A novel Hsp90 inhibitor AT13387 induces senescence in EBV-positive nasopharyngeal carcinoma cells and suppresses tumor formation
© Chan et al.; licensee BioMed Central Ltd. 2013
Received: 16 April 2013
Accepted: 2 October 2013
Published: 24 October 2013
Nasopharyngeal carcinoma (NPC) is an epithelial malignancy strongly associated with Epstein-Barr virus (EBV). AT13387 is a novel heat shock protein 90 (Hsp90) inhibitor, which inhibits the chaperone function of Hsp90 and reduces expression of Hsp90-dependent client oncoproteins. This study aimed to evaluate both the in vitro and in vivo antitumor effects of AT13387 in the EBV-positive NPC cell line C666-1.
Our results showed that AT13387 inhibited C666-1 cell growth and induced cellular senescence with the downregulation of multiple Hsp90 client oncoproteins EGFR, AKT, CDK4, and restored the protein expression of negative cell cycle regulator p27. We also studied the ability of AT13387 to restore p27 expression by downregulation of AKT and the p27 ubiquitin mediator, Skp2, using AKT inhibitor and Skp2 siRNA. In the functional study, AT13387 inhibited cell migration with downregulation of a cell migration regulator, HDAC6, and increased the acetylation and stabilization of α-tubulin. We also examined the effect of AT13387 on putative cancer stem cells (CSC) by 3-D tumor sphere formation assay. AT13387 effectively reduced both the number and size of C666-1 tumor spheres with decreased expression of NPC CSC-like markers CD44 and SOX2. In the in vivo study, AT13387 significantly suppressed tumor formation in C666-1 NPC xenografts.
AT13387 suppressed cell growth, cell migration, tumor sphere formation and induced cellular senescence on EBV-positive NPC cell line C666-1. Also, the antitumor effect of AT13387 was demonstrated in an in vivo model. This study provided experimental evidence for the preclinical value of using AT13387 as an effective antitumor agent in treatment of NPC.
Nasopharyngeal carcinoma (NPC) is a malignancy arising from the epithelial cells of the nasopharynx. It has a distinct geographic distribution with a remarkably high disease incidence in southern China and Southeast Asia with more than 50,000 new cases each year. Apparently, all NPC is associated with the Epstein-Barr virus (EBV) latent infection, indicating the role of EBV in NPC pathogenesis. However, most of the NPC cell lines had lost the EBV genome after a long time in vitro passage. C666-1 is the NPC cell line consistently maintaining the native EBV genome and referred as a suitable model for studies of EBV-associated NPC. Nowadays, combined radiotherapy and chemotherapy are used for the treatment of NPC patients[4, 5]. Most contemporary series reported very encouraging results with locoregional control exceeding 90%, but distant failure remains high and more potent systemic therapy is needed.
Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the maturation and stabilization of over 200 oncogenic client proteins crucial for oncogenesis[6–8]. Hsp90 inhibitors exert the antitumor effect by blocking the ATP binding domain of Hsp90 to abolish the Hsp90 chaperone function and leading to proteasomal degradation of the oncogenic client proteins. In tumor cells, the dependency of oncoproteins on the chaperone function of Hsp90 is much higher than in normal cells, and the binding affinity of Hsp90 inhibitor to Hsp90 was 100-fold higher in tumor cells than in normal cells[9–11]. For this reason, inhibition of the Hsp90 machinery is considered as a potent strategy in cancer therapies.
AT13387 is a small-molecule inhibitor of Hsp90 developed by Astex Pharmaceuticals Inc through fragment-based drug screening against the ATP-binding domain of Hsp90. Several studies also reported AT13387 as an effective antitumor agent in both the in vitro and in vivo cancer models, such as gastrointestinal stromal tumor (GIST) and non-small cell lung cancer (NSCLC)[14, 15]. AT13387 clinical activity against GIST was demonstrated in the Phase I and Phase II trials (ClinicalTrials.gov Identifier: NCT00878423 and NCT01294202, respectively), and further clinical trials in prostate (NCT01685268) and lung cancer (NCT01712217) in combination with standard of care are ongoing.
