NPC-TW01 Cells Research Articles

Treatment with autophagic inhibitors enhances oligonol‑induced apoptotic effects in nasopharyngeal carcinoma cells.

Although the combination of chemotherapy and radiotherapy has increased the survival rate of patients with nasopharyngeal carcinoma (NPC), certain patients do not respond well to the treatment and have a poor prognosis. Therefore, novel therapeutic drugs and strategies to improve prognosis of patients with NPC are required. As certain plant extracts can suppress the viability of cancer cells, the present study investigated whether oligonol, a polyphenolic compound primarily found in lychee fruit, exerts anticancer activities in NPC cells. MTT, ELISA and immunoblotting were performed to investigate cell survival, cytokeratin-18 fragment release, and the expression of apoptosis and autophagy markers, respectively. Oligonol decreased the viability of NPC-TW01 and NPC/HK1NPC cell lines. Oligonol increased the protein expression of several apoptosis markers, including cleaved caspase-8 and -3, cleaved PARP and cytokeratin 18 fragment. Moreover, it also increased expression of autophagy markers Beclin 1 and LC3-II, as well as LC3-II/LC3-I ratio in both NPC cell lines. Furthermore, treatment with autophagy inhibitors 3-methyladenine or LY294002 significantly increased oligonol-induced viability inhibition in NPC-TW01 cells. Combined treatment of oligonol + LY294002 reduced LC3-II expression and the LC3II/LC3I ratio while increasing cleaved caspase-8 and -3, cleaved PARP and cytokeratin 18 fragment expression in NPC-TW01 cells. These findings indicated autophagy inhibitors could enhance viability inhibition and apoptotic effects induced by oligonol in NPC cells.

METCAM/MUC18 Plays a Tumor Suppressor Role in the Development of Nasopharyngeal Carcinoma Type I.

From previous studies of negatively correlating the expression of human METCAM/MUC18 with the pathology of nasopharyngeal carcinoma (NPC), we have suggested that human METCAM/MUC18 (huMETCAM/MUC18) might play a tumor suppressor role in the development of nasopharyngeal carcinoma. To scrutinize this hypothesis, we investigated the effects of huMETCAM/MUC18's over-expression on in vitro cellular behavior and on the in vivo tumorigenesis of one NPC cell line (NPC-TW01). HuMETCAM/MUC18 cDNA was first transfected into the NPC-TW01 cell line, which was established from NPC type I, and many G418-resistant clones were obtained. Then, two NPC-TW01 clones, which expressed high and medium levels of huMETCAM/MUC18, respectively, and one empty vector (control) clone were used to test the effects of huMETCAM/MUC18's over-expression on in vitro behaviors and on in vivo tumorigenesis (via subcutaneous injection) in athymic nude mice (Balb/cAnN.Cg-Foxnlnu/Cr1Nar1). The time course of tumor proliferation and the final tumor weights were determined. Tumor sections were used for the histology and immunohistochemistry (IHC) studies. Tumor lysates were used for determining the expression levels of huMETCAM/MUC18 and various downstream key effectors. HuMETCAM/MUC18's over-expression reduced in vitro motility and invasiveness and altered growth behaviors in 3D basement membrane culture assays, and it decreased the in vivo tumorigenicity of the NPC-TW01 cells. The tumor cells from a high-expressing clone were clustered and confined in small areas, whereas those from a vector control clone were more spread out, suggesting that the tumor cells from the high-expressing clone appeared to stay dormant in micro-clusters. Expression levels of the proliferation index, an index of the metabolic switch to aerobic glycolysis, angiogenesis indexes, and survival pathway indexes were reduced, whereas the pro-apoptosis index increased in the corresponding tumors. The over-expression of huMETCAM/MUC18 in the NPC-TW01 cells decreased the epithelial-to-mesenchymal transition and the in vitro and in vitro tumorigenesis, suggesting that it plays a tumor suppressor role in the development of type I NPC, perhaps by increasing apoptosis and decreasing angiogenesis, proliferation, and the metabolic switch to aerobic glycolysis.

