探討超音波熱治療強化5-氟尿嘧啶及氯吡格雷聯合癌症治療之療效

Abstract

研究背景與目的：腫瘤細胞在增殖過程中表現出多樣的特性，以抵抗外來壓力誘導之死亡並能夠抵禦人體免疫系統之攻擊。其中惡性腫瘤因具有轉移之能力而難以治療，並為癌症相關死亡之主因，而黑色素瘤即為其中一種。雖為皮膚癌中較少見的一種，但黑色素瘤因其高轉移性而受到關注，且近年來其發病率亦有增加之趨勢。然而，目前用於黑色素瘤之治療如化學治療、標靶治療及免疫療法皆有各自之困境，因此，本研究期望聯合氯吡格雷(clopidogrel, CLO)、5-氟尿嘧啶(5-fluorouracil, 5-FU)及脈衝式超音波熱治療(pulsed-wave ultrasound hyperthermia, pUSHT)提供一可能之黑色素瘤治療方案，以多面向的策略抑制腫瘤進程，達到低副作用並具系統性之治療效果。 實驗材料與方法：本研究主要分為細胞實驗、動物實驗及腫瘤樣本分析此3大部分。細胞實驗使用B16F10黑色素瘤細胞株，並利用細胞存活率試驗探討CLO、5-FU及熱治療(hyperthermia, HT)對於此細胞株之生長抑制能力，其中CLO使用之濃度為0、0.1、0.5、1、1.5、2、2.5及5 mM ，5-FU為0、0.1、1、10、50、100、500及1000 μM，HT (43℃)之時間則為0、15、30、60分鐘。動物實驗使用C57BL/6J母鼠(周齡：6-8週；體重：18.98 ± 1.19 g)，並於其皮下接種B16F10細胞以建立黑色素瘤之小鼠模型。治療手段方面則分為3部分進行探討，分別為：(1) 不同劑量(35、70及105 mg/kg)之5-FU；(2) 合併CLO (10 mg/kg)與5-FU (35 mg/kg)之治療，及(3) 前述兩種藥物聯合pUSHT治療對於腫瘤生長之抑制效果。治療時pUSHT使用之參數如下：探頭發射頻率為3 MHz、強度為0.55 W/cm2、工作週期為50%，總施打時間則為15分鐘。動物實驗之治療成效皆以腫瘤體積及小鼠之存活率呈現，並以體重變化評估治療手段對小鼠造成之生存壓力。腫瘤樣本分析的部分，則於治療起始日後第9日將各組小鼠犧牲、並取下其背上之腫瘤，以進行後續之分析，包含蘇木精-伊紅染色(hematoxylin and eosin stain, H&E stain)、以免疫組織化學染色(immunohistochemistry, IHC)標定Ki-67與胱天蛋白酶-3(caspase-3)、末端脫氧核苷酸轉移酶脫氧尿苷三磷酸切口末端標記(terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL)檢測、及西方墨點法(Western blot, WB)。 實驗結果：細胞實驗之結果表明，單獨治療時，CLO之濃度達2.5 mM、5-FU之濃度達1 μM，或HT 15分鐘皆會與控制組(Control)達顯著性之統計差異(p < 0.05)，而合併治療雖皆可使B16F10細胞之存活率較單獨治療再降低，但CLO於細胞生長抑制之貢獻不如5-FU及HT多。動物實驗方面，第一部分動物實驗之結果顯示，35、70、105 mg/kg三種劑量之5-FU治療組的腫瘤體積差異皆與Control組達到統計顯著(p < 0.05)，其中35 mg/kg之抑制效果雖不如70及105 mg/kg，但因對小鼠造成之負擔較小，於存活率的表現上反而較另兩種劑量佳，並與Control組有顯著性之差異(p < 0.05)。第二部分動物實驗結果表明，單獨使用10 mg/kg之CLO、35 mg/kg之5-FU及兩者合併使用時於腫瘤體積上皆與Control組達到統計顯著(p < 0.05)，且藥物合併使用後之腫瘤抑制效果較單獨使用更好。最後一部分之動物實驗則說明，單獨使用pUSHT及pUSHT聯合兩種藥物治療與Control組相比，皆有顯著抑制腫瘤之效果(p < 0.05)，但在小鼠存活率之表現上聯合治療仍是最好的，並與其他組別達統計顯著(p < 0.05)。腫瘤樣本分析方面，由H&E染色可見Control組之腫瘤組織最為完整且分裂細胞多，其餘治療組別則或多或少對腫瘤組織產生破壞；IHC之染色結果則顯示Control組及5-FU組有較高之Ki-67表現，而CLO+5-FU+pUSHT組則較其他組別有更多且廣泛之caspase-3表現；而WB之結果表明，CLO+5-FU+pUSHT組於細胞凋亡相關蛋白如BAX、caspase-9及caspase-3之表現量最高；最後，TUNEL檢測之結果同樣顯示，藥物合併治療較單獨治療之細胞凋亡比例高，而增加pUSHT治療則可再提升凋亡細胞之比例，且CLO+5-FU+pUSHT組為Control組之3.5倍。 結論：本研究於細胞實驗中證實CLO、5-FU及HT之合併治療確實具有抑制B16F10黑色素瘤細胞生長之效果；動物模型之結果則顯示，pUSHT聯合CLO及5-FU治療展現出最佳之抑制腫瘤生長的能力，並顯著提升小鼠之存活率，且未對小鼠身體造成過大之負擔。腫瘤樣本分析的部分，則由H&E染色及Ki-67之標定觀察到Control組之腫瘤組織完整且細胞具有較高的增殖活性；並由caspase-3之標定、WB之半定量、以及TUNEL檢測之結果得知，藥物聯合治療及增加pUSHT之治療策略皆有效提升單獨治療誘導細胞凋亡之能力，並與實際於動物模型上所觀察到之抑制腫瘤生長及延長存活率之效益相呼應。

