End-of-treatment anti-HBs levels and HBeAg status identify durability of HBsAg loss after PEG-IFN discontinuation.

Background: Hepatitis B surface antigen (HBsAg) loss, namely, the functional cure, can be achieved through the pegylated interferon (PEG-IFN)-based therapy. However, it is an unignorable fact that a small proportion of patients who achieved functional cure develop HBsAg reversion (HRV) and the related factors are not well described. Methods: A total of 112 patients who achieved PEG-IFN-induced HBsAg loss were recruited. HBV biomarkers and biochemical parameters were examined dynamically. HBV RNA levels were assessed in the cross-sectional analysis. The primary endpoint was HRV, defined as the reappearance of HBsAg after PEG-IFN discontinuation. Results: HRV occurred in 17 patients during the follow-up period. Univariable analysis indicated that hepatitis B e antigen (HBeAg) status, different levels of hepatitis B surface antibody (anti-HBs), and hepatitis B core antibody (anti-HBc) at the end of PEG-IFN treatment (EOT) were significantly associated with the incidence of HRV through using the log-rank test. Additionally, time-dependent receiver operating characteristic (ROC) analysis showed that the anti-HBs was superior to anti-HBc in predictive power for the incidence of HRV during the follow-up period. Multivariable Cox proportional hazard analysis found that anti-HBs ≥1.3 log10IU/L (hazard ratio (HR), 0.148; 95% confidence interval (CI), 0.044-0.502) and HBeAg negativity (HR, 0.183; 95% CI, 0.052-0.639) at EOT were independently associated with lower incidence of HRV. Cross-sectional analysis indicated that the HBV RNA levels were significantly correlated with the HBsAg levels in patients with HRV (r=0.86, p=0.003). Conclusions: EOT HBeAg negativity and anti-HBs ≥1.3 log10IU/L identify the low risk of HRV after PEG-IFN discontinuation.

Alkalization of cellular pH leads to cancer cell death by disrupting autophagy and mitochondrial function.

We previously found that lactic acidosis in the tumor environment was permissive to cancer cell surviving under glucose deprivation and demonstrated that neutralizing lactic acidosis restored cancer cell susceptibility to glucose deprivation. We then reported that alternate infusion of bicarbonate and anticancer agent into tumors via tumor feeding artery markedly enhanced the efficacy of transarterial chemoembolization (TACE) in the local control of hepatocellular carcinoma (HCC). Here we sought to further investigate the mechanism by which bicarbonate enhances the anticancer activity of TACE. We propose that interfering cellular pH by bicarbonate could induce a cascade of molecular events leading to cancer cell death. Alkalizing cellular pH by bicarbonate decreased pH gradient (ΔpH), membrane potential (ΔΨm), and proton motive force (Δp) across the inner membrane of mitochondria; disruption of oxidative phosphorylation (OXPHOS) due to collapsed Δp led to a significant increase in adenosine monophosphate (AMP), which activated the classical AMPK-mediated autophagy. Meanwhile, the autophagic flux was ultimately blocked by increased cellular pH, reduced OXPHOS, and inhibition of lysosomal proton pump in alkalized lysosome. Bicarbonate also induced persistent mitochondrial permeability (MPT) and damaged mitochondria. Collectively, this study reveals that interfering cellular pH may provide a valuable approach to treat cancer.

Lactate dehydrogenases amplify reactive oxygen species in cancer cells in response to oxidative stimuli.

