Inhibitory effect of ubiquitin-proteasome pathway on proliferation of esophageal carcinoma cells

Abstract

AIM: To investigate the inhibitory effect of ubiquitin-proteasome pathway (UPP) on proliferation of esophageal carcinoma cells.

METHODS: Esophageal carcinoma cell strain EC9706 was treated with MG-132 to inhibit its UPP specificity. Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA synthesis was evaluated by ³H-thymidine (³H-TdR) incorporation. Morphologic changes of cells were observed under microscope. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) of PCR-ELISA. Cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. Expression of p27^kip1 was detected by immunocytochemical technique.

RESULTS: After exposed to MG-132, the growth and value of ³H-TdR incorporation of EC9706 cells were obviously inhibited. Cells became round, small and exfoliative under microscope. TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5 μmol/L of MG-132 for 24, 48, 72 and 96 h (P < 0.01). The percentage of cells at G₀/G₁ phase was increased and that at S and G₂/M was decreased (P < 0.01). The rate of apoptotic cells treated with 5 μmol/L of MG-132 for 48 and 96 h was 31.7% and 66.4%, respectively. Agarose electrophoresis showed marked ladders. In addition, the positive signals of p27^kip1 were located in cytoplasm and nuclei in MG-132 group in contrast to cytoplasm staining in control group.

CONCLUSION: MG-132 can obviously inhibit proliferation of EC9706 cells and induce apoptosis. The mechanisms include upregulation of p27^kip1 expression, G₁ arrest and depression of telomerase activity. The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.

Key Words: $[Keywords]

INTRODUCTION

Esophageal carcinoma is common in China. Previous studies have shown that its occurrence and progression are complicated, and are associated with the changes of multi-genes and molecules[1-3]. The ubiquitin-proteasome pathway (UPP) is the major system employed by eukaryotes for the selective degradation of cellular proteins that play key roles in cellular processes such as cell cycle regulation, differentiation, signal transduction, gene transcription, antigen presenting and transmembrance localization of proteins[4,5]. In this study, we investigated the inhibitory effect of UPP on proliferation of esophageal carcinoma cells by using specific ubiquitin proteasome to find a new strategy for esophageal carcinoma therapy.

MATERIALS AND METHODS

Materials

Esophageal carcinoma cell strain EC9706 was presented by professor Ming-Rong Wang, China Academy of Medical Sciences. MG-132 was purchased from Calbiochem Co. Ltd (USA), and dissolved in dimethylsulfoxide (DMSO) as a 40 mmol/L stock solution and stored at -20 °C. 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and DMSO were bought from Sigma Co. Ltd (USA). ³H-thymidine (³H-TdR) was provided by Beijing Atomic Power Research Institute. Telomeric repeat amplification protocol (TRAP) ELISA telomerase detection kit was obtained from Intergen Company (USA). Monoclonal mouse antibody of p27^kip1, ultra sensitive S-P kit, and 3,3-diaminobenaidine (DAB) kit were purchased from Fuzhou Maixin Biotechnology Co. Ltd. RPMI 1640 medium was obtained from GIBCOBRL Company (USA). Low melting-temperature agarose was obtained from Promega Company (USA). DNA-PREP^TM LPR and DNA-PREP^TM stain were obtained from America Beckman Coulter Company.

Cell culture

Human esophageal carcinoma cell strain EC9706 was maintained in RPMI 1640 medium supplemented with 100 mL/L fetal calf serum (FCS), 100 kU/L penicillin, 100 mg/L streptomycin and 2 mmol/L l-glutamine in a humidified incubator containing 50 mL/L CO₂ at 37 °C, the medium was changed every 2-3 d.

MTT assay

EC9706 cells growing exponentially were chosen. Cell density in the suspension was adjusted to 2 × 10⁵ cells/mL. After addition of 200 mL/well of the final cell suspension, 96-well plates were incubated for 24 h. Then the cells were treated with 0.5, 1.0, 2.5, 5.0, 10, 15, 20 μmol/L (DMSO ≤ 0.5 g/L) of MG-132, respectively, as the different observation groups. The cells treated with an equal amount of DMSO instead of MG-132 served as the control. The background was conducted using only cell-free culture medium. After cultivated for 24, 48, 72 and 96 h, 10 μL of stock MTT solution (5 g/L) was added to the cells in each well, followed by a further incubation at 37 °C for 4 h. The culture medium was carefully removed, 100 μL of DMSO was added to each well and culture was vibrated for 20 min. The absorbance of samples was measured three times for each group with three wells at a wavelength of 550 nm with the enzyme linked immunosorbent assay meter (PR 2100, SANOFI company, France). The inhibitory rate (IR) was calculated according to the formula: IR = [1-(absorbance of MG-132 group-absorbance of background group)/(absorbance of control group-absorbance of background group)] × 100%.

