AUY922(AUY)為近幾年由Vernalis與Novartis兩間藥廠所共同新開發的抗腫瘤藥物，目前已經證實AUY屬於一種強效型的熱休克蛋白90(heat shock protein-90)抑制劑。熱休克蛋白90是細胞內含量豐富的伴隨蛋白(chaperone)之一，對於細胞的存活、增生以及細胞凋亡都是不可或缺的。由於腫瘤細胞存活在極富挑戰的環境下，必須仰賴熱休克蛋白90使其生長並存活，因此，在治療癌症過程中，熱休克蛋白90是一個很重要的標的目標。更重要的是，雖然熱休克蛋白90抑制劑對於各種不同的實質腫瘤(solid tumors)具有不錯的治療潛力，然而，服用AUY後，卻出現一些並不是藉由抑制熱休克蛋白90機制產生的額外影響。換言之，儘管AUY可以強力的抑制熱休克蛋白90，卻也發現有副作用的產生，造成視覺上的毒害(ocular toxicities)就是其中之一。但是，由AUY所引起視覺傷害的詳細機轉，目前所知甚少。因此，在本篇研究中，我們利用AUY及其它相關化合物來探討藥物與感光細胞上離子通道之間的作用。我們利用細胞膜片箝制技術(patch-clamp technique)來評估661W細胞的電生理功能性。結果顯示，在完整細胞模式(whole-cell model)，AUY使非選擇性陽離子電流(non-selective [NS] cation current，INS)增加並呈現濃度依賴性的關係，進一步經由公式換算，得知藥物發揮一半效力時的濃度(EC50)為1.7 μM。此二藥物，iberiotoxin (200 nM)及apamin (200 nM)對於AUY誘發的INS沒有造成影響，而氯化鑭(LaCl3 100 μM)可以使其恢復。17-AAG (3 μM) 或 BIIB021 (3 μM)也都可以增加INS。其次，我們藉由將細胞膜處於過極化的電壓階，發現AUY會減緩INS不活化曲線的時間常數。在細胞接連片紀錄(cell-attached recordings)中，AUY可以增加NS陽離子通道的活性但並不會改變此通道的電導。接著藉由給予較長時間的斜坡脈衝式電壓，我們看到AUY導致NS陽離子通道的活化曲線傾向於較不去極化的電位，改變約18毫伏特，且對其曲線的斜率並沒有顯著改變。我們也觀察到AUY會增加NS陽離子通道打開的機率，LaCl3 (100 μM)則會減少，然而BAPTA-AM (10 μM)此藥物也可以增加。另外，AUY(10 μM)並沒有改變L-型鈣離子電流(L-type Ca2+ currents，ICa,L)電流-電壓關係(current-voltage relationship)的情況下，會抑制ICa,L。再利用電流箝制(current-clamp)記錄，也明確指出AUY可以造成細胞去極化的作用。最後，經由文獻的參考，我們進行對661W細胞進行轉染TRPM3 channel (transient receptor potential melastatin-3 channel) siRNA (short interfering RNA)的實驗，明確看到NS陽離子通道活性減少的結果。且AUY誘發的INS與熱休克蛋白90活性無關聯性。因此，AUY是直接與TRPM3交互作用，促使增加INS，接連呈現細胞膜去極化的現象。總而言之，於本篇研究中，證明了在感光受器細胞中，主要產生INS的源頭是NS陽離子通道中的TRPM3 channels。結論是，這些離子通道所共同組成的活化現象，在AUY造成視覺上敏銳度改變的情況中，扮演關鍵性的角色。

NVP-AUY922 (AUY) is currently recognized as a potent inhibitor of heat shock protein-90 (HSP-90). HSP-90, the most abundant molecular chaperone, is recognized to be essential for cell survival, proliferation, and apoptosis. HSP-90 is an important target for cancer therapeutics because tumor cells exist in a stressful environment and need depend on HSP-90 to grow and survive. Most importantly, the HSP-90 inhibitors such as AUY are thought to have strong therapeutic potential in a wide variety of tumor types, including solid tumors. However, AUY appears to possess an additional effect that does not require inhibition of HSP-90. Despite its inhibitory effect on HSP-90 activity, there are some adverse effects of this compound including ocular toxicities. The precise mechanism of action of AUY-induced ocular damage is still poorly understood. Therefore, in this study, we sought to investigate the possible effects of AUY and other related compounds on ion channels in a photoreceptor cell line (661W). The patch-clamp technique was used to evaluate electrophysiological properties of 661W photoreceptor cells. In whole-cell model, AUY increases the amplitude of non-selective (NS) cation current (INS) in a concentration-dependent fashion with an EC50 value of 1.7 μM. Neither iberiotoxin (200 nM) nor apamin (200 nM) had any effects on AUY-induced INS, while LaCl3 (100 μM) reversed it significantly. 17-AAG (3 μM) or BIIB021 (3 μM) increased the INS amplitude. AUY slowed the time course of INS inactivation elicited by membrane hyperpolarization. In cell-attached recordings, when AUY was applied to the bath, the activity of NS cation channels was significantly elevated with no change in single-channel conductance. With long-lasting ramp pulse protocol, addition of AUY produced a left shift in the activation curve of NS channels by 18 mV, with no change in the slope factor of the curve. The probability of NS-channel openings enhanced by AUY was decreased by LaCl3 (100 μM), while it was increased by BAPTA-AM (10 μM). AUY (10 μM) also suppressed L-type Ca2+ current with no change in the current-voltage relationship of this current. Under current-clamp conditions, addition of AUY caused membrane depolarization. In cells transfected with TRPM3 siRNAs, NS-channel activity was diminished. AUY-mediated stimulation of INS is unlinked to its inhibition of HSP-90 activity. Therefore, AUY interacts directly with the TRPM3 channel to increase INS and subsequently to depolarize the membrane in these cells. Taken together, our results suggested that TRPM3-encoded NS channels are primarily responsible for generation of INS in 661W cells. In conclusion, constitutive activation of these channels may play crucial roles in AUY-induced changes in visual acuity.

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