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Molecular mechanisms of anti-tumor properties of P276-00 in head and neck squamous cell carcinoma.

Mishra PB, Lobo AS, Joshi KS, Rathos MJ, Kumar GA, Padigaru M - J Transl Med (2013)

Bottom Line: P276-00 treatment suppressed cell proliferation through inhibition of CCND1 expression, reduced phosphorylation of retinoblastoma protein and abrogative transcription of E2F1 gene targets.It was also seen that P276-00 treatment reduced expression of tumor micro-environment proteins such as IL-6, secreted EGFR and HSPA8.Finally, P276-00 treatment resulted in significant tumor growth inhibition in xenograft tumor models via lowered proliferative activity of E2F1 and aggravated P53 mediated apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biomarker Discovery Group, Department of Pharmacology, Piramal Healthcare Ltd, 1-Nirlon Complex, Goregaon-East, Mumbai, Maharashtra 400063, India. prabha.mishra@piramal.com

ABSTRACT

Background: Tumors of the head and neck present aggressive pathological behavior in patients due to high expression of CDK/CCND1 proteins. P276-00, a novel CDK inhibitor currently being tested in clinic, inhibits growth of several cancers in vitro and in vivo. The pre clinical activity of P276-00 in head and neck cancer and its potential mechanisms of action at molecular level are the focus of the current studies.

Method: We have investigated the anti-cancer activity of P276-00 in head and neck tumors in vitro and in vivo. Candidate gene expression profiling and cell based proteomic approaches were taken to understand the pathways affected by P276-00 treatment.

Results: It was observed that P276-00 is cytotoxic across various HNSCC cell lines with an IC₅₀ ranging from 1.0-1.5 μmoles/L and culminated in significant cell-cycle arrest in G1/S phase followed by apoptosis. P276-00 treatment suppressed cell proliferation through inhibition of CCND1 expression, reduced phosphorylation of retinoblastoma protein and abrogative transcription of E2F1 gene targets. Further, we observed that apoptosis was mediated through P53 activation leading to higher BAX/BCL-2 ratio and cleaved caspase-3 levels. It was also seen that P276-00 treatment reduced expression of tumor micro-environment proteins such as IL-6, secreted EGFR and HSPA8. Finally, P276-00 treatment resulted in significant tumor growth inhibition in xenograft tumor models via lowered proliferative activity of E2F1 and aggravated P53 mediated apoptosis.

Conclusion: In summary, we have observed that P276-00 inhibits cyclin-D/CDK4/P16/pRB/E2F axis and induces apoptosis by increased P53 phosphorylation in HNSCC cells. These results suggest a novel indication for P276-00 in head and neck cancer with a potential role for IL-6 and HSPA8 as candidate serum biomarkers.

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Cytotoxic nature of P276-00 is due to phosphorylation of P53 in FaDu cells. (a) Western blot analysis of phosphorylated-P53 in FaDu cells treated with 1 and 3 μM P276-00 for the 24 hours. (b) Dose dependent relative mRNA expression of downstream target of P53 after 24 hours of treatment. Data in histograms represented on log2 scale as mean ± S.E.M. (n = 3). (c-d) Dose dependent treatment of P276-00 (8 different concentrations -range 0.03 to 10 μM) for 24 hours has shown induction of P53 phosphorylation at Ser392 and cleavage of CASP3 in FaDu cells detected by Automated fluorescence imaging (HCS reader) (c) Graphical representation of the data (d) Representative images.
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Figure 3: Cytotoxic nature of P276-00 is due to phosphorylation of P53 in FaDu cells. (a) Western blot analysis of phosphorylated-P53 in FaDu cells treated with 1 and 3 μM P276-00 for the 24 hours. (b) Dose dependent relative mRNA expression of downstream target of P53 after 24 hours of treatment. Data in histograms represented on log2 scale as mean ± S.E.M. (n = 3). (c-d) Dose dependent treatment of P276-00 (8 different concentrations -range 0.03 to 10 μM) for 24 hours has shown induction of P53 phosphorylation at Ser392 and cleavage of CASP3 in FaDu cells detected by Automated fluorescence imaging (HCS reader) (c) Graphical representation of the data (d) Representative images.

Mentions: The extent of apoptosis is maintained by a well regulated balance between pro- and anti-apoptotic factors. In this regard, we demonstrated that P276-00 induced the expression of proapoptotic gene BAX at 12 hours of treatment and attenuated expression of anti-apoptotic genes BCL2 and MCL1 (Figure 3b). Cleaved CASP3, critical for induction of apoptosis, was also elevated in response to P276-00 in a dose-dependent manner (Figure 3c, 3d). To further probe the apoptotic mechanism of P276-00, we observed that P53 phosphorylation was significantly elevated in vitro in response to P276-00 (Figure 3a, 3c, 3d). This observation is consistent with an increase in BAX and BCL2 genes (Figure 3b), since the latter are P53 target genes as well.


