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tNOX as a potential marker for cancer diagnosis and therapy
|作者:||闕斌如||關鍵字:||cancer;癌症;生物科學類, 基礎醫學類, 生物技術;tumor protein;protein degradation;apoptosis;RNA interference;腫瘤蛋白;蛋白降解;細胞凋亡;RNA甘擾技術;基礎研究||摘要:||
由衛生署公佈的國人十大死因可知，癌症仍為國人生命健康最大的殺手。當細胞的生長與死亡無法達到平衡時，常是癌症發展的開始。在許多的癌症細胞中，調控生長的許多蛋白質分子通常是過量表現，因而有不受調控的細胞生長;另一方面來說，促使細胞凋亡的眾多的蛋白質分子則是表現量不足，因此腫瘤細胞死亡減少，而導致過多的細胞增生。因此，生物醫學研究學者致力於找出抑制細胞生長的方法並用在癌症治療上，希望可以抑制體內的非正常細胞快速增生，同時也希望這些非正常細胞不致轉移到其它組織，進而影響正常細胞的生存及功能。本實驗室的研究以tNOX腫瘤蛋白為主題，tNOX的酵素活性被證明與腫瘤細胞的生長有著正相關性，tNOX的酵素活性高通常與腫瘤細胞的快速生長有密切關連;相反的，若是tNOX的酵素活性被抑制，則腫瘤細胞的生長則會延缓，甚至導致腫瘤細胞發生凋亡的現象。一般實驗室常用的腫瘤細胞株中可偵測到tNOX的酵素活性;tNOX的酵素活性也在癌症病人的血清及腫瘤組織檢體中可偵測的到;但在非癌症病人或健康者則無此酵素活性，可預見tNOX腫瘤蛋白在臨床研究的重要性。我們在1995年報導過辣椒素可有效地抑制tNOX酵素活性，同時也抑制腫瘤細胞的生長;但在非腫瘤細胞則無此現象。並在1997年發表由癌症病人的血清中純化出tNOX蛋白過程的研究。再於2002年報導過tNOX的cDNA序列及轉譯蛋白的發現，但有關tNOX的生物功能及其與癌症發展的關係的研究並不完善，至於tNOX是否在癌症發展扮演一個必要或直接的角色，也並不清楚，若是能夠確定tNOX的生物功能，並找出其與癌症的關聯點，我們將可利用其特性而發展出有效的癌症治療方式。為了確認tNOX 腫瘤蛋白基因是否具有特異性，我們利用Northern Blot Analysis來檢測不同的組織中tNOX蛋白基因的表現量。根據我們的實驗結果顯示，正常的脾臟、前列腺、睪丸、卵巢、心臟、腦、骨骼肌肉、腎臟和胰臟等組織都發現有著tNOX RNA的表現，因此，tNOX 癌蛋白的基因並非只在腫瘤組織中，也存在於正常組織中，但其酵素活性卻只在腫瘤組織偵測得到，我們認為所謂tNOX腫瘤蛋白與腫瘤之相關性可能並不在基因轉錄的層級上，而是由於tNOX蛋白在轉譯後的一些修飾，如蛋白磷酸化，而導致癌細胞中tNOX蛋白功能的轉變成為不受調控的。目前我們正在確認tNOX蛋白上可能被磷酸化的位置，並探討其磷酸化與tNOX蛋白功能間的關係。我們在第一年利用細菌系統表現的tNOX 蛋白來製造tNOX 的多株抗體。結果顯示tNOX 的多株抗體可辨識腫瘤細胞中的tNOX蛋白質，而在非癌化細胞中幾乎沒有可被tNOX的多株抗體辨識的蛋白質。也發現tNOX的多株抗體可抑制腫瘤細胞的生長並進而引發凋亡。我們並加以探討此tNOX 的多株抗體引發細胞凋亡的機制與氧化壓力的增加有關。這些研究結果已2006年中發表於國際期刊上。我們所建立的tNOX多株抗體可以用來輔助檢測tNOX蛋白在腫瘤細胞及各種組織的表現，對於本實驗室的研究，具有重要的意義。 我們進而利用RNA甘擾技術來探討癌蛋白(tNOX)在腫瘤細胞的生物功能，並探討內生性的tNOX 是否具有使細胞增生並抑制凋亡的能力。我們以tNOX 基因特定的序列放入pCDNA-HU6 vector中，所產生的small hairpin RNA (tNOX-shRNA)結構可以與tNOX mRNA結合而抑制tNOX蛋白的表現。我們在這個研究中採用人類子宮頸癌HeLa細胞株為研究模式，因為此細胞株有著較高的內生性tNOX腫瘤蛋白。利用隨機排列的DNA序列作為控制組，或將tNOX-shRNA導入HeLa細胞中造成基因靜默，並以G418處理十至十四天後，可篩選出穩定的具有低度tNOX 蛋白表現細胞株後，並用這些細胞株來研究其細胞特性。以Northern Blot analysis確認出，我們的tNOX-shRNA可以有效的抑制tNOX 的RNA表現量，控制組中則與未經處理的細胞無太大的差異。同樣的結果也在半定量的RT-PCR 分析中被觀察到。再以西方點墨法並利用tNOX多株抗體來檢測tNOX 蛋白表現量，tNOX-shRNA穩定的具有低度tNOX 蛋白表現細胞株中的tNOX 腫瘤蛋白表現量明顯地被抑制，而控制組則與未經處理的細胞無太大的差異。這些結果都證實tNOX-shRNA可同時在轉錄及轉譯層次上有效的抑制tNOX 腫瘤蛋白表現量。tNOX 腫瘤蛋白已被證明與腫瘤細胞的生長有所關連，因而，我們對於缺少tNOX腫瘤蛋白的腫瘤細胞的細胞特性非常有興趣。我們使用穩定的具有低度tNOX 蛋白表現細胞株，與控制組的細胞來比較其間的細胞生長能力。細胞生長能力的檢測可以利用Trypan blue exclusion試驗將細胞生長的數目作定量，結果顯示穩定的具有低度tNOX腫瘤蛋白表現細胞株的細胞比控制組細胞生長數目相對減少。也利用3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl-tetrazolium bromide (MTT) 試驗來判定細胞存活的能力，由實驗結果得知穩定的具有低度tNOX腫瘤蛋白表現細胞株的細胞比控制組細胞生長來得緩慢，尤其在四十八及七十二小時的培養時間下更為明顯。這些證據充分顯示tNOX腫瘤蛋白的存在與腫瘤細胞的生長是具有密切關係的。我們發現這些利用tNOX-shRNA技術所產生的穩定具低度tNOX腫瘤蛋白表現細胞株的細胞，除了細胞生長變得較慢，其增生能力也受到了抑制;並進一步的發現其細胞移動的能力也有缺陷。細胞移動在正常細胞中的轉移是正常的現象，例如：胚胎受精著床、胚胎發育生長過程、白血球趨光反應、以及受傷組織的修補能力等，而這些過程都是經由嚴謹的機制所調控的。在腫瘤細胞中，許多受到嚴謹的調控機制的路徑常會失去控制，尤其是調控細胞增生或轉移能力的因子再也不受控制，因此腫瘤細胞便會大量增生並且轉移到其他組織，並在這些組織中增生成腫瘤，造成癌症的轉移，也導致癌症治療上的困難。我們利用經過tNOX基因靜默後所產生的穩定的具低度tNOX腫瘤蛋白表現細胞株的細胞比控制組細胞比較，以傷口修補試驗來檢驗細胞傷口休復的能力及細胞移動的能力，發現具有低度tNOX腫瘤蛋白表現細胞株有著比控制組的細胞較低的細胞移動能力，我們認為降低tNOX的蛋白表現量直接或間接地調控了腫瘤細胞的移動能力，進而影響到腫瘤細胞的生長。