Effects Of Ganzheng Solution On Signaling Transduction Of TGF Beta 1/Smads On SMMC-7721 Cell Line

Objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. China is located the areas of high morbidity of HCC. About 1 million of new cases with HCC every year in the world and 45% patients are Chinese. The mortality rate of HCC is very high, and has become the second of all malignant tumors in china. The symptom of HCC is very latent and unspecific. Most of them lost the opportunity of surgical resection after diagnosis and can only receive transcatheter arterial chemoembolization (TACE), percutaneous ethanol injection (PEI), radiofrequency ablation (RFA) and other therapy. But the curative effect is unsatisfied. Chinese traditional medicine can suppress tumor progress and metastasis, improve the quality of live, and play an important role in treatment for later phase of HCC. According to the theory of Chinese traditional medicine and clinical study over ten years, Professor Yao Shukun had developed Ganzheng solution with the therapeutic principles of clearing away heat and toxic material, removing blood stasis and resolving lumps, strengthening the spleen and replenishing qi. Studies had proved that Ganzheng solution had obvious repressive effect on the genesis of HCC. Through mult–links, mult-targets and integral regulation mechanisms, Ganzheng solution can inhibit the progress and metastasis of HCC, Modern molecular biology confirmed that tumor is a type of gene diseases. Oncogene activation, antioncogene inactivation and signaling transduction disorder is associated with uncontrolled cell proliferation and decreased apoptosis. All of those play a central role in carcinogenesis. Transforming growth factor beta 1(TGF-β1)/Smads signaling pathway plays a very important role in controlling progress of many tumors. But the mechanisms of TGF-β1/Smads signaling pathway on HCC is unknown. In part 1 of this study we investigated the levels of serum TGF-β1 and other cytokines, analyze the relationship to other cytokies, explore the effect of TGF-β1 on HCC. In part 2 : we investigated the TGF-βreceptor and Smads expression on surgically resected HCC tissues, compare with normal liver tissue and liver cirrhosis tissue and adjacent nonneoplastic tissue of HCC, explore the mechanism of TGFβ1/Smads signaling pathway on HCC genesis. In patr 3: we observed the repressive effect of drug serum containing Ganzheng solution on TGF-β1/Smads signaling pathway of SMMC-7721 cells line. We also tried to clarity the molecular mechanism of Ganzheng solution treating HCC, and provide rational data for using Chinese herbal treating patients with HCC. Methods 1 The changes of TGF-β1 and other cytokines in patients with HCC and its correlation with disease severity 50 cases with HCC were enrolled to this study for 39 man and 11 women, and average age was 54.5±15.6 years. According to the diagnosing criterion of HCC in the 2001 Chinese liver cancer conference, 5 cases were clinical stageⅠb,13 cases were Ⅱa, 18 cases were Ⅱb,9 cases were Ⅲa and 5 were Ⅲb. 25 healthy volunteers were chosen as control group. T lymphocyte subset was detected by three-color flow cytometry. Concentration of serum TGFβ1, IL-2, IL-12, IFN-γ, TNFαwere detected by ELISA. 2 Alterations of the TGFβ1/Smads signaling pathway in patients with HCC and liver cirrosis To explore the mechanism of TGFβ1/Smads signaling pathway on HCC, ten surgically resected tissue were selected, including 4 surgically resected HCC tissue, 2 liver cirrhosis tissue, 2 tissue from liver hemangioma served as normal control, 2 adjacent nonneoplastic tissue of HCC. The TGF-βreceptor and Smads expression were detected. 2.1 The protein expression of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 and Smad7 in SMMC-7721 cell line were detected by Western blotting. 2.2 The expression of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 and Smad7mRNA in SMMC-7721 cell line were detected by RT-PCR. 3 Serapharmacological study of Ganzheng solution on proliferation, apoptosis and TGF-β1/Smads signaling pathway in SMMC-7721 cell line 10 patients with HCC were enrolled to this study for 7 man and 3 women, and average age was 57.8±13.9 years, and diagnosing criterion stageⅡb to stage Ⅲ, liver function was Child-Pugh B. 10 healthy volunteers were chosen as control group. All subjects were given Ganzheng solution 40ml per day for 8 weeks. Before medication and after the last medication the blood was drawn. The serum was separated and stored at -80℃for following study. The serum of patients with HCC before Ganzheng solution was taken as HCC serum. The serum of patients with HCC after eating Ganzheng solution was taken as HCC drug serum. The serum of healthy volunteers before Ganzheng solution was taken as volunteers serum. The serum of healthy volunteers after eating Ganzheng solution was taken as volunteers drug serum. 