Proteome Analysis of Antiproliferative Mechanism of 12-O

12-O-Tetradecanoylphorbol 13-Acetate on Cultured Nasopharyngeal ... 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is a plant derivative with multiple fu...
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Proteome Analysis of Antiproliferative Mechanism of 12-O-Tetradecanoylphorbol 13-Acetate on Cultured Nasopharyngeal Carcinoma CNE2 Cells Peizhou Jiang,†,‡ Ming Gan,§ Hua Huang,† Xinming Shen,† Shuang Wang,† and Kaitai Yao*,† Cancer Research Institute and Department of Histology and Embryology, Southern Medical University, Guangzhou 510515, People’s Republic of China and Department of Parasitology, Zhongshan Medical College, SUN Yat-sen University, Guangzhou 510080, People’s Republic of China Received December 11, 2004

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is a plant derivative with multiple function as tumor promoter, differentiation revulsant or leukemia therapy drug. The molecular mechanism of its function is perplexing. Many studies have focused on the mechanism of TPA stimulation in tumor promotion of mouse models or terminal differentiation of leukemia cells, but the effect of TPA on nasopharyngeal carcinoma (NPC) remains unclear, while TPA was considered to be associated with NPC development. In the present study, we employed proteomics techniques to study protein changes of a poorly differentiated squamous carcinoma cell linesCNE2 of human NPCs cells induced by TPA. Six significantly and reproducibly changed proteins were identified and their functional implications were discussed in some details. Keywords: 12-O-tetradecanoyl-phorbol-13-acetate • nasopharyngeal carcinoma • proteomics • apoptosis

Introduction 12-O-Tetradecanoyl-phorbol-13-acetate (TPA, also designated PMA by some workers), a plant derivative, is the most potent and most frequently used tumor promoter in mouse models of multistage carcinogenesis which indicates TPA can promote hyperplasia and carcinoma development in the skin of initiated animals.1 It is also a promising clinical drug for chemotherapy to induce myeloid leukemia cells or prostate cancer cells to growth arrest or differentiation.2,3 Moreover, TPA is considered to be closely associated with nasopharyngeal carcinoma(NPC) for its specific effect on the supposed primary pathogen of the lattersEpstein-Barr virus(EBV).4,5 It can facilitate EBV to infect human primary epithelial cells,6,7 then activate the EBV productive cycle,8 and even cause the malignant transformation of epithelial cells infected with EBV.6,7 The above reports indicated that the biological effect of TPA is multiplex, therefore, the molecular mechanism of its function is perplexing. Although many studies have focused on the mechanism of TPA stimulation in tumor promotion of mouse models9,10 or terminal differentiation of leukemia cells,11,12 the effect of TPA on nasopharyngeal epithelial cells remains few reports. In present study, a poorly differentiated squamous carcinoma cell linesCNE213 of human NPCs was treated with TPA or nontreated for control, and the cellular morphological * To whom correspondence should be addressed. Fax: +8620-61648225. E-mail: [email protected]. † Cancer Research Institute. ‡ Department of Histology and Embryology, Southern Medical University. § Zhongshan Medical College, SUN Yat-sen University. 10.1021/pr0497677 CCC: $30.25

 2005 American Chemical Society

changes induced by TPA were observed. Subsequently, the control and TPA-treated cells were compared at the level of cell dynamics by fluorescent activated cell sorting (FACS) and then at that of proteome by two-dimensional electrophoresis. Some proteins with altered expression were successfully identified by Matrix-Assisted Laser Desorption/Ionization Time-ofFlight Mass Spectrometer(MALDI-TOF-MS) and they might be involved in the biological changes of CNE2 cells induced by TPA.