Angiotensin II receptor blockers valsartan and losartan improve survival rate clinically and suppress tumor growth via apoptosis related to PI3K/AKT signaling in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a common type of head and neck cancer in Asia. Adverse effects occur in over 90% of NPC patients treated with radiotherapy or chemoradiation. Angiotensin II receptor blockers (ARBs) are commonly used to treat hypertension without serious adverse effects. However, the anticancer activity of ARBs in NPC remains unclear. We investigated the survival impacts of ARBs among NPC patients in a retrospective study. The anticancer effects and related signaling pathways of the ARBs valsartan and losartan were also evaluated in vitro and in vivo. A total of 927 patients with NPC who had hypertension were enrolled in the study, 272 (29.3%) of whom received ARBs. Kaplan-Meier analysis revealed that patients who used ARBs had higher rates of 5-year overall survival (OS; 87.8% vs 75.1%; P = .002) and disease-specific survival (DSS; 95.4% vs 77.7%; P < .001) than those who did not receive this treatment. Additionally, ARBs inhibited cell proliferation and induced apoptosis by increasing levels of cleaved caspase-3, cleaved caspase-9, and cytochrome C; the cell population in the sub-G1 phase; and caspase-3 activity in NPC-TW01 cells. ARBs inhibited tumor growth and angiogenesis via apoptosis in an NPC xenografts model. Interestingly, ARBs inhibited phosphorylation of PI3K/AKT signaling in vitro and in vivo, which is markedly attributed to their antitumor effects in NPC. These data indicate that ARBs not only improve 5-year OS and DSS among patients with NPC but also exert antiproliferative and antiangiogenesis effects by inducing apoptosis in NPC, supporting that ARBs may be promising agents for treatment of NPC.

Phenethyl Isothiocyanate Inhibits Nasopharyngeal Carcinoma Cell Growth and Invasion

The dietary compound phenethyl isothiocyanate (PEITC), is an active component of cruciferous vegetables and markedly inhibits the growth of a variety of tumors. However, its role in human nasopharyngeal carcinoma (NPC) is obscure. The aim of the present study was to elucidate the possible mechanisms whereby PEITC exhibited anticancer properties in human nasopharyngeal carcinoma NPC-TW01 cells in vitro. The experiment results exhibited that in a dose- and time-dependent manner treatment of NPC-TW01 cells with PEITC significantly inhibited cell proliferation, promoted apoptosis with concurrent G2/M cell cycle arrest and inhibited cell invasion in a dose-dependent manner. These effects were accompanied by significant alterations in the expression levels of key proteins associated with pro-survival signaling pathways, including PI3K, Akt, ERK, NF-κB, Bcl, Bax, cyclin B, CDK4 and CDK6. Significantly, these effects were not reflected in 16HBE normal human bronchial epithelial cells, indicating a safe range of treatment concentrations between 0 and 10 μM PEITC. In conclusion, PEITC exhibited significant anticancer effects against human nasopharyngeal carcinoma cells in vitro with low toxicological impact on normal bronchial epithelial cells. This was achieved through dysregulation of key proteins involved in the occurrence and development of tumors, and this approach may be applied to the clinical treatment of NPC and in drug screening.

Pre-treatment with angiotensin-(1-7) inhibits tumor growth via autophagy by downregulating PI3K/Akt/mTOR signaling in human nasopharyngeal carcinoma xenografts.

The highest incidence of nasopharyngeal carcinoma (NPC) is in southeast China, including Taiwan. Many side effects have been observed following radiation therapy with chemotherapy; hence, exploring new treatment modalities for NPC is an important future direction. Angiotensin-(1–7) [Ang-(1–7)] is an endogenous heptapeptide hormone and important component of the renin–angiotensin system that acts through both the Mas receptor and AT2 receptor, exhibiting anti-proliferative and anti-angiogenic properties in cancer cells. However, the anti-cancer activity of Ang-(1–7) related to autophagy in NPC remains largely debated. The effects and signaling pathway(s) involved in the Ang-(1–7)/Mas receptor axis in NPC were investigated both in vitro and in vivo. Ang-(1–7) inhibited cell proliferation, migration, and invasion in NPC-TW01 cells. Ang-(1–7) induced autophagy by increasing the levels of the autophagy marker LC3-II and by enhancing p62 degradation via activation of the Beclin-1/Bcl-2 signaling pathway with involvement of the PI3K/Akt/mTOR and p38 pathways in vitro study. In addition, pre-treatment with Ang-(1–7) inhibited tumor growth in NPC xenografts by inducing autophagy, suggesting a correlation between PI3K/Akt/mTOR pathway inhibition and the abovementioned anti-cancer activities. However, no autophagy was observed following Ang-(1–7) post-treatment. Taken together, these data indicate that Ang-(1–7) plays a novel role in autophagy downstream signaling pathways in NPC, supporting its potential as a therapeutic agent for alleviation the incidence of NPC and preventive treatment of recurrent NPC.Key messages Ang-(1–7) inhibits cell proliferation, migration, and invasion by activating autophagyAng-(1–7)pre-treatment inhibits tumor growth via autophagy by suppressing PI3K/Akt/mTOR pathway.Ang-(1–7) may provide a novel preventative treatment for NPC and recurrent NPC Electronic supplementary materialThe online version of this article (10.1007/s00109-018-1704-z) contains supplementary material, which is available to authorized users.

