Additional file 1: Figure S1. of Glucose-Reduced Graphene Oxide with Excellent Biocompatibility and Photothermal Efficiency as well as Drug Loading

Change of nGO, nGO-0, nrGO and nrGO-0 in mean diameter (Figure S1A), Zeta potential (Figure S1B) and Pdi (Figure S1C) can be found in supplementary materials. (DOC 10464 kb

Farnesylthiosalicylic acid sensitizes hepatocarcinoma cells to artemisinin derivatives

<div><p>Dihydroartemisinin (DHA) and artesunate (ARS), two artemisinin derivatives, have efficacious anticancer activities against human hepatocarcinoma (HCC) cells. This study aims to study the anticancer action of the combination treatment of DHA/ARS and farnesylthiosalicylic acid (FTS), a Ras inhibitor, in HCC cells (Huh-7 and HepG2 cell lines). FTS pretreatment significantly enhanced DHA/ARS-induced phosphatidylserine (PS) externalization, Bak/Bax activation, mitochondrial membrane depolarization, cytochrome <i>c</i> release, and caspase-8 and -9 activations, characteristics of the extrinsic and intrinsic apoptosis. Pretreatment with Z-IETD-FMK (caspase-8 inhibitor) potently prevented the cytotoxicity of the combination treatment of DHA/ARS and FTS, and pretreatment with Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited the loss of ΔΨm induced by DHA/ARS treatment or the combination treatment of DHA/ARS and FTS in HCC cells. Furthermore, silencing Bak/Bax modestly but significantly inhibited the cytotoxicity of the combination treatment of DHA/ARS and FTS. Interestingly, pretreatment with an antioxidant N-Acetyle-Cysteine (NAC) significantly prevented the cytotoxicity of the combination treatment of DHA and FTS instead of the combination treatment of ARS and FTS, suggesting that reactive oxygen species (ROS) played a key role in the anticancer action of the combination treatment of DHA and FTS. Similar to FTS, DHA/ARS also significantly prevented Ras activation. Collectively, our data demonstrate that FTS potently sensitizes Huh-7 and HepG2 cells to artemisinin derivatives via accelerating the extrinsic and intrinsic apoptotic pathways.</p></div

Caspase-8 is not activated by caspase-3.

<p>(<b>A</b>) Microscopic images of cells expressing FRET-Bid in the absence or presence of Ac-DEVD-CHO (caspase-3 inhibitor, 20 μM) in Huh-7 and HepG2 cells. (<b>B</b> and <b>C</b>) Statistical results of <i>I</i>_<i>CFP</i><i>/I</i>_<i>YFP</i> ratio from at least 80 Huh-7 cells (<b>B</b>) or HepG2 cells (<b>C</b>). Cells were transfected with FRET-Bid plasmid for 24 h and then pretreated with Ac-DEVD-CHO for 30 min before FTS/DHA/ARS treatment or the combination treatment of FTS and DHA/ARS for 24 h before fluorescence microscopic imaging. Scale bar: 10 μm. <i>NS</i> = no statistical significance, <i>P ></i> 0.05; <i>**P <</i> 0.01, <i>***P <</i> 0.001.</p

ROS is involved in the anticancer action of the combination treatment of DHA and FTS.

<p>(<b>A</b> and <b>B</b>) FTS significantly increased DHA-induced ROS generation but did not affect the ROS generation induced by ARS in Huh-7 cells (<b>A</b>), and FTS did not affect the ROS generation induced by DHA/ARS in HepG2 cells (<b>B</b>). Cells were treated with DHA/ARS for 2 h in the presence or absence of FTS and then incubated with DCF-DA before being analyzed by FCM. <i>NS</i> = no statistical significance, <i>P</i> > 0.05; <i>*P</i> < 0.05, <i>**P</i> < 0.01 and <i>***P</i> < 0.001, compared with control; ^<i>##</i><i>P</i> < 0.01. (<b>C</b> and <b>D</b>) Effects of NAC on the cytotoxicity of the combination treatment of DHA/ARS and FTS assessed by CCK-8 assays in both Huh-7 (<b>C</b>) and HepG2 (<b>D</b>) cells. Cells were pretreated with 10 mM NAC for 2 h or not, and then treated with DHA/ARS for 48 h in the presence or absence of FTS. <i>NS</i> = no statistical significance, <i>P</i> > 0.05; <i>*P</i> < 0.05 and <i>**P</i> < 0.01; ^$$ $<i>P</i> < 0.001; ^<i>##</i><i>P</i> < 0.01 and ^<i>###</i><i>P</i> < 0.001.</p

FTS enhances DHA/ARS-induced apoptosis in HCC cells.

