祛瘀化痰法抗肺癌转移的分子机制研究
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摘要
研究背景和目的：肺癌是发病率、死亡率最高的恶性肿瘤之一。80-90%的肺癌患者死亡是由转移所致。侵袭与转移是恶性肿瘤的本质特征之一,也是导致患者治疗失败和死亡的主要原因。因此,控制转移是决定癌症患者预后的关键因素。近些年来,国内外在肿瘤侵袭转移的研究上取得了较大进展,阐明了肿瘤侵袭转移的某些关键环节和机理,研制出了一些预防转移的药物,但至今尚无一种广泛用于临床预防肿瘤转移的药物。中医药疗法在肿瘤的治疗中有着悠久的历史和重要的地位。在长期医疗实践中,传统中医学形成了独特的恶性肿瘤防治的理论体系。祛瘀化痰法是恶性肿瘤尤其是肺癌的治疗大法之一。现代的有关实验研究显示：活血祛瘀、化痰散结药物是否有抗肿瘤及转移的作用仍存在较大的分歧。故本课题探讨祛瘀化痰法抗肺癌转移的分子机制有着现实意义。
本课题在传统中医理论的指导下,本着辨证与辨病相结合、宏观辨证与微观辨证相结合的原则,通过“以方示法、以方测证”的原理。采用传统中医经典的祛瘀方(桃红四物汤)合化痰方(二陈汤)的祛瘀化痰法分别对西医的经典小鼠Lewis肺癌转移模型及人高转移性肺癌95-D细胞进行体内实验和体外实验,探讨祛瘀化痰法抗肺癌转移的分子机制。同时运用传统中医“证治”反证的思路,尝试探寻肺癌痰证及血瘀证的生物学本质,为传统中医理论创新及中医现代化提供积极的思路和借鉴。
材料和方法：
第一部分体内动物实验
1、建立小鼠Lewis肺癌移植瘤模型
2、动物模型随机分为8组：生理盐水对照组、祛瘀化痰方高剂量组、祛瘀化痰方低级剂量组、祛瘀方高剂量组、祛瘀方低剂量组、化痰方高剂量组、化痰方低剂量组及顺铂组,每组10只小鼠。分别用药21天实验中定期测量移植瘤体积变化及各组小鼠体重变化情况。21天后处死各组小鼠,称小鼠体重,剥取瘤块,分别称瘤重,计算实验组抑瘤率,作移植瘤病理组织切片。取小鼠肺组织计算小鼠肺表面转移瘤结节数,计算实验组肺转移抑制率。作病理组织切片观察肺组织的病理改变。
3、SABC免疫组化法检测各组Lewis肺癌移植瘤组织CD31蛋白的表达,计数各组荷瘤小鼠瘤组织微血管密度(MVD)。
4、SABC免疫组化法检测各组Lewis肺癌移植瘤组织肿瘤转移相关分子(TF、MMP-2、VEGF、E-cad、CD44)蛋白水平的表达情况。
第二部分体外细胞实验
1、制备祛瘀化痰方、祛瘀方及化痰方的中药含药血清。体外培养人高转移性95-D细胞。
2、MTT法检测祛瘀化痰方对人肺癌95-D细胞增殖的影响
3、采用粘附分析实验检测祛瘀化痰方对人肺癌95-D细胞同质粘附、及其与人脐静脉内皮细胞异质粘附的影响。
4、细胞划痕(Wound healing)实验检测祛瘀化痰方对人肺癌95-D细胞迁移的影响
5、Transwell侵袭实验检测祛瘀化痰方对人肺癌95-D细胞侵袭的影响
6、实时荧光定量PCR法检测祛瘀化痰方对人肺癌95-D细胞肿瘤转移相关分子(TF、MMP-2、VEGF、E-cad、CD44) mRNA表达的影响
结果：
第一部分体内动物实验
1、各组小鼠体重变化：除顺铂组外其他各组小鼠体重均逐渐增加。而顺铂组总体趋势是体重逐渐下降后有上升趋势,但到实验结束时,体重仍没能回到实验开始时的体重。
2、各组小鼠移植瘤体积变化：顺铂组的移植瘤体积增长最慢,祛瘀化痰高剂量组次之,生理盐水对照组的移植瘤体积增长最快。
3、各实验组的抑瘤率：祛瘀化痰方高、低剂量组分别为38.02%、24.11%,祛瘀方高、低剂量组分别为19.28%、14.25%,化痰方高、低剂量组分别为10.74%、4.96%。且表现出一定的剂量依赖性。顺铂组的抑瘤率为59.22%。
4、各实验组的肺转移抑制率：祛瘀化痰方高、低剂量组的肺转移抑制率分别为39.77%、29.25%；化痰高、低剂量组的肺转移抑制率分别为20.92%、16.42%；且呈一定的量效关系。祛瘀高、低剂量组的肺转移抑制率分别是：11.91%、20.69%。
5、各实验组的小鼠移植瘤组织微血管的影响：祛瘀化痰方高、低剂量组,祛瘀方高剂量组和化痰方高剂量组有一定的抑制血管生成的作用。但祛瘀方低剂量组和化痰方低级量组却没有显示明显抑制肿瘤血管生成的作用。
6、各实验组移植瘤组织肿瘤转移相关分子(TF、MMP-2、VEGF、 E-cad、CD44)蛋白水平表达：祛瘀化痰方高低剂量组均可升高Lewis肺癌移植瘤组织E-cadherin蛋白的表达而降低CD44、MMP-2、VEGF、TF蛋白的表达。祛瘀方高、低剂量组主要能降低Lewis肺癌移植瘤组织TF、MMP-2、VEGF蛋白的表达。而化痰方高、低剂量组主要能升高E-cadherin蛋白的表达而降低CD44蛋白的表达。
第二部分体外细胞实验
1、各实验组对95-D细胞的增殖抑制率：祛瘀化痰方高、低剂量组分别是：31.49%(24h)、36.35%(48h)和25.32%(24h)、28.14%(48h)；祛瘀方高、低剂量组分别是：15.98%(24h)、21.96%(48h)和10.60%(24h)、19.72%(48h)；化痰方高、低剂量组分别是：4.43%(24h)、10.34%(48h)和0.79%(24h)、7.36%(48h)；
