臺灣博碩士論文加值系統

Thioridazine是一類phenothiazine的抗精神病藥物，過去在臨床上曾用於精神分裂症的治療，在本篇研究中透過microarray的分析發現thioridazine可作為癌症幹細胞（CSC）的抑制劑，thioridazine具有兩種不同的光學異構物（T1和T2），為了進行專利保護，希望透過本研究比較出T1及T2在抑制肺癌功效上及兩者所透過的機制有何差異，將T1及T2處理肺癌細胞之後透過L1000系統分析，將thioridazine處理後有變化的基因進一步進行pathway的分析，分析的結果中顯示：thioridazine可會影響AMPK及膽固醇生合成相關的代謝途徑，在 colony formation的結果也顯示thioridazine的其中一個異構物(T2)會透過抑制膽固醇的生合成進而抑制腫瘤的初始形成(tumor initiation)，而在肺癌幹細胞的結果也發現T2會透過抑制膽固醇的生合成進而抑制癌症幹細胞的幹性(stemness)及生長情形，除此之外，在本篇研究中也發現T2相較於T1對於KRAS突變的肺癌細胞有較好的抑制功效，在動物實驗中也顯示：以KRAS突變細胞(H441)所誘發的腫瘤在T2處理的組別相較於T1處理的組別有較明顯被抑制的情形，而在和臨床化療用藥(Alimta)合併使用的組別也可看到類似的結果，綜合上述結果顯示：T2相較於T1對於KRAS突變的肺癌細胞有較強的抑制功效，其主要透過抑制膽固醇生合成的機制來抑制肺癌幹細胞的幹性及生長。

Thioridazine is a class of anti-psychotic drug called phenothiazine for the treatment of schizophrenia. Recently, we and others have shown that thioridazine is a cancer stem cell (CSC) inhibitor. However, the molecular mechanism of thioridazine in anti-CSC effect has not been elucidated. Interestingly, thioridazine has R and S two enantiomes. In this study, we hypothesized that these two enantiomers (referred to as T1 and T2) of thioridazine might inhibit the lung cancer cells with distinct efficacy and through different mechanisms. Here, we employed the L1000 microarray to explore the differentially gene expression profiles and to compare pathways of thioridazine enantiomers. Using these gene expression signatures, the putative targets of thioridazine were predicted through a LINCS system, which contains ~1,320,000 L1000 profiles. The prioritized pathways affected by thioridazine are predicted to be AMPK and cholesterol-biosynthesis pathways. Consistent with the prediction, the colony formation of lung cancer cells was inhibited by thioridazine through disrupting cholesterol-biosynthesis pathway. Moreover, the cholesterol biosynthesis was also interfered by thioridazine and resulted in the stemness inhibition and cell viability reduction in lung cancer stem cells. More importantly, one of the enantiomers, T2, has better inhibitory effect on KRAS mutant cells than T1. Finally, the in vivo tumor suppressive effect of T2 and Alimta combined treatment groups appeared to be the most significant in H441 (KRAS mutant)-bearing mice. Taken together, thioridazine-T2 may have therapeutic benefits on anti-CSCs for NSCLC as compared to T1 on KRAS mutant lung cancer cells.

致謝……………………………………………………….....………i
Contents……………………………………………………….....………ii
Chinese Abstract………………………………………………………vii
English Abstract……………………………………………………..viii
Introduction ............................................................................................... 1
Lung cancer ................................................................................................................ 1
Thioridazine ............................................................................................................... 1
L1000.......................................................................................................................... 3
The relationship between the cholesterol and lipid raft ............................................. 4
Cholesterol and cancer .............................................................................................. 4
The role of the FDFT1 in cholesterol biosynthesis .................................................... 5
Ras and the cholesterol biosynthesis.......................................................................... 5
AMPK and cancer ...................................................................................................... 6
Specific aims .............................................................................................. 7
Materials and Methods ............................................................................. 8
Results ...................................................................................................... 15
The mechanism exploration of thioridazine via bioinformatics and resistant clones analysis ..................................................................................................................... 15
AMPK and the cholesterol AMPK and the cholesterol AMPK and the cholesterol AMPK and the cholesterol AMPK and the cholesterolAMPK and the cholesterol AMPK and the cholesterolAMPK and the cholesterol -related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced related pathway were induced by a lower concentration by a lower concentration by a lower concentration by a lower concentration by a lower concentration by a lower concentration by a lower concentration by a lower concentration by a lower concentration by a lower concentration by a lower concentration by a lower concentration by a lower concentration of thioridazine of thioridazine of thioridazine of thioridazine .......................................................................................................... 16
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The COSMIC and the colony formation analysis suggest that the KRAS mutant cells are more sensitive to thioridazine ............................................................................ 16
The activity of HMGThe activity of HMG The activity of HMG The activity of HMG The activity of HMGThe activity of HMG The activity of HMGThe activity of HMG The activity of HMG-CoA reduCoA reduCoA reduCoA redu CoA reduCoA redu ctase was inhibited by thioridazinectase was inhibited by thioridazine ctase was inhibited by thioridazine ctase was inhibited by thioridazine ctase was inhibited by thioridazinectase was inhibited by thioridazine ctase was inhibited by thioridazine ctase was inhibited by thioridazine ctase was inhibited by thioridazine ........................ 17
Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and Thioridazine inhibits the colony formation of lung cancer via mevalonate and cholesterolcholesterol cholesterolcholesterol -biosynthesis pathway biosynthesis pathway biosynthesis pathwaybiosynthesis pathway biosynthesis pathway ............................................................................. 17
Thioridazine reduces the cholesterol content in A549, and CL141 parental cells. . 18
Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant cells ..... 18
Thioridazine inhibits the protein expression of stemness markers and cholesterol-related enzymes in lung cancer stem cell. ............................................. 19