<p>(<b>A</b>) Typical FCM analysis of apoptosis induced by DHA/ARS in the presence or absence of FTS. Cells were treated with DHA/ARS for 48 h with or without the addition of FTS and then stained with Annexin V-FITC/PI before being analyzed by FCM. (<b>B</b> and <b>C</b>) Statistical results of three independent FCM analyses on apoptosis in Huh-7 (<b>B</b>) and HepG2 (<b>C</b>) cells. **<i>P</i> < 0.01 and ***<i>P</i> < 0.001, compared with control; ^#<i>P</i> < 0.05 and ^##<i>P</i> < 0.01.</p

FTS promotes DHA/ARS-induced caspase-8 and -9 activations.

<p>(<b>A</b> and <b>B</b>) <i>I</i>_CFP<i>/I</i>_YFP/Venus ratio images of cells expressing FRET-Bid or SCAT9 (left) and corresponding statistical results (right) from at least 250 cells in Huh-7 (<b>A</b>) and HepG2 (<b>B</b>) cells. Cells were transfected with FRET-Bid and SCAT9 plasmid for 24 h respectively and then treated with DHA/ARS for 24 h in the presence or absence of FTS before fluorescence microscopic imaging. *<i>P</i> < 0.05 and ***<i>P</i> < 0.001, compared with control; ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01 and ^###<i>P</i> < 0.001. (<b>C</b> and <b>D</b>) Cells were pretreated with Z-ITED-FMK (caspase-8 inhibitor, 20 μM) for 30 min before FTS/DHA/ARS treatment or the combination treatment of DHA/ARS and FTS for 48 h and then analyzed by CCK-8 assay. *<i>P</i> < 0.05, **<i>P</i> < 0.01, and ***<i>P</i> < 0.001.</p

IC₅₀ of FTS/DHA/ARS in both Huh-7 and HepG2 cells.

<p>IC₅₀ of FTS/DHA/ARS in both Huh-7 and HepG2 cells.</p

DHA/ARS inhibits Ras activation.

<p>(<b>A</b>) FRET efficiency images of HepG2 cells expressing Raichu-Ras plasmid quantified by PbFRET quantification method. D: fluorescence intensities images from donor channel (Emission 480/40 nm) during donor excitation (Excitation 435/20 nm); A: fluorescence intensities images from acceptor channel (Emission 550/40 nm) during acceptor excitation (Excitation 510/17 nm); DP: fluorescence intensities images from donor channel during donor excitation after photobleaching (Excitation 510/17 nm); AP: fluorescence intensities images from acceptor channel during acceptor excitation after photobleaching (Excitation 510/17 nm). Scale bar: 10 μm. (<b>B</b>) Statistical results of FRET efficiency from at least 90 living HepG2 cells expressing Raichu-Ras plasmid after different treatments. <i>NS</i> = no statistical significance, <i>P</i> > 0.05; *<i>P</i> < 0.05, compared with control from the group treated with FBS for 2 h; ^#<i>P</i> < 0.05, compared with control from the group treated with FBS for 24 h.</p

Synergistic cytotoxicity of the combination treatment of DHA/ARS and FTS in HCC cells.

<p>(<b>A</b>–<b>F</b>) Dose-response effects of FTS, DHA or ARS alone on Huh-7 cells (<b>A</b>–<b>C</b>) and HepG2 cells (<b>D</b>–<b>F</b>) growth. Cells were treated with increasing doses of FTS, DHA and ARS for 48 h and then analyzed by CCK-8 assay. (<b>G</b> and <b>H</b>) Morphological analysis on the anti-proliferation effect of DHA/ARS in the presence or absence of FTS in Huh-7 cells (<b>G</b>) and HepG2 cells (<b>H</b>). Original magnification: 100×.</p

Synergistic cytotoxicity of the combination treatment of DHA/ARS and FTS in HepG2 cells.

<p>Synergistic cytotoxicity of the combination treatment of DHA/ARS and FTS in HepG2 cells.</p