2、各实验组对95-D细胞同质粘附的影响：祛瘀化痰方低剂量组、化痰方高剂量组促进95-D细胞同质黏附(P<0.05)；祛瘀方高剂量组可降低95-D细胞同质黏附(P<0.05)。
3、各实验组对95-D细胞异质粘附的影响：祛瘀化痰方高、低剂量组、祛瘀方高、低剂量组及化痰方高、低剂量组均可抑制95-D与HUVEC的黏附(P<0.05),且存在一定的量效关系。祛瘀化痰方高低剂量组较祛瘀方和化痰方组抑制粘附作用更强。
4、各实验组对95-D细胞迁移运动的影响：祛瘀化痰方高、低剂量组、祛瘀高剂量组及化痰方高剂量组均能抑制95-D细胞的迁移运动(P<0.05,P<0.01)。
5、各实验组对95-D细胞侵袭能力的影响：祛瘀化痰方高低剂量组、祛瘀低剂量组、化痰高低剂量组均能够抑制95-D的侵袭能力(P<0.05,P<0.01)。
6、各实验组对95-D细胞肿瘤转移相关基因(TF、MMP-2、VEGF、 E-cad、CD44)转录水平的影响：
1)、祛瘀化痰方高剂量组、祛瘀方高剂量组及化痰方高剂量组均可抑制95-D细胞TF mRNA的表达(P<0.01)。化痰方低剂量组能促进95-D细胞TF mRNA的表达(P<0.01)。
2)、祛瘀化痰方高剂量组和祛瘀方低剂量组可以抑制95-D细胞MMP-2mRNA的表达(P<0.01)。祛瘀方高剂量组可以促进95-D细胞MMP-2mRNA的表达(P<0.01)。
3)、祛瘀化痰方高、低剂量组、祛瘀方高、低剂量组及化痰方高、低剂量组均抑制95-D细胞VEGF mRNA的表达(P<0.01)。且存在一定的一定剂量依赖性。
4)、祛瘀化痰方高、低剂量组、祛瘀方高、低剂量组及化痰方高、低剂量组均促进95-D细胞E-cad mRNA的表达(P<0.01)。但各方低剂量组作用较高剂量组明显。
5)、祛瘀化痰方低剂量组、祛瘀方低剂量组、化痰方低剂量组均促进95-D细胞CD44mRNA的表达(P<0.01)。祛瘀化痰方高剂量组能抑制95-D细胞CD44mRNA的表达(P<0.01)。
结论：
1、祛瘀化痰方可抑制肺癌的生长和转移,对肺癌转移的关键环节：肿瘤细胞增殖、肿瘤细胞黏附、细胞外基质降解、肿瘤细胞迁移和侵袭、肿瘤血管生成等均有不同程度的作用。
2、祛瘀方和化痰方在抗肺癌转移过程中并非作用于每一个肿瘤转移环节,不同方剂可能作用于不同的肿瘤转移环节,且同一方剂在不同肿瘤转移环节发挥作用不同,在有的环节可能是抑制转移,而在有的环节却可能是促进转移。但祛瘀化痰方整体上是抑制肺癌的生长和转移。
3、祛瘀化痰法抗肺癌转移作用机制可能在于影响肺癌细胞TFMMP-2、VEGF、E-cad、CD44蛋白合成和基因转录两个水平的表达,进而影响肿瘤细胞的增殖、粘附、迁移和侵袭,以及影响细胞外基质的降解和肿瘤血管的生成。祛瘀化痰法从多步骤、多环节、多靶点抑制肺癌转移。
4、肺癌血瘀证的实质可能为肺癌细胞异常表达的TF、VEGF、MMP-2所形成的分子网络。
5、肺癌痰证的实质可能是肺癌细胞异常表达的E-cad、CD44等粘附分子形成的分子网络。
Research Background and Objective:
Lung cancer is one of the malignant tumors with the highest rate of incidence and mortality, and80-90%death of patients with lung cancer is caused by the metastasis. Invasion&metastasis is one of the essential characteristics of malignant tumors, but also the major cause for the treatment failure and mortality.
Therefore, the metastasis control is a key factor to determine the prognosis of cancer patients. In recent years, the studies of tumor invasion and metastasis have made great progress both at home and abroad, they have clarified some of the key points and mechanism of tumor invasion and metastasis, and have developed several kinds of drugs to prevent diversion, but so far no drugs have been widely applied in the clinical prevention of tumor metastasis.
