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3,492,000 | Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species.-V -N -L -I -G -H -S -H -G -G --Y -N -L -I -G -H -S -Q -G -A --V -N -L -I -G -H -S -0 -G -A --V -N -L -V -G -H -S -Q -G -Q --V -H -L -V -G -H -S -M -G -G -. V . H . F -I -G -H -S -M -G -G -. V . M . I -F -G -E -S -A -G - | semantic_scholar |
3,492,001 | Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species.The deduced amino acid sequence contains a sequence, -Val-Asn-Leu-Ile-Gly-His-Ser-His-Gly-Gly-, which match almost exclusively to the consensus sequence of lipase (Fig. 5A). Comparison with known sequences of lipases, such as from P. fragi (8,9), P. cepuciu (7), Staphylococcus uureus (21), Staphylococcus hyicus (22), Geotrichum candidum (23), Candida cylindrucea (24), porcine pancreas (25), rat lingua (26), canine pancreas (27), human liver (28), and human pancreas (29), revealed that the lactonizing lipase have 38-40% homologies on the amino acid level with lipases of pseudomonads, but no overall homologies with amino acid sequences of other lipases. When the alignment of functionary similar amino acid residues are considered, similarities of lactonizing lipase toward lipases of pseudomonads increased (50% for lipase of P. fragi and 59% for P. cepacia, Fig. 5B). These high similarities on overall sequence together with the similarity in molecular weight suggest that those lipases from pseudomonads may have lactonizing activity, which lack in other lipases. For eucaryotic lipases, lipase activity has been reported to be inhibited by SH-blocking agents and the positions of Cys residues are well conserved (27,30). However, for procaryotic lipases, Cys residues are lacking or low in its content, and seems not to participate in the catalytic activity. The lactonizing lipase contains only 2 Cys residues and its esterase activity was not affected by treatment of iodoacetic acid or 5,5'-dithiobis(2-nitrobenzoic acid) (no inactivation after incubation at 5 mM reagent at 37 "C for 1 h). Thus, similar to other procaryotic lipases, Cys residues in lactonizing lipase are not involved in its catalytic function. No homologies were found when the nucleotide and predicted amino acid sequences were compared with the EMBL and GenBank nucleotide and National Biomedical Research Foundation and SWISSPROT protein sequences, except for the lipases | semantic_scholar |
3,492,002 | Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species.protein sequences, except for the lipases mentioned above and one hypothetical protein from Vibrio cholerue (SWISSPROT, accession number P15493). Although the latter only covered the NHZ-terminal part of the hypothetical protein, homology with lactonizing lipase is very high (58%), and, therefore, may represent a lipase-like enzyme of Vibrio origin. | semantic_scholar |
3,492,003 | Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species.The availability of genomic DNA clones for lactonizing lipase will provide tools for studying the mechanism involved in regulating the synthesis of the enzyme, and also make it possible to provide large quantities of highly purified enzyme, which is a prerequisite to initiate detailed investigations on the three-dimensional structural features which allow macrocyclic lactone formation.
FIG. 2 .
2Purity and molecular weight determination of the lactonizing lipase by SDS-PAGE. A , samples were run on a 4-20% linear gradient gel, and stained with Coomassie Brilliant Blue G-250. Lane a, 0.5 gg of crude lipase P after dialysis and centrifugation; lane b, 2 pg of HPLC fraction; lane M, marker proteins (phosphorylase b, M, = 94,000; bovine serum albumin, M, = 67,000; ovalbumin, M, = 43,000; carbonic anhydrase, M, = 30,000; soybean trypsin inhibitor, M, = 20,000). B, log(M,) versus relative mobility plot. 0, represent marker proteins; arrow, position of purified lipase.
"
ND, not determined. even by 50% (v/v) ethylene glycol, indicating the very hydrophobic nature of the lactonizing lipase. However, gel filtration afforded a good separation and activity yield. After open column gel filtration using Sepharose CL-GB and gel filtration HPLC, nearly 500-fold purification was attained on the basis of specific lactonizing activity
FIG. 3 .
3Southern blot analyses of genomic DNA of Pseudomonas nov. sp. 109 ( A ) and restriction endonuclease map of pUY45 and sequencing strategy for cloned lipL gene ( B ) . A, genomic DNA (5 pg/lane) digested completely by PstI (lanes 2 and 5), SalI | semantic_scholar |
3,492,004 | Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species.FIG. 4 .
