黑色素癌因為其高轉移性，而被視為最惡性的皮膚癌類型。然而，抗藥性導致的不良反應與療效降低仍是黑色素癌治療的瓶頸，為了獲得更好的臨床療效，開發新且有效的治療策略對於黑色素癌治療有其必要性。迄今為止，複合藥物療法已被證實可在多種癌症治療中展現協同抗癌作用。國外臨床研究指出，組蛋白去乙醯酶抑制劑Valproic acid (VPA) 極有潛力作為化療藥物增敏劑應用於多種轉移性癌症治療。因此，本研究旨在探討VPA是否可以作為化療藥物Etoposide (ETO)的增敏劑，以及其如何在黑色素癌細胞上展現協同抑制作用。
我們的研究結果顯示：相較於單一藥物治療，VPA與ETO的同時性複合療法可在小鼠黑色素癌細胞株B16-F10中產生更強的抑制作用。接著，我們進一步從DNA損傷反應與修復、細胞週期調控以及細胞凋亡層面探討此差異背後的作用機制。在B16-F10細胞中，我們發現VPA與ETO同時性複合療法可能透過G2/M 期停滯和降低同源重組(homologous recombination, HR)活性產生協同抑制作用，而ETO預處理的順序性複合療法可藉由增強細胞凋亡導致協同抑制作用。出乎意料的是，VPA預處理的順序性複合療法可透過增強HR活性使細胞對藥物產生拮抗作用。
綜上所述，我們的研究顯示VPA與ETO複合療法中的藥物施用順序與有效劑量可能導致黑色素癌細胞產生不同細胞反應。本研究可能有助於提升黑色素癌治療的效能，並增進我們對複合療法中藥物施用順序與有效劑量重要性的瞭解。

Among all skin cancers, melanoma is the most dangerous form owing to its high metastatic potential if left untreated. However, adverse event and reduced efficiency due to resistance are still major limitations in current melanoma therapies. To achieve better clinical outcomes, developing novel therapeutic strategies is still necessary for future melanoma treatment. According to the concept of precision medicine, combination drug therapy has demonstrated synergistic anti-cancer effects on several types of cancers. So far, the histone deacetylase inhibitor, valproic acid (VPA), has been indicated as a potential sensitizer of chemotherapy drugs on various metastatic cancers.
In this study, the objective is to explore whether and how VPA could work as an effective sensitizer of chemotherapy drug etoposide (ETO) on melanoma cells. Our results demonstrated that the simultaneous combined treatment of VPA and ETO could generate a higher inhibitory effectivity than single treatment of each drug in murine melanoma cell line B16-F10. We further explored such differential effects in terms of DNA damage response and repair, cell cycle arrest, and apoptosis. In cells under simultaneous combination treatment of VPA and ETO, we observed G2/M phase arrest and a compromised homologous recombination (HR) activity might contribute to the synergistic inhibitory effect. Besides, the sequential combination with ETO pre-treatment might lead to synergistic inhibitory effect via enhanced apoptosis. Surprisingly, the enhanced HR activity might result in the antagonistic effect in cells under VPA pre-treated sequential combined treatment.
In summary, our findings suggest sequential order and effective dose (ED) of drug administration in VPA-ETO combination therapy might contribute to different cellular responses in malignant melanoma cells. The present study might help to potentiate the effectiveness of melanoma treatment and advance our knowledge of the importance of sequential order and ED in drug combination therapy.