Claudin1 promotes the proliferation, invasion and migration of nasopharyngeal carcinoma cells by upregulating the expression and nuclear entry of β-catenin.

The aim of the present study was to measure the expression of Claudin (CLDN) 1 in nasopharyngeal carcinoma (NPC) and to determine its biological function and mechanism of action. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to measure the expression of CLDN1 mRNA and protein, respectively, in the immortalized human nasopharyngeal epithelial cell line NP69 and NPC-TW01 cells. Subsequently, small interfering RNA against CLDN1 and the LV-GFP-PURO-CLDN1 lentivirus were transfected into NPC-TW01 cells. Western blotting was used to determine the effects of CLDN1 down- and upregulation on the expression of the epithelial mesenchymal transition (EMT) markers E-cadherin and vimentin. In addition, the effect of CLDN1 on the expression of β-Catenin was determined. The results demonstrated that levels of CLDN1 mRNA and protein in NPC cells were significantly higher than in NP69 cells. Furthermore, the downregulation of CLDN1 inhibited the proliferation, invasion and migration of NPC-TW01 cells. The results of western blotting demonstrated that the downregulation of CLDN1 resulted in the upregulation of E-cadherin and inhibition of vimentin in NPC-TW01 cells. By contrast, the overexpression of CLDN1 resulted in the downregulation of E-cadherin and upregulation of vimentin in NPC-TW01 cells. The downregulation of β-catenin attenuated the cancer-promoting effect of CLDN1 on NPC-TW01 cells, whereas the upregulation of β-catenin reversed the tumor-suppressing effect of CLDN1 downregulation on NPC-TW01 cells. The results of the present study therefore demonstrate that CLDN1 expression is elevated in NPC cells. As an oncogene, CLDN1 promotes the proliferation, invasion and migration of NPC cells by upregulating the expression and nuclear entry of β-catenin.

Capsaicin Induces Autophagy and Apoptosis in Human Nasopharyngeal Carcinoma Cells by Downregulating the PI3K/AKT/mTOR Pathway.

Capsaicin is a potential chemotherapeutic agent for different human cancers. In Southeast China, nasopharyngeal carcinoma (NPC) has the highest incidence of all cancers, but final treatment outcomes are unsatisfactory. However, there is a lack of information regarding the anticancer activity of capsaicin in NPC cells, and its effects on the signaling transduction pathways related to apoptosis and autophagy remain unclear. In the present study, the precise mechanisms by which capsaicin exerts anti-proliferative effects, cell cycle arrest, autophagy and apoptosis were investigated in NPC-TW01 cells. Exposure to capsaicin inhibited cancer cell growth and increased G1 phase cell cycle arrest. Western blotting and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) were used to measure capsaicin-induced autophagy via involvement of the class III PI3K/Beclin-1/Bcl-2 signaling pathway. Capsaicin induced autophagy by increasing levels of the autophagy markers LC3-II and Atg5, enhancing p62 and Fap-1 degradation and increasing caspase-3 activity to induce apoptosis, suggesting a correlation of blocking the PI3K/Akt/mTOR pathway with the above-mentioned anticancer activities. Taken together, these data confirm that capsaicin inhibited the growth of human NPC cells and induced autophagy, supporting its potential as a therapeutic agent for cancer.

