Celecoxib Blocks Vasculogenic Mimicry via an Off-Target Effect to Radiosensitize Lung Cancer Cells: An Experimental Study.

The resistance to radiotherapy in lung cancer can be attributed to vasculogenic mimicry (VM) to some extent. Celecoxib (CXB), a selective inhibitor of cyclooxygenase-2 (COX-2), is reported as a radiosensitizer in non-small cell lung cancer (NSCLC). However, whether CXB can regulate VM formation via an off-target effect to radiosensitize NSCLC remains unclear. This study aimed to elucidate the mechanism underlying the radiosensitizing effect of CXB on NSCLC, i.e., whether CXB can inhibit VM formation via binding to newly identified targets other than COX-2. CXB radiosensitivity assay was performed in BALB/c mice bearing H460 xenografts and C57 mice bearing Lewis lung cancer (LLC) xenografts, which were divided into the control, CXB, irradiation (IR) treatment, and IR plus CXB groups. VM formation was observed using 3D Matrigel, periodic acid solution (PAS) staining, and immunofluorescence staining. The potential off-targets of CXB were screened using Protein Data Bank (PDB) database, MGLTools 1.5.6, and AutoDock Vina 1.1.2 and confirmed by Western blotting, enzyme activity assay, and RNA interference in vitro experiments and by immunohistochemistry in vivo experiments. CXB treatment almost eliminated the enhancement of VM formation by IR in vitro and in vivo, partially due to COX-2 inhibition. Four potential off-targets were predicted by molecular docking. Among them, aminopeptidase N (APN) and integrin alpha-V (ITAV) were remarkably inhibited in protein expression and enzyme activity in vitro or in vivo, consistent with the remarkable reduction of VM formation in H460 xenografts in BALB/c mice. In conclusion, CXB dramatically blocked VM through inhibiting newly identified off-targets APN and ITAV, other than COX-2, then radiosensitizing NSCLC.

Recruitment of CD11b+Ly6C+ monocytes in non-small cell lung cancer xenografts challenged by anti-VEGF antibody.

A series of antibodies against vascular endothelial growth factor (VEGF) have been developed for the treatment of various types of cancer, including non-small cell lung cancer (NSCLC) in recent years. However, tumors frequently demonstrate resistance to these strategies of VEGF inhibition. Efforts to better understand the mechanism underlying the acquired resistance to anti-VEGF antibodies are warranted. In the present study, in order to develop a xenograft model of acquired resistance to anti-VEGF antibody, xenografts of human adenocarcinoma A549 cells were generated through the successive inoculation of tumor tissue explants into first (F1), second (F2) and third (F3) generations of mice treated with the anti-VEGF antibody B20. Tumor growth rate and vessel-forming ability, assessed via cluster of differentiation (CD) 31 staining, were significantly lower in the F1, F2 and F3 groups compared with in the F0 control group (P<0.01), suggesting that drug resistance was not successfully acquired. The percentages of CD11b+ myeloid-derived suppressor cells and lymphocyte antigen 6C (Ly6C)+ subsets were significantly smaller in F1, F2 and F3 groups compared with in F0 (P<0.01). However, the ratio of Ly6C+ to CD11b+ cells was significantly higher in the F3 group compared with in F0 and F1 groups (P<0.01), indicating increasing recruitment of the Ly6C+ subset with successive challenges with the anti-VEGF antibody. In conclusion, the recruitment of CD11b+Ly6C+ monocytes increased with successive generations of NSCLC-xenografted mice challenged by B20, an anti-VEGF agent.

Induced cancer stem cells generated by radiochemotherapy and their therapeutic implications.

Local and distant recurrence of malignant tumors following radio- and/or chemotherapy correlates with poor prognosis of patients. Among the reasons for cancer recurrence, preexisting cancer stem cells (CSCs) are considered the most likely cause due to their properties of self-renewal, pluripotency, plasticity and tumorigenicity. It has been demonstrated that preexisting cancer stem cells derive from normal stem cells and differentiated somatic cells that undergo transformation and dedifferentiation respectively under certain conditions. However, recent studies have revealed that cancer stem cells can also be induced from non-stem cancer cells by radiochemotherapy, constituting the subpopulation of induced cancer stem cells (iCSCs). These findings suggest that radiochemotherapy has the side effect of directly transforming non-stem cancer cells into induced cancer stem cells, possibly contributing to tumor recurrence and metastasis. Therefore, drugs targeting cancer stem cells or preventing dedifferentiation of non-stem cancer cells can be combined with radiochemotherapy to improve its antitumor efficacy. The current review is to investigate the mechanisms by which induced cancer stem cells are generated by radiochemotherapy and hence provide new strategies for cancer treatment.

Plasma miRNAs in predicting radiosensitivity in non-small cell lung cancer.