Thioridazine reduces the cholesterol content in A549 and CL141 sphere cell lines ................................................................................................................................. .19
Thioridazine inhibits the stemness of the lung cancer stem cell through the cholesterol-biosynthesis pathway in A549 and CL141 sphere cell lines. ................ 19
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................................ ........... 20
Thioridazine induces the AMPK activation and inhibits the cholesterol-related pathway at lower concentration while induces the autophagy and apoptosis at higher concentration in A549, and CL141 parental cell lines ................................. 20 The autophagic-induced death is induced via thioridazine through cholesterol inhibition in A549 (KRAS mutant cells) ................................................................... 21
The KRAS wild-type cells sensitize to thioridazine through KRASG12D transfection ................................................................................................................................. .21
Thioridazine inhibits the viability of A549 through AMPK activation .................... 22
The viability inhibition via thioridazine is dependent on the FDFT1 inhibition rather than the inhibition of SREBP2 in A549 cells ................................................ 22
iv
Thioridazine significant inhibits the self-renewal of CL141 and CL97 cancer spheres...................................................................................................................... 23
Thioridazine reduces the proportion of side population cells and ALDH+ cells in CL141 and CL97 cell lines. ...................................................................................... 24
In vivo examination of tumor inhibitory effects of thioridazine and its enantiomers. .................................................................................................................................. 24 Discussions ............................................................................................... 26 References ................................................................................................ 30
Figures ...................................................................................................... 35
Figure 1. Figure 1.Figure 1.Figure 1. Treatment of thioridazine inhibits some the lung CSCTreatment of thioridazine inhibits some the lung CSCTreatment of thioridazine inhibits some the lung CSCTreatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSCTreatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSC Treatment of thioridazine inhibits some the lung CSCTreatment of thioridazine inhibits some the lung CSCTreatment of thioridazine inhibits some the lung CSC-related pathways related pathways related pathways related pathways related pathways related pathways related pathways related pathways at μM range. at μM range. at μM range. ............................................................................................................. 35
Figure 2. Thioridazine induces the AMPK activation and affects the cholesterol-related pathway at nM concentration. .................................................. 36
Figure 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS 3. The COSMIC and the colony formation analysis suggest that KRAS mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine.mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine.mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine. mutant cells are more sensitive to thioridazine. ....................................................... 37
Figure 4. The HMG Figure 4. The HMGFigure 4. The HMG Figure 4. The HMG Figure 4. The HMG Figure 4. The HMG-CoA reductase activity determination.CoA reductase activity determination. CoA reductase activity determination. CoA reductase activity determination.CoA reductase activity determination. CoA reductase activity determination. CoA reductase activity determination. CoA reductase activity determination.CoA reductase activity determination. CoA reductase activity determination.CoA reductase activity determination. CoA reductase activity determination.CoA reductase activity determination. CoA reductase activity determination. CoA reductase activity determination. .................................... 39
Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate Figure 5. Thioridazine inhibits the colony formation of lung cancer via mevalonate and cholesterol and cholesterol and cholesteroland cholesterol -biosynthesis pathway.. biosynthesis pathway..biosynthesis pathway.. biosynthesis pathway.. biosynthesis pathway.. biosynthesis pathway.. .................................................................... 40 Figure 6. Thioridazine reduces the cholesterol content in A549 and CL141 parental cell lines. .................................................................................................................. 41
Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant Figure 7. Thioridazine inhibits the cholesterol content of lipid raft in KRAS mutant cells.cells.cells. cells. ......................................................................................................................... 42
Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and Figure 8. Thioridazine inhibits the protein expression of stemness markers and cholesterolcholesterol cholesterolcholesterol -related enzymes in lung cancer stem cell..related enzymes in lung cancer stem cell..related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell..related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell..related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell..related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell..related enzymes in lung cancer stem cell..related enzymes in lung cancer stem cell.. related enzymes in lung cancer stem cell.. ............................................ 43
v
Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 Figure 9. Thioridazine reduces the cholesterol content in A549 and CL141sphere and CL141sphere and CL141sphere and CL141sphere and CL141sphere and CL141sphere and CL141sphere and CL141sphere and CL141sphere cell lines..cell lines..cell lines.. cell lines.. cell lines.. cell lines.. ................................................................................................................. 44