Mechanistic Study of Tetrahydrofuran- acetogenins In Triggering Endoplasmic Reticulum Stress Response-apotoposis in Human Nasopharyngeal Carcinoma

For past three decades, numerous studies have elucidated the antiproliferative effects of acetogenins in hopes of developing a new class of clinical anticancer agents. However, clear and definitive action mechanisms of acetogenins were less clarified. In the present study, three tetrahydrofuran (THF)-containing acetogenins were found to have potent and selective antiproliferative activity against human nasopharyngeal carcinoma (NPC) cell lines and their methotrexate-resistant counterparts. The THF-containing acetogenins induced G2/M phase arrest, mitochondrial damage and apoptosis, and increased cytosolic and mitochondrial Ca2+ in NPCs. Microarray analysis of NPC-TW01 cells treated with squamostatin A, a non-adjacent bis-THF acetogenin, demonstrated an increased endoplasmic reticulum (ER)-stress response (ESR). Enhanced ESR in squamostatin A-treated cells was confirmed by real-time PCR, Western blot and shRNA gene knockdown experiments. Although our results showed that squamostatin A-induced ESR was independent of extracellular Ca2+, the presence of extracellular Ca2+ enhanced the antiproliferative effect of acetogenins. In vivo analyses demonstrated that squamostatin A showed good pharmacokinetic properties and significantly retarded NPC tumor growth in the xenograft mouse model. Conclusively, our work demonstrates that acetogenins are effective and selective inducers of the ESR that can block NPC proliferation, and illustrate a previously unappreciated antitumor mechanism of acetogenins that is effective against nasopharyngeal malignancies.

WTC-01, a novel synthetic oxime-flavone compound, destabilizes microtubules in human nasopharyngeal carcinoma cells in vitro and in vivo.

Dynamic polymerization of microtubules is essential for cancer cell growth and metastasis, and microtubule-disrupting agents have become the most successful anti-cancer agents in clinical use. Besides their antioxidant properties, flavonoids also exhibit strong microtubule-disrupting activity and inhibit tumour growth. We have designed, synthesized and tested a series of oxime/amide-containing flavone derivatives. Here we report the evaluation of one compound, WTC-01 for its anti-proliferative effects in human cancer cells. We used a range of cancer cell lines including two human nasopharyngeal carcinoma (NPC) cell lines, measuring proliferation, cell cycle and apoptosis, along with caspase levels and mitochondrial membrane potentials. Assays of tubulin polymerisation in vitro and computer modelling of the colchicine binding site in tubulin were also used. In mice, pharmacokinetics and growth of NPC-derived tumours were studied. WTC-01 was most potent against proliferation of NPC cells (IC50 = 0.45 μM), inducing accumulation of cells in G2 /M and increasing apoptosis, time- and concentration-dependently. The colchicine competition-binding experiments and computer modelling results suggested that WTC-01 causes microtubule disruption via binding to the colchicine-binding site of tubulin resulting in mitochondrial membrane damage and cell apoptosis via activation of caspase-9/-3 without noticeable activation of the caspase-8. Notably, our in vivo studies demonstrated that at doses of 25 and 50 mg·kg(-1) , WTC-01 exhibited good pharmacokinetic properties and completely inhibited the growth of NPC-TW01 cells in a xenograft nude mouse model. WTC-01, a new synthetic oxime-containing flavone, exhibited potent anti-tumour activity against NPC cells and merits further investigation.

Abstract 3071: MicroRNA-486 (miR-486) promotes invasion by targeting RECK in nasopharyngeal carcinoma cells

Abstract Nasopharyngeal carcinoma (NPC) is a common cancer in Taiwanese and people living in the southeastern region of China. In order to investigate NPC pathogenesis, our laboratory has previously established NPC-TW01 cell line, which was derived from a Taiwanese NPC patient. We also established a NPC-TW01N1 cell line, which was derived from the NPC xenograft in SCID mouse. Both NPC-TW01 and NPC-TW01N1 cell lines have an identical DNA profile (STR). NPC-TW01N1 cell line has higher malignant potency than NPC-TW01 line. For example, the proliferation and invasion capabilities of NPC-TW01N1 line were higher than that of NPC-TW01 line. When we used microRNA microarray to compare the endogenous microRNA expression profiles between NPC-TW01 and NPC-TW01N1 lines, the miR-486, miR-200a and other miRNAs showed higher expression in NPC-TW01N1 cell. Transfection of exogenous miR-486 or anti-miR-486 into NPC-TW01 cells or NPC-TW01N1 cells revealed that miR-486 involves in the regulation of NPC cell progression and invasion. To determine whether miR-486 targeted gene involves in progression and invasion, we analyzed microRNA targeted gene database, Ingenuity pathway databases, and cDNA expression microarrays from miR-486 transfected cells. We found that the RECK gene is one of miR-486 candidate targeted genes that involves in NPC cell progression and invasion. We used pmirGLO luciferase reporter vector assays to demonstrate that miR-486 directly targets RECK 3′ UTR. When we transfected miR-486 or RECK siRNA into NPC cells, we found that they can accelerate tumor progression and invasion. It is concluded that miR-486 and RECK genes maybe potential targets for NPC therapy. Citation Format: Chin-Tarng Lin, Cheng-Der Wu. MicroRNA-486 (miR-486) promotes invasion by targeting RECK in nasopharyngeal carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3071. doi:10.1158/1538-7445.AM2015-3071

