中频交变电场对小鼠黑色素瘤离体细胞和皮下移植瘤的作用研究
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摘要
恶性黑色素瘤(malignant melanoma, MM)是一种起源于神经嵴黑色素细胞的恶性肿瘤,该病90%原发于皮肤,少数发生于皮肤以外的部位。MM的发病率和死亡率在全球均呈上升趋势,其中发病率更以每年4%的速度递增,增长速度远远超过其他恶性肿瘤。虽然MM的发病率仅占皮肤恶性肿瘤的3%左右,但死亡构成比却达到75%。早期MM经扩大切除术后90%以上可治愈,而一旦出现远处转移,则中位生存期一般不超过1年。本病5年生存率在发达国家较高,但发展中国家仅为40%。与欧美国家相比,我国MM发病率虽不高,但一般在初诊时疾病多为晚期,预后极差。
目前MM的临床常用治疗手段有：手术治疗、放射治疗、化学治疗、免疫治疗以及靶向治疗等,这些常规治疗方法对一部分患者是非常有效的,但其特定适应症和禁忌症,以及伴随的毒副反应限制了其临床使用,且临床上经常观察到一些患者对以上常规治疗的敏感性较差。目前针对无法手术的局部晚期或远处转移患者的治疗仍然非常棘手,对于这类患者,临床结果显示现有的常规治疗较少能提高其总生存率。因此,如何提高MM的总体疗效,研究新的,低毒高效的治疗方式,仍然是临床及基础研究者们长期研究的课题。
肿瘤电疗是将电工技术应用于肿瘤治疗的一种创新性物理治疗手段,随着近年来生物电学研究的蓬勃发展,利用电场作为肿瘤的治疗手段已显示出良好的应用前景。参数不同、性质各异的各类电场已被广泛试用于肿瘤治疗,并且取得了良好的效果。比较成熟的治疗肿瘤的电场类型是通过热效应杀灭肿瘤细胞的射频或微波装置。近年来关于单纯利用电场非热物理特性治疗肿瘤的报道逐渐增多,其中最具有代表性的是中频交变电场(intermediate-frequency alternating electricfield, IF-AEF)。一般认为,低频(<1kHz)电场的主要效应为刺激可兴奋组织,常用于刺激骨组织生长或促进骨折愈合；高频(MHz级以上)电场的电刺激作用基本消失而主要产生热效应,在以往的研究中常被用于热疗或消融治疗；而频率在100kHz-1MHz的IF-AEF,长期以来被认为不会产生明显的生物效应。但近期有报道指出在一定条件下IF-AEF也可产生一定的抗肿瘤效应,之所以以往未能发现其抗肿瘤效应,是由于其抗肿瘤效应具有极强的频率依赖性,对于特定的肿瘤细胞系,并非所有中频范围内的IF-AEF都可对其增殖造成影响,而是只有特定频率的IF-AEF才会明显影响肿瘤细胞的增殖,而在偏离这个最佳频率时,其生物学效应明显减弱。肿瘤治疗电场是以色列的一些研究者发现并命名的一种特殊IF-AEF,在低强度(<2V/cm)、中频(100-300kHz)条件下,可通过抗微管机制而抑制肿瘤细胞的生长。作为一种新的治疗肿瘤的物理手段,当电场频率被准确地选定时,这种IF-AEF已被证明对多种人类及啮齿类动物肿瘤细胞系的增殖有显著抑制作用。由于既往研究发现不恰当的处理(刀割、激光和冷冻等)有可能诱发MM的迅速生长,所以关于电场等物理手段治疗MM的研究较少。
侵袭与转移是恶性肿瘤最显著的生物学特征,也是恶性肿瘤预后差,并导致患者死亡的主要原因。MM容易侵袭局部组织,进而发生远处转移。因此抑制MM的侵袭是该病治疗的重要策略,也是临床治疗成败的关键。近年来的一些研究也发现一些电场对肿瘤的侵袭能力有一定的影响,但关于IF-AEF对肿瘤侵袭能力的影响,目前尚不清楚。肿瘤的发生、发展与细胞的凋亡异常密切相关,而且有观点认为细胞凋亡抑制比细胞过度增殖所起的作用更为重要。已有许多研究表明MM与其他恶性肿瘤相比,其自发凋亡水平较低,且对一般化疗药物所诱导的凋亡敏感性较低。因此,抗肿瘤技术若能诱发MM细胞凋亡,则可抑制肿瘤生长。维持肿瘤持续生长有赖于新生血管对其营养与代谢的支持,没有新生血管肿瘤将难以持续增殖,因为仅靠周围血管对肿瘤的弥散作用是远远不够的,肿瘤将只能处于休眠状态甚至发生退化,而血管一旦长入肿瘤组织中,肿瘤则呈现出快速生长的状态。因此靶向肿瘤血供的抗血管生成治疗正成为肿瘤治疗的一个研究热点。抗血管生成治疗的目的不是直接杀伤肿瘤细胞,而是通过抑制并破坏肿瘤的血管支持网络,从而抑制肿瘤的生长和转移。目前的研究已经显示,性质不同的电场对肿瘤新生血管的作用有着明显的不同,有些电场可以抑制新生血管的形成,有些电场则可促进新生血管的生长。而既往尚无关于IF-AEF对肿瘤新生血管作用的研究。
电场的生物效应及其作用机理是非常复杂的,同时电场对肿瘤的作用也受到多方面因素的制约,因此,对于IF-AEF抑制肿瘤的机制仍需进一步的探索。本课题从不同生物层次观察了IF-AEF对肿瘤细胞及组织的作用：首先通过体外实验观察IF-AEF对小鼠黑色素瘤B16F10细胞生长的抑制及对其侵袭能力的影响；然后通过体内实验观察IF-AEF对小鼠皮下移植瘤的生长抑制作用并探讨其可能的作用机制,从而进一步阐明IF-AEF抑制MM的分子机制,可能为MM提供一种新的治疗手段。
第一章中频交变电场对黑色素瘤B16F10细胞增殖及体外侵袭能力的影响
目的探讨IF-AEF对体外培养的B16F10细胞增殖及体外侵袭能力的影响。
方法恶性黑色素瘤B16F10细胞用于本实验研究。体外实验分为MTT (Methyl thiazolyl tetrazolium,四甲基偶氮唑蓝)实验和Traswell侵袭实验两部分：(1)MTT实验：将体外培养的B16F10细胞分为对照组和电场治疗组,电场治疗组中使用清华大学自制的IF-AEF装置,该装置可产生频率为10kHz-500kHz,正弦波的峰-峰值电压(Voltage of peak to peak, Vpp)为0-30V的IF-AEF。为明确IF-AEF作用与电场频率、电场强度以及作用时间的关系,实验又分为三小部分：①频率依赖性实验,设对照组和多个电场治疗组,电场组细胞均持续48h暴露于Vpp均为30V,频率分别为50kHz、100kHz.200kHz.300kHz.400kHz.500kHz的IF-AEF,通过改变频率,以明确IF-AEF作用的频率依赖性,并明确IF-AEF对B16F10细胞作用的最佳频率；②IF-AEF频率设定为最佳频率(100kHz),电场作用时间均为48h,电场治疗组又再细分为3个Vpp亚组(10V、20V、30V),通过梯度改变Vpp,评估IF-AEF作用效果与电场强度的关系；③采用特定参数(频率=100kHz,Vpp=30V)的IF-AEF对B16F10细胞进行治疗,电场治疗组又分为两个不同的时间处理组(24h、48h),通过梯度改变电场作用时间,评估IF-AEF作用效果与作用时间的关系。各组分别处理后,收集细胞并继续培养24h,然后MTT法测定细胞活性,计算出各组处理后的细胞增殖抑制率。(2)Traswell体外侵袭实验：将体外培养的B16F10细胞分为对照组和电场治疗组,电场治疗组又被分为两个时间亚组(24h、48h),采用特定参数(频率=100kHz,Vpp=30V)的IF-AEF对B16F10细胞进行治疗。各组分别处理后,应用Transwell侵袭实验检测肿瘤细胞体外侵袭能力的改变。
