Zhao, Yong-Qiang
(Department of Otolaryngology & Head and Neck Surgery, the Second Hospital of Shandong University)
,
Feng, Hui-Wei
(Department of Otolaryngology & Head and Neck Surgery, the Second Hospital of Shandong University)
,
Jia, Tao
(Department of Otolaryngology & Head and Neck Surgery, the Second Hospital of Shandong University)
,
Chen, Xue-Mei
(Department of Otolaryngology & Head and Neck Surgery, the Second Hospital of Shandong University)
,
Zhang, Hui
(Department of Otolaryngology & Head and Neck Surgery, the Second Hospital of Shandong University)
,
Xu, An-Ting
(Department of Otolaryngology & Head and Neck Surgery, the Second Hospital of Shandong University)
,
Zhang, Hai-Ling
(Department of Otolaryngology & Head and Neck Surgery, the Second Hospital of Shandong University)
,
Fan, Xian-Liang
(Department of Otolaryngology & Head and Neck Surgery, the Second Hospital of Shandong University)
Background: To investigate the effect of celecoxib on telomerase activity and apoptosis in a human laryngeal squamous carcinoma cell line (Hep-2 cells). Materials and Methods: The growth inhibition rate of Hep-2 cells in vitro was measured by MTT assay, and apoptosis by TUNEL assay and flow cytometr...
Background: To investigate the effect of celecoxib on telomerase activity and apoptosis in a human laryngeal squamous carcinoma cell line (Hep-2 cells). Materials and Methods: The growth inhibition rate of Hep-2 cells in vitro was measured by MTT assay, and apoptosis by TUNEL assay and flow cytometry (FCM). The TRAP-ELISA method was used to determine telomerase activity in Hep-2 cells. The mRNA expression of human telomerase RNA component(hTR), human telomerase reverse transcriptase (hTERT) and human telomerase-associated protein(hTEP1) was determined by RT-PCR assay. Expression of Bax and Bcl-2 proteins was assessed by Western blotting. Results: Celecoxib can inhibit proliferation and induce apoptosis in a dose- and time-dependent manner, repress telomerase activity, decrease hTERT mRNA and Bcl-2 protein expression and increase Bax protein expression, PGE2 had no effect on telomerase. Conclusions: Celecoxib had the antiproliferative and pro-apoptotic effect in Hep-2 cells. Apoptosis was accompanied by a decrease in telomerase activity which was directly correlated with hTERT mRNA and up-regulation of Bax/Bcl-2. Bcl-2 may thus play an important role in telomerase activity as well as apoptosis.
Background: To investigate the effect of celecoxib on telomerase activity and apoptosis in a human laryngeal squamous carcinoma cell line (Hep-2 cells). Materials and Methods: The growth inhibition rate of Hep-2 cells in vitro was measured by MTT assay, and apoptosis by TUNEL assay and flow cytometry (FCM). The TRAP-ELISA method was used to determine telomerase activity in Hep-2 cells. The mRNA expression of human telomerase RNA component(hTR), human telomerase reverse transcriptase (hTERT) and human telomerase-associated protein(hTEP1) was determined by RT-PCR assay. Expression of Bax and Bcl-2 proteins was assessed by Western blotting. Results: Celecoxib can inhibit proliferation and induce apoptosis in a dose- and time-dependent manner, repress telomerase activity, decrease hTERT mRNA and Bcl-2 protein expression and increase Bax protein expression, PGE2 had no effect on telomerase. Conclusions: Celecoxib had the antiproliferative and pro-apoptotic effect in Hep-2 cells. Apoptosis was accompanied by a decrease in telomerase activity which was directly correlated with hTERT mRNA and up-regulation of Bax/Bcl-2. Bcl-2 may thus play an important role in telomerase activity as well as apoptosis.
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제안 방법
PCR was carried out as follows: primer elongation (30 min, 25°C), telomerase inactivation (5 min, 94°C), and 30 cycles of amplification (94°C for 30 s, 50°C for 30 s, 72°C for 90 s).
대상 데이터
Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime, China) was used. Hep-2 cells (2×105) were cultured in a 6-well plate for 24 h prior to treatment.
데이터처리
All data were analyzed by the Student’s t-test.
이론/모형
Telomerase activity was determined by the telomeric repeat amplification protocol (TRAP assay) using the kit TeloTAGGG Telomerase PCR ELISA PLUS (Roche, Germany) according to manufacturer’s instructions.
The effect of celecoxib on cell proliferation was determined with the colorimetric MTT assay. Briefly, the cell suspension at an initial density of 8×104 cells/ml was seeded into 96-well plates and allowed to incubate for 24 h.
성능/효과
Hep-2 cells were treated with celecoxib at 50μM for 0, 3, 6, 12, 24 and 48 h, respectivel. A) hTR, hTEP1 and hTERT mRNA expression was decreased by RT-PCR. B)Bcl-2 and Bax protein expression was decreased by western blot
And telomerase activity may be involved in the regulated process of apoptosis. In the this study, concurrent suppression of hTERT mRNA, bcl-2 protein expression and telomerase activity was observed 6 h after celecoxib treatment, while the apoptotic cells did not detectable 6 h after celecoxib treatment, suggesting that the down-regulation of bcl-2 and telomerase may play a causative role in celecoxib-induced apoptosis. More over, it has been reported that the overexpression of Bcl-2 in human cancer cells resulted in increased telomerase activity and a resistance to apoptosis, indicating a link between Bcl-2 expression and telomerase activity in human cancer cells (Fu et al.
On the other hand, Hep-2 cells were treated with 50μM celecoxib for 24h. Result showed the TUNEL-positive cells appeared to be towards more as celecoxib treated hours increased. And all cells were stained with Hoechst 33342 as the total control (Figure 2B).
To determine whether hTR, hTEP1, and hTERT are involved in the mechanism of the inhibition of telomerase activity by celecoxib, those mRNA expressions were were measured by RT-PCR. We found that celecoxib did not inhibit expression of both hTR and hTEP-1 mRNA compared with control.
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