Treatment Of Nasopharyngeal Carcinoma Cells Research Articles

Targeting Glutamine Metabolism through Glutaminase Inhibition Suppresses Cell Proliferation and Progression in Nasopharyngeal Carcinoma.

Glutaminase (GLS), the key enzyme involved in glutamine metabolism, has been identified as a critical player in tumor growth and progression. The GLS inhibitor CB-839 has entered several clinical trials against a variety of tumors. Our study aimed to investigate the role and underlying mechanism of GLS and its inhibitor CB-839 in nasopharyngeal carcinoma (NPC). The expression, downstream genes, and signaling pathways of GLS in NPC were determined by real-time polymerase chain reaction (RT-PCR), PCR array, western blotting (WB), and immunohistochemical staining (IHC), and the phenotype of GLS was confirmed by in vivo experiments of subcutaneous tumor formation in mice and in vitro experiments of functional biology, including Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, transwell migration, and Boyden invasion assay. Finally, it was also verified whether the treatment of NPC cells by GLS inhibitor CB-839 can change various biological functions and protein expression to achieve the purpose of blocking tumor progression. GLS was remarkably overexpressed in NPC cells and tissues, predicting a poor overall survival of NPC patients. GLS promoted cell cycle, proliferation, colony formation, migratory, and invasive capacities by regulating Cyclin D2 (CCND2) via PI3K/AKT/mTOR pathway in NPC in vitro and in vivo. Notably, CB-839 showed an effective anti-NPC tumor effect by blocking the biological functions of the tumor. The first innovative proof is that GLS promotes cell proliferation by regulating CCND2 via PI3K/AKT/mTOR pathway in NPC, and GLS inhibitor CB-839 may serve as a new potential therapeutic target for NPC treatment.

TMEM130 regulates cell migration through DNA methylation in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC), the common malignant head and neck cancer, is highly prevalent in southern China. The molecular mechanism underlying NPC tumorigenesis is unclear. We used 5-Aza-CdR, a DNA methyltransferase inhibitor, to treat NPC cell lines and discovered that the expression of TMEM130 changed significantly compared with the untreatment cells. This study aimed to identify the relationship between the DNA methylation status of TMEM130 and NPC, and to explore the function of TMEM130 in NPC cell migration. qRT-PCR was performed to investigate the transcriptional expression of TMEM130 in NPC. Bisulfite sequencing PCR and 5-Aza-CdR treatment were used to detect the methylation level of the TMEM130 promoter. Gene Expression Omnibus (GEO) datasets were obtained to identifiy the methylation status and mRNA expression of TMEM130 in NPC and normal control tissues. Transwell and western blot analyses were used to detect cell migration ability after transfection of TMEM130/NC plasmids in NPC cells. The transcriptional expression of TMEM130 was decreased in NPC cell lines compared with in the NP69 cell line. TMEM130 promoter was significantly hyper methylated in three NPC cell lines (C666, CNE, and HONE) but hypo methylated in NP69 cells. The methylation level was higher in NPC than normal control tissues. Additionally, treatment of NPC cells with 5-Aza-CdR increased the TMEM130 mRNA expression level. Overexpression of TMEM130 in NPC cell lines suppressed cell migration ability and affected some epithelial-mesenchymal transition-associated gene expression. This study is the first to investigate the expression and function of TMEM130 in NPC. It was found that TMEM130 hyper methylation might contribute to NPC migration and this gene might act as a tumor suppressor gene. TMEM130 is a promising biomarker for NPC diagnosis.

ABT-199-mediated inhibition of Bcl-2 as a potential therapeutic strategy for nasopharyngeal carcinoma

