||Targeting cancer stem cell by small molecular compounds in esophageal cancer
||Institute of Clinical Medicine
ESCC (Esophageal Squamous cell carcinoma)
Cancer stem cell
CCRT-resistance (Concurrent Chemoradiation Therapy)
在台灣，食道癌是一個常見且致命的惡性腫瘤，鳞狀細胞癌是最常見的病理組織型態，食道癌病人即使接受手術與化學合併放射線治療，其五年存活率很少大於25%；有許多證據顯示食道癌病人對於合併化放療都出現抗藥性情形，而導致這個情況的原因有可能是因為癌症幹細胞具有抗藥性的特性進而導致腫瘤復發。在過去的研究指出癌症幹細胞在不同的癌症中參與腫瘤的形成、發展以及轉移，因此我們的研究目的是尋找出可以應用在食道癌中標靶癌症幹細胞的新穎小分子藥物或是已上市藥品，來提高病人的存活率。首先，我們已經建立了食道癌幹細胞球團的培養方式，利用WST-1去偵測新穎小分子藥物處理過後的癌症幹細胞的細胞存活率；在藥物篩選中，我們發現ILK (integrin-linked kinase)抑制劑-NCKU-1501對於癌症幹細胞具有高度抑制細胞存活率，而在功能性分析上發現，NCKU-1501可提高食道癌細胞對於合併化放療的療效，並且進一步發現，NCKU-1501會透過降低幹細胞以及抗藥性相關的蛋白質表現而抑制癌症幹細胞球團。同時也使用生物資訊資料庫-connectivity map (cmap)輸入先前微陣列矩晶片分析的基因變異結果，來預測可能影響幹細胞與非幹細胞基因變異的已知上市藥物，我們發現維他命B的衍生物-NCKU-150不會直接抑制細胞活性，但會透過抑制幹細胞特性以及抗藥性相關途徑影響癌症幹細胞球團形成，有趣的是，當癌症幹細胞處理NCKU-1507會促進細胞對於傳統藥物cisplatin的敏感度提高。綜合以上結果，在未來我們認為NCKU-1501是具有有效標靶癌症幹細胞的小分子藥物；此外NCKU-1507 可能會促進癌症幹細胞轉變成一般癌細胞進而提高細胞對於傳統化療藥物cisplatin的療效。
Esophageal cancer is a common and fatal malignancy. Squamous cell carcinoma is the predominant histological tumor type in Taiwan. Even after surgery and/or concurrent chemoradiation therapy (CCRT), 5-year survival rate of patients is less than 25%. Recently, there are more evidences showing CCRT-resistance in ESCC patients. One possible reason is because of cancer stem cells (CSCs) which have the characteristic of drug-resistance and lead to tumor relapse. Previous studies report that CSCs are responsible for tumor initiation, progression and metastasis in different cancers. In this study, we aimed to investigate novel compounds or defined compounds targeting CSCs in ESCC. First, we established the CSCs culture system which enriched CSCs in the tumor sphere of ESCC cell lines. CSCs were treated with novel compounds and their viabilities were determined by WST-1 assay. The results demonstrated that NCKU-1501, an ILK (integrin-link kinase) inhibitor, had a potency for targeting ESCC CSCs and non-CSCs from drug screening. Functional assays showed that NCKU-1501 increased the CCRT response of ESCC cell lines. Moreover, NCKU-1501 inhibited ESCC tumor sphere formation through down-regulating the stemness- and drug-resistance-associated genes expression. In addition, we used Connectivity Map to predict compounds which were able to convert CSCs specific genes expression to the levels of ESCC non-CSCs cell lines. NCKU-1507, vitamin B derivative, didn’t directly inhibit cell viability, but it suppressed the tumor sphere formation via down-regulating stemness- and drug-resistance-associated genes expression. Interestingly, NCKU-1507 could increase the cisplatin sensitivity of the ESCC tumor sphere. In conclusion, NCKU-1501 may be a potential therapeutic compound targeting ESCC CSCs. NCKU-1507 may reverse ESCC CSCs specific gene expression and increase ESCC sensitivity to cisplatin.
