Light-Trigerred Cellular Epigenetic Molecule Release To Reverse

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Light-trigerred Cellular Epigenetic Molecule Release to Reverse Tumor Multi-drug Resistance Leilei Shi, Li Xu, Qinghua Guan, Xin Jin, Jiapei Yang, and Xinyuan Zhu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00073 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Bioconjugate Chemistry

Light-trigerred Cellular Epigenetic Molecule Release to Reverse Tumor Multi-drug Resistance Leilei Shi, Li Xu, Qinghua Guan, Xin Jin,* Jiapei Yang, and Xinyuan Zhu School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China

ABSTRACT: Owing to the high spatial and temporal resolution of light, light related biotechnologies, for example, optogenetics, has wide ranging applications in neuroscience to control a subject’s behavior. Applying light to control tumors’ genetic behavior directly was still a challenge so far. Herein, we put forward a strategy of chemical optoepigenomics, in which epigenetic regulator (vorinostat) and paclitaxel (PTX) was conjugated onto a light-sensitive chemical molecule. The activity of vorinostat could be precisely controlled by the light, which could minimize the off-target effect. After UV irradiation under 350 nm, the photocagad epigenetic regulator (vorinostat) was selectively released from the conjugate in a spatiotemporal manner, inhibiting the activity of histone deacetylase (HDAC) then reversing PTX resistance of tumor cells effectively.

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION Owing to the high spatial and temporal resolution of light, light related biotechnologies, for example, optogenetics, has wide ranging applications in neuroscience to control a subject’s behavior.1-3 However, optogenetics highly relies on light-sensitive proteins, and it was mainly used for regulating neurons’ behaviors.4 Up to date, how to make optogenetics-related methodology be more general is still a challenge, for example, using light to control tumors’ behavior is still hard to be achieved. For malignant tumors, they could not express light sensitive proteins. Directly introducing typical light-sensitive ion channels into tumor cells via gene transfection is difficult to exert genetic regulation function.5 In addition, the gene transfection technology requires hostile conditions that sometimes alter cellular metabolism.6 To develop a general, free of light sensitive protein, light-triggered gene regulation method is highly desired. Herein, we put forward a strategy of chemical optoepigenomics, in which photochemical sensitive linkage was employed as light receptor to replace light-sensitive proteins. A genetic regulator, as well as a function group, was conjugated to the linker, conducting gene regulation cooperating with other functions after light irradiation. For detailed structure, (2-nitrophenyl)methanol was chosen in our work as the light receptor to release regulators after 350 nm short-time UV-light irradiation inspired by the pioneering work of Sivaguru and coworkers. Vorinostat (SAHA), which could induce epigenetic changes, was conjugated to light receptor via nucleophilic substitution as a gene regulator.7 Epigenetic changes, such as histone chemical 2


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modification and DNA methylation, are identified to be closely related to various biological behaviors, especially for multi-drug resistance (MDR).8-10 Therefore, a model drug paclitaxel (PTX) was conjugated via esterification, in order to detect reversing MDR efficiency. The whole conjugated chemical structure was indicated as SAHA-PTX for short. In our hypothesis, light can induce structure change of SAHA-PTX and result in gene regulation followed with a series of biological behavior changes. Under light irradiation, the caged gene regulator (SAHA) was firstly released from the conjugate and activated to exert epigenetic regulation (Scheme 1 and 2). The released SAHA is able to chelate Zn2+ and competitively binds with the active site of HDACs.11,12 Dysfunction of HDACs leads to repression of histone deacetylase, following activation of tumor suppressor genes.13-18 Under this condition, MDR can be reversed thus drug-resistant tumor cells become killable by PTX, which is released from the designed structure under the catalyzation of esterase. As a result, this chemical optoepigenomics strategy may regulate epigenetic changes of tumor cells, reverse tumor MDR and inhibit tumor growth precisely, while avoiding unwanted side effects.