4Nucleotide sequence of lipL encoding the lactonizing lipase from Pseudomonas nov. sp. 109. The deduced amino acid sequence is denoted under the nucleotide sequence in the standard one-letter code. Amino acids are numbered starting with the first inframe methionine as 1. A, amino-terminal end of mature lactonizing lipase. The translation termination codon is designated by asterisks (***). The amino acid sequences obtained by sequencingof the aminoterminal end are underlined. The putative ribosome-binding site is indicated by a broken line. The lipase consensus sequence is boxed.
.
A --V -T -L -F -G -E -S -A -G -S -. V -H -V -I -G -H -S -L -G -S --1 . N . Y -V -G -H -S -Q -G -T -. V . N . L -I -G -H -S -L -G -A -. V . H . L -I -G -Y -S -L -G -A -. V . H . V -I -G -H -S -L -G -. -G -H -S -X -G --? . H y -H y -G &-S -? -G -Hyd-FIG. 5. Comparison of lipase consensus sequences among various lipases ( A ) and overall amino acid sequences of lactonizing lipase from Pseudomonas nov. sp. 109 with those of lipases from P . frugi and P . cepuciu ( B ) . A , the amino acid sequences around the consensus region and the functional features were shown. The function of each amino acid are: Hyd, hdyrophobic;
The nucleotide sequence(s) reported i n this paper has been submitted to the GenBankTM/EMBL Data Bank with accession numberfs) 090398.$ T o whom correspondence and reprint requests should be addressed Dept. of Biotechnology, Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka 565, Japan. Tel.: 06-877-5111 (ext. 4357). | semantic_scholar |
3,492,005 | Purification, characterization, and molecular cloning of lactonizing lipase from Pseudomonas species.TABLE I
IPurification table of the lactonizing lipase from crude powder of lipase P Purification with 5 g of crude lipase P powder was summarized. Lactonizing activity was assayed with methyl 16-hydroxyhexadecanoate as substrate. Other experimental conditions are described under "Materials and Methods." Pseudomonas nov. sp. 109 in anhydrous organic solvents using methyl esters of w-hydroxy acids. I, substrate, n = 10-20; II, monomer lactone; III, dimer lactone (diolide).Purification step
Total
volume
Protein
Lactonizing
Specific
activity
activity
ml
mg
units
unitslmg
-fold
%
Purification
Yield
Crude
36.5
1717.7
108.6
0.063
1
100
Isoelectric focusing
3.6
107.5
47.6
0.422
7
44
Sepharose CL-GB
24.6
11.0
93.9
8.56
136
87
HPLC
430.5
5.5
172.2
31.30
497
158
I
I1
I11
FIG. 1. Lactonization reaction catalyzed by lipase
from
TABLE 111
111
Acknowledgments-We express our thanks to Nagase Biochemicals Ltd. for providing Pseudomonas nov. sp. 109, and also to Dr. H.Kataoka of the Department of Agricultural Chemistry, the University of Tokyo, for performing amino acid sequence analysis.
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3,492,011 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineSynregistic Potential of Gold Nanoparticles and Sorafenib Asian Pac
Department of Cancer Biology
National Cancer Institute (NCI)
Department of Medical Applications
National Institute of Laser-Enhanced Science
Cairo University
Giza
Department of Biochemistry
Faculty of Science
Ain Shams University
Cairo
Synregistic Potential of Gold Nanoparticles and Sorafenib Asian Pac
Asian Pacific Journal of Cancer Prevention
201110.31557/APJCP.2019.20.11.3369
Introduction
Haidy M Ebrahim 1 , Mahmoud N El-Rouby 1 , Mona E Morsy 2 , Mahmoud M Said 3 *, Magda K Ezz 3 regulation, chemotherapy and drug delivery, as well as in optical and electronic applications (Uboldi et al., 2009;Saw et al., 2018). Plasmonic photothermal therapy (PPTT) is a non-invasive technique for the treatment of cancer, where laser irradiation causes oscillation of electrons in the conduction band of plasmonic nanoparticles, thus simultaneously absorbing and scattering the laser light and resulting in absorbed light conversion to heat that kills cancer cells (hyperthermia) through either activation of intrinsic or extrinsic apoptotic pathways (Bonzon et al., 2006;Ahmed et al., 2015;Wang et al., 2018).
