| BackgroundLung cancer ranks first in the incidence and mortality all over the world cities. Current treatment of lung carcinoma including surgery, radiotherapy, chemotherapy and molecuLar targeted therapy is still unsatisfactory, the survival of patient without a breakthrough. Our study of National Tenth Five Year Plan and National Eleven Five Support Program showed that the therapy of integrative medicine could extend the median survival period of patients with stage III andⅣnon-small cell lung carcinoma to 3.57 months, especially in the Treatment of advanced NSCLC with splenic asthenia and phlegm-damp type through Yiqi Chutan Formula (median survival was extended to 401 days). Yiqi Chutan Formula has been proved by the long-term clinical efficacy, moreover, it has not significant adverse reactions. However, the treatment of traditional Chinese medicine is a multi-component, multi-target, multi-channel's overall adjustment process, then what is the possible functionary targets and efficacy of Yiqi Chutan Formula (YQCTF)? This research will study the molecuLar mechanisms of Yiqi Chutan Formula in lung carcinoma through differentiation proteomics and furthermore explore the scientific basis of Chinese traditional medicine on lung cancer.ObjectivesTo find out the therapeutic target of Yiqi Chutan Formula anti-LEWIS lung carcinoma by observing the impact of Yiqi Chutan Formula on LEWIS lung carcinoma tumor growth in C57BL/6J mice and screening the differential protein expression between Yiqi Chutan Formula treatment group and model group. To validate and explore the mechanism and significance of these differential proteins on the incidence and therapy of lung carcinoma by clinical pathology and further experimentation.Methods1 Lung carcinoma model was established by subcutaneously inocuLating LEWIS lung carcinoma cells in C57BL/6J mice.80 mice were randomLy divided into model group (saline) and treatment group (Yiqi Chutan Formula 3.0 g/kg/d). Yiqi Chutan Formula and saline was given daily by gavage for 21 days. The tumor tissues were harvested at day 22, tumor size, tumor weight and the numbers of lung metastases were detected, and the total protein was extracted from tumor tissue of treatment group and model group. The expressed differentially protein spots information were acquired by a two-way fluorescence difference gel electrophoresis (2D-DIGE) system, and the differential proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS). The differences in protein expression was finally verified by Western blot and fluorescence quantitative PCR.2 Clinical validation was achieved by immunohistochemical detection on Lhe expression of Vimentin of clinical lung carcinoma tissues 79cases(squamous cell carcinoma 10 cases,69 cases of adenocarcinoma),6 cases of adjacent cancer tissues, benign lung disease (inflammation, hyperplasia) tissue biopsy 20 cases. A549 cell line was treated with 20% YQCTF serum containing, wound healing assay was used to observe the mobility rate. The expression of MMP-1, MMP-2, MMP-9, MMP-14 mRNA were detected by RT-PCR. The immunohistochemical method was used to observe the expression of MMP-2, MMP-14 of the tumor tissues of C57BL/6J mice bearing LEWIS lung carcinoma.3 The expressions of prolyl 4-hydroxylase beta polypeptide (P4HB) was detected by immunohistochemistry with clinical lung carcinoma tissues 79cases (squamo(?) cell carcinoma 10 cases,69 cases of adenocarcinoma),6 cases of adjacent cancer tissues, benign lung disease (inflammation, hyperplasia) tissue biopsy 20 cases, and tissue microar rays were made up of 48 cases of lung cancer and 48 cases of adjacent cancer tissues,4 cases of normal lung tissue. Cultured human lung adenocarcinoma A549 cells, flow cytometry staining detected the A549 cell apoptosis of DDP 1.5μg/mL single PI group, 15% serum (?)ntaining of Yiqi Chutan Formula, and both parties combined group, celluLar immunity detected the expression of prolyl 4-hydroxylase beta polypeptide of the combination group and the DDP alone group, RT-PCR detected the expression of prolyl 4-hydroxylase beta polypeptide mRNA.Results1 In Yiqi Chutan Formula 3. Og/kg/d treatment group, tumor size, tumor weight and the numbers of lung metastases were significantly less than that in model group (P<0.01), and the tumor growth inhibition rate was 57.21%. For gel diagram analysis of 2D-DIGE system, compared with model group, there were 44 expressed differentially protein spots, of which 6 protein spots were up-reguLated and 38 protein spots were down-reguLated in the treatment group. Among these proteins,37 proteins were successfuLly identified by MALDI-TOF/TOF MS. The expression of protein and mRNA of Hsp-60, P4HB, PDI-A3 and EG433182, Hsp5p, Hsp9, STIP1 mRNA were verified down-reguLated respectively by Western blotting and qPCR in treatment group.2 Clinical pathological studies show that the expression of Vimentin were up-reguLated in the lung tumor tissue, down-reguLated in adjacent tissues, patients with inflammation and metastasis was detected significantly higher expression.20% YQCTF serum containing can significantly inhibit the metastasis of A549 cell and reduce the expression of MMP-2, MMP-14 mRNA compared with the control group (P<0.01), but