Experimental and Molecular Pathology 87 (2009) 42–47
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Experimental and Molecular Pathology
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Expression of Akt and Mdm2 in human esophageal squamous cell carcinoma
Ken Takahashi a,⁎, Masao Miyashita a, Hiroshi Makino a, Ichiro Akagi a, Hajime Orita b, Nobutoshi Hagiwara a,Tsutomu Nomura a, Edward W. Gabrielson c, Takashi Tajiri a
a Graduate School of Medicine, Surgery for Organ Function and Biological Regulation, Nippon Medical School, Tokyo, Japanb Department of Surgery, Juntendo University, Tokyo, Japanc Department of Pathology, Johns Hopkins Medical Institutions, USA
⁎ Corresponding author. Department of Surgery, SurgBiological Regulation, Nippon Medical School, 1-1-5 Se8603, Japan. Fax: +81 3 5685 0989.
lays an important role in carcinogenesis in a variety of malignant tumors.However, the Akt–Mdm2 pathway in esophageal squamous cell carcinoma (ESCC) has not been fullystudied. We investigated the proteins and mRNA expression of Akt and Mdm2 to elucidate the roles ofthese proteins in ESCC. We also examined the effect of Akt knockdown on Mdm2 expression in ESCC cells.ESCC tissue samples were obtained from 23 individuals who underwent surgical resection with nopreoperative treatment. Akt1–3 and Mdm2 gene and protein expression were analyzed. The effect ofsiRNA-mediated Akt knockdown on Mdm2 expression was also studied, using ESCC cell lines. Akt1 andMdm2 immunoreactivity was detected in 77.8 and 66.7% of tumor specimen from ESCC patients,respectively. Akt1 and Mdm2 mRNA expressions were correlated and significantly elevated in tumor tissue(pb0.0001 and pb0.05, respectively). The siRNA-targeted reduction of each Akt isoform reduced Mdm2protein expression. The overexpression of Akt1 and Mdm2 may be related to esophageal carcinogenesis.Furthermore, Akt expression regulates Mdm2 expression, which may in turn regulate the function of wild-type p53. These results may provide the basis for future preventative or clinical therapies for esophagealcancer.
Esophageal squamous cell carcinoma (ESCC) is the sixth mostcommon cause of cancer deaths worldwide. In Japan, ESCC causesnearly as many deaths as rectal carcinoma (Nishihira et al., 1993).Despite recent therapeutic advances, the prognosis remains poor forESCC patients. Recent research has shown that growth factors,cytokines, and certain oncogene products have powerful anti-apoptotic effects, which are mediated via the phosphatidylinositol3-OH-kinase (PI3K)-induced activation of Akt (Datta et al., 1999;Ogawara et al., 2002). In humans, there are three Akt isoforms, Akt1,Akt2, and Akt3, which are located on chromosomes 14q32, 19q13,and 1q44, respectively. Each member of the Akt family contains anamino-terminal pleckstrin homology (PH) domain, a short α-helicallinker, and a carboxy-terminal kinase domain. Although Akt1 mRNAis ubiquitous, Akt2 expression is markedly elevated in insulin-responsive tissues such as skeletal muscle and liver. Low-level Akt3expression is ubiquitous in skeletal muscle and liver (Bellacosa et
ery for Organ Dysfunction andndagi, Bunkyo-ku, Tokyo 113-
hi).
ll rights reserved.
al., 2004). The reason for this apparent functional redundancy isunclear (Testa and Bellacosa, 2001).
The murine double minute 2 (Mdm2) gene was originallyidentified and cloned by amplification in a transformed tumorigenicBALB/c3T3 fibroblast cell line (Cahilly-Snyder et al., 1987; Fakhar-zadeh et al., 1991; Oliner et al., 1992; Saito et al., 2002). Its productforms a tight complex with the wild-type p53 tumor suppressorprotein. This interaction inhibits p53 by masking its N-terminalacidic transactivating domain (Saito et al., 2002; Haines et al., 1994;Olson et al., 1993). Therefore, Mdm2 expression is thought to be akey factor in malignant transformation, tumor progression, and poorprognosis in various malignancies (Haitel et al., 2000). Akt enhancesthe ubiquitination-promoting function of Mdm2 by phosphorylationof Ser186, which eventually results in the inhibition of tumorsuppressor p53 protein (Ogawara et al., 2002).