Materials and Methods Reagents and Materials. Immobiline DryStrips (pH 4-7, 17 cm), DryStrip cover fluids, IPG buffer, were from BioRad (Hercules, CA,USA). Dimethyl sulfoxide(DMSO), Iodoacetamide, anti-Stathmin and anti-nucleophosmin antibody were from Sigma-aldrich (St. Louis, MO). Trifluoroacetic acid (TFA) were from Fluka (Switzerland). Trypsin (sequencing grade) was obtained from Boehringer Mannheim (Mannheim, Germany). TrizolTM Reagent and SuperScript II kit were from Gibco BRL Life Technologies. Annexin V FITC Apoptosis Detection Kit I were from BD Biosciences Pharmingen. The remaining chemicals were of analytical grade. All buffers were prepared with Milli-Q water. Cell Culture and Treatment. The CNE2 cell line was provided by Cancer Research Institute of Xiangya Medicine College, Central South University, Changsha, China. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 µg/mL of penicillin and 100 µg/ mL of streptomycin in a water-saturated, 5% CO2 atmosphere at 37 °C in 50 mL flasks. After allowing the cells to >60% Journal of Proteome Research 2005, 4, 599-605

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Table 1. Primers and PCR Condition for Each Gene

spot

accession

name

a

AK126419

reticulocalbin 1 precursor

b

BC020467

nucleophosmin

d e f g

primers

sense: 5′- tgtcataggcattctgtt-3′ antisense: 5′- aggatgataccactttcg-3′

sense: 5′-ttacgaaggcagtccaat-3′ antisense: 5′-tcatcagcagcaagtttt-3′ BC002676 matrix protein p1 precursor sense: 5′- tactggctcctcatctca-3′ antisense: 5′- tttctttgtctccgtttg-3′ BC015100 triosephosphate isomerase 1 sense: 5′- gacatcatcaatgccaaaca-3′ antisense: 5′- ggaacccaggagcaaatc-3′ NM_203401 stathmin sense: 5′-cgcttgtcttctattcacc-3′ antisense: 5′-gttcttccgcacttcttc-3′ AF029082 14-3-3 sigma sense: 5′-cctgcgaagagcgaaacc-3′ antisense: 5′-ctcgtagtggaagacggaaa-3′ BC002409 β-actin sense: 5′-gatgcagaaggagatcactg-3′ antisense: 5′- gggtgtaacgcaactaagtc-3′

confluence, experiments were initiated by changing the medium to RPMI-1640 with 20 nM TPA(Sigma, dissolved in DMSO). Control cells were treated in the same way with same amount of DMSO solution. After 48 h, control and TPA-treated samples were collected for FACS analysis, extraction of total protein and RNA, respectively. FACS Analysis. For FACS analysis, control and TPA-treated cells were respectively stained with annexin V and propidium iodide using the Annexin V FITC Apoptosis Detection Kit I (according to the protocols within kit) and then analyzed on flow cytometersEPICS Elite (Coulter, USA). Extract Preparation. Cells were harvested by scraper after 48 h and then washed with PBS buffer. 2 × 106 cells were added to 100 µL lysis buffer (8 M urea, 4% CHAPS, 40 mM Tris). After a few cycles of quick freezing in liquid nitrogen and subsequent thawing at room temperature, 20 µL RNase (20 µg/µL) was added. Extracts were centrifuged at 40 000 × g for 1.5 h at 10 °C. The supernatant was aspirated and protein concentrations were determined using the Bradford assay.14 2D Electrophoresis and Image Analysis. Iso-Electric Focusing(IEF) was carried out on an PROTEAN IEF CELL (Biorad) using precast 17 cm pH 4-7 IPG gel strips (Biorad). 600 µg total protein were mixed with a rehydration solution (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte 3/10 ampholyte, 0.001% Bromophenol Blue) to a total volume of 350 µL. Rehydration and IEF was performed as follows: 12 h of passive rehydration, IEF at 500 V for 30 min, 5000 V for 3 h, 10 000 V for 80 000 Vh. Current was limited to 50 µA per gel strip. After IEF separation, the gel strips were immediately equilibrated for two steps in equilibrium buffer including 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, and 2% SDS. In the first step, DTT (2% w/v) was included in the equilibrium buffer. Iodoacetamide (2.5% w/v) was added in the second step. The second dimension separation (13% T, 2.7% C, 20 × 20 × 1 mm) was performed using a Protean II xi 2-D cell (Bio-Rad) with the following procedure: 20 mA per gel until the bromophenol blue front reached the bottom of the gel. Then gels were stained with Coomassie Brilliant Blue R-250. Considering experimental variations, three batches of proteins extracted from experimental and control cells, respectively, were subjected to 2-DE and replicate gels were simultaneously run three times. Image analysis was performed using the Melanie 2D gel analysis software. 600