Materials and Methods 10
Tumor sphere showed drug-resistance of conventional cancer drug in ESCC 20
NCKU-1501 was a candidate compound for targeting CSCs in ESCC 20
NCKU-1501 increased the sensitivity of irradiation in ESCC 21
NCKU-1501 inhibited the capacity of CSC self-renewal 22
NCKU-1501 induced the cell death via increasing sub-G1 population 23
NCKU-1501 induced the cell death through apoptosis 24
NCKU-1501 suppressed stemness and drug-resistance protein expression in tumor sphere 24
ILK may play an important role in tumor sphere 25
NCKU-1507 (NCKU-1507) may reverse the mRNA expression of stemness and drug-resistance in tumor sphere 27
NCKU-1507 may increase the cisplatin sensitivity in ESCC tumor sphere 29
Conclusion and Discussion 30
Figure and Table 34
Figure 1. NCKU-1501 inhibited cell viability in tumor sphere and parental cell. 34
Figure 2. NCKU-1501 inhibited cell colony formation. 35
Figure 3. NCKU-1501 increased CCRT response 36
Figure 4. NCKU-1501 inhibited the capacity of CSC self-renewal in ESCC. 38
Figure 5. Cell cycle analysis in ESCC CSCs. 39
Figure 6. NCKU-1501 increased sub-G1 population leading to cell death in ESCC. 40
Figure 7. NCKU-1501 increased sub-G1 population leading to cell death in ESCC CSCs. 41
Figure 8. NCKU-1501 increased sub-G1 population leading to cell death in ESCC CSCs. 42
Figure 9. NCKU-1501 inhibited cell survival and induced cell death in ESCC. 43
Figure 10. NCKU-1501 inhibited cell survival and induced cell death in ESCC. 44
Figure 11. NCKU-1501 abolished the stemness and drug-resistance expression of protein level in CE48T tumor sphere. 45
Figure 12. NCKU-1501 abolished the stemness and drug-resistance expression of protein level in KYSE170 tumor sphere. 46
Figure 13. Knockdown of ILK expression decreased the tumor sphere formation ability. 47
Figure 14. Candidate compounds which were predicted form Cmap did not induce cell death in ESCC. 48
Figure 15. Gene expression profiles of stemness and drug-resistance of sphere in adhesion condition. 49
Figure 16. Gene expression of stemness and drug-resistance in NCKU-1508-treated cell. 50
Figure 17. Gene expression of stemness and drug-resistance in NCKU-1506-treated cell. 51
Figure 18. Gene expression of stemness and drug-resistance in NCKU-1510-treated cell. 52
Figure 19. Gene expression of stemness and drug-resistance in NCKU-1511-treated cell. 53
Figure 20. NCKU-1507 reverses the stemness and drug-resistance mRNA expression levels of tumor sphere in 24hr. 54
Figure 21. NCKU-1507 inhibited tumor sphere formation ability. 55
Figure 22. NCKU-1507 (NCKU-1507) increased the cisplatin sensitivity in NCKU-1507-treated sphere cell. 56
Figure 23. NCKU-1507 reversed the stemness and drug-resistance mRNA expression level of tumor sphere for 5days in KYSE170. 57
Figure 24. NCKU-1507 reversed the ABCG2 protein expression in KYSE170 for 5 days. 58
Table 1. The inhibitory concentration (μM) of 50% (IC50) of novel compounds in parental and tumor sphere cell in ESCC. 59
Table 2. Primers of quantitative reverse transcription PCR 60
Table 3 Antibodies of immunoblot 61
Appendix 1. 69
Appendix 2. 70
Appendix 3. 71
Appendix 4.. 72
Appendix 5.. 73
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