Quercetin及chrysin透過調節 TLR4/NF-kB信號路徑抑制鎳促進A549細胞之轉移

Quercetin and chrysin inhibited nickel-induce cell migration by regulating TLR4/NF-kB signaling pathway in A549 cells.

張致寧(Chih-Ning Chang)

指導教授 ： 葉姝蘭

中山醫學大學/健康管理學院/營養學系碩士班/碩士(2014年)

https://doi.org/10.6834/CSMU.2014.00176

摘要

細胞實驗部分
鎳是一種廣泛存在於環境中的重金屬汙染物，研究指出暴露於含鎳的環境中可能會透過TLR4信號路徑增加人類肺癌細胞的侵犯能力。Quercetin及chrysin屬於類黃酮的一種，廣泛存在於蔬果當中，先前研究發現quercetin及chrysin顯著的抑制鎳誘發的癌細胞轉移及侵犯，然而其可能的機制目前仍不清楚。故本研究主要的目的是探討quercetin及chrysin抑制鎳促進肺癌細胞移動性及侵犯性可能的機制途徑。我們將人類肺腺癌細胞株A549細胞與植化素 (2或5 μM)、CLI-095 (TLR4 inhibitor, 3 μM)及SC-514 (IKK inhibitor, 100 μM)預培養後，再以鎳 (NiCl2, 1 mM)培養一段時間，之後進行各項分析。結果發現，鎳顯著的增加TLR4、Myd88 mRNA及蛋白質的表現量，IκB kinase (IKK)的磷酸化作用，p65轉移至細胞核內，以及MMP-9活性及蛋白質的表現，而quercetin及chrysin則和TLR4及IKK的抑制劑相似的，能抑制鎳所誘發的TLR4/NF-κB訊號路徑中蛋白質表現，磷酸化作用及位置的轉移，和降低MMP-9活性及蛋白質的表現量，不過此二者的劑量效應並非全都顯著。綜合以上結果顯示，Quercetin及chrysin能藉由調控TLR4/NF-κB的訊號路徑，而抑制鎳誘發的肺癌細胞轉移能力。
動物實驗部分
此外，本研究探討quercetin在體內是否能抑制A549細胞之侵犯性。首先，以裸鼠為實驗模式，經由尾靜脈注射3次A549細胞，然而經鎳處理之A549細胞，其促進肺部腫瘤生長及轉移的能力並沒有比未經鎳處理之A549細胞好。與經鎳處理之A549細胞相比，結果發現腹腔注射(IP)給予quercetin(10 mg/kg, 3 times/week)能顯著降低肺臟促發炎激素含量、脂質過氧化程度及白血球的DNA損傷，也降低肺臟重量。為了確認quercetin之效果，本研究更進一步進行另一種動物實驗，裸鼠經由尾靜脈注射4次A549細胞 (未經鎳處理)。結果發現A549細胞會顯著增加肺臟重量，而給予quercetin能顯著降低肺臟重量、肺臟及血漿中促發炎激素TNF-α及IL-10含量、脂質過氧化程度及白血球DNA損傷。以上結果支持細胞實驗的結果，結果顯示quercetin能抑制癌細胞之侵犯能力，然而需更多的研究探討quercetin是否在體內能向下調控TLR4/NF-κB之訊號路徑。

關鍵字

A549 ； 鎳 ； 植化素

並列摘要

Part 1
Nickel (Ni) is a pollutant commonly found in atmosphere. Studies showed that Ni-exposure promotes the invasive potential of human lung cancer cells through TLR4 signaling. Quercetin and chrysin, two flavonoids, are ubiquitously present in vegetables and fruits. Our previous study found that quercetin and chrysin significantly inhibited Ni-induced invasion and migration in human lung cancer cells. However the possible mechanisms are unclear. Thus, the present study investigated whether quercetin and chrysin inhibit the effects of Ni through the regulation of TLR4 signaling. Human lung adenocarcinoma A549 cells were pre-incubated with quercetin or chrysin (2 or 5 μM), CLI-095 (TLR4 inhibitor, 3μM) or SC-514 (IKK inhibitor, 100 μM), and then, were treated with NiCl2 (1 mM). The results showed that Ni-exposure significantly increased the mRNA and protein expression of TLR4 and Myd88, the phosphorylation of IκB kinase (IKK) and IκB, the translocation of p65 and the MMP-9 expression and activation. Similar to TLR4 and IKKβ inhibitors, quercetin and chrysin suppressed all the effects of Ni in A549 cells. However, the dose effects of quercetin and chrysin were not significant in all parameters. These results indicate that quercetin and chrysin inhibits Ni-promoted lung cancer cell invasion and migration through downregulation of TLR4/NF-κB signaling, at least in part.

Part2
Furthermore, we investigated whether quercetin inhibits the invasive of A549 cells in vivo. Fist, the nude mice were injected with A549 cells 3 times intravenously. However, the growth of tumor cells in the lung was not marked and the metastatic ability of Ni-treated A549 cells did not better than that of A549 cells without Ni-treatment. Comparing the mice bearing Ni-treated A549 cells, we found that quercetin (IP, 10 mg/kg, 3 times/week)administration tended to decrease the secretion of cytokines, lipid peroxidation, DNA damage in the lymphocytes and the weight of lung. To confirm the effect of quercetin, we further performed another animal study. In the study, the nude mice were injected with original A549 cells (without Ni-treatment) 4 times intravenously. We found that the growth of tumor cells was marked companying with a significant increase of lung weight. In consistent with the above findings, quercetin administration significantly decreased lung weights, the levels of TNF-α and IL-10 in the plasma and lung, lipid peroxidation and DNA damage in lymphocyte. These results support our in vitro findings, that is, quercetin suppresses the invation of cancer cells. However, it warrants more studies to investigate whether quercetin down-regulate the activation of TLR4/NF-κB pathway in vivo.

並列關鍵字

A549 ； Nickel ； quercetin ； chrysin

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