We hypothesized that Akt and Mdm2 are associated withmalignant transformation, tumor progression and poor prognosis inESCC. Therefore, we investigated the expression of Akt1–3 and Mdm2in ESCC tumor tissue, using immunohistochemistry and real-timesemi-quantitative reverse-transcription polymerase chain reaction(RT-PCR). To determine whether the Akt gene regulates Mdm2expression, we suppressed Akt expression in human ESCC usingsmall interfering RNA (siRNA). The manipulating of Akt expression toinhibit the Mdm2 gene may provide future therapeutic options forpatients with ESCC.
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Materials and methods
Surgical specimens
ESCC tissue samples were obtained from 23 individuals whounderwent surgical resection at Nippon Medical School Hospitalduring April, 1995 and August, 2005. Their follow up was done from32 months to 156 months. It was confirmed that all specimens in thisexperiment were squamous cell carcinoma by our hospital's pathol-ogists. Induction or adjuvant therapy was not administered to anypatient in this study. The study population included 16 males (69.6%)and seven females (30.4%) with a median age of 63 years (range, 42 to81 years). The TNM (tumor, nodes, and metastasis) staging system(Japanese Society for Esophageal, D., 1999) was used to classify resec-ted specimens and lymph nodes. Among the 23 patients, two patientswere in Stage 0, eight in Stage I, eight in Stage II, four in Stage III, andone in Stage IVa. To compare superficial tumors with more invasivecancers, the specimens were divided into two groups based on tumorinvasion (T factor), T1 (superficial tumors) and T2/T3 (advancedtumors). Similarly, specimens were divided into two groups, N0 andN1, based on lymph node metastasis. Tissue samples were obtainedimmediately after the surgical resection and stored at−80 °C until use.Written informed consent was obtained from each patient.
Cell lines and culture conditions
The human ESCC cell lines TE-1 and TE-5 were kindly provided byDr. Tetsuro Nishihira (Seta Clinic Group, Tokyo, Japan) and the CellResource Center for Biomedical Research at the Institute of Develop-ment, Aging and Cancer, Tohoku University. The cells were cultured inRPMI1640 (Sigma-Aldrich Co., St. Louis, MO) supplemented with100 U/ml penicillin, 100 μg/ml streptomycin (GIBCO), and 10% fetalcalf serum (FCS, PAA Laboratories GmbH, Pasching, Austria). The cellswere grown as sub-confluent monolayers in a humidified atmospherecontaining 5% CO2 at 37 °C. The cells were passaged using 0.25%trypsin, every 5 days.
Immunohistochemistry
We analyzed specimens from 18 patients who underwent surgicalresection without any preoperative treatment at Nippon MedicalSchool Hospital. Five out of 23 specimens could not meet the criteriafor evaluating immunohistochemistry due to its poor condition informalin. Formalin-fixed, paraffin-embedded specimens were sub-jected to antigen retrieval techniques followed by the avidin–biotin–peroxidase complex-immunoperoxidase method (Nichirei Co., Tokyo,Japan). Our immunocytochemical analyses used the following specificantibodies: mousemonoclonal anti-Akt1 (1:50 dilution; Cell SignalingTechnology, Beverly, MA), goat polyclonal anti-Akt2 (1:150 dilution;Cell Signaling Technology), goat polyclonal anti-Akt3 (1:500 dilution;Santa Cruz Biotechnology, Santa Cruz, CA), and mouse monoclonalanti-Mdm2 (1:100 dilution; Santa Cruz Biotechnology). Tissuesections were incubated with the designated antibody overnight at4 °C. Positive immunostaining was detected with diaminobenzene(DAB). After light counterstaining with Mayer's hematoxylin, theslides were dehydrated, covered with a glass slip, and observed underan Olympus DP70 microscope (Olympus Optical, Tokyo, Japan). Whenpositive staining, regardless of the intensity, was noted in thecytoplasm of tumor cells, the cells were designated positive. Tumorspecimens were deemed immunopositive when positively stainedcells were observed in more than 10% of the tumor (Lee et al., 2005).