Chinese medicine has a long history and an important position in the treatment of tumors. In the long-term medical practice, traditional Chinese medicine formed a unique theoretical system of prevention and treatment of malignant tumors. The blood-stasis-removing and phlegm-resolving therapy is an important method for the treatment of malignant tumors, especially for the lung cancer treatment.
Modern experimental studies have shown that controversies still exist over the anti-tumor and metastasis effects of the medicines for blood-stasis removing and phlegm resolving. Thus the exploration of the molecular mechanism of blood-stasis-removing and phlegm-resolving therapy's resistance to the lung cancer metastasis is practically significant.
This study was conducted under the guidance of traditional Chinese medicine theory, and followed the principle of the combination of the syndrome differentiation and disease differentiation, and combination of macro syndrome and microcosmic syndrome.
We conducted in vivo and in vitro experiments with the blood-stasis-removing therapy (Taohong Siwu decoction) and phlegm-resolving therapy (Erchen decotion) to study the classical mouse Lewis lung carcinoma metastasis model and human highly metastatic lung carcinoma95-D cells, and to explore the molecular mechanism of blood-stasis-removing and phlegm-resolving therapy's resistance to the lung cancer metastasis.
Meanwhile, we applied the idea of disproval of the'syndrome and therapy' in Traditional Chinese Medicine (TCM) to the attempt at searching for the biological essence of lung cancer with blood stasis syndrome and phlegm syndrome, offering positive method and reference to the innovation and modernization of TCM
Materials and Methods:
The first section:Experiments in vivo
1、Building mouse Lewis lung carcinoma metastasis model
2、Animal models were randomly divided into eight groups,10mice per group:saline control group, high-dose blood-stasis-removing and phlegm-resolving therapy group, low-dose blood-stasis-removing and phlegm-resolving therapy group, high-dose blood-stasis-removing therapy group, low-dose blood-stasis-removing therapy group, high-dose phlegm-resolving therapy group, low-dose phlegm-resolving therapy group, and cisplatin group.
3Medications were respectively given to the8groups for21days. Periodic measurements of changes in the transplanted tumor's volume and mice's weights in each group were conducted during the experiment. After21days, mice in each group were killed and weighed, then, tumor masses were taken off and weighed for calculating the inhibition rates of the experimental groups and histopathological section of transplanted tumor were made.
4The lungs of mice were taken to calculate the number of metastases tumor nodules on lung surface, and then, the inhibition rates of lung metastasis in experimental groups were calculated. The histopathological sections of lungs were made to observe the pathological changes in lungs.
5With the SABC immunohistochemical method, detection of expression of CD31in Lewis lung carcinoma transplanted tumor was conducted and the micro vessel density (MVD) of the tumor in tumor-bearing mice is calculated.
6The expressions of molecules (TF and MMP-2and VEGF in E-cad, CD44) related to transplanted tumor in Lewis lung carcinoma on protein level was detected with SABC immunohistochemical method.
The second section:Experiment in vitro
^Preparations for the serums containing the blood-stasis-removing and phlegm-resolving therapy or blood-stasis-removing therapy or phlegm-resolving therapy were made. Human highly metastatic95-D cell were cultivated in vitro.
2,The impact that the blood-stasis-removing and phlegm-resolving therapy might have on human lung cancer95-D cell proliferation was detected with MTT method.
3, Adhesive analysis experiment was conducted to detect the effect that the blood-stasis-removing and phlegm-resolving therapy might have on the human lung cancer95-D cell's homotypic adhesion and on the heterotypic adhesion to human umbilical vein endothelial cells (HUVEC).
4, Wound healing experiment was applied to the detection of the impact on the human lung cancer95-D cell's migration made by the blood-stasis-removing and phlegm-resolving therapy
5, Transwell invasiveness method was used to detect the impact that the blood-stasis-removing and phlegm-resolving therapy might have on human lung cancer95-D cell.
6, The impact on the expression of mRNA of the molecules (TF and MMP-2and VEGF in E-cad, CD44) related to transplanted tumor in human lung cancer95-D cells made by the blood-stasis-removing and phlegm-resolving therapy was investigated by the real-time PCR
Results
The first section:Experiment in vitro
1, Weight changes of the mice in every group:except the cisplatin group, the weights of the mice in each group increased gradually. The overall trend of weight in the cisplatin group was that it deceased first and that it tended to increase later, but the weight hasn't returned to that of the beginning of the experiment till the end.
2, Changes in the volumes of transplanted tumors in each group:volume of transplanted tumor in saline control group was the fastest-growing one, followed by high-dose blood-stasis-removing and phlegm-resolving therapy group and cisplatin group.
3, The tumor inhibition rate in each group:the rates of the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups were38.02%and24.11%respectively and those of the high-and low-dose blood-stasis-removing therapy groups were19.28%and14.25%, while the high-and low-dose phlegm-resolving therapy groups got the inhibition rates of10.74%and4.96%, presenting certain dose-dependent manner. The inhibition rate in Cisplatin group was59.22%.
4, Lung metastasis inhibition rates in each experimental group:the Lung metastasis inhibition rates in the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups were39.77%and29.25%and those of the high-and low-dose phlegm-resolving therapy groups were20.92%and16.42%, and it also presented certain dose-dependent manner. The lung metastasis inhibition rates in high-and low-dose the blood-stasis-removing therapy groups were11.91%and20.69%respectively.
