Xuyu Zu

Aldo-keto reductase family 1 B10 gene silencing results in growth inhibition of colorectal cancer cells: Implication for cancer intervention.

Aldo–keto reductase family 1 B10 (AKR1B10), a member of aldo–keto reductase superfamily, is overexpressed in human hepatocellular carcinoma, lung squamous cell carcinoma and lung adenocarcinoma. Our previous study had demonstrated that the ectopic expression of AKR1B10 in 293T cells promotes cell proliferation. To evaluate its potential as a target for cancer intervention, in the current study we knocked down AKR1B10 expression in HCT‐8 cells derived from a colorectal carcinoma, using chemically synthesized small interfering RNA (siRNA). The siRNA 1, targeted to encoding region, downregulated AKR1B10 expression by more than 60%, and siRNA 2, targeted to 3′ untranslational region, reduced AKR1B10 expression by more than 95%. AKR1B10 silencing resulted in approximately a 50% decrease in cell growth rate and nearly 40% suppression of DNA synthesis. More importantly, AKR1B10 downregulation significantly reduced focus formation rate and colony size in semisolid culture, indicating the critical role of AKR1B10 in HCT‐8 cell proliferation. Recombinant AKR1B10 protein showed strong enzymatic activity to acrolein and crotonaldehyde, with Km = 110.1 ± 12.2 μM and Vmax = 3,122.0 ± 64.7 nmol/mg protein/min for acrolein and Km = 86.7 ± 14.3 μM and Vmax = 2,647.5 ± 132.2 nmol/mg protein/min for crotonaldehyde. AKR1B10 downregulation enhanced the susceptibility of HCT‐8 cells to acrolein (25 μM) and crotonaldehyde (50 μM), resulting in rapid oncotic cell death characterized with lactate dehydrogenase efflux and annexin‐V staining. These results suggest that AKR1B10 may regulate cell proliferation and cellular response to additional carbonyl stress, thus being a potential target for cancer intervention.

Aldo-keto Reductase Family 1 B10 Affects Fatty Acid Synthesis by Regulating the Stability of Acetyl-CoA Carboxylase-α in Breast Cancer Cells

Recent studies have demonstrated that aldo-keto reductase family 1 B10 (AKR1B10), a novel protein overexpressed in human hepatocellular carcinoma and non-small cell lung carcinoma, may facilitate cancer cell growth by detoxifying intracellular reactive carbonyls. This study presents a novel function of AKR1B10 in tumorigenic mammary epithelial cells (RAO-3), regulating fatty acid synthesis. In RAO-3 cells, Sephacryl-S 300 gel filtration and DEAE-Sepharose ion exchange chromatography demonstrated that AKR1B10 exists in two distinct forms, monomers (∼40 kDa) bound to DEAE-Sepharose column and protein complexes (∼300 kDa) remaining in flow-through. Co-immunoprecipitation with AKR1B10 antibody and protein mass spectrometry analysis identified that AKR1B10 associates with acetyl-CoA carboxylase-α (ACCA), a rate-limiting enzyme of de novo fatty acid synthesis. This association between AKR1B10 and ACCA proteins was further confirmed by co-immunoprecipitation with ACCA antibody and pulldown assays with recombinant AKR1B10 protein. Intracellular fluorescent studies showed that AKR1B10 and ACCA proteins co-localize in the cytoplasm of RAO-3 cells. More interestingly, small interfering RNA-mediated AKR1B10 knock down increased ACCA degradation through ubiquitination-proteasome pathway and resulted in >50% decrease of fatty acid synthesis in RAO-3 cells. These data suggest that AKR1B10 is a novel regulator of the biosynthesis of fatty acid, an essential component of the cell membrane, in breast cancer cells.

AKR1B10 overexpression in breast cancer: Association with tumor size, lymph node metastasis and patient survival and its potential as a novel serum marker†

