Decreased tumorigenicity in vivo when transforming growth factor beta treatment causes cancer cell senescence

We have previously reported that transforming growth factor beta (TGF-beta) triggers two independent senescence programs, 1) replicative senescence dependent upon telomere shortening and 2) premature senescence independent of telomere shortening, in the cell line of A549 human lung adenocarcinoma. In this study, we examined the possibility that cancer cell tumor phenotypes could be suppressed by forced senescence. We used A549 cells treated with TGF-beta for a long time (over 50 days), where senescence was induced in a telomere-shortening-dependent or an independent way. Fully senescent A549 cells were elongated, acquired contact inhibition capabilities when reaching confluence, and secreted the senescence-associated cytokine IL-6. Furthermore, senescent A549 cells had no tumorigenicity in nude mice. These results indicate that the forced induction of senescence in cancer cells may be a novel and potentially powerful method for advancing anti-cancer therapy.     

Expression of Prostate-Specific Membrane Antigen in Lung Cancer Cells and Tumor Neovasculature Endothelial Cells and Its Clinical Significance

BackgroundProstate-specific membrane antigen (PSMA) has been found in tumor neovasculature endothelial cells (NECs) of non-prostate cancers and may become the most promising target for anti-tumor therapy. To study the value of PSMA as a potential new target for lung cancer treatment, PSMA expression in non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) tissues and its relationship with clinicopathology were investigated in the current study.MethodsImmunohistochemistry was used to detect PSMA expression in a total of 150 lung specimens of patients with lung cancer. The data were analyzed using univariate and multivariate statistical analyses.ResultsThe percentages of NSCLC patients who had PSMA (+) tumor cells and PSMA (+) NECs were 54.02% and 85.06%, respectively. The percentage of patients younger than 60 years old who had PSMA (+) tumor cells was 69.05%, which was significantly greater than the percentage of patients aged 60 years or older (40.00%, p<0.05). A significant difference was observed in the percentage of NSCLC patients with PMSA (+) NECs and stage I or II cancer (92.98%) and those patients with stage III or IV cancer (76.77%). In the SCLC tissues, NEC PSMA expression (70.00%) did not differ significantly from NSCLC. SCLC tumor cells and normal lung tissues cells were all negative. There was no significant correlation between the presence of PSMA (+) NECs in SCLC patients and the observed clinicopathological parameters.ConclusionsPSMA is expressed not only in NECs of NSCLC and SCLC but also in tumor cells of most NSCLC patients. The presence of PSMA (+) tumor cells and PSMA (+) NECs in NSCLC was negatively correlated with age and the clinicopathological stage of the patients, respectively.     

Synthesis and biological evaluation of PSMA-targeting paclitaxel conjugates

Prostate cancer (PC) is the second most commonly occurring cancer in men. Conventional chemotherapy has wide variety of disadvantages such as high systemic toxicity and low selectivity. Targeted drug delivery is a promising approach to decrease side effects of therapy. Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer cells while low level of expression is observed in normal cells. In this study we describe the development of Glu-urea-Lys based PSMA-targeting conjugates with paclitaxel. A series of new PSMA targeting conjugates with paclitaxel was designed and synthesized. The cytotoxicity of conjugates was evaluated against prostate (LNCaP, 22Rv1 and PC-3) and non-prostate (Hek293T, VA13, A549 and MCF-7) cell lines. The most promising conjugate 21 was examined in vivo using 22Rv1 xenograft mice model. It demonstrated good efficiency comparable with paclitaxel, while reduced toxicity. 3D molecular docking study was also performed to understand underlying mechanism of binding and further optimization of the linker substructure and conjugates structure for improving the target affinity. These conjugates may be useful for further design of novel PSMA targeting delivery systems for PC.     

PSMA2 knockdown impacts expression of proteins involved in immune and cellular stress responses in human lung cells

Proteasome subunit alpha type-2 (PSMA2) is a critical component of the 20S proteasome, which is the core particle of the 26S proteasome complex and is involved in cellular protein quality control by recognizing and recycling defective proteins. PSMA2 expression dysregulation has been detected in different human diseases and viral infections. No study yet has reported PSMA2 knockdown (KD) effects on the cellular proteome. Methods: We used SOMAScan, an aptamer-based multiplexed technique, to measure >1300 human proteins to determine the impact of PSMA2 KD on A549 human lung epithelial cells. Results: PSMA2 KD resulted in significant dysregulation of 52 cellular proteins involved in different bio-functions, including cellular movement and development, cell death and survival, and cancer. The immune system and signal transduction were the most affected cellular functions. PSMA2 KD caused dysregulation of several signaling pathways involved in immune response, cytokine signaling, organismal growth and development, cellular stress and injury (including autophagy and unfolded protein response), and cancer responses. Conclusions: In summary, this study helps us better understand the importance of PSMA2 in different cellular functions, signaling pathways, and human diseases.     

