類別:

鼻咽癌專一性單株抗體的製備與功能之探討

2021-06-13T01:24:52Z

標題: 鼻咽癌專一性單株抗體的製備與功能之探討; Generation and Characterization of Monoclonal Antibodies Against Nasopharyngeal Carcinoma 作者: Shin-Yi Yu; 游欣頤摘要: 鼻咽癌為中國人特有的癌症。在台灣鼻咽癌佔國內癌症的第十四位。目前鼻咽癌的治療方式有手術、放射線治療及化學治療，但以放射治療為主。而這種癌症對放射線極為敏感，故療效很好。經治療之結果，大部分病人五年之存活率超過90%，但轉移嚴重的病人五年存活率低於50%，其中局部復發及遠端轉移是治療的主要失敗原因，加上一般療法常會帶給病人相當大的副作用，因此我們嘗試找出其他更好的治療方式。近年來，因治療性抗體發展與生物技術的進步，使抗體在癌症治療上逐漸扮演重要的角色。目前已有不少治療性抗體已通過美國食品藥物管理局認可，在臨床治療上使用。本篇研究，我們以NPC TW01免疫BALB/c老鼠後，利用單株抗體融合的技術，產生十四株專一性對抗到鼻咽癌的單株抗體，進行一連串的功能測試。我們選擇其中五株單株抗體來進行ELISA、流式細胞儀、西方墨漬法、免疫化學染色等研究，結果發現這五株抗體皆能專一對抗鼻咽癌細胞，卻和正常鼻咽細胞沒有反應。在西方墨漬法中觀察到NPC-Ab 1-1、 NPC-Ab 6-1、NPC-Ab 9-2 和NPC-Ab 14-1可偵測到NPC 細胞的蛋白質，分子量分別為 47 kDa、170 kDa、47 kDa、220 kDa。除此之外，這些抗體也可辨認到其他癌細胞的蛋白質，例如胰臟癌、口腔癌、大腸癌、肺癌等，對於鼻黏膜細胞、血球細胞和人類內皮細胞則無反應。除此，從細胞流式儀的實驗中，發現NPC-Ab 4-2 和 NPC-Ab 6-1對於鼻咽癌的細胞膜蛋白有較佳的結合能力。在細胞凋亡實驗中，更證明其中一株抗體NPC-Ab 4-2 可以造成鼻咽癌細胞進行細胞凋亡，並且也進一步證明具有抑制鼻咽癌細胞增生的能力。最後我們也研究鼻咽癌的標的治療，以NPC-Ab 6-1 的抗體接上Lipo-Dox來治療鼻咽癌腫瘤的SCID mice，結果發現含有此抗體的標的微脂體對老鼠腫瘤生長的抑制，明確比其他控制組更具療效。經過以上結果顯示，我們所製備的單株抗體，有其專一性對抗癌症的特性，可用來偵測鼻咽癌抗原表現及運用在鼻咽癌治療上。; Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in southern China, Taiwan, and Singapore. The 5-year survival rate has been improved for localized NPC cases to > 90% in some hospital, but for advanced cases, the survival rate remains below the 50% margin. Radiotherapy, surgical removal, and chemotherapy have been used for decades with varying degrees of success. However, the side-effects of radiotherapy are substantial, including mucositis, central nervous system toxicity, alterations in taste, and problems with saliva and trismus. Furthermore, chemotherapy can easily induce chemoresistant tumor cells. There is a great need for developing new modalities of therapy such as targeted therapy. Over the last few years, monoclonal antibodies (mAbs) have repeatedly made the successful transition from the bench to the bedside by the US Food and Drug Adminstration (FDA) for use in various clinical settings, including cancer therapy. In this study, we generated 14 mAbs specific against NPC cells. Five mAbs NPC-Ab 1-1, NPC-Ab 4-2, NPC-Ab 6-1, NPC-Ab 9-2 and NPC-Ab 14-1 revealed different binding pattern to nine cancer and two normal cells were further characterized. The specificity of these antibodies was further confirmed by the ELISA, Western blot, immunohistochemistry stain and flow cytometric assays. In Western blot analysis, NPC-Ab14-1 can recognize a protein of 220 kDa; NPC-Ab 9-2, a protein of 47 kDa; NPC-Ab 1-1, protein of 47 kDa; NPC-Ab 6-1 can bind a protein of 170 kDa. These target proteins were also identified in other cancer cell lines, such as PaCa-2, SAS, HCT116 and H460. Results of FACS analysis further demonstrated that the NPC-Ab 4-2 and NPC-Ab 6-1 can detect the antigens expressed on the cell surface of NPC but not NNM cells. Furthermore, the NPC-Ab 4-2 can even inhibit NPC cell proliferation and induce apoptosis by MTT and flow cytometry assays. Finally, to develop of ligand-targeted therapy using diagnostic animal experiments, we found that NPC-Ab 6-1 antibody-conjugated targeting liposome can significantly enhanced the therapeutic efficacy of the drug against NPC. These data suggest that these mAbs might be useful to identify tumor antigens, and developing ligand-targeted therapy in the treatment of nasopharyngeal carcinoma.