Sorafenib, an oral multikinase inhibitor drug with anti-proliferative and anti-angiogenic effects, has been approved by the US Food and Drug Administration (FDA) as a unique target drug for advanced HCC (Parsons et al., 2017). Although it represents a much-required treatment decision for HCC patients, it produces toxicities that affect patients' quality of life (Morisaki et al., 2013).
The current study aimed at investigating the cytotoxic effect of sorafenib combination with laser-irradiated AuNPs against HepG2 cells growth, as to explore some of the underlying mechanisms that contribute to resulting cytotoxicity.
Materials and Methods
Chemicals | semantic_scholar |
3,492,012 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineMaterials and Methods
Chemicals
Sorafenib was obtained in a pure powder form from Santa Cruz Biotechnology (TX, USA). Chlorauric acid (HAuCl 4 ), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), absolute methanol, trisodium citrate dihydrate (Na 3 C 6 H 5 O 7 .2H 2 O) and phosphate-buffered saline (PBS, pH 7.2) were provided from Sigma-Aldrich (Darmstadt, Germany). RPMI-1640 cell culture medium, enriched with L-glutamine and fetal bovine serum (FBS), was purchased from Gibco ® (Thermo Fisher Scientific, UK), whereas penicillin-streptomycin (1×) and trypsin/ EDTA (1×) were provided by Biowest ® (South Africa).
Cell line
A hepatocellular carcinoma HepG2 cell line (ATCC ® HB-8065™) was used throughout this study. Cells were grown in RPMI-1640 L-glutamine medium enriched with 10% FBS and penicillin-streptomycin antibiotic in a humidified 5% CO 2 incubator at 37ºC. Sub-culturing was routinely carried out twice a week using trypsin/EDTA.
Preparation and characterization of gold nanoparticles (AuNPs) | semantic_scholar |
3,492,013 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineSpherical gold nanoparticles (AuNPs) were chemically prepared in an aqueous medium by citrate reduction of HAuCl 4 .3H 2 O, where sodium citrate serves also as a capping material to prevent aggregation and further growth of particles (Ojea-Jiménez et al., 2010). Briefly, a volume of 2 ml 1% trisodium citrate solution was added quickly to 50 ml boiling 1 mM HAuCl 4 solution. The color of the solution changed from yellow to black and finally to red, which was considered as an indication for the formation of AuNPs. The boiling was continued for an additional 10 min, then the heater was turned off and the solution was stirred for 30 min. The colloidal gold nanoparticles solution was stored in a dark bottle at room temperature. The absorption spectrum of prepared gold nanoparticles was measured using a T80+UV/Vis Spectrophotometer (PG Instruments Ltd, England), whereas AuNPs shape and size were characterized using Transmission Electron Microscopy (JEM-2100 LaB6, JOEL, MA, USA).
Compounds preparation
Sorafenib was dissolved in DMSO at 10 mmol/L concentration and sterilized before use by filtration through 0.22 µm filters (Millipore ® , Merck, Germany) to physically remove solution-suspended bacteria. The following concentrations (5, 10, 20, 40 and 80 µmol/L) were prepared by diluting in a complete RPMI-1640 cell culture medium. In addition, five ascending concentrations of AuNPs (50,100,150,200 and 250 µmol/L) were prepared by diluting a stock AuNPs solution in a complete RPMI-1640 medium.
Cytotoxicity assay | semantic_scholar |
3,492,014 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineCytotoxicity assay
A number of 10×10 3 HepG2 cells per well was seeded in a 96-well tissue culture plate containing complete RPMI-1640 growth medium and allowed to attach for 24 hrs in an incubator at 37ºC. After cell attachment, the culture medium was aspirated and replaced with 200 µl of fresh complete growth medium containing different sorafenib and AuNPs concentrations (3 wells per dose) and allowed to grow for 48 hrs in a humidified 5% CO 2 . The cell viability was measured using MTT assay. Briefly, a volume of 20 µl MTT (5 mg/ml in PBS) was added to each well and the plate was incubated at 37ºC for 3 hrs. After a careful aspiration of the culture medium, a volume of 100 µl DMSO was added to each well to elaborate the formazan crystal and the plate was left to stand for 1 hr, then the absorbance was read at 570 nm against blank (DMSO). The percentage of cell viability was calculated by multiplying sample ratio absorbance versus the control by 100. Sorafenib and AuNPs median inhibition concentrations (IC 50 ) against the growth of HepG2 cells were then determined (Marks et al., 1992).
