奈米劑型紫杉醇對非小細胞肺癌與神經母細胞瘤細胞株的代謝影響研究

Abstract

紫杉醇 (paclitaxel) 常用於治療非小細胞肺癌、乳癌、與卵巢癌等，由於溶解度低，傳統劑型以polyoxyl 35 castor oil為主要溶媒，可能引起嚴重的過敏反應與周邊神經病變。以白蛋白包覆紫杉醇製成奈米劑型 (nab-paclitaxel)，除了減少過敏反應，亦提升療效，但對周邊神經病變的影響尚無定論。為了解奈米劑型對療效及安全性影響之潛在原因，本研究以人類非小細胞肺癌細胞株 (A549細胞株) 及神經母細胞瘤細胞株 (SH-SY5Y 細胞株) 為研究對象，利用質譜儀分析比較奈米劑型 (Abraxane®) 與傳統劑型 (Phyxol) 對細胞代謝物之影響，並探討影響代謝物含量變化之機轉。 Sulforhodamine B (SRB) assay結果顯示paclitaxel、Phyxol、及Abraxane®於本研究測試條件下，對A549細胞存活率之影響未有顯著差異。考量臨床使用下之血中濃度範圍及致使大約50%細胞存活率之濃度，選擇給藥時間為24小時且投予濃度為100 nM的條件進行後續研究。以質譜儀分析代謝物含量，Phyxol及Abraxane®對A549細胞carnitine及部分acylcarnitines的含量影響具顯著差異 (P < 0.05)。由於carnitine及acylcarnitines與長鏈脂肪酸氧化相關，故分析長鏈脂肪酸含量變化。結果顯示palmitic acid與linoleic acid含量在Abraxane®處理的A549細胞中較以Phyxol處理者上升幅度較高 (P < 0.05)。由於carnitine和長鏈脂肪酸為carnitine palmitoyltransferase 1 (CPT1) 的受質，而acylcarnitines為產物，因此利用西方點墨法檢測A549細胞株表現之兩種CPT1亞型的含量變化。雖然蛋白質表現量變化於加藥組間並無顯著差異，然而，給予Phyxol者之CPT1活性較其他加藥組低，可能與其acylcarnitines上升幅度較其他加藥組低相關。給予Phyxol者之medium-chain acyl-CoA dehydrogenase (MCAD) 之表現量較其他加藥組低，可能因此使其octanoylcarnitine含量上升幅度較其他加藥組大。 SRB assay結果顯示以paclitaxel、Phyxol、及Abraxane® 濃度100 nM給藥24小時，對分化或未分化SH-SY5Y細胞存活率之影響皆未有顯著差異。以質譜儀分析代謝物含量，Phyxol及Abraxane®對未分化SH-SY5Y細胞carnitine及部分acylcarnitines的含量影響具顯著差異，但對分化SH-SY5Y細胞僅少數acylcarnitines含量變化具顯著差異。分析長鏈脂肪酸結果顯示，未分化SH-SY5Y細胞中具顯著變化差異的長鏈脂肪酸數目比分化SH-SY5Y細胞多，且於未分化SH-SY5Y細胞中，以Abraxane®處理者之長鏈脂肪酸含量較以Phyxol處理者高。由西方點墨法結果顯示，以Abraxane®及Phyxol處理分化或未分化SH-SY5Y細胞，CPT1C含量皆無顯著差異。 Carnitine之重要功能為促使長鏈脂肪酸進入粒線體進行氧化，此氧化過程為癌細胞生存之關鍵。研究結果顯示，紫杉醇奈米劑型，不僅避免使用與過敏反應相關之油性溶劑，其影響A549細胞及SH-SY5Y細胞能量代謝之程度，亦與傳統劑型相異。奈米劑型臨床療效較傳統劑型提升，可能與此能量代謝之影響差異相關。由分化與未分化SH-SY5Y細胞之代謝物含量變化差異，推測紫杉醇奈米劑型與傳統劑型於神經分化程度不同時影響程度相異。 綜上所述，由紫杉醇奈米劑型與傳統劑型對A549細胞與分化或未分化SH-SY5Y細胞造成的影響顯示，兩者脂肪酸氧化程度不同，且因細胞而異。相關詳細機制及此能量代謝途徑對療效和安全性之影響，值得進一步研究探討。