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Scheme 1. Schematic representation of UV light triggers the release of gene regulator (SAHA). Under the UV irradiation, the caged gene regulator would be released and activated, then PTX would be also released from the conjugate through esterase.

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Scheme 2. Vorinostat (SAHA) and paclitaxel (PTX) were conjugated on light-sensitive chemical molecule 3-(bromomethyl)-4-nitrobenzoic acid through esterification and nucleophilic substitution.

RESULTS AND DISCUSSION Syntheis and Characterization of SAHA-PTX conjugate. Firstly, PTX was conjugated with 4-(bromomethyl)-3-nitrobenzoic in anhydrous dichloromethane, the conjugate was term as PTX-NO2. Then, SAHA-PTX conjugate was obtained via nucleophilic substitution of PTX-NO2 and SAHA. The synthesized conjugate was characterized using 1H-nuclear magnetic resonance (1H-NMR),

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C-nuclear magnetic

resonance (13C-NMR), and high resolution mass spectrum (HRMS), by which the successful synthesis of SAHA-PTX conjugate was confirmed (Figure S8-10). In vitro drug release of SAHA-PTX conjugate. Herein, we first evaluated the light response behavior of SAHA-PTX. It was found that the gene regulator SAHA could be released rapidly under light irradiation with accumulative release reaching 85% within 60 min, as analyzed by high performance liquid chromatograph (HPLC). In comparison, SAHA-PTX conjugate is quite stable without light irradiation in PBS buffer (Figure 1a). In addition, low pH could promote the release of SAHA from the conjugate to a certain extent (Figure S1). Furthermore, PTX could also be released from SAHA-PTX conjugate under the catalyze of esterase, and it is worth noting that weakly acidic environment could promote the release of PTX from the conjugate under the catalyze of esterase (Figure 1b). This result demonstrated that SAHA-PTX

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conjugate has good UV light and esterase dual responsibility. In vitro cytotoxicities. With the good light responsive property, how SAHA-PTX acted to MDR tumor cells was examined. PTX resistant HeLa cells and PTX resistant MCF-7 cells were incubated with SAHA, PTX, SAHA-PTX conjugates, SAHA/PTX drug mixtures at different concentrations for 72 h, cells without any treatment were used as negative control. For SAHA-PTX conjugate group, cells were irradiated with UV light (350 nm) for 10 min, with no-irradiated SAHA-PTX group as control. Based on MTT results, PTX was not able to inhibit the growth of both drug resistant tumor cells, and no-irradiated SAHA-PTX group showed poor tumor inhibition rate. For light-irradiated SAHA-PTX group, however, a dose-dependent cytotoxicity to both drug resistant tumor cells was observed, following with 50% cellular growth inhibition (IC50) value 0.86±0.002 µM, 1.21±0.014 µM for PTX resistant HeLa and PTX resistant MCF-7 cells, respectively (Figure 1c, d). UV light did not perform anti-proliferation effect to both cells under the same irradiation condition (Figure S2, 3). In addition, SAHA-PTX conjugate under UV irradiation could also exhibit superior inhibition rate to HeLa cells (Figure S4). However, SAHA-PTX conjugate performed the relatively low inhibition rate to normal L929 cells in contrast to other formulations (Figure S5). The cytotoxicity results demonstrated that SAHA-PTX only acted under light-irradiation to activate PTX of killing drug resistant tumor cells and decreased the side effect brought by traditional chemotherapeutics to normal cells.

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Figure 1. In vitro drug release and cell viability analysis of PTX resistant MCF-7 cells treated with different agents. a) Accumulative release profile of SAHA from the conjugate under 350 nm irradiation. b) Accumulative release profile of PTX from the conjugate under different pH with or without esterase. c) Cell viability analysis of PTX-resistant HeLa cells treated with different agents for 72 h. d) Cell viability analysis of PTX-resistant MCF-7 cells treated with different agents for 72 h. (** = p< 0.01, *** = p< 0.001). Cell cycle and apoptosis determination, relative expression levels of proteins. PTX is a cell cycle specific agent, the activation of PTX to drug resistant cells were further proved by observe cell cycle change. Firstly, MCF-7/PTX cells were treated with light-irradiated SAHA-PTX (5 µM), no-irradiated SAHA-PTX (5 µM), SAHA (5 µM), PTX (5 µM) and SAHA/PTX (5 µM) for 48 h and then stained with PI. The 7