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annealing temperature (°C)

PCR protocols

product length

50.5

96 °C, 5 min 431 bp 30 cycles with (95 °C, 1 min specific annealing, 1 min 72°C, 1 min) 72 °C, 10 min 288 bp

49.5

495 bp

53.5

308 bp

54.5

427 bp

57.5

437 bp

55.5

221 bp

49.5

In-Gel Digestion. Spots were excised to 1-2 mm2 slices using a blade, destained with freshly prepared 15 mM potassium ferricyanide/50 mM sodium thiosulfate, washed with 25 mM ammonium bicarbonate/50% acetonitrile, and dried in a SpeedVac Plus SC110A (Savant, Holbook, NY) vacuum concentrator. The dried gel pieces were rehydrated with 3-10 µL of 20 ng/µL trypsin solution, the solution volume being enough for the dried gel to be reswelled. Digestion was continued at 37 °C for 14-18 h. Tryptic peptides were first extracted using 5% TFA at 40 °C for 1 h, then 2.5% TFA/50% acetonitrile at 30 °C for 1 h. The extracted solutions were mixed in an Eppendorf tube, and dried in a vacuum concentrator. MALDI-TOF MS Identification of Peptide Mixtures. The peptides mixture was solubilized with 0.5% TFA, with saturated R-cyano-4-hydroxy-trans-cinnamic (CHCA) solution in 0.1% TFA/50% acetonitrile as matrix and analyzed by M@LDI R (Micromass, Manchester, UK). Mass spectra was externally calibrated with lock mass 2465.199 Da and internally calibrated with autodigested peaks of trypsin (MH+: 2211.105 Da). Database Searching. The protein identification was performed by searching protein databases of Swiss-Prot/TrEMBL (http://www.expasy.ch/tools/peptident.html) and Mascot (http:// www.matrixscience.com/). The error for peptide mass was set as 50 ppm and possible missed cleavage of trypsin was set as 1. The proteins with more than 4 matched peptides were considered significant. Confirmation with RT-PCR. Total RNA was isolated from control and TPA-treated cells respectively using TrizolTM reagent and the First strand cDNA was reversely transcribed according to the manufactures’ instructions. Six genes identified by mass spectrometry were amplified by each primers for validation in a gradient DNA Thermal cycler (Biometra, USA) with 50 µL reaction mixtures containing equal cDNA, primers and other components, respectively. At the same time, the β-actin gene was chosen to be control for each RT-PCR. All of the PCRs were operated at the same time in gradient PCR instrument from 49.5 °C to 60.5 °C with 1 °C interval in 12 ranks. An aliquot (10 µL) of each PCR products was detected by 1% agarose gel with ethidium bromide. The primers and PCR condition for each gene were listed in Table 1. Confirmation with Western Blotting. We selected stathmin (STMN1) and nucleophosmin (NPM1) from the six genes for confirmation of TPA-induced altered expression in CNE2 cells

Antiproliferative Mechanism of TPA on C-NPC

Figure 1. Morphological features of control and TPA-treated CNE2 cells under microscope. In contrasted with control (picture marked with “C” on the lower left), CNE2 cells treated with TPA showed the following characteristics(pictures marked with “T1” and “T2”): cell-cell contact was drastically reduced with excessive margin around each cell; cells became less flattened and shrinked; cytoplasmic-conjugation between two cells by slim duct(pointed by white arrows in “T2”) appeared; a proportion of cells detached from bottom and then conglobated together(enclosed by black circles in “T1”).