Real-time semi-quantitative RT-PCR
The surgical specimens were stored at −80 °C before mRNAextraction. Total RNA was isolated from the frozen specimens of 23
patients, using ISOGEN reagent (Nippon Gene Co., Toyama, Japan)according to the manufacturer's instructions. Total RNA (0.2 μg/μl)was reverse transcribed using an mRNA-selective PCR kit (Ver.1.1;Takara Bio, Shiga, Japan). Taqman probes for human Akt1–3, Mdm2,and beta glucuronidase (GUS; TaqMan Gene Expression Assays) werepurchased from Applied Biosystems. The samples were amplified in atotal volume of 20 μl containing 1 μl of each specific probe and 10 μl ofTaqMan PCR Core Reagent Kit (Universal PCR Master Mix; AppliedBiosystems). Amplification was performed for 45 cycles using aGeneAmp 5700 sequence detection system (Applied Biosystems), andthe level of expression was calculated by the comparable cyclenumber method as recommended by the manufacturer. Semi-quantitative mRNA levels were calculated based on the ratio of targetmRNA expression to GUS expression. We also investigated therelationship of Akt andMdm2mRNA expressionwith clinical variablessuch as the T factor and regional lymph nodes (N factor), andcompared the relative levels of Akt or Mdm2 mRNA expressionbetween tumors and healthy tissues. The corresponding healthytissues were obtained from surgically resected tissues which werelocated at least 5 cm away from the tumors.
Introduction of siRNA into TE-1 and TE-5 cells
To investigate the role of Akt in Mdm2 expression, we silencedAkt1, Akt2, and Akt3 mRNA expression in ESCC cells using Silencer®Validated siRNA (Ambion, Ontario, Canada). Cells from each line(5.0×104 cells) were seeded in separate 24-well plates and wereallowed to incubate for 24 h at 37 °C under 5% CO2. Then the cells weretransfected with 200 pmol of siRNA per well using Lipofectamine2000(Invitrogen, Carlsbad, CA) and Opti-MEM reduced-serum medium(GIBCO). The control was transfected with non-silencing siRNA. Aftertransfection, the cells were incubated in serum-free medium for 48 hand then detached from the plates using 0.25% trypsin. Mdm2expression in the transfected cells was analyzed using real-time semi-quantitative RT-PCR. In addition, total cell lysates were subjected toWestern blotting with specific antibodies against Akt, Mdm2, andGAPDH.
Western blotting
Approximately 5×104 cells from each line were cultured overnightin growth medium before analyzing Mdm2 protein expression amongthree groups: untreated cells, cells treated with non-specific siRNA,and cells treated with specific siRNAs for Akt1, Akt2, or Akt3. Afterthree washes in phosphate-buffered saline (PBS), the cells were lysedin M-PER protein extraction reagent (Pierce Biotechnology, Rockford,IL) containing Halt protease inhibitor cocktail (Pierce Biotechnology).The whole cell lysates were subjected to SDS-polyacrylamide gelelectrophoresis (PAGE) and transferred to membranes. After applyingthe primary antibodies against Akt1, Akt2, Akt3, or Mdm2, the blotswere treated with horseradish peroxidase-conjugated polyclonalsecondary antibody (1:2000; Medical and Biological LaboratoriesCo., Nagoya, Japan). Immunodetection was performed using anenhanced chemiluminescence kit for Western blot analysis (Amer-sham Biosciences, Buckinghamshire, UK). The immunoblots wereexposed to an X-ray film for approximately 3 min, and the auto-radiograms were scanned using Adobe Photoshop (Adobe System Inc.,San Jose, CA). The blots were adjusted for brightness and contrast tominimize the background, and the mean density of each band wasdetermined.
Statistical analysis
The Mann–Whitney U test was used to assess the differencebetween mRNA expression in tumors and healthy tissue. Spearman'srank correlation coefficient was used to examine the relationship
Fig. 1. Positive rate of immunohistochemical expression of Akt1–3 and Mdm2 in ESCC tissue. Akt1 immunoreactivity was observed in 14 of 18 specimens (77.8%). Akt2, Akt3, andMdm2 overexpressions were also observed in 7 of 18 (38.9%), 5 of 18 (27.8%), and 12 of 18 specimens (66.7%), respectively.
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between the relative levels of Akt and Mdm2 mRNA expression.Fisher's exact test was used to assess the relationship between Akt andMdm2 protein expression. The T test was performed to assess thedifferences in total cell count. Computations were performed usingthe StatView J software package (Version 5.0; SAS Institute, Inc., Cary,NC, USA). A p value of less than 0.05 was considered significant in allanalysis.
