Quercetin abrogates chemoresistance in melanoma cells by modulating deltaNp73.
BACKGROUND
The alkylating agent dacarbazine (DTIC) has been used in the treatment of melanoma for decades, but when used as a monotherapy for cancer only moderate response rates are achieved. Recently, the clinical use of temozolomide (TMZ) has become the more commonly used analog of DTIC-related oral agents because of its greater bioavailability and ability to cross the blood brain barrier. The response rates achieved by TMZ are also unsatisfactory, so there is great interest in identifying compounds that could be used in combination therapy. We have previously demonstrated that the bioflavonoid quercetin (Qct) promoted a p53-mediated response and sensitized melanoma to DTIC. Here we demonstrate that Qct also sensitizes cells to TMZ and propose a mechanism that involves the modulation of a truncated p53 family member, deltaNp73.


METHODS
DB-1 melanoma (p53 wildtype), and SK Mel 28 (p53 mutant) cell lines were treated with TMZ (400 microM) for 48 hrs followed by Qct (75 microM) for 24 hrs. Cell death was determined by Annexin V-FITC staining and immunocytochemical analysis was carried out to determine protein translocation.


RESULTS
After treatment with TMZ, DB-1 cells demonstrated increased phosphorylation of ataxia telangiectasia mutated (ATM) and p53. However, the cells were resistant to TMZ-induced apoptosis and the resistance was associated with an increase in nuclear localization of deltaNp73. Qct treatment in combination with TMZ abolished drug insensitivity and caused a more than additive induction of apoptosis compared to either treatment alone. Treatment with Qct, caused redistribution of deltaNp73 into the cytoplasm and nucleus, which has been associated with increased p53 transcriptional activity. Knockdown of deltaNp73 restored PARP cleavage in the TMZ treated cells, confirming its anti-apoptotic role. The response to treatment was predominantly p53 mediated as the p53 mutant SK Mel 28 cells showed no significant enhancement of apoptosis.


CONCLUSIONS
This study demonstrates that Qct can sensitize cells to TMZ and that the mechanisms of sensitization involve modulation of p53 family members.
Thangasamy
Thilakavathy
T
Department of Nutritional Sciences, University of Arizona, Tucson, AZ 85721, USA.
Sittadjody
Sivanandane
S
Mitchell
Geoffrey C
GC
Mendoza
Erin E
EE
Radhakrishnan
Vijayababu M
VM
Limesand
Kirsten H
KH
Burd
Randy
R
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D006801:Humans; D008543:Melanins / Q000096:biosynthesis; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D011794:Quercetin / Q000494:pharmacology*; D034622:RNA Interference; D000077204:Temozolomide; D013997:Time Factors; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D008543:Melanins; D009687:Nuclear Proteins; C495901:TP53 protein, human; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C000606369:delta Np73 protein, human; D003606:Dacarbazine; D011794:Quercetin; D011065:Poly(ADP-ribose) Polymerases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000077204:Temozolomide