Aldo‐keto reductase 1B10 (AKR1B10) is a secretory protein that is upregulated with tumorigenic transformation of human mammary epithelial cells. This study demonstrated that AKR1B10 was overexpressed in 20 (71.4%) of 28 ductal carcinomas in situ, 184 (83.6%) of 220 infiltrating carcinomas and 28 (87.5%) of 32 recurrent tumors. AKR1B10 expression in breast cancer was correlated positively with tumor size (p = 0.0012) and lymph node metastasis (p = 0.0123) but inversely with disease‐related survival (p = 0.0120). Univariate (p = 0.0077) and multivariate (p = 0.0192) analyses both suggested that AKR1B10, alone or together with tumor size and node status, is a significant prognostic factor for breast cancer. Silencing of AKR1B10 in BT‐20 human breast cancer cells inhibited cell growth in culture and tumorigenesis in female nude mice. Importantly, AKR1B10 in the serum of breast cancer patients was significantly increased to 15.18 ± 9.08 ng/ml [n = 50; 95% confidence interval (CI), 12.60–17.76], with a high level up to 58.4 ng/ml, compared to 3.34 ± 2.27 ng/ml in healthy donors (n = 60; 95% CI, 2.78–3.90). In these patients, AKR1B10 levels in serum were correlated with its expression in tumors (r = 0.8066; p < 0.0001). Together our data suggests that AKR1B10 is overexpressed in breast cancer and may be a novel prognostic factor and serum marker for this deadly disease.

Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma

Protein N‐arginine methyltransferases (PRMTs) participate in a number of cellular processes, including cell growth, nuclear/cytoplasmic protein shuttling, differentiation, RNA splicing and post‐transcriptional regulation. PRMT2 (also known as HRMT1L1) is clearly involved in lung function, the inflammatory response, apoptosis promotion, Wnt signaling and leptin signaling regulation through different mechanisms. In this study, we report the molecular and cell biological characterization of three novel PRMT2 splice variants isolated from breast cancer cells and referred to as PRMT2α, PRMT2β and PRMT2γ. Compared with the wild‐type PRMT2, these variants lack different motifs and therefore generate distinct C‐terminal domains. Confocal microscopy scanning revealed a distinct intracellular localization of PRMT2 variants, suggesting that the alternatively spliced C‐terminus of PRMT2 can directly influence its subcellular localization. Our findings reveal that these variants are capable of binding to estrogen receptor alpha (ERα) both in vitro and in vivo, and the N‐terminal regions of these variants contribute to ERα–PRMT2 interactions. Furthermore, these variants were proved to be able to enhance ERα‐mediated transactivation activity. Luciferase reporter assays showed that PRMT2s could modulate promoter activities of the ERα‐targeted genes of Snail and E‐cadherin. In addition, PRMT2 silencing could enhance 17β‐estradiol‐induced proliferation by regulating E2F1 expression and E2F1‐responsive genes in ERα‐positive breast cancer cells. Real‐time PCR and immunohistochemistry showed that overall PRMT2 expression was upregulated in breast cancer tissues and significantly associated with ERα positivity status both in breast cancer cell lines and breast cancer tissues. We speculate that PRMT2 and its splice variants may directly modulate ERα signaling and play a role in the progression of breast cancer.

Synthesis and potent antileukemic activities of 10-benzyl-9(10H)-acridinones

A novel series of 10-benzyl-9(10H)-acridinones and 1-benzyl-4-piperidones were synthesized and tested for their in vitro antitumor activities against CCRF-CEM cells. Assay-based antiproliferative activity study using CCRF-CEM cell lines revealed that the acridone group and the substitution pattern on the benzene unit had significant effect on cytotoxicity of this series of compounds, among which 10-(3,5-dimethoxy)benzyl-9(10H)-acridinone (3b) was found to be the most active compound with IC(50) at about 0.7 microM. Compound 3b was also found to have antiproliferative activity against two other human leukemic cell lines K562 and HL60 using the MTT assay. The antitumor effect of 3b is believed to be due to the induction of apoptosis, which is further confirmed by PI (Propidium iodide) staining and Annexin V-FITC/PI staining assay using flow cytometry analysis.

Transforming growth factor-β signaling in tumor initiation, progression and therapy in breast cancer: an update

Transforming growth factor-β (TGF-β) is a ubiquitous cytokine playing an essential role in cell proliferation, differentiation, apoptosis, adhesion and invasion, as well as in cellular microenvironment. In malignant diseases, TGF-β signaling features a growth inhibitory effect at an early stage but aggressive oncogenic activity at the advanced malignant state. Here, we update the current understanding of TGF-β signaling in cancer development and progression with a focus on breast cancer. We also review the current approaches of TGF-β signaling-targeted therapeutics for human malignancies.

