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Protein and Signaling Pathway Responses to rhIL-6 Intervention before Lobaplatin Treatment in Osteosarcoma Cells using Quantitative Phosphoproteome Analysis

ABSTRACT: IL-6 plays a pivotal role in the process of drug resistance to kinds of chemotherapeutics including lobaplatin. However, the underlying changes of phosphoproteins and related signaling pathways in the posttranslational modification level is still uncover. In our study, a quantitative phosphoproteome analysis of the responses to rhIL-6 & lobaplatin treatment was performed in osteosarcoma cells. Cells were divided into three goups: the control group, the lobaplatin group, and the rhIL-6 & lobaplatin group. Three biological replicates were performed in each group. A total of 3373 proteins and 12183 peptides was identified and quantified, of which 3232 phosphorylated proteins and 11358 phosphorylated peptides were obtained. Between the rhIL-6 & lobaplatin group and the lobaplatin group, twenty-three statistically differentially expressed phosphorylated peptides were identified. The characteristics of differentially expressed phophoproteins were analyzed by GO and KEGG enrichment, kinases of the phosphoproteins were predicted and protein-protein interaction was illustrated/demonstrated using STRING. Conservative motifs that surround the phosphorylated serine, threonine and tyrosine residues were analyzed using the Motif-x algorithm. Western blotting were performed to verify the differentially expressed phosphorylated FLNC and the signaling pathway it involved in. Immunohistochemistry staining showed that p-FLNC and its kinase AKT1 as well as ERK1/2 are positively expressed in the platinum-resistant specimens, while these proteins are negative or weak in those of platinum-sensitive specimens. In conclusion, p-FLNC and the MAPK signaling pathway plays a crucial role in the process of IL-6 induced chemoresistance in osteosarcoma. It is the first phosphoproteomic study which reveals the signature of IL-6 intervention before lobaplatin treatment in human osteosarcoma cells.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Bone Cancer

SUBMITTER: Qiong Ma

LAB HEAD: Qiong Ma

PROVIDER: PXD025970 | Pride | 2022-02-17

REPOSITORIES: Pride

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Quantitative phosphoproteomic analysis reveals chemoresistance-related proteins and signaling pathways induced by rhIL-6 in human osteosarcoma cells.

Zhang Rui R Wang Huan H Li Erliang E Wu Yonghong Y Wen Yanhua Y Li Chenyu C Liao Bo B Ma Qiong Q

Cancer cell international 20211030 1

<h4>Background</h4>IL-6 plays a pivotal role in resistance to chemotherapeutics, including lobaplatin. However, the underlying mechanisms are still unclear. This study was to investigate the changes in phosphoproteins and their related signaling pathways in the process of IL-6-induced chemoresistance to lobaplain in osteosarcoma cells.<h4>Methods</h4>We performed a quantitative phosphoproteomic analysis of the response of SaOS-2 osteosarcoma cells to recombinant human IL-6 (rhIL-6) intervention ...[more]

PMID: 34717622

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Project description:Lobaplatin is a third-generation platinum-based antineoplastic agent and is widely used for osteosarcoma treatment before and after tumor removal. However, treatment failure often results from lobaplatin drug resistance. In our study, we found that SaOS-2 and SOSP-9607 osteosarcoma cells became less sensitive to lobaplatin after treatment with exogenous interleukin (IL)-6. Quantitative proteomic analysis was performed to elucidate the underlying mechanism in SaOS-2 osteosarcoma cells. Cells were divided into a control group (CG), a lobaplatin treatment group (LG) and a recombinant human IL-6 (rhIL-6) and lobaplatin group (rhILG). We performed three biological replicates in each group to compare the differential protein expression between groups using tandem mass tag (TMT) labeling technology based on liquid chromatography–tandem mass spectrometry (LC-MS/MS). A total of 1,313 proteins with significantly differential expression were identified and quantified. The general characterizations of significantly enriched proteins were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and protein-protein interaction (PPI) analysis was conducted using IntAct and STRING. In total, 31 proteins were further verified by parallel reaction monitoring (PRM), in which Ras GTPase-activating protein-binding protein 1 (G3BP1), fragile X mental retardation syndrome-related protein 1 (hFXR1p) and far upstream element-binding protein 1 (FUBP1) were significantly differentially expressed. Immunohistochemistry results showed that these three proteins are highly expressed in specimens of platinum-resistant osteosarcoma patients, while the proteins are negatively or weakly expressed in specimens of platinum-sensitive osteosarcoma patients. The results of immunofluorescence staining were in accordance with those of immunohistochemistry staining. This is the first proteomic study on rhIL-6 intervention before lobaplatin treatment in osteosarcoma cells.

