Dissertation
Dissertation > Medicine, health > Oncology > Respiratory system tumors > Lung tumors

Influence of NF-κB Inhibitor (PDTC) on Protein Expression of ASPP1 and ASPP2 of Non-small Cell Lung Cancer

Author ZhangYueYan
Tutor ShiGuangXia
School Dalian Medical University
Course Pathology and Pathophysiology
Keywords ASPP protein family p53 Cisplatin Of NF- κB inhibitor PDTC
CLC R734.2
Type Master's thesis
Year 2011
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Objective: Constitutive activation of nuclear factor-κB (NF-κB) and the dysfunction of the tumor suppressor gene p53 are common molecular changes in the lung cancer. Insufficent apoptosis resulted in by them play an impotent role in the process of development, progression and treatment of lung cancer. Tumor cell DNA damage caused by chemotherapeutic drugs may activate p53 pathway inducing apoptosis. However, the activation of NF-κB may be against apoptosis. Apoptosis-stimulating proteins of p53 (ASPP) includes three members: ASPP1, ASPP2 and iASPP.They can specifically regulate the protein of p53. When ASPP1 and ASPP2 bind with the p53 they can activate the proapoptotic function of p53 and play an anti-tumor role, and iASPP is inhibitory for proapoptosis function of p53 and function of Rel A of NF-κB family. The relationships among these regulatory pathways for apoptosis and their role in occurrence, progression and treatment would to be further investigated. In this experimental the NF-κB inhibitor pyrrolidine dithio-carbamate (PDTC) was used to block NF-κB-mediated inhibition of apoptosis functions. Non-small cell lung cancer cell lines A549 (p53 wild type) and NCI-157 (p53 mutant) were treated with only PDTC or comined with conventional chemotherapy drug cisplatin (CDDP). The proliferation and relative expression of ASPP1 and ASPP2 were measured to analyze the role of ASPP family in crosstalk between p53 and NF-κB.Methods: The MTT method was used to quantitize the minimum effective concentration of conventional chemotherapy drug cisplatin (CDDP) and PDTC, Non-small cell lung cancer cell lines A549 and NCI-157 were treated with CDDP or PDTC only at first and inhibition rate of proliferation was calculated. Then these two cell lines were treated with the minimum effective concentration of drugs only or combined for 24 hours, their proliferation of A549 and NCI-157 was measured with MTT assay. ASPP1 and ASPP2 protein expression was assayed with Western blot method. Experimental data were analyzed with analysis of variance (ANOVA) in SPSS13.0 statistical software, P<0.05 was considered statistically significant.Results:⑴. Inhibitory effect on proliferation of PDTC and CDDP single and combination treatment: Both PDTC and CDDP exhibited growth inhibitory effect on two cell lines A549 and NCI-157, and the inhibition rate increased with the concentration of drugs. The inhibition rate of the two cell lines treated by combined PDTC and CDDP was higher than that of either of two drugs used single. Extent of increase in inhibition rate of A549 cells was significantly higher than that of NCI-157.⑵Influence of PDTC on the protein expression of ASPP1 and ASPP2: The result of Western Blot analysis showed both A549 and NCI-157 expressed ASPP1 and ASPP2. In untreated A549 cells relative expression of ASPP1 was 0.809±0.053,that of ASPP2 was 0.502±0.024。In the A549 cells cultured with PDTC or CDDP for 24 hours the relative expression of ASPP1 was 0.898±0.062 and 1.468±0.239,that of ASPP2 increased to 1.020±0.132 and 0.679±0.058. In A549 cells cultured with PDTC for 6 hours followed by CDDP for 24 hours relative expression of ASPP1 and ASPP2 was 1.804±0.298 and 0.882±0.188. In untreated NCI-157 cells relative expression of ASPP1 was 0.653±0.100,that of ASPP2 was 2.692±0.224. In the NCI-157 cells cultured with PDTC or CDDP for 24 hours the relative expression of ASPP1 was 0.784±0.131 and 0.848±0.099,that of ASPP2 increased to 3.280±0.511 and 4.355±0.547. In NCI-157 cells cultured with PDTC for 6 hours followed by CDDP for 24 hours relative expression of ASPP1 and ASPP2 was 0.819±0.101 and 5.225±0.187.Conclusion: NF-κB inhibitor PDTC exhibited inhibitiory effects on proliferation of A549 and NCI-157 lung cancer cells and increased their expression of ASPP2. Preculture with PDTC increased inhibitory effect of proliferation of CDDP and expression of ASPP1 and ASPP2 of CDDP cultured cells.

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