结果(1)与对照组比较,频率分别为50kHz.100kHz.200kHz.300kHz.400kHz、500kHz的IF-AEF(Vpp=30V)持续作用48h后,B16F10细胞的增殖能力均受到不同程度的抑制,各组细胞增殖抑制率分别为16.43±2.17%、27.59±3.44%、13.30±2.25%、7.08±1.03%、3.23±0.35%、2.69±0.33%,各频率间比较其差异具有显著性(P<0.05),IF-AEF对B16F10细胞增殖抑制作用的最佳频率为100kHz。当Vpp分别为10V、20V、30V的IF-AEF(频率=100kHz)电场作用48h后,与对照组比较,B16F10细胞的增殖能力均受到不同程度的抑制,各组细胞增殖抑制率分别为4.37±0.51%、12.43±1.46%、27.10±3.49%,电场组各组间比较其差异也具有显著性(P<0.05)。当固定IF-AEF的两个参数(频率=100kHz,Vpp=30V)时,24h处理组和48h处理组中细胞增殖能力均受到抑制,其中IF-AEF作用48h后B16F10细胞增殖抑制率为27.10±3.49%,明显高于24h组的16.42±1.80%,两组之间差异也具有显著性(P<0.05)。(2)细胞体外侵袭实验显示,频率为100kHz的IF-AEF作用后,B16F10细胞侵袭穿过Matrigel胶到达Transwell小室膜背面的细胞数明显减少。而B16F10细胞体外侵袭能力大小以穿过Matrigel膜的细胞数与无Matrigel时穿过膜的细胞数的百分比表示。实验结果显示,阴性对照组的侵袭率为49.79±9.12%,电场24h组和48h组的侵袭率分别为37.97±7.31%和27.64±5.09%,电场组与阴性对照组之间以及两电场组者间比较其差异均有统计学意义(P<0.05)。
结论(1)IF-AEF对B16F10细胞增殖的抑制作用具有明显的频率依赖性,频率100kHz时其抑制作用最为明显,偏离此频率时,其抑制作用明显减弱。随着作用时间的延长,电场强度的增加,其抑制作用也增强,即IF-AEF对B16F10细胞增殖的抑制呈现频率-时间-电场强度依赖性；(2)频率100kHz的IF-AEF能够明显地抑制B16F10细胞的体外侵袭能力。
第二章中频交变电场对小鼠皮下移植瘤生长的抑制作用及可能的机制
目的研究IF-AEF对小鼠皮下移植瘤生长的抑制作用并探讨其可能的作用机制。
方法将成功地在皮下接种B16F10细胞的荷瘤C57/BL6小鼠随机进行分组,每组各10只小鼠：实验组从皮下接种B16F10细胞后第3天开始施加IF-AEF(频率=100kHz,Vpp=30V),电场连续作用7天,每天上下午各施加电场4h(8h/天)。对照组小鼠用同样的方法固定在一样的电场装置中,但未施加电场。从电场治疗后第一天开始隔两天(治疗后d1,4,7天)记录肿瘤体积,并计算抑制率。电场作用7天后,各组均处死两只实验小鼠,并行常规病理检查,Bax(作为Bc1相关的x蛋白)、Bcl-2(B细胞淋巴瘤／白血病基因2)、MVD(微血管密度)、VEGF (血管内皮生长因子)的免疫组化检测和凋亡检测,同时运用RT-PCR(逆转录-聚合酶链反应)检测对照组和IF-AEF组肿瘤组织中VEGF mRNA的表达。剩下的小鼠继续培养并观察肿瘤生长情况和两组荷瘤小鼠的长期生存情况。
结果(1)实验组IF-AEF治疗7天后小鼠肿瘤平均体积明显小于对照组(26.33±9.82mm3vs.263.79±107.34mm3),两者之间有统计学差异(P<0.05)。IF-AEF治疗后小鼠生存期与对照组比较明显延长(29.10±3.70天vs.23.60±3.70天),两者之间有统计学差异(P<0.05)。(2)HE染色显示IF-AEF治疗后肿瘤组织中的坏死区域比对照组明显要大,但电场范围内的正常组织并未受到明显的损伤,Tunel分析显示电场治疗后肿瘤细胞出现大量凋亡,与对照组比较差异有显著性(31.74±6.62%vs.4.51±1.34%,P<0.05)。与对照组相比,电场治疗后肿瘤组织中Bcl-2表达下降,而Bax蛋白表达增加。(3)与对照组相比,IF-AEF治疗后小鼠肿瘤组织中微血管密度明显下降(23.42±5.53vs.13.46±2.97,P<0.05),同时,可见肿瘤组织中VEGF蛋白表达明显下降。电场组中肿瘤组织中VEGF mRNA的表达较对照组明显减少。
结论(1)IF-AEF对皮下移植小鼠MM的生长有显著的抑制作用,从而导致电场治疗后荷瘤小鼠的生存期延长,且IF-AEF对正常组织并无明显的损伤。(2)IF-AEF可诱导体内B16F10细胞凋亡,其机制可能为通过下调Bcl-2同时上调Bax的表达来实现。(3)IF-AEF作用后肿瘤组织中微血管密度明显下降,其机制至少部分可能是通过抑制VEGF的功能实现的。
Background:Malignant melanoma is a kind of cancer generating from melanocytes,90%of which arises in the skin and a few cases occur in other sites. Statistical data showed that the incidence and mortality of malignant melanoma are continuously increasing worldwide and its incidence is rising steadily at a rate of4%per year, which is sharper than any other cancers. Melanoma constitutes only approximately3%of all skin cancers, but is the most deadly, accounting for approximately75%of all skin cancer-related death. When identified early and fully excised, long-term survival can be achieved. However, the median survival is less than1year in metastatic disease. The5-year survival rate of malignant melanoma is universally good in developed countries, but only40%in developing countries. Compared to the European Union and United States, malignant melanoma is rare in China, but it is usually present at advanced disease at diagnosis.
