Anti-Cancer Roles of Probiotic-Derived P8 Protein in Colorectal Cancer Cell Line DLD-1.

A novel probiotics-derived protein, P8, suppresses the growth of colorectal cancer (CRC). P8 can penetrate the cell membrane via endocytosis and cause cell cycle arrest in DLD-1 cells through down-regulation of CDK1/Cyclin B1. However, neither the protein involved in the endocytosis of P8 nor the cell cycle arrest targets of P8 are known. We identified two P8-interacting target proteins [importin subunit alpha-4 (KPNA3) and glycogen synthase kinase-3 beta (GSK3ß)] using P8 as a bait in pull-down assays of DLD-1 cell lysates. Endocytosed P8 in the cytosol was found to bind specifically to GSK3ß, preventing its inactivation by protein kinases AKT/CK1ε/PKA. The subsequent activation of GSK3ß led to strong phosphorylation (S33,37/T41) of ß-catenin, resulting in its subsequent degradation. P8 in the cytosol was also found to be translocated into the nucleus by KPNA3 and importin. In the nucleus, after its release, P8 binds directly to the intron regions of the GSK3ß gene, leading to dysregulation of GSK3ß transcription. GSK3ß is a key protein kinase in Wnt signaling, which controls cell proliferation during CRC development. P8 can result in a cell cycle arrest morphology in CRC cells, even when they are in the Wnt ON signaling state.

Phosphorylation of the kinase domain regulates autophosphorylation of myosin IIIA and its translocation in microvilli.

Motor activity of myosin III is regulated by autophosphorylation. To investigate the role of the kinase activity on the transporter function of myosin IIIA (Myo3A), we identified the phosphorylation sites of kinase domain (KD), which is responsible for the regulation of kinase activity and thus motor function. Using mass spectrometry, we identified six phosphorylation sites in the KD, which are highly conserved among class III myosins and Ste20-related misshapen (Msn) kinases. Two predominant sites, Thr¹84 and Thr¹88, in KD are important for phosphorylation of the KD as well as the motor domain, which regulates the affinity for actin. In the Caco2 cells, the full-length human Myo3A (hMyo3AFull) markedly enlarged the microvilli, although it did not show discrete localization within the microvilli. On the other hand, hMyo3AFull(T184A) and hMyo3AFull(T188A) both showed clear localization at the microvilli tips. Our results suggest that Myo3A induces large actin bundle formation to form microvilli, and phosphorylation of KD at Thr¹84 and Thr¹88 is critical for the kinase activity of Myo3A, and regulation of Myo3A translocation to the tip of microvilli. Retinal extracts potently dephosphorylate both KD and motor domain without IQ motifs (MDIQo), which was inhibited by okadaic acid (OA) with nanomolar range and by tautomycetin (TMC) with micromolar range. The results suggest that Myo3A phosphatase is protein phosphatase type 2A (PP2A). Supporting this result, recombinant PP2Ac potently dephosphorylates both KD and MDIQo. We propose that the phosphorylation-dephosphorylation mechanism plays an essential role in mediating the transport and actin bundle formation and stability functions of hMyo3A.

Toxicological Evaluation of a Probiotic-Based Delivery System for P8 Protein as an Anti-Colorectal Cancer Drug.