Results
Immunohistochemical expression of Akt isoforms and Mdm2 in ESCCcells
Akt1 immunoreactivity was observed in the cytoplasm of ESCCcells in 14 of 18 specimens (77.8%). Akt2, Akt3, and Mdm2 over-expressions were also observed in the cytoplasm of ESCC cells in 7 of18 (38.9%), 5 of 18 (27.8%) and 12 of 18 specimens (66.7%),respectively (Fig. 1). Mdm2 and Akt1 overexpressions were signifi-cantly correlated in ESCC cells (p=0.005). However, no correlation
Table 1Correlation between Akt1–3 and Mdm2 protein
There was a statistical correlation between the overexpression of Akt1 protein and thatof Mdm2 protein in ESCC cells (p=0.005).
a p=0.005 correlation between Akt1 and Mdm2 Fisher's exact probability test.
was observed between Akt2 or Akt3 and Mdm2 protein overexpres-sion in ESCC cells (Table 1).
Correlation of the T and N factors with immunoreactivity
We observed immunopositive staining for Akt1 in 8 in 11 super-ficial tumors (72.7%) and in 6 of 7 advanced tumors (85.7%), with nosignificant difference between these two groups. In superficial tumors,we observed positive immunoreactivity for Akt2, Akt3, andMdm2 in 4of 11 (36.4%), 2 of 11 (18.2%), and 7 of 11 cases (63.6%), respectively. Inadvanced tumors, Akt2, Akt3, and Mdm2 immunoreactivity wasevident in 3 of 7 (42.9%), 3 of 7 (42.9%), and 5 of 7 cases (71.4%),respectively. As with Akt1, there were no significant differences inAkt2, Akt3, and Mdm2 expression between superficial and advancedtumors. Lymph node metastasis was not associated with immunor-eactivity for these genes (Table 2).
Table 2Correlation between T factor, N factor and positive rate of immunoreactivity of each Aktand Mdm2 in ESCC
There was no significant difference between superficial tumor and advanced tumor.Lymph node metastasis was not associated with the immunoreactivity of each gene.
Fig. 2. The relative level of Akt and Mdm2 mRNA in the tumor tissue. The relative levelof non-tumor tissue is 1.0. The relative level of mRNA in Akt1 and Mdm2 wassignificantly high (pb0.0001 and pb0.05, respectively). The Mann–Whitney U test wasused in this examination.
Table 3Clinicopathological factors and the level of each mRNA
Variable n Akt1 mRNA(T/N)
Akt2 mRNA(T/N)
Akt3 mRNA(T/N)
Mdm2 mRNA(T/N)
T factorTis, T1 14 1.683±0.721a 1.025±0.905 0.787±0.725 1.412±0.876T2, T3 9 1.910±0.698 1.113±0.409 0.959±0.646 1.232±0.509
T factor, N factor, lymphatic invasion and blood vessel invasionwere not associated withthe relative mRNA expression of each gene. The Mann–Whitney U test was used toassess the difference between mRNA expression in each factor.
a Mean±S.D.
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Expression of Akt isoforms and Mdm2 mRNA in ESCC cells
The relative level of Akt1 mRNA expression ranged from 0.68 to4.11 arbitrary units (AU). In 19 of 23 tumor specimens (82.6%), therelative level of Akt1 mRNA was higher than 1.0 AU. The relativemRNA levels of Akt2, Akt3, and Mdm2 ranged from 0.22 to 2.65, 0.09to 2.26, and 0.46 to 4.00 AU, respectively, and were higher than1.0 AU in 7 of 23 (30.4%), 5 of 23 (21.7%), and 15 of 23 cases (65.2%),respectively. The relative levels of Akt1 and Mdm2 mRNA were sig-nificantly elevated in tumor specimens (pb0.0001 and pb0.05,respectively) (Fig. 2). Furthermore, the relative level of Mdm2mRNA was significantly correlated with Akt1 expression (p=0.009)but not with Akt2 or Akt3 expression (Fig. 3).
Fig. 3. Correlation between each Akt and Mdm2 mRNA. There was a statistical correlationcorrelation coefficient was used in this examination.
Correlation of the T and N Factors with mRNA expression
As shown in Table 3, Akt1 mRNA expression was not significantlydifferent between superficial and advanced tumors (1.683±0.721 vs.1.910±0.698 AU, respectively). Likewise, the levels of Akt2, Akt3, andMdm2 expression were not significantly different between superficialtumors (1.025±0.905 AU, 0.787±0.725 AU, and 1.412±0.876 AU,respectively) and advanced tumors (1.113±0.409AU, 0.959±0.646AU,and 1.232±0.509 AU, respectively). Lymph nodemetastasis, lymphaticinvasion and blood vessel invasion were not associated with theexpression of the target genes (Table 3).
