Restoration of tumor suppressor p53 by differentially regulating pro- and anti-p53 networks in HPV-18-infected cervical cancer cells

Abstract

Abrogation of functional p53 is responsible for malignant cell transformation and maintenance of human papilloma virus (HPV)-infected cancer cells. Restoration of p53 has, therefore, been regarded as an important strategy for molecular intervention of HPV-associated malignancies. Here we report that differential regulation of pro-and anti-p53 setups not only upregulates p53 transcription but also stabilizes and activates p53 protein to ensure p53-induced apoptosis in HPV-18-infected cervical cancer. Functional restoration of p53 can be achieved by nonsteroidal anti-inflammatory drug celecoxib via multiple molecular mechanisms: (i) inhibition of p53 degradation by suppressing viral oncoprotein E6 expression, (ii) promoting p53 transcription by downmodulating cycloxygenase-2 (Cox-2) and simultaneously retrieving p53 from Cox-2 association and (iii) activation of p53 via ataxia telangiectasia mutated-/p38 mitogen-activated protein kinase-mediated phosphorylations at serine-15/-46 residues. That restored p53 is functional has been confirmed by its ability of transactivating Bax and p53-upregulated modulator of apoptosis, which in turn switch on the apoptotic machinery in these cells. Studies undertaken in biopsy samples of cervical carcinoma further validated celecoxib effect. Our approaches involving gene manipulation and pharmacological interference finally highlight that celecoxib alters pro-and anti-p53 networks, not in isolation but in concert, to rejuvenate p53-dependent apoptotic program in HPV-infected cervical cancer cells.

Nuclear Matrix Protein SMAR1 Represses c-Fos-mediated HPV18 E6 Transcription through Alteration of Chromatin Histone Deacetylation

“…The cells were routinely maintained in complete Dulbecco's modified Eagle's medium at 37°C in a humidified incubator containing 5% CO 2 (26). Cells were allowed to reach confluence before use.…”

“…Cells were allowed to reach confluence before use. Viable cell numbers were determined by trypan blue exclusion test (26). Cells were treated with different concentrations of curcumin (Sigma) for different time points to select the optimum dose and time required for cancer cell apoptosis.…”

“…RT-PCR-Two g of the total RNA extracted from cells with TRIzol Q16 reagent (Invitrogen) was reverse transcribed and then subjected to PCR with enzymes and reagents of the RTplusPCR system (Eppendorf, Hamburg, Germany) using GeneAmpPCR 2720 (Applied Biosystems) (26). The cDNAs were amplified with primers specific for BAX (5Ј-TTTGCTTCAGGGTTTCATCC-3Ј/5Ј-CAG-TTGAAGTTGCCGTCAGA-3Ј), E6 (5Ј-AAGCTACCTGATCTG-TGCACGG-3Ј/5Ј-GCTGGATTCAACGGTT-TCTGG-3Ј), c-FOS (5Ј-AGACAGACCAACTAGAAGATGA-3Ј/5Ј-AGCTCTGTGG-CCATGGGCCCC-3Ј), SMAR1 (5Ј-GCATTGAGGCCAAGCTG-AAAGCTC-3Ј/5Ј-CGGAGTTCAGGGTGATGAGTGTGAC-3Ј), PUMA (5Ј-CCACCACCATCTCAGGAAAG-3Ј/5Ј-ACGTTTGG-CTCATTTGCTCT-3Ј), and GAPDH (internal control) (5Ј-CAG AACATCATCCTGCCTCT-3Ј/5Ј-GCTTGACAAAGTGGTCG-TTGAG-3Ј).…”

“…SMAR1 Up-regulates p53-SMAR1 Feedback Loop to Suppress E6 and Mediate Apoptosis-Because E6 mediates the accelerated proteosomal degradation of p53 tumor suppressor (12,26), our next attempt was to check the status of p53 in these HPV18-infected cervical carcinoma cells. Our results indicate that with curcumin-induced up-regulation of SMAR1 (Fig.…”

Sulphur alters NFκB-p300 cross-talk in favour of p53–p300 to induce apoptosis in non-small cell lung carcinoma

“…Therefore, targeting NFκB pathway may serve as a novel approach to regress NSCLC cells. Conversely, tumor suppressor p53, the 'guardian of genome' translates stress signals into cell cycle arrest or apoptosis, depending on the balance between pro-apoptotic and anti-proliferative genes (29)(30)(31). Thus, drugs reviving tumor suppressor functions of p53 will be proficient for targeted cancer therapy.…”