5, The effect on micro vessel of the transplanted tumor in the every experimental group:high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups, high-dose blood-stasis-removing therapy and high-dose phlegm-resolving therapy groups could inhibit the angiogenesis. Whereas the inhibitory effects of the low-dose blood-stasis-removing therapy group and phlegm-resolving therapy group were not conspicuous.
6, The expressions of molecules (TF and MMP-2and VEGF in E-cad, CD44) related to transplanted tumor on protein level:both the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups could improve the expression of E-cadherin of transplanted tumor in lung cancer and lower that of the CD44, MMP-2, VEGF and TF.
7, The high-and low-dose blood-stasis-removing therapy groups mainly reduced the expression of TF, MMP-2and VEGF in transplanted tumor in Lewis lung carcinoma; The high-and low-dose phlegm-resolving therapy groups mainly enhanced the expression of E-cadherin whereas lessened that of CD44.
The second section:Experiment in vitro
1, Inhibition rates for95-D cell proliferation in each experimental group: In the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups, the rates were31.49%(24h),36.35%(48h) and25.32%(24h),28.14%(48h), and those of high-and low-dose blood-stasis-removing therapy groups were15.98%(24h),21.96%(48h) and10.60%(24h),19.72%(48h), and in the high-and low-dose phlegm-resolving therapy groups, the rates decreased to4.43%(24h),10.34%(48h) and0.79%(24h),7.36%(48h)
2, The impact on the95-D cell's homotypic adhesion in each experimental group:the95-D cell's homotypic adhesion was improved in the low-dose blood-stasis-removing and phlegm-resolving therapy group and the high-dose phlegm-resolving therapy groups (P<0.01), while the high-dose blood-stasis-removing therapy group reduced the95-D cell's homotypic adhesion (P<0.05).
3, The impact on the95-D cell's heterotypic adhesion in each experimental group:The high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-and low-dose phlegm-resolving therapy groups restrained the adhesion of95-D and HUVEC (P<0.01), and there existed the certain dose-effect relationship. Compared with the blood-stasis-removing therapy group and the phlegm-resolving therapy group, the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups had a stronger effect on inhibiting the adhesion.
4, The impact on the migration of95-D cell in each experimental group: the high-dose and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-dose blood-stasis-removing therapy and phlegm-resolving therapy groups inhibited the migration of95-D cell (P<0.05, P<0.01).
5, The impact on the invasiveness of the95-D cell in each experimental group:the high-dose and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-dose blood-stasis-removing therapy and phlegm-resolving therapy groups all repressed the invasiveness of95-D cell (P<0.05, P<0.01).
6, The Impact on the transcription of genes(TF、MMP-2、VEGF、E-cad. CD44) related to the95-D cell tumor metastasis:
1), The expression of TF mRNA in95-D cell was inhibited in the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-dose blood-stasis-removing therapy and phlegm-resolving therapy groups(P<0.01). The low-dose phlegm-resolving therapy group facilitated the expression of TF mRNA in95-D cell (P<0.01).
2), The high-dose blood-stasis-removing and phlegm-resolving therapy group and the low-dose blood-stasis-removing therapy group inhibited the expression of MMP-2mRNA in95-D cell (P<0.01). The high-dose blood-stasis removing therapy promoted the expression of MMP-2mRNA in95-D cell (P<0.01)
3), The expression of VEGF mRNA in95-D cell was inhibited in the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-and low-dose blood-stasis-removing therapy groups and the high-and low-dose phlegm-resolving therapy groups (P<0.01), and there existed certain dose-dependent manner.
4), The expression of E-cad mRNA in95-D was improved in the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-and low-dose blood-stasis-removing therapy groups and the high-and low-dose phlegm-resolving therapy groups (P<0.01). The low-dose therapy groups, however, presented more obvious effects than high-dose groups.
5),The expression of CD44mRNA was improved in low-dose blood-stasis-removing and phlegm-resolving therapy and low-dose blood-stasis-removing therapy and low-dose phlegm-resolving therapy groups (P<0.01). The expression of CD44mRNA was inhibited in the high-dose blood-stasis-removing and phlegm-resolving therapy group (P<0.01).
Conclusion
1, The blood-stasis-removing and phlegm-resolving therapy can inhibit the growth and metastasis of lung cancer, and have different degrades of effects on the tumor cell proliferation which is the key point in metastasis of lung cancer, on adhesion of tumor cell, on the degradation of extracellular matrix, on the migration and invasion of tumor cell, and on tumor angiogenesis.
2, Blood-stasis-removing therapy and phlegm-resolving therapy not act on every step during the tumor metastasis, and different therapy may act on different steps of the metastasis. Besides, the same therapy exerts dissimilar function in different steps of the metastasis:it inhibits the metastasis in some steps while improves in others. On the whole, however, the blood-stasis-removing and phlegm-resolving therapy inhibits the growth and metastasis of lung cancer.
3,The mechanism of the blood-stasis-removing and phlegm-resolving therapy' resistance to the lung cancer metastasis probably is that the therapy affects the expressions of TF, MMP-2, VEGF, E-cad and CD44in lung tumor cells on the level of protein synthesis and gene transcription, thus impacting on the proliferation, adhesion, migration and invasion of tumor cell, and on the tumor angiogenesis and degradation of extracellular matrix. The therapy can inhibit the lung tumor metastasis through multi-process, multi-step, multi-target.