Akt silencing in TE-1 and TE-5 cells
Messenger RNA from three Akt isoforms and Mdm2was expressedin TE-1 and TE-5 cells. Real-time semi-quantitative RT-PCR demon-
of the relative mRNA levels between Akt1 and Mdm2 (p=0.009). Spearman's rank
Fig. 4. Suppression of each Akt expression by siRNA. Synthetic siRNAs directed against Akt1, Akt2 or Akt3 suppressed each Akt isoform expression in TE-1 and TE-5 cells.Downregulation of each Akt isoform mRNA was not observed in cells transfected with a control non-silencing siRNA. The Mann–Whitney U test was used in this examination.
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strated that synthetic siRNAs directed against Akt1, Akt2, and Akt3suppressed each isoform in TE-1 and TE-5 cells. Akt downregulationwas not observed in control cells transfectedwith non-silencing siRNA.Compared with control levels, mRNA levels at 48 h after siRNAtransfectionwere reduced at 64% for Akt1, 73.5% for Akt2 and 72.3% forAkt3 in TE-1 cells, and at 75% for Akt1, 72% for Akt2, and 73.5% for Akt3in TE-5 cells (Fig. 4).
Effect of Akt mRNA suppression on Mdm2 protein expression in TE-1 andTE-5 cells
Akt was downregulated in cells transfected with specific siRNAscorresponding to each Akt isoform, and all siRNAs resulted in thedownregulation of Mdm2 protein in TE-1 and TE-5 cells (Fig. 5). Incontrast, Akt suppression and Mdm2 downregulation were notobserved in cells transfected with non-silencing siRNA.
Discussion
We found that Akt1 mRNA expression was significantly higher inESCC tumors than in normal esophageal tissue. Akt1 protein was alsooverexpressed more frequently in tumor specimens. Akt, also knownas protein kinase B, represents a subfamily of serine/threonineprotein kinases. Three Akt kinase family members have emerged asimportant players in the signaling cascades that regulate cell growth,proliferation, survival, and various aspects of intermediary metabo-lism (Bellacosa et al., 2004). Akt expression has been examined in
Fig. 5. Effect for the expression of Mdm2 protein in TE-1 and TE-5 cells by suppression ofAkt isoform protein with siRNA. Mdm2 protein was downregulated in TE-1 and TE-5cells by transfection of siRNAs for each Akt isoform.
cancers of the stomach, breast, ovary, and pancreas and in malignantmelanoma, but no report has investigated Akt expression in ESCC.Elevated Akt1 expression is observed in a human gastric cancer (Staal,1987). Akt1 kinase activity is also frequently found in breast, ovary,and prostate cancers, where it portends a poor prognosis (Sun et al.,2001). The elevation and overexpression of Akt2 occur in 10–20% ofhuman cancers of the ovary and pancreas (Bellacosa et al., 1995;Cheng et al., 1996). Other reports have indicated that Akt3 mRNA isupregulated in estrogen receptor-negative breast tumors and thatincreased Akt3 enzymatic activity is present in estrogen receptor-deficient breast cancer cell lines (Nakatani et al., 1999). Based on ourresults, Akt1 may play a greater role than Akt2 or Akt3 in humanESCC, although it is still unclear whether Akt1 performs a specificfunctional role. Recent research has indicated that PTEN down-regulation and the activation of phosphoinositide-3-OH-kinase (PI3-kinase) are related to Akt1 activation and cell proliferation (Sun et al.,2001; Stambolic et al., 1998). However, PTEN is rarely associated withcarcinogenesis in ESCC (Ding et al., 2000; Negoro et al., 2000;Tachibana et al., 2002), and there have been no reports regarding therole of PI3-kinase in ESCC. We showed that Akt1 mRNA and proteinexpression are elevated in ESCC. Therefore, it may be useful toinvestigate the activation of PI3-kinase as a mechanism of Akt1overexpression in ESCC.