PURPOSE: This study aimed to toxicological evaluate a probiotics-based delivery system for p8 protein as an anti-colorectal cancer drug. INTRODUCTION: Lactic acid bacteria (LAB) have been widely ingested for many years and are regarded as very safe. Recently, a Pediococcus pentosaceus SL4 (PP) strain that secretes the probiotic-derived anti-cancer protein P8 (PP-P8) has been developed as an anti-colorectal cancer (CRC) biologic by Cell Biotech. We initially identified a Lactobacillus rhamnosus (LR)-derived anti-cancer protein, P8, that suppresses CRC growth. We also showed that P8 penetrates specifically into CRC cells (DLD-1 cells) through endocytosis. We then confirmed the efficacy of PP-P8, showing that oral administration of this agent significantly decreased tumor mass (~42%) relative to controls in a mouse CRC xenograft model. In terms of molecular mechanism, PP-P8 induces cell-cycle arrest in G2 phase through down-regulation of Cyclin B1 and Cdk1. In this study, we performed in vivo toxicology profiling to obtain evidence that PP-P8 is safe, with the goal of receiving approval for an investigational new drug application (IND). METHODS: Based on gene therapy guidelines of the Ministry of Food and Drug Safety (MFDS) of Korea, the potential undesirable effects of PP-P8 had to be investigated in intact small rodent or marmoset models prior to first-in-human (FIH) administration. The estimated doses of PP-P8 for FIH are 1.0×1010 - 1.0×1011 CFU/person (60 kg). Therefore, to perform toxicological investigations in non-clinical animal models, we orally administered PP-P8 at doses of 3.375 × 1011, 6.75 × 1011, and 13.5×1011 CFU/kg/day; thus the maximum dose was 800-8000-fold higher than the estimated dose for FIH. RESULTS: In our animal models, we observed no adverse effects of PP-P8 on clinicopathologic findings, relative organ weight, or tissue pathology. In addition, we observed no inflammation or ulceration during pathological necropsy. CONCLUSION: These non-clinical toxicology studies could be used to furnish valuable data for the safety certification of PP-P8.

A synthetic probiotic engineered for colorectal cancer therapy modulates gut microbiota.

BACKGROUND: Successful chemoprevention or chemotherapy is achieved through targeted delivery of prophylactic agents during initial phases of carcinogenesis or therapeutic agents to malignant tumors. Bacteria can be used as anticancer agents, but efforts to utilize attenuated pathogenic bacteria suffer from the risk of toxicity or infection. Lactic acid bacteria are safe to eat and often confer health benefits, making them ideal candidates for live vehicles engineered to deliver anticancer drugs. RESULTS: In this study, we developed an effective bacterial drug delivery system for colorectal cancer (CRC) therapy using the lactic acid bacterium Pediococcus pentosaceus. It is equipped with dual gene cassettes driven by a strong inducible promoter that encode the therapeutic protein P8 fused to a secretion signal peptide and a complementation system. In an inducible CRC cell-derived xenograft mouse model, our synthetic probiotic significantly reduced tumor volume and inhibited tumor growth relative to the control. Mice with colitis-associated CRC induced by azoxymethane and dextran sodium sulfate exhibited polyp regression and recovered taxonomic diversity when the engineered bacterium was orally administered. Further, the synthetic probiotic modulated gut microbiota and alleviated the chemically induced dysbiosis. Correlation analysis demonstrated that specific bacterial taxa potentially associated with eubiosis or dysbiosis, such as Akkermansia or Turicibacter, have positive or negative relationships with other microbial members. CONCLUSIONS: Taken together, our work illustrates that an effective and stable synthetic probiotic composed of P. pentosaceus and the P8 therapeutic protein can reduce CRC and contribute to rebiosis, and the validity and feasibility of cell-based designer biopharmaceuticals for both treating CRC and ameliorating impaired microbiota. Video abstract.

MiR-921 directly downregulates GPx3 in A549 lung cancer cells.

Glutathione peroxidase 3 (GPx3), a major antioxidant enzyme in plasma, catalyzes the reduction of H2O2, lipid peroxides and organic hydroperoxides by reducing glutathione (GSH). Hypermethylation of the GPx3 promoter and suppression of GPx3 expression are associated with inflammation, tumorigenesis, and response to chemotherapy in various types of cancer. We previously reported the possibility of GPx3 as a serological marker for lung cancer. In this study, we assessed the role of the microRNA (miRNA) hsa-miR-921 (miR-921) in the regulation of GPx3 expression in A549 lung cancer cells. The expression patterns of the miRNAs of A549, H1650, and H1975 cells were compared and analyzed. Of 25 miRNAs from the A549 cell line, the expression of 10 decreased and the expression of 15 increased in comparison to the miRNAs from the other cell lines. Of the miRNAs with reduced expression, the most reduced miRNA was miR-921 and the expected binding site of which is in the 3'-untranslated region (UTR) of GPx3. We found that miR-921 inhibited the expression of GPx3 and bound directly to the 3'-UTR of GPx3.

