Tao, Yue
(Institute of Dermatology, Chinese Academy of Medical Science and Peking Union Medical College)
,
Zhang, Meng-Li
(Institute of Dermatology, Chinese Academy of Medical Science and Peking Union Medical College)
,
Ma, Peng-Cheng
(Institute of Dermatology, Chinese Academy of Medical Science and Peking Union Medical College)
,
Sun, Jian-Fang
(Institute of Dermatology, Chinese Academy of Medical Science and Peking Union Medical College)
,
Zhou, Wu-Qing
(Institute of Dermatology, Chinese Academy of Medical Science and Peking Union Medical College)
,
Cao, Yu-Ping
(Institute of Dermatology, Chinese Academy of Medical Science and Peking Union Medical College)
,
Li, Ling-Jun
(Institute of Dermatology, Chinese Academy of Medical Science and Peking Union Medical College)
Triptolide, a diterpenoid obtained from Tripteryglum wilfordii Hook.f, has attracted interest for its antitumor activities against human tumor cell lines in recent years. This report focuses on anti-proliferative and pro-apoptotic activities in human melanoma A375 cells assessed by CCK8 assay, Hoech...
Triptolide, a diterpenoid obtained from Tripteryglum wilfordii Hook.f, has attracted interest for its antitumor activities against human tumor cell lines in recent years. This report focuses on anti-proliferative and pro-apoptotic activities in human melanoma A375 cells assessed by CCK8 assay, Hoechst 33258 staining and flow cytometry. In addition, triptolide-induced arrest in the S phase was also observed. Caspase assays showed the apoptosis induced by triptolide was caspase-dependent and probably through intrinsic apoptotic pathways. Furthermore, expression of NF-${\kappa}B$ (p65) and its downstream factors such as Bcl-2, Bcl-$X_L$ was down-regulated. Taken together, the data indicate that triptolide inhibits A375 cells proliferation and induces apoptosis by a caspase-dependent pathway and through a NF-${\kappa}B$-mediated mechanism.
Triptolide, a diterpenoid obtained from Tripteryglum wilfordii Hook.f, has attracted interest for its antitumor activities against human tumor cell lines in recent years. This report focuses on anti-proliferative and pro-apoptotic activities in human melanoma A375 cells assessed by CCK8 assay, Hoechst 33258 staining and flow cytometry. In addition, triptolide-induced arrest in the S phase was also observed. Caspase assays showed the apoptosis induced by triptolide was caspase-dependent and probably through intrinsic apoptotic pathways. Furthermore, expression of NF-${\kappa}B$ (p65) and its downstream factors such as Bcl-2, Bcl-$X_L$ was down-regulated. Taken together, the data indicate that triptolide inhibits A375 cells proliferation and induces apoptosis by a caspase-dependent pathway and through a NF-${\kappa}B$-mediated mechanism.
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제안 방법
While staining, cells were placed in dark for 30 min at room temperature. Analysis was performed on a FACSCalibur flow cytometer (Becton Dickinson, CA, USA) within 1 h of staining using Cellquest and ModFit software.
Next, we evaluated the activity of caspase-3 and caspase-9 to investigate the possible mechanisms underlying the pro-apoptotic action of triptolide in A375 cells. Caspase-3 and caspase-9 activity was measured at 48 h after treatment with 10, 20, 30 nM of triptolide.
To investigate the inhibitory effect of triptolide on growth and survival of melanoma cells, we treated human melanoma A375 cells with the triptolide concentrations of 12.5, 25, 50, 100 nM for 24, 48 or 72 h, respectively. As shown in Figure 1, triptolide brought about a dosedependent inhibitory effect of cell proliferation with statistical significance (P < 0.
, 2009). To investigate whether this regulatory mechanism is also involved in the process of apoptosis of A375 cells, we evaluated the protein level of the p65 and some of their downregulating genes.
대상 데이터
Human CMM cell line A375 cells were provided by Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College.
Triptolide (purity> 99.0%, Institute of Dermatology, Chinese Academy of Medical Sciences, Nanjing, China) was prepared in dimethyl sulfoxide (DMSO) to obtain 1 mM stock solution and then was added in medium at required concentrations for a certain period of time.
데이터처리
The Statistical Package for Social Sciences (SPSS) 13.0 software was used for Student’s t-test.
성능/효과
In conclusion, the present study demonstrated that human melanoma A375 cells are susceptible to triptolideinduced apoptosis and inhibition of proliferation. Our results indicate that triptolide exert anticancer activity against A375 cells by inducing S cell cycle rest, thereby causing apoptosis, which is mediated in part by inhibition of NF-κB expression and caspases activation.
The results of cell viability assay demonstrated that cell growth inhibitory effect of triptolide was time- and concentration-dependent during a 0-100 nM range in A375 cells. The cell inhibitory rate was increased over gradually increasing time and concentration.
With the increasing dose of triptolide, the early apoptosis rate increased from 19.5±0.32%, 36.6±3.85% and 58.7±2.03% to the exposure of 10, 20 and 30 nM of triptolide, respectively.
후속연구
In view of these data, triptolide may be considered as a potential chemotherapy agent to deal with refractory human malignant melanoma. However, further studies are needed to explore more detailed mechanisms of triptolide on human melanoma cells.
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