We also demonstrated that Mdm2 expression is elevated in ESCC.The Mdm2 gene encodes a negative regulator of the p53 tumorsuppressor. Increased Mdm2 expression may be linked to thesuppression of the essential functions of p53, including apoptosisand cell cycle arrest, which are common features of carcinogenesis(Iwakuma and Lozano, 2003). Mdm2 transcription is induced by p53(Testa and Bellacosa, 2001). In addition, Mdm2 expression correlateswith wild-type p53 status in esophageal adenocarcinoma (Soslowet al., 1999). Mutant p53 is frequently expressed in ESCC (Sarbia et al.,1994), but this mutant-type p53 protein does not activate the Mdm2gene (Piette et al., 1997). It is also reported that p53 mutation and theoverexpression of Mdm2 occur independently (Florenes et al., 1994).Therefore, it is most probable that Akt1 overexpression inducesMdm2mRNA overexpression in ESCC. Consequently, it may be expected thatMdm2 overexpression inhibits the tumor suppressing activities ofwild-type p53, allowing neoplastic cells to continue growing. Unfor-tunately, there is no way to distinguish wild-type p53 from mutantp53 using the commercially available antibody for p53. However, ithas been reported that Mdm2 abrogates the function of p53. Thisreference allows us to speculate that Mdm2 overexpression inhibitsthe tumor suppressing activities of wild-type p53 in ESCC. Furtherstudies are required to investigate other functions of the Akt gene,including the regulation of p21, p27, and forkhead transcription factors(Zhou et al., 2001).
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Our immunohistochemical analysis revealed frequent overexpres-sion of Akt and Mdm2 protein in ESCC tumors. In this study, each Aktand Mdm2 were stained in the cytoplasm only. Usually, Mdm2 isthought to be located in both cytoplasm and nucleus. The serine/threonine kinase Akt phosphorylates Mdm2 at serine 166 and 186 andcauses Mdm2 shift to the nucleus where it is considered to play a rolein p53 degradation. Primarily, we selected the antibody for totalMdm2. In this study, cytoplasmic Mdm2 immunoreactivity was detec-ted in 66.7% of cases. Whereas low grade nuclear Mdm2was detected,which was grouped into negative staining. The reason simply comesfrom the fact that Mdm2 abrogates the function of p53 either bydegrading the protein in the cytoplasm or by repressing p53-mediatedtranscription activity in the nucleus. Similarly, the antibody wasselected for not only phosphorylated Akt but also non-phosphorylatedAkt because Akt is thought to be located in both cytoplasm andnucleus. We could detect only cytoplasmic Akt in this study. Now theantibody specific for phosphorylated Akt is commercially available, wecould detect nuclear Akt if a specific one was used.
The overexpression of these proteins is related to tumor progres-sion and prognosis in other cancers (Pinto et al., 2005; Yu et al., 2007;Lim et al., 2007), we did not observe a correlation between Akt orMdm2 overexpression and pathological factors of ESCC, includingtumor depth, invading blood or lymph vessels, and lymph nodemetastasis. Therefore, our results suggest that the Akt–Mdm2 path-way may be more important in the malignant transformation of pre-neoplastic esophageal cells than in tumor progression in human ESCC.
Akt activates Mdm2 via phosphorylation. However, we observed acorrelation between the relative levels of Akt1 and Mdm2 mRNAexpression. Furthermore, our immunohistochemical data revealed astatistical correlation between Akt1 and Mdm2 protein overexpres-sion in ESCC cells. Certainly, each commercially available siRNA cansuppress its targets, including Akt1, Akt2 and Akt3. In this study, onlyAkt1 was highly expressed in both mRNA status and protein,comparing to Akt2 and Akt3. Then, we obtained the statisticallysignificant relationship between Akt1 and Mdm2. Whereas Akt2 andAkt3 are not commonly expressed in ESCC. This is why the lattercould not reach statistical significance. For these reasons, weexamined whether Akt regulates Mdm2 expression and found thatMdm2 protein expression was downregulated in ESCC cells aftertransfection with specific siRNAs against Akt isoforms. Although theAkt1 isoform appears to play a more crucial role in ESCC, it is possiblethat simultaneously inhibiting all Akt isoforms would downregulateMdm2 expression in ESCC. The inhibition of Mdm2 expression wouldhelp to maintain wild-type p53 function in ESCC.
Conclusions
In conclusion, the overexpression of Akt1 and Mdm2 may berelated to early stages of human esophageal carcinogenesis. Further-more, the regulation of Mdm2 expression by the blockade of Aktexpression may preserve the function of wild-type p53 in ESCC. Theseresults may prove useful in future preventive or clinical therapies foresophageal cancer.
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