4,The essence of lung cancer with blood-stasis syndrome may lie in the molecular network formed by abnormal expressions of TF, VEGF and MMP-2in lung tumor cells.
5,The essence of lung cancer with phlegm syndrome may be the molecular network formed by the abnormal expressions of adhesion molecules such as E-cadherin and CD44.
本课题在传统中医理论的指导下,本着辨证与辨病相结合、宏观辨证与微观辨证相结合的原则,通过“以方示法、以方测证”的原理。采用传统中医经典的祛瘀方(桃红四物汤)合化痰方(二陈汤)的祛瘀化痰法分别对西医的经典小鼠Lewis肺癌转移模型及人高转移性肺癌95-D细胞进行体内实验和体外实验,探讨祛瘀化痰法抗肺癌转移的分子机制。同时运用传统中医“证治”反证的思路,尝试探寻肺癌痰证及血瘀证的生物学本质,为传统中医理论创新及中医现代化提供积极的思路和借鉴。
材料和方法：
第一部分体内动物实验
1、建立小鼠Lewis肺癌移植瘤模型
2、动物模型随机分为8组：生理盐水对照组、祛瘀化痰方高剂量组、祛瘀化痰方低级剂量组、祛瘀方高剂量组、祛瘀方低剂量组、化痰方高剂量组、化痰方低剂量组及顺铂组,每组10只小鼠。分别用药21天实验中定期测量移植瘤体积变化及各组小鼠体重变化情况。21天后处死各组小鼠,称小鼠体重,剥取瘤块,分别称瘤重,计算实验组抑瘤率,作移植瘤病理组织切片。取小鼠肺组织计算小鼠肺表面转移瘤结节数,计算实验组肺转移抑制率。作病理组织切片观察肺组织的病理改变。
3、SABC免疫组化法检测各组Lewis肺癌移植瘤组织CD31蛋白的表达,计数各组荷瘤小鼠瘤组织微血管密度(MVD)。
4、SABC免疫组化法检测各组Lewis肺癌移植瘤组织肿瘤转移相关分子(TF、MMP-2、VEGF、E-cad、CD44)蛋白水平的表达情况。
第二部分体外细胞实验
1、制备祛瘀化痰方、祛瘀方及化痰方的中药含药血清。体外培养人高转移性95-D细胞。
2、MTT法检测祛瘀化痰方对人肺癌95-D细胞增殖的影响
3、采用粘附分析实验检测祛瘀化痰方对人肺癌95-D细胞同质粘附、及其与人脐静脉内皮细胞异质粘附的影响。
4、细胞划痕(Wound healing)实验检测祛瘀化痰方对人肺癌95-D细胞迁移的影响
5、Transwell侵袭实验检测祛瘀化痰方对人肺癌95-D细胞侵袭的影响
6、实时荧光定量PCR法检测祛瘀化痰方对人肺癌95-D细胞肿瘤转移相关分子(TF、MMP-2、VEGF、E-cad、CD44) mRNA表达的影响
结果：
第一部分体内动物实验
1、各组小鼠体重变化：除顺铂组外其他各组小鼠体重均逐渐增加。而顺铂组总体趋势是体重逐渐下降后有上升趋势,但到实验结束时,体重仍没能回到实验开始时的体重。
2、各组小鼠移植瘤体积变化：顺铂组的移植瘤体积增长最慢,祛瘀化痰高剂量组次之,生理盐水对照组的移植瘤体积增长最快。
3、各实验组的抑瘤率：祛瘀化痰方高、低剂量组分别为38.02%、24.11%,祛瘀方高、低剂量组分别为19.28%、14.25%,化痰方高、低剂量组分别为10.74%、4.96%。且表现出一定的剂量依赖性。顺铂组的抑瘤率为59.22%。
4、各实验组的肺转移抑制率：祛瘀化痰方高、低剂量组的肺转移抑制率分别为39.77%、29.25%；化痰高、低剂量组的肺转移抑制率分别为20.92%、16.42%；且呈一定的量效关系。祛瘀高、低剂量组的肺转移抑制率分别是：11.91%、20.69%。
5、各实验组的小鼠移植瘤组织微血管的影响：祛瘀化痰方高、低剂量组,祛瘀方高剂量组和化痰方高剂量组有一定的抑制血管生成的作用。但祛瘀方低剂量组和化痰方低级量组却没有显示明显抑制肿瘤血管生成的作用。
6、各实验组移植瘤组织肿瘤转移相关分子(TF、MMP-2、VEGF、 E-cad、CD44)蛋白水平表达：祛瘀化痰方高低剂量组均可升高Lewis肺癌移植瘤组织E-cadherin蛋白的表达而降低CD44、MMP-2、VEGF、TF蛋白的表达。祛瘀方高、低剂量组主要能降低Lewis肺癌移植瘤组织TF、MMP-2、VEGF蛋白的表达。而化痰方高、低剂量组主要能升高E-cadherin蛋白的表达而降低CD44蛋白的表达。
第二部分体外细胞实验
1、各实验组对95-D细胞的增殖抑制率：祛瘀化痰方高、低剂量组分别是：31.49%(24h)、36.35%(48h)和25.32%(24h)、28.14%(48h)；祛瘀方高、低剂量组分别是：15.98%(24h)、21.96%(48h)和10.60%(24h)、19.72%(48h)；化痰方高、低剂量组分别是：4.43%(24h)、10.34%(48h)和0.79%(24h)、7.36%(48h)；
2、各实验组对95-D细胞同质粘附的影响：祛瘀化痰方低剂量组、化痰方高剂量组促进95-D细胞同质黏附(P<0.05)；祛瘀方高剂量组可降低95-D细胞同质黏附(P<0.05)。
3、各实验组对95-D细胞异质粘附的影响：祛瘀化痰方高、低剂量组、祛瘀方高、低剂量组及化痰方高、低剂量组均可抑制95-D与HUVEC的黏附(P<0.05),且存在一定的量效关系。祛瘀化痰方高低剂量组较祛瘀方和化痰方组抑制粘附作用更强。
4、各实验组对95-D细胞迁移运动的影响：祛瘀化痰方高、低剂量组、祛瘀高剂量组及化痰方高剂量组均能抑制95-D细胞的迁移运动(P<0.05,P<0.01)。
5、各实验组对95-D细胞侵袭能力的影响：祛瘀化痰方高低剂量组、祛瘀低剂量组、化痰高低剂量组均能够抑制95-D的侵袭能力(P<0.05,P<0.01)。