Chemoselective synthesis, antiproliferative activities, and SAR study of 1H-pyrazol-5-yl-N,N-dimethylformamidines and pyrazolyl-2-azadienes

Chemoselective microwave-assisted amidination was successfully developed to synthesize 1H-pyrazol-5-yl-N,N-dimethylformamidines and pyrazolyl-2-azadienes. All of the starting materials and resulting products were tested against NCI-H226, NPC-TW01, and Jurkat cancer cells to evaluate their antiproliferative activities. 1H-Pyrazol-5-yl-N,N-dimethylformamidines 2b, 2c, and 2d were most potent with IC50 values in low micromolar range. The formyl group at C-4 position and the grafted amidinyl group in the main core of pyrazolic molecule were necessary for the inhibitory activity.

Nucleolin antisense oligodeoxynucleotides induce apoptosis and may be used as a potential drug for nasopharyngeal carcinoma therapy

Nucleolin (C23, NCL) mRNA was up-regulated in nasopharyngeal carcinoma (NPC) cells compared to that of normal nasomucosal (NNM) cells using a cDNA microarray approach. The level of nucleolin protein was also up-regulated in 13 NPC cell lines, 30 biopsy specimens and nine other cancer cell lines compared to five NNM cells or normal stromal cells, which were analyzed using immunoblotting or immunohistochemistry. We transfected nucleolin antisense oligodeoxynucleotides (phosphorothioate-modified oligodeoxynucleotides; S-ODNs) into NPC-TW01 cells to knockdown nucleolin expression to evaluate the function of nucleolin in cancer cells. Nucleolin knockdown induced NPC cells but not NNM cells to undergo apoptosis. Furthermore, treatment of NPC-TW01 xenograft tumors with nucleolin antisense oligodeoxynucleotides suppressed the growth of xenograft tumors without obvious side effects. Therefore, we suggest that nucleolin may be a potential cancer therapeutic target and that nucleolin antisense oligodeoxynucleotides may be used as a potential drug for therapy in NPC.

Magnetically actuated micromechanical tweezers

Two planar actuators with magnetic thin films are used for magnetic tweezers. The planar actuators consisting of a pair of a 75 × 0.8 × 0.3 μm 3 silicon oxide beam and a 72 × 13 × 0.3 μm 3 silicon oxide plate deposited with a 65 × 4 × 0.1 μm 3 Ni magnetic thin film are successfully fabricated and successfully gripped to a single NPC-tw01 cell consisting of Fe 3O 4 magnetic nanoparticles under a vertical magnetic field. The planar actuator bends under an external magnetic field because of the high shape magnetic anisotropy of the Ni magnetic thin film and a highly sensitive microcantilever. NPC-tw01 cells, which are adherent cells, are cultivated in a culture solution. The two planar actuators are placed in water to move and grip a living cell.

Abstract C236: A novel oxime-containing flavone anticancer agent, WTC01, exhibits potent microtubule disruption ability