A variety of effective treatments (including surgery, radiotherapy, chemotherapy, immunotherapy, targeted therapy, etc.) have been successfully applied to malignant melanoma. The indications, contraindications and side effects of these treatments limit patient accessibility to some extent. In addition, some melanoma cases respond poorly to the traditional therapies. No standard treatments can be identified for patients with locally advanced or metastatic diseases not considered for surgery. Therefore, available current therapies do not add any survival benefit to these patients. In this regard, how to improve the overall survival of melanoma and develop new effective treatments with low toxicity continues to be a long-term topic for clinical and basic researchers.
Cancer electrotherapy is a novel physical cancer treatment utilizing the electrical technology. Recently, with the rapid development of biological study of electricity, a series of new electric fields based physical therapies hold great promise for cancer therapy. Different electric fields with different parameters have been widely used to cancer treatments, in which the most used was the electric field used as radiofrequency or microwave devices killing the cells via hyperthermia. Recently, there are gradually increasing reports on the electric fields as physical therapies for cancer utilizing the non-thermal properties, in which the representative method is intermediate-frequency alternating electric field (IF-AEF). Data have been provided that at very low frequencies (under1kHz), alternating electric field stimulates excitable tissues, so that it has been claimed to stimulate bone growth and accelerate fracture healing. However, as the frequency of the electric field increases above1MHz, the stimulatory effect diminishes and heating becomes dominant. This phenomenon serves as the basis for some commonly used medical treatment modalities including diathermy and radiofrequency tumor ablation. Alternating electric field of intermediate frequencies (100kHz to1MHz) was considered not to have any meaningful biological effects. But some new reports showed that IF-AEF can also result in a number of anti-tumor effects. The reason why in the past we failed to find its anti-tumor effects was due to the strong frequency dependence of such effects. Not all IF-AEF can exert impact on the proliferation of special tumor cells, but only the IF-AEF at specific frequency will significantly affects the proliferation. When it deviates from the optimum frequency, its biological effects significantly reduce. Tumor-treating field is a kind of special IF-AEF found and termed by some researches from Israel. This IF-AEF at very low-intensity (<2V/cm) and intermediate-frequency (100-300kHz) can inhibit cancerous cell growth in vitro by an anti-microtubule mechanism. As a new physical cancer treatment modality, IF-AEF is effective when applied to human and animal cancer if the frequency is accurately selected. Previous studies have found that inappropriate treatment (knife, laser and freezing) may induce rapid growth of melanoma, so few studies were reported on the treatment of melanoma utilizing electric field or other physical technologies.
Invasion and metastases are the most significant biological features leading to poor prognosis and the major cause of mortality. Malignant melanoma is accustomed to burrow into surrounding tissue, and then results in distant metastases. Therefore, inhibiting invasion is an important strategy for the cancer treatment and the key to therapeutic efficacy. Recently, some studies have reported that some electric fields have effects on cancer invasion. However, to our knowledge, the effects of IF-AEF on cancer invasion remain unclear. The occurrence and development of cancer are associated with abnormal apoptosis, in which the deficient apoptosis play a more important role than the excessive proliferation of cells. Many studies show that the levels of spontaneous apoptosis of melanoma cells are lower than many other malignant tumors, and melanoma cells are not susceptible to apoptosis induced by chemotherapy. Hence, if the anti-tumor technology is effective at initiating the process of apoptosis, then it can effectively inhibit cancer growth. It is well known that tumor development relies on a functioning vascular network to provide nutrient and oxygen, and tumors are unable to develop without tumor angiogenesis. Tumors can only remain dormant or even degradation with the support of blood vessels around them, because it is not enough. Initiation of angiogenesis, however, allows rapid tumor growth and ongoing tumor progression. Therefore, vascular-targeted antiangiogenic therapy is becoming a hot topic in cancer therapy. Rather than targeting the tumor cells per se, antiangiogenic therapy is intended to target the tumor blood vessels and disrupt support networks, and therefore inhibits tumor growth and metastasis. The current study has shown that the effects of different electric fields on tumor angiogenesis are significantly different, some inhibiting angiogenesis, whereas others may induce angiogenesis. The effects of IF-AEF on tumor angiogenesis have not been reported previously.
Because the biological effects and underlying mechanisms of the electric fields are complex and restricted by many factors, therefore, further studies were needed to explore the mechanisms of IF-AEF. In the present study, we observed the effects of IF-AEF on the tumor cells and tissues from different biological levels. The effects of IF-AEF on the cell proliferation and invasion inhibition of malignant melanoma cells cultured were initially determined in vitro. Subsequently, the effects of IF-AEF on melanoma in mouse B16F10melanoma model and the underlying mechanisms were explored. Thus, this study will further elucidate the molecular mechanisms of IF-AEF, likely find novel therapeutic targets for malignant melanoma.
Part I Effects of IF-AEF on the Proliferation and Invasion of B16F10cells
Objective To investigate the effects of IF-AEF on proliferation and invasion of malignant melanoma cells cultured in vitro.