EGF enhances the migration of cancer cells by up-regulation of TRPM7

Ion channels involved in the migration of tumor cells that is required for their invasion and metastasis. In this paper, we describe the interaction of TRPM7 channel and epidermal growth factor (EGF), an important player in cancer development in the migration of lung cancer cells. The TRPM7 currents in A549 cells were first characterized by means of electrophysiology, pharmacology and RNA interference. Removing Ca²⁺ from the extracellular solution not only potentiated a large inward current, but also abolished the outward rectification. 200 μM 2-APB inhibited the outward and the inward TRPM7 currents and at the same time restored the property of outward rectification. EGF greatly enhanced the migration of A549 cells, and also markedly up-regulated the membrane protein expression of TRPM7 and the amplitude of TRPM7 currents. Depressing the function of TRPM7 with RNA interference or pharmacological agents not only reversed the EGF-enhanced migration of A549 cells but also inhibited the basal migration of A549 cells in the absence of EGF. Thus it seems that TRPM7 plays a pivotal role in the migration of A549 cells induced by EGF and thus could be a potential therapeutic target in lung cancers.     

Generation of a baculovirus recombinant prostate-specific membrane antigen and its use in the development of a novel protein biochip quantitative immunoassay

Prostate-specific membrane antigen (PSMA) is a 100-kDa transmembrane glycoprotein identified by the monoclonal antibody 7E11-C5.3 from the human prostate tumor cell line LNCaP. Because of its significant upregulation in androgen refractory and metastatic prostate cancers, PSMA may be a useful prognostic biomarker and a target for developing novel therapeutic strategies. However, the lack of abundant pure PSMA protein and the low efficacy in immunoaffinity isolation from LNCaP cells have hampered the development of clinical assays. In order to obtain a renewable and reliable source of pure antigen, we used the baculovirus/insect cell system to express and purify a recombinant PSMA. A recombinant baculovirus containing a 6x histidine-tagged PSMA gene was generated, from which rPSMA was expressed and purified using cobalt-chelating affinity chromatography. The purity and correct molecular size of rPSMA were demonstrated by gel electrophoresis and mass spectrometry. Glycosidase digestions showed that the oligosaccharides on rPSMA are primarily N-linked high-mannose type. Although the glycosylation is different from the native PSMA, it did not affect the immunoreactivity of rPSMA to antibodies specific for either the intra- or the extracellular domains of PSMA. Finally, the purified rPSMA was successfully used to develop a quantitative PSMA immunoassay using the novel ProteinChip surface-enhanced laser desorption/ionization mass spectrometry technology.     

Biosynthesis of d-Erythroascorbic Acid by Candida

Epigallocatechin 3-gallate (EGCG) has cytotoxic effects in many cancer cells. It has been reported that A549 lung cancer cells are markedly resistant to cell death induced by EGCG. In the present study, the effects of EGCG on A549 lung cancer cell growth and angiogenesis were studied. We found that EGCG dose-dependently suppressed A549 cell growth, while A549 cells were markedly resistant to cell death in vitro. Next we found that EGCG increased endostatin expression and suppressed vascular endothelial growth factor (VEGF) expression. We further studied to determine whether EGCG would suppress A549 tumor growth in nude mouse and angiogenesis. EGCG in drinking water significantly suppressed A549 tumor growth in nude mice. Histological analysis revealed that the number of CD34 positive vessels had a tendency to decrease in the tumor. In sum, EGCG had anti-proliferative effects of A549 on tumor growth and showed a tendency to suppress angiogenesis.     

Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.     

