| The incidence and mortality of primary lung cancer rank first in malignant tumors around the world,About 85% of lung cancers are non-small cell lung cancer(NSCLC).Invasion and metastasis are major causes of treatment failure in patients with lung carcinoma,tumor angiogenesis plays an important role in the occurrence, development,invasion, metastasis and recurrence of cancer,Tumor invasion and metastasis is a polygenic,multistep process that depends on the characteristics of the tumor and its host organs. Understanding the molecular targets involved and their corresponding inhibitors will provide promising new approaches for the treatment of cancer. Therefore, the blocking of angiogenesis in cancer therapy has become a focus of cancer research.A disintegrin and metallopeptidase with thrombospondin motif type 1(ADAMTS1) is a recently discovered metalloproteinase with antiangiogenic activity. The function of ADAMTS1 in lung cancer remains unknown.It is well known that vascular endothelial growth factor(VEGF)is recognized as the most pro-angiogenic factor in regulating angiogenesis,it can be a good response of vascular endothelial growth state, reducing the expression of VEGF can degrade network of blood vessels and inhibit tumor growth. CD105 involves in tumor angiogenesis and development process, and it plays an crucial role in the biological behavior of malignant tumors.Tetramethylpyrazine(TMP), one of the major bioactive components of traditional Chinese medicine Chuanxiong, has been applied in the treatment of cerebralvascular and cardiovascular diseases.Tetramethylpyrazine(TMP) has been found to inhibit the growth and the metastasis of certain solid tumors by promoting immune function and inducing apoptosis of the cell.Cisplatin(DDP) is one of the most effective single-drug in the current treatment of small cell lung cancer.It has such characteristics as a broad spectrum of anti-cancer effect, a variety of antineoplastic synergistic effect and no cross-resistance.In the newly recent studies have shown that the combination of certain chemotherapeutic drugs and TMP can enhance the effectiveness of tumor treatment.The experiment through applying TMP and DDP on Lewis lung cancer C57BL/6 mice model,observation of the impact on mice tumor growth after the drug effect by measuring tumor weight and tumor size in different groups, changes in the expression of ADAMTS1、VEGF、CD105, and the change of the tumor cells and tumor vascular morphology can explain that TMP combined with DDP may improve the therapeutic effect, and can describe the possible mechanism, so as to provide an experimental basis for research.The contents of study as follows:56 cases of healthy male rats were adapted to live in the room for one week after bought back. The models were established by subcutaneously injecting Lewis lung cancer cells of logarithmic growth phase(cell confluence 80%) and cell density was adjusted to 1 × 107/L with saline solution from the right axilla of C57BL/6 mice, per 0.2m L. Animal experiments were divided into four groups:saline group,TMP group,DDP group, combined TMP and DDP group.Each group of model rats were conventional fed after vaccinating Lewis lung cancer cells.The drugs were given two weeks after the inoculation could touch the the size of about8 mm tumor mass at subcutaneous inoculation.The Control group:the saline group were given with saline 0.2 m L,by intraperitoneal injection, once daily,for 14 days.The TMP group:the TMP group were given with TMP100 mg ﹒ kg-1,0.2 m L,by intraperitoneal injection, once daily,for 14 days.The DDP group :the DDP group were given with DDP 2 mg ﹒ kg-1,0.2 m L,once daily,for 14 days.Combined TMP and DDP group: the combination group in accordance with the above mentioned method.Before dosing every time, adjusted the dosage leading to weight.All the Mice were sacrificed at the nineteenth day of the experiment,he tumor blocks were taken out and fixed.Subcutaneous tumors were processed for tumor weight,the volume of the tumors and tumor growth inhibition rate and tumor necrosis rate of each group were calculated. Observed the ultrastructure of tumor tissue under electron microscopy and photographed the record.The expression levels of a disintegrin and metallopeptidase with thrombospondin motif type 1(ADAMTS1), vascular endothelial growth factor(VEGF) were measured by immunohistochemistry and Western Blot after treatment.The microvascular density(MVD) was determined by CD105 staining.1 The toxic side reaction of chemotherapy and the life quality of the Lewis lung cancer miceAfter the treatment according to the plan, we Selected the general condition of Control group mice as standard, and observed the indicators of mouse’s spirit state,activity, diet, weight and tumor volume changes to evaluate chemotherapy side effects and life quality.Compared with Control group, in TMP group, the mice spirit state、eating、activities were basically normal, the growth of weight and tumor volume slowed down. The DDP group mice gradually performed the poor mental state,diet and activities significantly reduced、gray color without burnish、huddled、got together,Compared with