藉由克服抗藥性以提升Doxorubicin在犬淋巴癌的抗癌效果

Abstract

犬淋巴癌為犬隻中最常發生的惡性腫瘤之一。除了提早檢查預防之外，腫瘤治療的進步及改善也是相當重要的，有些癌細胞在一開始使用化學治療時即對化療藥物具有抗性，另一部分的抗藥性則是在療程時逐漸產生。這些具有抗藥性的癌細胞，通常會高度表現和藥物排出有關的運輸蛋白，這樣的機制在多重抗藥性的細胞中是相當常見的。這些運輸蛋白中，ABC transporters 尤其是P-glycoprotein (P-gp, ABCB1)，breast cancer resistant protein (BCRP, ABCG2)，以及 multidrug resistant protein (MRP1, ABCC1)在對艾黴素(doxorubicin)具有抗藥性的細胞當中常常有高度表現的現象，因此使得化療藥物的效果降低，因此這些蛋白在抗藥性中扮演了重要的角色。在我們的研究中，酪胺酸激酶抑制劑基利克(imatinib)能夠有效的藉由促使細胞進行凋亡，以提升具有過度表現P-glycoprotein的doxorubicin抗藥性犬淋巴癌細胞對於doxorubicin的敏感度。並且當同時使用imatinib以及doxorubicin時，可以藉由減少doxorubicin的排出以提高其在抗藥性細胞內的累積量，而非藉由改變比起母細胞還要過度表現的P-glycoprotein之表現量。然而，Akt在同時給予兩種藥物後的24小時有被活化的現象，這可能是在癌細胞中特有的性質以避免細胞死亡的機制之一。但經過48小時後，活化的Akt表現量則降低，這樣的結果可與先前實驗互相對照，此時細胞已經經由細胞凋亡的機制而死亡，因此調控細胞存活的磷酸化蛋白激酶B (Protein kinase B/Akt) 表現量因此降低。總結來說，在過度表現P-glycoprotein的doxorubicin抗藥性犬淋巴癌細胞當中，使用imatinib可以藉由抑制藥物的排出以反轉doxorubicin的抗藥性。而此結果顯示未來在臨床上imatinib 或許可以用來反轉抗藥性，並提高doxorubicin的抗癌效果。

Canine lymphoma is one of most common malignant tumors to occur in dogs with high incidence around the world. Despite advances in the cancer prevention, the treatment of tumor diseases also has to be improved. Some cancer cells may resist the effect of chemotherapeutic agents by upregulating drug transporters which efflux the drugs intrinsically or develop during treatments, this kind of mechanism is commonly seen in doxorubicin resistant tumor cells. ABC transporters, especially P-glycoprotein (P-gp, ABCB1), breast cancer resistant protein (BCRP, ABCG2), and multidrug resistant protein (MRP1, ABCC1) are mostly involved in doxorubicin resistant cancer cells. In our studies, CLBL1-8.0, a doxorubicin resistant B type lymphoma cell line was created from CLBL-1 by increasing doxorubicin concentration during culturing, it shows highly expressed of P-glycoprotein (P-gp, ABCB1), the protein which are mostly involved in doxorubicin resistant cancer cells. Also, we demonstrated that imatinib, a tyrosine kinase inhibitor, significantly potentiated the sensitivity of doxorubicin in P-gp overexpressing resistant cells by promoting cells into apoptosis. And also combination of these two drugs may increase the accumulation of doxorubicin by decreasing the efflux of doxorubicin, but not by altering the protein expression of P-glycoprotein, which was highly expressed than its parental cells. While the Akt was activated after 24 hours treatment of imatinib and doxorubicin to suppress apoptosis, it may be one of a mechanism reflects a cellular defense of cancer cells in order to prevent doxorubicin-induced cytotoxicity effects. While after 48 hour treatment, activated Akt was decreased, it corresponds to the results of cell viability assay that imatinib may induce cell death after 48 hour of treatment combined with doxorubicin. In conclusion, imatinib can reverse doxorubicin resistant by decreasing the drug efflux in ABCB1 overexpressed canine lymphoma doxorubicin resistant cells. These results suggest an optimal to use this strategy of combining doxorubicin, one of the mostly used chemotherapeutic drugs in the treatment of canine lymphoma, with imatinib in clinic to overcome its resistance.