Previous studies demonstrated that superoxide could initiate and amplify LDH-catalyzed hydrogen peroxide production in aqueous phase, but its physiological relevance is unknown. Here we showed that LDHA and LDHB both exhibited hydrogen peroxide-producing activity, which was significantly enhanced by the superoxide generated from the isolated mitochondria from HeLa cells and patients' cholangiocarcinoma specimen. After LDHA or LDHB were knocked out, hydrogen peroxide produced by Hela or 4T1 cancer cells were significantly reduced. Re-expression of LDHA in LDHA-knockout HeLa cells partially restored hydrogen peroxide production. In HeLa and 4T1 cells, LDHA or LDHB knockout or LDH inhibitor FX11 significantly decreased ROS induction by modulators of the mitochondrial electron transfer chain (antimycin, oligomycin, rotenone), hypoxia, and pharmacological ROS inducers piperlogumine (PL) and phenethyl isothiocyanate (PEITC). Moreover, the tumors formed by LDHA or LDHB knockout HeLa or 4T1 cells exhibited a significantly less oxidative state than those formed by control cells. Collectively, we provide a mechanistic understanding of a link between LDH and cellular hydrogen peroxide production or oxidative stress in cancer cells in vitro and in vivo.

Determining the quantitative relationship between glycolysis and GAPDH in cancer cells exhibiting the Warburg effect.

Previous studies have identified GAPDH as a promising target for treating cancer and modulating immunity because its inhibition reduces glycolysis in cells (cancer cells and immune cells) with the Warburg effect, a modified form of cellular metabolism found in cancer cells. However, the quantitative relationship between GAPDH and the aerobic glycolysis remains unknown. Here, using siRNA-mediated knockdown of GAPDH expression and iodoacetate-dependent inhibition of enzyme activity, we examined the quantitative relationship between GAPDH activity and glycolysis rate. We found that glycolytic rates were unaffected by the reduction of GAPDH activity down to 19% ± 4.8% relative to untreated controls. However, further reduction of GAPDH activity below this level caused proportional reductions in the glycolysis rate. GAPDH knockdown or inhibition also simultaneously increased the concentration of glyceraldehyde 3-phosphate (GA3P, the substrate of GAPDH). This increased GA3P concentration countered the effect of GAPDH knockdown or inhibition and stabilized the glycolysis rate by promoting GAPDH activity. Mechanistically, the intracellular GA3P concentration is controlled by the Gibbs free energy of the reactions upstream of GAPDH. The thermodynamic state of the reactions along the glycolysis pathway was only affected when GAPDH activity was reduced below 19% ± 4.8%. Doing so moved the reactions catalyzed by GAPDH + PGK1 (phosphoglycerate kinase 1, the enzyme immediate downstream of GAPDH) away from the near-equilibrium state, revealing an important biochemical basis to interpret the rate control of glycolysis by GAPDH. Collectively, we resolved the numerical relationship between GAPDH and glycolysis in cancer cells with the Warburg effect and interpreted the underlying mechanism.

Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells.

Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 µm (yeast PGK1) and 6.86 µm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 µm (yeast PGK1) and 186 µm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 µm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.

Evidence of drug-response heterogeneity rapidly generated from a single cancer cell.

One cancer cell line is believed to be composed of numerous clones with different drug sensitivity. We sought to investigate the difference of drug-response pattern in clones from a cell line or from a single cell. We showed that 22 clones derived from 4T1 cells were drastically different from each other with respect to drug-response pattern against 11 anticancer drugs and expression profile of 19 genes associated with drug resistance or sensitivity. Similar results were obtained using daughter clones derived from a single 4T1 cell. Each daughter clone showed distinct drug-response pattern and gene expression profile. Similar results were also obtained using Bcap37 cells. We conclude that a single cancer cell can rapidly produce a population of cells with high heterogeneity of drug response and the acquisition of drug-response heterogeneity is random.

Next-generation sequencing of colorectal cancers in chinese: identification of a recurrent frame-shift and gain-of-function Indel mutation in the TFDP1 gene.

