Possible target-related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ-based and label-free proteomic analysis.

Bufalin (BF) exhibited antiproliferation and antimigration effects on human A549 lung cancer cells. To search its target-related proteins, protein expression profiles of BF-treated and control cells were compared using two quantitative proteomic methods, iTRAQ-based and label-free proteomic analysis. A total of 5428 proteins were identified in iTRAQ-based analysis while 6632 proteins were identified in label-free analysis. The number of common identified proteins of both methods was 4799 proteins. By application of 1.20-fold for upregulated and 0.83-fold for downregulated cutoff values, 273 and 802 differentially expressed proteins were found in iTRAQ-based and label-free analysis, respectively. The number of common differentially expressed proteins of both methods was 45 proteins. Results of bioinformational analysis using Metacore(TM) showed that the two proteomic methods were complementary and both suggested the involvement of oxidative stress and regulation of gene expression in the effects of BF, and fibronectin-related pathway was suggested to be an important pathway affected by BF. Western blotting assay results confirmed BF-induced change in levels of fibronectin and other related proteins. Overexpression of fibronectin by plasmid transfection ameliorated antimigration effects of BF. Results of the present study provided information about possible target-related proteins and signal network of BF.

Bufalin derivative BF211 inhibits proteasome activity in human lung cancer cells in vitro by inhibiting ß1 subunit expression and disrupting proteasome assembly.

AIM: Bufalin is one of the active components in the traditional Chinese medicine ChanSu that is used to treat arrhythmia, inflammation and cancer. BF211 is a bufalin derivative with stronger cytotoxic activity in cancer cells. The aim of this study was to identify the putative target proteins of BF211 and the signaling pathways in cancer cells. METHODS: A549 human lung cancer cells were treated with BF211. A SILAC-based proteomic analysis was used to detect the protein expression profiles of BF211-treated A549 cells. Cellular proteasome activities were examined using fluorogenic peptide substrates, and the binding affinities of BF211 to recombinant proteasome subunit proteins were evaluated using the Biacore assay. The expression levels of proteasome subunits were determined using RT-PCR and Western blotting, and the levels of the integral 26S proteasome were evaluated using native PAGE analysis. RESULTS: The proteomic analysis revealed that 1282 proteins were differentially expressed in BF211-treated A549 cells, and the putative target proteins of BF211 were associated with various cellular functions, including transcription, translation, mRNA splicing, ribosomal protein synthesis and proteasome function. In A549 cells, BF211 (5, 10, and 20 nmol/L) dose-dependently inhibited the enzymatic activities of proteasome. But BF211 displayed a moderate affinity in binding to proteasome ß1 subunit and no binding affinity to the ß2 and ß5 subunits. Moreover, BF211 (0.1, 1, and 10 nmol/L) did not inhibit the proteasome activities in the cell lysates. BF211 (5, 10, and 20 nmol/L) significantly decreased the expression level of proteasome ß1 subunit and the levels of integral 26S proteasome in A549 cells. Similarly, knockdown of the ß1 subunit with siRNA in A549 cells significantly decreased integral 26S proteasome and proteasome activity. CONCLUSION: BF211 inhibits proteasome activity in A549 cells by decreasing ß1 subunit expression and disrupting proteasome assembly.

Carnosol ameliorated cancer cachexia-associated myotube atrophy by targeting P5CS and its downstream pathways.

Introduction: Carnosol exhibited ameliorating effects on muscle atrophy of mice developed cancer cachexia in our previous research. Method: Here, the ameliorating effects of carnosol on the C2C12 myotube atrophy result from simulated cancer cachexia injury, the conditioned medium of the C26 tumor cells or the LLC tumor cells, were observed. To clarify the mechanisms of carnosol, the possible direct target proteins of carnosol were searched using DARTS (drug affinity responsive target stability) assay and then confirmed using CETSA (cellular thermal shift assay). Furthermore, proteomic analysis was used to search its possible indirect target proteins by comparing the protein expression profiles of C2C12 myotubes under treatment of C26 medium, with or without the presence of carnosol. The signal network between the direct and indirect target proteins of carnosol was then constructed. Results: Our results showed that, Delta-1-pyrroline-5-carboxylate synthase (P5CS) might be the direct target protein of carnosol in myotubes. The influence of carnosol on amino acid metabolism downstream of P5CS was confirmed. Carnosol could upregulate the expression of proteins related to glutathione metabolism, anti-oxidant system, and heat shock response. Knockdown of P5CS could also ameliorate myotube atrophy and further enhance the ameliorating effects of carnosol. Discussion: These results suggested that carnosol might ameliorate cancer cachexia-associated myotube atrophy by targeting P5CS and its downstream pathways.

Paraptosis accompanied by autophagy and apoptosis was induced by celastrol, a natural compound with influence on proteasome, ER stress and Hsp90.