我們的研究證實tNOX腫瘤蛋白與腫瘤細胞生長的密切關係，也研究其與腫瘤細胞移動有關的分子機制。許多可抑制腫瘤細胞生長的藥物如辣椒素及綠茶素，也被發現可抑制tNOX腫瘤蛋白酵素活性，而在抑制tNOX活性的同時，並引發腫瘤細胞的凋亡。在今年度的研究中，我們進一步探討在人類大腸癌細胞中，辣椒素及綠茶素是如何抑制tNOX腫瘤蛋白的活性。首先我們確認辣椒素及綠茶素的處理，是否可降低大腸癌細胞的生長，利用3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl-tetrazolium bromide (MTT) 試驗來判定細胞存活的能力，由實驗結果得知在辣椒素及綠茶素的處理下，大腸癌細胞的生長被有效的抑制。我們也利用流式細胞儀來偵測細胞死亡的狀況，發現辣椒素及綠茶素的處理引發了大腸癌細胞的凋亡。我們另外也使用UVC的刺激，發現UVC的刺激會引發大腸癌細胞的凋亡。進一步的研究發現，在這些藥物或在UVC的刺激下，tNOX腫瘤蛋白的表現有下降的趨勢，並且是隨著藥物濃度及處理時間增加而增加。這是第一篇報導探討tNOX腫瘤蛋白的表現量因外在刺激而受到抑制，我們認為抗癌藥物的處理導致tNOX腫瘤蛋白受到降解，而tNOX腫瘤蛋白的表現量不足影響了腫瘤細胞的生長。為了驗證這個假說，我們將大腸癌細胞以蛋白合成抑制劑 (cycloheximide) 前處理後，再以UVC的刺激下來觀察腫瘤細胞中tNOX蛋白質的表現，發現tNOX腫瘤蛋白的半生期很明顯的因UVC的處理而減短;很顯然的，tNOX腫瘤蛋白因為外在刺激而影響到其蛋白的穩定度。另外我們也發現，UVC的刺激可在轉錄的層級上抑制tNOX腫瘤蛋白的表現。以不同的蛋白酶抑制劑前處理後，可發現由UVC刺激引發的tNOX腫瘤蛋白表現下降可得到舒緩;而同樣的結果也發生在辣椒素及綠茶素的處理下。我們也在tNOX-shRNA技術所產生的穩定具低度tNOX腫瘤蛋白表現細胞株系統下，來探討tNOX腫瘤蛋白表現量與在腫瘤細胞發生凋亡間的關係，我們發現具低度tNOX腫瘤蛋白表現細胞株對細胞凋亡的刺激變得較為敏感，以流式細胞儀及共軛式螢光顯微鏡都可觀察到，在具低度tNOX腫瘤蛋白表現細胞株中有比較高比例的細胞發生凋亡。再次驗證我們的假說，tNOX腫瘤蛋白的表現與腫瘤細胞的生長及細胞凋亡之發生與否具有密切的關係。
Unbalanced cell survival and cell death can result in the development of cancer. In many cancer cells, the survival proteins are up-regulated whereas the pro-apoptotic proteins are down-regulated to result in uncontrolled cell growth. Therefore, recent interests have been focused on identifying the anti-survival pathways for the development of cancer therapy. Tumor-associated NADH oxidase (tNOX) belongs to a family of growth-related NADH (or hydroquinone) oxidases. tNOX cDNA has been cloned and functional motifs of tNOX have been identified using site-directed mutagenesis. These structural elements include a quinine-binding site, an adenine-nucleotide-binding site, and a CXXXC cysteine pair important for its activity. tNOX was originally identified as a plasma membrane protein in rat hepatoma, and has since been identified in numerous cancer cell lines. It has also been found in sera of cancer patients, suggesting its potential in clinical applications. Moreover, accumulating evidence supports an important role for tNOX in regulating cancer cell growth. In addition, the survival of transformed cells is inhibited by antisera raised against bacterially expressed tNOX protein, a cytotoxic effect that is associated with apoptosis; in contrast, non-transformed cells are unaffected. A key role for tNOX in regulating cell growth is further supported by the observation that the growth rate of MEF cells from tNOX-overexpressing transgenic mice is approximately two-fold greater than that of wild-type cells. A recent report has further demonstrated that suppression of tNOX using antisense oligonucleotides decreased HeLa cell colony formation, whereas tNOX overexpression increased the invasiveness of non-cancerous MCF-10A cells, suggesting that tNOX increased the ability