there was no significant difference in the expression of MMP-1, MMP-9 mRNA (P>0.05). Furthermore, after treated with YQCTF 3.0 g/kg/d, the tumor's volume, weight, and the number of lung metastases in mice bearing LEWIS lung carcinoma were significantly decreased (P<0.01), and the expression of MMP-2, MMP-14 in tumor were significantly decreased compared with the model group (P<0.01)2 The immunohistochemical staining of the clinical pathological studies show that the strong positive expression rate of Vimentin protein in the patients of lung adenocarcinoma was 34.78%, lung squamous cell carcinoma was 33.33%, adjacent tissues was 25%, benign lung disease was 25%. Syndrome of splenic asthenia and phlegm-damp of lung cancer was 51.31%, Syndrome of Qi stagnation with phlegm blokage and blood stasis was 49.66%, Syndrome of phlegm heat due to Yin deficiency was 49.51%, Syndrome of deficiency of Qi and Yin was 50.22%. Clinico-pathological differences between patients with and without multiple metastases was 62.5%,21.74%, with and without infection was 58.33%,41.86%. The results suggest that strong expression of Vimentin were up-regulated in the lung tumor tissue, down-regulated in adjacent tissues, there were weak expressions of the normal lung tissues (P<0.05). The high expressions of Vimentin was correlated with patients with inflammation and metastasis (P<0.05). There was no significant differences in the 4 syndromes of TCM syndromes (P>0.05).20% YQCTF serum containing can significantly inhibit the metastasis of A549 cell and reduce the expression of MMP-2, MMP-14 mRNA compared with the control group (P<0.01), but there was no significant difference in the expression of MMP-1, MMP-9 mRNA (P>0.05). Furthermore, after treated with YQCTF 3.0 g/kg/d, the tumor's volume, weight, and the number of lung metastases in mice bearing LEWIS lung carcinoma were significantly decreased (P<0.01), and the expression of MMP-2, MMP-14 in tumor were significantly decreased compared with the model group (P<0.01)3 The immunohistochemical staining of the clinical pathological studies show that the stained area percentage of P4HB in the patients of lung adenocarcinoma was36.22±6.83%, lung squamous cell carcinoma was 35.01±7.25%, adjacent tissues was 21.93±7.69%, pulmonary benign lesions (inflammation, hyperplasia) was 27.87±5.73%, the results suggest that strong expression of P4HB were up-regulated in the lung tumor tissue, down-regulated in adjacent tissues, there were weak expressions of the normal lung tissues (P<0.05) Syndrome of splenic asthenia and phlegm-damp of lung cancer was 39.01±7.11%, Syn(?) ome(?) Qi stagnation with phlegm blokage and blood stasis was 38.29±6.93%, Syndrome of phlegm heat due to Yin deficiency was 38.89±6.58%, Syndrome of defic(?)y of Qi and Yin was 38.91±5.87%. The expressions of P4HB in lung cancer was significantly higher than that of the adjacent cancer tissues and norma(?)ng tissues (P<0.05). There was no significant differences in the 4(?) of TCM syndromes (P>0.05). Immunohistochemical detection of the tissue (?)icroarray show that, staining area percentage of P4HB in the lung ad(?)noma was 36.96±7.89%, and its corresponding adjacent tissues was 23(?)5.62%, normal lung tissue was 12.82±5.60%. Staining area percentage of P4HB in the lung squamous cell carcinoma was 34.23±6.81%, and the co(?)esp nding adjacent tissues was 21.75±7.02%. The expressions of P4HB in (?) tissue was significantly higher than that of the adjacent cancer ti (?)normal lung tissues (P<0.05). The results indicate that the e(?) P4HB in adenocarcinoma was slightly stronger than that of the sq(?) carcinoma, but there was no statistically significant difference (P>0.05). Flow cytometry staining detected that the A549 cell apoptosis rate of DDP1.5μg/mL combined with 15% serum containing of Yiqi Chutan Formula was 37.00±1.13%, serum containing of Yiqi Chutan Formula was 9.90+1.86%, DDP alone was 15.80±2.75%. Cell Immunohistochemistry shows that the expression of prolyl 4-hydroxylase beta polypeptide of A549 cells decreased in the combination group. Also RT-PCR shows that the expression of prolyl 4-hydroxylase beta polypeptide mRNA of A549 cells decreased significantly in the combination group.ConclusionYQCTF can inhibit the metastasis of A549 cell and also the LEWIS lung carcinoma's growth and its metastases. Proteomics research discovers two new antitumor mechanism of traditional Chinese medicine. One-third of differential proteins which include prolyl 4-hydroxylase beta polypeptide, protein disuLfide-isomerase A3 precursor, stress-induced phosphoprotein 1, heat shock protein 5 precursor, heat shock protein 9 are molecuLar chaperones or related proteins, suggesting that anti-cancer medicine might take effect through reguLating endoplasmic reticuLum unfolded protein response (unfolded protein response, UPR). Furthermore, its mechanism of inhibiting the incidence and growth process of lung carcinoma is based on this. Vimentin is the most significant protein which elucidate the mechanism of YQCTF on lung carcinoma might be highly related to its effection of controling metastasis which might also correlate epithelial-mesenchymal transition (EMT)...
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