6、各实验组对95-D细胞肿瘤转移相关基因(TF、MMP-2、VEGF、 E-cad、CD44)转录水平的影响：
1)、祛瘀化痰方高剂量组、祛瘀方高剂量组及化痰方高剂量组均可抑制95-D细胞TF mRNA的表达(P<0.01)。化痰方低剂量组能促进95-D细胞TF mRNA的表达(P<0.01)。
2)、祛瘀化痰方高剂量组和祛瘀方低剂量组可以抑制95-D细胞MMP-2mRNA的表达(P<0.01)。祛瘀方高剂量组可以促进95-D细胞MMP-2mRNA的表达(P<0.01)。
3)、祛瘀化痰方高、低剂量组、祛瘀方高、低剂量组及化痰方高、低剂量组均抑制95-D细胞VEGF mRNA的表达(P<0.01)。且存在一定的一定剂量依赖性。
4)、祛瘀化痰方高、低剂量组、祛瘀方高、低剂量组及化痰方高、低剂量组均促进95-D细胞E-cad mRNA的表达(P<0.01)。但各方低剂量组作用较高剂量组明显。
5)、祛瘀化痰方低剂量组、祛瘀方低剂量组、化痰方低剂量组均促进95-D细胞CD44mRNA的表达(P<0.01)。祛瘀化痰方高剂量组能抑制95-D细胞CD44mRNA的表达(P<0.01)。
结论：
1、祛瘀化痰方可抑制肺癌的生长和转移,对肺癌转移的关键环节：肿瘤细胞增殖、肿瘤细胞黏附、细胞外基质降解、肿瘤细胞迁移和侵袭、肿瘤血管生成等均有不同程度的作用。
2、祛瘀方和化痰方在抗肺癌转移过程中并非作用于每一个肿瘤转移环节,不同方剂可能作用于不同的肿瘤转移环节,且同一方剂在不同肿瘤转移环节发挥作用不同,在有的环节可能是抑制转移,而在有的环节却可能是促进转移。但祛瘀化痰方整体上是抑制肺癌的生长和转移。
3、祛瘀化痰法抗肺癌转移作用机制可能在于影响肺癌细胞TFMMP-2、VEGF、E-cad、CD44蛋白合成和基因转录两个水平的表达,进而影响肿瘤细胞的增殖、粘附、迁移和侵袭,以及影响细胞外基质的降解和肿瘤血管的生成。祛瘀化痰法从多步骤、多环节、多靶点抑制肺癌转移。
4、肺癌血瘀证的实质可能为肺癌细胞异常表达的TF、VEGF、MMP-2所形成的分子网络。
5、肺癌痰证的实质可能是肺癌细胞异常表达的E-cad、CD44等粘附分子形成的分子网络。
Research Background and Objective:
Lung cancer is one of the malignant tumors with the highest rate of incidence and mortality, and80-90%death of patients with lung cancer is caused by the metastasis. Invasion&metastasis is one of the essential characteristics of malignant tumors, but also the major cause for the treatment failure and mortality.
Therefore, the metastasis control is a key factor to determine the prognosis of cancer patients. In recent years, the studies of tumor invasion and metastasis have made great progress both at home and abroad, they have clarified some of the key points and mechanism of tumor invasion and metastasis, and have developed several kinds of drugs to prevent diversion, but so far no drugs have been widely applied in the clinical prevention of tumor metastasis.
Chinese medicine has a long history and an important position in the treatment of tumors. In the long-term medical practice, traditional Chinese medicine formed a unique theoretical system of prevention and treatment of malignant tumors. The blood-stasis-removing and phlegm-resolving therapy is an important method for the treatment of malignant tumors, especially for the lung cancer treatment.
Modern experimental studies have shown that controversies still exist over the anti-tumor and metastasis effects of the medicines for blood-stasis removing and phlegm resolving. Thus the exploration of the molecular mechanism of blood-stasis-removing and phlegm-resolving therapy's resistance to the lung cancer metastasis is practically significant.