results showed that PTX and no-light-irradiated SAHA-PTX groups exhibited a similar cell cycle to that of control group. However, the cell cycle obviously changed after incubation with SAHA, SAHA/PTX mixture and light-irradiated SAHA-PTX groups, especially for drug conjugate group. Particularly for light-irradiated SAHA-PTX group, the percentage of G0/G1 phase decreased to 5.47%, the percentage of S phase and G2/M phase increased to 43.17% and 48.52% (Figure 2). To determine whether the inhibition of cancer cell proliferation by drug conjugate was a consequence of SAHA and PTX induced apoptosis, the FITC-Annexin V/PI double-staining assay in PTX resistant MCF-7 cells was conducted. First, cells were incubated with PTX (5 µM), SAHA (5 µM), drug mixture (5 µM), SAHA-PTX conjugate (5 µM) with or without UV irradiation for 48 h followed with FITC-Annexin V/PI staining. At the same time, cells without any treatment were used as negative control. Flow cytometry analysis indicates that the frequencies of apoptotic cells are 19.2%, 31.5%, 37.4%, 19% and 61.9% induced by PTX, SAHA, drug mixture, SAHA-PTX conjugate without or with UV irradiation, respectively (Figure 3). In addition, as shown in Figure 4a, obvious destroys of the action filaments inside the cells showed in immunofluorescence staining for light-irradiated SAHA-PTX (5 µM). Combined all these results together with cytotoxicity results, SAHA-PTX could significantly change biological behavior of tumor cell and reverse MDR under light irradiation. To provide a clear version of the regulation mechanism, a series of characterizations was designed to prove it.

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Figure 2. Cell cycle distribution histograms of PTX resistant MCF-7 cells treated with PTX, SAHA, SAHA/PTX mixture, SAHA-PTX conjugate, SAHA-PTX conjugate under UV irradiation at the same concentration (5 µM) for 48 h. (* = p< 0.05, ** = p< 0.01, *** = p< 0.001).

Figure 3. Apoptosis of PTX-resistant MCF-7 cells incubated with PTX, SAHA, drug mixture, SAHA-PTX conjugate with or without UV irradiation for 48 h by flow cytometry analysis. Inserted numbers in the profiles present the percentage of the cells in this area. Lower left: living cells; upper left: necrotic cells; lower right: early apoptotic cells; upper right: late apoptotic cells. Each experiment group is repeated three times. Firstly, SAHA-PTX could inhibit HDACs and up-regulate the acetylation level of histone after light irradiation. As shown in Figure 4b, the acetylation level of histone is obviously raised after MCF-7/PTX cells were treated with SAHA-PTX under UV 9



irradiation. In comparison, for cells without any treatment or treated by no-irradiated SAHA-PTX, no up-regulated level of histone acetylation could be observed. Acetylation of histone could promote the gene transcription and expression, especially for tumor suppressor genes such as p53. p53 has been regarded as the most important tumor suppressor gene to inhibit tumor growth and reset cell cycle.19, 20 Therefore, the expression level of acetylation p53 was then assayed via western blot analysis. It could be found that the expression of acetylation p53 was increased as expected for light-irradiated SAHA-PTX cells (Figure 4b, 5a). By contrast, the level of acetylation p53 is quite low when cells without any treatment or with only SAHA-PTX. From these results, we preliminary inferred that gene regulation could be triggered by light through designed chemical structure SAHA-PTX conjugate.