BackgroundAberrant overexpression of Bcl-2 protein has been detected in 80% of nasopharyngeal carcinoma (NPC), and Bcl-2 family proteins are implicated in both NPC oncogenesis and chemotherapy resistance. Previous studies have shown that while treatment of NPC cells with Bcl-2 family inhibitors alone is rarely effective, concomitant treatment with a cytotoxic reagent such as cisplatin can increase efficacy through a synergistic effect. The aim of the current work was to determine how we might increase the efficacy of Bcl-2 family inhibitors in the absence of cytotoxic reagents, which are associated with negative side effect profiles. MethodsWe assessed cell proliferation in Bcl-2 high-expressing NPC cells by CCK-8 assay after treatment with the Bcl-2 inhibitor ABT-199 and/or the Mcl-1 inhibitor S63845. Apoptotic induction by ABT-199 was evaluated by Annexin V-FITC and PI double staining. We also evaluated Bcl-2 family protein expression (Bim, Mcl-1, Bcl-xL, Noxa) after treatment with ABT-199 by western blotting. Finally, xenografted Balb/c nude mice were used to test ABT-199 efficacy in vivo, H&E and immunohistochemistry assay were used to analyze tumor samples. ResultsABT-199 effectively induced NPC cell apoptosis in vitro and in the xenograft model. Following ABT-199 treatment in NPC cells, upregulation of Mcl-1 and Bcl-xL can lead to drug resistance, while concomitant Noxa overexpression partially neutralized the Mcl-1-caused resistance. Given that ABT-199 induces apoptosis in NPC cells through the Bcl-2/Noxa/Mcl-1 axis, treatment avenues further targeting this pathway should be promising. Indeed, the newly developed Mcl-1 inhibitor S63845 in combination with ABT-199 had a synergistic effect on NPC cell apoptosis. ConclusionBcl-2 inhibition in NPC cells with ABT-199 triggers apoptosis through the Bcl-2/Noxa/Mcl-1 axis, and dual inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2 and Mcl-1 provided a strong synergistic effect without the need for adjunctive cytotoxic agent treatment with cisplatin.

Liquiritigenin Enhances the Radiosensitivity of Nasopharyngeal Carcinoma Cell by Affecting Its Autophagy

Objective: To investigate the effect of licorice on radiosensitization of nasopharyngeal carcinoma cells and its mechanism. Methods: MTT assay was used to detect the effect of different concentrations of glycyrrhizin on the activity of nasopharyngeal carcinoma cells. The changes of autophagy were observed by transmission electron microscopy (TEM) in the treatment of nasopharyngeal carcinoma cells. Western blotting was used to detect the effect of glycyrrhizin on the level of autophagy protein in nasopharyngeal carcinoma. Flow cytometry was used to detect the changes of apoptosis rate of nasopharyngeal carcinoma cell line after radiotherapy. Results: After successfully constructing CNE-2-RR radiation resistant cell line, MTT assay was used to detect the best inhibition rate of 20 mmol/L glycyrrhizin on nasopharyngeal carcinoma (58.86 ± 5.02)%. Transmission electron microscopy showed that the number of autophagosomes increased and the mitochondria and nuclei were abnormal in the treatment of nasopharyngeal carcinoma cells. Western Blotting was used to detect the increase of LC3-II level and the decrease of LC3-I level in nasopharyngeal carcinoma cells of 20 mmol/L concentration group. Conclusion: Glycyrrhizin enhances its sensitivity to radiotherapy by influencing autophagy and DNA repair ability of nasopharyngeal carcinoma cells.

Chloroquinone Inhibits Cell Proliferation and Induces Apoptosis in Nasopharyngeal Carcinoma Cell Lines

Purpose: To demonstrate the role of chloroquinone (CQ) in inducing apoptosis in HONE-1 and HNE-1, nasopharyngeal carcinoma (NPC) cell lines.Methods: Water-soluble tetrazolium salt (WST)-1 assay was used for the determination of cell proliferation while an inverted microscope was employed for the analysis of alterations in the morphology of the cells.Results: CQ treatment led to a significant reduction in the rate of cell proliferation in NPC cells after 48 h. In HONE-1 and HNE-1 cell lines viability was reduced to 89 and 82 %, respectively on treatment with 10 μΜ concentration of CQ without affecting normal human skin keratinocyte cell line, K38. The expression of Ki67, a marker for proliferation as well as proliferating cell nuclear antigen (PCNA), decreased in the CQ-treated NPC cells. Morphological examination of NPC cells revealed cell apoptosis on treatment with CQ after 48 h. Treatment of NPC cells with CQ induced activation of caspases and DNA was damaged which further confirmed CQ mediated induction of apoptosis. The level of apoptotic cells in CQ treated and untreated control HONE-1 cell cultures was 53.67 and 3.78 %, respectively (p < 0.05). In addition, CQ treatment decreased reactive oxygen species (ROS) generation in NPC cells.Conclusion: CQ inhibits cell proliferation of NPC cells by inducing apoptosis via DNA damage, and may be of therapeutic use for the treatment of NPC. However, this requires clinical investigation to ascertain its therapeutic potential.Keywords: Chloroquinone, Caspases, Apoptosis, Nuclear antigen, Nasopharyngeal carcinoma

The natural compound gambogic acid radiosensitizes nasopharyngeal carcinoma cells under hypoxic conditions.