This study was conducted under the guidance of traditional Chinese medicine theory, and followed the principle of the combination of the syndrome differentiation and disease differentiation, and combination of macro syndrome and microcosmic syndrome.
We conducted in vivo and in vitro experiments with the blood-stasis-removing therapy (Taohong Siwu decoction) and phlegm-resolving therapy (Erchen decotion) to study the classical mouse Lewis lung carcinoma metastasis model and human highly metastatic lung carcinoma95-D cells, and to explore the molecular mechanism of blood-stasis-removing and phlegm-resolving therapy's resistance to the lung cancer metastasis.
Meanwhile, we applied the idea of disproval of the'syndrome and therapy' in Traditional Chinese Medicine (TCM) to the attempt at searching for the biological essence of lung cancer with blood stasis syndrome and phlegm syndrome, offering positive method and reference to the innovation and modernization of TCM
Materials and Methods:
The first section:Experiments in vivo
1、Building mouse Lewis lung carcinoma metastasis model
2、Animal models were randomly divided into eight groups,10mice per group:saline control group, high-dose blood-stasis-removing and phlegm-resolving therapy group, low-dose blood-stasis-removing and phlegm-resolving therapy group, high-dose blood-stasis-removing therapy group, low-dose blood-stasis-removing therapy group, high-dose phlegm-resolving therapy group, low-dose phlegm-resolving therapy group, and cisplatin group.
3Medications were respectively given to the8groups for21days. Periodic measurements of changes in the transplanted tumor's volume and mice's weights in each group were conducted during the experiment. After21days, mice in each group were killed and weighed, then, tumor masses were taken off and weighed for calculating the inhibition rates of the experimental groups and histopathological section of transplanted tumor were made.
4The lungs of mice were taken to calculate the number of metastases tumor nodules on lung surface, and then, the inhibition rates of lung metastasis in experimental groups were calculated. The histopathological sections of lungs were made to observe the pathological changes in lungs.
5With the SABC immunohistochemical method, detection of expression of CD31in Lewis lung carcinoma transplanted tumor was conducted and the micro vessel density (MVD) of the tumor in tumor-bearing mice is calculated.
6The expressions of molecules (TF and MMP-2and VEGF in E-cad, CD44) related to transplanted tumor in Lewis lung carcinoma on protein level was detected with SABC immunohistochemical method.
The second section:Experiment in vitro
^Preparations for the serums containing the blood-stasis-removing and phlegm-resolving therapy or blood-stasis-removing therapy or phlegm-resolving therapy were made. Human highly metastatic95-D cell were cultivated in vitro.
2,The impact that the blood-stasis-removing and phlegm-resolving therapy might have on human lung cancer95-D cell proliferation was detected with MTT method.
3, Adhesive analysis experiment was conducted to detect the effect that the blood-stasis-removing and phlegm-resolving therapy might have on the human lung cancer95-D cell's homotypic adhesion and on the heterotypic adhesion to human umbilical vein endothelial cells (HUVEC).
4, Wound healing experiment was applied to the detection of the impact on the human lung cancer95-D cell's migration made by the blood-stasis-removing and phlegm-resolving therapy
5, Transwell invasiveness method was used to detect the impact that the blood-stasis-removing and phlegm-resolving therapy might have on human lung cancer95-D cell.
6, The impact on the expression of mRNA of the molecules (TF and MMP-2and VEGF in E-cad, CD44) related to transplanted tumor in human lung cancer95-D cells made by the blood-stasis-removing and phlegm-resolving therapy was investigated by the real-time PCR
Results
The first section:Experiment in vitro
1, Weight changes of the mice in every group:except the cisplatin group, the weights of the mice in each group increased gradually. The overall trend of weight in the cisplatin group was that it deceased first and that it tended to increase later, but the weight hasn't returned to that of the beginning of the experiment till the end.
2, Changes in the volumes of transplanted tumors in each group:volume of transplanted tumor in saline control group was the fastest-growing one, followed by high-dose blood-stasis-removing and phlegm-resolving therapy group and cisplatin group.
3, The tumor inhibition rate in each group:the rates of the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups were38.02%and24.11%respectively and those of the high-and low-dose blood-stasis-removing therapy groups were19.28%and14.25%, while the high-and low-dose phlegm-resolving therapy groups got the inhibition rates of10.74%and4.96%, presenting certain dose-dependent manner. The inhibition rate in Cisplatin group was59.22%.
4, Lung metastasis inhibition rates in each experimental group:the Lung metastasis inhibition rates in the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups were39.77%and29.25%and those of the high-and low-dose phlegm-resolving therapy groups were20.92%and16.42%, and it also presented certain dose-dependent manner. The lung metastasis inhibition rates in high-and low-dose the blood-stasis-removing therapy groups were11.91%and20.69%respectively.
5, The effect on micro vessel of the transplanted tumor in the every experimental group:high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups, high-dose blood-stasis-removing therapy and high-dose phlegm-resolving therapy groups could inhibit the angiogenesis. Whereas the inhibitory effects of the low-dose blood-stasis-removing therapy group and phlegm-resolving therapy group were not conspicuous.
6, The expressions of molecules (TF and MMP-2and VEGF in E-cad, CD44) related to transplanted tumor on protein level:both the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups could improve the expression of E-cadherin of transplanted tumor in lung cancer and lower that of the CD44, MMP-2, VEGF and TF.