Regulatory role of KEAP1 and NRF2 in PPARγ expression and chemoresistance in human non-small-cell lung carcinoma cells

The nuclear factor-E2-related factor 2 (NRF2) serves as a master regulator in cellular defense against oxidative stress and chemical detoxification. However, persistent activation of NRF2 resulting from mutations in NRF2 and/or downregulation of or mutations in its suppressor, Kelch-like ECH-associated protein 1 (KEAP1), is associated with tumorigenicity and chemoresistance of non-small-cell lung carcinomas (NSCLCs). Thus, inhibiting the NRF2-mediated adaptive antioxidant response is widely considered a promising strategy to prevent tumor growth and reverse chemoresistance in NSCLCs. Unexpectedly, stable knockdown of KEAP1 by lentiviral shRNA sensitized three independent NSCLC cell lines (A549, HTB-178, and HTB-182) to multiple chemotherapeutic agents, including arsenic trioxide (As(2)O(3)), etoposide, and doxorubicin, despite moderately increased NRF2 levels. In lung adenocarcinoma epithelial A549 cells, silencing of KEAP1 augmented the expression of peroxisome proliferator-activated receptor γ (PPARγ) and genes associated with cell differentiation, including E-cadherin and gelsolin. In addition, KEAP1-knockdown A549 cells displayed attenuated expression of the proto-oncogene cyclin D1 and markers for cancer stem cells (CSCs) and reduced nonadherent sphere formation. Moreover, deficiency of KEAP1 led to elevated induction of PPARγ in response to As(2)O(3). Pretreatment of A549 cells with PPARγ agonists activated PPARγ and augmented the cytotoxicity of As(2)O(3). A mathematical model was formulated to advance a hypothesis that differential regulation of PPARγ and detoxification enzymes by KEAP1 and NRF2 may underpin the observed landscape changes in chemosensitivity. Collectively, suppression of KEAP1 expression in human NSCLC cells resulted in sensitization to chemotherapeutic agents, which may be attributed to activation of PPARγ and subsequent alterations in cell differentiation and CSC abundance.     

Design,synthesis, and biological evaluation of a cephalosporin-monohydroguaiaretic acid prodrug activated by a monoclonal antibody-beta-lactamase conjugate

A novel cephalosporin derivative of monohydroguaiaretic acid (cephem-M(3)N, 7) was synthesized and found to possess anticancer activity against human leukemia (K562), breast carcinoma (MCF7), human lung cancer (A549), human colon cancer (Colo205) and pancreatic cancer cells (Capan2 and MiaPaCa2). A tumor targeting fusion protein (dsFv3-beta-lactamase) was also used in conjunction with cephem-based M(3)N 7 and its potency toward K562, MCF7, A549, Colo205, Capan2, and MiaPaCa2 was found to approach that of the free M(3)N (4). In the presence of dsFv3-beta-lactamase, tumor cells were found to be much more susceptible to conjugate 7 than normal human embryonic lung (HEL) cells and normal fibroblasts (Hef522). These notions provide a new approach to the use of nordihydroguaiaretic acid (NDGA) and its derivatives for antitumor therapy.     

Regulation of Endoribonuclease Activity of Alpha-Type Proteasome Subunits in Proerythroleukemia K562 Upon Hemin-Induced Differentiation

The proteasome is the main intracellular proteolytic machine involved in the regulation of numerous cellular processes, including gene expression. In addition to their proteolytic activity, proteasomes also exhibit ATPase/helicase (the 19S particle) and RNAse (the 20S particle) activities, which are regulated by post-translational modifications. In this report we uncovered that several 20S particle subunits: α1 (PSMA6), α2 (PSMA2), α4 (PSMA7), α5 (PSMA5), α6 (PSMA1) and α7 (PSMA3) possess RNAse activity against the p53 mRNA in vitro. Furthermore, we found that the RNAse activity of PSMA1 and PSMA3 was regulated upon hemin-induced differentiation of K562 proerythroleukemia cells. The decrease in RNAse activity of PSMA1 and PSMA3 was paralleled by changes in their status of phosphorylation and ubiquitylation. Collectively, our data support the notion that proteasomal RNAse activity may be functionally important and provide insights into the potential mechanism of p53 repression in erythroleukemia cells by RNAse activity of the 20S α-type subunits.     

肿瘤坏死因子α和β对电离辐射诱导细胞凋亡的效应

为探讨肿瘤坏死因子（tumor necrosis foctor)α和β（TNFα和β）对电离辐射诱发细胞凋亡的效应及其机理,采用DNA琼脂糖凝胶电泳和FACS分析等方法,观察了人肿瘤坏死因子α（hTNFα)和β（hTNFβ)对^60Co-γ射线诱发细胞凋亡的形态学,生化学变化。结果显示：hTNFα或hTNFβ均可明显抑制^60Co-γ射线诱发正常人胚肺二倍体细胞（2BS）的凋亡,而相同剂量的hTNFα能促进^60Co-γ射线诱发的人体肺腺癌细胞系A549细胞凋亡,而对另一株人体肺癌SPC细胞的效应比A549降低1倍;hTNFβ能分别增强A549和SPC的细胞凋亡频率。由此认为,hTNFα和hTNFβ均可通过调节细胞的生理生化反应来改变细胞对电离辐射的敏感性,可保护正常细胞免受辐射损伤,而增加某些肿瘤细胞对辐射的敏感性。     