鼻咽癌基因NOLC1 調控腫瘤生長之機轉

2021-06-15T00:17:57Z

標題: 鼻咽癌基因NOLC1 調控腫瘤生長之機轉; Mechanism of NOLC1 gene regulating nasopharyngeal carcinoma progression 作者: Yu-Chyi Hwang; 黃鈺琪摘要: 鼻咽癌是中國南方、新加坡、香港、台灣等地的中國人好發的癌症。然而，它的病源因子尚未完全被研究清楚。目前被認為EB病毒 (Epstein-Barr virus)很可能和鼻咽癌有密切關係。然而，EB病毒和它的宿主鼻咽癌的基因之間所牽涉到的分子機轉仍然不很明確。為了釐清引起鼻咽癌而改變的基因表現，我們設計了兩組全面性的基因篩選的實驗來鑑定可能參與鼻咽癌細胞變化的基因。第一個是兩組互相比較的基因晶片實驗：一組是比較鼻咽癌（無EB病毒）和正常上皮細胞的基因晶片，另一組是比較有EB病毒感染和沒有EB病毒感染的鼻咽癌基因晶片。我們利用交叉比較這兩組基因晶片所得到的資料而鑑定它們彼此間的相關性。結果顯示EB病毒似乎比較傾向於調節在鼻咽癌細胞中已經有差異表現的基因，對於無異於正常細胞表現的基因則沒有什麼調控現象。綜合這兩組基因的資料可以顯示利用全面性的基因體掃瞄方式研究鼻咽癌以及EB病毒感染的鼻咽癌細胞的轉錄基因量，可以成功的呈現EB病毒感染具有調控有差異表現的鼻咽癌基因的能力。第二個實驗是結合PCR-Select™ cDNA 相減雜交（subtractive hybridization）技術和基因晶片分析來鑑定鼻咽癌細胞株和正常鼻咽細胞之間的差異基因表現。我們發現在大部份的鼻咽癌細胞株和病人檢體中發現NOLC1基因的表現比正常細胞高出許多。利用NOLC1基因的干擾RNA質體（shRNA）轉染鼻咽癌細胞的實驗顯示減少NOLC1基因表現會降低鼻咽癌細胞的生長速率。在帶有異種移植（xenografts）鼻咽癌細胞的嚴重混合性免疫缺乏症（severe combined immune deficiency，簡稱SCID）老鼠的實驗中顯示：減少NOLC1基因表現的shRNA轉染鼻咽癌細胞在SCID老鼠體內第十一週時，腫瘤大小比控制組小了百分之八十二，而且腫瘤內呈現出嚴重的細胞凋亡（apoptosis）。在檢測與細胞生長、細胞凋亡、和血管新生相關的基因表現後，發現轉染NOLC1基因shRNA的鼻咽癌細胞內，MDM2、MMP9、VEGF等基因表現降低。相反的，TNF、BAX、CASP1等基因的表現被調節後上升。在染色體免疫沉澱（Chromatin Immunoprecipitation）和共同轉染的實驗中顯示TP53蛋白質調控MDM2基因的表現需要NOLC1蛋白質的共同活化。以上這些發現顯示NOLC1在鼻咽癌細胞中扮演一個促進轉錄調控的角色，進而調控與細胞凋亡和細胞生長相關的基因，並且證明了NOLC1蛋白質與TP53蛋白質在鼻咽癌細胞中合力活化MDM2基因的啟動子（promoter）。; Nasopharyngeal carcinoma (NPC) is one of the most common cancers among Chinese living in South China, Singapore, and Taiwan. Its etiological factors are not yet well defined. It was proposed that Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC). However, the molecular mechanisms involved in the effect of EBV on NPC host genes remained vague to date. To clarify the possible genetic alteration, we used two approaches to globally screen for genes involved in NPC. The first one was designed by two sets of microarray experiments; NPC (EBV-free) compared with normal epithelial cells and EBV+ compared with EBV-free NPC arrays. We