3.1 The inhibitory effect of Ganzheng solution drug serum on SMMC-7721 cell cultured in vitro was detected by MTT. 3.2 The effect of Ganzheng solution drug serum on cell cycle and cell apoptosis of SMMC-7721 cell cultured in vitro were observed by flow cytometry. 3.3 The effect of Ganzheng solution drug serum on protein expression of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 and Smad7 of SMMC-7721 cell cultured in vitro was examined by Western blotting. 3.4 The effect of Ganzheng solution drug serum on mRNA expression of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 and Smad7 of SMMC-7721 cell cultured in vitro was investigated by RT-PCR. Statistical methods: data were analysed using the SPSS 9.0 statistical package and were expressed as mean ±standard ( x ±S). Statistical significance of the results was calculated using the student's t-test with a p value of <0.05 considered statistically signaficant. Spearman's correlation test was used to test the relationship between two sets of data. All tests ofsignificance were two tailed. Results 1 The changes of TGF-β1 and other cytokines in patients with HCC and its correlation with disease severity 1.1 The serum levels of TGF-β1,TNF-α,IFN-γ,IL-2 and IL-12 in patients with HCC Compared with control, TGF-β1 (0.79±0.43 vs 0.53±0.42, ng/ml, p=0.014), TNF-α(257.61±68.03 vs 151.60±115.78, pg/ml, p=0.0001) level were significantly increased and the levels of IFN-γ(78.60±17.58 vs 95.74±37.26, pg/ml, p=0.008), IL-2 (1.72±1.30 vs 2.43±0.93, pg/ml, p=0.018) and IL-12 ( 53.72 ±47.15 vs 93.43 ±77.32, pg/ml, p=0.007) were significantly decreased in patients with HCC. 1.2 The correlation of TGF-β1,TNF-α,IFN-γ,IL-2,IL-12,ALT and BIL TGF-β1 was negatively correlated with TNF-α(r=-0.350, P<0.05) and IL-2(r=-0.476, P<0.01). IL-12 was positively correlated with IFN-γ(r=0.612, P<0.01) and BIL (r=0.439, P<0.01). 1.3 The changes of T lymphocyte subsets in patients with HCC Compared with NC, the quantity of CD3+ cell (59.80±5.80 vs 66.01±9.80, P<0.05), CD4+ cell (34.38±6.25 vs 41.00±4.73, P<0.01)and CD4+/ CD8+ ratio(0.91±0.28 vs 1.56±0.28, P<0.01) were decreased and CD8+ cells (37.67±7.83 vs 26.74±4.27, P<0.01) were increased in patients with HCC. 1.4 The concentration of TGF-β1 and other serums cytokines in different stage of HCC In Ⅱb and Ⅲstage of HCC patients, the concentration of TGF-β1 (0.890±0.408 vs 0.645±0.406, ng/ml, P<0.05) and TNF-α(273.10±78.23 vs 230.06±30.44, pg/ml, P<0.05) were higher than that in Ⅰand Ⅱa stage, and the concentration of IFN-γ(72.75±16.52 vs 89.00±14.63, pg/ml, P<0.05), IL-2 (1.43±0.74 vs 2.05±1.45, pg/ml, P<0.05) and IL-12 (42.98±38.06 vs 69.77±50.69, pg/ml, P<0.05) were lower than that in Ⅰand Ⅱa stage. These data showed that T lymphocyte subsets and cytokines levels weredisordered in patients with HCC, and the network of cytokines were imbalance, and anti-tumor immune function were inhibited, especially medium-later period patients with HCC. It might be one of the causative factors of the tumor progress. 2 Alterations of the TGFβ1/Smads signaling pathway in patients with HCC and liver cirrhosis 2.1 The expression of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 mRNA and protein in tissues of liver cirrhosis were significantly higher than normal control liver, especially TGFβRⅡand Smad4 mRNA and protein. The expression of Smad7 mRNA and protein and the ratio of Smad7/Smad4 were significantly lower than that in the liver of normal contral. 2.2 The expression of TGFβRⅠ, TGFβRⅡ, Smad2 and Smad4 mRNA in HCC liver tissues were significantly lower than normal control liver, liver cirrhosis and adjacent nonneoplastic tissues. The expression of Smad7 mRNA and the ratio of Smad7/Smad4 in HCC liver were significantly higher than normal control, liver cirrhosis and adjacent nonneoplastic tissues. The tendency of protein expression of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 and Smad7 were similar to mRNA expression in HCC liver tissues. The expression of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 and Smad7 mRNA in one patient with HCC were significantly higher than normal, liver cirrhosis and adjacent nonneoplastic tissues. The ratio of Smad7/Smad4 was slightly increased. The protein expression