Project description:To elucidate the effect of the polyphenols contained in alcoholic beverages on the metabolic stress induced by ethanol consumption, four groups of mice were fed for five weeks on Lieber's diet with or without ethanol, with ethanol plus ellagic acid, and with ethanol plus trans-resveratrol. Alcoholic fatty liver was observed in the group fed the ethanol diet but not in those fed the ethanol plus polyphenol diets. Liver transcriptome analysis revealed that the addition of the polyphenols suppressed the expression of the genes related to cell stress that were up-regulated by ethanol alone. Conversely, the polyphenols up-regulated the genes involved in bile acid synthesis, unsaturated fatty acid elongation, and tetrahydrofolate synthesis that were down-regulated by ethanol alone. Because parts of these genes were known to be regulated by the constitutive androstane receptor (CAR), we performed the same experiment in the CAR-deficient mice. As a result, fatty liver was observed not only in the ethanol group but also with the ethanol plus polyphenol groups. In addition, there was no segregation of the gene expression profiles among these groups. These results provide a molecular basis for the prevention of alcohol-induced stress by the polyphenols in alcoholic beverages. Five-week-old C3H/HeN female mice (CLEA, Japan) were acclimated to the maintenance condition (25Â°C, 8:00-20:00 day / 20:00-8:00 night cycle and 35~40 % humidity), fed a CE-2 diet (CLEA, Japan), and given water ad libitum for one week. Each group of mice (n=4 for wild type mice analysis and n=3 for CAR decficient mice analysis) was fed Lieber's isocaloric diet (Oriental yeast, Japan) containing water, containing ethanol, containing ethanol and ellagic acid (Fluka Biochemika, Switzerland), or containing ethanol and trans-resveratrol (Sigma, USA) (Supplementary Table 1) for one week at 10:00 ad libitum. Then, the mice were fed each diet at 12 g / day for four weeks (Supplementary Fig. 1A). The approximate intake of each polyphenol was 50 mg / kg body weight / day. At 10:00 of the final day of the experimental period, the animals were anesthetized by diethyl ether, sacrificed by cervial fracture, and the heart blood, and the liver were collected.

Project description:Gene expression analyses of three-week old soil grown wild-type (Col-3) and apum23-1 mutant. Expression profiling was conducted as an attempt to identify the genes differentially regulated in apum23-1 mutant This study was under taken to characterize a member of pumilio family, APUM23 in Arabidopsis. Pumilio, an RNA-binding protein (RBP) containing tandem repeat PUF domains, has been known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their roles in plants are unknown. We detail the characterization of an Arabidopsis Pumilio gene, APUM23. APUM23 is constitutively expressed with higher level in metabolically active tissues, and upregulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth with serrated and scrunched leaves, abnormal venation pattern, and distorted organization of the palisade parenchyma cells, a phenotype reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on the localization, rRNA processing was examined in apum23 mutant. In apum23, 35S pre-rRNA and unprocessed 18S and 5.8S poly(A) rRNAs were accumulated without affecting steady-state levels of mature rRNAs, indicating that APUM23 participates in the degradation of rRNA by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 snoRNAs, and accumulated RNAs within nucleolus. Our results suggest that APUM23 plays an important role in plant development via rRNA processing and ribosome biogenesis. To identify the genes affected in apum23-1 as compared to wild-type (Col-3) under normal growth conditions using Arabidopsis ATH1 Genome arrays (Affymetrix). RNA samples from these plants were used to generate biotin labelled (cDNA) probes, which were then hybridized to the microarray. For data set generation, three independent samples were used, three from Col-3 and three from apum23-1 plants grown on soil for 21 days.