In NPC, many of the aberrantly overexpressed oncoproteins such as EGFR, AKT, and CDK4 are known Hsp90 client proteins[12, 18, 19]. We hypothesize that targeting the chaperone function of Hsp90 in NPC cells can lead to downregulation of multiple crucial oncoproteins and regression of tumor. Therefore, we aim to study the tumor suppressive efficacy of AT13387 in the C666-1 EBV-positive NPC cell line and provide preclinical evidence of using AT13387 as a novel antitumor agent in treatment of NPC.
Growth inhibitory effect of AT13387 on the EBV-positive NPC cell line C666-1
AT13387 induces senescence in C666-1
Western-blotting analysis of senescence and growth associated Hsp90 client oncoproteins and the re-expression of p27 after AT13387 treatment
The negative cell cycle regulator p27 has previously been reported as a commonly downregulated tumor suppressive protein in NPC. In order to further study the mechanism of resotration of p27 protein expression in AT13387-treated C666-1 cells, we first measured the p27 mRNA expression by real-time quantitative PCR. However, the p27 mRNA level was unchanged by 72 hours treatment with AT13387 (data not shown), then we focused on the regulation of p27 at the protein level. The degradation of p27 protein is known to require the interaction between p27 and the F-box protein S-phase kinase 2 (Skp2) in the SCFskp2 complex. Since p27 is a normal physiological target of Skp2 for ubiquitination, we then studied the inversed expression of Skp2 and p27 by treating C666-1 cells with Skp2 siRNA. Results in Figure 3B showed that the expression of p27 proteins was increased in the Skp2 siRNA-treated C666-1. It has previously been shown that Skp2 is highly expressed in NPC tumor with poor prognosis[25, 26], and the stability of Skp2 is regulated by AKT. We then measured the protein expression of Skp2 after adding the AKT inhibitor SH-6 in C666-1. Results in Figure 3C showed with the downregulation of p-AKT, the Skp2 is coordinately downregulated in SH-6 treated C666-1. We then further determined the expression of Skp2 and AKT in AT13387-treated C666-1 cells. Figure 3D showed that the expression of Skp2, AKT, and phosphorylated form of AKT (p-AKT) were all reduced in the AT13387-treated C666-1 cells. This observation suggested that the ability of AT13387 to restore p27 protein expression may due to downregulation of p27 ubiquitination mediator Skp2 through downregulating AKT and p-AKT.
Apart from AKT, EGFR is one of the most commonly overexpressed oncoproteins in NPC[18, 19]. Targeting EGFR has been suggested as a new therapeutic treatment in NPC and EGFR is also a known Hsp90 client oncoprotein. In this study, AT13387 significantly reduced EGFR and its downstream target p-STAT3 in C666-1 (Figure 3D). It is worthy to note that AT13387 is designed to block the Hsp90 chaperon function, therefore the expression level of Hsp90 was not affected by AT13387. Taken together with the downregulation of CDK4, AKT, and Skp2, AT13387 can deplete multiple oncoproteins and restore the tumor suppressive protein p27 in EBV-positive NPC cell line. This result supported the potential use of AT13387 as an antitumor agent in NPC by simultaneously targeting multiple NPC oncoproteins.
Inhibition of tumor cell migration
AT13387 inhibits the tumor spheres formation and growth, accompanied by reduction of CD44 and SOX2 expression
AT13387 suppressed NPC tumor formation in nude mouse tumorigenicity assay
Cancer is a complex disease, with multiple aberrantly overexpressed oncogenic proteins involving activation of multiple signaling pathways. The stability of most of these oncoproteins depends heavily on the chaperon function of Hsp90. For this reason, the molecular chaperone Hsp90 is an attractive therapeutic target in cancer therapy. In the present study, we demonstrated both the in vitro and in vivo antitumor effects of a novel Hsp90 inhibitor, AT13387, on C666-1 EBV-positive NPC cells. First of all, AT13387 was found to inhibit cell growth and induce cellular senescence in the C666-1 EBV-positive NPC cells. Inhibition of cell growth and induction of cellular senescence, instead of induction of cell death through Hsp90 inhibition has also been reported in small cell lung cancer as a mode of cancer cell response to Hsp90 inhibitor. Cellular senescence is a permanent and irreversible process in the induction of cell growth arrest without massive cell death[20, 21]. The induction of cellular senescence has recently been proposed as a novel approach to improve cancer therapy with less severe side effects than cytotoxic therapies and high dose radiation[36, 37].