Figure 2. Flow cytometric analysis of annexin V- and propidium iodide-stained control(marked with “C” on the upper right) and TPA-treated(marked with “T”) CNE2 cells after 48 h of cell culture. Quantitation of the proportion of apoptotic cells for control and TPA-treated group was described in the text.

because the antibody of anti-Stathmin and anti-nucleophosmin was purchasable. The equal total protein from control and TPAtreated CNE2 cells respectively was loaded into two immediate lanes in a 13% polyacrylamide gel (BioRad, Richmond, CA), separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane using standard techniques.15 Blots were blocked with 10 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween50 with 10% nonfat dry milk, and then incubated with antibody. The identified bands were visualized with horseradish peroxidase-linked secondary antibody.

Results Morphological Changes of CNE2 Cells Treated with TPA. Morphological changes of CNE2 cells could be induced in vitro by TPA treatment. Figure 1 illustrated the microscopic morphological differences between control and TPA-treated cells. FACS Analysis Results. TPA treatment in CNE2 cells resulted in significant increases of apoptotic cells in contrast to control cells as determined by flow cytometry of immunofluorescent cells labeled with annexin V. Mean apoptotic event of CNE2 cells with FITC+ was 34 ( 0.17%, and FITC+/PI- was 21.57 ( 0.35% for control; that with FITC+ was 54.43 ( 0.84%, and FITC+/PI- was 41 ( 0.46% for TPA treatment. The FACS data were representative of 3 reproductive experiments per group. Statistically significant changes were indicated (*P < 0.01) for both FITC+ and FITC+/PI-. These results indicated that TPAmediated regulation constituted an anti-proliferative and proapoptotic signaling pathway in CNE2 cells. See Figure 2. Proteins Expression Patterns of Control and TPA-Treated CNE2 Cells. The proteome of the control and TPA-treated CNE2

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Figure 3. 2D gel images of protein expression in control and TPAtreated CNE2 cells. The image marked with “C” in upper left was from control sample, “T” from TPA-treat one. The areas enclosed by black frames in two images indicated the distribution of altered proteins identified successfully. Each area was denoted at the side of black frames with number 1 to 5 in parallel. The numbers between two images showed molecular marker. The pH gradient was denoted on the top of each image.

Figure 4. Discrepant protein spots in enlarged images. Each image was integrated with 6 small pictures aligned in two rows with 3 pictures a row up and down. The small picture marked with C(1 to 5) and T(1 to 5) in parallel corresponded to the area (in frame 1 to 5) in image C and T from Figure 3, respectively. Discrepant protein spots were arrowed and marked with lowercase (a-g). The other two small figures in the same row of C(1 to 5) and T(1 to 5) were from reduplicate experiments in which the same spots were arrowed likewise.

cells were compared using 2D electrophoresis. Three gels per sample were processed simultaneously and analyzed with Melanie 2-D software. The discrepant protein spots were found and the significance of protein changes was evaluated using the Student T-test. Some spots were found to be significantly altered (p < 0.05) or absent/emergent by TPA treatment and 7 of them were identified successfully. Figure 3 showed two representative 2D gel images and the distribution of identified protein spots in which 2 were upregulated and 5 downregulated by TPA (see Figure 4). Identification of Proteins with Differential Expressions by PMF. Discrepant protein spots induced by TPA were exised from the 2D gels and subjected to trypsin digestion and MALDI mass spectrometry. Seven spots were identified successfully (see Table 2 and Figure 5). RT-PCR Results. Six genes identified by mass spectrometry were successfully amplified from equal cDNA of control and TPA-treated samples by specific primers(see Table 1). Referring to β-actin, the variation of each gene between control and TPAtreated samples showed to be consistent with its appearance in 2D electrophoresis images(see Figure 6). Western Blotting Results. STMN1 and NPM1 were detected in both control and TPA-treated samples. As expected, the expression of each protein in TPA-treated CNE2 cell was Journal of Proteome Research • Vol. 4, No. 2, 2005 601