Methods Malignant melanoma B16F10cells were used. There are two parts, including MTT assay and Transwell invasion assay, in this study:(1) MTT assay:B16F10cells cultured in vitro were divided into control and IF-AEF groups. For this study we developed a special apparatus producing IF-AEF at the frequency from10kHz to500kHz and Vpp (voltage of peak to peak) from0V to30V. The IF-AEF parameters (frequency, Vpp and exposure time) were analysed in order to determine whether any effects was frequency, electrical intensity, or time-dependent. The experiment was divided into three parts. In the first part, IF-AEF inhibitory efficacy vs. frequency was studied. The Vpp of IF-AEF was set at30V and exposure time was48h. The IF-AEF groups were further divided into six frequency subgroups (50kHz,100kHz,200kHz,300kHz,400kHz, and500kHz). We try to identify the frequency dependent of IF-AEF inhibitory efficacy and select the optimum frequency for next studies. In the second part, the optimum frequency (100kHz) of IF-AEF used and exposure time was48h. the IF-AEF groups were further divided into three Vpp subgroups (10V,20V,30V), in which the time of IF-AEF exposure was set at48h. In the third part, the IF-AEF groups were further divided into two different time subgroups (24h,48h), in which the Vpp of IF-AEF (100kHz) was set at30V. After exposure, plated samples were harvested and the cells were incubated in supplemented DMEM for24h before MTT assay and then the inhibitive rates of the cells were calculated.(2) Transwell invasion assay:B16F10cells cultured in vitro were divided into two groups: control and IF-AEF group. The IF-AEF groups were further divided into two time subgroups (24h,48h). B16F10cells cultured in vitro were exposured to IF-AEF (100kHz,30V) or sham exposure. Transwell chamber in vitro invasion assay was applied to observe the changes of invasion capability after treatment.
Results (1) After treated with IF-AEF (Vpp=30V) for48h, the proliferation ability of B16F10cells was arrested at six six frequency subgroups (50kHz,100kHz,200kHz,300kHz,400kHz, and500kHz). The inhibitive rates of cell growth were16.43±2.17%,27.59±3.44%,13.30±2.25%,7.08±1.03%,3.23±0.35%, and2.69±0.33%respectively. There is significant difference between six frequency subgroups (P<0.05) and the maximal growth inhibition was found at100kHz. So the optimum frequency of IF-AEF on the inhibition of B16F10cells growth was100kHz. After treated with IF-AEF (100kHz) for48h, the proliferation ability of B16F10cells was arrested at three Vpp subgroups (10V,20V,30V). The inhibitive rates of cell growth at Vpp of10V,20V and30V were4.37±0.51%,12.43±1.46%and27.10±3.49%respectively. There is significant difference between three Vpp subgroups (P<0.05). After treated with IF-AEF (100kHz) at Vpp of30V, the proliferation ability of B16F10cells was inhibited at two time subgroups. The inhibitive rate of cell growth at48h group was27.1±3.49%, which was remarkably higher than that of16.4±1.80%at24h group (P<0.05).(2) The result of transwell chamber in vitro invasion assay showed that in the transwell cell culture chambers, invading B16F10cells were significantly lower than those in the control group after treated with IF-AEF. The invasion ability of B16F10cells was expressed as the percentage of invading B16F10cells in the transwell chamber with or without matrigel. The invasion rate was49.79±9.12%,37.97±7.31%and27.64±5.09%at negative control group,24h group and48h group respectively. There is significant difference between each other (P<0.05).
Conclusion (1) IF-AEF has inhibitory effects on proliferation of B16F10cells and100kHz IF-AEF cause maximal inhibition of B16F10cells growth. The effects of IF-AEF are frequency, intensity and time dependent.(2) IF-AEF can remarkably decrease the invasion ability of B16F10cells cultured in vitro.
Part II Effects of IF-AEF on Subcutaneous Tumor Model of B16F10Cells in C57/BL6Mice and Underlying Mechanisms
Objective To observe the effects of IF-AEF on subcutaneous tumor model of B16F10cells in C57/BL6mice and explore the underlying mechanisms.
Methods C57/BL6mice model bearing B16F10melanoma were successfully established and then the tumor-bearing mice were randomly divided into the two groups of control (n=10) and IF-AEF treatment (n=10). From2days after inoculation, the IF-AEF group was stimulated by the IF-AEF (sinusoidal wave,100kHz,30V) twice a day,4hr each time. The treatment was continued for7days, consecutively. Animals in the control group underwent sham treatment under the same schedule. Tumors were measured every3days after treatment, i.e. on days1,4and7after treatment. After7days, two mice from each group were sacrificed, the tumors were excised and samples were collected and processed immeditately for histopathological and immunohistochemical examination. The level of apoptosis was detected and we investigated the involvement of Bcl-2and Bax using immunohistochemistry on sections from both IF-AEF and untreated tumors. The tumor microvessel density and VEGF of the tumors were also tested by immunohistochemical staining. The level of expression of VEGF mRNA in the tumor of IF-AEF group and control group was tested by reverse transcription-polymerase chain reaction. To observe the survival rate, the remaining mice were kept alive and tumor growth was monitored.
Results (1) After7days of IF-AEF exposure, there was a significant decrease in mean tumor volume in mice treated with IF-AEF compared with the control group (26.33±9.82mm3vs.263.79±107.34mm3, P<0.05). The mean survival time of the tumor-bearing mice in IF-AEF group was significantly longer than in control-treated animals (29.10±3.70days vs.23.60±3.70days, P<0.05).(2) Note marked differences in the extent of tumor necrosis regions between IF-AEF group and control group in HE staining examination. Tunel analysis showed that Tumors treated with IF-AEF exhibited significantly greater numbers of apoptotic cells than control tumors (31.74±6.62%vs.4.51±1.34%, P<0.05). In mice treated with IF-AEF, the expression of Bcl-2significantly decreased compared with the control group (P<0.05), as well as an increase in the expression of Bax (P<0.05).(3) IF-AEF treatment signficantly reduced the number of CD34-positive cells compared with controls group (23.42±5.53vs.13.46±2.97, P<0.05). During the reduction of MVD, we determined a decrease in the expression of VEGF. The level of expression of the VEGF mRNA decreased significantly as determined by RT-PCR in mice treated with IF-AEF compared with control-treated animals.