中文摘要-----------------------------------------------------------I
Abstract----------------------------------------------------------II
致謝辭------------------------------------------------------------IV
Table of contents-------------------------------------------------VI
List of abbreviations---------------------------------------------XI
1. Introduction-------------------------------------------------1
1.1 Melanoma and its epidemiology-------------------------------1
1.2 Current treatments and clinical challenges of melanoma------2
1.3 Synergistic effects of combination therapy------------------3
1.4 Histone deacetylase inhibitor on melanoma-------------------4
1.5 Chemotherapy on melanoma------------------------------------5
1.6 The objectives of this study--------------------------------7
2. Materials and methods----------------------------------------8
2.1 Cell lines and cell culture---------------------------------8
2.2 Drugs-------------------------------------------------------8
2.3 Cell viability assay and IC50 determination-----------------8
2.4 Analysis of combination index-------------------------------9
2.5 Cell cycle analysis----------------------------------------10
2.6 Caspase-3 apoptosis analysis-------------------------------10
2.7 Western blot analysis--------------------------------------11
2.8 Statistics-------------------------------------------------12
3. Results-----------------------------------------------------13
3.1 Exponential two-phase decay model fits the dose-response curves under single treatments of VPA or ETO on B16-F10 cells-----13
3.2 Simultaneous and sequential combined treatments of VPA and ETO contribute to synergistic inhibitory effects or antagonistic effect on B16-F10 cells-------------------------------------------13
3.3 DNA double strand breaks (DSBs) induced by single treatment,
simultaneous, and sequential combined treatments of VPA and ETO may not be the major cause of synergistic inhibitory effects in B16-F10 cells-------------------------------------------------------------16
3.4 DNA DSBs induced by VPA pre-treated sequential combined treatment may be predominantly repaired via homologous recombination in B16-F10 cells--------------------------------------------------17
3.5 Different sequential orders of VPA and ETO administration may influence the cell cycle regulation in B16-F10 cells--------------19
3.6 ETO pre-treated sequential combined treatment augments apoptosis via caspase-3 up-regulation in B16-F10 cells------------20
4. Discussion--------------------------------------------------21
4.1 Summary----------------------------------------------------21
4.2 Sequential order and effective dose of VPA and ETO administration might influence inhibitory effects on B16-F10 cells ------------------------------------------------------------------21
4.3 Different sequential orders of VPA and ETO administration might lead to different cell fate decisions in B16-F10 cells------22
4.3.1 The enhanced HR activity might repair the DNA DSBs and contribute to antagonistic effect in B16-F10 cells under VPA pre-treated sequential combined treatment-----------------------------22
4.3.2 G2/M phase arrest and the compromised HR activity might contribute to the synergistic inhibitory effect on B16-F10 cells under simultaneous combined treatment of VPA and ETO--------------24
4.3.3 Enhanced apoptosis might underlie the synergistic inhibitory effect on B16-F10 cells under sequential combination with ETO pre-treatment-------------------------------------------------25
4.4 Time-course analysis is needed to reveal the progression of DNA damage responses under combination treatments with different drug sequential orders--------------------------------------------26
4.5 Clinical significance of drug sequential order in combination therapy of VPA and ETO--------------------------------------------26
4.6 Future perspectives of this study--------------------------28
5. Acknowledgement---------------------------------------------29
6. References--------------------------------------------------30
7. Tables------------------------------------------------------36
Table 1. IC50 values of valproic acid (VPA) or etoposide (ETO) single treatment on B16-F10 cells---------------------------------36
Table 2. Categorization of combination index (CI) of drug combination to corresponding drug effect and grading--------------37
Table 3. Combination index (CI) values and gradings of simultaneous and sequential combined treatments of VPA and ETO on B16-F10 cells ------------------------------------------------------------------38
Table 4. List of antibodies used in western blot------------------39
8. Figures-----------------------------------------------------41
Figure 1. Dose-response curves of single treatments of VPA or ETO on B16-F10 cells-----------------------------------------------------41
Figure 2. Fitting curves of dose-response toward single treatments of VPA or ETO on B16-F10 cells------------------------------------42
Figure 3. Cell inhibition of single treatments versus simultaneous combined treatment of VPA and ETO on B16-F10 cells----------------43
Figure 4. Cell inhibition of simultaneous versus sequential combined treatments of VPA and ETO on B16-F10 cells------------------------44
Figure 5. Synergistic inhibitory and antagonistic effects of VPA and ETO on B16-F10 cells are demonstrated by Fa-CI plot---------------45
Figure 6. Synergistic inhibitory and antagonistic effects of VPA and ETO on B16-F10 cells are demonstrated by isobolograms-------------46
Figure 7. DNA double strand breaks induced by single, simultaneous, and sequential combined treatments of VPA and ETO in B16-F10 cells ------------------------------------------------------------------47
Figure 8. Expression of homologous recombination (HR) related proteins under single, simultaneous, and sequential combined treatments of VPA and ETO in B16-F10 cells------------------------48
Figure 9. Expression of non-homologous end joining (NHEJ) related proteins under single, simultaneous, and sequential combined treatments of VPA and ETO in B16-F10 cells------------------------49
Figure 10. Cell cycle analysis of single, simultaneous, and sequential combined treatments of VPA and ETO in B16-F10 cells----50
Figure 11. Caspase-3 activity analysis of single, simultaneous, and sequential combined treatments of VPA and ETO in B16-F10 cells----52
9. Supplemental information------------------------------------53
Supplemental table 1. IC50 values of valproic acid (VPA) or etoposide (ETO) single treatment on SK-MEL-2-Luc cells------------53
Supplemental table 2. Combination index (CI) values and gradings of simultaneous and sequential combined treatments of VPA and ETO on SK-MEL-2-Luc cells------------------------------------------------54
Supplemental figure 1. Expression of G2/M phase checkpoint controlling proteins under single, simultaneous, and sequential combined treatments of VPA and ETO on B16-F10 cells---------------55
Supplemental figure 2. Dose-response curves of single treatments of VPA or ETO on SK-MEL-2-Luc cells----------------------------------56
Supplemental figure 3. Fitting curves of dose-response toward single treatments of VPA or ETO on SK-MEL-2-Luc cells--------------------57
Supplemental figure 4. Cell inhibition of single treatments versus simultaneous combined treatment of VPA and ETO on SK-MEL-2-Luc cells ------------------------------------------------------------------58
Supplemental figure 5. Cell inhibition of simultaneous versus sequential combined treatments of VPA and ETO on SK-MEL-2-Luc cells ------------------------------------------------------------------59
Supplemental figure 6. Synergistic inhibitory and antagonistic effects of VPA and ETO on SK-MEL-2-Luc cells are demonstrated by Fa-CI plot-----------------------------------------------------------60
Supplemental figure 7. Synergistic inhibitory and antagonistic effects of VPA and ETO on SK-MEL-2-Luc cells are demonstrated by isobolograms------------------------------------------------------61

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