子宮頸神經內分泌腫瘤之細胞株的建立、特徵鑑定與應用

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研究生：	陳亞澤 Chen, Ya-Tse
論文名稱：	子宮頸神經內分泌腫瘤之細胞株的建立、特徵鑑定與應用 Establishment and Characterization of a New Cell Line (HM-1) from Neuroendocrine Tumor of the Uterine Cervix
指導教授：	莊永仁 Chuang, Yung-Jen
口試委員:	張壯榮 Chang, Chuang-Rung 詹鴻霖 Chan, Hong-Lin
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	52
中文關鍵詞：	子宮頸神經內分泌腫瘤
外文關鍵詞：	Neuroendocrine tumor of uterine cervix, Forkhead box M1, MDM2, nutlin-3, CCRT-EP5+
相關次數：	點閱：3 下載：0
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子宮頸神經內分泌腫瘤是子宮頸癌中相當罕見的一種。和過去常見的子宮頸癌類型，如鱗狀上皮癌及腺癌相比，子宮頸神經內分泌腫瘤大多較具侵犯性，疾病的進展相當快速且預後不佳。我們研究團隊與新竹馬偕醫院婦癌科合作，於先前計畫中得知了一位75歲的婦人疑似患有這種罕見的癌症，也在病人的同意下取得了這個子宮頸腫瘤組織活體樣本。我們透過細胞分離選殖方式，成功的將此子宮頸腫瘤組織分離成原代培養細胞，並且命名此細胞株為HM-1，而STR鑑定的結果顯示，HM-1確實為一個全新的子宮頸腫瘤細胞株。HM-1的細胞型態不同於過去常見的子宮頸癌細胞，較近似於纖維母細胞，呈現較細長的紡錘狀。其細胞倍增時間約為34小時，相較於過去常見的子宮頸癌HeLa細胞株的19小時高出許多。此外我們發現HM-1細胞表現神經內分泌細胞的生物標記SYP，說明了HM-1可能是由神經內分泌細胞演化而來。我們也發現HM-1表現HPV16，這也代表了HM-1這個子宮頸神經內分泌腫瘤的發生可能和HPV感染具有關聯性。
為了觀察HM-1形成腫瘤的能力，我們將HM-1細胞以皮下注射的方式打入小鼠體內。結果顯示HM-1確實可以在小鼠體內形成腫瘤，其腫瘤倍增時間約為13天，較過去文獻指出HeLa細胞株的腫瘤倍增時間5天緩慢許多。而在抗癌藥物反應方面，測試結果顯示Docetaxel對於抑制HM-1細胞存活率具有最好的效果，依次為Cisplatin和Etoposide，而Gemcitabine則幾乎沒有效果。此外我們也驗證Cisplatin和Etoposide兩種藥物的結合能夠有效的抑制HM-1細胞的存活率，與前人研究相符，顯示了多種藥物結合在臨床上對於治療子宮頸神經內分泌腫瘤的可能性。
進一步探討多重藥物療法的增益方案，我們發現以nutlin-3去抑制MDM2，會使子宮頸癌細胞Caski、HeLa以及HM-1細胞中FoxM1的表現量被抑制。且發現Caski和HeLa細胞的移動能力也會受到抑制。因此我們認為MDM2和FoxM1的抑制劑在未來治療子宮頸癌甚至是子宮頸神經內分泌腫瘤或許會是個相當重要的候選藥物。相信我們所建立的子宮頸神經內分泌腫瘤細胞株HM-1於未來可以提供我們進行更多子宮頸神經內分泌腫瘤的相關研究，也期望在未來能夠找到對這種罕見疾病最有效的治療方法。

Neuroendocrine tumor of uterine cervix (NTUC) is a relative rare subtype of cervical cancer. In comparison to other subtypes of gynecologic cancers, such as squamous cell carcinoma and adenocarcinoma, the NTUC is much more aggressive. Consequently, patients of NTUC have poor prognosis.
In collaboration with gynecologic oncologists at Hsinchu branch of MacKay memorial hospital, we obtained a tissue sample of suspected NTUC from a 75-year-old patient. Using enzymatic and mechanical dissociation methods, we established the primary culture (HM-1) from this specimen, and the STR analysis result showed that HM-1 is a novel cancer cell line. HM-1 cells are bipolar or multipolar and showed fibroblast-like, elongated and spindle shape, which is different to common types of cervical cancer. In addition, we found HM-1 has a cell doubling time of 34 hours, which is significantly longer than the 19 hours cell doubling time of the common cervical cancer HeLa cells. Immunocytochemical staining validated that high expression level of the neuroendocrine marker, neuroendocrine synaptophysin (SYP), was present in the HM-1 cells, which suggested that HM-1 cells were originated from neuroendocrine cells. In addition, the presence of human papillomavirus 16 (HPV16) was detected in HM-1 cells, suggesting its potential association with HPV infection. To test its tumorigenesis capacity, the HM-1 cells were subcutaneously inoculated into BALB/c female nude mice. The result showed the HM-1 cells could proliferate in vivo, with a tumor volume doubling time of 13 days, which was higher than that HeLa.
In term of anti-cancer drug sensitivity, Docetaxel has the highest inhibitory effect on HM-1 cell viability, followed by Cisplatin and Etoposide respectively, while the cells was shown to be least sensitive to Gemcitabine. On the other hand, HM-1 cells were highly sensitive to the combination treatment of etoposide and cisplatin.
In order to develop a more effective treatment, we found that inhibition of MDM2 by nutlin-3 would decreased FoxM1 expression in cervical cancer cell lines CasKi, HeLa and NTUC. Inhibition of MDM2 can also decrease the migration ability of cervical cancer cells. Our ﬁndings suggested that the inhibition of MDM2 pathways and FoxM1-dependent pathway may be novel therapeutic targets for cervical cancers, including NTUC. We believe this new neuroendocrine cancer cell line HM-1 will be useful as a research model of NTUC. HM-1-based assay may help in developing effective diagnosis test or therapy for this rare subtype of gynecological malignancy.