Conclusion (1) IF-AEF causes a significant reduction in tumor growth rate without any significant side effects and extends survival period of C57/BL6mice bearing B16F10melanoma.(2) IF-AEF therapy of subcutaneous tumors results in a quantitative increase of B16F10cells apoptosis. The results of expression profile of apoptosis-relate gene suggest that the result may be associated with the decrease of Bcl-2and increase of Bax.(3) The application of IF-AEF to the tumors leads to a reduction in tumor MVD. The mechanism of action of the fields is, at least in part, dependent on inhibiting the functions of VEGF.
目前MM的临床常用治疗手段有：手术治疗、放射治疗、化学治疗、免疫治疗以及靶向治疗等,这些常规治疗方法对一部分患者是非常有效的,但其特定适应症和禁忌症,以及伴随的毒副反应限制了其临床使用,且临床上经常观察到一些患者对以上常规治疗的敏感性较差。目前针对无法手术的局部晚期或远处转移患者的治疗仍然非常棘手,对于这类患者,临床结果显示现有的常规治疗较少能提高其总生存率。因此,如何提高MM的总体疗效,研究新的,低毒高效的治疗方式,仍然是临床及基础研究者们长期研究的课题。
肿瘤电疗是将电工技术应用于肿瘤治疗的一种创新性物理治疗手段,随着近年来生物电学研究的蓬勃发展,利用电场作为肿瘤的治疗手段已显示出良好的应用前景。参数不同、性质各异的各类电场已被广泛试用于肿瘤治疗,并且取得了良好的效果。比较成熟的治疗肿瘤的电场类型是通过热效应杀灭肿瘤细胞的射频或微波装置。近年来关于单纯利用电场非热物理特性治疗肿瘤的报道逐渐增多,其中最具有代表性的是中频交变电场(intermediate-frequency alternating electricfield, IF-AEF)。一般认为,低频(<1kHz)电场的主要效应为刺激可兴奋组织,常用于刺激骨组织生长或促进骨折愈合；高频(MHz级以上)电场的电刺激作用基本消失而主要产生热效应,在以往的研究中常被用于热疗或消融治疗；而频率在100kHz-1MHz的IF-AEF,长期以来被认为不会产生明显的生物效应。但近期有报道指出在一定条件下IF-AEF也可产生一定的抗肿瘤效应,之所以以往未能发现其抗肿瘤效应,是由于其抗肿瘤效应具有极强的频率依赖性,对于特定的肿瘤细胞系,并非所有中频范围内的IF-AEF都可对其增殖造成影响,而是只有特定频率的IF-AEF才会明显影响肿瘤细胞的增殖,而在偏离这个最佳频率时,其生物学效应明显减弱。肿瘤治疗电场是以色列的一些研究者发现并命名的一种特殊IF-AEF,在低强度(<2V/cm)、中频(100-300kHz)条件下,可通过抗微管机制而抑制肿瘤细胞的生长。作为一种新的治疗肿瘤的物理手段,当电场频率被准确地选定时,这种IF-AEF已被证明对多种人类及啮齿类动物肿瘤细胞系的增殖有显著抑制作用。由于既往研究发现不恰当的处理(刀割、激光和冷冻等)有可能诱发MM的迅速生长,所以关于电场等物理手段治疗MM的研究较少。
侵袭与转移是恶性肿瘤最显著的生物学特征,也是恶性肿瘤预后差,并导致患者死亡的主要原因。MM容易侵袭局部组织,进而发生远处转移。因此抑制MM的侵袭是该病治疗的重要策略,也是临床治疗成败的关键。近年来的一些研究也发现一些电场对肿瘤的侵袭能力有一定的影响,但关于IF-AEF对肿瘤侵袭能力的影响,目前尚不清楚。肿瘤的发生、发展与细胞的凋亡异常密切相关,而且有观点认为细胞凋亡抑制比细胞过度增殖所起的作用更为重要。已有许多研究表明MM与其他恶性肿瘤相比,其自发凋亡水平较低,且对一般化疗药物所诱导的凋亡敏感性较低。因此,抗肿瘤技术若能诱发MM细胞凋亡,则可抑制肿瘤生长。维持肿瘤持续生长有赖于新生血管对其营养与代谢的支持,没有新生血管肿瘤将难以持续增殖,因为仅靠周围血管对肿瘤的弥散作用是远远不够的,肿瘤将只能处于休眠状态甚至发生退化,而血管一旦长入肿瘤组织中,肿瘤则呈现出快速生长的状态。因此靶向肿瘤血供的抗血管生成治疗正成为肿瘤治疗的一个研究热点。抗血管生成治疗的目的不是直接杀伤肿瘤细胞,而是通过抑制并破坏肿瘤的血管支持网络,从而抑制肿瘤的生长和转移。目前的研究已经显示,性质不同的电场对肿瘤新生血管的作用有着明显的不同,有些电场可以抑制新生血管的形成,有些电场则可促进新生血管的生长。而既往尚无关于IF-AEF对肿瘤新生血管作用的研究。
电场的生物效应及其作用机理是非常复杂的,同时电场对肿瘤的作用也受到多方面因素的制约,因此,对于IF-AEF抑制肿瘤的机制仍需进一步的探索。本课题从不同生物层次观察了IF-AEF对肿瘤细胞及组织的作用：首先通过体外实验观察IF-AEF对小鼠黑色素瘤B16F10细胞生长的抑制及对其侵袭能力的影响；然后通过体内实验观察IF-AEF对小鼠皮下移植瘤的生长抑制作用并探讨其可能的作用机制,从而进一步阐明IF-AEF抑制MM的分子机制,可能为MM提供一种新的治疗手段。
第一章中频交变电场对黑色素瘤B16F10细胞增殖及体外侵袭能力的影响
目的探讨IF-AEF对体外培养的B16F10细胞增殖及体外侵袭能力的影响。
方法恶性黑色素瘤B16F10细胞用于本实验研究。体外实验分为MTT (Methyl thiazolyl tetrazolium,四甲基偶氮唑蓝)实验和Traswell侵袭实验两部分：(1)MTT实验：将体外培养的B16F10细胞分为对照组和电场治疗组,电场治疗组中使用清华大学自制的IF-AEF装置,该装置可产生频率为10kHz-500kHz,正弦波的峰-峰值电压(Voltage of peak to peak, Vpp)为0-30V的IF-AEF。为明确IF-AEF作用与电场频率、电场强度以及作用时间的关系,实验又分为三小部分：①频率依赖性实验,设对照组和多个电场治疗组,电场组细胞均持续48h暴露于Vpp均为30V,频率分别为50kHz、100kHz.200kHz.300kHz.400kHz.500kHz的IF-AEF,通过改变频率,以明确IF-AEF作用的频率依赖性,并明确IF-AEF对B16F10细胞作用的最佳频率；②IF-AEF频率设定为最佳频率(100kHz),电场作用时间均为48h,电场治疗组又再细分为3个Vpp亚组(10V、20V、30V),通过梯度改变Vpp,评估IF-AEF作用效果与电场强度的关系；③采用特定参数(频率=100kHz,Vpp=30V)的IF-AEF对B16F10细胞进行治疗,电场治疗组又分为两个不同的时间处理组(24h、48h),通过梯度改变电场作用时间,评估IF-AEF作用效果与作用时间的关系。各组分别处理后,收集细胞并继续培养24h,然后MTT法测定细胞活性,计算出各组处理后的细胞增殖抑制率。(2)Traswell体外侵袭实验：将体外培养的B16F10细胞分为对照组和电场治疗组,电场治疗组又被分为两个时间亚组(24h、48h),采用特定参数(频率=100kHz,Vpp=30V)的IF-AEF对B16F10细胞进行治疗。各组分别处理后,应用Transwell侵袭实验检测肿瘤细胞体外侵袭能力的改变。
