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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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Effects of P276-00 on FaDu xenograft. Average tumor growth of HNSCC (FaDu) xenograft over a period of 18 day after randomization. Group treated with P276-00 (shaded square), control group (open square) (n = 6).
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Figure 5: Effects of P276-00 on FaDu xenograft. Average tumor growth of HNSCC (FaDu) xenograft over a period of 18 day after randomization. Group treated with P276-00 (shaded square), control group (open square) (n = 6).

Mentions: Given the significant anti-proliferative and pro-apoptotic effects of P276-00 in HNSCC in vitro, we investigated the anti-tumor efficacy in a HNSCC tumor xenograft mouse model. P276-00 showed significant (p < 0.001) in vivo activity with a 48% tumor growth inhibition (TGI) on day 18 (Figure 5). Consistent with the in vitro evidence, P276-00 inhibited cell cycle gene expression in the tumor tissue including CCND1, CCNE1, cMYC, KI67 and PCNA (>1.8 fold inhibition or fold change in gene expression compared to control group Figure 6a). Immunohistochemical analysis of tumor tissue for molecular targets of cell cycle such as CCND1, CCNE1, pRB and PCNA demonstrated a significant decrease in expression level accompanied by a moderate decrease in KI67 in response to P276-00 treatment (Figure 6b, 6c). Similar to in vitro studies, FaDu xenograft tumors showed increased levels of phosphorylated P53 in response to P276-00 administration (Figure 6b, 6c).


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)

Effects of P276-00 on FaDu xenograft. Average tumor growth of HNSCC (FaDu) xenograft over a period of 18 day after randomization. Group treated with P276-00 (shaded square), control group (open square) (n = 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effects of P276-00 on FaDu xenograft. Average tumor growth of HNSCC (FaDu) xenograft over a period of 18 day after randomization. Group treated with P276-00 (shaded square), control group (open square) (n = 6).
Mentions: Given the significant anti-proliferative and pro-apoptotic effects of P276-00 in HNSCC in vitro, we investigated the anti-tumor efficacy in a HNSCC tumor xenograft mouse model. P276-00 showed significant (p < 0.001) in vivo activity with a 48% tumor growth inhibition (TGI) on day 18 (Figure 5). Consistent with the in vitro evidence, P276-00 inhibited cell cycle gene expression in the tumor tissue including CCND1, CCNE1, cMYC, KI67 and PCNA (>1.8 fold inhibition or fold change in gene expression compared to control group Figure 6a). Immunohistochemical analysis of tumor tissue for molecular targets of cell cycle such as CCND1, CCNE1, pRB and PCNA demonstrated a significant decrease in expression level accompanied by a moderate decrease in KI67 in response to P276-00 treatment (Figure 6b, 6c). Similar to in vitro studies, FaDu xenograft tumors showed increased levels of phosphorylated P53 in response to P276-00 administration (Figure 6b, 6c).

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