14-3-3 zeta is a molecular target in guggulsterone induced apoptosis in head and neck cancer cells.
BACKGROUND
The five-year survival rates for head and neck squamous cell carcinoma (HNSCC) patients are less than 50%, and the prognosis has not improved, despite advancements in standard multi-modality therapies. Hence major emphasis is being laid on identification of novel molecular targets and development of multi-targeted therapies. 14-3-3 zeta, a multifunctional phospho-serine/phospho-threonine binding protein, is emerging as an effector of pro-survival signaling by binding to several proteins involved in apoptosis (Bad, FKHRL1 and ASK1) and may serve as an appropriate target for head and neck cancer therapy. Herein, we determined effect of guggulsterone (GS), a farnesoid X receptor antagonist, on 14-3-3 zeta associated molecular pathways for abrogation of apoptosis in head and neck cancer cells.


METHODS
Head and neck cancer cells were treated with guggulsterone (GS). Effect of GS-treatment was evaluated using cell viability (MTT) assay and apoptosis was verified by annexin V, DNA fragmentation and M30 CytoDeath antibody assay. Mechanism of GS-induced apoptosis was determined by western blotting and co-IP assays using specific antibodies.


RESULTS
Using in vitro models of head and neck cancer, we showed 14-3-3 zeta as a key player regulating apoptosis in GS treated SCC4 cells. Treatment with GS releases BAD from the inhibitory action of 14-3-3 zeta in proliferating HNSCC cells by activating protein phosphatase 2A (PP2A). These events initiate the intrinsic mitochondrial pathway of apoptosis, as revealed by increased levels of cytochrome c in cytoplasmic extracts of GS-treated SCC4 cells. In addition, GS treatment significantly reduced the expression of anti-apoptotic proteins, Bcl-2, xIAP, Mcl1, survivin, cyclin D1 and c-myc, thus committing cells to apoptosis. These events were followed by activation of caspase 9, caspase 8 and caspase 3 leading to cleavage of its downstream target, poly-ADP-ribose phosphate (PARP).


CONCLUSIONS
GS targets 14-3-3 zeta associated cellular pathways for reducing proliferation and inducing apoptosis in head and neck cancer cells, warranting its investigation for use in treatment of head and neck cancer.
1Department of Biochemistry, All India Institute of Medical Sciences, New Delhi-110029, India.
Muzafar A
MA
Macha
Ajay
A
Matta
Ss
S
Chauhan
Kw Michael
KM
Siu
Ranju
R
Ralhan
D048948:14-3-3 Proteins / Q000378:metabolism*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000378:metabolism* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006258:Head and Neck Neoplasms / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D047468:Immunoprecipitation; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011282:Pregnenediones / Q000494:pharmacology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000378:metabolism; D013997:Time Factors; D051025:bcl-Associated Death Protein / Q000378:metabolism; D000099150:Receptor, Farnesoid X-Activated
D048948:14-3-3 Proteins; D000972:Antineoplastic Agents, Phytogenic; D020169:Caspases; D018797:Cell Cycle Proteins; D011282:Pregnenediones; D018160:Receptors, Cytoplasmic and Nuclear; D051025:bcl-Associated Death Protein; D000099150:Receptor, Farnesoid X-Activated; C494819:BAD protein, human; C023617:pregna-4,17-diene-3,16-dione