Figure 4. The immunofluorescence staining of micro-tubulin and western blot 10


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analysis of expression level of proteins. a) Fluorescence images of PTX resistant MCF-7 cells stained with Alexa Fluor 633 Phalloidin (F-actin) and Hoechst (nuclei) after treatment of SAHA-PTX conjugate under UV irradiation at the concentration of 5 µM. The scale bar is 25 µm. b) The expression level of acetylation of histone and p53 after PTX resistant MCF-7 cells being treated with SAHA-PTX conjugate under UV irradiation for 24 h. c) The expression level of BCl-2, Caspase-3 (Cas-3) and p-gp after PTX resistant MCF-7 cells being treated with SAHA-PTX conjugate under UV irradiation for 24 h.

Figure 5. The relative expression level of acetylation p53 and histone, BCL-2, caspase-3, ABCC 10 after MCF-7/PTX cells being treated with SAHA-PTX conjugate and SAHA-PTX conjugate under UV irradiation. (* = p< 0.05, ** = p< 0.01, *** = p< 0.001). Ferroptosis signal pathway study. The up-regulated the level of histone acetylation and p53 has highly relative to MDR reversion. For further investigation, we chose some routine MDR-related biomarkers, such as p-glycoprotein (p-gp), B-cell lymphoma-2 (Bcl-2) and cleaved caspase-3, and evaluated their expression levels. Surprisingly, the levels of all the three proteins did not perform significant 11



difference between drug treated groups and control groups (Figure 4c, Figure 5b). These results gave us a hint that acetylation p53 might regulate other targets to reverse MDR, other than down-regulating the expression level of pgp and BCL-2. As a result, we had to hypothesis that chemoepigenomics strategy adopted in this work might regulate other signal pathways to reverse MDR and induce cell death in a caspase-independent way. Gu and coworkers have recently reported that p53 acetylation was crucial for ferroptosis and tumor suppression.21, 22 When ferroptosis occurs, reactive oxygen species (ROS) was highly accumulated and mitochondria would be damaged then the ATP production was blocked. Inspired by their research, we inferred that the light irradiation released SAHA firstly increased the level of acetylation p53, then induced ferroptosis and resulted in energy deficiency. Eventually, the lack of ATP could block the activity of p-gp and induce accumulated drug content inside the cells. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS) results showed that light-irradiated SAHA-PTX group remained the highest cellular PTX concentration comparing to other groups, indicating a ferroptosis pathway might happen (Figure 6a). In ferroptosis cells, acetylation of p53 would down-regulate the level of cysteine/glutamate antiporter (xCT) and glutathione peroxidase, then generating soluble and lipid ROS and blocking the ATP production. For further conformation, identification of several ferroptosis related biomarkers was conducted, including ROS, malondialdehyde (MDA) and ATP. As shown in Figure 6b and c, the light-irradiated SAHA-PTX group performed the highest level of ROS (Figure S6) and MDA, as well

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as the lowest content of ATP. In addition, western blot analysis indicated that the expression level of xCT and GPx4 was decreased after the treatment of SAHA-PTX conjugate with UV irradiation (Figure 6d, Figure S7). All these data demonstrated a ferroptosis pathway has been induced to reverse MDR by chemical optoepigenomics regulation.

Figure 6. The content of cellular MDA, APT, PTX and western blot analysis of expression level of xCT and GPx4. a) MDA concentration inside the PTX resistant MCF-7 cells after they were treated with light-irradiated SAHA-PTX or without UV light. b) The content of ATP in PTX resistant MCF-7 cells after being treated with light-irradiated SAHA-PTX or without UV light. c) The concentration of PTX inside the PTX resistant MCF-7 cells after they were treated with light-irradiated 13



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SAHA-PTX or without UV light. d) The expression level of xCT and GPx4 in PTX resistant MCF-7 cells after they were treated with light-irradiated SAHA-PTX or without UV light. (* = p< 0.05, ** = p< 0.01, *** = p< 0.001).