In the present study, AT13387 was found to downregulate several cell growth and cellular senescence associated Hsp90 client oncoproteins, including CKD4, AKT and EGFR. Also, we reported the correlation between restoration of p27 protein expression and the downregulation of S-phase kinase associated protein 2 (Skp2). Skp2 is the F-box protein responsible for substrate recognition in the Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase and specifically targeting the tumor suppressive proteins such as p27 for ubiquitination and proteasomal degradation. The role of the Skp2 in the regulation of cellular senescence has recently been reported and reviewed. In the present study, we found that AT13387 induced senescence in C666-1 cells and the effect was correlated with the reduction of the Skp2 and the increased expression of p27. The stability of Skp2 has been reported to be dependent on the phosphorylation by AKT. We further demonstrated that the loss of Skp2 was correlated with the reduced expression of the Hsp90 client proteins AKT in the treated C666-1 cells. These findings suggested that AT13387 inhibit cell growth and induce cellular senescence in C666-1 by downregulating cell growth and cellular senescence associated Hsp90 client proteins and also restored the tumor suppressive protein p27 by downregulating Skp2 through downregulation of Hsp90 client protein AKT and p-AKT.
The downregulation of Skp2 by AT13387 showed an important clinical relevance in the treatment of NPC which is worthy to discuss. Recent studies on the clinical samples from Taiwan and South China showed that Skp2 was overexpressed in 80% NPC tumor and the expression was correlated with poor prognosis[25, 26]. The overexpression of Skp2 in NPC clinical samples may explain the commonly loss of p27 in NPC tissues[41, 42]. The oncogenic role of Skp2 in NPC pathogenesis has been studied in NPC cells transfected with Skp2 of showing higher colony forming ability and the side population of NPC cells showed higher level of Skp2[25, 26]. However, up until now, no pharmacological Skp2 inhibitor has yet been available for clinical use. In our study, we demonstrated Skp2 can be downregulated by AT13387 in C666-1. This observation suggested that NPC patients with a high Skp2 expression might benefit from AT13387 for personalized therapy.
As mentioned above, AT13387 can target on multiple oncoproteins simultaneously. We studied the depletion of a very important NPC oncoprotein EGFR in AT13387-treated C666-1. EGFR has been reported to be overexpressed in 85% of NPC tissues and the expression is associated with poor prognosis. EGFR is the receptor tyrosine kinase of the natural ligand EGF and TGF. Activation of EGFR was associated with proliferation, migration, and drug resistance, which play an important role in NPC pathogenesis. In recent years, EGFR has been proposed as a new therapeutic target for NPC. EGFR inhibitors such as cetuximab and gefitinib, which are the monoclonal antibody and the small molecule against EGFR, respectively, are currently under NPC clinical evaluations. However, targeting a single oncoprotein is unlikely to be effective enough to eliminate the disease, as the tumor cells may switch from utilization of one signaling pathway to another signaling pathway for growth. Despite the promising effect of EGFR inhibitors in the preclinical and clinical studies, not all the patients respond and benefit from the treatment in clinical studies[46, 47]. In one third of gefitinib non-responsive NPC patients, AKT was found to be overexpressed. The activation of AKT pathway in gefitinib-resistant cells may take over the EGFR pathway and therefore maintain the tumorigenicity and escape from the EGFR targeted therapy. In the present study, we observed the simultaneous downregulation of EGFR, EGFR downstream signaling molecules p-STAT3, AKT and p-AKT. Hence, targeting multiple oncoproteins using AT13387 alone or in combination with specific antitumor agents may serve as a potential solution to overcome the development of drug resistance in NPC targeted therapy.
One of the current problems in the treatment of NPC is the development of distant metastasis and tumor recurrence. HDAC6, also a client protein of Hsp90, is a key modulator involved in the regulation of cell migration through the deacetylation of tubulins in the cytoplasm[28, 30, 49]. Overexpression of HDAC6 is frequently correlated with the tumor development, and hence HDAC6 is considered to be a target for cancer therapy. However, the role of HDAC6 in NPC has not been demonstrated. In the present study, we found that the expression of HDAC6 was downregulated by AT13387. The effect was correlated with the increase in the acetylation of α-tubulin and the decrease in the tumor cell migration. This finding indicates that AT13387 may reduce metastasis through the disruption of microtubules dynamics.