Project description:Supplement S1: Figure S1. (a) Effect of culture age of Euglena gracilis on gene expression, determined with gene arrays analysis. Total number of differentially expressed genes classified into groups of fold-change-ranges in Euglena gracilis cells of different culture age (5 d, 9 d, 11 d) compared to culture age of six days. Underlying data are presented under the figure or in “FC in classes”, respectively. The total number of investigated genes on microarray was n = 20296. Data were retrieved from four independent cell cultures. (b) Effect of culture age of Euglena gracilis on gene expression. Fraction of differentially expressed genes in percent of all investigated transcripts [n = 20,396] classified into groups of fold-change-ranges in Euglena gracilis cells of different culture age (5 d, 9 d, 11 d) compared to culture age of six days. Underlying data are presented under the figure or in “FC in classes”, respectively. For more details, please see the legend Figure S1 a. (c) Effect of culture age of Euglena gracilis on gene expression. Fraction differentially expressed genes (DEGs) above (“upregulation”), below (“downregulation”) or above AND below (“all”), respectively a certain threshold (th). In contrast to (b), where the fold changes are provided in classes, all DEGs above, below, or above AND below as certain threshold are summarized. In addition, Supplement S1 contains all raw data about all DEGs detected and a rankling of the genes regarding their fold changes (FCs). Supplement S2: Determination of differential gene expression with respect to culture age in Euglena gracilis, obtained with micro-arrays. The table “Overlapping DEGs” contains all transcripts, which were found to be differentially expressed in Euglena gracilis at all three investigated days of culture age (5, 9, or 11 days, respectively), against the 6-day samples. In the table “Overlapping DEGs ranked”, the data are ranked according to their fold change. Supplement S3: Transcription of Euglena gracilis cells of different culture age (5, 9, and 11 d, respectively) was compared with the transcription of cells after 6 days of culturing. All data were obtained in quadruplicate. This supplementary data show the possible functions of the proteins encoded by the DEGs (differentially expressed genes), which were differentially expressed for the corresponding days. Table “Diff transcripts funct.” gives a general overview about gene expression changes for all days. The other tables list the possible function of the DEGs for each particular day. The lists only contain genes where a function could be assigned. Complete lists of DEGs also including transcripts with unknown function are provided in Supplement S1. Supplement S4: Determination of gene expression changes with respect to culture age in Euglena gracilis samples of cells were analyzed after 5 d, 9 d, and 11 days of growth in batch cultures, and their gene expression was compared with the gene expression of cells with a culture age of 5 d. For each day, the number of differentially expressed genes (DEGs) each showing the same GO terms were summed up. In order to correlate these changes with the number of all genes with the same GO term in Euglena gracilis, data from sequencing projects were employed [1,2]. Genes with the same GO terms were counted and correlated with the number of observed DEGs in Euglena gracilis. In the first table, GO terms of DEGs were assigned to corresponding days of culturing and correlated with the data of [1, 2] (total numbers and ratios). Diagram “Ratio of DEGs compared to Cordoba et al. [%]” visualizes the ratio of DEGs with total data of [7]. The second diagram shows the number of GO terms of DEGs, which were found at day 9 AND day 11. The change between the two days is provided. Data are visualized in the diagram “Fraction of DEGs of a certain GO over time”. Ref. [2]: Additional file 2: Table S1 “proteome”. Ref. [1]: Supplementary Data, Table S2. Supplement S5: Amino acid sequences of proteins, which were found to become differentially expressed with respect to culture age of Euglena gracilis cultures. [1] Cordoba, J.; Perez, E.; Van Vlierberghe, M.; Bertrand, A.R.; Lupo, V.; Cardol, P.; Baurain, D. De novo transcriptome meta-assembly of the mixotrophic freshwater microalga Euglena gracilis. Genes 2021, 12, 842. https://doi.org/10.3390/genes12060842. [2] Ebenezer, T.E.; Zoltner, M.; Burrell, A.; Nenarokova, A.; Novák Vanclová, A.M.G.; Prasad, B.; Soukal, P.; Santana-Molina, C.; O’Neill, E.; Nankissoor, N.N.; et al. Transcriptome, proteome and draft genome of Euglena gracilis. BMC Biol. 2019, 17, 11. https://doi.org/10.1186/s12915-019-0626-8.

Project description:Euglena gracilis is a unicellular freshwater flagellate, which uses the gravitational vector for orientation in the water column in the dark. This allows the cell to reach areas in the water column for reproduction and growth. How exactly the gravitational vector is perceived, and which intracellular pathways are involved in the signaling is not very well understood so far. In the past, parabolic flight campaigns were used to study the swimming behavior of Euglena gracilis under altered gravitational accelerations. It was shown that cells adapt their swimming direction very fast: in the dark under 1xg cell show negative gravitaxis, i.e. they move upwards in the water column of the experiment hardware and against the gravitational vector. With onset of the first hypergravity period of 1.8xg the precision of upward swimming increases slightly. This first hyper gravity lasts for only 20 seconds and is followed by 22 sec of microgravity. During this period no gravitational vector is perceived by the cells, therefore they lack a cue for orientation and move randomly in all direction. In the subsequent hyper gravity period of 1.8xg, which also lasts for 20 sec, cells direct their movement again and swim upwards. Over different parabolic flight campaigns and other experiments it was shown that this gravitactic behavior is linked to changes in membrane potential, calcium and cAMP concentration. However, due to the lack of genomic and transcriptomic data, it was so far not possible to link the differential movement to the abundance of distinct mRNA transcripts. In contrast, other model organisms, such as Arabidopsis thaliana, have been analyzed by means of gene expression with respect to the effects of altered gravitational accelerations. Also various human cell lines have shown to adapt their gene expression in dependence of the prevailing acceleration. With the recently published Euglena gracilis transcriptome, we now aimed at analyzing effects of altered acceleration on the gene expression in the flagellate. Therefore, Euglena gracilis samples were taken in the course of the 29th DLR parabolic flight campaign during parabola 1 and 31 (time difference of 2 hours and 30 additional parabolas). During both parabolas samples were fixed with TRIzol at 1xg just before onset of the first hyper gravity period, 20 sec into hyper gravity (1.8xg), 20 sec into microgravity (µg) and 20 sec into the last hypergravity period.

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