CONCLUSIONS In summary, we firstly adopted the chemical optoepigenomics strategy of employing well-designed chemical structure to achieve light regulation of genetic and biological behavior in non-neuron cells. SAHA-PTX was designed and used as a model structure, where a fast light-response of epigenetic regulation of tumor cells and reversed MDR status were observed and proved. Technically, chemical optoepigenomics strategy realizes light regulation of gene biological behavior through a chemical structure, other than photosensory cell or light sensitive protein. We hope that this study could make optoepigenetic technology be more universal and promote this strategy being used for non neurological disease like cancer treatment. Furthermore, to solve the low penetration of UV light, SAHA-PTX conjugate could be loaded into the up-conversion nanoparticles (UCNP) and the further in vivo study is ongoing in our laboratory. EXPERIMENTAL SECTION General Remarks. Vorinostat (SAHA) and paclitaxel (PTX) were purchased from Meilune

Biotech.

4-(bromomethyl)-3-nitrobenzoic

acid,

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N, N-dimethylpyridin-4-amine (DMAP) were purchased from Adamas. All reagents were 14


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of

analytical

grade,

and

used

without

purfication.

3-(4,

5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchase from sigma. Dulbecco’s Modified Eagle Medium (DMEM) and RMPI-1640 were purchased from Gibco. Fetal bovine serum (FBS), penicillin and streptomycin were obtained from Gibco. Monoclonal antibodies used for western blot analysis were purchased from Abcam. All reagents were of analytical grade, and used as received. 1

H and

13

C nuclear magnetic resonance spectroscopy (1H and

13

C NMR) were

recorded using a Mercury plus 400 MHz spectrometer (Varian, USA) with dimethyl sulfoxide-d6 (DMSO-d6) and chloroform-d3 (CDCl3) as solvents. Ultraviolet-visible (UV-Vis) absorption of the sample solutions was determined by Thermo Electron-EV300 UV-Vis spectrophotometer. Confocal imaging of microtubule was determined via Super-resolution Multiphoton Confocal Microscope (Leica/TCS SP8 STED 3X). Synthesis and characterization. PTX (0.5 mmol, 426.5 mg), EDCI (0.6 mmol, 114.8 mg), DMAP (0.6 mmol, 72.1 mg) was suspended in anhydrous dichloromethane (20 mL). The mixture was then cooled in an ice bath. A solution of 4-(bromomethyl)-3-nitrobenzoic (0.55 mmol, 143 mg) in anhydrous dichloromethane (5 mL) was added dropwise over a period of 20 min under stirring. The resulting solution was allowed to react at room temperature for 12 h. The reaction solvent was removed via rotary evaporators and the residue was partitioned between distilled water (20 mL) and ethyl acetate (EA) (40 mL). The organic layer was separated and dried over anhydrous MgSO4, filtered, and concentrated via rotary evaporators to 15



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obtain crude product, which was further purified via silica gel chromatography (hexane: EA = from 5:1 to 1:1, v:v) to afford 370 mg of conjugate named as PTX-NO2, yield 67.5%. 1H-NMR (400 MHz, CDCl3): δ = 8.71 (s, 1H), 8.26-8.28 (m, 1H), 8.04-8.07 (m, 2H), 7.81-7.83 (m, 1H), 7.73-7.75 (m, 2H), 7.54-7.56 (m, 1H), 7.41-7.49 (m, 6H), 7.22-7.25 (m, 3H), 7.11-7.13 (m, 1H), 6.31-6.34 (m, 2H), 6.01-6.03 (m, 1H), 5.71-5.74 (m, 1H), 4.98 (s, 2H), 4.46-4.48 (m, 1H), 4.39-4.41 (m, 1H), 4.07-4.13 (m, 2H), 3.62-3.65 (m, 1H), 2.42-2.45 (m, 1H), 2.41 (s, 3H), 2.37-2.39 (m, 2H), 2.26 (s, 3H), 2.11-2.14 (m, 2H), 1.95 (s, 3H), 1.90-1.93 (m, 1H), 1.73 (s, 3H), 1.23-1.25 (m, 2H), 1.21 (s, 3H), 1.18 (s, 3H) ppm;

