標靶奈米順鉑傳輸技術應用於口腔癌移轉模型治療之研究

轉移為癌症嚴重性重要指標，因 90 ％癌症死亡與癌症轉移有相關性。於口腔癌病例中，約50 ％的轉移行口腔癌病患，在5年內預後生存率下降約30 ％。因此本次研究，計畫模擬口腔癌轉移，因此將6.3×105個SAS-G418細胞接種到裸鼠的舌頭上，建立模擬原位轉移性口腔癌模型。並在SAS-G418細胞接種後第7天，觀察到宮頸區域包括淺表型頸淋巴結（SCL），深部頸淋巴結（DCL）和頜下腺（SG），是否有近端轉移發生。建立的轉移模型目的為評估脂質 - 氯化鉑納米顆粒（LPC NP），是否對原位轉移性口腔癌模型有達到治療效果。於本次實驗所採用的LPC NPs粒徑為23.8±1.2 nm，LPC NPs外圍接上標靶物質氨基乙基茴香酰胺(aminoethyl anisamide)，目的靶向的SAS細胞模表面所過度表達的sigma 受體。在本次研究中，預合成 LPC NP的CDDP前趨物產率為91.6±6.7 wt％，LPC NPs的CDDP包封率和藥物載量％分別為2.4±0.1wt％和89.14±8.91wt％。體外細胞存活率顯示，LPC NPs與CDDP相比顯著誘導細胞死亡（p <0.05），IC50數據顯示對於口腔癌細胞珠LPC NP 比CDDP 有效治療2.4~2.8倍(SAS: IC50 7±1.7μM LPC，17±1.7μM CDDP；SCC4 IC50: 5±4.6μM LPC，14±1.7μM CDDP)。使用Kaplan-Meier生存分析，LPC_AEAA(-) 和LPC_AEAA（+）與CDDP或PBS組相比，顯示與動物存活率有顯著性差異（P <0.001）。然而LPC_AEAA(-) 組與LPC_AEAA（+）組相比，動物存活率無顯著性差異（P> 0.05），PBS組與CDDP組相比無統計學意義（P> 0.05）。此外與PBS（19天），CDDP（22天）相比，LPC_AEAA（-）和LPC_AEAA（+）的動物存活平均值顯著延長 (P<0.001），其中LPC_AEAA（-）和LPC_AEAA（-）分別為80天和89天。 LPC治療組在細胞增殖（Ki-67），腫瘤微血管（CD31），DNA損傷（TP53）和凋亡（切割的半胱天冬酶-3和TUNEL測定）標記物的IHC分析中顯示出顯著差異（P <0.001）。在LPC處理組中，總P53的蛋白質水平和P53對絲氨酸15的磷酸化在單個小鼠上變化。此外與CDDP或PBS組相比，LPC_AEAA（-）（P <0.05）和LPC_AEAA（+）（P <0.001），觀察到cleaved caspase-3蛋白水平的顯著誘導。 LPC NPs治療組肝、腎、肌肉功能各項指標差異無統計學意義（P>0.05），但CDDP組在肝臟（天冬氨酸氨基轉移酶，AST）表達差異有統計學意義（P <0.05），腎臟（血尿素氮，BUN）和肌肉（乳酸脫氫酶，LDH）功能。結果表明，LPC NPs與CDDP相比具有最小的副作用。通過酶聯免疫吸附測定（ELISA）檢測細胞因子炎症因子（IL-6，IL-12和INF-γ）的活化無差異（P> 0.05）。總體而言，我們證明LPC NPs可以大大提高順鉑的抗癌作用，延長動物存活率，可能是治療晚期HOSCC的潛在抗轉移藥物。

