FBPAII and rpoBC, the Two Novel Secreted Proteins Identified by the Proteomic Approach from a Comparative Study between Antibiotic-Sensitive and Antibiotic-Resistant Helicobacter pylori-Associated Gastritis Strains.

Helicobacter pylori infection is the leading cause of chronic gastritis, which can develop into gastric cancer. Eliminating H. pylori infection with antibiotics achieves the prevention of gastric cancer. Currently, the prevalence of H. pylori resistance to clarithromycin and metronidazole, and the dual resistance to metronidazole and clarithromycin (C_R, M_R, and C/M_R, respectively), remains at a high level worldwide. As a means of exploring new candidate proteins for the management of H. pylori infection, secreted proteins from antibiotic-susceptible and antibiotic-resistant H. pylori-associated gastritis strains were obtained by in-solution tryptic digestion coupled with nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS). A total of 583, 582, 590, and 578 differential expressed proteins were identified from C_R, M_R, C/M_R, and antibiotic-sensitive strain (S_S) samples, respectively. Of these, 23 overlapping proteins were found by Venn diagram analysis. Based on heat map analyses, the most and least differing protein expressions were observed from C/M_R strains and S_S strains, respectively. Of the proteins secreted by the S_S strain, only nine were found. After predicting the protein interaction with metronidazole and clarithromycin via the STITCH database, the two most interesting proteins were found to be rpoBC and FBPAII. After quantitative real-time reverse transcription PCR (qRT-PCR) analysis, a downregulation of rpoB from M_R strains was observed, suggesting a relationship of rpoB to metronidazole sensitivity. Inversely, an upregulation of fba from C_R, M_R, and C/M_R strains was noticed, suggesting the paradoxical expression of FBPAII and the fba gene. This report is the first to demonstrate the association of these two novel secreted proteins, namely, rpoBC and FBPAII, with antibiotic-sensitive H. pylori-associated gastritis strains.

mRNA Expression of Bax, Bcl-2, p53, Cathepsin B, Caspase-3 and Caspase-9 in the HepG2 Cell Line Following Induction by a Novel Monoclonal Ab Hep88 mAb: Cross-Talk for Paraptosis and Apoptosis.

Monoclonal antibodies with specific antigens have been widely used as targeted therapy for cancer. Hep88 mAb is a monoclonal antibody which shows specific binding with anti-cancer effects against the HepG2 cell line. However, its mechanisms of action are still not completely understood. We examined cell cycling and apoptosis by flow cytometry and mRNA expression of factors involved in apoptosis and paraptosis in Hep88 mAb-treated HepG2 cells by real-time PCR. The cell-cycle analysis demonstrated that growth-inhibitory activity was associated with G2/M cell cycle arrest. Hep88 mAb induced a significant increase in apoptotic cell populations in a dose- and time-dependent manner. The mRNA expression results also suggested that the process triggered by Hep88 mAb involved up-regulation of tumor suppressor p53, pro-apoptotic Bax, Cathepsin B, Caspase-3 and Caspase-9, with a decrease of anti-apoptotic Bcl-2 - thus confirming paraptosis and apoptosis programmed cell death. These findings represent new insights into the molecular mechanisms underlying the anti-cancer properties of Hep88 mAb in liver cancer cells.

Hep88 mAb-mediated paraptosis-like apoptosis in HepG2 cells via downstream upregulation and activation of caspase-3, caspase-8 and caspase-9.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Presently, targeted therapy via monoclonal antibodies to specific tumor-associated antigens is being continuously developed. Hep88 mAb has proven to exert tumoricidal effects on the HepG2 cell via a paraptosis-like morphology. To verify the pathway, we then demonstrated downstream up-regulation of caspase-3, caspase-8 and caspase-9, assessingmRNA expression by real-time PCR and associated enzyme activity by colorimetric assay. Active caspase-3 determination was also accomplished by flow cytometry. Active caspase-3 expression was increased by Hep88 mAb treatment in a dose-and time-dependent manner. All of the results indicated that Hep88 mAb induced programmed cell death in the HepG2 cell line from paraptosis-like to apoptosis by downstream induction of caspases. These conclusions imply that Hep88mAb might be a promising tool for the effective treatment of HCC in the future.