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C-NMR (100 MHz,

CDCl3): δ = 203.9, 171.6, 169.9, 168.0, 167.8, 167.1, 163.6, 148.1, 142.6, 138.0, 136.6, 134.7, 133.9, 133.7, 133.2, 132.4, 130.4, 130.2, 129.5, 129.4, 128.9, 127.3, 126.9, 126.7, 84.7, 81.3, 79.2, 76.6, 75.7, 75.3, 72.5, 72.3, 58.7, 53.4, 45.6, 43.2, 42.5, 35.3, 26.7, 23.0, 22.3, 20.9, 14.6, 9.9 ppm; HRMS: m/z (ESI) calcd for C55H55BrN2O17 (M+H+), 1096. 9324, Found 1097.2860. SAHA (0.11 mmo, 30 mg) and Cs2CO3 (0.11 mmol, 36 mg) were dissolved in anhydrous methanol and stirred at room temperature for 1 h, then methanol was removed by rotary evaporator to obtain the activated SAHA. PTX-NO2 (0.1 mmol, 110 mg) was dissolved in anhydrous N, N-dimethylformamide (DMF, 5 mL), activated SAHA was also dissolved in DMF and added dropwise to PTX-NO2 solution, then the reaction mixture was stirred at room temperature for 24 h. 15 mL water was added to the reaction mixture, then centrifugation to obtain the solid residue. Eventually, the residue was purified via silica gel chromatography (DCM:

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Methanol = from 500:1 to 50:1, v:v) to afford 55 mg SAHA-PTX conjugate, yield 43%. Finally, the SAHA-PTX conjugate was purified via preparative reversed-phase high performance liquid chromatography, with elution CH3CN/H2O from 5% to 95%. The flow rate is 15 mL/min. HPLC purity: 98.44%. 1H-NMR (400 MHz, CDCl3): δ = 8.14-8.17 (m, 2H), 8.09-8.11 (m, 2H), 7.70-7.72 (m, 2H), 7.61-7.62 (m, 2H), 7.50-7.53 (m, 7H), 7.37-7.48 (m, 5H), 7.17 (d, J = 8.0 Hz, 1H), 6.77-6.81 (m, 2H), 6.23-6.25 (m, 1H), 5.59-5.77 (m, 3H), 4.90-4.93 (m, 2H), 4.75-4.78 (m, 1H), 4.66-4.69 (m, 1H), 4.49 (s, 2H), 3.98-4.01 (m, 1H), 3.81-3.84 (m, 1H), 3.75-3.77 (m, 1H), 3.53-3.57 (m, 1H), 2.49 (s, 3H), 2.36-2.47 (m, 3H), 2.34 (s, 3H), 2.14-2.33 (m, 4H), 2.13 (s, 3H), 1.97-2.01 (m, 2H), 1.77-1.79 (m, 1H), 1.54-1.65 (m, 3H), 1.52 (s, 3H), 1.25-1.28 (m, 3H), 1.21 (s, 3H), 1.12-1.18 (m, 3H), 1.10 (s, 3H) ppm; 13C-NMR (100 MHz, CDCl3): δ = 207.6, 207.4, 172.9, 172.7, 172.5, 169.8, 167.4, 167.3, 144.1, 140.0, 138.2, 133.9, 133.8, 133.5, 133.2, 130.5, 130.3, 129.2, 129.0, 128.9, 128.5, 127.3, 127.1, 82.9, 79.3, 79.1, 78.3, 77.9, 76.0, 75.8, 75.5, 75.4, 73.4, 72.4, 67.9, 57.9, 57.8, 55.2, 42.9, 40.9, 40.5, 38.8, 36.4, 35.3, 26.7, 26.2, 22.8, 21.6, 21.2, 21.1, 20.6, 16.5, 15.9, 15.0 ppm; HRMS: m/z (ESI) calcd for C69H74N4O20 (M+H+), 1279. 4896, Found 1279.5067. In vitro drug release of SAHA-PTX conjugate under UV irradiation.23 SAHA-PTX conjugate was dissolved in a mixture of DMSO/PBS (5:1) solutions and irradiated with 350 nm UV light for 60 min. The solution was monitored by HPLC to detect release of SAHA. Percent conversion was calculated by analyzing standards of SAHA-PTX conjugate and SAHA by HPLC, using integration of the area under the

