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Guggulsterone targets smokeless tobacco induced PI3K/Akt pathway in head and neck cancer cells.

Macha MA, Matta A, Chauhan SS, Siu KW, Ralhan R - PLoS ONE (2011)

Bottom Line: Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins--Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway.In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST/nicotine on PI3K/Akt pathway in head and neck cancer cells.These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST/nicotine associated head and neck cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

ABSTRACT

Background: Epidemiological association of head and neck cancer with smokeless tobacco (ST) emphasizes the need to unravel the molecular mechanisms implicated in cancer development, and identify pharmacologically safe agents for early intervention and prevention of disease recurrence. Guggulsterone (GS), a biosafe nutraceutical, inhibits the PI3K/Akt pathway that plays a critical role in HNSCC development. However, the potential of GS to suppress ST and nicotine (major component of ST) induced HNSCC remains unexplored. We hypothesized GS can abrogate the effects of ST and nicotine on apoptosis in HNSCC cells, in part by activation of PI3K/Akt pathway and its downstream targets, Bax and Bad.

Methods and results: Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins--Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway. ST and nicotine treatment also resulted in induction of Bad and Bax phosphorylation, increased the association of Bad with 14-3-3ζresulting in its sequestration in the cytoplasm of head and neck cancer cells, thus blocking its pro-apoptotic function. Notably, GS pre-treatment inhibited ST/nicotine induced activation of PI3K/Akt pathway, and inhibited the Akt mediated phosphorylation of Bax and Bad.

Conclusions: In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST/nicotine on PI3K/Akt pathway in head and neck cancer cells. These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST/nicotine associated head and neck cancer.

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Guggulsterone and PI3K-specific inhibitor LY294002 block ST and nicotine induced Bad and Bax phosphorylation.SCC4 cells (2–3×106) were treated with (A) ST (20 µg/ml) or (B) Nicotine (10 µM) for the indicated time intervals. SCC4 cells were pre-treated with LY294002 (10 µM) for 60 min, or 50 µM GS for 4 h, followed by ST (20 µg/ml) for 4 h, or with nicotine (10 µM) for 4 h, and whole-cell extracts were prepared. Sixty microgram proteins from whole-cell extracts were resolved on 10% SDS-PAGE, electrotransferred to a PVDF membrane followed by blocking with 5% non-fat milk overnight. Protein expression was determined using enhanced chemiluminescence method and probed by antibodies against pBad and pBax. The blots were stripped and reprobed for Bax and Bad proteins.
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pone-0014728-g004: Guggulsterone and PI3K-specific inhibitor LY294002 block ST and nicotine induced Bad and Bax phosphorylation.SCC4 cells (2–3×106) were treated with (A) ST (20 µg/ml) or (B) Nicotine (10 µM) for the indicated time intervals. SCC4 cells were pre-treated with LY294002 (10 µM) for 60 min, or 50 µM GS for 4 h, followed by ST (20 µg/ml) for 4 h, or with nicotine (10 µM) for 4 h, and whole-cell extracts were prepared. Sixty microgram proteins from whole-cell extracts were resolved on 10% SDS-PAGE, electrotransferred to a PVDF membrane followed by blocking with 5% non-fat milk overnight. Protein expression was determined using enhanced chemiluminescence method and probed by antibodies against pBad and pBax. The blots were stripped and reprobed for Bax and Bad proteins.

Mentions: Both ST and nicotine potently stimulated serine phosphorylation of Bax and Bad in a time dependent manner (Figure 4A and 4B) resulting in abrogation of their pro-apoptotic function. To demonstrate a functional role of Akt as the physiological ST and nicotine activated Bax and Bad kinase, LY294002, a PI3K specific inhibitor that can block the PI3K/Akt signaling pathway, was used. SCC4 cells were pre-treated with LY294002 (10 µM) for 60 min or 50 µM GS for 4 h followed by ST (20 µg/ml) for 6 h or with nicotine (10 µM) for 4 h. GS as well as LY294002 blocked ST and nicotine induced Bax and Bad phosphorylation (Figure 4A and 4B). No effect on expression of total Bad and Bax was observed at these time points. These findings suggest that inhibition of ST and nicotine induced Bax and Bad phosphorylation by GS may restore the pro-apoptotic function of these molecules.


Guggulsterone targets smokeless tobacco induced PI3K/Akt pathway in head and neck cancer cells.