Hypoxia is an important factor that causes decreased local disease control as well as increased distant metastases and resistance to radiotherapy in patients with advanced nasopharyngeal carcinoma (NPC). Gambogic acid (GA), the major active ingredient of gamboge, exerts antitumor effects in vitro and in vivo. However, the molecular mechanism by which GA inhibits tumor radioresistance remains unclear. The present study aimed to investigate the radiosensitizing effects of GA on NPC and explore the underlying mechanisms. CNE-1 and CNE-2 cells exposed to hypoxia and radiation were treated with GA at different concentrations. CCK-8 assay, clonogenic assay, and flow cytometry were performed to analyze cell proliferation, colony formation, apoptosis, and cell cycle. The expression levels of hypoxia-inducible factor-1α (HIF-1α), Bcl-2, Bax, caspase-3, cyclin B1/p-cdc2 and γ-H2AX were assessed using Western blot and/or immunofluorescence analysis. Results of the CCK-8 assay, clonogenic assay, and flow cytometry showed that treatment of NPC cells with growth-suppressive concentrations of GA resulted in G2/M phase arrest and apoptosis. Western blot analysis demonstrated that GA-induced cell cycle arrest and apoptosis in CNE-2 cells was associated with upregulated expression of caspase-3 and Bax and downregulated expression of Bcl-2 and cyclin B1/p-cdc2 in hypoxia. Treatment with GA markedly decreased the expression of HIF-1α under hypoxic conditions. The results of this study suggest that GA efficiently radiosensitizes NPC cells and the effect may be significant in hypoxic conditions.

Chemotherapeutic effect of a novel temozolomide analog on nasopharyngeal carcinoma in vitro and in vivo.

BackgroundMany patients with nasopharyngeal carcinoma (NPC) face poor prognosis. Due to its hidden anatomical location, the tumor is usually diagnosed quite late, and despite initially successful treatment with radiation and cisplatin, many patients will relapse and succumb to the disease. New treatment options are urgently needed. We have performed preclinical studies to evaluate the potential NPC therapeutic activity of a newly developed analog of temozolomide (TMZ), an alkylating agent that is the current chemotherapeutic standard of care for patients with malignant glioma.ResultsTMZ was covalently conjugated to the natural monoterpene perillyl alcohol (POH), creating the novel fusion compound NEO212. Its impact on two NPC cell lines was studied through colony formation assays, cell death ELISA, immunoblots, and in vivo testing in tumor-bearing mice. In vitro, NEO212 effectively triggered tumor cell death, and its potency was significantly greater than that of its individual components, TMZ or POH alone. Intriguingly, merely mixing TMZ with POH also was unable to achieve the superior potency of the conjugated compound NEO212. Treatment of NPC cells with NEO212 inactivated the chemoprotective DNA repair protein MGMT (O6-methylguanine methyltransferase), resulting in significant chemosensitization of cells to a second round of drug treatment. When tested in vivo, NEO212 reduced tumor growth in treated animals.ConclusionOur results demonstrate anticancer activity of NEO212 in preclinical NPC models, suggesting that this novel compound should be evaluated further for the treatment of patients with NPC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0175-6) contains supplementary material, which is available to authorized users.

TFAP2A Regulates Nasopharyngeal Carcinoma Growth and Survival by Targeting HIF-1α Signaling Pathway

TFAP2A is a transcription factor that orchestrates a variety of cell processes, including cell growth and tissue differentiation. However, the regulation of TFAP2A in human nasopharyngeal carcinoma tumorigenesis and its precise mechanism of action remain largely unknown. In this study, we investigated the biologic role and clinical significance of TFAP2A in nasopharyngeal carcinoma growth and progression and identified the underlying molecular mechanisms. We found that TFAP2A was highly expressed in various nasopharyngeal carcinoma cell lines and tumor tissue specimens and was significantly correlated with hypoxia-inducible factor-1α (HIF-1α) expression. A positive correlation of TFAP2A overexpression with advanced tumor stage, local invasion, clinical progression, and poor prognosis of patients with nasopharyngeal carcinomas were also observed. Moreover, we found that knockdown of TFAP2A expression by siRNA significantly inhibited tumor cell growth in nasopharyngeal carcinoma cell lines and in a subcutaneous xenograft mouse model by targeting the HIF-1α-mediated VEGF/pigment epithelium-derived factor (PEDF) signaling pathway. Treatment of nasopharyngeal carcinoma cells with TFAP2A siRNA dramatically inhibited the expression and the release of VEGF protein but did not change the level of PEDF protein, resulting in a significant reduction of the ratio of VEGF/PEDF. Pretreatment with a HIF-1α siRNA did not significantly change the TFAP2A siRNA-mediated inhibition in cell viability. Our results indicate that TFAP2A regulates nasopharyngeal carcinoma growth and survival through the modulation of the HIF-1α-mediated VEGF/PEDF signaling pathway, and suggest that TFAP2A could be a potential prognostic biomarker and therapeutic target for nasopharyngeal carcinoma treatment.

