the cellular sensitivity to oxaliplatin may provide potential targets for development of novel compounds that may be useful in enhancement of oxaliplatin cytotoxicity in p53 deficient colon cancer.

Secretin is a neuropeptide that is expressed in distinct central neurones. As there is no information on how the secretin gene is regulated in neuronal cells, a well established neuronal differentiation cell model, SH-SY5Y, was used to study transcriptional regulation of the human secretin gene. High secretin transcript and peptide levels were found in this cell, and secretin gene expression and promoter activity were up-regulated upon all-trans retinoic acid (RA) treatment. Within the promoter, a functional GC-box 1 (-131 from ATG, relative to the ATG initiation codon) was found to be regulated by a brain-specific Sp protein, Sp4, and ubiquitous factors Sp1 and Sp3. The human secretin gene in SH-SY5Y cells is controlled by the (Sp1 + Sp4)/Sp3 ratio and the RA-induced activation is a partial result of a decrease in Sp3 levels. In addition to the GC-box 1, an N1 motif in close proximity was also responsible for RA-induced secretin gene activation. Competitive gel mobility shift and southwestern blot studies revealed binding of Nuclear Factor I (NFI) with the N1 motif. Overexpression of NFI-C increased promoter activity upon RA treatment. Consistent with this observation, NFI-C transcript levels were augmented after RA treatment. We conclude that RA induction of the secretin gene in neuronal cells is regulated by the combined actions of reducing Sp3 and increasing NFI-C expression.

Background and Aim: Products of the arachidonic acid metabolizing enzyme, 5-lipoxygenase (5-LOX), stimulate the growth of several cancer types. Inhibitors of 5-LOX and 5-LOX-activating protein (FLAP) induce apoptosis in some cancer cells. Here, the authors investigated the effect of a FLAP inhibitor, MK-886, on the inhibition of proliferation and induction of apoptosis in gastric cancer. Methods: Cell proliferation in gastric cancer cells was measured using an 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was measured using acridine orange staining and flow cytometry. Protein expression of apoptosis-related genes p53, p21waf1, p27kip1, bcl-2 families, cytochrome c, and the caspases were examined using Western blotting. Caspase-3 activity was measured using colorimetric assay of substrate cleavage. Results: MK-886 inhibited cell growth in a dose- and time-dependent manner. Apoptosis was induced in gastric cancer cells and was characterized by upregulation of p27kip1 and bax, with release of cytochrome c from mitochondria into cytosol, which initiated caspase-3 activation. Specific caspase-3 inhibitors partially blocked MK-886-induced apoptosis. Conclusion: The present results suggest that MK-886 induces apoptosis in gastric cancer cells through upregulation of p27kip1 and bax, and that MK-886 is a potentially useful drug in gastric cancer prevention and therapy.

The most severe form of brain glioma, glioblastoma (GBM), is highly malignant and usually resistant to chemotherapy. Therefore, discovery of new targets for gene therapy is important. Using subtraction cloning, we identified the human N-Myc downstream-regulated gene 2 (hNDRG2), located at chromosome 14q11.2, as a gene that is significantly suppressed in GBM tissues. Semiquantitative RT-PCR showed that the hNDRG2 gene transcript is expressed in normal brain tissue and low-grade gliomas but is present at low levels in 15 of 27 (56%) human GBM tissues and all of the 6 human glioblastoma cell lines examined. Furthermore, transfection of human glioblastoma U373 and U138 cells with a cDNA encoding hNDRG2 markedly reduced the cell proliferation. Our findings provide the first evidence to suggest that hNDRG2 may play a role in glioblastoma carcinogenesis. © 2003 Wiley-Liss, Inc.

Despite extensive annotation by two independent teams, the Helicobacter pylori genome appeared to lack a complete secretion machinery. The use of clinical isolates to substantiate in silico annotation is used here to identify the missing secE component of the major secretion machinery of Helicobacter pylori., published_or_final_version

Triptolide, a major component in the extract of Chinese herbal plant Tripterygium wilfordii Hook f (TWHf), has potential anti-neoplastic effect. In the present study we investigated the potential therapeutic effects and mechanisms of triptolide against human gastric cancer cells. Four gastric cancer cell lines with different p53 status, AGS and MKN-45 (wild type p53); MKN-28 and SGC-7901 (mutant p53) were observed as to cell growth inhibition and induction of apoptosis in response to triptolide treatment. We showed that triptolide inhibited cell growth, induced apoptosis and suppressed NK-kappaB and AP-1 transactivation in AGS cells with wild-type p53. Triptolide induced apoptosis by stimulating the expressions of p53, p21(waf1/cip1), bax protein, and increased the activity of caspases. In addition, it caused cell cycle arrest in the G(0)/G(1) phase. To examine the role of p53 in these functions, we showed that suppression of p53 level with antisense oligonucleotide abrogated triptolide-induced apoptosis and over-expression of dominant negative p53 abolished the inhibitory effect on NF-kappaB activation. Furthermore, we demonstrated that triptolide had differential effects on gastric cancer cells with different p53 status. We showed that triptolide also inhibited cell growth and induced apoptosis in MKN-45 with wild-type p53, whereas it had no significant growth-inhibition and apoptosis induction effects on the MKN-28 and SGC-7901 cells with mutant p53. Our data suggest that triptolide exhibits anti-tumor and anti-inflammatory effects by inhibiting cell proliferation, inducing apoptosis and inhibiting NF-kappaB and AP-1 transcriptional activity. However, a functional p53 is required for these proapoptotic, anti-inflammatory and anti-tumor effects.