Background: Melanoma is the most aggressive skin cancer and its incidence has continued to rise during the past decades. Surgical resection is the standard therapeutic option for melanoma, while chemotherapy is also needed to slow down the progression of the disease. However, melanoma is often resistant to commonly used anticancer drugs like 5-fluorouracil (5-FU), therefore the treatment effectiveness compromises. Our study aimed to combine 5-FU, Clopidogrel (CLO) and pulsed-wave ultrasound hyperthermia (pUSHT) to provide a potential treatment for melanoma. Materials and Methods: This research included in-vitro and in-vivo studies and analysis of tumor samples. For in-vitro studies, MTT assay was used to determine the viability of B16F10 murine melanoma cells for the treatment of CLO, 5-FU and 43℃ hyperthermia (HT). The concentrations of CLO were 0, 0.1, 0.5, 1, 1.5, 2, 2.5 and 5 mM, while 5-FU were 0, 0.1, 1, 10, 50, 100, 500 and 1000 μM and the durations of HT were 0, 15, 30 and 60 minutes. For in-vivo studies, 105 B16F10 cells were subcutaneously inoculated on the right back of C57BL/6 mice and the treatment started 7 days later. There were three parts of in-vivo studies. To ensure the treatment efficacy of 5-FU on melanoma, we tested three dosages of 5-FU (35, 70, 105 mg/kg) firstly. Consequently, 5-FU at 35 mg/kg was used in combination with CLO (10 mg/kg) as a dual-drug therapy. At last, 5-FU (35 mg/kg) and CLO (10 mg/kg) were administered together with pUSHT sonication as a triple therapy. The parameters of pUSHT were set at 3 MHz of frequency, 0.55 W/cm2 of intensity, 50% of duty cycle and 15 minutes of treatment duration. Tumor volume and survival rate were assessed to evaluate the treatment efficacy, and body weight of mice was also recorded to test toxicity of treatment. For sample analysis, mice were sacrificed on the next day of final treatment and tumor tissues were collected for analysis, including hematoxylin and eosin (H&E) stain, immunohistochemistry (IHC) stain, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and Western blot (WB). Results: In-vitro results showed that CLO, 5-FU and HT individually could inhibit the growth of B16F10 melanoma cells, while co-treatment reduced the viability of cells more effectively. As for in-vivo results, 5-FU did inhibit tumor growth and 35 mg/kg was safer than 70 and 105 mg/kg. The dual-drug treatment was expected to improve the antitumor effect of 5-FU alone, but with limited improvement. The triple therapy showed that there were significant improvements in terms of tumor growth and survival and no excessive burden to mice (body weight of mice treated with triple therapy did not drop more than 20% during the treatment regimen). H&E stain showed that there were more proliferative tumor cells in the control group. IHC stain revealed more expression of Ki-67 on the control and 5-FU groups, while the group treated with triple therapy had more expression of caspase-3. Furthermore, we performed WB to quantify proteins associated with apoptosis. The results of WB demonstrated that caspase-3 was significantly upregulated in the dual-drug group compared to the groups with single treatment, while pUSHT could further increase the expression of caspase-3 of the dual-drug group. Through quantitative comparison of the apoptotic rate using TUNEL assay, we found that the percentage of cells undergoing apoptosis in the tumor tissues was the greatest (1.85%) in the group treated with triple therapy. Conclusion: In in-vitro study, the combination of CLO, 5-FU and HT inhibits the growth of B16F10 cells. In in-vivo study and tumor sample analysis, the triple therapy induces tumor cell apoptosis by upregulating caspase-3 expression and results in tumor growth retardation effectively. These results suggest that the triple therapy may provide a potential strategy that can reduce tumor progression and prolong survival.