Colorectal Cancer Therapy Using a Pediococcus pentosaceus SL4 Drug Delivery System Secreting Lactic Acid Bacteria-Derived Protein p8.

Despite decades of research into colorectal cancer (CRC), there is an ongoing need for treatments that are more effective and safer than those currently available. Lactic acid bacteria (LAB) show beneficial effects in the context of several diseases, including CRC, and are generally regarded as safe. Here, we isolated a Lactobacillus rhamnosus (LR)-derived therapeutic protein, p8, which suppressed CRC proliferation. We found that p8 translocated specifically to the cytosol of DLD-1 cells. Moreover, p8 down-regulated expression of Cyclin B1 and Cdk1, both of which are required for cell cycle progression. We confirmed that p8 exerted strong anti-proliferative activity in a mouse CRC xenograft model. Intraperitoneal injection of recombinant p8 (r-p8) led to a significant reduction (up to 59%) in tumor mass when compared with controls. In recent years, bacterial drug delivery systems (DDSs) have proven to be effective therapeutic agents for acute colitis. Therefore, we aimed to use such systems, particularly LAB, to generate the valuable therapeutic proteins to treat CRC. To this end, we developed a gene expression cassette capable of inducing secretion of large amounts of p8 protein from Pediococcus pentosaceus SL4 (PP). We then confirmed that this protein (PP-p8) exerted anti-proliferative activity in a mouse CRC xenograft model. Oral administration of PP-p8 DDS led to a marked reduction in tumor mass (up to 64%) compared with controls. The PP-p8 DDS using LAB described herein has advantages over other therapeutics; these advantages include improved safety (the protein is a probiotic), cost-free purification, and specific targeting of CRC cells.

Anti-Colorectal Cancer Effects of Probiotic-Derived p8 Protein.

Recently, we reported a novel therapeutic probiotic-derived protein, p8, which has anti-colorectal cancer (anti-CRC) properties. In vitro experiments using a CRC cell line (DLD-1), anti-proliferation activity (about 20%) did not improve after increasing the dose of recombinant-p8 (r-p8) to >10 µM. Here, we show that this was due to the low penetrative efficiency of r-p8 exogenous treatment. Furthermore, we found that r-p8 entered the cytosol through endocytosis, which might be a reason for the low penetration efficiency. Therefore, to improve the therapeutic efficacy of p8, we tried to improve delivery to CRC cells. This resulted in endogenous expression of p8 and increased the anti-proliferative effects by up to 2-fold compared with the exogenous treatment (40 µM). Anti-migration activity also increased markedly. Furthermore, we found that the anti-proliferation activity of p8 was mediated by inhibition of the p53-p21-Cyclin B1/Cdk1 signal pathway, resulting in growth arrest at the G2 phase of the cell cycle. Taken together, these results suggest that p8 is toxic to cancer cells, shows stable expression within cells, and shows strong cancer suppressive activity by inducing cell cycle arrest. Therefore, p8 is a strong candidate for gene therapy if it can be loaded onto cancer-specific viruses.

GPx3-mediated redox signaling arrests the cell cycle and acts as a tumor suppressor in lung cancer cell lines.