Figure 10. Thioridazine inhibits the stemness of the lung cancer stem cell through the cholesterol-biosynthesis pathway in A549 and CL141 sphere cell lines ........... 45
Figure 11. Thioridazine inhibits the viability of A549 and CL141 spheres through cholesterol-biosynthesis pathway............................................................................. 46
Figure 12. Thioridazine dysregulates the cholesterol-related pathway and induces the autophagy or apoptosis in A549 and CL141… .................................................. 47
Figure 13. Thioridazined-elicited autophagic death is through cholesterol inhibition. ................................................................................................................. 48
Figure 14. The KRAS wild-type cells are sensitized to thioridazine through KRASG12D transfection.. ............................................................................................ 49
Figure 15. Thioridazine inhibits the viability of A549 through AMPK activation... 50
Figure 16. The viability inhibition via thioridazine is dependent on FDFT1 but not SREBP2 inhibition in A549 ...................................................................................... 51
Tables ....................................................................................................... 52
Table 1. The potential pathways might be affected by the enantiomers of thioridazine via L1000 and LINCS analysis. ........................................................... 52
Table 2. The possible candidates may interact with thioridazine. ........................... 53
Supplementary figures and tables Supplementary figures and tables Supplementary figures and tablesSupplementary figures and tablesSupplementary figures and tables Supplementary figures and tablesSupplementary figures and tablesSupplementary figures and tablesSupplementary figures and tablesSupplementary figures and tables Supplementary figures and tablesSupplementary figures and tablesSupplementary figures and tables Supplementary figures and tables Supplementary figures and tables Supplementary figures and tablesSupplementary figures and tables ......................................................... 55
Supplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of nonSupplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of non Supplementary figure 1. Thioridazine reduces the percentage of non –small cell small cell small cell small cell small cell
lung cancerstem lung cancerstemlung cancerstem lung cancerstemlung cancerstem-like cells. like cells.like cells.like cells. like cells.like cells. like cells. ....................................................................................... 55
Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its Supplementary figure 2. Comparative efficacy between thioridazine and its enantiomers and examination of thiorenantiomers and examination of thior enantiomers and examination of thior enantiomers and examination of thior enantiomers and examination of thior enantiomers and examination of thiorenantiomers and examination of thiorenantiomers and examination of thior enantiomers and examination of thior enantiomers and examination of thior enantiomers and examination of thiorenantiomers and examination of thior enantiomers and examination of thior idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. idazine’s efficacy in combination with Alimita. .................................................................................................................................. 56
vi
Supplementary figure 3 Supplementary figure 3Supplementary figure 3 Supplementary figure 3 Supplementary figure 3 Supplementary figure 3Supplementary figure 3Supplementary figure 3. Western blot and the migration assay of the TGF-beta
signaling pathways analysis. .................................................................................... 58
Supplementary figure 4 Supplementary figure 4Supplementary figure 4 Supplementary figure 4 Supplementary figure 4Supplementary figure 4 . Western blot of the IGF1R signaling pathway analysis.Western blot of the IGF1R signaling pathway analysis.Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis.Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis.Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis. Western blot of the IGF1R signaling pathway analysis. ... 59
Supplementary figure 5 Supplementary figure 5Supplementary figure 5 Supplementary figure 5 Supplementary figure 5 Supplementary figure 5Supplementary figure 5Supplementary figure 5. Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which Structures and effects of various phenothiazines which suppressed CSC suppressed CSCsuppressed CSC -like side population of A549 lung cancer cells. like side population of A549 lung cancer cells.like side population of A549 lung cancer cells.like side population of A549 lung cancer cells. like side population of A549 lung cancer cells. like side population of A549 lung cancer cells. like side population of A549 lung cancer cells.like side population of A549 lung cancer cells. like side population of A549 lung cancer cells.like side population of A549 lung cancer cells. like side population of A549 lung cancer cells.like side population of A549 lung cancer cells. like side population of A549 lung cancer cells.like side population of A549 lung cancer cells. like side population of A549 lung cancer cells. ............................ 60
Supplementary figure 6. Cell cycle progression analysis via flow cytometry .......... 62
Supplementary table 1. The Ingenuity Canonical Pathway analysis of T1 treatment. .................................................................................................................................. 64
Supplementary table 2. The Ingenuity Canonical Pathway analysis of T2 treatment. .................................................................................................................................. 65
Supplementary table 3. The Ingenuity Canonical Pathway analysis of T3 treatment. .................................................................................................................................. 67
Supplementary table 4. The top 10 genes affected via T1 treatment. ...................... 68
Supplementary table 5. The top 10 genes affected via T2 treatment. ...................... 69

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