Evaluation on covalent and noncovalent linking of peptide to graphene oxide for MMP-9 detection

<p>FITC-labeled peptide (Pep-FITC) containing the cleavage site of matrix metalloproteinase-9 (MMP9) was non-covalently or covalently linked to graphene oxide (GO) to form non-covalent or covalent nanoprobes (nGO/Pep-FITC and nGO-Pep-FITC) for MMP9 detection. nGO-Pep-FITC was prepared by formation of amide bonds between the carboxyl groups of c-nGO and the amine groups on Pep-FITC with the addition of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC). Pep-FITC was physically adsorbed onto the surface of c-nGO through π-π stacking and electrostatic interactions to produce nGO/Pep-FITC. Both nGO/Pep-FITC and nGO-Pep-FITC exhibited good selectivity for MMP9 detection. nGO/Pep-FITC and nGO-Pep-FITC showed 29.61 and 32.53 pM of detection limits for MMP9, respectively. nGO-Pep-FITC was much more resistant to bovine serum albumin (BSA) than nGO/Pep-FITC, thus may be applicable to analyze clinical samples especially containing proteins.</p

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

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 Huh-7 cells.

<p>Synergistic cytotoxicity of the combination treatment of DHA/ARS and FTS in Huh-7 cells.</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

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 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

Bak/Bax plays a modest but significant role in inducing apoptosis by the combination treatment.

<p>(<b>A</b> and <b>B</b>) Combination treatment of DHA/ARS and FTS enhanced the activation of Bak/Bax compared with single drugs treatment in both Huh-7 (<b>A</b>) and HepG2 (<b>B</b>) cell lines. Cells were treated with DHA/ARS for 48 h in the presence or absence of FTS and then incubated with 6A7 monoclonal anti-Bax antibody or Ab-2 monoclonal anti-Bak antibody before being analyzed by FCM. (<b>C</b> and <b>D</b>) Effects of silencing Bak or Bax on the cytotoxicity of the combination treatment assessed by CCK-8 assays in both Huh-7 (<b>C</b>) and HepG2 (<b>D</b>) cell lines. Cells were transfected with shBak and shBax expression vectors respectively before treatment with DHA/ARS for 48 h in case of FTS pretreatment. Cells with shNC were used as negative control. <i>*P</i> < 0.05, <i>**P</i> < 0.01 and <i>***P</i> < 0.001, compared with control; ^<i>#</i><i>P</i> < 0.05, ^<i>##</i><i>P</i> < 0.01 and ^<i>###</i><i>P</i> < 0.001; ^<i>$</i><i>P</i> < 0.05, ^<i>$ $</i><i>P</i> < 0.01 and ^{<i>$ $$</i>}<i>P</i> < 0.001.</p