Glutathione peroxidase 3 (GPx3), a major scavenger of reactive oxygen species (ROS) in plasma, acts as a redox signal modulator. However, the mechanism underlying GPx3-mediated suppression of cancer cell growth is unclear. The aim of this study was to identify these mechanisms with respect to lung cancer. To enhance the redox modulating properties of GPx3, lung cancer cells were subjected to serum starvation for 12 h, resulting in ROS generation in the absence of oxidant treatment. We then investigated whether suppression of tumorigenesis under conditions of oxidative stress was dependent on GPx3. The results showed that GPx3 effectively suppressed proliferation, migration, and invasion of lung cancer cells under oxidative stress. In addition, GPx3 expression led to a significant reduction in ROS production by cancer cells and induced G2/M phase arrest. We also found that inactivation of cyclin B1 significantly suppressed by nuclear factor-κB(NF-κB) inactivation in lung cancer cells was dependent on GPx3 expression. To further elucidate the mechanism(s) underlying GPx3-medited suppression of tumor proliferation, we next examined the effect of GPx3-mediated redox signaling on the ROS-MKP3-extracellular signal-regulated kinase (Erk)-NF-κB-cyclin B1 pathway and found that GPx3 strongly suppressed activation of the Erk-NF-κB-cyclin B1 signaling cascade by protecting MKP3 (an Erk-specific phosphatase) from the effects of ROS. Thus, this study demonstrates for the first time that the GPx3 suppresses proliferation of lung cancer cells by modulating redox-mediated signals.

Integrative analysis for the discovery of lung cancer serological markers and validation by MRM-MS.

Non-small-cell lung cancer (NSCLC) constitutes approximately 80% of all diagnosed lung cancers, and diagnostic markers detectable in the plasma/serum of NSCLC patients are greatly needed. In this study, we established a pipeline for the discovery of markers using 9 transcriptome datasets from publicly available databases and profiling of six lung cancer cell secretomes. Thirty-one out of 312 proteins that overlapped between two-fold differentially expressed genes and identified cell secretome proteins were detected in the pooled plasma of lung cancer patients. To quantify the candidates in the serum of NSCLC patients, multiple-reaction-monitoring mass spectrometry (MRM-MS) was performed for five candidate biomarkers. Finally, two potential biomarkers (BCHE and GPx3; AUC = 0.713 and 0.673, respectively) and one two-marker panel generated by logistic regression (BCHE/GPx3; AUC = 0.773) were identified. A validation test was performed by ELISA to evaluate the reproducibility of GPx3 and BCHE expression in an independent set of samples (BCHE and GPx3; AUC = 0.630 and 0.759, respectively, BCHE/GPx3 panel; AUC = 0.788). Collectively, these results demonstrate the feasibility of using our pipeline for marker discovery and our MRM-MS platform for verifying potential biomarkers of human diseases.

Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells.

Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7-8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-inflammatory signaling in lung cancer cells.

An additional cysteine in a typical 2-Cys peroxiredoxin of Pseudomonas promotes functional switching between peroxidase and molecular chaperone.

Peroxiredoxins (Prx) have received considerable attention during recent years. This study demonstrates that two typical Pseudomonas-derived 2-Cys Prx proteins, PpPrx and PaPrx can alternatively function as a peroxidase and chaperone. The amino acid sequences of these two Prx proteins exhibit 93% homology, but PpPrx possesses an additional cysteine residue, Cys112, instead of the alanine found in PaPrx. PpPrx predominates with a high molecular weight (HMW) complex and chaperone activity, whereas PaPrx has mainly low molecular weight (LMW) structures and peroxidase activity. Mass spectrometry and structural analyses showed the involvement of Cys112 in the formation of an inter-disulfide bond, the instability of LMW structures, the formation of HMW complexes, and increased hydrophobicity leading to functional switching of Prx proteins between peroxidase and chaperone.

Engineering of 2-Cys peroxiredoxin for enhanced stress-tolerance.