In the present study, we found that celastrol, a natural compound with well-known apoptosis-inducing effect, could also induce paraptosis-like cytoplasmic vacuolization in cancer cell lines including HeLa cells, A549 cells and PC-3 cells derived from cervix, lung and prostate, respectively. Further study using HeLa cells indicated that the vacuoles induced by celastrol might be derived from dilation of endoplasmic reticulum. And, in celastrol-treated cells, markers of autophagy such as transformation of microtubule-associated protein 1 light chain 3 (LC3)I to LC3II and LC3 punctates formation were identified. Interestingly, autophagy inhibitors could not interrupt but enhance the induction of cytoplasmic vacuolization. Furthermore, MAPK pathways were activated by celastrol and inhibitors of MEK and p38 pathways could prevent the formation of cytoplasmic vacuolization. Celastrol treatment also induced G2/M cell cycle arrest and apoptosis in HeLa cells. In conclusion, celastrol induced a kind of paraptosis accompanied by autophagy and apoptosis in cancer cells. The coincidence of apoptosis and autophagy together with paraptosis might contribute to the unique characteristics of paraptosis in celastrol-treated cells such as the dependence of paraptosis on MAPK pathways and dynamic change of LC3 proteins. Both paraptosis and apoptosis could contribute to the cell death induced by celastrol while autophagy might serve as a kind of survival mechanism. The potency of celastrol to induce paraptosis, apoptosis and autophagy at the same dose might be related to its capability to affect a variety of pathways including proteasome, ER stress and Hsp90.

Clarifying the signal network of salvianolic acid B using proteomic assay and bioinformatic analysis.

Salvianolic acid B (SB) is a natural compound with protective effect against ischemia-reperfusion heart injury. However, the signal network of SB including both direct target proteins and downstream signal-related proteins has not been clarified. In the present study, epidermal growth factor receptor (EGFR) was predicted to be the most possible direct protein target of SB by INVDOCK, a ligand-protein inverse-docking algorithm. Possible signal-related proteins of SB in H9C2 cells, including both under normal condition and under ischemia-reperfusion injury, were searched using 2-DE analysis. Totally, 14 signal-related proteins were found. Finally, signal network from EGFR to the signal-related proteins was established using bioinformatic analysis. Interestingly, 9 of the 14 signal-related proteins could be included in a network together with EGFR through direct interaction or only one intermediate partner. The signal cascade from EGFR to heat shock protein 27 (HSP27) and mitofilin (IMMT, inner membrane mitochondrial protein) might be the most important cascade. The signal network was certified by measuring the binding affinity of SB to EGFR in vitro, the effect of SB on internalization and phosphorylation of EGFR, the effect of SB on viability and proliferation of H9C2 cells, and the expression of inner membrane mitochondrial protein in the presence of EGFR inhibitor AG 1478.

A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice.

BF211 is a synthetic molecule derived from bufalin (BF). The apoptosis-inducing effect of BF211 was stronger than that of BF while the acute toxicity of BF211 was much lower than that of BF. BF211 exhibited promising concentration-dependent anti-cancer effects in nude mice inoculated with A549 cells in vivo. The growth of A549 tumor xenografts was almost totally blocked by treatment with BF211 at 6 mg/kg. Notably, BF and BF211 exhibited differences in their binding affinity and kinetics to recombinant proteins of the α subunits of Na+/K+-ATPase. Furthermore, there was a difference in the effects of BF or BF211 on inhibiting the activity of porcine cortex Na+/K+-ATPase and in their time-dependent effects on intracellular Ca2+ levels in A549 cells. The time-dependent effects of BF or BF211 on the activation of Src, which was mediated by the Na+/K+-ATPase signalosome, in A549 cells were also different. Both BF and BF211 could induce apoptosis-related cascades, such as activation of caspase-3 and the cleavage of PARP (poly ADP-ribose polymerase) in A549 cells, in a concentration-dependent manner; however, the effects of BF211 on apoptosis-related cascades was stronger than that of BF. The results of the present study supported the importance of binding to the Na+/K+-ATPase α subunits in the mechanism of cardiac steroids and also suggested the possibility of developing new cardiac steroids with a stronger anti-cancer activity and lower toxicity as new anti-cancer agents.

Nano-LC-ESI MS/MS analysis of proteins in dried sea dragon Solenognathus hardwickii and bioinformatic analysis of its protein expression profiling.

The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components of the sea dragon, shotgun proteomic analysis of its protein expression profiling was conducted in the present study. Proteins were extracted from dried sea dragon using a trichloroacetic acid/acetone precipitation method and then separated by SDS-PAGE. The protein bands were cut from the gel and digested by trypsin to generate peptide mixture. The peptide fragments were then analyzed using nano liquid chromatography tandem mass spectrometry (nano-LC-ESI MS/MS). 810 proteins and 1 577 peptides were identified in the dried sea dragon. The identified proteins exhibited molecular weight values ranging from 1 900 to 3 516 900 Da and pI values from 3.8 to 12.18. Bioinformatic analysis was conducted using the DAVID Bioinformatics Resources 6.7 Gene Ontology (GO) analysis tool to explore possible functions of the identified proteins. Ascribed functions of the proteins mainly included intracellular non-membrane-bound organelle, non-membrane-bounded organelle, cytoskeleton, structural molecule activity, calcium ion binding and etc. Furthermore, possible signal networks of the identified proteins were predicted using STRING (Search Tool for the Retrieval of Interacting Genes) database. Ribosomal protein synthesis was found to play an important role in the signal network. The results of this study, to best of our knowledge, were the first to provide a reference proteome profile for the sea dragon, and would aid in the understanding of the expression and functions of the identified proteins.