Background and Aims: Cyclo-oxygenase-2 (COX-2) is the inducible enzyme in the gastric mucosa responsible for prostaglandin production during inflammation and ulcer healing. The regulation of COX-2 gene expression in gastric epithelial cells is not well understood. In this study, we investigated the effect of interleukin (IL)-1β on COX-2 expression in the human gastric cancer cell, and explored the signaling pathways involved. Methods: Gastric cancer cell line AGS was treated with IL-1β or the inhibitors of mitogen-activated protein-Erk kinase (MEK) and p38 mitogen-activated protein (MAP) kinase prior to the addition of IL-1β. The COX-2 mRNA or protein levels were measured by using RT-PCR or western blot analysis, respectively. Prostaglandin E2 (PGE2) production/secretion was determined by using the prostaglandin E2 EIA assay. The phosphorylation/activation of p44/42 and p38 MAP kinases were determined by using western blot analysis and using phospho-specific antibodies. Results: Interleukin-1β treatment dose- and time-dependently increased COX-2 mRNA and protein expression levels, and enhanced PGE2 production/secretion in AGS cells. In contrast, IL-1β had no effect on the level of the constitutively expressed COX-1. In parallel to the increase of COX-2, we showed that p44/42 and p38 MAP kinase activities were also upregulated by IL-1β treatment. To demonstrate the cause–effect relationship, we showed that inhibition of MEK and p38 MAP kinase with specific inhibitors suppressed IL-1β-mediated increases in COX-2 mRNA and protein levels, and the PGE2 production. Conclusions: Our results demonstrated that in human gastric cancer cells, IL-1β upregulates the COX-2 gene expression through the activation of MEK/p44/42 and p38 MAP kinases pathway.

Arachidonic acid release from membrane phospholipids is essential for tumour cell proliferation. Lipoxygenases constitute a pathway for arachidonate metabolism. The present study investigated the expression of 12-lipoxygenase and its effect on cell proliferation as well as survival in two human gastric cancer cell lines (AGS and MKN-28). RT-PCR and western blots, respectively, showed 12-LOX mRNA and protein expression in both AGS and MKN-28 cell lines. Treatment with a 12-LOX inhibitor, baicalein, significantly inhibited cancer cell proliferation, but a metabolite of 12-LOX activity, 12 hydroxyeicosatetraenoic acid (12-HETE) reversed baicalein-induced growth inhibition. Furthermore, the blockade of the 12-LOX pathway through a 12-LOX inhibitor and antisense induced apoptosis of gastric cancer cell lines. The biochemical characteristics of apoptosis were p53-independent combined with a decrease in bcl-2 expression. Caspase-7 was proteolytically activated and responsible for the apoptosis execution.

The ubiquitin-proteasome pathway plays a critical role in the degradation of cellular proteins and cell cycle control. Dysregulating the degradation of such proteins should have profound effects on tumor growth and causes cells to undergo apoptosis. The aims of this study are to evaluate the ubiquitin-proteasome pathway in gastric cancer and the potential role of pharmacological inhibition of proteasome on induction of apoptosis in gastric cancer cells. Gastric cancer cell lines AGS (p53 wild-type) and MKN-28 (p53 mutant) were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in AGS and MKN-28 cells in a time- and dose-dependent manner. The inhibition of cell proliferation was caused by apoptosis which was also time- and dose-dependent. AGS cells were more responsive to MG132 than MKN-28 cells. Induction of apoptosis was preceded by the activation of caspase-3, as measured by a colorimetric caspase-3 cellular activity and Western blotting of the cleavage of caspase-3 and its substrate PARP. Activation of caspase-7 was also exhibited. In addition, z-VAD-fmk, a broad spectrum caspase inhibitor, reversed apoptosis induced by MG132 in AGS and MKN28 cells. Although z-DEVD-fmk, a specific caspase-3 inhibitor, suppressed MG132-induced apoptosis in MKN28 cells, it only partially rescued the apoptotic effect in AGS cells. Caspase-3 activation was the result of release of cytochrome c from mitochondria into the cytosol, as a consequence of upregulation of bax. There were overexpressions of all the proteasome-related proteins p53, p21(waf1) and p27(kip1) at 4 hr after proteasome inhibition which was identified by the accumulation of ubiquitin-tagged proteins. This was accompanied by accumulation of cells at G(1) phase. Our present study suggests that inhibition of proteasome function in gastric cancer cells induces apoptosis and proteasomal inhibitors have potential use as novel anticancer drugs in gastric cancer.

hTERTC27 is a newly constructed polypeptide that can induce telomere dysfunction. To study the synergistic antitumor effects of the hTERTC27 polypeptide driven by the early growth response protein-1 (Egr-1) promoter and chemotherapeutic 5-flurorouracil (5-FU) on nasopharyngeal carcinoma, a series of in vitro and in vivo experiments were performed. The results showed that hTERTC27 expression was significantly increased up to 7.21-folds by the 5-FU-activated Egr-1 promoter in C666-1 cells. Overexpressed hTERTC27 made the cells more sensitive to 5-FU, and additionally, inhibited cell proliferation about 20.41%. Combinational therapy of overexpressed hTERTC27 driven by the 5-FU-activated Egr-1 promoter and 5-FU synergistically inhibited cell proliferation and promoted apoptosis of C666-1 cells for about 4.75-fold and 1.76-fold in comparison with a sole therapy of hTERTC27 or 5-FU in vitro. In vivo experiments showed that overexpressed hTERTC27 driven by 5-FU-activated Egr-1 promoter and 5-FU synergistically reduced tumor volume, tumor weight, and local infiltration, which may be relative to tumor cell apoptosis. These results suggest that combinational therapy of overexpressed hTERTC27, which is driven by the 5-FU-activated Egr-1 promoter, and 5-FU may provide a novel approach to treat nasopharyngeal cancer.