Resveratrol inhibits human nasopharyngeal carcinoma cell growth via blocking pAkt/p70S6K signaling pathways

Resveratrol (trans-3,4',5-trihydroxystilbene) has been shown to exert potent anticancer effects on various types of cancer through its anti-proliferative, anti-angiogenic, antioxidant and pro-apoptotic functions. There is still a lack of experimental evidence regarding whether resveratrol has potential anticancer activity in human nasopharyngeal carcinoma (NPC) cells. The purpose of this study was to explore the anticancer activity of resveratrol in human NPC cells both invitro and invivo. Our results indicated that treatment with resveratrol led to a time- and dose-dependent decrease in cell proliferation in NPC cells. A dose-dependent increase in apoptosis in response to resveratrol treatment was also observed in NPC cells. Flow cytometric analysis showed that treatment of NPC cells with resveratrol led to cell cycle arrest at the S and G2/M phases. Mechanistically, resveratrol treatment downregulated the expression of Bcl-2 and hypoxia-inducible factor-1α (HIF-1α) proteins and upregulated the expression of caspase-3 protein. In addition, resveratrol treatment also significantly decreased the phosphorylation levels of Akt1, p70S6K and p-4E-BP-1 and the protein expression of several cyclins involved in cell cycle regulation. Invivo studies further showed that resveratrol was able to significantly inhibit the growth of NPC tumor xenografts in nude mice. Collectively, our findings suggest that resveratrol exerts potent anti-prolife-rative and pro-apoptotic effects on human NPC cells possibly through interfering with the pAkt1/p70S6K signaling pathways, thus it may potentially be developed as an effective agent for chemoprevention and chemotherapy of human NPC.

Protocadherin8 is a functional tumor suppressor frequently inactivated by promoter methylation in nasopharyngeal carcinoma

Protocadherin8 (PCDH8), an integral membrane protein, was reported to be a tumor suppressor involved in tumorigenesis in cancers. We aimed to investigate the epigenetic inactivation of PCDH8 and its tumor-suppressor function in nasopharyngeal carcinoma (NPC). Frequent downregulation/silencing of PCDH8 was found in NPC cell lines using semiquantitative PCR. Promoter methylation of PCDH8 was observed in 100% (5/5) of cell lines and 85.3% (35/41) of primary tumors by methylation-specific PCR, but not in normal nasopharyngeal tissues. Treatment of NPC cells with 5-aza-2'-deoxycytidine and/or trichostatin A could restore PCDH8 expression. Ectopic expression of PCDH8 in silenced NPC cells significantly inhibited cell colony formation and cell migration. For the first time, our study demonstrates the epigenetic inactivation of PCDH8 by promoter methylation and its tumor-suppressive function in NPC. Thus, PCDH8 could be identified as a tumor suppressor in NPC.

Nicotine Promotes Proliferation of Human Nasopharyngeal Carcinoma Cells by Regulating α7AChR, ERK, HIF-1α and VEGF/PEDF Signaling

