Zhen-Zhou Yang

Metformin sensitizes EGFR-TKI-resistant human lung cancer cells in vitro and in vivo through inhibition of IL-6 signaling and EMT reversal

Purpose: The EGF receptor tyrosine kinase inhibitors (EGFR-TKI) have become a standard therapy in patients with EGFR-activating mutations. Unfortunately, acquired resistance eventually limits the clinical effects and application of EGFR-TKIs. Studies have shown that suppression of epithelial–mesenchymal transition (EMT) and the interleukin (IL)-6/STAT3 pathway may abrogate this acquired mechanism of drug resistance of TKIs. This study aims to investigate the effect of metformin on sensitizing EGFR-TKI–resistant human lung cancer cells in vitro and in vivo through inhibition of IL-6 signaling and EMT reversal. Experimental Design: The effect of metformin on reversing TKI resistance was examined in vitro and in vivo using MTT, BrdUrd incorporation assay, invasion assay, flow cytometry analysis, immunostaining, Western blot analysis, and xenograft implantation. Results: In this study, metformin, a widely used antidiabetic agent, effectively increased the sensitivity of TKI-resistant lung cancer cells to erlotinib or gefitinib. Metformin reversed EMT and decreased IL-6 signaling activation in TKI-resistant cells, while adding IL-6 to those cells bypassed the anti-TKI-resistance effect of metformin. Furthermore, overexpression or addition of IL-6 to TKI-sensitive cells induced TKI resistance, which could be overcome by metformin. Finally, metformin-based combinatorial therapy effectively blocked tumor growth in xenografts with TKI-resistant cancer cells, which was associated with decreased IL-6 secretion and expression, EMT reversal, and decreased IL-6–signaling activation in vivo. Conclusion: Metformin, generally considered nontoxic and remarkably inexpensive, might be used in combination with TKIs in patients with non–small cell lung cancer, harboring EGFR mutations to overcome TKI resistance and prolong survival. Clin Cancer Res; 20(10); 2714–26. ©2014 AACR.

Identification and characterization of mitochondrial targeting sequence of human apurinic/apyrimidinic endonuclease 1.

Dually targeted mitochondrial proteins usually possess an unconventional mitochondrial targeting sequence (MTS), which makes them difficult to predict by current bioinformatics approaches. Human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the cellular response to oxidative stress. It is a dually targeted protein preferentially residing in the nucleus with conditional distribution in the mitochondria. However, the mitochondrial translocation mechanism of APE1 is not well characterized because it harbors an unconventional MTS that is difficult to predict by bioinformatics analysis. Two experimental approaches were combined in this study to identify the MTS of APE1. First, the interactions between the peptides from APE1 and the three purified translocase receptors of the outer mitochondrial membrane (Tom) were evaluated using a peptide array screen. Consequently, the intracellular distribution of green fluorescent protein-tagged, truncated, or mutated APE1 proteins was traced by tag detection. The results demonstrated that the only MTS of APE1 is harbored within residues 289–318 in the C terminus, which is normally masked by the intact N-terminal structure. As a dually targeted mitochondrial protein, APE1 possesses a special distribution pattern of different subcellular targeting signals, the identification of which sheds light on future prediction of MTSs.

Evaluation of Selected Flavonoids as Antiangiogenic, Anticancer, and Radical Scavenging Agents: An Experimental and In Silico Analysis

Developing antiangiogenic agents using natural products has remained a significant hope in the mainstream of anticancer research. In the present investigation series of flavonoids possessing di-, tri-, tetra-, and penta-hydroxy substitutions were evaluated as antiangiogenic agents using in vivo choriallantoic membrane model. The MTT-based cytotoxicity against selected cancer cell lines was carried out to determine the anticancer potential. The kinetics of free radical scavenging activities of these compounds was demonstrated using 2,2-diphenyl-1-picryl hydrazine (DPPH) and superoxide anion radicals (SORs). To understand the possible antiangiogenic mechanism, the selected flavonoids were docked in silico onto the proangiogenic peptides such as vascular endothelial growth factor (VEGF), hypoxia inducible factor (HIF-1α), and vascular endothelial growth factor receptor-2 (VEGFR2) from human origin. The results of the study shows that amongst the tested flavonoids, genistein (87.1%), kaempferol, (86.3%), and quercetin (84.7%) were found to be effective inhibitors of angiogenesis in CAM model. The antiangiogenic, cytotoxic, and antioxidant activities are discussed in light of structure–activity relationship using in silico approach and other drug-related properties were also calculated using BioMed CAChe V. 6.1.10. The results of the present study focus the isoflavone genistein, kaempferol, and quercetin as lead molecules for designing novel anti-tumor/antioxidant agents targeting angiogenesis.

