Alendronate-anchored PEGylation Of Ceria Nanoparticles Promotes Human Hepatoma Cell Proliferation Via AKT/ERk Signaling Pathways

Background:Previous studies have shown that ceria nanoparticles(CNPs)are of much interest due to their regenerative antioxidant property;this characteristic has encouraged researchers to consider CNPs as therapeutic agents to treat a number of diseases,including cancer.Recent studies have shown CNPs to be toxic to cancer cells,to inhibit invasion and to sensitize cancer cells to radiotherapy.In addition,several hydrophilic polymers have been used in attempts to form CNP surface coatings that enhance the CNP properties of extensive biocompatibility and systemic non-toxicity to normal cells and tissues.However,the results of previous studies are based on high CNP doses(e.g.,10 μg/ml or more),and these doses may cause serious side effects in future clinical applications.However,the impact of low CNP doses on tumor cells remains unknown.In this study,we found that CNPs-AL-PEG600,a type of surface-modified CNP that is more stable and less toxic than traditional CNPs,could promote hepatoma cell proliferation in a dose-dependent manner.In addition,further research showed that a low dose(0.01 μg/ml)of CNPs-AL-PEG600 could reduce hepatoma cell apoptosis and activate AKT/ERK signaling pathways.These results may provide information that is important for using CNPs-AL-PEG600 as a therapeutic agent in clinical cancer treatments.Objectives:1.Low concentration of CNPs-AL-PEG600 promoted human hepatoma cell proliferation.2.CNPs-AL-PEG600 promoted human hepatoma cell proliferation via AKT/ERK signaling pathways.Methods:1.Cell culture:The human hepatocellular carcinoma HepG2,Huh7,and SMMC-7721 cell lines,the human osteogenic sarcoma U2 OS cell line,the human breast cancer MCF-7 cell line,and the human colon carcinoma HCT116 cell line were purchased from American Type Culture Collection(ATCC,Manassas,USA)and stored in our laboratory.All these cells were cultured in Dulbecco’s modified Eagle medium(DMEM)(HyClone,Logan,UT,USA)containing 10% fetal bovine serum(FBS)(HyClone,Logan,UT,USA)and kept at 37°C in a humidified atmosphere containing 5% CO2.2.Cell proliferation assay:Cell proliferation was assessed using the Cell Counting Kit-8(CCK-8)method.In brief,six types of human cancer cells were cultured with the CNPs-AL-PEG600 at 0,0.005,0.01,0.05,0.1 and 1 μg/ml at 37°Cfor 24 h.Then,CCK-8 was added to each sample and incubated at 37°C for an additional 2 h.The absorbance of each solution was recorded at 450 nm with a Thermo microplate reader.3.Quantitative real-time PCR:Total RNA were extracted from the cells with Trizol reagent(Invitrogen,USA).The RNA concentration and purity were determined spectrophotometrically using the NanoDropND-1000(NanoDrop Technologies,DE).RNA(100ng)was reverse transcribed into cDNA with PrimeScript RT MasterMix(Takara Co.Ltd,Dalian,China)in a 20-μl final reactionvolume,according to the manufacturer’s protocol.The primer sequences(5′to 3′)were as follows: BAX(forward)TCA gg A TgC gTC CAC CAAgAAg,(reverse)TgT gTC CACgg CggC AAT CAT C;BCL-2(forward)ATC gCC CTg Tgg ATg ACT gAg T;(reverse)gCC Agg AgA AAT CAA ACA gAg gC.All the RT-PCR sam-ples were performed using SYBR Green PCR Master Mix SYBR Premix Ex TaqTM II(Takara Co.Ltd,Dalian,China).4.Gene microarray analysis:A microarray analysis was performed using 5 mg of total RNA from sixsamples,including three separate samples of untreated HepG2 cells or HepG2 cells treated with 0.01 μg/ml of CNPs-AL-PEG600.Cells were harvested,washed with cold PBS,centrifuged,mixed with the RNA extraction kit and stored at-80°C.Then,the samples were submitted to Gminix Biological Technology Limited Corp(Shanghai,China)for microarray analysis.5.Gene ontology(GO)and pathway analysis:Gene ontology(GO)and pathway analysis with two-sided Fisher’s exact tests andχ2 tests were used to classify GO categories,and false discovery rates(FDRs)were calculated to correct the P-values.We chose only GOs that had a P-value of <0.01 and a FDR of <0.05.6.Western Blot:HepG2 cells were collected and lysed in RIPA buffer.Protein was loaded onto 10% polyacrylamide gels and transferred to PVDF membranes(Immobilon-P,Millipore).Membranes were blocked for nonspecific antibody binding in 5% nonfat milk and incubated with the corresponding primary and secondary antibodies.AKT,pAKT,ERK and pERK antibodies were obtained from Epitomics(Cell Signal Technology,Danvers,USA).GAPDH antibodies were purchased from Santa Cruz Biotechnology Inc.(Santa Cruz,CA,USA).7.HepG2 nude mice xenograft model:HepG2 cells(1*106)were suspended in 100 μl of PBS and subcutaneously injected into the right flank of 6–7-week-old BALB/c nude mice.Ten mice were chosen,and equal numbers were assigned to two groups(i.e.,n=5 per group).The mice in the treatment group were intraperitoneally injected with CNPs-AL-PEG600 every alternate day at a dose of 0.01 mg/kg body weight for 30 days.Negative control group animals were given PBS intraperitoneally at the same dose.All mice were sacrificed on day 31,after the last drug administration,and the tumors were dissected and weighed.8.Statistical analysis:All data in this article are expressed as the mean ± standard deviation(SD),and analyses were performed using Prism 5.0 software(Graph Pad,San Diego,CA,USA).The independent-sample tests were used to compare two groups,and ANOVA was used for multiple comparisons;differences with P<0.05 were considered statistically significant.Results:1.CNPs-AL-PEG600 promoted human hepatoma cell proliferationin a dose-dependent manner.2.CNPs-AL-PEG600 reduced apoptosis in human hepatoma cells.3.CNPs-AL-PEG600 promoted human hepatoma cell proliferation via AKT/ERK signaling pathways.4.CNPs-AL-PEG600 promoted tumor growth in vivo.Conclusions:1.The results of the CCK-8 assay showed that treatment of cells with CNPs-AL-PEG600(0.01 μg/ml)for 24 h stimulated proliferation.2.the expression of Bax showed little change,the ratio of Bax to Bcl-2 increased significantly,and this suggested that the CNPs-AL-PEG600 reduced apoptosis in human hepatoma cells.3.GO enrichment and pathway analysis showed that the CNPs-AL-PEG600 could activate AKT/ERK signal pathways.4.The results of the western blot showed that CNPs-AL-PEG600 promoted hepatoma cell proliferation by activating AKT/ERK signal pathways.5.10 μg/kg concentration of CNPs-AL-PEG600 should promote tumor growth in vivo.