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Triptolide-Mediated Apoptosis by Suppression of Focal Adhesion Kinase through Extrinsic and Intrinsic Pathways in Human Melanoma Cells.

Kwon HY, Kim KS, Baik JS, Moon HI, Lee JW, Kim CH, Cho YS, Jeong YK, Lee YC - Evid Based Complement Alternat Med (2013)

Bottom Line: TPL increased the levels of Fas and Fas-associated death domain (FADD) and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3.Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK) and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression.We also found that TPL mediated the dissociation of receptor-interacting protein (RIP) from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death.

View Article: PubMed Central - PubMed

Affiliation: College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea ; Medi-Farm Industrialization Research Center, Dong-A University, Busan 604-714, Republic of Korea.

ABSTRACT
Triptolide (TPL) has been shown to inhibit cell proliferation and induce apoptosis in various human cancer cells; however, the precise mechanism of apoptosis induced by TPL in human melanoma cells has not yet been elucidated. In this study, we investigated the precise mechanism underlying cytocidal effects of TPL on human melanoma cells. Treatment of human melanoma cells with TPL significantly inhibited cell growth and induced apoptosis, as evidenced by flow cytometry and annexin V-fluorescein isothiocyanate analyses. TPL increased the levels of Fas and Fas-associated death domain (FADD) and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3. Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK) and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression. We also found that TPL mediated the dissociation of receptor-interacting protein (RIP) from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death. In conclusion, our data firstly demonstrated that TPL induces apoptosis by both extrinsic and intrinsic apoptosis pathways in human melanoma cells and identified that RIP shuttles between Fas and FAK to mediate apoptosis.

No MeSH data available.


Related in: MedlinePlus

Effects of TPL on caspases activation in SK-MEL-2 cells. Human SK-MEL-2 cells were treated with 0, 30, 50, and 70 nM of TPL for 24 h, and then cells were collected. (a) Cell lysates were subjected to Western blotting analysis to detect caspase-8, -9, and -3 and PARP. (b and c) Effects of TPL on protein expressions related to apoptosis through Fas death receptor. (b) The cell lysates were subjected to Western blot analysis. Each protein was detected using Fas, FasL, FADD and Bid antibodies. (c) Total RNA was extracted in SK-MEL2 cells treated with TPL. The mRNA levels of Fas receptor and Fas ligand were examined by RT-PCR. (d and e) Effects of TPL on protein expressions related to apoptosis through mitochondria. (d) Each protein was detected using Bcl-2, Bax. (e) The cytosolic and mitochondrial fraction proteins were collected and then detected using cytochrome c antibody. GAPDH and β-actin were used as internal controls.
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fig3: Effects of TPL on caspases activation in SK-MEL-2 cells. Human SK-MEL-2 cells were treated with 0, 30, 50, and 70 nM of TPL for 24 h, and then cells were collected. (a) Cell lysates were subjected to Western blotting analysis to detect caspase-8, -9, and -3 and PARP. (b and c) Effects of TPL on protein expressions related to apoptosis through Fas death receptor. (b) The cell lysates were subjected to Western blot analysis. Each protein was detected using Fas, FasL, FADD and Bid antibodies. (c) Total RNA was extracted in SK-MEL2 cells treated with TPL. The mRNA levels of Fas receptor and Fas ligand were examined by RT-PCR. (d and e) Effects of TPL on protein expressions related to apoptosis through mitochondria. (d) Each protein was detected using Bcl-2, Bax. (e) The cytosolic and mitochondrial fraction proteins were collected and then detected using cytochrome c antibody. GAPDH and β-actin were used as internal controls.