In addition to the mechanistic study, two biological end-point assays, namely the in vitro 3D tumor sphere formation assay and the in vivo NPC xenograft, were used to evaluate the efficacy of AT13387 for NPC. The tumor sphere assay is frequently used to measure the in vitro self-renewal capability of cancer stem cells and to assess the effectiveness of the drug on the cells in the presence of growth factors[31–33]. Our results clearly showed that AT13387 not only reduced the in vivo tumor formation, but also reduced the formation and growth of NPC tumor spheres accompanied by reduced expression of cancer stem-like cells markers CD44 and SOX2. Lo KW and co-workers have recently demonstrated that CD44 and SOX2 expression are enriched in C666-1 tumor sphere forming cells which may serve as the potential candidate stem cell markers for the NPC C666-1 cells. CD44 is a well known cell surface marker involved in the signal transduction of multiple oncogenic pathways[51–53]. SOX2 is a well known master transcription factor of stem cells. Decreased expression of CD44 and SOX2 might reduce the oncogenic potential of the tumor cells. The result revealed the potential of AT13387 on targeting the CD44-and SOX2-overexpressing NPC subpopulation. Taken together, results from the present study suggest that targeting on multiple oncogenic pathways by AT13387 is a novel approach in the treatment of NPC. Further development will focus on the evaluation of using AT13387 as a single agent or in combination with other current therapies in the treatment of NPC.
Our study demonstrated the in vitro and in vivo antitumor effect of a novel Hsp90 inhibitor, AT13387, on the EBV-positive NPC cell line C666-1. AT13387 inhibited cell growth, cell migration, tumor sphere formation and induced cellular senescence in C666-1. The ability of AT13387 to target multiple NPC oncoproteins, make it a potent antitumor agent in treatment of NPC. Together with the tumor suppressive effect of AT13387 in nude mice tumorigenicity assay, this study provided preclinical evidence of using AT13387 as a new therapeutic agent in treatment of NPC.
Chemical and antibodies
AT13387 was synthesized and provided by Astex Pharmaceuticals Inc. AKT inhibitor SH-6 was purchased from Calbiochem, San Diego, CA. Primary antibodies for Western blotting analysis include Caspase-3, BAX, Bcl-2, Bcl-xl, CDK2, CDK4, p16, p21, p27, Rb, STAT3, p-STAT3 (Tyr705), AKT, p-AKT (Ser473), Skp2 (Cell Signaling, Danvers, MA); EGFR, Hsp90, and HDAC6 (Santa Cruz Biotechnology, Santa Cruz, CA); and acetylated α-tubulin and β-tubulin (Sigma-Aldrich, St. Louis, MO). Antibodies for immunofluorescence staining were Alexa Fluor® 488 conjugated CD44 and Alex Fluor® 647 conjugated SOX2 (Cell Signaling, Danvers, MA).
Cell line and cell culture
C666-1, an EBV-positive NPC cell line still carrying the native EBV genomes, were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 1% penicillin and streptomycin (P/S) (Invitrogen, Carlsbad, CA). Cells were cultured at 37°C with 5% CO2 in humidified incubator.
MTT cell viability assay
Viable cells treated with various dose of AT13387 were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described. Briefly, C666-1 (3×104) were seeded in 96-well microplates and treated with serial diluted AT13387 (0.001 μM-10 μM) for 48 hours. MTT solution (Sigma-Aldrich, St. Louis, MO) (0.25 mg⁄ml) was added to cells and incubated for 3 hours in 37°C. The optical densities (OD) were measured at absorbance 550 nm with reference to absorbance 690 nm. The OD is directly proportional to the number of living cells and the percentage of viable cells compared to control wells was calculated.
Cell growth assay
The kinetic effect of AT13387 on proliferation of C666-1 was studied using a cell growth assay. C666-1 cells (3×105) were seeded onto 35 mm culture dishes. The cells were then treated with AT13387 (1 μM and 10 μM) for 2 to 7 days. The total number of viable cells determined by trypan blue staining was counted on day 2, 4, and 7 after AT13387 treatment.