關鍵字

轉移, 口腔癌,藥物傳遞,微脂體,標靶治療, 順鉑

並列摘要

Metastasis is one hallmark of cancers responsible for 90% of cancer-related deaths. In oral cancer, ~50% of metastases have found at the prognosis declining ~30% of the 5-year survival rate. In this study, orthotopic metastatic oral cancer model was established by inoculation 6.3 x 105 SAS-G418 cells onto tongue of nude mice. The metastasis was observed to cervical area including superficial cervical lymph node (SCL), deep cervical lymph node (DCL) and submandibular gland (SG) on day 7 post SAS-G418 cells inoculation. The established metastasis model allowed to further therapeutic evaluation of lipid-platinum-chloride nanoparticles (LPC NPs). The measured LPC NPs size was 23.8±1.2nm and enhanced with outer leaflet of aminoethyl anisamide, a ligand of overexpressed sigma receptor on the surface of SAS cells for targeted therapy. The %yield of CDDP precursor was 91.6±6.7wt%, but the %encapsulation efficiency and %drug loading of LPC NPs were 2.4±0.1wt% and 89.14±8.91wt%, respectively. In vitro cell viability showed that LPC NPs significantly induced cell death (p<0.05) and promoted 2.4 or 2.8 folds stronger to reach IC50 on SAS (7±1.7µM LPC, 17±1.7µM CDDP) and SCC4 (5±4.6µM LPC, 14±1.7µM CDDP) cells, respectively. LPC_AEAA(-) and LPC_AEAA(+) showed a significant difference (P<0.001) in animal survival rate analyzed using Kaplan-Meier survival analysis compared to CDDP or PBS group. However, LPC_AEAA(-) group showed no significant difference (P>0.05) in animal survival rate compared to LPC_AEAA(+) group and so did PBS group compared to CDDP group. In addition, the average of animal survival in LPC_AEAA(-) and LPC_AEAA(+) were significantly prolonged (P<0.001) compared to PBS (19 days), CDDP (22 days), where animal survival days for LPC_AEAA(-) and LPC_AEAA(-) were 80 and 89 days, respectively. LPC-treated groups showed significantly different (P<0.001) on IHC analysis against cell proliferation (Ki-67), tumor microvessel (CD31), DNA damage (TP53), and apoptosis (cleaved caspase-3 and TUNEL assay) markers. The protein levels of total P53 and phosphorylation of P53 on serine 15 enhanced on LPC-treated groups but varied on individual mouse. In addition, a significant induction of cleaved caspas-3 protein level was observed on LPC_AEAA(-) (P<0.05) and LPC_AEAA(+) (P<0.001) compared to CDDP or PBS group. LPC NPs-treated groups showed no difference (P>0.05) in all parameters of liver, kidney, and muscle functions compared to PBS group, but CDDP group showed significant difference (P<0.05) on some markers including liver (aspartate aminotransferase, AST), kidney (blood urea nitrogen, BUN) and muscle (lactate dehydrogenase, LDH) functions. Results suggest that LPC NPs served minimum side effects compared to CDDP. No difference (P>0.05) activation of cytokines inflammatory factors (IL-6, IL-12 and INF-γ) were detected by enzyme-linked immunosorbent assay (ELISA). Overall, we demonstrated that LPC NPs highly improved the anticancer effect of cisplatin, prolonged animal survival rate, and could be a potential antimetastatic drug for treatment against advanced HOSCC

並列關鍵字

Metastasis ； oral cancer ； drug delivery ； liposome ； targeted therapy ； cisplatin

參考文獻

67. Hsu, L.-P. and P.-R. Chen, Distant metastases of head and neck squamous cell carcinomas-experience from eastern Taiwan. Tzu Chi Med J, 2005. 17(9).

1. American Society of Clinical Oncology (ASCO). Head and Neck Cancer 2016 [cited 2016 May 3]; Available from: http://www.cancer.net/cancer-types/head-and-neck-cancer/overview.

3. Siegel, R., D. Naishadham, and A. Jemal, Cancer statistics, 2013. CA: A Cancer Journal for Clinicians, 2013. 63(1): p. 11-30.

4. Wyganowska-Swiatkowska, M. and J. Jankun, Plasminogen activation system in oral cancer: Relevance in prognosis and therapy (Review). Int J Oncol, 2015. 47(1): p. 16-24.

7. Sano, D. and J.N. Myers, Xenograft models of head and neck cancers. Head & Neck Oncology, 2009. 1: p. 32-32.

國際替代計量

標靶奈米順鉑傳輸技術應用於口腔癌移轉模型治療之研究

未授權

主題瀏覽