结果(1)与对照组比较,频率分别为50kHz.100kHz.200kHz.300kHz.400kHz、500kHz的IF-AEF(Vpp=30V)持续作用48h后,B16F10细胞的增殖能力均受到不同程度的抑制,各组细胞增殖抑制率分别为16.43±2.17%、27.59±3.44%、13.30±2.25%、7.08±1.03%、3.23±0.35%、2.69±0.33%,各频率间比较其差异具有显著性(P<0.05),IF-AEF对B16F10细胞增殖抑制作用的最佳频率为100kHz。当Vpp分别为10V、20V、30V的IF-AEF(频率=100kHz)电场作用48h后,与对照组比较,B16F10细胞的增殖能力均受到不同程度的抑制,各组细胞增殖抑制率分别为4.37±0.51%、12.43±1.46%、27.10±3.49%,电场组各组间比较其差异也具有显著性(P<0.05)。当固定IF-AEF的两个参数(频率=100kHz,Vpp=30V)时,24h处理组和48h处理组中细胞增殖能力均受到抑制,其中IF-AEF作用48h后B16F10细胞增殖抑制率为27.10±3.49%,明显高于24h组的16.42±1.80%,两组之间差异也具有显著性(P<0.05)。(2)细胞体外侵袭实验显示,频率为100kHz的IF-AEF作用后,B16F10细胞侵袭穿过Matrigel胶到达Transwell小室膜背面的细胞数明显减少。而B16F10细胞体外侵袭能力大小以穿过Matrigel膜的细胞数与无Matrigel时穿过膜的细胞数的百分比表示。实验结果显示,阴性对照组的侵袭率为49.79±9.12%,电场24h组和48h组的侵袭率分别为37.97±7.31%和27.64±5.09%,电场组与阴性对照组之间以及两电场组者间比较其差异均有统计学意义(P<0.05)。
结论(1)IF-AEF对B16F10细胞增殖的抑制作用具有明显的频率依赖性,频率100kHz时其抑制作用最为明显,偏离此频率时,其抑制作用明显减弱。随着作用时间的延长,电场强度的增加,其抑制作用也增强,即IF-AEF对B16F10细胞增殖的抑制呈现频率-时间-电场强度依赖性；(2)频率100kHz的IF-AEF能够明显地抑制B16F10细胞的体外侵袭能力。
第二章中频交变电场对小鼠皮下移植瘤生长的抑制作用及可能的机制
目的研究IF-AEF对小鼠皮下移植瘤生长的抑制作用并探讨其可能的作用机制。
方法将成功地在皮下接种B16F10细胞的荷瘤C57/BL6小鼠随机进行分组,每组各10只小鼠：实验组从皮下接种B16F10细胞后第3天开始施加IF-AEF(频率=100kHz,Vpp=30V),电场连续作用7天,每天上下午各施加电场4h(8h/天)。对照组小鼠用同样的方法固定在一样的电场装置中,但未施加电场。从电场治疗后第一天开始隔两天(治疗后d1,4,7天)记录肿瘤体积,并计算抑制率。电场作用7天后,各组均处死两只实验小鼠,并行常规病理检查,Bax(作为Bc1相关的x蛋白)、Bcl-2(B细胞淋巴瘤／白血病基因2)、MVD(微血管密度)、VEGF (血管内皮生长因子)的免疫组化检测和凋亡检测,同时运用RT-PCR(逆转录-聚合酶链反应)检测对照组和IF-AEF组肿瘤组织中VEGF mRNA的表达。剩下的小鼠继续培养并观察肿瘤生长情况和两组荷瘤小鼠的长期生存情况。
结果(1)实验组IF-AEF治疗7天后小鼠肿瘤平均体积明显小于对照组(26.33±9.82mm3vs.263.79±107.34mm3),两者之间有统计学差异(P<0.05)。IF-AEF治疗后小鼠生存期与对照组比较明显延长(29.10±3.70天vs.23.60±3.70天),两者之间有统计学差异(P<0.05)。(2)HE染色显示IF-AEF治疗后肿瘤组织中的坏死区域比对照组明显要大,但电场范围内的正常组织并未受到明显的损伤,Tunel分析显示电场治疗后肿瘤细胞出现大量凋亡,与对照组比较差异有显著性(31.74±6.62%vs.4.51±1.34%,P<0.05)。与对照组相比,电场治疗后肿瘤组织中Bcl-2表达下降,而Bax蛋白表达增加。(3)与对照组相比,IF-AEF治疗后小鼠肿瘤组织中微血管密度明显下降(23.42±5.53vs.13.46±2.97,P<0.05),同时,可见肿瘤组织中VEGF蛋白表达明显下降。电场组中肿瘤组织中VEGF mRNA的表达较对照组明显减少。
结论(1)IF-AEF对皮下移植小鼠MM的生长有显著的抑制作用,从而导致电场治疗后荷瘤小鼠的生存期延长,且IF-AEF对正常组织并无明显的损伤。(2)IF-AEF可诱导体内B16F10细胞凋亡,其机制可能为通过下调Bcl-2同时上调Bax的表达来实现。(3)IF-AEF作用后肿瘤组织中微血管密度明显下降,其机制至少部分可能是通过抑制VEGF的功能实现的。
Background:Malignant melanoma is a kind of cancer generating from melanocytes,90%of which arises in the skin and a few cases occur in other sites. Statistical data showed that the incidence and mortality of malignant melanoma are continuously increasing worldwide and its incidence is rising steadily at a rate of4%per year, which is sharper than any other cancers. Melanoma constitutes only approximately3%of all skin cancers, but is the most deadly, accounting for approximately75%of all skin cancer-related death. When identified early and fully excised, long-term survival can be achieved. However, the median survival is less than1year in metastatic disease. The5-year survival rate of malignant melanoma is universally good in developed countries, but only40%in developing countries. Compared to the European Union and United States, malignant melanoma is rare in China, but it is usually present at advanced disease at diagnosis.