7, The high-and low-dose blood-stasis-removing therapy groups mainly reduced the expression of TF, MMP-2and VEGF in transplanted tumor in Lewis lung carcinoma; The high-and low-dose phlegm-resolving therapy groups mainly enhanced the expression of E-cadherin whereas lessened that of CD44.
The second section:Experiment in vitro
1, Inhibition rates for95-D cell proliferation in each experimental group: In the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups, the rates were31.49%(24h),36.35%(48h) and25.32%(24h),28.14%(48h), and those of high-and low-dose blood-stasis-removing therapy groups were15.98%(24h),21.96%(48h) and10.60%(24h),19.72%(48h), and in the high-and low-dose phlegm-resolving therapy groups, the rates decreased to4.43%(24h),10.34%(48h) and0.79%(24h),7.36%(48h)
2, The impact on the95-D cell's homotypic adhesion in each experimental group:the95-D cell's homotypic adhesion was improved in the low-dose blood-stasis-removing and phlegm-resolving therapy group and the high-dose phlegm-resolving therapy groups (P<0.01), while the high-dose blood-stasis-removing therapy group reduced the95-D cell's homotypic adhesion (P<0.05).
3, The impact on the95-D cell's heterotypic adhesion in each experimental group:The high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-and low-dose phlegm-resolving therapy groups restrained the adhesion of95-D and HUVEC (P<0.01), and there existed the certain dose-effect relationship. Compared with the blood-stasis-removing therapy group and the phlegm-resolving therapy group, the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups had a stronger effect on inhibiting the adhesion.
4, The impact on the migration of95-D cell in each experimental group: the high-dose and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-dose blood-stasis-removing therapy and phlegm-resolving therapy groups inhibited the migration of95-D cell (P<0.05, P<0.01).
5, The impact on the invasiveness of the95-D cell in each experimental group:the high-dose and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-dose blood-stasis-removing therapy and phlegm-resolving therapy groups all repressed the invasiveness of95-D cell (P<0.05, P<0.01).
6, The Impact on the transcription of genes(TF、MMP-2、VEGF、E-cad. CD44) related to the95-D cell tumor metastasis:
1), The expression of TF mRNA in95-D cell was inhibited in the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-dose blood-stasis-removing therapy and phlegm-resolving therapy groups(P<0.01). The low-dose phlegm-resolving therapy group facilitated the expression of TF mRNA in95-D cell (P<0.01).
2), The high-dose blood-stasis-removing and phlegm-resolving therapy group and the low-dose blood-stasis-removing therapy group inhibited the expression of MMP-2mRNA in95-D cell (P<0.01). The high-dose blood-stasis removing therapy promoted the expression of MMP-2mRNA in95-D cell (P<0.01)
3), The expression of VEGF mRNA in95-D cell was inhibited in the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-and low-dose blood-stasis-removing therapy groups and the high-and low-dose phlegm-resolving therapy groups (P<0.01), and there existed certain dose-dependent manner.
4), The expression of E-cad mRNA in95-D was improved in the high-and low-dose blood-stasis-removing and phlegm-resolving therapy groups and the high-and low-dose blood-stasis-removing therapy groups and the high-and low-dose phlegm-resolving therapy groups (P<0.01). The low-dose therapy groups, however, presented more obvious effects than high-dose groups.
5),The expression of CD44mRNA was improved in low-dose blood-stasis-removing and phlegm-resolving therapy and low-dose blood-stasis-removing therapy and low-dose phlegm-resolving therapy groups (P<0.01). The expression of CD44mRNA was inhibited in the high-dose blood-stasis-removing and phlegm-resolving therapy group (P<0.01).
Conclusion
1, The blood-stasis-removing and phlegm-resolving therapy can inhibit the growth and metastasis of lung cancer, and have different degrades of effects on the tumor cell proliferation which is the key point in metastasis of lung cancer, on adhesion of tumor cell, on the degradation of extracellular matrix, on the migration and invasion of tumor cell, and on tumor angiogenesis.
2, Blood-stasis-removing therapy and phlegm-resolving therapy not act on every step during the tumor metastasis, and different therapy may act on different steps of the metastasis. Besides, the same therapy exerts dissimilar function in different steps of the metastasis:it inhibits the metastasis in some steps while improves in others. On the whole, however, the blood-stasis-removing and phlegm-resolving therapy inhibits the growth and metastasis of lung cancer.
3,The mechanism of the blood-stasis-removing and phlegm-resolving therapy' resistance to the lung cancer metastasis probably is that the therapy affects the expressions of TF, MMP-2, VEGF, E-cad and CD44in lung tumor cells on the level of protein synthesis and gene transcription, thus impacting on the proliferation, adhesion, migration and invasion of tumor cell, and on the tumor angiogenesis and degradation of extracellular matrix. The therapy can inhibit the lung tumor metastasis through multi-process, multi-step, multi-target.
4,The essence of lung cancer with blood-stasis syndrome may lie in the molecular network formed by abnormal expressions of TF, VEGF and MMP-2in lung tumor cells.
5,The essence of lung cancer with phlegm syndrome may be the molecular network formed by the abnormal expressions of adhesion molecules such as E-cadherin and CD44.
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