Incorporation test of ³H-TdR

As described above, experimental group and control group were cultured for 3 h with 100 mL/L FCS RPMI 1640, and then for 12 h with FCS-free RPMI 1640. MG-132 and 1μCi ³H-TdR were added to each group, which was rinsed with PBS after 24, 48, 72 and 96 h and fixed with methyl alcohol and absolute ethyl alcohol for 10 min each. Finally, 200 μL of 0.1 mol/L NaOH was added, then 200 μL of each was taken after blowing, and mixed in 5 mL scintillation liquid for overnight. On the following day, the count per minute (CPM) of ³H was tested three times for each group with three wells.

Morphologic changes of cells

Morphologic changes of EC9706 cells were observed under microscope 24, 48, 72 and 96 h after treated with MG-132 (5 μmol/L).

Telomerase assay

The cells (10⁵-10⁶) treated with MG-132 (5 μmol/L) for 24, 48, 72 and 96 h were collected, respectively. After addition of 300 μL telomerase assay lysis buffer (1 × CHAPS), the cells were lysed on ice. The lysate was incubated on ice for 30 min and then centrifuged at 13000 g for 25 min at 4 °C. The supernatant (2 μL) was added to reaction solution containing 10 μL of TRAP buffer, 2 units of Taq polymerase and 48 μL of DH₂Oqs. PCR was carried out through 33 amplification cycles, each cycle consisting of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. The amplified product was added to block/dilution buffer (250 μL), and incubated at 37 °C for 30 min, and 5 μL of TRAP reactant was then added and mixed. After incubated at 37 °C for 60 min, 100 μL working solution of anti-DNP Ab was added and incubated for 30 min, then 100 μL of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution and 100 μL of stop reagent were added. The absorbance value in each well was read at the wave lengths of 450 nm and 690 nm on an enzyme linked immunosorbent assay meter. Telomerase activity was considered positive when the absorbance value of a sample was at least 0.8 units. When those were lower than 0.2 units, they were regarded as negative.

Flow cytometry detection

After cell cycle was synchronic, the cells of experiment group were treated with MG-132 (5 μmol/L) for 48 h and 96 h. The collected cells were added with DNA-PREP^TM LPR and DNA-PREP^TM stain, respectively, after they were washed with PBS and centrifuged. Cell cycle and apoptosis were detected by flow cytometry (Epics XL, Beckman Coulter Company, USA) and SYSTEM II^TM software was used to dispose the data.

DNA ladder demonstration

As described above, the cells (7 × 10⁶/sample, both attached and detached cells) were lyzed with hypotonic lysis buffer (10 mmol/L edetic acid, 5 g/L Triton X-100, Tris-HCl, pH7.4) for 15 min on ice and precipitated with 25 g/L polyethylene glycol and 1 mol/L NaCl for 15 min at 4 °C. After centrifugation at 16000 g for 10 min at room temperature, the supernatant was treated with proteinase K (0.3 g/L) at 37 °C for 1 h and precipitated with isopropanol. After centrifugation, each pellet was dissolved in 10 μL of Tris-EDTA (pH7.6) and electrophoresed on a 17 g/L agarose gel containing ethidium bromide. DNA ladder pattern was identified under ultraviolet light.

Immunocytochemical staining

EC9706 cells cultured with MG-132 (5 μmol/L) for 48 h were fixed with dimethyl ketone at 4 °C. The cells carrying the detected antigen were stained following SP immunocytochemical staining method using anti-p27kip1 as primary antibody[6]. PBS was substituted for primary antibody as negative control.

Statistics

The data were expressed as mean ± SD. The difference between each group was analyzed by t-test. P < 0.05 was considered statistically significant.

RESULTS

Inhibitory effect of MG-132 on EC9706 cell growth

The growth of EC9706 cells treated with 0.5-20 μmol/L of MG-132 was significantly inhibited compared with that of control group. While the cells exposed to MG-132 for 24 h produced a certain inhibitory effect, but only exceeding 48 h did MG-132 show significant effect. When the dose of MG-132 exceeded 5 μmol/L, only slight increases in IR of the cells were observed (Figure 1).

Figure 1 MTT assay of EC9706 cells after exposed to MG-132.

Inhibition of DNA synthesis

The values of ³H-TdR incorporation of MG-132 group were decreased compared with the control group (Table 1).

Table 1 Evaluation of DNA synthesis by ³H-TdR incorporation (mean ± SD).