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Table 2. MALDI-TOF-MS Identification of Protein Molecules with Altered Expression Induced by TPA in CNE2 Cells spot

AC

ID

description

matched peptides

theoretical pI/Mr (kDa)

sequence coverage (%)

a b c d e f g

Q15293 P06748 P06748 P10809 P00938 P16949 P31947

RCN1•HUMAN NPM•HUMAN NPM•HUMAN CH60•HUMAN TPIS•HUMAN STHM•HUMAN 143S•HUMAN

reticulocalbin 1 precursor (RCN1) nucleophosmin (NPM1) nucleophosmin (NPM1) mitochondrial matrix protein p1 precursor (HSPD1) triosephosphate isomerase (TPI1) stathmin (STMN1) 14-3-3 protein sigma (SFN)

19 12 10 32 18 6 15

4.9/38.872 4.7/32.557 4.7/32.557 5.8/61.036 6.9/26.52 6.0/17.153 4.7/27.756

59.52 39.80 30.61 59.69 87.50 31.08 33.07

visually much lower than that in control (see Figure 7), which was consistent with results of identification of mass spectrometry and RT-PCR confirmation.

Figure 5. Spectra of MALDI-TOF-MS obtained from spot “b” matched with the tryptic peptide sequences of nucleophosmin.

Figure 6. The RT-PCR results of six genes from control and TPAtreated samples. In this figure, 2 lanes labeled with “M” were molecular marker named “DL2000” containing different fragment size listed on the left. The other lanes with 2 stripes showed amplified results of upper interesting gene and lower reference β-actin; “C” and “T” indicated the cDNAs was from “control” and “TPA-treated” samples respectively; “a” to “g” denoted the same genes of Figure 4. and Table 1.

Figure 7. TPA-induced decrease in expression of STMN1 (left) and NPM1 (right) by western blotting confirmation. “T” and “C” indicated “TPA-treated” and “control” samples, respectively.

Discussion TPA has attracted great interests because of their high potency as tumor promoters and a variety of effects in cells. Former studies showed that a number of cultured cell lines were responsive to TPA to fall into two categories, viz. stimulation of quiescent cells into growth and subsequent mitosis or 602

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inhibition of cell proliferation and stimulation of terminal differentiation.16,17 It was worthy to mention that TPA could be found in many Chinese medicinal herbs and food-types which suggested that the diet of Chinese individual might make them more vulnerable to NPC.4,5,18,19 So the original intention of our present study was to use proteomics technology to seek some key proteins which could help to clarify the mechanism of TPA promoting NPC. In view of the enormous quantity of cells for experimental manipulation, we followed the former researchers20 who had preferred epithelial cell line from NPC to primary normal nasopharyngeal epithelial cells because the source of the latter was very difficult. However, we obtained unexpected results. In the present study, CNE2 cell line from NPC proved to be a cell type which responses to exposure to TPA by distinct changes in cell morphology (see Figure 1). FACS analysis revealed that growth arrest and then apoptosis to death were concurrent with the above changes. Subsequently, differential proteome analysis showed that TPA treatment could upregulate the expression of “triosephosphate isomerase (TPI1)”, “14-3-3 protein sigma (SFN)”, and downregulate that of “reticulocalbin 1 precursor (RCN1)”, “nucleophosmin (NPM1)”, “mitochondrial matrix protein p1 precursor (HSPD1)”, “stathmin (STMN1)” in CNE2 cells. TPI1 is a homodimeric enzyme with subunit polypeptides of around 27 kDa.21 Some reports revealed that TPI1 could be induced as a stress protein in MBEC cells22 and up-regulated through an AP-1-dependent pathway.23 Likewise, we speculated that the increase of TPI1 expression might be due to cellular stress to TPA, and then the regulation by AP-1 which could be activated by TPA.24 SFN, a member of the 14-3-3 superfamily, was first identified as an epithelium specific markersHME1 which was inactivated during neoplastic transformation.25 Its protein level was advised to constitute a marker and should be tested for clinical efficacy.26 Former researchs showed that SFN is an important gene which can influence many biological processes, including cell cycle control, regulation of cell death,27 signal transduction,28 cellular dedifferentiation and act as a tumor suppressor.29 In present study, the upregulated expression of SFN as differential marker of epithelium in TPA-treated CNE2 cells suggested that TPA could prevent the high cellular proliferation characteristic of the transformed phenotype so as to induce this cell line to differentiate to end in death. In addition, we guessed that that a proportion of TPA-treated CNE2 cells detached from plate to suspension and conglobated together (see Figure 1) was partly caused by cell apoptosis to death (supported by FACS analysis, see Figure 2), which might be resulted from increased expression of SFN by TPA treatment. RCN1 is an endoplasmic reticulum resident Ca2+-binding protein and involved in protein synthesis, modification, and intracellular transport.30 It was reported that RCN1 was ex-