of cancer cells to acquire an aggressive phenotype. We also have constructed the RNA interference to specifically knock down the expression of tNOX protein in cancer cells. Gene-silencing by RNA interference experiments were conducted in HeLa (cervical carcinoma) cells using the shRNA system in pCDNA-HU6 with Bam HI and Hind III sites. Stable clones were selected with G418 for 10-14 days and used for further characterization. The phenotypes of clones were verified with both Northern blot and Western blot analyses. The Northern analyses revealed that the level of RNA of tNOX was repressed by RNA interference (tNOX-shRNA) in stable clones. Similar patterns were also observed in semi-quantification RT-PCR. Furthermore, the reduction of RNA was correlated with the reduction of tNOX protein expression as in the Western blot analysis. The viability of HeLa wild type, shRNA control, and tNOX-shRNA clones, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl- tetrazolium bromide (MTT) test. We found that tNOX-knockdown cells have shown a decreased cell proliferation when compared with HeLa wild type and shRNA control, in particularly, at the 48 h and 72h culturing time. In addition, the capsaicin-inhibition was suppressed in the knock-down cells. To further analyze the inhibited cell viability observed in tNOX-shRNA cells, we have utilized flow cytometry to analyze the cell death pattern, particularly in apoptosis. tNOX-knockdown by RNA interference did not induce spontaneous apoptosis in HeLa cells.To further investigate the effects of tNOX-shRNA, we conducted a search for tNOX targets using microarray analysis. Among the genes most down-regulated by tNOX-shRNA are Rho proteins, which are believed to primarily regulate cell motility. Consistent with these observations, our results from wound healing assay and Boyden chamber assay suggest that tNOX-shRNA mediates a decrease in cell migration. To search for a possible mechanism to account for the tNOX-shRNA-mediated decrease in cell motility, we next measured expression of FAK and Rac in HeLa cells. Western blot analysis showed that there were slight decreases in the levels of both Rac and FAK proteins in tNOX-shRNA-treated cells. To further address whether the activation of Rho proteins could explain the tNOX-shRNA-dependent decrease in cell migration, we examined the membrane versus cytosolic distribution of Rho proteins. In tNOX-shRNA cells, Rac was predominantly decreased in the membrane fractions, with only a minor reduction observed in the cytosolic fractions. The consequence of these changes was a reduction in the membrane:cytosolic ration of Rac from 1.0 in wild type, to 0.69 and 0.33 in tNOX-knockdown cells. Similarly, the membrane fraction of Rho was drastically diminished in all three tNOX-shRNA clones. In contrast, tNOX-knockdown had no significant