DNA content analysis
DNA content analysis was performed using propidium iodide (PI) staining and flow cytometry analysis as previously described. Briefly, C666-1 (3×105) were seeded in 6-well plates and treated for 48 hours with 1 μM ATT13387 (the minimum concentration that had shown maximum cell growth inhibition in MTT assay). Both adherent cells and floating cells were collected for analysis. The cells were fixed in 70% cold ethanol, stained with 1 mg/ml propidium iodide (PI) and analyzed by FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Fluorescence profiles represent the DNA content of the PI stained cells.
Nucleus and SAHF staining with DAPI
DAPI nucleus staining was used to identify the apoptotic cells with chromatin condensation and fragmentation and/or senescence cells with senescence-associated heterochromatic foci (SAHFs) formation as previously described[22, 57]. For the apoptotic nucleus staining, 3×105 cells were seeded in 6-well plates and treated with 1 μM AT13387 for 48 hours. For the SAHF staining, 3×105 cells were seeded in 6-well plates and treated with 1 μM and 10 μM AT13387 for 96 hours. Both adherent cells and floating cells were collected onto slides by cytospin. The cells were fixed with 2% paraformaldehyde and permeablized with 0.2% Triton-X. The cells were then stained with DAPI (1 μg/ml) and the nuclear images were captured under a fluorescence microscope (ECLIPSE Ti, Nikon) equipped with camera. At least 200 cells were counted from different microscopic fields.
Senescence-associated β-Galactosidase cell staining
Senescence-associated β-galactosidase (SA-β-gal) activation was detected by cytochemical staining with the X-Gal according to the protocol of the Cell Signaling Senescence β-Galactosidase Staining Kit #9860. Briefly, C666-1 (8×104) cells were seeded onto wells of a 24-well plate and the cells were treated with 1 μM and 10 μM of AT13387 for 72 hours. Both adherent cells and floating cells were collected and stained with X-gal (pH6) overnight in the dark. The senescent cells were stained with blue color. Cells images were captured under microscope with a camera.
Western blotting analysis
Western blotting analysis was performed as previously described. In brief, C666-1 cells (5×105) were seeded onto the 6-well plate and the cells were treated with 1 μM and 10 μM of AT13387 for 48 hours, 72 hours and 96 hours. Both adherent cells and floating cells were collected and lysed with ice-cold lysis buffer (250 mM Tris–HCl, pH 8; 1% NP-40 and 150 mM NaCl containing 1% phosphatase inhibitors cocktail (Calbiochem, San Diego, CA) and 0.25% protease inhibitors cocktail (Sigma, St. Louis, MO). Samples were resolved on SDS–polyacrylamide gel and transferred to PVDF membrane (Millipore, Billerica, MA). The membrane was blocked with 5% non-fat milk, incubated with primary antibodies (1:1000) followed by corresponding secondary antibodies (1:4000). A Western blotting substrate (Labfrontier Co. Ltd., Bio Division) was added and chemiluminescence signal was detected on the X-ray film. β-actin primary antibody (1:4000) was probed and served as an internal control.
Knockdown with siRNA and transfection were performed according to the manufacturer’s instruction. In brief, C666-1 cells (5×105cells/well) were seeded onto fibronectin-coated 35-mm dishes for 24 hours. Cells were transfected with 5 nM of si-Skp2 RNA (a pool of 4 siRNA: GGAUGUGACUGGUCGGUUG; GGUAUCGCCUAGCGUCUGA; UCGGUGCUAUGAUAUAAUA; UGUCAAUACUCUCGCAAAA), or si-Control RNA (UGGUUUACAUGUCGACUAA) (Dharmacon, Lafayette, CO) using Lipofectamine Reagent 2000 (Invitrogen, Carlsbad, CA). After 6 hours of transfection, the medium was then replaced by fresh medium. Cells were harvested for western-blotting analysis after 72 hours of transfection.
The migration capability of AT13387-treated C666-1 cells was analyzed using the transwell migration assay. C666-1 cells (5×105) were seeded on the 6-well plate and treated with 1 μM and 10 μM AT13387 for 72 hours. Cells were then harvested and 2×105 viable cells were seeded on the upper chamber of the transwell. After 24 hours of incubation, the cells that had migrated through the membrane were fixed in 2% paraformaldehyde, permeablized with 0.2% Triton-X, and stained with 1 μg/ml DAPI. The stained cell images were captured under fluorescence microscopy (ECLIPSE Ti, Nikon). At least 100 cells were counted from different microscopic fields.