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Antiproliferative Mechanism of TPA on C-NPC

pressed in the highly invasive breast cancer and colorectal carcinoma cell lines but was not or low expressed in poorly invasive ones,31 and might play an important role in hepatoma cell progression.32 This protein might play an important role in cell adhesion,33 thus be associated with tumor proliferation and invasiveness. Here, the appearance of excessive margin around each cell and many cells detaching from bottom to suspension due to TPA treatment indicated that the adhesion of cell to cell and cell to plate lessened. So we speculated that TPA might reduce adhesion of cells to impact them in morphology by decreasing the expression of RCN1. NPM1 is a major nucleolar phosphoprotein which is more abundant in cancer cells and proliferating cells than in normal resting cells34 and might be applied for a marker of proliferation activity.35 NPM1 can interact with c-myc,36 p53,37 interferon regulatory factor 138 and NF-κB39 to probably play an important role in control of cellular apoptosis and immortalization. Moreover, NPM1 appears to be particularly important in oncogenesis. It can transform NIH/3T3 fibroblasts,38 and be involved in the oncogenic conversion of various associated proteins.37 Therefore, the overexpression of NPM1 should be responsible for malignant phenotype in nontreated CNE2 cells. Moreover, we suggested that the down-regulation of NPM1 by TPA treatment be one pivotal reason for growth arrest and then apoptosis of CNE2 cell. It was worthy to mention that two closely adjacent spots marked with “b” and “c” in foregoing 2D images were samely identified as NPM1. We speculated that they were two isoforms according to former reports.40 HSPD1 belongs to the chaperonin (hsp60) family.41 Former studies showed that HSPD1 has been identified in several database of some human cancer cells42,43 and normal cells with vigorous metabolism,44 which implied its function might be associated with cell metabolism. So we surmised that the high expression of HSPD1 in control CNE2 cells should be also associated with cellular rapid proliferation, and its downregulation be linked to growth arrest and apoptosis of cells induced by TPA. STMN1 is a soluble, ubiquitous cytoplasmic phosphoprotein.45 It was expressed at high level in wide variety of human tumors.46,47 Inhibition or decrease of STMN1 expression in malignant cells interfered with their orderly progression through the cell cycle and even abrogated their transformed phenotype.48 STMN1 might also play a role in alteration of epithelial cell shape and size by modulating the microtubules to impact cytoskeleton.49 Thus, STMN1 provides an attractive molecular target for new antineoplastic drugs50 to disrupt the mitotic apparatus and arrest the growth of malignant cells.51 Therefore, it was not surprising that STMN1 was highly expressed in untreated CNE2 cells such as NPM1. Similarly, we inferred that the decrease of STMN1 was also another crucial reason for cellular antiproliferation and apoptosis in TPA-treated CNE2 cells. In addition, evidences from microscopy indicated that TPA could drastically impact CNE2 cells in morphology such as less flattening and cytoplasmicconjugation between two cells by slim duct. We surmised that the above changes were due to microtubules modulation by STMN1;49 the downregulation of STMN1 expression altered the cytoskeleton through destroying microtubules by unknown mechanism to cause a cell in mitosis epilogue unable to split to 2 thoroughly, then that two cells conjugated with a slim duct came forth (see Figure 1).