analyzed the datasets by cross-comparison gene clusters involved in EBV targeting and the NPC host gene expression profiles to identify the correlation between them. Result from the statistical analysis showed that EBV regulates genes from the differentially expressed group in NPC cells than those from the unchanged expression group. Taken together, the genome-wide comparative scanning of transcription levels of genes in EBV+ and EBV－ of NPC has successfully demonstrated that EBV infection transduces signals involved in NPC gene expression. The second approach was combined the PCR-Select™ cDNA subtractive hybridization with microarray analysis to identify differential gene expression between NPC cell lines and normal nasomucosal cells. We identified that human NOLC1 (nucleolar and coiled-body phosphoprotein 1) gene expression was upregulated in most NPC cell lines and biopsy specimens. NOLC1 knockdown NPC cells showed reduced cell proliferation rates. In SCID mice bearing NPC xenografts derived from these transfected cell lines, the NPC xenograft tumors had diminished about 82% in size with marked tumor cell apoptosis compared with the control at week 11. To understand the contribution of NOLC1 in controlling tumor cell proliferation, a screening was performed to measure the expression of 26 genes related to apoptosis and angiogenesis in the transfectants showed that MDM2, MMP9, and VEGF expression were downregulated, whereas the expression of TNF, BAX, and CASP1 was upregulated connection. Chromatin immunoprecipitation and cotransfection experiments showed that TP53-regulated MDM2 expression requires NOLC1 coactivation. These findings suggest that NOLC1 plays a role as a transcriptional regulator to regulate apoptosis-related and proliferation-related genes, and demonstrate a relationship between NOLC1 and TP53 in the synergistic activation of the MDM2 promoter in NPC cells.