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curve for normalization. Cell Culture. The breast cancer line MCF-7/ADR and cervical cancer line HeLa/PTX were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and RMPI-1640, respectively. The culture medium contains 10% fetal bovine serum (FBS) and antibiotics (50 units mL-1 and 50 units mL-1 streptomycin) at 37oC under a humidified atmosphere containing 5% CO2. In vitro cytotoxicity studies. HeLa cells, L929 cells, both PTX resistant HeLa cells and MCF-7 cells were seeded in the 96-well plates. 12 hours later, cells were treated by PTX, SAHA, SAHA-PTX conjugate, SAHA/PTX mixture with different concentrations and incubated for 72 h. Meanwhile, for one of SAHA-PTX group, UV light (350 nm, 10000 µw/cm2) was used to irradiate cells for 10 min. Cells without any treatment or only with UV light irradiation were used for negative control. Then, 20 µL MTT solutions (5 mg/mL) were added into 96-well plates. After 4 h incubation at 37oC, the medium was replaced by 200 µL DMSO. The obtained solution was measured in a Bio Tech Synergy H4 at a wavelength of 570 nm. Cell cycle arrest study.24 MCF-7/PTX cells (5×105) were seeded in 6-well plates cultured for 12 h in complete DMEM culture medium. Cells were treated with PTX, SAHA, SAHA-PTX conjugate, SAHA/PTX mixture at the concentration 5 µM. Meanwhile, for one of SAHA-PTX group, UV light (350 nm) was used to irradiate cells for 10 min. Cells without any treatment were used as the control. After that, cells were washed with PBS three times, and fixed with 70% ethanol at 4oC overnight and treated with RNAnase A for 45 min at 37oC, followed by PI staining for 30 min in the

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dark. The samples were analyzed by flow cytometry (BD FACSSalibur, USA), and 1×104 cells events per sample were counted. Cell apoptosis. Apoptosis of PTX-resistant MCF-7 cells was measured by FITC-Annexin V/propidium iodide (PI) dual-stainging method. PTX-resistant MCF-7 cells were incubated with PTX, SAHA, drug mixture, SAHA-PTX conjugate with or without UV irradiation. After 48 h incubation, FITC-Annexin V/PI was used to stain the cells. Cells without any treatment were used as negative control. The samples were analyzed with a BD LSR Fortessa flow cytometer. Western blot analysis. MCF-7/PTX cells were seeded in the culture dish at a density of 3.0×106 cells in 10 mL of complete DMEM culture medium. After being adhered, 5 µM SAHA-PTX conjugate was added into the culture medium. Meanwhile, for one of SAHA-PTX group, UV light (350 nm) was used to irradiate cells for 10 min. Cells without any treatment was used as negative control. Then, the cellular proteins were extracted in Laemmli buffer and the protein content in the extracts was quantified using a bicinchoninic acid (BCA) protein assay kit (Pierce, USA). Equal amounts

of

proteins

(20

µL/lane)

were

separated

on

sodium

dodecyl

sulfate-poly-acrylamide gels (SDS-PAGE) and electrotransferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% nonfat milk in TBST (Tris buffered saline supplemented with 0.1% Tween-20) and probed with antibodies against GADPH (1:1000 dilution), anti-acetylation histone-3 (1:1000 dilution), anti-acetylation p53, anti-BCL-2, anti-ABCC10, anti-caspase-3 followed by HRP-conjugated (HRP = horseradish peroxidase)