Laser irradiation source
Continuous-wave (CW) laser irradiation of AuNPs was done using a low power diode laser (532 nm DPSS laser-LSR-PS-1, Lasever Inc., China). The laser spot size was 1 cm 2 . Samples were irradiated with 80 mW and the resulted energy was 75 J/cm 2 for each sample.
Study design | semantic_scholar |
3,492,015 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineStudy design
HepG2 cells were left either intact without any treatment (Control), treated with sorafenib (Sora; 5 µmol/L), gold nanoparticles (AuNPs; 100 µmol/L), or both compounds (Sora+AuNPs), or irradiated with a single diode laser dose (75 J/cm 2 ) then incubated for 48 hrs at 37ºC in a humidified 5% CO 2 incubator. In addition, HepG2 cells were first incubated with AuNPs for 24 hrs, then exposed to a single diode laser dose (75 J/cm 2 ) (AuNPs+Laser) and incubated for another 24 hrs at 37ºC in a humidified 5% CO 2 incubator. Finally, HepG2 cells were treated with sorafenib and AuNPs, then irradiated after a 24-hr incubation with a single diode laser dose (75 J/cm 2 ) (Sora+AuNPs+Laser) and further incubated for an additional 24 hr at 37ºC in a humidified 5% CO 2 incubator.
Assay of lactate dehydrogenase level
After 48 hrs of incubation with different treatments, the cultured medium was aspirated from attached HepG2 cells, and then centrifuged at 200×g for 5 min. The supernatant was used for the immediate analysis of lactate dehydrogenase (LDH) concentration using an immunoassay kit provided by Mybiosource (SD, USA).
Flow cytometry | semantic_scholar |
3,492,016 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineFlow cytometry
HepG2 cells incubated with different treatments were washed with PBS, trypsinized and collected in a fresh medium containing 10% FBS into a tube that was centrifuged at 200×g for 5 min at 4ºC. The supernatant was discarded and the cell pellets were washed with PBS, then centrifuged for 5 min at 500×g at 4ºC for flow cytometry analysis using ANNEXIN V-FITC using Graphpad Prism version 5.0 software (Graphpad Software Inc., San Diego, USA). The Shapiro-Wilks test for normality (p>0.05) showed that all data was normally distributed (Shapiro and Wilk, 1965). Statistical analysis of difference between means was carried out using one-way analysis of variance (ANOVA). In case of a significant F-ratio, posthoc Duncan's and Bonferroni tests for multiple comparisons were used to evaluate statistical significance between treated groups at p<0.05 level of significance. All statistical analysis was done using Statistical Package for Social Science (SPSS) version 20.0 (SPSS Inc., Chicago, IL, USA).
Results
Characterization of synthesized gold nanoparticles (AuNPs)
Transmission electron microscopy (TEM) of prepared AuNPs using citrate reduction method revealed an average particle size of 19±0.37 nm ( Figure 1A). On the other hand, spectral analysis of synthesized nanoparticles demonstrated an optimum absorption peak at 521 nm ( Figure 1B).
Apoptosis kit (Beckman Coulter, Paris, France). Briefly, cell suspensions were incubated with FITC-conjugated annexin V and propidium iodide in the dark for 15 min on ice, followed by the addition of ice-cold binding buffer. The cell preparations were analyzed within 30 min in a flow cytometer (COULTER ® , EPICS ® , Beckman, USA).
Assay of caspases | semantic_scholar |
3,492,017 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineAssay of caspases
The culture media of HepG2-treated cells were aspirated, then cells were washed once with PBS, and harvested by scraping into a centrifuged falcon tube at 200×g for 3 min. After centrifugation, the PBS was aspirated and the cell pellet was resuspended in 250 µl lysis buffer. Caspases 3 and 8 were determined in HepG2 cell lysate using ELISA kits purchased from eBioscience (SD, USA), whereas caspase 9 level was assayed using a kit provided by IBL International (Hamburg, Germany).