TMP group, tumor volume were smaller and weight-loss were the most obvious. Combined TMP and DDP group mice’s general condition was better than DDP group, but worse than TMP group.The results showed that TMP group were no obvious toxicity reaction, life quality was the best, Combination group were significantly lower than the toxicity of cisplatin, life quality was better than cisplatin group, while the cisplatin group toxic side reaction was heaviest, life quality was the worst.Suggesting that combination therapy can reduce the toxic side reaction of cisplatin,application TMP after DDP in the treatment of Lewis lung cancer mice could improve the quality of life in mice.2 Calculating tumor growth inhibition rate and tumor necrosis rateCalculated tumor growth inhibition rate and tumor necrosis rate after treatment:TMP group(35.58%), DDP group(40.41%)and Combination group(62.48%).The inhibition rate of tumors in the groups above were all higher than the inhibition rate in control group.The tumor necrosis rate of mice:TMP group(15.58±3.19),DDP group( 24.64±11.96)and combined TMP and DDP therapy group(31.76±15.20).The tumor necrosis rate in the groups above were all higher than those in control group(9.37 ±1.36).Compared with DDP group,the tumor growth inhibition rate and tumor necrosis rate in combination group were significantly higher(P <0.05),and the DDP group was better than the TMP group(P<0.05).Showing that combined TMP and DDP therapy group Can more enhance anti-tumor efficacy than DDP group.3 Expression of VEGF, ADAMTS1 and MVD in Lewis lung carcinoma TissuesAfter removed all the tumor organization, the expression of VEGF and ADAMTS1 were observed by immunohistochemistry and Western Blot.The results showed with immunohistochemical methods : the four groups were visible the positive expression of VEGF.Compared with Control group(56367.82±7441.12),TMP group(28674.58±2331.40),DDP group(23932.76 ± 3151.59) and Combination group(19183.99 ±1453.64)decreased significantly. Combination group expressed the lowest,there was a significant difference(P< 0.05).So as ADAMTS1 positiveexpression in the four groups, TMP group(15475.27 ± 2064.88),DDP group( 18382.79±2162.08)and combined Combination g group(26403.85± 3293.36)were higher than those in control group(1646.74 ± 212.96).Combination group expressed the highest, and Control group expressed the lowest,there was a significant difference(P< 0.05). The result showed by Western Blot assay:the expression of ADAMTS1 after treatment,TMP group(0.57±0.02,DDP group(0.79±0.01) and combined Combination g group(0.98±0.02),compared with Control group(0.48±0.01),there was a significant difference(P< 0.05),so as VEGF in Combination group expressed the highest(0.87±0.03), and Control group expressed the lowest(0.28 ± 0.01).Showing that they had the same trend between immunohistochemistry and Western Blot assay,the results demonstrated that TMP, DDP could decreased VEGF expression and increased ADAMTS1 expression, their combination might further enhance ADAMTS1 expression and inhibit VEGF expression.4 Detection of MVD in tumor origantionDetected CD105 with immunohistochemistry. Obseved the blood vessels within the tumor tissue with light microscope, and the last counted microvascular density(MVD) with the Weidner method. Having taken the average of the five high power fields,the results suggested : Control group expressed the highest(4.9×104 ± 7.3×103)and Combination group expressed the lowest(1.4×104±6.2×103).5 The observation of the tumor cells and vascular structures with electron microscopeThe results were observed through electron microscopy examination,in Control group the tumor cells could be seen more, large nucleus with obvious nucleoli,karyogram of irregular, there were abundant cellar organs in the cytoplasm,such as mitochondria, endoplasmic reticulum,the tumor cell apoptosis rared.in TMP group we found more fatty degeneration in the tumor cells.Lipid droplets in the cytoplasm increased,the cell membrane uncompleted,mitochondrias being vacuolated,we could found more Necrotic cells and its debris,reduction of cellular organ.In DDP group and Combination group,the tumor cells rared,mitochondria suspension in part of the tumor cells in cytoplasm,more cell debris could be seen.Tumor cell apoptosis was common,casual formation of apoptotic bodies.In conclusion, the Lewis lung carcinoma model of C57BL/6 were produced by intraperitoneal injection of Lewis lung cancer cells in right axillary, all could develop tumor about 8~10 days.Observed the general condition of the mice, weight, volume and microvascular density change,the expression of angiogenesis-related factor, tumor cell and the comparison of tumor necrosis rateand tumor inhibition rate, it is proved that application TMP after DDP in the treatment of nude mice can significantly improve the quality of life in Lewis lung cancer mice,. TMP has synergetic inhibitory effect with DDP on transplanted tumor growth of Lewis lung carcinoma in mice,the action mechanism may be due to down-regulate the VEGF expression and up-regulate the ADAMTS1 expression,inhibiting tumor angiogenesis. |
PDF Full Text Request