適用於多種亞型的肺癌標的胜肽於複合式藥物傳輸與分子磁振照影之應用

2021-06-08T01:38:35Z

標題: 適用於多種亞型的肺癌標的胜肽於複合式藥物傳輸與分子磁振照影之應用; Lung Cancer-Targeting Peptides with Multi-subtype Indication for Combinatorial Drug Delivery and Molecular Magnetic Resonance Imaging 作者: Yi-Hsuan Chi; 紀怡亘摘要: 背景：肺癌位居全世界癌症死亡率之首。目前已有的肺癌標靶藥物主要是針對肺腺癌的表皮細胞生長因子 (EGFR) 或間變性淋巴瘤激酶 (ALK) 所設計的的酪胺酸激酶抑制劑 (TKIs) 。到目前為止，還沒有針對大細胞癌和小細胞肺癌有效的標靶藥物。因此，我們的研究目標希望尋找能廣泛辨識小細胞肺癌和非小細胞肺癌各種亞型的標的胜肽，可應用於肺癌的偵檢和治療。結果：由於近期的研究將大細胞癌歸類為各種肺癌亞型中“未分化”的腫瘤型態。因此我們運用噬菌體顯現法，針對H460大細胞癌細胞株，期望篩選出廣效型的肺癌標的胜肽。三條標的胜肽 (HSP1、HSP2和HSP4) 及其所對應的的噬菌體 (HPC1、HPC2和HPC4) 可以結合小細胞肺癌和非小細胞肺癌的細胞株和臨床病人檢體，且不會與正常細胞結合。在活體的噬菌體光學照影及胜肽接合超順磁氧化鐵奈米粒子的磁振照影實驗顯示，HSP1最適合發展為多重模式的分子影像探針。HSP1可以導引氧化鐵在H460腫瘤中累積並造成T2磁振訊號下降42%。在T2的磁振造影模式中，HSP2的水分子自旋子吸引效應及其卓越的腫瘤標的能力造成奈米粒子周圍局部磁場不均勻的現象，使HSP2-HDex-Fe3O4奈米粒子成為有前景的顯影劑，可以將細微的腫瘤結構顯像並可運用於未來臨床中於低信號的肺實質中偵測肺癌病灶。HSP1可與肺癌細胞表面緊密結合，相反的，HSP4則傾向於導引內噬作用與細胞內的藥物傳輸，因此顯著的增進活體的治療指數。在非小細胞肺癌 (H460和H1993) 的動物模式中，HSP1、HSP2和HSP4接合的小紅莓微脂體顯著地增進非標的微脂體藥物的療效。在更惡性的A549原位癌移植動物模式中，HSP4接合的穩定型長春瑞濱微脂體及HSP4接合的小紅莓微脂體的複合式療法能更近一步地增進非標的微脂體藥物的中位存活率，由84天延長至131天( P值0.0248)。結論：總結而言，在未來的應用上，這三條標的胜肽可以導引針對各樣的肺癌亞型特製之“治療診斷試劑”，對於發展標靶治療、非侵入式照影、小細胞肺癌和非小細胞肺癌的偵檢極具臨床潛力。; Background: Lung cancer is the leading cause of cancer-related death worldwide. Most targeting drugs approved for lung cancer treatment are tyrosine kinase inhibitors (TKIs) of EGFR or ALK, mainly for adenocarcinoma. At present, there is no effective or tailored targeting agent for large cell carcinoma (LCC) or small cell lung cancer (SCLC); we therefore aimed to identify targeting peptides with broad subtype specificity for SCLC and non-small cell lung cancer (NSCLC) for diagnostic and therapeutic purposes. Results: We performed phage display biopanning of H460 LCC cells to select “broad-spectrum” lung cancer-binding peptides, since LCC has recently been categorized as an undifferentiated tumor type within other histological subcategories of lung cancer. Three targeting phage (HPC1, HPC2, and HPC4) and their respective displayed peptides (HSP1, HSP2, and HSP4) were able to bind to both SCLC and NSCLC cell lines and clinical specimens, but not to normal pneumonic tissues. In vivo optical imaging of phage homing and magnetic resonance imaging (MRI) of peptide-SPIONs revealed that HSP1 was the most favorable probe for multimodal molecular imaging, which resulted in a decrease of T2-weighted MR signal of up to 42% in H460 xenografts. The water spin collection effect and prominent tumor homing capability of HSP2 contribute to an inhomogeneous local magnetic field around the nanoparticles, making HSP2-HDex-Fe3O4 a promising contrast agent for visualizing subtle tumor structures and for detecting foci of lung cancer in hypointense lung parenchyma clinically by using common T2 MRI. Contrary to the tight binding of HSP1 to cancer cell surfaces, HSP4 favors endocytosis and intracellular drug delivery, thereby significantly improving the therapeutic index in vivo. Liposomal doxorubicin (LD) conjugated to HSP1, HSP2, or HSP4 had significantly greater therapeutic efficacy than non-targeting liposomal drugs in NSCLC (H460 and H1993) animal models. Combined therapy with an HSP4-conjugated stable formulation of liposomal vinorelbine (sLV) further improved median overall survival (131 vs. 84 days; P = 0.0248), even in aggressive A549 orthotopic models. Conclusions: Overall, these peptides have the potential to guide a wide variety of tailored “theranostic agents” for targeting therapy, non-invasive imaging, and clinical detection of SCLC and NSCLC.