Group	24 h	48 h	72 h	96 h
Control MG-132 (μmol/L)	4295.52 ± 136.32	5236.17 ± 221.36	5642.92 ± 105.41	5863.43 ± 206.58
0.5	3764.68 ± 97.37a	2879.83 ± 86.25b	1918.73 ± 76.49b	1759.29 ± 89.23b
1.0	3526.14 ± 101.42a	2643.29 ± 79.38b	1547.25 ± 68.94b	1366.18 ± 52.49b
2.5	3402.34 ± 93.44a	2567.76 ± 68.21b	1260.37 ± 51.27b	910.25 ± 45.37b
5.0	3324.78 ± 65.43a	2411.56 ± 69.34b	840.79 ± 41.17b	517.83 ± 41.26b
10.0	3301.29 ± 59.28a	2360.40 ± 49.28b	820.56 ± 39.76b	498.71 ± 40.14b
15.0	3294.12 ± 67.33a	2324.25 ± 47.30b	810.17 ± 45.61b	485.26 ± 37.56b
20.0	3280.54 ± 62.46a	2320.60 ± 46.83b	804.63 ± 51.34b	476.90 ± 38.41b

^aP < 0.05,

^bP < 0.01 vs control.

Morphologic changes of cells

EC9706 cells became round, small and exfoliative after exposed to MG-132 under microscope (Figure 2).

Figure 2 Morphologic changes of EC9706 cells observed un-der microscope after treated with 5 μmol/L of MG-132. A: control group; B: EC9706 cells treated with MG-132 for 24 h; C: EC9706 cells treated with MG-132 for 48 h; D: EC9706 cells treated with MG-132 for 72 h; E: EC9706 cells treated with MG-132 for 96 h (× 200).

Inhibition of telomerase activity

After treated with MG-132 (5 μmol/L) for 24, 48, 72 and 96 h, respectively, EC9706 cells showed a gradual decrease in values of A compared with the control group (P < 0.01). Furthermore, telomerase activity was negative (Table 2).

Table 2 Effect of MG-132 on telomerase activity of EC9706 cells (mean ± SD).

Groups	Light absorption (A)
	24 h	48 h	72 h	96 h
Control	1.871 ± 0.061	2.234 ± 0.092	2.907 ± 0.113	3.025 ± 0.120
MG-132	0.154 ± 0.008b	0.085 ± 0.006b	0.072 ± 0.004b	0.067 ± 0.003b

^bP < 0.01 vs control group.

Changes of cell cycle and apoptosis

The percentage of cells at G₀/G₁ phase was increased and that at G₂/M and S was decreased (P < 0.01). The rate of apoptotic cells treated with 5 μmol/L of MG-132 for 48 and 96 h was 31.7% and 66.4%, respectively (Table 3, Figure 3).

Table 3 Effect of MG-132 on cell cycle of EC9706 cells (mean ± SD, %).

Groups	48 h			96 h
	G0/G1	S	G2/M	G0/G1	S	G2/M
Control	40.4 ± 3.9	46.5 ± 4.3	13.1 ± 1.0	44.6 ± 4.1	40.9 ± 2.3	14.5 ± 1.4
MG-132	67.5 ± 5.1b	29.3 ± 2.8b	3.2 ± 0.2b	73.1 ± 5.2b	24.6 ± 2.1b	2.3 ± 0.1b

^bP < 0.01 vs control group.

Figure 3 Cell cycle and apoptosis of EC9706 cells exposed to 5 μmol/L of MG-132. A: control group. Apoptotic sub-G₁ peak was not found; B: EC9706 cells exposed to MG-132 for 48 h. The ratio of apoptotic cells was 31.7%; C: EC9706 cells ex-posed to MG-132 for 96 h. The ratio of apoptotic cells was 66.4%.

DNA ladder

Agarose electrophoresis showed marked ladders in MG-132 group, while the ladder was not detected in control group. Furthermore, DNA fragmentation was more apparent at 96 h (Figure 4). Immunohistochemical staining of p27^kip1 protein located in cy-toplasm and nuclei (× 200).

Figure 4 Results of DNA agarose electrophoresis. Lane1: con-trol group; Lane 2: EC9706 cells exposed to 5 μmol/L of MG-132 for 48 h; Lane 3: EC9706 cells exposed to 5 μmol/L of MG-132 for 96 h; Lane 4: 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000 bp ladder markers.

Expression of p27kip1

In the control group, the cytoplasm was stained in brownish yellow and the nuclei were stained in blue. In the experiment group, both the cytoplasm and nuclei were stained in brownish yellow, indicating that the expression of p27^kip1 in EC9706 cells was increased after treated with MG-132 (Figure 5).

