超音波熱治療時二甲雙胍與阿斯匹靈對原位腫瘤生長與腫瘤轉移的形成之影響

Abstract

研究背景與目的：癌症的共同特徵概念在2011年被提出，希望能藉由抑制越多的癌腫瘤共同特徵來達到抑制腫瘤的生長和轉移。普遍來說癌腫瘤的轉移是導致死亡的主要原因，導致癌細胞轉移的因素有很多種，其詳細的機制目前尚不明瞭，但目前多數認為癌細胞的轉移早在癌症初期就開始了，因此在抑制癌細胞的轉移上是目前諸多研究的方向。阿斯匹靈與二甲雙胍都是發展歷史悠久老藥，近年來的研究發現他們在抗癌上有不錯的效果。超音波的熱效應與非熱效應有對癌腫瘤有不錯的療效，也能提升全身性抗癌免疫反應。本研究主要探討阿斯匹靈與二甲雙胍結合超音波熱治療，對於原位腫瘤與癌細胞肺臟轉移的影響。 實驗材料與方法：本研究有細胞實驗與動物實驗兩方面，細胞實驗細胞株使用乳癌腫瘤細胞4T1，進行細胞存活率分析與傷口癒合試驗，細胞存活率分析的治療分組為阿斯匹靈組、二甲雙胍組、阿斯匹靈＋二甲雙胍組以及阿斯匹靈＋二甲雙胍＋熱治療組。阿斯匹靈組的藥物濃度為0、0.25、0.5、1、2、4和8 mM，二甲雙胍組的藥物濃度為0、1、2、4、8、16、32和64 mM，之後再結合熱治療和不同濃度的阿斯匹靈與二甲雙胍藥物。傷口癒合試驗的治療分組為阿斯匹靈組、二甲雙胍組、阿斯匹靈＋二甲雙胍組。阿斯匹靈組的藥物濃度為0.25 mM，二甲雙胍組的藥物濃度為1 mM。動物實驗分為兩部分，細胞株都是使用乳癌腫瘤細胞4T1；小鼠品系為BALB/cByNarl。主要治療方式為每天管餵藥物阿斯匹靈與二甲雙胍以及超音波熱治療，阿斯匹靈劑量為20 mg/kg；二甲雙胍劑量為200 mg/kg。超音波熱治療使用物理治療用超音波探頭，第一部分設定參數為發射頻率：1 MHz；發射強度：3 W/cm2；工作週期：50%；治療時間：15分鐘。有效發射面積：0.9 cm2；第二部分設定參數為發射頻率：3 MHz；發射強度：0.55 W/cm2；工作週期：50%；治療時間：15分鐘。有效發射面積：5 cm2。實驗流程為先在小鼠右背上種腫瘤，經過10天後開始治療以及尾靜脈注射腫瘤細胞，超音波治療在第0、3、6、9天，每天管餵藥物，15天後犧牲小鼠觀察肺臟腫瘤結節數。治療分組有控制組、阿斯匹靈＋二甲雙胍組、超音波組、阿斯匹靈＋超音波治療組以及合併治療組。兩部分的實驗差異性主要為尾靜脈注射的腫瘤細胞數目的不同。 實驗結果：在細胞實驗上本研究所選用的阿斯匹靈與二甲雙胍，在對於4T1癌腫瘤細胞都有生長抑制效果，這兩種藥物的結合對於4T1細胞生長有加乘的抑制效果（p<0.0001），且熱治療與藥物也對4T1細胞生長有加乘的抑制效果（p<0.0001）。傷口癒合試驗結果顯示阿斯匹靈+二甲雙胍，最能有效抑制4T1細胞移行能力（p<0.05）。在動物實驗第一與第二部分上，小鼠右背上的原位腫瘤體積，合併治療組相較於單獨藥物組與控制組來說，腫瘤體積較小（p<0.05）；在肺臟腫瘤結節數上的表現，第一部分實驗合併治療組較其他三組來的少（p<0.05）。第二部分實驗結果，可能由於尾靜脈注射癌細胞數量過少，導致各組別間的肺臟腫瘤結節數皆很少，故無統計上的差異。H&E切片染色可以看出合併治療組的腫瘤組織結構較為鬆散，TUNEL檢測的結果顯示合併治療組的細胞凋亡比例較高。 結論：在細胞實驗上，阿斯匹靈與二甲雙胍結合熱治療能有效抑制4T1腫瘤細胞生長，而阿斯匹靈結合二甲雙胍能有效抑制4T1細胞移行能力。在動物實驗方面，阿斯匹靈合併二甲雙胍與熱治療能有效降低原位腫瘤生長速度，以及肺臟轉移腫瘤形成。