Mentions: It is known that TPL induces cancer cell apoptosis directly via the mitochondrial (intrinsic) and/or the death receptor-mediated (extrinsic) pathways [9]. Caspase-8 and -9 play important roles in induction of apoptosis through intrinsic and extrinsic pathways, respectively [22]. To clarify the pathway by which TPL induces apoptosis in SK-MEL-2 cells, protein levels of caspase-8 and caspase-9 were analyzed by Western blot analysis after the cells were exposed to the indicated concentration of TPL for 24 h. As shown in Figure 3(a), protein levels of both pro-caspase-8 and pro-caspase-9 were decreased in dose-dependent manner, whereas those of their cleaved forms were increased, indicating activation of both caspases by TPL. Caspase-3 is activated through cleavage into two smaller subunits by caspase-8 or caspase-9 when the cells undergo apoptosis and cleaves a number of proteins that are essential for cell survival, such as PARP [22]. TPL also caused the activation of caspase-3 and subsequent cleavage of PARP. This result indicates that TPL-induced apoptosis in SK-MEL-2 cells was mediated by activations of caspase-3, -8, and -9, suggesting that TPL affects two pathways. Therefore, we checked downstream of extrinsic and intrinsic pathways in TPL-treated cells. In the extrinsic pathway, Fas aggregation is known to recruit FADD protein to the plasma membrane, which in turn activates pro-caspase-8 [9]. To verify TPL-mediated apoptosis through extrinsic pathway in SK-MEL-2 cells, levels of Fas, FasL, and FADD were investigated. As shown in Figures 3(b) and 3(c), the expression levels of Fas and FADD were increased in TPL-treated cells, whereas FasL showed no appreciable change in the expression levels of mRNA and protein. This result suggests that TPL-induced Fas aggregation is independent of its ligand. This result suggests that TPL-induced Fas aggregation is independent of its ligand. Moreover, the level of Bid was decreased, which presumably resulted in production of truncated Bid form (tBid). Bid is cleaved by active caspase-8, resulting in generating tBid, and translocates to the mitochondria and then enhances cytochrome c release by its interaction with Bax or Bak [22]. Thus, this result suggests that TPL-induced apoptosis in SK-MEL-2 cells can be mediated through intrinsic pathway via extrinsic pathway mediated by tBid. To confirm TPL-mediated apoptosis through intrinsic pathway in SK-MEL-2 cells, levels of Bcl-2, Bax, and cytochrome c were investigated. As shown in Figure 3(d), the level of Bcl-2, antiapoptotic protein, was decreased, whereas the level of Bax, proapoptotic protein, was increased after TPL exposure. Furthermore, cytochrome c was released from mitochondria to cytosol in a dose-dependent manner by TPL treatment (Figure 3(e)). This result indicates that TPL-induced apoptosis in SK-MEL-2 cells was mediated through intrinsic (mitochondrial-mediated) pathway.


Triptolide-Mediated Apoptosis by Suppression of Focal Adhesion Kinase through Extrinsic and Intrinsic Pathways in Human Melanoma Cells.

Kwon HY, Kim KS, Baik JS, Moon HI, Lee JW, Kim CH, Cho YS, Jeong YK, Lee YC - Evid Based Complement Alternat Med (2013)