A variety of effective treatments (including surgery, radiotherapy, chemotherapy, immunotherapy, targeted therapy, etc.) have been successfully applied to malignant melanoma. The indications, contraindications and side effects of these treatments limit patient accessibility to some extent. In addition, some melanoma cases respond poorly to the traditional therapies. No standard treatments can be identified for patients with locally advanced or metastatic diseases not considered for surgery. Therefore, available current therapies do not add any survival benefit to these patients. In this regard, how to improve the overall survival of melanoma and develop new effective treatments with low toxicity continues to be a long-term topic for clinical and basic researchers.
Cancer electrotherapy is a novel physical cancer treatment utilizing the electrical technology. Recently, with the rapid development of biological study of electricity, a series of new electric fields based physical therapies hold great promise for cancer therapy. Different electric fields with different parameters have been widely used to cancer treatments, in which the most used was the electric field used as radiofrequency or microwave devices killing the cells via hyperthermia. Recently, there are gradually increasing reports on the electric fields as physical therapies for cancer utilizing the non-thermal properties, in which the representative method is intermediate-frequency alternating electric field (IF-AEF). Data have been provided that at very low frequencies (under1kHz), alternating electric field stimulates excitable tissues, so that it has been claimed to stimulate bone growth and accelerate fracture healing. However, as the frequency of the electric field increases above1MHz, the stimulatory effect diminishes and heating becomes dominant. This phenomenon serves as the basis for some commonly used medical treatment modalities including diathermy and radiofrequency tumor ablation. Alternating electric field of intermediate frequencies (100kHz to1MHz) was considered not to have any meaningful biological effects. But some new reports showed that IF-AEF can also result in a number of anti-tumor effects. The reason why in the past we failed to find its anti-tumor effects was due to the strong frequency dependence of such effects. Not all IF-AEF can exert impact on the proliferation of special tumor cells, but only the IF-AEF at specific frequency will significantly affects the proliferation. When it deviates from the optimum frequency, its biological effects significantly reduce. Tumor-treating field is a kind of special IF-AEF found and termed by some researches from Israel. This IF-AEF at very low-intensity (<2V/cm) and intermediate-frequency (100-300kHz) can inhibit cancerous cell growth in vitro by an anti-microtubule mechanism. As a new physical cancer treatment modality, IF-AEF is effective when applied to human and animal cancer if the frequency is accurately selected. Previous studies have found that inappropriate treatment (knife, laser and freezing) may induce rapid growth of melanoma, so few studies were reported on the treatment of melanoma utilizing electric field or other physical technologies.
Invasion and metastases are the most significant biological features leading to poor prognosis and the major cause of mortality. Malignant melanoma is accustomed to burrow into surrounding tissue, and then results in distant metastases. Therefore, inhibiting invasion is an important strategy for the cancer treatment and the key to therapeutic efficacy. Recently, some studies have reported that some electric fields have effects on cancer invasion. However, to our knowledge, the effects of IF-AEF on cancer invasion remain unclear. The occurrence and development of cancer are associated with abnormal apoptosis, in which the deficient apoptosis play a more important role than the excessive proliferation of cells. Many studies show that the levels of spontaneous apoptosis of melanoma cells are lower than many other malignant tumors, and melanoma cells are not susceptible to apoptosis induced by chemotherapy. Hence, if the anti-tumor technology is effective at initiating the process of apoptosis, then it can effectively inhibit cancer growth. It is well known that tumor development relies on a functioning vascular network to provide nutrient and oxygen, and tumors are unable to develop without tumor angiogenesis. Tumors can only remain dormant or even degradation with the support of blood vessels around them, because it is not enough. Initiation of angiogenesis, however, allows rapid tumor growth and ongoing tumor progression. Therefore, vascular-targeted antiangiogenic therapy is becoming a hot topic in cancer therapy. Rather than targeting the tumor cells per se, antiangiogenic therapy is intended to target the tumor blood vessels and disrupt support networks, and therefore inhibits tumor growth and metastasis. The current study has shown that the effects of different electric fields on tumor angiogenesis are significantly different, some inhibiting angiogenesis, whereas others may induce angiogenesis. The effects of IF-AEF on tumor angiogenesis have not been reported previously.
Because the biological effects and underlying mechanisms of the electric fields are complex and restricted by many factors, therefore, further studies were needed to explore the mechanisms of IF-AEF. In the present study, we observed the effects of IF-AEF on the tumor cells and tissues from different biological levels. The effects of IF-AEF on the cell proliferation and invasion inhibition of malignant melanoma cells cultured were initially determined in vitro. Subsequently, the effects of IF-AEF on melanoma in mouse B16F10melanoma model and the underlying mechanisms were explored. Thus, this study will further elucidate the molecular mechanisms of IF-AEF, likely find novel therapeutic targets for malignant melanoma.
Part I Effects of IF-AEF on the Proliferation and Invasion of B16F10cells
Objective To investigate the effects of IF-AEF on proliferation and invasion of malignant melanoma cells cultured in vitro.
