副乾酪乳酸桿菌副乾酪亞種 NTU 101 發酵產物對抑制結直腸癌與改善化療副作用之研究

本研究旨在探討副乾酪乳酸桿菌副乾酪亞種 NTU 101 (Lactobacillus paracasei subsp. paracasei NTU 101, NTU 101) 發酵產物於體內及體外模式中，結合化療藥物抗結直腸癌與改善化療副作用之效果。首先以結直腸癌原位誘導小鼠模式探討 NTU 101 發酵脫脂乳 (NTU 101FM) 結合化療藥物抑制腫瘤與改善化療藥物副作用之效果。結果顯示，每日給予化療藥物 (uracil- tegafur) 40 mg/kg bw 之小鼠出現體重下降、食慾衰退與血液血球減少等化療副作用，若同時給予 NTU 101FM 四週後，則可顯著改善化療所造成之體重下降與食慾衰退 (p < 0.05)，且血液中之白血球顯著增加約 30%，紅血球亦增加 9%，並顯著提升脾臟中之介白素-10 (interleukin-10) 和顆粒性白血球群落刺激因子 (granulocyte colony-stimulating factor) 含量 (p < 0.05)。與化療組小鼠相比，NTU 101FM 結合化療藥物小鼠之腫瘤體積與腫瘤重量分別顯著下降為 1/3 與 1/4 倍 (p < 0.05)。根據動物實驗結果可知，NTU 101FM 可藉由調節發炎細胞激素、血球分群與免疫細胞分化，改善化療副作用，並具有協同化療藥物達抗腫瘤之效果。進一步探討 NTU 101FM 萃取物結合化療藥物 5-氟尿嘧啶 (5-FU) 於結直腸癌細胞模式中抗腫瘤效果。結果顯示，NTU 101FM 水萃取物或乙醇萃取物，皆可有效降低結直腸癌細胞存活率達 24% (p < 0.05)，但不會對結腸上皮細胞產生毒殺性，此外亦可顯著促使 RAW 264.7 細胞增殖達 150% (p < 0.05)，表示 NTU 101FM 萃取物具有潛在之免疫調節能力。值得注意的是，相較於單獨 5-FU 試驗組，5-FU 結合 NTU 101FM 乙醇萃取物抑制結直腸癌細胞增殖能力增加 25% (p < 0.05)，且 NTU 101FM 乙醇萃取物亦能顯著減弱 CT26 遷移能力達 60% (p < 0.05)。進一步探討 NTU 101FM 乙醇萃取物中具有抗結直腸癌活性之有效成分，經分配萃取、管柱層析取得細分層後，再以核磁共振儀 (nuclear magnetic resonance, NMR) 與超高效液相層析串聯質譜儀 (ultra-performance liquid chromatography-mass spectrometry, UPLC-MS) 之結果鑑定其為棕櫚酸 (palmitic acid, P)、硬脂酸 (stearic acid, S) 與 glyceryl 1,3-dipalmitate (G) 之混合物，其混合比例為 1:1.5:6.3 (PSG)。PSG 於對正常腸道上皮細胞或巨噬細胞無細胞毒殺性的濃度下，可顯著抑制 CT26 細胞之存活率；此外，PSG 合併 5-FU 試驗組抑制結直腸癌細胞增殖之能力顯著優於單獨 5-FU 試驗組 (p < 0.05)。與控制組相比，PSG 可顯著增加結直腸癌細胞中細胞凋亡相關蛋白活性，並顯著抑制核因子-κB (nuclear factor-κB, NF-κB) 含量，值得注意的是，PSG 合併 5-FU 試驗組調控細胞凋亡相關蛋白活性與 NF-κB 含量之效果顯著優於單獨 5-FU 試驗組，表示 PSG 可藉由誘導結直腸癌細胞凋亡與減緩化療藥物藥劑使用量之能力，達到加強化療藥物抗結直腸癌之能力與改善化療副作用之效果。綜上所述，NTU 101FM 具開發成輔助抗結直腸癌或減緩化療副作用食品補充劑之潛能。

關鍵字

Lactobacillus paracasei subsp. paracasei NTU 101 ；結直腸癌；原位誘導；協同作用；化療副作用；棕櫚酸、硬脂酸與 glyceryl 1,3-dipalmitate 混合物

並列摘要

The purpose of this study is to investigate the chemotherapeutic combination effects and improve chemotherapy side effects of Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) fermentation products in the cell model and orthotopic tumor mouse model. In the first part, we investigated the chemotherapeutic combination effect of NTU 101-fermented skim milk (NTU 101FM) and ameliorate chemotherapeutic side effects in orthotopic colorectal cancer mouse model. The result shows oral administration of chemotherapy drugs (uracil-tegafur) 40 mg/kg bw daily could induce chemotherapeutic side effects such as appetite loss, body weight loss and circulating blood cells reduction. However, oral administration of NTU 101FM for 4 weeks could significantly improve the appetite and weight loss which induced by chemotherapeutic. And the white blood cells significantly increased by about 30%, and the red blood cells also increased by 9 % (p < 0.05). NTU 101FM also significantly improve interleukin-10 and granulocyte colony-stimulating factor in spleen. In addition, we also found that compared to the chemotherapy-only group, NTU 101FM combine chemotherapy treatment will significantly reduce the tumor volume and tumor weight to 1/3 and 1/4, respectively (p < 0.05). Next, we investigated the anti-cancer effects of NTU 101FM extracts combination with the chemotherapeutic drug 5-fluorouracil (5-FU) in the CRC cell model. The results show that the extracts of NTU 101FM could effectively reduce CRC cell viability to 76% (p < 0.05), but it did not induce cytotoxicity to colon epithelial cells. Moreover, they significantly increased RAW 264.7 cell viability up to 150% (p < 0.05). This result demonstrated that the extracts of NTU 101FM have favorable preliminary immunomodulatory ability. Notably, 5-FU combination with the NTU 101FM extracts treated group decreased the cell viability of CRC cells by 25% compared to the 5-FU alone group (p < 0.05). An ethanol extract of NTU 101FM effectively attenuated 60% CT26 cell migration (p < 0.05). We further investigated the anti-CRC effect of bioactive compound(s) in NTU 101FM ethanol extract. The bioactive compound(s) was identified as a mixture of palmitic acid (P), stearic acid (S) and glyceryl 1,3-dipalmitate (G) using nuclear magnetic resonance (NMR) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and the ratio of this mixture was about 1:1.5:6.3 (PSG). PSG significantly decreased CRC cell viability at dosages that were not cytotoxic to normal colon epithelial cells or macrophages. Moreover, the inhibitory effect of the combination of PSG and 5-fluorouracil (5-FU) was significantly greater than that of 5-FU alone (p < 0.05). PSG up-regulated the activities of apoptosis-related proteins and down-regulated the nuclear factor-κB (NF-κB) signaling pathway compared to levels in the control group. It is worth noting that the effect of PSG combined with 5-FU group on the regulation of apoptosis-related protein activity and NF-κB level was superior to that of 5-FU-only group. These findings indicatied that PSG might ameliorate the 5-FU-induced side effects and enhance the treatment effect of 5-FU by reducing the dosage of chemotherapy drugs and inducing apoptosis of CRC cells. In conclusion, NTU 101FM would possibly be a favorable candidate in food supplement for auxiliary anti-colorectal cancer or moderation chemotherapeutic side effects.