Exploration of (S)-3-aminopyrrolidine as a potentially interesting scaffold for discovery of novel Abl and PI3K dual inhibitors

Based on the literature-reported compensatory effect of PI3K on Abl inhibition and the improved preclinical effect of drug combination of Abl and PI3K inhibitors, a series of compounds bearing novel scaffold of (S)-3-aminopyrrolidine was identified as Abl and PI3K dual inhibitors through support vector machine screening tool, which were subsequently synthesized and tested. Most compounds demonstrated promising cytoxicity against a CML leukemia cell-line K562 and moderate inhibition against Abl and PI3K kinases. These compounds induced no apoptosis in K562 cell-line, suggesting that their cytotoxic activities are unlikely duo to other known anti-CML mechanisms. Molecular docking study further showed that the compound 5k could bind with both Abl and PI3K, but the weaker binding with Abl compared to Imatinib is consistent with its low kinase inhibitory rates. These plus literature-reported evidences suggest that the promising cytotoxic effect of our novel compounds might be due to the collective effect of Abl and PI3K inhibition.

Pro-oncogene Pokemon promotes breast cancer progression by upregulating survivin expression

IntroductionPokemon is an oncogenic transcription factor involved in cell growth, differentiation and oncogenesis, but little is known about its role in human breast cancer. In this study, we aimed to reveal the role of Pokemon in breast cancer progression and patient survival and to understand its underlying mechanisms.MethodsTissue microarray analysis of breast cancer tissues from patients with complete clinicopathological data and more than 20 years of follow-up were used to evaluate Pokemon expression and its correlation with the progression and prognosis of the disease. DNA microarray analysis of MCF-7 cells that overexpress Pokemon was used to identify Pokemon target genes. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were utilized to determine how Pokemon regulates survivin expression, a target gene.ResultsPokemon was found to be overexpressed in 158 (86.8%) of 182 breast cancer tissues, and its expression was correlated with tumor size (P = 0.0148) and lymph node metastasis (P = 0.0014). Pokemon expression led to worse overall (n = 175, P = 0.01) and disease-related (n = 79, P = 0.0134) patient survival. DNA microarray analyses revealed that in MCF-7 breast cancer cells, Pokemon regulates the expression of at least 121 genes involved in several signaling and metabolic pathways, including anti-apoptotic survivin. In clinical specimens, Pokemon and survivin expression were highly correlated (n = 49, r = 0.6799, P < 0.0001). ChIP and site-directed mutagenesis indicated that Pokemon induces survivin expression by binding to the GT boxes in its promoter.ConclusionsPokemon promotes breast cancer progression by upregulating survivin expression and thus may be a potential target for the treatment of this malignancy.

The design, synthesis, and anti-tumor mechanism study of N-phosphoryl amino acid modified resveratrol analogues

A novel series of trans-N-phosphoryl amino acid modified resveratrol analogues were synthesized and evaluated in vitro for their cytotoxic effects against CNE-1 and CNE-2 cell lines. These analogues showed good anti-proliferative activity, among which 8d, 8e, 8j, and 9d displayed much stronger inhibition effect than resveratrol and 8d showed the most potent activity with IC(50) value at 3.45+/-0.82microM. The anti-tumor effects of 8d, 8e, 8j, and 9d were due to the induction of apoptosis, confirmed by the DNA fragmentation and flow cytometry analysis using PI (propidium iodide) staining and Annexin-V-FITC/PI staining assay. The PI staining assay also showed that 8d, 8e, 8j, and 9d caused cell cycles arrest at G(0)-G(1) phase which finally led to cell apoptosis. Further mechanism study on compound 8d against CNE-2 cells has shown the PARP cleavage, which is a hallmark of caspase-3 activation, as well as the activation of caspase-9, and the intracellular ROS generation. These results all suggest that 8d induced a mitochondrial-dependent apoptosis pathway.

ATP Citrate Lyase Inhibitors as Novel Cancer Therapeutic Agents

ATP citrate lyase (ACL or ACLY) is an extra-mitochondrial enzyme widely distributed in various human and animal tissues. ACL links glucose and lipid metabolism by catalyzing the formation of acetyl-CoA and oxaloacetate from citrate produced by glycolysis in the presence of ATP and CoA. ACL is aberrantly expressed in many immortalized cells and tumors, such as breast, liver, colon, lung and prostate cancers, and is correlated reversely with tumor stage and differentiation, serving as a negative prognostic marker. ACL is an upstream enzyme of the long chain fatty acid synthesis, providing acetyl-CoA as an essential component of the fatty acid synthesis. Therefore, ACL is a key enzyme of cellular lipogenesis and potent target for cancer therapy. As a hypolipidemic strategy of metabolic syndrome and cancer treatment, many small chemicals targeting ACL have been designed and developed. This review article provides an update for the research and development of ACL inhibitors with a focus on their patent status, offering a new insight into their potential application.