Methods Malignant melanoma B16F10cells were used. There are two parts, including MTT assay and Transwell invasion assay, in this study:(1) MTT assay:B16F10cells cultured in vitro were divided into control and IF-AEF groups. For this study we developed a special apparatus producing IF-AEF at the frequency from10kHz to500kHz and Vpp (voltage of peak to peak) from0V to30V. The IF-AEF parameters (frequency, Vpp and exposure time) were analysed in order to determine whether any effects was frequency, electrical intensity, or time-dependent. The experiment was divided into three parts. In the first part, IF-AEF inhibitory efficacy vs. frequency was studied. The Vpp of IF-AEF was set at30V and exposure time was48h. The IF-AEF groups were further divided into six frequency subgroups (50kHz,100kHz,200kHz,300kHz,400kHz, and500kHz). We try to identify the frequency dependent of IF-AEF inhibitory efficacy and select the optimum frequency for next studies. In the second part, the optimum frequency (100kHz) of IF-AEF used and exposure time was48h. the IF-AEF groups were further divided into three Vpp subgroups (10V,20V,30V), in which the time of IF-AEF exposure was set at48h. In the third part, the IF-AEF groups were further divided into two different time subgroups (24h,48h), in which the Vpp of IF-AEF (100kHz) was set at30V. After exposure, plated samples were harvested and the cells were incubated in supplemented DMEM for24h before MTT assay and then the inhibitive rates of the cells were calculated.(2) Transwell invasion assay:B16F10cells cultured in vitro were divided into two groups: control and IF-AEF group. The IF-AEF groups were further divided into two time subgroups (24h,48h). B16F10cells cultured in vitro were exposured to IF-AEF (100kHz,30V) or sham exposure. Transwell chamber in vitro invasion assay was applied to observe the changes of invasion capability after treatment.
Results (1) After treated with IF-AEF (Vpp=30V) for48h, the proliferation ability of B16F10cells was arrested at six six frequency subgroups (50kHz,100kHz,200kHz,300kHz,400kHz, and500kHz). The inhibitive rates of cell growth were16.43±2.17%,27.59±3.44%,13.30±2.25%,7.08±1.03%,3.23±0.35%, and2.69±0.33%respectively. There is significant difference between six frequency subgroups (P<0.05) and the maximal growth inhibition was found at100kHz. So the optimum frequency of IF-AEF on the inhibition of B16F10cells growth was100kHz. After treated with IF-AEF (100kHz) for48h, the proliferation ability of B16F10cells was arrested at three Vpp subgroups (10V,20V,30V). The inhibitive rates of cell growth at Vpp of10V,20V and30V were4.37±0.51%,12.43±1.46%and27.10±3.49%respectively. There is significant difference between three Vpp subgroups (P<0.05). After treated with IF-AEF (100kHz) at Vpp of30V, the proliferation ability of B16F10cells was inhibited at two time subgroups. The inhibitive rate of cell growth at48h group was27.1±3.49%, which was remarkably higher than that of16.4±1.80%at24h group (P<0.05).(2) The result of transwell chamber in vitro invasion assay showed that in the transwell cell culture chambers, invading B16F10cells were significantly lower than those in the control group after treated with IF-AEF. The invasion ability of B16F10cells was expressed as the percentage of invading B16F10cells in the transwell chamber with or without matrigel. The invasion rate was49.79±9.12%,37.97±7.31%and27.64±5.09%at negative control group,24h group and48h group respectively. There is significant difference between each other (P<0.05).
Conclusion (1) IF-AEF has inhibitory effects on proliferation of B16F10cells and100kHz IF-AEF cause maximal inhibition of B16F10cells growth. The effects of IF-AEF are frequency, intensity and time dependent.(2) IF-AEF can remarkably decrease the invasion ability of B16F10cells cultured in vitro.
Part II Effects of IF-AEF on Subcutaneous Tumor Model of B16F10Cells in C57/BL6Mice and Underlying Mechanisms
Objective To observe the effects of IF-AEF on subcutaneous tumor model of B16F10cells in C57/BL6mice and explore the underlying mechanisms.
Methods C57/BL6mice model bearing B16F10melanoma were successfully established and then the tumor-bearing mice were randomly divided into the two groups of control (n=10) and IF-AEF treatment (n=10). From2days after inoculation, the IF-AEF group was stimulated by the IF-AEF (sinusoidal wave,100kHz,30V) twice a day,4hr each time. The treatment was continued for7days, consecutively. Animals in the control group underwent sham treatment under the same schedule. Tumors were measured every3days after treatment, i.e. on days1,4and7after treatment. After7days, two mice from each group were sacrificed, the tumors were excised and samples were collected and processed immeditately for histopathological and immunohistochemical examination. The level of apoptosis was detected and we investigated the involvement of Bcl-2and Bax using immunohistochemistry on sections from both IF-AEF and untreated tumors. The tumor microvessel density and VEGF of the tumors were also tested by immunohistochemical staining. The level of expression of VEGF mRNA in the tumor of IF-AEF group and control group was tested by reverse transcription-polymerase chain reaction. To observe the survival rate, the remaining mice were kept alive and tumor growth was monitored.
Results (1) After7days of IF-AEF exposure, there was a significant decrease in mean tumor volume in mice treated with IF-AEF compared with the control group (26.33±9.82mm3vs.263.79±107.34mm3, P<0.05). The mean survival time of the tumor-bearing mice in IF-AEF group was significantly longer than in control-treated animals (29.10±3.70days vs.23.60±3.70days, P<0.05).(2) Note marked differences in the extent of tumor necrosis regions between IF-AEF group and control group in HE staining examination. Tunel analysis showed that Tumors treated with IF-AEF exhibited significantly greater numbers of apoptotic cells than control tumors (31.74±6.62%vs.4.51±1.34%, P<0.05). In mice treated with IF-AEF, the expression of Bcl-2significantly decreased compared with the control group (P<0.05), as well as an increase in the expression of Bax (P<0.05).(3) IF-AEF treatment signficantly reduced the number of CD34-positive cells compared with controls group (23.42±5.53vs.13.46±2.97, P<0.05). During the reduction of MVD, we determined a decrease in the expression of VEGF. The level of expression of the VEGF mRNA decreased significantly as determined by RT-PCR in mice treated with IF-AEF compared with control-treated animals.
Conclusion (1) IF-AEF causes a significant reduction in tumor growth rate without any significant side effects and extends survival period of C57/BL6mice bearing B16F10melanoma.(2) IF-AEF therapy of subcutaneous tumors results in a quantitative increase of B16F10cells apoptosis. The results of expression profile of apoptosis-relate gene suggest that the result may be associated with the decrease of Bcl-2and increase of Bax.(3) The application of IF-AEF to the tumors leads to a reduction in tumor MVD. The mechanism of action of the fields is, at least in part, dependent on inhibiting the functions of VEGF.
引文
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