Abstract Flavonoids and its derivatives are common components found in human diet. Several studies indicated flavonoids could benefit in cancer prevention or therapy, but their structure-activity relationships (SAR) remain poorly defined. In order to further understand the SAR, a series of oxime-containing flavone derivatives were designed, synthesized and evaluated for their biological anti-cancer potential. The anti-proliferative ability of these compounds was measured by several human cancer cell lines from different origins including nasopharyngeal cancer (NPC-TW01), leukemia (MT-2) and lung carcinoma (H661). Among over 30 different oxime-containing flavone derivatives, (Z)-6-[2-hydroxyimino-2-(4-methoxyphenyl)ethoxy]-2-phenyl -4H-1- benzopyran-4-one (WTC01) possessed potent activity against NPC-TW01 growth with IC50 of 400 nM. As determined by flow cytometry, WTC01 treatment results in an accumulation of NPC-TW01 cells in G2-M phase following a 6-h exposure with amaximum accumulation observed by 18 h. Furthermore, Annexin-V/propidium iodide (PI) binding assay, Hoechst 33258/PI double staining, and PARP cleavage indicates that cell death proceeded through an apoptotic pathway. Interestingly,WTC01 also causes mitochondrial transmembrane potential loss and activation of caspase-9 and caspase-3 without noticeable activation of the caspase-8. These results suggest that the WTC01-mediated apoptotic signaling pathway depends on the mitochondria and caspase-9/-3 cascades. Further studies showed that WTC01 inhibits microtubule assembly in a concentration-dependent manner, this data suggests that tubulin may serve as the cellular target for WTC01. Taken together, these findings indicated that WTC01, a novel synthetic oxime-containing flavone, exhibits potent activity against cancer growth through the disruption of microtubule and has potential for management of nasopharyngeal malignancies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C236.

A G-Quadruplex Ligand 3,3′-Diethyloxadicarbocyanine Iodide Induces Mitochondrion-Mediated Apoptosis But Not Decrease of Telomerase Activity in Nasopharyngeal Carcinoma NPC-TW01 Cells

The G-quadruplex ligand 3,3'-diethyloxadicarbocyanine iodide (DODC) was reported to enhance the apoptotic potency of pheochromocytoma PC-12 and leukemia HL-60 cells through the inhibition of telomerase activity. In this study, a mitochondrion-mediated apoptotic pathway was demonstrated as another cytotoxic mechanism for DODC action. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DNA laddering assays were performed to exhibit the cytotoxicity and apoptosis-inducing activity of DODC. Telomeric repeat amplification protocol (TRAP) assay was used to evaluate the effect of DODC on cellular telomerase. The mitochondrial uptake of probe 3,3'-dihexyloxacarbocyanine iodide was measured by flow cytometry. The mitochondrial proteomes were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Western blot analyses were adopted to demonstrate the change of the distribution of mitochondrial proteins. DODC alone was able to induce apoptotic cell death but not decrease of telomerase activity in nasopharyngeal carcinoma NPC-TW01 cells. Instead, we found evidence that DODC significantly affected cellular mitochondria. DODC inhibited the uptake of another mitochondrial probe 3,3'-dihexyloxacarbocyanine iodide. By proteomic comparative analysis, we found that DODC induced the increase of prohibitin level in the mitochondria, indicating the occurrence of mitochondrial perturbation. Moreover, DODC was found to induce the levels of p53 and an 18-kDa truncated Bax on mitochondria, which in turn potentiated the release of cytochrome c for activation of caspases. DODC induces NPC-TW01 cell apoptosis via a mitochondrion-mediated mechanism. This paper demonstrates another cytotoxic mechanism of DODC other than inhibition of telomerase.

Evaluation of GL331 in combination with paclitaxel: GL331's interference with paclitaxel-induced cell cycle perturbation and apoptosis.

Combination of selecting agents that act on different cellular mechanisms is a common strategy in cancer chemotherapy. GL331 is a new potent topoisomerase II (Topo II) poison; distinctly, paclitaxel is a microtubule-interfering cancer chemotherapeutic agent. In this study, we intended to evaluate the efficacy of combining GL331 with paclitaxel in cell killing and apoptotic induction in nasopharyngeal carcinoma NPC-TW01 cells. By MTT and internucleosomal DNA cleavage assays, we found that pretreatment or simultaneous treatment of NPC-TW01 cells with GL331 could significantly interfere with paclitaxel's cell killing and apoptosis-inducing activity. When the administration schedule was reversed, the cytotoxicity of GL331 was attenuated by paclitaxel pretreatment. The anti-cancer activity produced by combining GL331 with paclitaxel was obviously lower than the addition of the activities of two individual agents. NPC-TW01 cells were treated with GL331 and 3H-labeled paclitaxel simultaneously or with GL331 before 3H-labeled paclitaxel. In both conditions, GL331 did not reduce the [3H]paclitaxel level in the cells, suggesting that GL331's interference with paclitaxel's cell-killing and apoptosis-inducing efficacy did not result from any inhibition of cellular uptake or retention of paclitaxel. In addition, we found that GL331-induced perturbation of cell cycle progression dramatically over-rode the patterns of mitotic arrest induced by paclitaxel, and the mechanism could be the inhibition of cyclin B1/CDC2 kinase and MAD2 checkprotein activities.

