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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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In vitro efficacy of P276-00 caused by G1 arrest in HNSCC cells (a) Anti-proliferative effect of P276-00 on HNSCC cells using CCK-8 assay. Cells (Detroite-562, filled circles; SCC25, filled triangles, and FaDu, filled boxes) were treated to 5 different concentrations of P276-00 (range: 0.1-10 μM) and measured after 48 hours. Here data is expressed in % cytotoxicity mean ± S.E.M. (n = 3). The IC50 of each cell line was calculated. (b) Flow cytometric analysis of asynchronous population of FaDu cells exposed to either P276-00 (1 and 3 μM) or DMSO for different time points (12, 16, 24, 48 and 72 hours). Histograms of cellular DNA content obtained by flow cytometry have been represented. The (%) DNA content for each phase of the cell cycle is indicated, which was determined by ModFit analysis.
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Figure 1: In vitro efficacy of P276-00 caused by G1 arrest in HNSCC cells (a) Anti-proliferative effect of P276-00 on HNSCC cells using CCK-8 assay. Cells (Detroite-562, filled circles; SCC25, filled triangles, and FaDu, filled boxes) were treated to 5 different concentrations of P276-00 (range: 0.1-10 μM) and measured after 48 hours. Here data is expressed in % cytotoxicity mean ± S.E.M. (n = 3). The IC50 of each cell line was calculated. (b) Flow cytometric analysis of asynchronous population of FaDu cells exposed to either P276-00 (1 and 3 μM) or DMSO for different time points (12, 16, 24, 48 and 72 hours). Histograms of cellular DNA content obtained by flow cytometry have been represented. The (%) DNA content for each phase of the cell cycle is indicated, which was determined by ModFit analysis.

Mentions: Anti-cancer properties of P276-00 were tested across multiple HNSCC cell lines (FaDu, Detroits-562, SCC-25) by measuring dehydrogenase based tetrazolium salt reduction produced by viable cells (Figure 1a). The results showed significant inhibition of cell viability for all three cell lines with IC50 value ranging from 0.8 to 1.7 μM depending on specific cell lines.


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)

In vitro efficacy of P276-00 caused by G1 arrest in HNSCC cells (a) Anti-proliferative effect of P276-00 on HNSCC cells using CCK-8 assay. Cells (Detroite-562, filled circles; SCC25, filled triangles, and FaDu, filled boxes) were treated to 5 different concentrations of P276-00 (range: 0.1-10 μM) and measured after 48 hours. Here data is expressed in % cytotoxicity mean ± S.E.M. (n = 3). The IC50 of each cell line was calculated. (b) Flow cytometric analysis of asynchronous population of FaDu cells exposed to either P276-00 (1 and 3 μM) or DMSO for different time points (12, 16, 24, 48 and 72 hours). Histograms of cellular DNA content obtained by flow cytometry have been represented. The (%) DNA content for each phase of the cell cycle is indicated, which was determined by ModFit analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: In vitro efficacy of P276-00 caused by G1 arrest in HNSCC cells (a) Anti-proliferative effect of P276-00 on HNSCC cells using CCK-8 assay. Cells (Detroite-562, filled circles; SCC25, filled triangles, and FaDu, filled boxes) were treated to 5 different concentrations of P276-00 (range: 0.1-10 μM) and measured after 48 hours. Here data is expressed in % cytotoxicity mean ± S.E.M. (n = 3). The IC50 of each cell line was calculated. (b) Flow cytometric analysis of asynchronous population of FaDu cells exposed to either P276-00 (1 and 3 μM) or DMSO for different time points (12, 16, 24, 48 and 72 hours). Histograms of cellular DNA content obtained by flow cytometry have been represented. The (%) DNA content for each phase of the cell cycle is indicated, which was determined by ModFit analysis.
Mentions: Anti-cancer properties of P276-00 were tested across multiple HNSCC cell lines (FaDu, Detroits-562, SCC-25) by measuring dehydrogenase based tetrazolium salt reduction produced by viable cells (Figure 1a). The results showed significant inhibition of cell viability for all three cell lines with IC50 value ranging from 0.8 to 1.7 μM depending on specific cell lines.

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