Table of contents
中文摘要    I
Abstract    III
致謝    V
List of Abbreviations    XI
1. Introduction    1
1.1 Cervical cancer    1
1.2 Neuroendocrine tumor of uterine cervix and its treatment strategies    2
1.3 Forkhead box M1 (FoxM1) is a tumor progressive and metastatic factor associated with chemotherapy resistance    3
1.4 MDM2/p53/FoxO3 pathway are potential therapeutic targets for various types of cancer    4
1.5 Objectives and significant finding of this study    5
2. Materials and methods    7
2.1 Cancer cell isolation    7
2.2 Cell and culture    8
2.3 STR (Short tandem repeat) analysis    8
2.4 Doubling time assay    8
2.5 Reverse transcription-PCR (RT-PCR for HPV detection)    9
2.6 Western blot    9
2.7 Immunocytochemistry (ICC for HM-1)    9
2.8 Xenotransplantation    10
2.9 Histological analysis    11
2.10 Drug treatment    11
2.11 MTT assay    11
2.12 Transwell migration assay    12
2.13 Wound healing assay    12
3. Results    14
3.1 Establishment of in vitro HM-1 cells culture    14
3.2 HM-1 is a novel cervical cancer cell line    14
3.3 Morphologic and growth characteristics of HM-1 cells    16
3.4 HM-1 cells show expression of HPV-16 and neuroendocrine markers    16
3.5 Xenotransplantation of HM-1    17
3.6 Combination of etoposide and cisplatin may be potential NETCx treatment    18
3.6.1 Drug sensitivity of HM-1 to anti-cancer drugs    18
3.6.2 Drug sensitivity of HM-1 to combination treatment of etoposide and cisplatin    20
3.7 MDM2 regulate FoxM1 expression to affect mobility of cervical cancer cells    20
4. Discussion    23
4.1 Neuroendocrine tumor are rare neoplasm arising from cells of neuroendocrine system    24
4.2 Specific biochemical markers for neuroendocrine tumor    25
4.3 HPV is the most important risk factor of cervical cancer    26
4.4 Potential chemotherapeutic drugs to NTUC    27
4.5 MDM2/FoxO3/FoxM1 pathway may be a novel treatment strategy for neuroendocrine tumor of uterine cervix.    28
4.6  Perspective of this study    29
Reference :    30
 
List of figures
Figure 1. : Morphological presentation of HM-1 and other common cervical cancer cells.    33
Figure 2. : A cell growth curve analysis of HM-1.    34
Figure 3. : HPV detection.    35
Figure 4. Western blot and immuno-cytochemical staining showed that HM-1 cells express the neuroendocrine marker, neuroendocrine synaptophysin (SYP).    36
Figure 5. Xeno-transplantation and in vivo tumor growth curve of HM-1 cells.    37
Figure 6. HM-1 xenograft showed histological morphology similar to that of squamous cell carcinoma, Caski.    38
Figure 7. Dose response curves of HM-1 cells exposed to anti-cancer drug.    39
Figure 8. Drug response assay of HM-1 cells exposed to combination treatment of anti-cancer drugs.    40
Figure 9. Hypothesis for the relationship between MDM2-p53 pathway and FoxM1 axis in cancer progression    41
Figure 10. Inhibition of p53-Mdm2 interaction would decrease 2D cell mobility in cervical cancer.    43
Figure 11. Inhibition of MDM2-p53 and MDM2-FoxO3 interaction would decrease 3D cell mobility in cervical cancer.    45
Figure 12. Inhibition of MDM2-p53 and MDM2-FoxO3 interaction would decrease FoxM1 expression in cervical cancer and HM-1 cells.    47

 
List of Tables
Table 1. RT-PCR primers list    48
Table 2. Antibodies list    49
Table 3. Allele table for the HM-1 cells    50
Table 4. Comparison the STR profile between HM-1 cells and other NET cell lines    51
Table 5. Comparison the STR profile between HM-1 cells and ATCC database    52

                                

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