Elevated Expression of APE1/Ref-1 and its Regulation on IL-6 and IL-8 in Bone Marrow Stromal Cells of Multiple Myeloma

A number of growth factors secreted by bone marrow stromal cells (BMSCs), including interleukin-6 and -8 (IL-6/8), are important for the initiation and progression of multiple myeloma (MM). However, the mechanisms that regulate the production of IL-6/8 by BMSC have not yet been well characterized. Human dual functional protein apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is essential for cell survival and proliferation. Previous studies showed that APE1/Ref-1 was overexpressed in tumor cells, but few studies showed its expression in supportive cells in the tumor microenvironment. We first detected APE1/Ref-1 expression in BMSCs of normal, initial, and recurrent MM patients, and then explore the correlation between APE1/Ref-1 level and IL-6/8 secretion of BMSCs. A marked increase of APE1/Ref-1 expression and abnormal subcellular distribution were observed in MM BMSCs. APE1/Ref-1 overexpression was related to higher secretary level of IL-6/8 by MM BMSCs and the IL-6/8 secretion was blocked significantly by adenovirus-mediated APE1/Ref-1-specific (small interfering RNA) siRNA. Our results also demonstrated that APE1/Ref-1-specific siRNA significantly inhibited DNA binding activity of AP-1 and nuclear factor-κB (NF-κB), 2 important transcription factors in the regulation IL-6/8 secretion in MM BMSCs. The results provided by the present study indicate APE1/Ref-1, which plays a regulatory role in IL-6/8 production by BMSCs, may be a potential therapeutic target of MM.

Human apurinic/apyrimidinic endonuclease 1 translocalizes to mitochondria after photodynamic therapy and protects cells from apoptosis

Photodynamic therapy (PDT) is an effective therapeutic regime for lung cancer. Mitochondrial functional failure is considered to be one of the most important factors causing cell death after PDT. However, the detailed mechanisms that are involved are still unclear. We previously reported that apurinic/apyrimidinic endonuclease (APE1) plays a critical role in regulating sensitivity to PDT in the lung cancer A549 cell line. An important mitochondrial regulatory role for APE1 has recently been reported, so therefore we explored the role of APE1 in cell survival after PDT‐induced oxidative stress through regulation of mitochondrial function. We first observed that photoirradiation induced the mitochondrial translocation of APE1. The ability of APE1 to regulate mitochondrial membrane potential and reactive oxygen species (ROS) production after photoirradiation was tested in APE1 knockdown A549 cells. APE1‐deficient A549 cells were characterized as having a lower mitochondrial membrane potential and higher ROS production, which led to increased apoptosis through the mitochondrial pathway after PDT. Additionally, unexpected activity of APE1 was observed in mitochondria: the control of mitochondrial transcriptional activity by redox regulation of mitochondrial transcription factor A (TFAM). Furthermore, two dominant‐negative mutants of APE1 were overexpressed to enhance their individual activities in mitochondria. The results suggest that both these APE1 activities play a role in the regulation of mitochondrial function but through different mechanisms. The present study not only provides possible mechanisms for APE1 in regulating survival after photoirradiation but also uncovers a new activity of APE1 in mitochondria. (Cancer Sci 2012; 103: 882–888)

Nm23-H1 Protein Binds to APE1 at AP Sites and Stimulates AP Endonuclease Activity Following Ionizing Radiation of the Human Lung Cancer A549 Cells

Non-metastatic protein-23 homolog-1 (Nm23-H1) is a multifunctional protein with DNase and histidine protein kinase activities. Human apurinic endonuclease-1 (APE1) is the AP endonuclease DNA base excision repair (BER) enzyme involved in several important cellular functions. Since the relationship between Nm23-H1 and APE1 proteins is unclear, we evaluated their interaction at different time points after irradiating human lung cancer A549 cells with X-rays. We found that Nm23-H1 and APE1 overexpression was induced by irradiation in a dose- and time-dependent manner. Subcellular distribution pattern of both proteins was reversed after irradiation. After irradiation, APE1 that initially showed nuclear localization was gradually increased in the cytoplasm, whereas Nm23-H1 that mainly showed cytoplasmic localization was gradually increased in the nuclei of A549 cells. Nm23-H1 and APE1 interaction was demonstrated by His-pull-down and co-immunoprecipitation assays. The presence of Nm23-H1/APE1 complex in X-ray-irradiated A549 cells was also detected by DNA affinity precipitation analysis of a DNA fragment containing an AP site. Although the AP endonuclease activity of Nm23-H1 was too weak to be detected, the AP endonuclease activity of APE1 was increased with the enhanced Nm23-H1 expression. In conclusion, our data point to a mechanism by which Nm23-H1 protects cells against oxidative stress through the engagement of DNA BER enzyme APE1.