Tumor sphere formation assay
Tumor sphere formation assay was performed as previously described. C666-1 cells were dissociated into single cells and seeded in low cell density (2×103) on a 24-well ultra-low attachment plate (Corning, Acton, MA), and cultured with serum-free DMEM/F-12 (Invitrogen, Carlsbad, CA), 20 ng/ml EGF (Sigma, St. Louis, MO), 20 ng/ml bFGF (Cell Signaling, Danvers, MA), and 20 ng/ml insulin (Cell Signaling, Danvers, MA). The cultures were fed with fresh serum-free DMEM/F12 supplemented with growth factors every other day. For studying the effect of AT13387 on the tumor sphere forming ability, AT13387 was added to the culture on the same day of seeding the dissociated C666-1 single cells. After 7-days of incubation, the images of cells were captured under an inverted microscope equipped with camera. Tumor spheres having diameter >20 μm were counted using Image J software. Total numbers of tumor spheres formed in AT13387-treated and-untreated cultures were compared. In order to study the effect of AT13387 on the growth of established tumor spheres, tumor spheres were first allowed to grow for 7 days, followed by incubation with AT13387 for further 7 days. Then the images of AT13387-treated and–untreated tumor spheres were captured under an inverted microscope equipped with camera. Tumor spheres with diameter >20 μm were measured and counted using Image J software. Data from each treatment were presented as size distribution profile with mean diameter.
Immunofluorescence staining and FACS analysis of spheroid cells
Tumor spheres for CD44 and SOX2 immunofluorescence staining and FACS analysis were established as described above. Briefly, C666-1 cells were incubated in serum-free DMEM/F12 supplemented with growth factors for 7 days to allow tumor sphere formation. Then, AT13387 was added to the tumor spheres culture and incubated in serum-free DMEM/F12 supplemented with growth factors for another 7 days. For CD44 and SOX2 immunofluorescence staining, the tumor spheres were carefully collected and fixed with 2% paraformaldehyde and permeablized with 0.2% Triton-X. Tumor spheres were then incubated with Alexa Fluor® 488 conjugated CD44 and Alex Fluor® 647 conjugated SOX2 antibodies in the dark. The immunofluorescence signals were visualized and imaged using an Olympus Fluoview 1000 confocal scanning laser microscope. For FACS analysis, tumor spheres were collected and the spheroid cells were resuspended in PBS and fixed in 1.6% paraformaldehyde. Then the cells were pelleted and resuspended in ice-cold methanol. The cells were washed twice in incubation buffer [0.5% bovine serum albumin (BSA) in PBS], and stained with Alexa Fluor® 488 conjugated CD44 and Alex Fluor® 647 conjugated SOX2 antibodies in the dark. Respective mouse or rabbit IgG isotypic controls were included as negative controls. For each sample, 10,000 cells were acquired and analyzed by FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
Nude mice tumorigenicity assay
Nude mice were supplied and housed by Laboratory Animal Unit of the University of Hong Kong. Experiments were conducted under license from the Hong Kong Department of Health and approved by Committee on the Use of Live Animals in Teaching and Research (CULTAR) at the University of Hong Kong. AT13387 drug formulation used in a previous publication was used in the nude mice tumorigenicity assay. In brief, 1×107 C666-1 cells were subcutaneously (s.c.) injected into the flank of 8-10 week old female athymic BALB/c nu/nu mice. Immediately after cell inoculation, the mice were randomly divided into two groups (three mice per group) for either treatment with AT13387 or vehicle. For the drug treatment group, AT13387 formulated in 17.5% hydroxy-propyl-β-cyclodextrin in sterile water was administrated at 50 mg/kg by intraperitoneal (i.p.) injection at a dose volume of 10 ml/kg twice per week (on Days 2 and 5 of each week). For the control group, the drug vehicle alone was given through i.p. injection. The tumor volume in mm3 (length × width × height) and the mice body weight were measured weekly until tumor volume reached 1000 mm3.
All results were representative results from at least two independent experiments. Each data points with error bars were the arithmetic mean ± SE of three replicates (n = 3). The p-values were calculated using Student’s t-test, p-value < 0.05 was considered as statistically significant.
This project is funded by the Research Grants Council of the HKSAR for the NPC Area of Excellence (AoE/M 06/08 Center for Nasopharyngeal Carcinoma Research).
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