Macha MA, Matta A, Chauhan SS, Siu KW, Ralhan R - PLoS ONE (2011)

Guggulsterone and PI3K-specific inhibitor LY294002 block ST and nicotine induced Bad and Bax phosphorylation.SCC4 cells (2–3×106) were treated with (A) ST (20 µg/ml) or (B) Nicotine (10 µM) for the indicated time intervals. SCC4 cells were pre-treated with LY294002 (10 µM) for 60 min, or 50 µM GS for 4 h, followed by ST (20 µg/ml) for 4 h, or with nicotine (10 µM) for 4 h, and whole-cell extracts were prepared. Sixty microgram proteins from whole-cell extracts were resolved on 10% SDS-PAGE, electrotransferred to a PVDF membrane followed by blocking with 5% non-fat milk overnight. Protein expression was determined using enhanced chemiluminescence method and probed by antibodies against pBad and pBax. The blots were stripped and reprobed for Bax and Bad proteins.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044714&req=5

pone-0014728-g004: Guggulsterone and PI3K-specific inhibitor LY294002 block ST and nicotine induced Bad and Bax phosphorylation.SCC4 cells (2–3×106) were treated with (A) ST (20 µg/ml) or (B) Nicotine (10 µM) for the indicated time intervals. SCC4 cells were pre-treated with LY294002 (10 µM) for 60 min, or 50 µM GS for 4 h, followed by ST (20 µg/ml) for 4 h, or with nicotine (10 µM) for 4 h, and whole-cell extracts were prepared. Sixty microgram proteins from whole-cell extracts were resolved on 10% SDS-PAGE, electrotransferred to a PVDF membrane followed by blocking with 5% non-fat milk overnight. Protein expression was determined using enhanced chemiluminescence method and probed by antibodies against pBad and pBax. The blots were stripped and reprobed for Bax and Bad proteins.
Mentions: Both ST and nicotine potently stimulated serine phosphorylation of Bax and Bad in a time dependent manner (Figure 4A and 4B) resulting in abrogation of their pro-apoptotic function. To demonstrate a functional role of Akt as the physiological ST and nicotine activated Bax and Bad kinase, LY294002, a PI3K specific inhibitor that can block the PI3K/Akt signaling pathway, was used. SCC4 cells were pre-treated with LY294002 (10 µM) for 60 min or 50 µM GS for 4 h followed by ST (20 µg/ml) for 6 h or with nicotine (10 µM) for 4 h. GS as well as LY294002 blocked ST and nicotine induced Bax and Bad phosphorylation (Figure 4A and 4B). No effect on expression of total Bad and Bax was observed at these time points. These findings suggest that inhibition of ST and nicotine induced Bax and Bad phosphorylation by GS may restore the pro-apoptotic function of these molecules.

Bottom Line: Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins--Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway.In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST/nicotine on PI3K/Akt pathway in head and neck cancer cells.These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST/nicotine associated head and neck cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India.

ABSTRACT

Background: Epidemiological association of head and neck cancer with smokeless tobacco (ST) emphasizes the need to unravel the molecular mechanisms implicated in cancer development, and identify pharmacologically safe agents for early intervention and prevention of disease recurrence. Guggulsterone (GS), a biosafe nutraceutical, inhibits the PI3K/Akt pathway that plays a critical role in HNSCC development. However, the potential of GS to suppress ST and nicotine (major component of ST) induced HNSCC remains unexplored. We hypothesized GS can abrogate the effects of ST and nicotine on apoptosis in HNSCC cells, in part by activation of PI3K/Akt pathway and its downstream targets, Bax and Bad.

Methods and results: Our results showed ST and nicotine treatment resulted in activation of PI3K, PDK1, Akt, and its downstream proteins--Raf, GSK3β and pS6 while GS induced a time dependent decrease in activation of PI3K/Akt pathway. ST and nicotine treatment also resulted in induction of Bad and Bax phosphorylation, increased the association of Bad with 14-3-3ζresulting in its sequestration in the cytoplasm of head and neck cancer cells, thus blocking its pro-apoptotic function. Notably, GS pre-treatment inhibited ST/nicotine induced activation of PI3K/Akt pathway, and inhibited the Akt mediated phosphorylation of Bax and Bad.

Conclusions: In conclusion, GS treatment not only inhibited proliferation, but also induced apoptosis by abrogating the effects of ST/nicotine on PI3K/Akt pathway in head and neck cancer cells. These findings provide a rationale for designing future studies to evaluate the chemopreventive potential of GS in ST/nicotine associated head and neck cancer.

Show MeSH
Related in: MedlinePlus