Paclitaxel is commonly used for the treatment of non-small cell lung cancer, ovarian cancer, and breast cancer. Due to low solubility of paclitaxel, polyoxyl 35 castor oil is utilized as the main solvent in the traditional formulation, which may induce severe hypersensitivity reactions and peripheral neuropathy. The paclitaxel formulated as albumin-bound nanoparticles (nab-paclitaxel) was developed for mitigating hypersensitivity reactions related to the solvent. Improved efficacy has also been reported, however, diverse findings regarding peripheral neuropathy were presented. In order to investigate the potential reasons of the impact on efficacy and safety exerted by nanoparticle formulations, the human non-small cell lung cancer cell line, A549 cells, and the human neuroblastoma cell line, SH-SY5Y cells, were used in the present study. The mass spectrometry study was conducted for the comparison between the nanoparticle formulation, Abraxane®, and the traditional formulation, Phyxol, with respect to altered levels of cell metabolites, and the related mechanisms were further investigated. No significant difference of cell viability evaluated by sulforhodamine B (SRB) assay was found in paclitaxel, Phyxol, and Abraxane®-treated A549 cells at the test conditions of the present study. Considering the range of serum concentrations of paclitaxel in clinical use and the concentration resulting in approximately 50% of cell growth inhibition, the concentration of 100 nM and the treatment period of 24 hours were selected for subsequent studies. Fold changes normalized to the control group for the levels of carnitine and some acylcarnitines were significantly different (P < 0.05) between A549 cells treated with Phyxol and Abraxane® analyzed by mass spectrometry. Since carnitine and acylcarnitines are related to long-chain fatty acid oxidation, levels of long-chain fatty acids were analyzed. The increase in the levels of palmitic acid and linoleic acid in A549 cells treated with Abraxane® were significantly higher (P < 0.05) than that treated with Phyxol. Carnitine and long-chain fatty acids are the substrates of carnitine palmitoyltransferase 1 (CPT1), and acylcarnitines are the products. Therefore, western blots were utilized to test the level changes of the two subtypes of CPT1 expressed in A549 cells. Although no significant difference was found in the protein level changes between treatment groups, the CPT1 activity was lower in A549 cells treated with Phyxol in comparison with other treatment groups, which might be related to the less increase in the levels of acylcarnitines. Moreover, the protein level of medium-chain acyl-CoA dehydrogenase (MCAD) was lower in A549 cells treated with Phyxol in comparison with other treatment groups, which might contribute to the greater increase in the level of octanoylcarnitine. SRB assay results revealed no significant difference in undifferentiated and differentiated SH-SY5Y cells between treatments of 100 nM of paclitaxel, Phyxol, and Abraxane® for 24 hours. Levels of carnitine and some acylcarnitines were significantly affected in undifferentiated SH-SY5Y cells due to Phyxol and Abraxane® treatments analyzed by mass spectrometry. However, for differentiated SH-SY5Y cells, few acylcarnitines were significantly influenced. The number of long-chain fatty acids with significantly altered levels in undifferentiated SH-SY5Y cells was higher than that in differentiated SH-SY5Y cells. Levels of long-chain fatty acids in undifferentiated SH-SY5Y cells treated with Abraxane® were higher than that treated with Phyxol. Western blots showed that levels of CPT1C were not significantly different between SH-SY5Y cells treated with Phyxol and that with Abraxane®, either undifferentiated and differentiated. The important function of carnitine is to promote the entry of long-chain fatty acids into mitochondria for oxidation, which is a key to survival for cancer cells. Results demonstrated that nab-paclitaxel not only eliminates the use of the lipophilic solvent related to hypersensitivity, but also affects energy metabolism in A549 cells and SH-SY5Y cells to an extent different from that induced by the traditional formulation. The higher clinical efficacy of nab-paclitaxel may be due to the different effects on energy metabolism. The varied levels of metabolites in differentiated and undifferentiated SH-SY5Y cells suggested the different impact of nab-paclitaxel and the traditional formulation on SH-SY5Y cells with diverse differentiation extents. In summary, the effects of nab-paclitaxel and the traditional formulation on A549 cells and undifferentiated or differentiated SH-SY5Y cells revealed the difference in the extent of fatty acid oxidation between the two formulations, which was also dependent on cell lines. The related detailed mechanisms and the influence of the pathways of energy metabolism on efficacy and safety are worth further investigation.