Periostin mediates human adipose tissue-derived mesenchymal stem cell-stimulated tumor growth in a xenograft lung adenocarcinoma model

Mesenchymal stem cells stimulate tumor growth in vivo through a lysophosphatidic acid (LPA)-dependent mechanism. However, the molecular mechanism by which mesenchymal stem cells stimulate tumorigenesis is largely elusive. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells (A549 CM) induces expression of periostin, an extracellular matrix protein, in human adipose tissue-derived mesenchymal stem cells (hASCs). A549 CM-stimulated periostin expression was abrogated by pretreatment of hASCs with the LPA receptor 1 (LPA(1)) inhibitor Ki16425 or short hairpin RNA-mediated silencing of LPA(1), suggesting a key role of the LPA-LPA(1) signaling axis in A549 CM-stimulated periostin expression. Using a xenograft transplantation model of A549 cells, we demonstrated that co-injection of hASCs potentiated tumor growth of A549 cells in vivo and that co-transplanted hASCs expressed not only periostin but also α-smooth muscle actin (α-SMA), a marker of carcinoma-associated fibroblasts. Small interfering RNA- or short hairpin RNA-mediated silencing of periostin resulted in blockade of LPA-induced α-SMA expression in hASCs. In addition, silencing of periostin resulted in blockade of hASC-stimulated growth of A549 xenograft tumors and in vivo differentiation of transplanted hASCs to α-SMA-positive carcinoma-associated fibroblasts. Conditioned medium derived from LPA-treated hASCs (LPA CM) potentiated proliferation and adhesion of A549 cells and short interfering RNA-mediated silencing or immunodepletion of periostin from LPA CM abrogated proliferation and adhesion of A549 cells. These results suggest a pivotal role for hASC-secreted periostin in growth of A549 xenograft tumors within the tumor microenvironment.     

A novel compound T7 (N-{4′-[(1E)-N-hydroxyethanimidoyl]-3′,5,6-trimethoxybiphenyl-3-yl}-N′-[4-(3-morpholin-4-ylpropoxy)phenyl]urea) screened by tissue angiogenesis model and its activity evaluation on anti-angiogenesis

A tissue model for angiogenesis that imitated new blood vessels formation in vivo had been established in the previous study. Here, it was used to screen and evaluate a series of synthesized compounds and the results indicated that compound T7 (N-{4′-[(1E)-N-hydroxyethanimidoyl]-3′,5,6-trimethoxybiphenyl-3-yl}-N′-[4-(3-morpholin-4-ylpropoxy)phenyl]urea) could effectively inhibit the blood vessels formation. Then the anti-angiogenic potential of T7 and its related molecular mechanisms against lung carcinoma in vitro and in vivo were investigated. Treatment with T7 significantly inhibited human umbilical vein endothelial cells and A549 cells proliferation and migration. T7 reduced human umbilical vein endothelial cells tube formation as well. Western blotting analysis of cell signaling molecules indicated that T7 reduced phosphorylation of KDR and its downstream signaling players AKT and ERK1/2 activation in endothelial cells and A549 cells. Moreover, T7 inhibited tumor growth in A549 xenografted model of athymic mice and reduced CD34 expression levels in tumor-bearing mice by immunohistochemistry. In sum, our findings showed that T7 was a candidate of tumor angiogenesis inhibitors, and it functioned by interrupting the autophosphorylation of KDR, AKT and ERK1/2.     

Comparative study of p53 expression in human carcinoma cell lines A549 and MCF7 under anticancer drug treatment

The effect of anticancer drugs on the expression of p53 protein in tumor cells was studied using the Western Blot analysis. Human lung carcinoma cell line A549 and human breast carcinoma cell line MCF7 sensitive (WT) and resistant (DOX/R) to doxorubicin were used. An increase in p53 protein expression was found in A549 and MCF7 (WT) cells treated with cisplatin, methotrexate, and doxorubicin, whereas the level of p53 was not statistically significantly changed in the MCF7 DOX/R cells. In the untreated MCF7 DOX/R cells the level of p53 protein was markedly higher than in the untreated WT MCF7 cells. A potential role of p53 protein in the development of doxorubicin-resistance in carcinoma cells is discussed.