研究電位閘控型鈉離子通道對神經性疼痛及腫瘤細胞之影響

Effects of Voltage-Gated Sodium Channels on Neuropathic Pain and Tumor Cells

黃伯元(Po-Yuan Huang)

指導教授 ： 李銘仁

國立臺灣大學/生命科學院/生命科學系/博士(2015年)

https://doi.org/10.6342/NTU.2015.01583

摘要

由於電位閘控型鈉離子通道異常造成的鈉離子通道病變被認為和神經性病變疼痛及腫瘤細胞轉移相關，在本篇論文我們探討鈉離子通道病變對這兩種疾病的影響。我們對Nav1.7鈉離子通道突變引起的原發性肢端紅痛症進行電生理特性分析，同時也評估lidocaine對高度表現鈉離子通道的惡性週邊神經髓鞘瘤的影響。原發性肢端紅痛症的症狀為肢端燒灼疼痛感及皮膚上的紅斑，環境溫度升高或運動會觸發疼痛。我們對三個突變型鈉離子通道I136V、I848T和V1316A進行電生理特性的探討。這些突變型鈉離子通道皆在電壓依賴性活化產生過極化偏移，在穩定快速去活化表現出去極化偏移，且比起原生型，突變型鈉離子通道能更快從去活化恢復。溫度升高是造成成病人疼痛加劇的關鍵因素，我們比較25°C和35°C對原生型與突變鈉離子通道的影響。在35°C，野生型、I136V及V1316A突變通道在電壓依賴性活化皆產生過極化偏移，但是突變型通道仍比野生型通道有更顯著之過極化偏移。升溫則在三個突變型通道之穩定去活化皆產生顯著去極化偏移，但野生型通道則不受影響。這些特性改變有助於解釋臨床上觀察到的現象，特別是在溫度升高造成的影響。我們同時也測試lidocaine及mexiletine這兩種鈉離子通道阻斷劑對三個突變通道的抑制效果，作為治療上的參考。分析lidocaine及mexiletine對突變型與野生型Nav1.7通道抑制效果的IC50後，我們發現lidocaine很可能不適合用來治療帶有這些突變的病人，而如同臨床觀察所示，mexiletine可減緩帶有I848T突變的病人的症狀，和我們的研究結果I848T突變型通道具有對mexiletine較低的IC50相符合。惡性週邊神經髓鞘瘤是一種十分罕見的軟組織肉瘤，發生於週邊神經分枝或週邊神經纖維，且多源自於許旺氏細胞。惡性週邊神經髓鞘瘤缺乏有效的治療方式，因此病人的五年存活率非常低。大約百分之二十至五十的惡性週邊神經髓鞘瘤病人患有神經纖維瘤。神經纖維瘤是一種因NF1基因突變造成的自體顯性遺傳疾病。我們發現NF1基因突變的惡性週邊神經髓鞘瘤細胞株的鈉離子通道表現量增加，我們進一步研究鈉離子通道各亞型在S462這個細胞株上的表現，發現Nav1.2、1.3、1.5、1.6和1.7這幾個亞型和正常人類許旺氏細胞相比有顯著增加。為了瞭解鈉離子通道表現量增加對S462細胞的影響，我們使用TTX和lidocaine這兩種鈉離子通道阻斷劑處理細胞。結果發現TTX對S462細胞的入侵作用或細胞增生皆無影響，然而lidocaine卻具有細胞毒殺作用，且隨劑量增加毒殺作用越強。然而正常人類許旺氏細胞具有較高的lidocaine耐受性。我們也發現lidocaine能抑制S462細胞的核糖體蛋白S6激酶，及其受質S6和eIF4B活性。同時我們的研究也顯示lidocaine不影響凋亡蛋白酶-3之活性，但會增強細胞自噬作用。在本篇研究我們發現lidocaine能抑制的惡性週邊神經髓鞘瘤細胞的蛋白質合成，同引發細胞自噬現象。