Summary and Conclusions In summary, TPA could induce CNE2 cells to growth arrest and then differentiation to death with concurrently distinct changes in morphology by upregulation of TPI1 and SFN and downregulation of RCN1, NPM1, HSPD1 and STMN1. Among these identified proteins, we suggested the following: (1) SFN should be mostly responsible for cellular differentiation and then death; (2) the decrease of NPM1 and STMN1 should be pivotal reason for growth arrest and then apoptosis of CNE2 cells; and (3) Some morphological changes might be result from scarcity of STMN1 and RCN1. Some reports implied that the effect of TPA on cells was different and specific to the type of cells.52,53 As we knowed, primary nasopharyngeal epithelial cells must be different from transformed epithelial cells of NPCs, which might be the reason former researchers considered TPA as a promoter for primary epithelial cells while we concluded that it was an antiproliferative chemical for NPC cells. On the other hand, most of former reports about TPA promoting transformation of noninitiated epithelial cells were based on cooperation with EBV infection.6,7 Therefore, there was a possibility that TPA promoting NPC should need the cooperation from EBV, or else it might perform as an antiproliferative or differentiative revulsant in noninitiated cells. Therefore, TPA seemed to be two-edged in carcinogenesis. In addition, TPA has been considered to activate various PKC isozymes subject to different biochemical regulation for many years. However, with the isolation of new receptors responsing to phorbol esters one by one54 in recent years, the multiplicity of TPA effects seems to be more perplexing but attractive to us. In conclusion, more and deeper studies should be done to understand the mechanism of TPA effects on cells about proliferation or antiproliferation, susceptibility to EBV even transformation, and its potential as an anticancer drug.

Acknowledgment. We thank Dr. Wantao Ying and Professor Xiaohong Qian (Beijing Institute of Radiation Medicine, Beijing 100850, China) for supports of mass spectrometry analysis. Our study was supported by grants from Natural Science Foundation of Guangdong Province, P. R. China No. 13050. References (1) Yuspa, S. H. The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesissthirty-third G. H. A. Clowes Memorial Award Lecture. Cancer Res. 1994, 54, 11781189. (2) Zheng, X.; Chang, R. L.; Cui, X. X.; Avila, G. E.; Lee, S.; Lu, Y. P.; Lou, Y. R.; Shih, W. J.; Lin, Y.; Reuhl, K.; Newmark, H.; Rabson, A.; Conney, A. H. Inhibitory effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) alone or in combination with all-trans retinoic acid on the growth of LNCaP prostate tumors in immunodeficient mice. Cancer Res. 2004, 64, 1811-1820. (3) Zheng, X.; Chang, R. L.; Cui, X. X.; Kelly, K. A.; Shih, W. J.; Lin, Y.; Strair, R.; Suh, J.; Han, Z. T.; Rabson, A.; Conney, A. H. Synergistic effects of clinically achievable concentrations of 12-O-tetradecanoylphorbol-13-acetate in combination with all-trans retinoic acid, 1alpha,25-dihydroxyvitamin D3, and sodium butyrate on differentiation in HL-60 cells. Oncol. Res. 2000, 12 (9-10), 419427. (4) Fedder, M.; Gonzalez, M. F. Nasopharyngeal carcinoma. Brief review. Am. J. Med. 1985, 79, 365-369. (5) Guy, G. R.; Gordon, J. Epstein Barr virus and a tumor promoting phorbol ester use similar mechanisms in the stimulation of human B-cell proliferation. Int. J. Cancer 1989, 43, 703-708.

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