A typical 2-cysteine peroxiredoxin (2-Cys Prx)-like protein (PpPrx) that alternatively acts as a peroxidase or a molecular chaperone in Pseudomonas putida KT2440 was previously characterized. The dual functions of PpPrx are regulated by the existence of an additional Cys(112) between the active Cys(51) and Cys(171) residues. In the present study, additional Cys residues (Cys(31), Cys(112), and Cys(192)) were added to PpPrx variants to improve their enzymatic function. The optimal position of the additional Cys residues for the dual functionality was assessed. The peroxidase activities of the S31C and Y192C mutants were increased 3- to 4-fold compared to the wild-type, while the chaperone activity was maintained at > 66% of PpPrx. To investigate whether optimization of the dual functions could enhance stress-tolerance in vivo, a complementation study was performed. The S31C and Y192C mutants showed a much greater tolerance than other variants under a complex condition of heat and oxidative stresses. The optimized dual functions of PpPrx could be adapted for use in bioengineering systems and industries, such as to develop organisms that are more resistant to extreme environments.

Improvement of chaperone activity of 2-Cys peroxiredoxin using gamma ray.

A typical 2-cysteine peroxiredoxin (2-Cys Prx) PaPrx can act alternatively as thioredoxin (Trx)-dependent peroxidase and molecular chaperone in Pseudomonas aeruginosa PAO1. In addition, the functional switch of PaPrx is regulated by its structural change which is dependently induced by stress conditions. In the present study, we examined the effect of gamma ray on structural modification related to chaperone activity of PaPrx. The structural change of PaPrx occupied with gamma ray irradiation (2 kGy) based on polyacrylamide gel electrophoresis (PAGE) analysis and the functional change also began. The enhanced chaperone activity was increased about 3-4 folds at 30 kGy gamma irradiation compared with nonirradiated PaPrx, while the peroxidase activity was significantly decreased. We also investigated the influence of the gamma ray on protein hydrophobicity as related to chaperone function. The exposure of hydrophobic domains reached a peak at 30 kGy gamma ray and then decreased dependently with increasing gamma irradiation. Our results suggest that highly enhanced chaperone activity could be adapted for use in bio-engineering systems and industrial applications such as enzyme stabilization during industrial process (inactivation protection), improvement of useful protein productivity (refolding and secretion) and industrial animal cell cultivation (stress protection).

Functional switching of a novel prokaryotic 2-Cys peroxiredoxin (PpPrx) under oxidative stress.

Many proteins have been isolated from eukaryotes as redox-sensitive proteins, but whether these proteins are present in prokaryotes is not clear. Redox-sensitive proteins contain disulfide bonds, and their enzymatic activity is modulated by redox in vivo. In the present study, we used thiol affinity purification and mass spectrometry to isolate and identify 19 disulfide-bond-containing proteins in Pseudomonas putida exposed to potential oxidative damages. Among these proteins, we found that a typical 2-Cys Prx-like protein (designated PpPrx) displays diversity in structure and apparent molecular weight (MW) and can act as both a peroxidase and a molecular chaperone. We also identified a regulatory factor involved in this structural and functional switching. Exposure of pseudomonads to hydrogen peroxide (H(2)O(2)) caused the protein structures of PpPrx to convert from high MW complexes to low MW forms, triggering a chaperone-to-peroxidase functional switch. This structural switching was primarily guided by the thioredoxin system. Thus, the peroxidase efficiency of PpPrx is clearly associated with its ability to form distinct protein structures in response to stress.

Global analysis of disulfide bond proteins in Pseudomonas aeruginosa exposed to hydrogen peroxide and gamma rays.

