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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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P276-00 inhibit growth signaling proteins and cytokine in FaDu cells. (a-b) P276-00 inhibits mRNA level of IL-6, EGFR and HSPA8 in FaDu cells. The relative expression level of these genes was measured in comparison with control sample (a) Time dependent response (b) dose response. (c-d) P276-00 potently suppresses in vitro production of IL-6 and HSPA8 in FaDu cells. (c) IL-6 (d) HSPA8. All values are average ± S.E.M. (n=3-5) * indicates p-val < = 0.05 as compared to DMSO pre-treated control cells.
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Figure 4: P276-00 inhibit growth signaling proteins and cytokine in FaDu cells. (a-b) P276-00 inhibits mRNA level of IL-6, EGFR and HSPA8 in FaDu cells. The relative expression level of these genes was measured in comparison with control sample (a) Time dependent response (b) dose response. (c-d) P276-00 potently suppresses in vitro production of IL-6 and HSPA8 in FaDu cells. (c) IL-6 (d) HSPA8. All values are average ± S.E.M. (n=3-5) * indicates p-val < = 0.05 as compared to DMSO pre-treated control cells.

Mentions: We examined growth and immune signalling proteins such as EGFR, HSPA8 and cytokines IL-6 that are involved in the survival of tumor. There was a marked inhibition of HSPA8 and IL-6 genes at mRNA level in dose- and time-dependent manner (Figure 4a, 4b). In congruence with the gene expression, P276-00 significantly inhibited IL-6 and HSPA8 protein levels measured by ELISA with IC50 at 1.4 μM and 1.2 μM respectively (Figure 4c, 4d). EGFR gene expression was significantly reduced by 1 μM P276-00 in a time-dependent manner with maximal inhibition at 24 h (Figure 4b). Additional file 1: Figure S1 showed a marked dose-dependent inhibition of EGFR gene expression at 24 h with a not so robust dose–response effect at 12 h (Figure 4a).


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 inhibit growth signaling proteins and cytokine in FaDu cells. (a-b) P276-00 inhibits mRNA level of IL-6, EGFR and HSPA8 in FaDu cells. The relative expression level of these genes was measured in comparison with control sample (a) Time dependent response (b) dose response. (c-d) P276-00 potently suppresses in vitro production of IL-6 and HSPA8 in FaDu cells. (c) IL-6 (d) HSPA8. All values are average ± S.E.M. (n=3-5) * indicates p-val < = 0.05 as compared to DMSO pre-treated control cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: P276-00 inhibit growth signaling proteins and cytokine in FaDu cells. (a-b) P276-00 inhibits mRNA level of IL-6, EGFR and HSPA8 in FaDu cells. The relative expression level of these genes was measured in comparison with control sample (a) Time dependent response (b) dose response. (c-d) P276-00 potently suppresses in vitro production of IL-6 and HSPA8 in FaDu cells. (c) IL-6 (d) HSPA8. All values are average ± S.E.M. (n=3-5) * indicates p-val < = 0.05 as compared to DMSO pre-treated control cells.
Mentions: We examined growth and immune signalling proteins such as EGFR, HSPA8 and cytokines IL-6 that are involved in the survival of tumor. There was a marked inhibition of HSPA8 and IL-6 genes at mRNA level in dose- and time-dependent manner (Figure 4a, 4b). In congruence with the gene expression, P276-00 significantly inhibited IL-6 and HSPA8 protein levels measured by ELISA with IC50 at 1.4 μM and 1.2 μM respectively (Figure 4c, 4d). EGFR gene expression was significantly reduced by 1 μM P276-00 in a time-dependent manner with maximal inhibition at 24 h (Figure 4b). Additional file 1: Figure S1 showed a marked dose-dependent inhibition of EGFR gene expression at 24 h with a not so robust dose–response effect at 12 h (Figure 4a).

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