Background and Purpose: The hallmarks of cancer were proposed in 2011, in hopes of inhibiting these characteristics to prevent tumor growth and migration. In general, the metastasis of tumor is the primary cause of patient death. Many factors are involved in tumor metastasis. It is believed that metastasis of tumor cells starts at early cancer stage. Therefore, inhibition of tumor cell migration is the major research and therapeutic direction. Aspirin (Asp) and metformin (Met) are common drugs used for anti-inflammation and diabetes, respectively; and their anti-tumor effects have been reported. The thermal and non-thermal effects of ultrasound (US) can be used for tumor treatment. US can also enhance immune response against cancer. This research primarily investigated the influence of combining US with Asp/Met on primary tumor growth and metastatic tumor formation. Materials and Methods: The research involved in-vitro and in-vivo studies. For in-vitro studies, breast cancer cell line 4T1 was used. The MTT assay was conducted along with the wound healing assay. The treatment groups of MTT assay included Asp alone, Met alone, Asp plus Met, Asp plus Met with hyperthermia (HT) groups. The concentrations of Asp were 0, 0.25, 0.5, 1, 2, 4 and 8 mM. The concentrations of Met were 0, 1, 2, 4, 8, 16, 32 and 64 mM. In addition, HT was performed along with different concentrations of Asp and Met. The treatment groups of wound healing assay included Asp alone, Met alone and Asp (0.25 mM) plus Met (1 mM) groups. There were two parts of in-vivo studies, and breast cancer cell strain 4T1 and BALB/cByNarl mice were used. Primary treatment included daily oral feeding of Asp (20 mg/kg) and Met (200 mg/kg), and pulsed-wave ultrasound hyperthermia (pUSHT). The setting for the first part included: ultrasound frequency 1 MHz, intensity 3 W/cm2, duty cycle 50%, duration 15 minutes and effective radiating area 0.9 cm2. The parameters for the second part included: ultrasound frequency 3 MHz, intensity 0.55 W/cm2, duty cycle 50%, duration 15 minutes and effective radiating area 5 cm2. The procedure was as follows: Tumor cells were implanted on the right back of the mice. Ten days later, treatment started with tumor cell injected via tail vein, with pUSHT on day 0, 3, 6 and 9 and oral feeding the drugs. Fifteen days after the first treatment, the mice were euthanized and dissected to observe tumor nodules in the lungs. There are control, Asp plus Met, pUSHT alone, Asp plus pUSHT and Asp plus Met plus pUSHT groups. The difference between these two parts of experiment is the number of cancer cells injected into the tail vein. Results: In-vitro study showed that Asp and Met had inhibitory effects on the growth of 4T1 cancer cells. The combination of these two drugs had a multiplicatively inhibitory effects on 4T1 cell growth (p<0.0001), while HT plus drugs also had a multiplicatively inhibitory effects on the growth of 4T1 cells (p<0.0001). Wound healing assay showed that Asp plus Met had the most effective inhibition on 4T1 cell migration ability (p<0.05). In the first and second parts of the animal experiment, the tumor volume on the right back of mice in the combined treatment group was smaller than those of the drugs and the control groups (p<0.05). Furthermore, the combined treatment group also showed less tumor nodules in the lungs than the other three groups (p<0.05), but the number of tumor nodules of the second part of experiment were all small and had no statistical difference among the groups. H&E staining displayed that tumor tissue structure in the combined treatment group was relatively loose. The TUNEL assay showed that the proportion of apoptotic cells in the combined treatment group was higher. Conclusion: In in-vitro studies, the combination of Asp and Met could effectively inhibit the growth and the migration of 4T1 tumor cells. In in-vivo studies, pUSHT combined with Asp and Met could effectively reduce the growth of primary tumor and the metastasis of tumor formation in the lung.