PURPOSE: To isolate and characterise the oxidative stress-responsible disulfide bond proteins (DSBP) from Pseudomonas aeruginosa, under hydrogen peroxide (H(2)O(2)) and gamma ray-induced stress conditions. MATERIALS AND METHODS: Oxidative stress in P. aeruginosa was induced by H(2)O(2) or (60)Co gamma-rays. DSBP of P. aeruginosa were isolated by using a thiol affinity purification method and liquid chromatography-mass spectrometry (LC-MS/MS). Induction of DSBP and their transcriptional levels were analysed by Western blot and quantitative-polymerase chain reaction (q-PCR), respectively. RESULTS: We isolated and identified 40 DSBP in P. aeruginosa. A comparison of DSBP patterns under different stress conditions, including exposure to 0.5 mM H(2)O(2) and 30 Gy of gamma rays, was carried out. We selected 13 DSBP based on their increased intensity under oxidative stress and investigated their expression levels using q-PCR. Of these 13 proteins, P. aeruginosa (PA) 0126 and PA0277 transcriptional levels were strongly induced (4- to 9-fold) by gamma ray. A corresponding induction in the translational levels of PA0126 was confirmed by Western blot analysis. PA0126 was identified is a hypothetical protein with 206 amino acids and contained three cysteine residues. A shift in molecular weight of PA0126 under oxidised/reduced state indicated its homo-oligomeric structure under non-reducing condition. CONCLUSION: PA0126, a hypothetical protein with disulfide bonds was identified as a key responder to oxidative stress along with other known DSBP of P. aeruginosa. We determined that all DSBP underwent a redox cycling. Furthermore, our results give deeper insight into the relationship between oxidative stress and DSBP in P. aeruginosa.

A new antioxidant with dual functions as a peroxidase and chaperone in Pseudomonas aeruginosa.

Thiol-based peroxiredoxins (Prxs) are conserved throughout all kingdoms. We have found that a conserved typical 2-Cys Prx-like protein (PaPrx) from Pseudomonas aeruginosa bacteria displays diversity in its structure and apparent molecular weight (MW), and can act alternatively as a peroxidase and molecular chaperone. We have also identified a regulatory factor involved in this structural and functional switching. Exposure of P. aeruginosa to hydrogen peroxide (H2O2) causes PaPrx to convert from a high MW (HMW) complex to a low MW (LMW) form, which triggers a chaperone to peroxidase functional switch. This structural switching is primarily guided by either the thioredoxin (Trx) or glutathione (GSH) systems. Furthermore, comparison of our structural data [native and non-reducing polyacrylamide gel electrophoresis (PAGE) analysis, size exclusion chromatography (SEC) analysis, and electron microscopy (EM) observations] and enzymatic analyses (peroxidase and chaperone assay) revealed that the formation of oligomeric HMW complex structures increased chaperone activity of PaPrx. These results suggest that multimerization of PaPrx complexes promotes chaperone activity, and dissociation of the complexes into LMW species enhances peroxidase activity. Thus, the dual functions of PaPrx are clearly associated with their ability to form distinct protein structures.

cDNA cloning and expression pattern of cinnamate-4-hydroxylase in the Korean black raspberry.

Cinnamate-4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway, which is responsible for synthesizing a variety of secondary metabolites that participate in development and adaptation. In this study, we isolated a full-length cDNA of the C4H gene from the Korean black raspberry (Rubus sp.) and found that this gene existed as a single gene. By comparing the deduced amino acid sequence of Rubus sp. C4H with other sequences reported previously we determined that this sequence was highly conserved among widely divergent plant species. In addition, quantitative real time PCR studies indicated that the C4H gene had a differential expression pattern during fruit development, where gene expression was first detected in green fruit and was then remarkably reduced in yellow fruit, followed by an increase in red and black fruit. To investigate the two peaks in expression observed during fruit development and ripening, we measured the flavonoid content. The content of the major flavanol of Korean black raspberry fruits was determined to be highest at the beginning of fruit development, followed by a gradually decrease according to the developmental stages. In contrast, the content of anthocyanins during the progress of ripening was dramatically increased. Our results suggest that the C4H gene in Korean black raspberry plays a role during color development at the late stages of fruit ripening, whereas the expression of C4H gene during the early stages may be related to the accumulation of flavanols.