Statistical analysis
The median inhibition concentrations (IC 50 ) of sorafenib and AuNPs against the growth of HepG2 cells were determined by plotting the mean percentage of viability of HepG2 versus logarithmic concentrations and the resulting plot was fitted to a nonlinear regression curve
Cytotoxicity Assay for Sorafenib
Exposure of HepG2 cells for 48 hrs to increasing levels of sorafenib concentration (5-80 µmol/L) gave rise to a dose-dependent significant decrease in cellular growth, as manifested by the significant decrease in MTT absorbance at 570 nm, compared to untreated HepG2 control ( Figure 2A). Furthermore, addition of increasing concentrations of sorafenib in HepG2 cells culture media reduced the percentage of cell viability in a significant dose-dependent manner, where a complete inhibition of cell viability (99.93%) was obtained at a sorafenib concentration of 80 µmol/L ( Figure 2B). The half inhibition concentration (IC 50 ) following incubation of HepG2 cells with different concentrations of sorafenib for 48 hrs was found to be 8.74 μmol/L ( Figure 2C).
Cytotoxicity Assay for Synthesized Gold Nanoparticles | semantic_scholar |
3,492,018 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineExposure of HepG2 cells for 48 hrs to increasing levels of AuNPs concentration (50-250 µmol/L) gave rise to a dose-dependent significant decrease in cellular growth, as manifested by the significant decrease in MTT absorbance at all AuNPs concentrations, compared to untreated HepG2 control ( Figure 3A). Furthermore, addition of increasing concentrations of AuNPs in HepG2 cells culture media reduced the percentage of cell viability in a significant dose-dependent manner, where the highest AuNPs dose (250 μmol/L) recorded the greatest cytotoxicity on HepG2 cells (68.85%) ( Figure 3B). The half inhibition concentration (IC 50 ) following incubation of HepG2 cells with different concentrations of AuNPs for 48 hrs was found to be 176.6 μmol/L ( Figure 3C).
Cytotoxicity assay of combined treatments
A significant decrease in HepG2 cellular growth was noticed following exposure to sorafenib and/or AuNPs, laser-irradiated AuNPs either alone or combined with sorafenib, as evidenced by significant decrease in MTT absorbance, compared to untreated HepG2 cells ( Figure 4A). Similarly, a parallel inhibition in HepG2 viability percentage was recorded following treatment with sorafenib (5 μmol/L) or AuNPs (100 μmol/L). On the other hand, a more pronounced decrease in the percentage of HepG2 cell viability was demonstrated after treatment of HepG2 cells with AuNPs and sorafenib or laser-irradiated AuNPs. The most notable reduction in HepG2 viability was recorded following treatment of HepG2 cells with sorafenib and laser-irradiated AuNPs ( Figure 4B).
Effect of different treatments on lactate dehydrogenase level | semantic_scholar |
3,492,019 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineTreatment of HepG2 cells with sorafenib or AuNPs induced a marked cytotoxicity as demonstrated by the significant increase in LDH level in the culture media supernatant (142.86 and 118.56%, respectively), compared to untreated HepG2 cells. On the other hand, treatment of HepG2 cells with AuNPs along with sorafenib or laser irradiation exaggerated cytotoxicity and LDH release (357.14 and 214.28%, respectively), compared to untreated HepG2 cells. The combination of sorafenib with laser-irradiated AuNPs caused the most pronounced significant increase in LDH level (644.28%), compared to untreated HepG2 cells ( Figure 5).
Effect of different treatments on caspase 3, 8 and 9 levels
Treatment of HepG2 cells with sorafenib or AuNPs induced apoptosis as manifested by the significant increase in caspase-3, -8 and -9 levels in HepG2 cell lysates (48.51 & 76.89%, 140.65 & 130.32%, 58.82 & 85.62%, respectively), compared to untreated HepG2 cells. On the other hand, treatment of HepG2 cells with AuNPs, along with sorafenib or laser irradiation, caused a marked increase in the concentration of caspase-3 (270.95 and 185.15%, respectively), caspase-8 (448.39 and 308.39%, respectively) and caspase-9 (301.96 and 158.17%, respectively), compared to untreated HepG2 cells. Interestingly, the combination of sorafenib treatment with laser-irradiated AuNPs induced the outmost apoptotic insult, where the highest significant increase in caspase-3, -8 and -9 levels (452.14, 583.87 and 404.25%, respectively) were recorded, compared to untreated HepG2 cells ( Figure 6A-C).
