Limits...
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.

Show MeSH

Related in: MedlinePlus

P276-00 targeting CCND1/pRB mediated E2F1 pathway and causing G1 arrest in FaDu cells. (a) Western blot analysis of CCND1, pRb, CCNE1 and E2F1 in FaDU cells treated with 1 and 3 μM P276-00 for 3, 6, 12 and 24 hours.(b-c) RTQPCR based transcript profiling of various downstream targets of E2F1 pathway. Data is represented on log2 scale as mean ± S.E.M. (n=3-5). (b) Dose response - FaDu cells were exposed to 5 different concentrations of P276-00 (range: 0.1-10 μM) for 12 hours in FaDu cells. (c) Time response- cells were exposed to 4 different time points (3, 6, 12 and 24 hours) with 1 μΜ of P276-00 (d-e) Automated fluorescence imaging based dose dependent protein profiling of CCND1/pRB mediated E2F1 pathway and its downstream targets. FaDu cells (1–2 × 104 cells/ml) were treated with 8 different concentrations of P276-00 (range 0.03 to 10 μM) for 24 hours and observed using ArrayScan HCS reader after staining with respective antibody (d) Quantification of the automated fluorescence imaging data has been represented in sigmoidal dose response curve where compound concentration. is represented in log2 scale. One thousand cells were counted for each replicate well and the results were presented as an average ± S.E.M. (n = 3). EC50 values have been generated for all the 5 proteins. (e) Panel of representative images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3672051&req=5

Figure 2: P276-00 targeting CCND1/pRB mediated E2F1 pathway and causing G1 arrest in FaDu cells. (a) Western blot analysis of CCND1, pRb, CCNE1 and E2F1 in FaDU cells treated with 1 and 3 μM P276-00 for 3, 6, 12 and 24 hours.(b-c) RTQPCR based transcript profiling of various downstream targets of E2F1 pathway. Data is represented on log2 scale as mean ± S.E.M. (n=3-5). (b) Dose response - FaDu cells were exposed to 5 different concentrations of P276-00 (range: 0.1-10 μM) for 12 hours in FaDu cells. (c) Time response- cells were exposed to 4 different time points (3, 6, 12 and 24 hours) with 1 μΜ of P276-00 (d-e) Automated fluorescence imaging based dose dependent protein profiling of CCND1/pRB mediated E2F1 pathway and its downstream targets. FaDu cells (1–2 × 104 cells/ml) were treated with 8 different concentrations of P276-00 (range 0.03 to 10 μM) for 24 hours and observed using ArrayScan HCS reader after staining with respective antibody (d) Quantification of the automated fluorescence imaging data has been represented in sigmoidal dose response curve where compound concentration. is represented in log2 scale. One thousand cells were counted for each replicate well and the results were presented as an average ± S.E.M. (n = 3). EC50 values have been generated for all the 5 proteins. (e) Panel of representative images.

Mentions: To establish the anti-proliferative properties of P276-00 via G1 arrest, we have investigated the status of CCND1/pRB mediated E2F1 signaling components in FaDu cells treated with or without P276-00 (Figure 2a). Treatment of FaDu cells with P276-00 showed a reduction in pRB and CCND1 protein levels as early as 6 hours and was maintained upto 24 hours. P276-00 also reduced E2F1 and CCNE1 protein expression by 24 hours (Figure 2a).


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)

P276-00 targeting CCND1/pRB mediated E2F1 pathway and causing G1 arrest in FaDu cells. (a) Western blot analysis of CCND1, pRb, CCNE1 and E2F1 in FaDU cells treated with 1 and 3 μM P276-00 for 3, 6, 12 and 24 hours.(b-c) RTQPCR based transcript profiling of various downstream targets of E2F1 pathway. Data is represented on log2 scale as mean ± S.E.M. (n=3-5). (b) Dose response - FaDu cells were exposed to 5 different concentrations of P276-00 (range: 0.1-10 μM) for 12 hours in FaDu cells. (c) Time response- cells were exposed to 4 different time points (3, 6, 12 and 24 hours) with 1 μΜ of P276-00 (d-e) Automated fluorescence imaging based dose dependent protein profiling of CCND1/pRB mediated E2F1 pathway and its downstream targets. FaDu cells (1–2 × 104 cells/ml) were treated with 8 different concentrations of P276-00 (range 0.03 to 10 μM) for 24 hours and observed using ArrayScan HCS reader after staining with respective antibody (d) Quantification of the automated fluorescence imaging data has been represented in sigmoidal dose response curve where compound concentration. is represented in log2 scale. One thousand cells were counted for each replicate well and the results were presented as an average ± S.E.M. (n = 3). EC50 values have been generated for all the 5 proteins. (e) Panel of representative images.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: P276-00 targeting CCND1/pRB mediated E2F1 pathway and causing G1 arrest in FaDu cells. (a) Western blot analysis of CCND1, pRb, CCNE1 and E2F1 in FaDU cells treated with 1 and 3 μM P276-00 for 3, 6, 12 and 24 hours.(b-c) RTQPCR based transcript profiling of various downstream targets of E2F1 pathway. Data is represented on log2 scale as mean ± S.E.M. (n=3-5). (b) Dose response - FaDu cells were exposed to 5 different concentrations of P276-00 (range: 0.1-10 μM) for 12 hours in FaDu cells. (c) Time response- cells were exposed to 4 different time points (3, 6, 12 and 24 hours) with 1 μΜ of P276-00 (d-e) Automated fluorescence imaging based dose dependent protein profiling of CCND1/pRB mediated E2F1 pathway and its downstream targets. FaDu cells (1–2 × 104 cells/ml) were treated with 8 different concentrations of P276-00 (range 0.03 to 10 μM) for 24 hours and observed using ArrayScan HCS reader after staining with respective antibody (d) Quantification of the automated fluorescence imaging data has been represented in sigmoidal dose response curve where compound concentration. is represented in log2 scale. One thousand cells were counted for each replicate well and the results were presented as an average ± S.E.M. (n = 3). EC50 values have been generated for all the 5 proteins. (e) Panel of representative images.
Mentions: To establish the anti-proliferative properties of P276-00 via G1 arrest, we have investigated the status of CCND1/pRB mediated E2F1 signaling components in FaDu cells treated with or without P276-00 (Figure 2a). Treatment of FaDu cells with P276-00 showed a reduction in pRB and CCND1 protein levels as early as 6 hours and was maintained upto 24 hours. P276-00 also reduced E2F1 and CCNE1 protein expression by 24 hours (Figure 2a).

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