Knockdown of Apolipoprotein E Enhanced Sensitivity of Hep3B Cells to Cardiac Steroids via Regulating Na+/K+-ATPase Signalosome.

This study compared the sensitivity of human hepatoma Hep3B, SK-HEP-1, SMMC-7721, and BEL-7402 cells to cardiac steroids, including bufalin (BF), a bufalin derivative (BF211), ouabain (OUA), and digitoxin (DIG). Hep3B cells exhibited relatively low sensitivity to cardiac steroids. Expression levels of subunits of Na+/K+-ATPase were high in Hep3B cells. However, colocalization of Na+/K+-ATPase and caveolin was nearly undetectable in Hep3B cells. By using RNA-Seq technology, we found a total of 36 genes to be differentially expressed between Hep3B cells and SK-HEP-1 cells, which are highly sensitive to cardiac steroids. Our bioinformatics analysis determined that these genes were mostly comprised of extracellular space, protein binding, and extracellular region. Among these 36 genes, apolipoprotein E (APOE) played a critical role, as knockdown APOE expression induced colocalization of Na+/K+-ATPase and caveolin and increased sensitivity of Hep3B cells to both proliferation-inhibiting and cytotoxic effects of BF or BF211. Also, the effects of BF on PI3K/AKT/GSK3ß and apoptosis signal cascades were enhanced in APOE knockdown cells. The results of our study confirmed the role of Na+/K+-ATPase signalosome in cytotoxicity of cardiac steroids and suggested that APOE regulated the sensitivity of cells to cardiac steroids by affecting formation and function of Na+/K+-ATPase signalosome. In addition, intercellular interaction with high level of Na+/K+-ATPase ß1 subunit may be also a factor in the low sensitivity of Hep3B cells to cardiac steroids. Mol Cancer Ther; 15(12); 2955-65. ©2016 AACR.

Agglutinin isolated from Arisema heterophyllum Blume induces apoptosis and autophagy in A549 cells through inhibiting PI3K/Akt pathway and inducing ER stress.

Arisaema heterophyllum Blume is one of the three medicinal plants known as traditional Chinese medicine Rhizoma Arisaematis (RA). RA has been popularly used to treat patients with convulsions, inflammation, and cancer for a long time. However, the underlying mechanisms for RA effects are still unclear. The present study was designed to determine the cytotoxicity of agglutinin isolated from Arisema heterophyllum Blume (AHA) and explore the possible mechanisms in human non-small-cell lung cancer A549 cells. AHA with purity up to 95% was isolated and purified from Arisaema heterophyllum Blume using hydrophobic interaction chromatography. AHA dose-dependently inhibited the proliferation of A549 cells and induced G1 phase cell cycle arrest. AHA induced apoptosis by up-regulating pro-apoptotic Bax, decreasing anti-apoptotic Bcl-2, and activating caspase-9 and caspase-3. In A549 cells treated with AHA, the PI3K/Akt pathway was inhibited. Furthermore, AHA induced increase in the levels of ER stress markers such as phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), inositol-requiring enzyme 1α (IRE1α), and phosphorylated c-Jun NH2-terminal kinase (p-JNK). AHA also induced autophagy in A549 cells. Staining of acidic vesicular organelles (AVOs) and increase in the levels of LC3II and ATG7 were observed in AHA-treated cells. These findings suggested that AHA might be one of the active components with anti-cancer effects in Arisaema heterophyllum Blume. In conclusion, cytotoxicity of AHA on cancer cells might be related to its effects on apoptosis and autophagy through inhibition of PI3K/Akt pathway and induction of ER stress.

Inhibition on Proteasome ß1 Subunit Might Contribute to the Anti-Cancer Effects of Fangchinoline in Human Prostate Cancer Cells.

Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome ß1 subunit and also inhibited its activity in vitro. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome ß1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome ß1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome ß1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting ß1 subunit.

Proteomic and bioinformatic analyses of possible target-related proteins of gambogic acid in human breast carcinoma MDA-MB-231 cells.

Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.

Cytotoxicity of 9,11-dehydroergosterol peroxide isolated from Ganoderma lucidum and its target-related proteins.

The cytotoxicty of 9,11-dehydroergosterol peroxide (DHEP) isolated from the fruiting bodies of Ganoderma lucidum on HeLa cells was studied. DHEP treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC50-value of 8.58 +/- 0.98 microM. Morphological changes of DHEP-treated cells indicated that DHEP induced apoptosis in HeLa cells. To identify the cellular targets of DHEP, two-dimensional electrophoresis analysis was performed to compare the protein expression profiles of DHEP-treated cells with that of control cells. Proteins altered in expressional level after DHEP exposure were identified by MALDI-TOF MS/MS. The cytotoxic effect of DHEP was associated with regulated expression of 6 proteins. Stathmin 1 might be an important target-related protein of DHEP. The regulation of stathmin 1 by DHEP treatment was also confirmed by Western blotting.