Hep88 mAb-initiated paraptosis-like PCD pathway in hepatocellular carcinoma cell line through the binding of mortalin (HSPA9) and alpha-enolase.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most prevalent hepatic cancer worldwide. Currently, a targeted therapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is undergoing continual development in HCC treatment. METHODS: In this regard, after establishing and consequently exploring Hep88 mAb's tumoricidal effect on hepatocellular carcinoma cell line (HepG2 cell line), the Hep88 mAb's specific antigens from both membrane and cytoplasmic fractions of HepG2 cell line were identified by 2-D gel electrophoresis and western blot analysis. After in-gel digestion and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS), mortalin (HSPA9) and alpha-enolase were identified. The recombinant proteins specific to Hep88 mAb were cloned and expressed in E. coli BL21(DE3). Moreover, alteration of HepG2 and Chang liver cell line after being induced by Hep88 mAb for 1-3 days was investigated using a transmission electron microscope. RESULTS: The result demonstrated that Hep88 mAb can bind to the recombinant mortalin (HSPA9) and alpha-enolase. In addition, the gradual appearing of mitochondria vacuolization and endoplasmic reticulum dilatation were observed. Those characteristics might be explained by the paraptosis-like program cell death (PCD), which is induced by the binding of Hep88 mAb to mortalin (HSPA9). Mortalin depletion resulting from the formation of Hep88 mAb-mortalin (HSPA9) complex might initiate transcription-independence of p53-mediated apoptosis. Additionally, Hep88mAb-alpha-enolase complex might initiate HepG2 cells energy exhaustion by glycolysis pathway obstruction. CONCLUSION: These fascinating results imply that Hep88 mAb might be a promising tool for the development of an effective treatment of HCC in the next decade.

Antioxidant activity and ultrastructural changes in gastric cancer cell lines induced by Northeastern Thai edible folk plant extracts.

BACKGROUND: Phytochemical products have a critical role in the drug discovery process. This promising possibility, however, necessitates the need to confirm their scientific verification before use. Hence, this study aims to evaluate (1) the antioxidant activity, (2) cytotoxicity potential, and (3) the effect on ultrastructural alteration in gastric cancer cell lines through exposure to fractions of three local Northeastern Thai edible plants. METHODS: Plants, Syzygium gratum, Justicia gangetica and Limnocharis flava were extracted with ethyl acetate, and each crude extract analysed for their total phenolics content by Folin-Ciocalteu method. Their antioxidant activity was assessed using the ABTS system. The extracts were then assayed for cytotoxicity on two gastric cancer cell lines Kato-III and NUGC-4, and compared with Hs27 fibroblasts as a control using the MTT assay. The cell viability (%), IC50 values, as well as the ultrastructural alterations were evaluated after treatment with one way analysis of variance (ANOVA). RESULTS: The total phenolic values of the ethyl acetate extracts were well correlated with the antioxidant capacity, with extracted product of S. gratum displaying the highest level of antioxidant activity (a 10-fold greater response) over J. gangetica and L. flava respectively. Exposure of S. gratum and J. gangetica extracts to normal cell lines (Hs27) resulted in marginal cytotoxicity effects. However, through a dose-dependent assay S. gratum and J. gangetica extracts produced cytotoxicological effects in just over 75 percent of Kato-III and NUGC-4 cell lines. In addition, apoptotic characteristic was shown under TEM in both cancer cell lines with these two extracts, whereas characteristics of autophagy was found in cell lines after post exposure to extracts from L. flava. CONCLUSIONS: From these three plants, S. gratum had the highest contents of phenolic compounds and antioxidant capacity. All of them found to contain compound(s) with cytotoxicity in vitro on cancer cells but not on normal cell lines as resolved in tissue culture and ultrastructural analysis. This is the first report to show the effect on cellular alteration as apoptosis of an ethyl acetate extract of S. gratum and J. gangetica. Further studies are now focused on individual isolates and their function, prioritizing on S. gratum and J. gangetica for the development of novel therapeutics and combatants against cancer.

Hep 88 mAB induced ultrastructural alteration through apoptosis like program cell death in hepatocellular carcinoma.

Hep88 mAbs, a novel monoclonal antibodies against hepatocellular carcinoma cell line from Thai patient, has been proved earlier for its tumoricidal effect on HepG2 cell line. In the present study, we investigated not only Hep88 mAb's targeted proteins from HepG2 cell line by western blot analysis but also its inhibitory activity on those cells by MTT assay. Moreover the ultrastructural alteration induced by Hep88 mAb of HepG2 cell line compare with Chang liver cell line was also examined. The results demonstrated that Hep88 mAb had cytotoxic effect on HepG2 cell line but not Chang liver cell line. Additionally, recognizing proteins against Hep88 mAb have been found on both cell lines. The ultrastructural alteration detected from transmission electron microscopy included the appearing of intracellular vacuolization as well as the dilatation of endoplasmic reticulum and mitochondria have been observed. These findings are suggested that the death of HepG2 cell line after treatment with Hep88 mAb might be involved by an apoptosis-like program cell death (PCD) pathway. From all of these remarks, it is possible that Hep88 mAb can injure HCC cells by binding with its membrane-bound antigen and activated downstream intracellular signals which is finally leading cell to be death via apoptosis-like PCD.