Flow cytometric analysis of HepG2-treated cells | semantic_scholar |
3,492,020 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineTreatment of HepG2 cells with sorafenib or AuNPs resulted in a slight significant decrease in viable cells percentage (8.4 and 6.09%, respectively), and by contrast the early apoptotic cells percentage was significantly increased (232.29 and 165.44%, respectively), compared to untreated cells. On the other hand, treatment of HepG2 cells with AuNPs and sorafenib or laser-irradiated AuNPs caused a marked decrease in viable cells percentage (36.53 and 22.17%, respectively), whereas the percentage of early apoptotic cells showed significant elevations (991.5 and 584.70%, respectively), as well as the percentage of late apoptosis for AuNPs and laser-treated HepG2 cells (265%), compared to untreated HepG2 cells. In addition, the combination of sorafenib treatment with laser-irradiated AuNPs produced the most pronounced significant increase not only in early apoptotic cells percentage (1256%) but also in late apoptotic cells percentage (7850%), compared to untreated HepG2 cells (Figures 7 and 8A-C).
Discussion
HCC remains one of the challenging health problems in the world (Morisaki et al., 2013). Therapeutic options in advanced irresectable HCC are limited to sorafenib, which is the available conventional chemotherapy for HCC treatment. However, the acquired resistance to sorafenib, in addition to its severe side effects and toxicity, limits its beneficial effects (Loutfy et al., 2015;Abdel Hamid et al., 2018;Raoul et al., 2018). Therefore, the current study aimed at minimizing the toxic effects of sorafenib through investigating additive or synergistic potential of photothermal therapy (PTT) by using laser-irradiated gold nanoparticles. | semantic_scholar |
3,492,021 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineIn agreement with the current finding, a cytotoxic effect for non-irradiated AuNPs (Paino et al., 2012) or sorafenib (Cervello et al., 2012;Wei et al., 2015) against HepG2 cells growth had been previously reported. By contrast, Wang et al., (2018) reported a little damage to HepG2 cells incubated with AuNPs. The cytotoxic effect of AuNPs prepared using citrate method may be due to particles surface coating acidic nature (Vijayakumar and Ganesan, 2012). One of the most common approaches to measure cell viability and cell membrane integrity is based on substances leakage; such as lactate dehydrogenase (LDH) that normally reside inside cells, to the external environment and its subsequent release in extracellular media (Fard et al., 2015). A cytotoxic effect for different treatments against HepG2 cells viability was demonstrated in the current study, and the most notable reduction in viability, along with highest significant release in LDH level, was recorded following treatment of HepG2 cells with sorafenib and laser-irradiated AuNPs.
Reactive oxygen species (ROS) are critical signaling molecules that regulate many signal transduction pathways, and excessive generation of ROS may interfere with cellular signaling pathways and activate subsequent apoptotic and autophagy processes (Kaminskyy and Zhivotovsky, 2014). Sorafenib has been reported to stimulate ROS production and induce caspase-dependent cell apoptosis, inhibit the activation of Bcl-2 family members, NF-κβ, AKT, leading to the inhibition of cell growth and proliferation via PI3K/AKT/mTOR and ERK signaling pathways (Huang et al., 2010a;Fecteau et al., 2012;Park et al., 2014;Pal et al., 2015). | semantic_scholar |
3,492,022 | The Synergistic Cytotoxic Effect of Laser-Irradiated Gold Nanoparticles and Sorafenib Against the Growth of a Human Hepatocellular Carcinoma Cell LineFurthermore, Huang et al., (2016) reported that AuNPs synthesized by citrate reduction method of HAuCl 4 induced cytotoxicity and ROS-independent mitochondrial apoptosis in rabbit articular chondrocyte primary cultures. Among the cytotoxic mechanisms of nanoparticles (NPs) is the formation of free radicals and ROS generation. In fact, oxidative stress occurs as a part of cellular responses to NPs that result from oxidant-generating properties of NPs themselves as well as their ability to stimulate generation of ROS because of their surfaces interaction with biological system (Fard et al., 2015).
Hyperthermia, in combination with lower chemotherapeutic or radioactive agents, can substitute the usage of large toxic doses from the latter treatment modalities. This is the reason that strived us to use sorafenib and AuNPs at doses lower than their calculated IC 50 in the current study. Hyperthermia is connected to cell death with three mechanisms, cell apoptosis, necrosis and necroptosis, which is a type of programmed necrosis (Jaque et al., 2014;Mouratidis et al., 2015). Cellular apoptosis takes place when the heating temperatures range from 41ºC to 47ºC. Excessive cell necrosis occurs by heat shock for temperatures usually higher than 50ºC, producing a much quicker cell death than apoptosis and is based on protein denaturing (Cherukuri et al., 2010). | semantic_scholar |
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