Nicotine, the major component in cigarette smoke, can promote tumor growth and angiogenesis, but the precise mechanisms involved remain largely unknown. Here, we investigated the mechanism of action of nicotine in human nasopharyngeal carcinoma (NPC) cells. Nicotine significantly promoted cell proliferation in a dose and time-dependent manner in human NPC cells. The mechanism studies showed that the observed stimulation of proliferation was accompanied by the nicotine-mediated simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling. Treatment of NPC cells with nicotine markedly upregulated the expression of α7AChR and HIF-1α proteins. Transfection with a α7AChR or HIF-1α-specific siRNA or a α7AChR-selective inhibitor significantly attenuated the nicotine-mediated promotion of NPC cell proliferation. Nicotine also promoted the phosphorylation of ERK1/2 but not JNK and p38 proteins, thereby induced the activation of ERK/MAPK signaling pathway. Pretreatment with an ERK-selective inhibitor effectively reduced the nicotine-induced proliferation of NPC cells. Moreover, nicotine upregulated the expression of VEGF but suppressed the expression of PEDF at mRNA and protein levels, leading to a significant increase of the ratio of VEGF/PEDF in NPC cells. Pretreatment with a α7AChR or ERK-selective inhibitor or transfection with a HIF-1α-specific siRNA in NPC cells significantly inhibited the nicotine-induced HIF-1α expression and VEGF/PEDF ratio. These results therefore indicate that nicotine promotes proliferation of human NPC cells in vitro through simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling and suggest that the related molecules such as HIF-1α might be the potential therapeutic targets for tobacco-associated diseases such as nasopharyngeal carcinomas.

Abstract LB-385: The role of tumor suppressing microRNA let-7 in undifferentiated nasopharyngeal carcinoma

Abstract AIMS:This study aimed at evaluating the potential anti-proliferative effects of the microRNA let-7 family in nasopharyngeal carcinoma (NPC) cells. In addition, the association between let-7 suppression and DNA hypermethylation is examined. MATERIALS AND METHODS:Levels of mature let-7 family members (-a, -b, -d, -e, -g, and -i) in normal nasopharyngeal cells (NP69 and NP460) and nasopharyngeal carcinoma cells (HK1 and HONE1) were measured by real-time quantitative PCR. Cell-proliferation assay and c-Myc immunohistochemical staining were performed on NPC cells transfected with let-7 precursor molecules. In addition, expression changes in let-7 family members in response to demethylating agents (5-azacytidine and zebularine) were also examined. RESULTS:In comparison with the normal nasopharyngeal cells, let-7 (-a, -b, -d, -e, -g, and -i) levels were reduced in nasopharyngeal carcinoma cells. Ectopic expression of the let-7 family in nasopharyngeal carcinoma cells resulted in inhibition of cell proliferation through downregulation of c-Myc expression. Demethylation treatment of nasopharyngeal carcinoma cells caused activation of let-7 expression in poorly differentiated nasopharyngeal carcinoma cells only. CONCLUSION:Our results suggested that miRNA let-7 might play a role in the proliferation of NPC. DNA methylation is a potential regulatory pathway, which is affected when let-7 is suppressed in NPC cells. However, the extent of DNA hypermethylation/hypomethylation in regulating let-7 expression requires further elucidation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-385. doi:1538-7445.AM2012-LB-385

Suppression of the notch signaling pathway by γ-secretase inhibitor GSI inhibits human nasopharyngeal carcinoma cell proliferation

Notch signaling has been suggested to be required for many human cancers. However, the role of Notch signaling in human nasopharyngeal carcinoma cells (NPC) remains unknown. Here, we report that Notch-1, Notch-2, Notch-3 and Notch-4 are all detected in NPC cells. Notch inhibitor, GSI, suppresses the levels of Notch-1, Notch-2 and Notch-4, but not Notch-3. In addition, GSI inhibits NPC cell proliferation by inducing the cell cycle arrest and apoptosis. Furthermore, GSI inhibits the AKT and MEK signaling, without affecting P38 and JNK1/2. Thus, NPC cells may up-regulate Notch signaling to maintain cell proliferation and targeting the Notch signaling pathway may offer a potential alternative strategy for the treatment of NPC.

Down‐regulation of MMP‐2 through the p38 MAPK‐NF‐κB‐dependent pathway by aloe‐emodin leads to inhibition of nasopharyngeal carcinoma cell invasion