軟組織肉瘤ATRX表現喪失及端粒替代性延長和腫瘤分類的關係

2021-06-16T06:41:47Z

標題: 軟組織肉瘤ATRX表現喪失及端粒替代性延長和腫瘤分類的關係; Loss of ATRX expression and alternative lengthening of telomere in soft tissue sarcoma: the relationship with tumor classification 作者: Tsung-Lin Liu; 劉宗霖摘要: 從基因分型角度來看，肉瘤可被分為兩大類：一類是簡單染色體組型肉瘤；另一類是複雜色體組型肉瘤。端粒的縮短為細胞分裂時必然發生的現象，當端粒縮短至一定程度後，細胞即停止分裂或死亡，腫瘤細胞要無限制地生長必須克服此一限制。目前已知克服端粒縮短的機制主要有兩種：一是活化端粒酶；另一則是透過端粒替代性延長（alternative lengthening of telomere, ALT）。在胰臟神經內分泌瘤（pancreatic neuroendocrine tumor, p-NET）中，此端粒替代性延長機制是由ATRX或DAXX失去表現所導致。我們共收集了195例的肉瘤檢體，其中簡單染色體組型肉瘤共62例，及複雜染色體組型肉瘤133例。透過ATRX、DAXX免疫組織染色及端粒特異性螢光原位雜交，我們發現在複雜染色體組型的軟組織肉瘤中，常帶有ATRX表現量喪失（P = 0.049）及端粒替代性延長（P = 0.004）的現象。所有195例肉瘤中，DAXX並無表現喪失。在58例多形性未分化肉瘤中，共21例為經放射線治療後續發之惡性肉瘤。ATRX表現量喪失（P = 0.007）及端粒替代性延長（P < 0.001）常表現在多形性未分化肉瘤，但經放射線治療後續發之惡性肉瘤並無此現象發生。我們的研究結果顯示，在複雜染色體組型的軟組織肉瘤中，帶有ATRX表現量喪失的腫瘤常伴有表現端粒替代性延長機制，且在多形性未分化肉瘤當中，ATRX表現量喪失及端粒替代性延長是其維持端粒長度的主要機制。; From a genetic perspective, sarcomas tend to fall into two groups: one is sarcomas with simple karyotypes and other one is sarcomas with complex karyotypes. Telomere shortening is an inevitable phenomenon during cell division. Once telomere has shortened to a critical length, cells undergo replicative senescence. Tumor cells must overcome this limitation to become immortal. There are two major mechanisms for telomere length maintenance; one is the activation of telomerase. The other is the alternative lengthening of telomeres, which in pancreatic neuroendocrine tumor is caused by mutation and loss of expression of either ATRX or DAXX. We collected 195 cases of sarcoma which were composed of 62 type I sarcomas and 133 type II sarcomas. By immunohistochemical stain of ATRX/DAXX and telomere-specific fluorescence in situ hybridization, we found ATRX was frequently loss (P = 0.049) and ALT usually occurred in karyotype-complex sarcomas (P = 0.004). None of the 195 cases lost DAXX expression. Within the 58 cases of undifferentiated pleomorphic sarcoma, 21 cases were post-irradiation sarcoma. Both ATRX loss of expression (P = 0.007) and ALT (P < 0.001) occur in undifferentiated pleomorphic sarcoma but not in post-irradiation sarcoma. Our results indicate that loss of ATRX is associated with ALT phenotype in karyotype-complex sarcoma and ATRX loss and ALT is a major mechanism of telomere preservation in undifferentiated pleomorphic sarcoma.