of TGFβRⅡ, Smad2 and Smad4 were significantly higher than normal, liver cirrhosis and adjacent nonneoplastic tissues and the ratio of Smad7/Smad4 was significantly decreased in this patient. These data showed that the TGFβ1/Smads signaling pathway was over-activated in patients of cirrhosis, the expression of Smad7 was inhibited, the negative regulation of Smad7 in TGFβ1/Smads signaling pathway was repressed. All of these may account for the development of liver fibrosis. In most patients with HCC, there are over-expression of Smad7, and lower-expression of TGFβRⅠ, TGFβRⅡ, Smad2 and Smad4. The ratio ofSmad7/Smad4 was significantly increased, and the TGFβ1/Smads signaling pathway was repressed. TGFβ1 signal of growth inhibitory could not be transmitted and the proliferation of cells were enhanced. It may be one of the factors of HCC pathogenesis. 3 Serapharmacological study of Ganzheng solution on proliferation, apoptosis and TGF-β1/Smads signaling pathway in SMMC-7721 cell line 3.1 The inhibitory effects of Ganzheng solution drug serum on SMMC-7721 cell proliferation SMMC-7721 cells were cultured by HCC drug serum, HCC serum, volunteers drug serum and volunteers serum respectively for 48 hours in vitro, and the proliferation rate was observed. HCC drug serum could significantly inhibit the proliferation of SMMC-7721 cells and the inhibition rate is 13.97%(p<0.01). Volunteers drug serum could slightly inhibit the proliferation of Smmc-7721 cells and the inhibition rate is 5.57%(p>0.05). The result demonstrated that Ganzheng solution drug serum of HCC patients could significantly inhibit the proliferation of SMMC-7721 cells. 3.2 The effect of Ganzheng solution drug serum on cell cycle and cell apoptosis of SMMC-7721 cell SMMC-7721 cells were cultured by HCC serum and HCC drug serum respectively, the apoptosis rate of SMMC-7721 cell were 2.31±0.76 vs 7.49±0.48(%, P<0.01), and G0/1 phase cell 51.00±2.10 vs 61.50±9.94(%, P<0.05), and G2M phase cell 33.63±3.00 vs 19.97±3.70(%,P<0.05), and S phase cell 15.37±2.57 vs 11.87±3.77(%,P<0.05). The result demonstrated that Ganzheng solution drug serum of HCC patients could increase the apoptosis of SMMC-7721 cell and decrease S phase cells and leads to growth arrest in G0/G1 phase of the cell cycle. 3.3 The effect of Ganzheng solution drug serum on the protein expression of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 and Smad7 of SMMC-7721 cell Cultured by HCC drug serum, the protein expression of TGFβRⅠ, TGFβRⅡ, Smad2 and Smad4 in SMMC-7721 cells were significantly increased,especially TGFβRⅡand Smad4 protein. The ratios of TGFβRⅠ/β-actin, TGFβRⅡ/β-actin, Smad2/β-actin, Smad4/β-actin were 0.812±0.213 vs 0.945±0.644(p>0.05),1.135±0.357 vs 1.467±0.590(p<0.05), 0.771±0.533 vs 0.796±0.374(p>0.05), 0.895±0.318 vs 1.171±0.133(p<0.05) respectively. The expression of Smad7 protein was significantly decreased, and the ratio of Smad7 OD value/β-actin OD value was significantly decreased (1.354±0.661 vs 0.885±0.309, p<0.05). These results demonstrate that Ganzheng solution drug serum of HCC patients could enhance the expression of TGFβRⅡand Smad4 protein and repress the Smad7 expression in SMMC-7721 cells. Thus, It activate the TGFβ1/Smads signaling pathway, inhibite the progress of HCC. 3.4 The effect of Ganzheng solution drug serum on the mRNA expression of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 and Smad7 of SMMC-7721 cell line cultured in vitro was examined by RT-PCR Cultured by HCC drug serum, the mRNA expression of TGFβRⅠ, TGFβRⅡ, Smad2 and Smad4 in SMMC-7721 cells were significantly increased, especially TGFβRⅡand Smad4 mRNA. The ratios of OD values of TGFβRⅠ, TGFβRⅡ, Smad2, Smad4 mRNA and β-actin OD value were 0.482±0.134 vs 0.678±0.545(p>0.05), 1.019±0.810 vs 1.717±0.959(p<0.05), 0.992±0.121 vs 1.123±0.217(p>0.05), 1.195±0.554 vs 1.665±0.432(p<0.05)respectively. The expression of Smad7 mRNA was significantly decreased in SMMC-7721 cells. The ratio of Smad7 OD value/β-actin OD value was 1.619±0.778 vs 0.192±0.324(p<0.01). Cultured by volunteers drug serum , the mRNA expression of Smad7 in SMMC-7721 cells were significantly decreased, the ratio of Smad7 OD value/β-actin OD value was 1.250±0.899 vs 0.459±0.332(p<0.05). These results demonstrate that Ganzheng solution drug serum of HCC patients could enhance the expression of TGFβRⅡand Smad4 mRNA and repress the Smad7 expression in SMMC-7721 cells. Activated TGFβ1/Smads signaling pathway could inhibited the progress of HCC, which might be one of the mechanism of anticarcinoma of Ganzheng solution.