Abstract Re-sequencing of target genes is a highly effective approach for identifying mutations in cancers. Mutations, including indels (insertions, deletions, and the combination of the two), play important roles in carcinogenesis. Combining genomic DNA capture using high-density oligonucleotide microarrays (NimbleGen, Inc.) with next-generation high-throughput sequencing, we identified approximately 1600 indels for colorectal cancers in the Chinese population. Among them, 5 indels were localized to exonic regions of genes, including the TFDP1 (transcription factor Dp-1) gene. TFDP1 is an important transcription factor that coordinates with E2F proteins, thereby promoting transcription of E2F target genes and regulating the cell cycle and differentiation. We report here the identification of a recurrent frame-shift indel mutation (named indel84) in the TFDP1 gene in colorectal cancers by next-generation sequencing. We found in a validation set that TFDP1 indel84 is present in 70% of colorectal cancer (CRC) tissues. Wild-type TFDP1 encodes a protein of 410 amino acids with a potential DNA binding site at its N-terminal followed by several functional protein domains. The TFDP1 indel cDNA would generate an alternative TFDP1 protein missing the first 120 amino acids and potentially affecting the DNA binding domain. We further demonstrated that the TFDP1 indel84 mutation generated a gain-of-function phenotype by increasing cell proliferation, migration, and invasion of CRC cells. Our study identified a key molecular event for CRC that might have great diagnostic and therapeutic potentials.

Next generation sequencing analysis of miRNAs: MiR-127-3p inhibits glioblastoma proliferation and activates TGF-ß signaling by targeting SKI.

Glioblastoma (GBM) proliferation is a multistep process during which the expression levels of many genes that control cell proliferation, cell death, and genetic stability are altered. MicroRNAs (miRNAs) are emerging as important modulators of cellular signaling, including cell proliferation in cancer. In this study, using next generation sequencing analysis of miRNAs, we found that miR-127-3p was downregulated in GBM tissues compared with normal brain tissues; we validated this result by RT-PCR. We further showed that DNA demethylation and histone deacetylase inhibition resulted in downregulation of miR-127-3p. We demonstrated that miR-127-3p overexpression inhibited GBM cell growth by inducing G1-phase arrest both in vitro and in vivo. We showed that miR-127-3p targeted SKI (v-ski sarcoma viral oncogene homolog [avian]), RGMA (RGM domain family, member A), ZWINT (ZW10 interactor, kinetochore protein), SERPINB9 (serpin peptidase inhibitor, clade B [ovalbumin], member 9), and SFRP1 (secreted frizzled-related protein 1). Finally, we found that miR-127-3p suppressed GBM cell growth by inhibiting tumor-promoting SKI and activating the tumor suppression effect of transforming growth factor-ß (TGF-ß) signaling. This study showed, for the first time, that miR-127-3p and its targeted gene SKI, play important roles in GBM and may serve as potential targets for GBM therapy.

UCHL1 Is a Putative Tumor Suppressor in Ovarian Cancer Cells and Contributes to Cisplatin Resistance.

Ubiquitin carboxyl terminal hydrolase 1 (UCHL1) catalyzes the hydrolysis of COOH-terminal ubiquityl esters and amides. It has been reported as either an oncogene or a tumor suppressor in cancers. However, UCHL1's role in ovarian cancer is still unclear. Therefore, we conducted an analysis to understand the role of UCHL1 in ovarian cancer. Firstly, we detected UCHL1 promoter methylation status in 7 ovarian cancer cell lines. 4 of them with UCHL1 silencing showed heavy promoter methylation while the other 3 with relative high UCHL1 expression showed little promoter methylation. Then we reduced UCHL1 expression in ovarian cancer cell line A2780 and IGROV1 and found that inhibition of UCHL1 promoted cell proliferation by increasing cells in S phases of cell cycle. Knockdown of UCHL1 also reduced cell apoptosis and contributed to cisplatin resistance. Furthermore, the expression level of UCHL1 in several ovarian cancer cell lines correlated negatively with their cisplatin resistance levels. Microarray data revealed that UCHL1 related genes are enriched in apoptosis and cell death gene ontology (GO) terms. Several apoptosis related genes were increased after UCHL1 knockdown, including apoptosis regulator BCL2, BCL11A, AEN and XIAP. Furthermore, we identified up-regulation of Bcl-2 and pAKT as well as down-regulation of Bax in UCHL1 knockdown cells, while no significant alteration of p53 and AKT1 was found. This study provides a new and promising strategy to overcome cisplatin resistance in ovarian cancer via UCHL1 mediated pathways.