Molecular mechanisms of anti-tumor properties of P276-00 in head and neck squamous cell carcinoma.

Mishra PB, Lobo AS, Joshi KS, Rathos MJ, Kumar GA, Padigaru M - J Transl Med (2013)

Cytotoxic nature of P276-00 is due to phosphorylation of P53 in FaDu cells. (a) Western blot analysis of phosphorylated-P53 in FaDu cells treated with 1 and 3 μM P276-00 for the 24 hours. (b) Dose dependent relative mRNA expression of downstream target of P53 after 24 hours of treatment. Data in histograms represented on log2 scale as mean ± S.E.M. (n = 3). (c-d) Dose dependent treatment of P276-00 (8 different concentrations -range 0.03 to 10 μM) for 24 hours has shown induction of P53 phosphorylation at Ser392 and cleavage of CASP3 in FaDu cells detected by Automated fluorescence imaging (HCS reader) (c) Graphical representation of the data (d) Representative images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672051&req=5

Figure 3: Cytotoxic nature of P276-00 is due to phosphorylation of P53 in FaDu cells. (a) Western blot analysis of phosphorylated-P53 in FaDu cells treated with 1 and 3 μM P276-00 for the 24 hours. (b) Dose dependent relative mRNA expression of downstream target of P53 after 24 hours of treatment. Data in histograms represented on log2 scale as mean ± S.E.M. (n = 3). (c-d) Dose dependent treatment of P276-00 (8 different concentrations -range 0.03 to 10 μM) for 24 hours has shown induction of P53 phosphorylation at Ser392 and cleavage of CASP3 in FaDu cells detected by Automated fluorescence imaging (HCS reader) (c) Graphical representation of the data (d) Representative images.
Mentions: The extent of apoptosis is maintained by a well regulated balance between pro- and anti-apoptotic factors. In this regard, we demonstrated that P276-00 induced the expression of proapoptotic gene BAX at 12 hours of treatment and attenuated expression of anti-apoptotic genes BCL2 and MCL1 (Figure 3b). Cleaved CASP3, critical for induction of apoptosis, was also elevated in response to P276-00 in a dose-dependent manner (Figure 3c, 3d). To further probe the apoptotic mechanism of P276-00, we observed that P53 phosphorylation was significantly elevated in vitro in response to P276-00 (Figure 3a, 3c, 3d). This observation is consistent with an increase in BAX and BCL2 genes (Figure 3b), since the latter are P53 target genes as well.

Bottom Line: P276-00 treatment suppressed cell proliferation through inhibition of CCND1 expression, reduced phosphorylation of retinoblastoma protein and abrogative transcription of E2F1 gene targets.It was also seen that P276-00 treatment reduced expression of tumor micro-environment proteins such as IL-6, secreted EGFR and HSPA8.Finally, P276-00 treatment resulted in significant tumor growth inhibition in xenograft tumor models via lowered proliferative activity of E2F1 and aggravated P53 mediated apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biomarker Discovery Group, Department of Pharmacology, Piramal Healthcare Ltd, 1-Nirlon Complex, Goregaon-East, Mumbai, Maharashtra 400063, India. prabha.mishra@piramal.com

ABSTRACT

Background: Tumors of the head and neck present aggressive pathological behavior in patients due to high expression of CDK/CCND1 proteins. P276-00, a novel CDK inhibitor currently being tested in clinic, inhibits growth of several cancers in vitro and in vivo. The pre clinical activity of P276-00 in head and neck cancer and its potential mechanisms of action at molecular level are the focus of the current studies.

Method: We have investigated the anti-cancer activity of P276-00 in head and neck tumors in vitro and in vivo. Candidate gene expression profiling and cell based proteomic approaches were taken to understand the pathways affected by P276-00 treatment.

Results: It was observed that P276-00 is cytotoxic across various HNSCC cell lines with an IC₅₀ ranging from 1.0-1.5 μmoles/L and culminated in significant cell-cycle arrest in G1/S phase followed by apoptosis. P276-00 treatment suppressed cell proliferation through inhibition of CCND1 expression, reduced phosphorylation of retinoblastoma protein and abrogative transcription of E2F1 gene targets. Further, we observed that apoptosis was mediated through P53 activation leading to higher BAX/BCL-2 ratio and cleaved caspase-3 levels. It was also seen that P276-00 treatment reduced expression of tumor micro-environment proteins such as IL-6, secreted EGFR and HSPA8. Finally, P276-00 treatment resulted in significant tumor growth inhibition in xenograft tumor models via lowered proliferative activity of E2F1 and aggravated P53 mediated apoptosis.

Conclusion: In summary, we have observed that P276-00 inhibits cyclin-D/CDK4/P16/pRB/E2F axis and induces apoptosis by increased P53 phosphorylation in HNSCC cells. These results suggest a novel indication for P276-00 in head and neck cancer with a potential role for IL-6 and HSPA8 as candidate serum biomarkers.

Show MeSH
Related in: MedlinePlus