關鍵字

肢端紅痛症 ； SCN9A ； 電位閘控型鈉離子通道 ； 神經纖維瘤 ； NF1 ； S6激酶

並列摘要

Sodium channelopathy caused by dysfunction of voltage-gated sodium channel (VGSC) has been implicated in neuropathic pain and metastasis of tumor cells. In the thesis, we investigated two different effects of sodium channelopathy in pain disorder and tumor cells. We characterized the biophysical properties of mutant Nav1.7 channel, which causes primary erythromelalgia (PE), and effect of lidocaine on NF1-derived malignant peripheral nerve sheath tumor (MPNST) cells with expression of sodium channels. The symptoms of PE are extreme burning pain and erythema in the extremities upon heat challenge or exercise. We examined the electrophysiological properties of three mutant Nav1.7 channels, I136V, I848T, and V1316A. Mutant channels displayed hyperpolarizing shift of voltage dependence activation, depolarizing shift in steady state fast inactivation, and faster recovery from inactivation as compared to wild type channel. Elevating temperature is critical for inducing pain attack in PE. We compared the temperatures at 25 and 35°C to evaluate the effect on wild type and mutant channels. Wild type, I136V and V1316A mutant channels exhibit a further hyperpolarizing shift in activation at 35°C than that at 25°C, nevertheless, mutant channels still produce hyperpolarizing shift compared to wild type. Increasing temperature caused depolarizing shift in steady state fast inactivation among three mutant channels except for the wild type channel. These results suggest mutant channels may contribute to hyperexcitability of sensory neurons that explains part of the symptoms of PE, especially at high temperature. We tested whether selective sodium channel blockers such as lidocaine and mexiletine exert differential antagonistic effect on mutant sodium channels. The mutant channels exhibited higher IC50 values for lidocaine compared to wild type channel, which suggests that lidocaine would not be effective medication for the patients carrying these mutations. However, IC50 for mexiletine is lower for I848T mutant channel. The result consists with the clinical observations that mexiletine alleviates symptoms in the patient with I848T mutations. MPNST is a rare soft tissue sarcoma originated from Schwann cells. Twenty to fifty percent of the MPNSTs occur in patients with neurofibromatosis type 1 (NF1). NF1 is an autosomal dominant disorder resulted from a mutation of human NF1 gene. In our NF1-derived MPNST cell lines (S462, ST8814 and T265), the expression of VGSC increased significantly as compared to normal human Schwann cells. Further analysis demonstrated that the mRNA level of Nav-1.3, -1.5, -1.6 and -1.7 were significantly increased in S462 MPNST cells. The overexpression of Nav channels in MPNST cells brings about the idea that blockade of sodium channel might implicate in the cell phenotypes of MPNST. The tetrodotoxin has been used to treat without a positive effect in inhibiting the cell proliferation and invasiveness. However, lidocaine has shown a cytotoxic effect in a dose dependent manner whereas the normal Schwann cells were much resistant than the MPNST. Concerning the decrease of cell viability after treatment of lidocaine, cell death pathways were investigated. The activity of caspase-3 was not changed in treatment cells; however, the increase of autophagy by high level of transformation from LC3-I to LC3-II was identified in MPNST cells treated with lidocaine. Further studies on the molecular mechanism involving the lidocaine induced cell death identified that the treatment resulted in reduction the level ribosomal protein S6 kinase as well as its substrates, S6 and eIF4B. The S6 and eIF4B play a role in protein translation machinery. These findings suggest that lidocaine, a sodium channel blocker can induce the autophagy process of MPNST associated with inhibition of ribosomal protein kinase S6 which can hinder the protein synthesis in MPNST. In this study, we demonstrated that two different mechanisms result in the sodium channelopathies. It suggests that voltage-gated sodium channels might play a role in progression of diseases.

並列關鍵字

Erythromelalgia ； SCN9A ； Nav1.7 ； Neurfibromatosis type 1 ； NF1 ； S6 kinase

參考文獻

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