SOX2 targets fibronectin 1 to promote cell migration and invasion in ovarian cancer: new molecular leads for therapeutic intervention.

Abstract Ovarian cancer ranks as the second most common tumor of the female reproductive system, with a large burden on global public health. Therefore, the identification of novel molecular targets and diagnostics is an urgent need for many women affected by this disease. To this end, the human transcription factor SOX2 is involved in a wide range of pathophysiological roles, such as the maintenance of stem cell characteristics and carcinogenesis. To date, in most studies, SOX2 has been shown to promote the development of cancer, although its inhibitory roles in cancer have also been reported. However, to the best of our knowledge, the role of SOX2, specifically in ovarian cancer cells, has not been examined in detail. In this article, we report, for the first time, that SOX2 promotes migration, invasion, and clonal formation of ovarian cancer cells. We further observed that SOX2 targeted FN1, a key gene that regulates cell migration in ovarian cancer. Our findings collectively suggest that the SOX2-FN1 axis is a key pathway in mediating the migration and invasion of ovarian cancer cells. This pathway offers crucial molecular insights and promises to develop putative candidate therapeutic interventions in women with ovarian cancer.

Developing a highly stable PLGA-mPEG nanoparticle loaded with cisplatin for chemotherapy of ovarian cancer.

BACKGROUND: Cisplatin is a potent anticancer drug, but its clinical application has been limited due to its undesirable physicochemical characteristics and severe side effects. Better drug formulations for cisplatin are highly desired. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we have developed a nanoparticle formulation for cisplatin with high encapsulation efficiency and reduced toxicity by using cisplatin-crosslinked carboxymethyl cellulose (CMC) core nanoparticles made from poly(lactide-co-glycolide)-monomethoxy-poly(polyethylene glycol) copolymers (PLGA-mPEG). The nanoparticles have an average diameter of approximately 80 nm measured by transmission electron microscope (TEM). The encapsulation efficiency of cisplatin in the nanoparticles is up to 72%. Meanwhile, we have also observed a controlled release of cisplatin in a sustained manner and dose-dependent treatment efficacy of cisplatin-loaded nanoparticles against IGROV1-CP cells. Moreover, the median lethal dose (LD(50)) of the cisplatin-loaded nanoparticles was more than 100 mg/kg by intravenous administration, which was much higher than that of free cisplatin. CONCLUSION: This developed cisplatin-loaded nanoparticle is a promising formulation for the delivery of cisplatin, which will be an effective therapeutic regimen of ovarian cancer without severe side effects and cumulative toxicity.

Global analysis of DNA methylation by Methyl-Capture sequencing reveals epigenetic control of cisplatin resistance in ovarian cancer cell.

Cisplatin resistance is one of the major reasons leading to the high death rate of ovarian cancer. Methyl-Capture sequencing (MethylCap-seq), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein with NGS, global and unbiased analysis of global DNA methylation patterns. We applied MethylCap-seq to analyze genome-wide DNA methylation profile of cisplatin sensitive ovarian cancer cell line A2780 and its isogenic derivative resistant line A2780CP. We obtained 21,763,035 raw reads for the drug resistant cell line A2780CP and 18,821,061reads for the sensitive cell line A2780. We identified 1224 hyper-methylated and 1216 hypomethylated DMRs (differentially methylated region) in A2780CP compared to A2780. Our MethylCap-seq data on this ovarian cancer cisplatin resistant model provided a good resource for the research community. We also found that A2780CP, compared to A2780, has lower observed to expected methylated CpG ratios, suggesting a lower global CpG methylation in A2780CP cells. Methylation specific PCR and bisulfite sequencing confirmed hypermethylation of PTK6, PRKCE and BCL2L1 in A2780 compared with A2780CP. Furthermore, treatment with the demethylation reagent 5-aza-dC in A2780 cells demethylated the promoters and restored the expression of PTK6, PRKCE and BCL2L1.