Aloe-emodin (AE), extracted from the rhizome of Rheum palmatum, has an anti-proliferative effect on different human cancer cell lines. Nonetheless, the underlying mechanism by which AE inhibits nasopharyngeal carcinoma (NPC) cell invasion is still unclear. The results of this study show that treatment of NPC cells with growth suppressive concentrations of AE caused cell cycle arrest at the S-G(2)/M phase. Coimmunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE-induced cell cycle arrest in NPC cells was associated with increasing levels of cyclin B1 bound to cyclin-dependent kinase 1. The inhibition of NPC cell invasion by AE was evidenced through the suppression of matrix metalloproteinases-2 (MMP-2) expression. MMP-2 promoter activity and cell invasion were inhibited by p38 mitogen-activated protein kinase (MAPK) siRNA, inhibitor 4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole (SB203580), and AE, but not by JNK siRNA and inhibitor 1,9-pyrazoloanthrone. Treatment with AE, SB203580, NF-kappaB inhibitors N-p-tosyl-(L)-phenylalanine chloromethyl ketone (TPCK) and pyrrolidine dithiocarbamate (PDTC) or transfection with p38 MAPK siRNA significantly inhibited NF-kappaB transcriptional activity. In addition, TPCK and PDTC treatment inhibited the expression and promoter activity of MMP-2 and thereby significantly inhibited cell invasion activity. The involvement of p38 MAPK activity in NF-kappaB-mediated MMP-2 function was further confirmed through the attenuation of p38 MAPK by SB203580 and NF-kappaB ectopic expression. Collectively, our results indicate that AE inhibits invasion of NPC cells by suppressing the expression of MMP-2 via the p38 MAPK-NF-kappaB signaling pathway.

MicroRNA let-7 suppresses nasopharyngeal carcinoma cells proliferation through downregulating c-Myc expression

AimsThis study aimed at evaluating the potential anti-proliferative effects of the microRNA let-7 family in nasopharyngeal carcinoma (NPC) cells. In addition, the association between let-7 suppression and DNA hypermethylation is examined.Materials and methodsLevels of mature let-7 family members (-a, -b, -d, -e, -g, and -i) in normal nasopharyngeal cells (NP69 and NP460) and nasopharyngeal carcinoma cells (HK1 and HONE1) were measured by real-time quantitative PCR. Cell-proliferation assay and c-Myc immunohistochemical staining were performed on NPC cells transfected with let-7 precursor molecules. In addition, expression changes in let-7 family members in response to demethylating agents (5-azacytidine and zebularine) were also examined.ResultsIn comparison with the normal nasopharyngeal cells, let-7 (-a, -b, -d, -e, -g, and -i) levels were reduced in nasopharyngeal carcinoma cells. Ectopic expression of the let-7 family in nasopharyngeal carcinoma cells resulted in inhibition of cell proliferation through downregulation of c-Myc expression. Demethylation treatment of nasopharyngeal carcinoma cells caused activation of let-7 expression in poorly differentiated nasopharyngeal carcinoma cells only.ConclusionOur results suggested that miRNA let-7 might play a role in the proliferation of NPC. DNA methylation is a potential regulatory pathway, which is affected when let-7 is suppressed in NPC cells. However, the extent of DNA hypermethylation/hypomethylation in regulating let-7 expression requires further elucidation.

Aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase-8-mediated activation of the mitochondrial death pathway

Aloe-emodin (AE), a natural, biologically active compound from the rhizome of Rheum palmatum, has been shown to induce apoptosis in several cancer cell lines in vitro. However, its molecular mechanism of action in the apoptosis induction of human nasopharyngeal carcinoma (NPC) cells has not been explored. This study shows that AE induced G 2/M phase arrest by increasing levels of cyclin B1 bound to Cdc2, and also caused an increase in apoptosis of NPC cells, which was characterized by morphological changes, nuclear condensation, DNA fragmentation, caspase-3 activation, cleavage of poly (ADP-ribose) polymerase (PARP) and increased sub-G 1 population. Treatment of NPC cells with AE also resulted in a decrease in Bcl-X L and an increase in Bax expression. Ectopic expression of Bcl-X L but not Bcl-2 or small interfering RNA (siRNA)-mediated attenuation of Bax suppressed AE-induced apoptotic cell death. AE-induced loss of mitochondrial membrane potential (MMP) and increase in cellular Ca ++ content, reactive oxygen species (ROS) and apoptotic cell death were suppressed by the treatment of cyclosporin A (CsA) or caspase-8 inhibitor Z-IETD-FMK. Co-treatment with caspase-9 inhibitor Z-LEHD-FMK could inhibit AE-induced cell death and the activation of caspase-3 and -9. In addition, suppression of caspase-8 with the specific inhibitor Z-IETD-FMK inhibited AE-induced the activation of Bax, the cleavage of Bid, the translocation of tBid to the mitochondria and the release of cytochrome c, apoptosis-inducing factor (AIF) and Endo G from the mitochondria and subsequent apoptosis. Taken together, these results indicate that the caspase-8-mediated activation of the mitochondrial death pathway plays a critical role in AE-induced apoptosis of NPC cells.