Anti-tumor Effects of All-trans Retinoic Acid are Enhanced by Genistein

The effects of all-trans retinoic acid (ATRA) on cancer are complex. ATRA has anti-cancer effects as it promotes cancer cell differentiation. However, ATRA also up-regulates expression of vascular endothelial growth factor (VEGF) in cancer cells, which leads to angiogenesis and can, thus, facilitate cancer growth. Genistein, a crucial non-nutrient component in soybean, exhibits anti-cancer effects by inhibiting protein tyrosine kinase that is involved in up-regulation of VEGF. We hypothesized that genistein, applied simultaneously with ATRA, would counter its undesired angiogenic effects and, thus, enhance the anti-cancer effects of ATRA. The purpose of this study was to document potential synergistic effects of genistein and ATRA in A549 lung adenocarcinoma cells. We further explored the role of genistein on countering the ATRA-induced VEGF expression. We demonstrate that genistein enhances the ATRA-induced growth inhibition of A549 cells by promoting apoptosis. Further, the combined use of ATRA and genistein leads to cancer cell arrest in G0/G1 and G2/M cell cycle phases. Finally, expression of VEGF (both mRNA and protein) was diminished in A549 cells exposed to both ATRA and genistein. In conclusion, our results demonstrate that genistein effectively enhances anti-cancer effects of ATRA, particularly, by countering the ATRA-induced up-regulation of VEGF. Our study provides an experimental basis for combined use of ATRA and genistein in the treatment of lung cancer.

Genistein induces apoptosis by stabilizing intracellular p53 protein through an APE1-mediated pathway.

Genistein (GEN) has been previously shown to have a proapoptotic effect on cancer cells through a p53-dependent pathway, the mechanism of which remains unclear. One of its intracellular targets, APE1, protects against apoptosis under genotoxic stress and interacts with p53. In this current study, we explored the mechanism of the proapoptotic effect of GEN by examining the APE1-p53 protein-protein interaction. We initially showed that the p53 protein level was elevated in GEN-treated human non-small lung cancer A549 cells and cervical cancer HeLa cells. By examining both protein synthesis and degradation, we found that GEN enhances p53 intracellular stability by interfering with the interaction of APE1 and p53, which provided a plausible explanation for how GEN initiates apoptosis. Furthermore, we found that the interaction between APE1 and p53 is important for the degradation of p53 and is dependent on the redox domain of APE1 by utilizing the redox domain mutant APE1 C65A. Our data suggest that the degradation of wild-type p53 is blocked when the redox domain of APE1 is masked or interrupted. Based on this evidence, we hereby report a novel mechanism of p53 degradation through an APE1-mediated, redox-dependent pathway.

High-incidence of PTEN mutations in Chinese patients with primary small cell carcinoma of the esophagus

BackgroundPrimary small cell carcinoma of the esophagus (PSCCE) is a rare and aggressive tumor with poor prognosis. The aim of this study was to investigate the existence of EGFR, KRAS, PIK3CA and PTEN mutations in PSCCE.MethodsClinical–pathological data and paraffin-embedded specimens were collected from 38 patients. Exons 18 to 21 of EGFR, KRAS and PIK3CA status were analyzed by real-time PCR based on ARMS and Scorpion technology in all patients, and the PTEN gene was also screened using real-time PCR and high-resolution melting curve analysis (HRMA).ResultsOnly 1 (2.63%) out of 38 patients had EGFR mutations in L858R missense, and KRAS and PIK3CA were not found in the mutational spot in all patients. However, PTEN mutations presented in 14 (36.84%) out of 38 patients, including exon 5 coding for PTEN missense mutation (n =4, 10.53%), exon 6 (n =7, 18.42%), concurrent exon 5 and exon 6 (n =2, 5.26%), and exon 8 (n =1, 2.63%). Concurrent mutations of these genes were not detected in all samples. No statistically significant associations were found between the clinicopathological features and the mutation status of PTEN.ConclusionsThe incidence of PTEN mutations in Chinese patients with PSCCE was higher than that of previous reports in other histological subtypes of esophageal cancer.

Killing Effect of Ad5/F35-APE1 siRNA Recombinant Adenovirus in Combination with Hematoporphrphyrin Derivative-Mediated Photodynamic Therapy on Human Nonsmall Cell Lung Cancer

The main goal of this work is to investigate the killing effects and molecular mechanism of photodynamic therapy (PDT) mediated by the Ad5/F35-APE1 siRNA recombinant adenovirus in combination with a hematoporphrphyrin derivative (HpD) in the A549 human lung adenocarcinoma cell line in vitro to provide a theoretical reference for treating lung cancer by HpD-PDT. By using the technologies of MTT, flow cytometry, ELISA, and western blot, we observed that the proliferation inhibition and apoptosis of the A549 cells were significantly higher than the control group (P < 0.05) after HpD-PDT was performed. The inhibitory efficiency is dependent on the HpD concentration and laser intensity dose. The inhibitory effect on the proliferation of A549 cells of Ad5/F35-APE1 siRNA is more significant after combining with PDT, as indicated by a significant elevation of the intracellular ROS level and the expression of inflammatory factors (P < 0.05). The HpD-PDT-induced expression of the APE1 protein reached the peak after 24 h in A549 cells. The inhibition of APE1 expression in A549 cells was most significant after 48 hours of infection by Ad5/F35-APE1 siRNA recombinant adenovirus (10 MOI). In conclusion, the Ad5/F35-APE1 siRNA recombinant adenovirus could efficiently inhibit the HpD-PDT-induced APE1 expression hence could significantly enhance the killing effect of HpD-PDT in lung cancer cells.