探討超音波熱治療與氯喹對癌腫瘤治療之綜效

Abstract

背景與研究目的：在現行的癌症治療中除了常見的手術、化療或放射線治療之外，熱治療(Hyperthermia)也是選項之一，然而文獻指出雖然熱治療對周邊組織的傷害較低，但在過程中癌細胞受到代謝壓力(Metabolic Stress)會進行細胞自噬(Macroautophagy)作用，分解受損胞器以維持細胞恆定。因此本研究主要是探討針對小鼠癌腫瘤進行脈衝式超音波熱治療，並輔以氯喹(Chloroquine)作為自噬抑制劑，觀察是否在抑制癌細胞自噬後可以對癌腫瘤及小鼠生存率造成影響。 材料與方法：本研究分成細胞實驗及動物實驗。細胞實驗方面使用4T1 乳癌腫瘤細胞進行細胞存活率分析，治療組分為氯喹組、脈衝式超音波熱治療組、氯喹合併熱治療組。氯喹的藥物濃度為0μM、1μM、2μM、3μM、4μM、5μM、6μM、7μM、8μM、10μM，再結合熱治療做分析。動物實驗則是使用週齡6-7 週的BALB/cByJNarl 小鼠；使用的腫瘤細胞為4T1 乳腺癌細胞；主要治療方式為結合超音波以及氯喹。超音波熱治療設定的參數為頻率：3MHz；強度：0.55W/cm2；工作週期：50%；治療時間：15 分鐘。實驗分成控制組、單獨氯喹治療組、單獨超音波治療組和超音波結合氯喹組。實驗流程先在小鼠右後背注射106 個4T1 腫瘤細胞，七天後腫瘤成長到約50-100 mm3 開始治療，並將治療起始日定義為第0天，單獨氯喹組在第0 天開始連續12 天以腹腔注射方式給予藥物，劑量為50mg/kg；單獨超音波治療組分別在第0、3、6、9 天以上述參數進行治療；合併治療組則是合併氯喹及超音波做治療。同樣的實驗重複進行三次以求再現性。 實驗結果：在細胞實驗上可以看到當氯喹濃度較低時，反而會使細胞存活率上升；單獨使用熱治療已經可以將細胞存活率降到低於50%，結合氯喹後可以再讓細胞存活率隨著氯喹濃度上升而下降。在動物實驗方面，單獨氯喹組、超音波熱治療組及合併治療組都有減緩腫瘤成長的趨勢，而三個治療組中又以合併治療組的效果最為顯著。在生存率上，單獨氯喹組及合併治療組相較於控制組都能提高小鼠的生存率。而TUNEL 檢測顯示合併治療組有最高細胞凋亡的趨勢，且西方墨點法也顯示Bax 表現量顯著上升，而CD 31 的高表現量則顯示氯喹可能有讓腫瘤周邊血管正常化的趨勢，進而降低了腫瘤轉移的風險。 結論：在細胞實驗上，熱治療及較高濃度的氯喹分別可以有效抑制4T1 乳癌細胞的生長，若將這兩種療法結合可以再降低4T1 乳癌細胞的活性。在動物實驗方面，氯喹合併超音波熱治療可以有效抑制腫瘤體積大小及腫瘤的成長幅度。

Background and Purpose: Currently, surgery, chemotherapy, radiotherapy, and hyperthermia are commonly used for cancer treatments. Hyperthermia causes less damage to the surrounding normal tissues. Recent studies showed that cancer cells may use macroautophagy as a surviving mechanism when they are under strong metabolic stress. It also showed that chloroquine (CQ) can serve as autophagy inhibitor in various cancer treatments. The purpose of this study is to investigate the effects of combining pulsed-wave ultrasound hyperthermia (pUSHT) and CQ on tumor growth. Materials and Methods: This research consists of in-vitro and in-vivo studies and 4T1 breast cancer cell line was used. For in-vitro study, MTT assay was conducted to assess cell viability. The MTT assay study included CQ group, hyperthermia (HT) group and HT combined CQ group with CQ concentration 0μM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM and 10μM. As for in-vivo study, BALB/cByJNarl mice between 6 to 8 weeks old were used, and there included control, CQ, pUSHT and CQ + pUSHT groups. The parameters of pUSHT are set at 3MHz of frequency, 0.55W/cm2 of intensity, 50% of duty cycle and 15 mins of treatment duration. 4T1 cells 106 were subcutaneously implanted on mouse’s right back. Treatment started 7 days after cancer cells implanted when the tumor grows approximately to 50-100 mm3 and the day is set as Day 0. pUSHT was given on Day 0, 3, 6 and 9, and CQ was given via i.p. injection at a dose of 50mg/kg for 12 consecutive days starting from Day 0. Results: The results of MTT assay showed that cell viability was significantly higher at a lower concentration of CQ and HT alone could reduce cell viability to be lower than 50%. The combination of CQ and HT tended to gradually reduce cell viability in a CQ concentration-dependent manner. The in-vivo experiments showed treatment with CQ alone, pUSHT or CQ + pUSHT could all retard tumor growth, especially with the combined therapy. Both CQ alone and combined therapy could improve the mouse survival rate. TUNEL assay revealed that combined therapy had the highest tendency for causing tumor cells apoptosis. Western Blot analysis further demonstrated that Bax protein was significantly expressed in combined therapy group. On the other hand, the high expression of CD 31 indicated that CQ might also possess the function of tumor vessel normalization which could alleviate the risk of tumor metastasis. Conclusion: In in-vitro study, both HT and high concentrations of CQ could effectively reduce the viability of 4T1 breast tumor cells. Combination of CQ and HT could further lower cell viability. In in-vivo studies, the combination of CQ and pUSHT could potently suppress both tumor volume and tumor growth rate.