Effects of TPL on caspases activation in SK-MEL-2 cells. Human SK-MEL-2 cells were treated with 0, 30, 50, and 70 nM of TPL for 24 h, and then cells were collected. (a) Cell lysates were subjected to Western blotting analysis to detect caspase-8, -9, and -3 and PARP. (b and c) Effects of TPL on protein expressions related to apoptosis through Fas death receptor. (b) The cell lysates were subjected to Western blot analysis. Each protein was detected using Fas, FasL, FADD and Bid antibodies. (c) Total RNA was extracted in SK-MEL2 cells treated with TPL. The mRNA levels of Fas receptor and Fas ligand were examined by RT-PCR. (d and e) Effects of TPL on protein expressions related to apoptosis through mitochondria. (d) Each protein was detected using Bcl-2, Bax. (e) The cytosolic and mitochondrial fraction proteins were collected and then detected using cytochrome c antibody. GAPDH and β-actin were used as internal controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Effects of TPL on caspases activation in SK-MEL-2 cells. Human SK-MEL-2 cells were treated with 0, 30, 50, and 70 nM of TPL for 24 h, and then cells were collected. (a) Cell lysates were subjected to Western blotting analysis to detect caspase-8, -9, and -3 and PARP. (b and c) Effects of TPL on protein expressions related to apoptosis through Fas death receptor. (b) The cell lysates were subjected to Western blot analysis. Each protein was detected using Fas, FasL, FADD and Bid antibodies. (c) Total RNA was extracted in SK-MEL2 cells treated with TPL. The mRNA levels of Fas receptor and Fas ligand were examined by RT-PCR. (d and e) Effects of TPL on protein expressions related to apoptosis through mitochondria. (d) Each protein was detected using Bcl-2, Bax. (e) The cytosolic and mitochondrial fraction proteins were collected and then detected using cytochrome c antibody. GAPDH and β-actin were used as internal controls.
Mentions: It is known that TPL induces cancer cell apoptosis directly via the mitochondrial (intrinsic) and/or the death receptor-mediated (extrinsic) pathways [9]. Caspase-8 and -9 play important roles in induction of apoptosis through intrinsic and extrinsic pathways, respectively [22]. To clarify the pathway by which TPL induces apoptosis in SK-MEL-2 cells, protein levels of caspase-8 and caspase-9 were analyzed by Western blot analysis after the cells were exposed to the indicated concentration of TPL for 24 h. As shown in Figure 3(a), protein levels of both pro-caspase-8 and pro-caspase-9 were decreased in dose-dependent manner, whereas those of their cleaved forms were increased, indicating activation of both caspases by TPL. Caspase-3 is activated through cleavage into two smaller subunits by caspase-8 or caspase-9 when the cells undergo apoptosis and cleaves a number of proteins that are essential for cell survival, such as PARP [22]. TPL also caused the activation of caspase-3 and subsequent cleavage of PARP. This result indicates that TPL-induced apoptosis in SK-MEL-2 cells was mediated by activations of caspase-3, -8, and -9, suggesting that TPL affects two pathways. Therefore, we checked downstream of extrinsic and intrinsic pathways in TPL-treated cells. In the extrinsic pathway, Fas aggregation is known to recruit FADD protein to the plasma membrane, which in turn activates pro-caspase-8 [9]. To verify TPL-mediated apoptosis through extrinsic pathway in SK-MEL-2 cells, levels of Fas, FasL, and FADD were investigated. As shown in Figures 3(b) and 3(c), the expression levels of Fas and FADD were increased in TPL-treated cells, whereas FasL showed no appreciable change in the expression levels of mRNA and protein. This result suggests that TPL-induced Fas aggregation is independent of its ligand. This result suggests that TPL-induced Fas aggregation is independent of its ligand. Moreover, the level of Bid was decreased, which presumably resulted in production of truncated Bid form (tBid). Bid is cleaved by active caspase-8, resulting in generating tBid, and translocates to the mitochondria and then enhances cytochrome c release by its interaction with Bax or Bak [22]. Thus, this result suggests that TPL-induced apoptosis in SK-MEL-2 cells can be mediated through intrinsic pathway via extrinsic pathway mediated by tBid. To confirm TPL-mediated apoptosis through intrinsic pathway in SK-MEL-2 cells, levels of Bcl-2, Bax, and cytochrome c were investigated. As shown in Figure 3(d), the level of Bcl-2, antiapoptotic protein, was decreased, whereas the level of Bax, proapoptotic protein, was increased after TPL exposure. Furthermore, cytochrome c was released from mitochondria to cytosol in a dose-dependent manner by TPL treatment (Figure 3(e)). This result indicates that TPL-induced apoptosis in SK-MEL-2 cells was mediated through intrinsic (mitochondrial-mediated) pathway.

Bottom Line: TPL increased the levels of Fas and Fas-associated death domain (FADD) and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3.Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK) and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression.We also found that TPL mediated the dissociation of receptor-interacting protein (RIP) from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death.

View Article: PubMed Central - PubMed

Affiliation: College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea ; Medi-Farm Industrialization Research Center, Dong-A University, Busan 604-714, Republic of Korea.

ABSTRACT
Triptolide (TPL) has been shown to inhibit cell proliferation and induce apoptosis in various human cancer cells; however, the precise mechanism of apoptosis induced by TPL in human melanoma cells has not yet been elucidated. In this study, we investigated the precise mechanism underlying cytocidal effects of TPL on human melanoma cells. Treatment of human melanoma cells with TPL significantly inhibited cell growth and induced apoptosis, as evidenced by flow cytometry and annexin V-fluorescein isothiocyanate analyses. TPL increased the levels of Fas and Fas-associated death domain (FADD) and induced cleavage of Bid by activation of caspase-8 and cytochrome c release from mitochondria to the cytosol, which resulted in activation of caspase-9 and caspase-3. Moreover, TPL-induced apoptosis in SK-MEL-2 cells was mediated through dephosphorylation of focal adhesion kinase (FAK) and its cleavage by caspase-8-mediated caspase-3 activation via upregulation of Fas expression. We also found that TPL mediated the dissociation of receptor-interacting protein (RIP) from FAK and enhanced the formation of RIP/Fas complex formation initiating cell death. In conclusion, our data firstly demonstrated that TPL induces apoptosis by both extrinsic and intrinsic apoptosis pathways in human melanoma cells and identified that RIP shuttles between Fas and FAK to mediate apoptosis.

No MeSH data available.


Related in: MedlinePlus