Epstein-Barr virus nuclear antigen 2 retards cell growth, induces p21 WAF1 expression, and modulates p53 activity post-translationally

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) has been shown to be required for promotion of cell-cycle progression in EBV-immortalized B-lymphocytes. However, other studies have indicated that EBNA2 alone, in the absence of other EBV genes, may retard cell growth. To resolve this discrepancy, we investigated the effect of EBNA2 on the growth of various cells, including EBV target nasopharyngeal carcinoma cells, NPC-TW01 and NPC-TW04. We found that EBNA2 could retard cell growth, in stable Vero, HEp-2, and U2OS cell clones expressing EBNA2, and in Vero, 293, NPC-TW01, and NPC-TW04 cells transiently transfected with EBNA2. While investigating the mechanism underlying the growth-retarding function of EBNA2, we found that EBNA2 induced p21 WAF1 expression in these cells. This induction of p21 WAF1 expression was mediated through p53. EBNA2 was found to stimulate p53 to bind to the p53-response element within the p21 WAF1 promoter, possibly by promoting p53 phosphorylation. This enhancement of p53 sequence-specific DNA-binding activity may be the mechanism through which EBNA2 activates the expression of p53-regulated genes, including p21 WAF1 and mdm-2. Together, these studies reveal a possible intrinsic function of EBNA2 in cell-growth regulation and elucidate a novel mechanism by which EBNA2 modulates transcription.

In vitro evaluation of GL331's cancer cell killing and apoptosis-inducing activity in combination with other chemotherapeutic agents.

GL331 is a novel podophyllotoxin-derived compound and is more efficacious than its congener VP-16 in killing several types of cancer cells, that has promoted considerable interest in its possibility of clinical use. In this study, we found that the higher cytotoxicity of GL331 in nasopharyngeal carcinoma NPC-TW01 cells was attributed to the elevated ability to induce apoptotic cell death. In addition to evaluation of GL331's single agent activity, the use of GL331 in combination with other established therapeutic agents was also evaluated. We found that GL331-induced cell cycle perturbation occurred upon initial 8-h exposure, and pretreatment of NPC-TW01 cells with GL331 for 8 h significantly interfered with the cytotoxicities of VP-16, cisplatin, 5-fluorouracil and adriamycin. When the schedule of drug administration was reversed, high-toxic concentrations of these agents revealed an antagonistic effect on GL331; however, their low-toxic doses had the additive or even more-than-additive effect on the cytotoxicity induced by GL331 at 0.1 microM or less, but for GL331 concentrations of greater than 1 microM, the effect became less than additive. These data suggest that overlapping mechanisms could be elicited by GL331 and other agents, and additional preclinical studies are needed to determine the optimal dose combination and administration schedule that will enhance, rather than interfere with, the efficacy of GL331 in combination with other anti-cancer agents.

GL331-induced disruption of cyclin B1/CDC 2 complex and inhibition of CDC 2 kinase activity

GL331, a new homologue of etoposide (VP-16), was developed to cope with the multiple drug resistance occurring in certain malignant tumours. We previously indicated that GL331, like VP-16 and other major cancer chemotherapeutic agents, induced apoptosis in a variety of human cancer cell lines including nasopharyngeal carcinoma (NPC) NPC-TW01 and NPC-TW04 cells. In this study, we further explored the effect of GL331 on the cell cycle progression of NPC cells. Flow cytometric analysis of DNA content was first used to demonstrate the ability of GL331 to induce cell growth arrest at S-G2 phase in most NPC cells. Besides acting as a topoisomerase II inhibitor, GL331 inhibited cellular cyclin B1-associated CDC 2 kinase activity 6 h after treatment, accounting partly at least for its induction of the cell cycle arrest. As with cyclin A, D1, E, CDK 2 and PCNA, the levels of cyclin B1 and CDC 2 proteins were not changed after GL331 treatment; however, the ability to form complex between cyclin B1 and CDC 2 was obviously affected in GL331-treated NPC cells, which associates with the inhibition of cyclin B1/CDC 2 kinase activity elicited by GL331. These data could provide more principal bases for future therapeutic application of this potential anti-cancer agent.