effect on the membrane:cytosolic ratio of Cdc42. Together, these results suggest that a reduction in tNOX expression attenuates cell motility through a reduction in the membrane association of Rac and Rho, but not Cdc42.The presence of tNOX protein has been demonstrated in several lines of cancer cells where it is associated with cell growth. Capsaicin, an important component of chili pepper, has been shown to induce apoptosis in various cancer cell lines; however, with the exception of two reports using HT-29 cells, this has not been shown in colon cancer lines. EGCg, also a natural chemopreventative compound, has a well-established role in suppressing colon carcinogenesis via various pathways. Using viability assays, we found that both EGCg and capsaicin induced significant cytotoxicity in HCT116 cells at concentrations of 100 M and 250 M, respectively; notably, these cells exhibit a higher tolerance to capsaicin. In this current study, the concentration of EGCg required to induce cytotoxicity is consistent with other reports. We have previously shown that the cytotoxic effects of capsaicin and EGCg are associated with inhibition of tNOX activity and apoptosis in HeLa and BT-20 cells; here, we verified their cytotoxic effects in HCT116 cells. At 24-hours post-exposure, the percentage of apoptotic cells increased by 54.5% and 27.3% after treatment with 100 M EGCg and 250 M capsaicin, respectively. In addition, exposure to DNA-damaging UVC radiation for 18 hours also induced apoptosis in HCT116 cells. These results demonstrate that diverse stress stimuli induced a concentration- and time-dependent increase in cytotoxicity and apoptosis. To further analyzing the mechanism of EGCg-induced apoptosis, we investigated the levels of PARP cleavage and p53 phosphorylation and our results indicated there were increases over time on these two molecules in HCT116 cells treated with EGCg. Using an antiserum generated in our laboratory that recognizes multiple tNOX forms, we showed that the increase in PARP cleavage occurred concomitantly with a decrease in tNOX levels; moreover, this EGCg-induced reduction in tNOX occurred in a time-dependent manner. At lower EGCg concentrations, an increase in tNOX was observed. Similarly, UVC (40 J/m2) irradiation led to p53 activation and a time- and dose-dependent decline in tNOX levels. To extend these observations to another cancer cell line, we exposed A549 cells to UVC irradiation. In these cells, UVC treatment also induced tNOX down-regulation, indicating that this response is not specific to a single cancer cell line. In this first-reported use of UV radiation to assess tNOX expression, we found that tNOX was significantly down-regulated by DNA-damaging stress. To verify that UVC-mediated tNOX down-regulation occurred at the protein level, we pretreated cells with cycloheximide (CHX) for 30 minutes and then evaluated tNOX levels. tNOX levels progressively decreased in CHX/UVC-treated cells to an extent greater than seen after CHX treatment alone. A densitometric analysis based on equal amount of total protein of each sample confirmed that tNOX deteriorated more rapidly in the CHX/UVC-treated cells, suggesting that UVC exposure decreased the half-life of tNOX. Additionally, a reduction in the tNOX RNA level was observed after UVC exposure; this was also observed in EGCg-treated cells. We also confirmed that UVC irradiation induced a reduction in tNOX RNA transcription. To determine whether protein degradation might contribute to tNOX down-regulation, we tested various protease inhibitors and showed that they rescued UVC-induced tNOX down-regulation. In addition, pretreatment with the caspase-8 inhibitor Z-IETD or the caspase-9 inhibitor Z-LEHD partially recovered tNOX degradation. Using shRNA-mediated silencing of tNOX in HEK293 cells, we showed that tNOX knockdown enhanced capsaicin-mediated apoptosis, as indicated by increased PARP cleavage, an elevated sub-G1 population, and augmented Annexin V-staining, indicating that the loss of tNOX sensitized transformed cells to apoptosis. Additional evidence for apoptosis in tNOX-knockdown cells was provided by confocal microscopy, which showed that cytochrome c was released from mitochondria into the cytosol by treatment with 100 M EGCg, whereas cytochrome c remained in the mitochondria in untreated cells. Re-expression of EGFP-tNOX in tNOX-knockdown cells partially recovered cell proliferation, however, failed to exhibit the anti-apoptotic effect, possibly reflecting a less-than-optimal functional configuration of the fusion protein as suggested in oxidase activity assay. Alternatively, because exogenous tNOX requires a prolonged expression interval (> 96 hours) to exert a significant biological effect as suggested by our proliferation data, the absence of an anti-apoptotic effect may simply reflect limitations inherent in the transient transfection system used. It is also possible that other function of tNOX protein, besides NADH oxidase, might be involved in regulating cell proliferation. tNOX is known to have a role in transformed cell growth, although this may possibly be due to regulation of proliferation rather than inhibition of apoptosis. In conclusion, we report the novel finding that tNOX, which is required for transformed cell growth, is suppressed during apoptosis. Capsaicin and EGCg have been shown to preferentially inhibit tNOX activity, and our results here suggest that this inhibitory effect is caused by down-regulation of tNOX. The down-regulation of tNOX results in decreased cell proliferation and renders cells sensitive to apoptosis, leading to decreased growth.
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