Figure 5 Results of immunohistochemical staining of EC9706 cells. A: control group. Immunohistochemical staining of p27^kip1 protein located in cytoplasm of EC9706 cells; B: MG-132 group.

DISCUSSION

Proteolytic degradation by ubiquitin-proteasome system involves ATP-dependent covalent attachment of a macromolecular chain of ubiquitin (Ub) molecules to the target protein, followed by degradation through the multicatalytic 26s proteasome. The conjugation of Ub, a highly conserved 8.6 kDa protein, to its target protein is mediated by the serial actions of three enzymes. E1, the Ub-activating enzyme, activates Ub in an ATP-dependent manner. E2, the Ub-conjugating enzyme, catalyzes the attachment of Ub to the substrate protein. E3, the Ub-ligase, serves as a scaffold between E2 and the substrate and provides recognition specificity of the substrate[7-10]. A protein tagged with a polyubiquitin chain is recognized and degraded by the 26S proteasome complex. This complex is composed of a 19S regulatory subcomplex and two 20S catalytic subcomplexes. UPP is extensively involved in physiological and biochemical processes. Some experiments showed that the low expression of some anti-oncogene including p53, p27^kip1 in tumor cells was associated with the increasing activity of ubiquitin proteasome which leads to degradation of expression products of anti-oncogene, and have proved that deubiquitination of p53 is an important pathway for p53 stabilization[11,12]. Moreover, the degradation accommodation of some transcription factors was regulated by UPP, such as NF-κB, c-fos, c-jun, c-mos, c-myc and MATa[13-17]. So UPP is closely associated with the occurrence and development of malignant tumor.

Ubiquitin proteasome inhibitors include peptide aldehyde, borofax peptide and 3, 4-dichloro isocoumarin. MG-132, also known as carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, a reversible, effective and specific peptide aldehyde inhibitor of ubiquitin-proteasome, could block UPP through inhibiting ubiquitin-mediated proteolysis by binding to and inactivating 20S and 26S proteasomes[18-22].

In our study, esophageal cancer cell line EC9706 was exposed to MG-132 to observe whether UPP could be inhibited. We found that the proliferation of cells was obviously inhibited in a dose- and time-dependent manner. The results also revealed some anti-tumor mechanisms of MG-132. First, MG-132 could up-regulate the expression of p27^kip1. p27^kip1 was recently found[23] as a anti-oncogene with function of negative regulation of cell cycle, getting involved in the inhibitive reaction of cytokines, inducing cell differentiation and apoptosis, enhancing cell’s adherence and regulating the resistance to medicines for noumenal tumors. p27^kip1 protein is a cyclin dependent kinase inhibitor (CDKI) that could block G₁/S transition of cell cycle by inhibiting the action of cyclin E-CDK2 complex and cyclin D-CDK4 complex[24-26]. p27^kip1 expression decreases in esophageal cancer and it may correlate with the histologic differentiation. Reduction of p27^kip1 has been considered to be an independent prognostic indicator of esophageal cancer[27-30]. The nuclear localization signal of p27^kip1 contains a protein kinase B (PKB/Akt) consensus site at threonine 157, and phosphorylation of p27^kip1 by PKB/Akt has been found to impair its nuclear import[31,32], which is a key procedure to play its functional role[33]. We found that p27^kip1 protein localized in cytoplasm of EC9706 cells showed low expression, but that localized both in cytoplasm and nuclei of EC9706 cells showed high expression after treated with MG-132. Our previous studies[6,34] demonstrated that the growth of EC9706 cells and tumors implanted in nude mice was obviously inhibited, apoptosis was induced and cell cycle was arrested in G₁ phase by up-regulating p27^kip1. Second, MG-132 could depress telomerase activity. The activation of telomerase was closely associated with cyclin. It has been reported that inhibition of UPP could not only increase the expression of p27^kip1, but also increase the expression of p53[35]. Moreover p27^kip1 and p21 regulated by p53 could inhibit cyclin and result in decreased telomerase activity[36,37]. Third, MG-132 could cause G₁ arrest, which may be involved in changes of cell cycle regulatory factors such as p27^kip1. Fan et al[38] obtained the same results as ours. But Ling et al[39] tended to consider ubiquitin proteasome inhibitors to cause G₂ arrest. The difference may be involved in the different types of cells. The last, MG-132 could induce apoptosis, which may be closely associated with the functions mentioned above.

In conclusion, MG-132 can obviously inhibit proliferation of EC9706 cells and induce apoptosis. The mechanisms include upregulation of p27^kip1 expression, G₁ arrest and depression of telomerase activity. The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.