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anti-rabbit immunoglobulin-G (IgG, 1:5000 dilution). GADPH was used as internal control. Protein bands were observed by chemiluminescent HRP Substrate S13 according to the manufacture’s protocol and analyzed using the ChemiDoc MP imaging System (Bio-Rad, USA). Cellular accumulation of PTX. MCF-7/PTX cells were seeded in 100 mm culture dish and incubated until the cell confluency reached at the density of 85%. Then 5 µM SAHA-PTX conjugate were added to the culture medium at the concentration of 20 µM. Meanwhile, for one of SAHA-PTX group, UV light (350 nm) was used to irradiate cells for 10 min. After 12 h exposure, cells were collected and washed 3 times with ice-cold PBS. Cells were crashed via cell scraper, then centrifugation and extraction by ethyl acetate to obtain the supernatant. The supernatant was analyzed by HPLC and calculated by analyzing standards of PTX. Cellular ATP content determination.25 Cellular ATP levels were measured using a firely luciferase ATP assay kit (Beyotime, China) according to the manufacture’s instructions. PTX resistant MCF-7 cells were firstly incubated with 5 µM SAHA-PTX conjugate for 24 h. Cells without any treatment were used as negative controls. Meanwhile, for one of SAHA-PTX group, UV light (350 nm) was used to irradiate cells for 10 min. Then, the cells were lysised and centrifugeled at 12000 rpm at 40oC for 5 min. 100 µL of each supernatant was mixed with 100 µL of ATP detection working dilution. Luminance was determined by GloMax 20/20 luminometer. Intracellular LPO measurement.26 Thiobarbituric acid reaction was used here to measure the intracellular LPO content. The PTX resistant MCF-7 cells were lysed

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with freeze thaw. Then, lysates were centrifuged at 12,000 g for 15 min at 4oC to collect the supernatant. Intracellular total proteins were normalized according to their concentrations and subjected to MDA assay as described in the Lipid Peroxidation MDA assay kit (Beyotime, Jiangsu, China). Lipid peroxidation was calculated as nanomoles of MDA per milligram of protein. ROS content determination.27 PTX resistant cells (4×105) with culture medium were seed in a culture dish. After 12 h, cells were treated with 5 µM SAHA-PTX conjugate and incubated for 24 h, then washed with PBS and incubated with 100 µL DCFH-DA probe at 37oC for 30 min. After incubation, cells were washed with PBS again. Fluorescence data was measure by flow cytometry using excitation wavelength 488 nm and emission wavelength 525 nm. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Figures showing measurement of in vitro release of SAHA from SAHA-PTX conjugate in different pH conditions. Cell viability assays, relative expression level of proteins, cellular ROS concentration measurement, 1H-NMR and of SAHA-PTX conjugate. AUTHOR INFORMATION Corresponding Authors [email protected]; phone, 086-21-34203400;

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13

C-NMR, HRMS


Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (51690151, 51503122, 51473093), National Basic Research Program (2015CB931801) and Shanghai Rising-Star Program（17QC1401100）. ABBREVIATIONS HDAC, Histone deacetylase; SAHA, vorinostat; PTX, paclitaxel; NMR, nuclear magnetic resonance; HPLC, high-performance liquid chromatography; HRMS, high resolution mass spectrum; ROS, reactive oxygen species; LPO, lipid peroxide; UV, ultraviolet; MDA, malondialdehyde; DMEM, Dulbecco’s Modified Eagle Medium; FBS, Fetal bovine serum; MTT, 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

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Herein, we put forward a strategy of chemical optoepigenomics, in which epigenetic regulator (vorinostat) and paclitaxel (PTX) was conjugated onto a light-sensitive chemical molecule. The activity of vorinostat could be precisely controlled by the light, which could minimize the off-target effect. After UV irradiation under 350 nm, the photocagad epigenetic regulator (vorinostat) was selectively released from the conjugate in a spatiotemporal manner, inhibiting the activity of histone deacetylase (HDAC) then reversing PTX resistance of tumor cells effectively.

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Light-Trigerred Cellular Epigenetic Molecule Release To Reverse

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