BACKGROUND: Radioresistance of thoracic radiotherapy is a major bottleneck in the treatment of non-small cell lung cancer (NSCLC). Until now, there have been no effective biomarkers to predict the radiosensitivity. PURPOSES: Based on miRNA profile screened from NSCLC cell lines with different radiosensitivity, this study was conducted to explore the correlation between plasma miRNAs and radiotherapy response in NSCLC patients, and to identify biomarkers of the radiosensitivity in NSCLC. METHODS: Differentially expressed genes were acquired from time-series gene expression profiles of radioresistant H1299 and radiosensitive H460 lung cancer cells (GSE20549). Potential miRNAs were screened from these differentially expressed genes by combining bioinformatics with GO analysis, pathway analysis, and miRNA prediction. A clinical observational study was performed to explore the correlation between candidate miRNAs and radiotherapy response. Stage IIIa-IV NSCLC patients who received two to four cycles of previous chemotherapy and underwent thoracic radiotherapy alone were included. Total RNA was purified from peripheral blood before radiotherapy, and plasma miRNAs were detected by real-time PCR (qRT-PCR). Then, tumor response, progression-free survival (PFS), and overall survival (OS) were acquired. Four miRNAs significantly different between effective and ineffective groups were further analyzed to obtain cutpoints from receiver operating characteristic (ROC) curves and the predictive value of radiosensitivity. RESULTS: Candidate miRNAs included 14 miRNAs screened from radioresistant genes and five from radiosensitive genes. From Jan., 2013 to Dec., 2014, 54 eligible patients were enrolled with a median follow-up of 15.3 months (range 4.6 to 31.4) by the deadline of Aug. 31, 2015. Totally, there were no case of complete response (CR), 15 of partial response (PR), 35 of stable disease (SD), and 4 of progressive disease (PD). Eight patients had no progression and 19 patients were still alive. The median PFS and OS were 6.6 months (range 2.3 to 29.3) and 15.3 months (range 4.6 to 31.4), respectively. Four miRNAs (hsa-miR-98-5p, hsa-miR-302e, hsa-miR-495-3p, and hsa-miR-613) demonstrated a higher expression in effective group (CR + PR, 15 cases) than in ineffective group (SD + PD, 39 cases). Based on each cutpoint, objective response rate (ORR) was higher in miR-high group than in miR-low group. No miRNA showed correlation with median PFS or OS. CONCLUSION: Bioinformatical analysis and clinical verification reveal the correlation between plasma miRNAs and radiosensitivity in NSCLC patients. Plasma miRNAs represent novel biomarkers to predict radiotherapy response clinically.

Essential role of miR-200c in regulating self-renewal of breast cancer stem cells and their counterparts of mammary epithelium.

BACKGROUND: Breast cancer stem cells (BCSCs) have been reported as the origin of breast cancer and the radical cause of drug resistance, relapse and metastasis in breast cancer. BCSCs could be derived from mutated mammary epithelial stem cells (MaSCs). Therefore, comparing the molecular differences between BCSCs and MaSCs may clarify the mechanism underlying breast carcinogenesis and the targets for gene therapy. Specifically, the distinct miRNome data of BCSCs and MaSCs need to be analyzed to find out the key miRNAs and reveal their roles in regulating the stemness of BCSCs. METHODS: MUC1(-)ESA(+) cells were isolated from normal mammary epithelial cell line MCF-10A by fluorescence-activated cell sorting (FACS) and tested for stemness by clonogenic assay and multi-potential differentiation experiments. The miRNA profiles of MaSCs, BCSCs and breast cancer MCF-7 cells were compared to obtain the candidate miRNAs that may regulate breast tumorigenesis. An miRNA consecutively upregulated from MaSCs to BCSCs to MCF-7 cells, miR-200c, was chosen to determine its role in regulating the stemness of BCSCs and MaSCs in vitro and in vivo. Based on bioinformatics, the targets of miR-200c were validated by dual-luciferase report system, western blot and rescue experiments. RESULTS: In a 2-D clonogenic assay, MUC1(-)ESA(+) cells gave rise to multiple morphological colonies, including luminal colonies, myoepithelial colonies and mixed colonies. The clonogenic potential of MUC1(-)ESA(+) (61.5 ± 3.87 %) was significantly higher than that of non-stem MCF-10A cells (53.5 ± 3.42 %) (P < 0.05). In a 3-D matrigel culture, MUC1(-)ESA(+) cells grew into mammospheres with duct-like structures. A total of 12 miRNAs of interest were identified, 8 of which were upregulated and 4 downregulated in BCSCs compared with MaSCs. In gain- and lost-of-function assays, miR-200c was sufficient to inhibit the self-renewal of BCSCs and MaSCs in vitro and the growth of BCSCs in vivo. Furthermore, miR-200c negatively regulated programmed cell death 10 (PDCD10) in BCSCs and MaSCs. PDCD10 could rescue the tumorigenesis inhibited by miR-200c in BCSCs. DISCUSSION: Accumulating evidence shows that there is a milignant transformation from MaSCs into BCSCs. The underlying mechanism remains unclear. In present study, miRNA profiles between MaSCs and BCSCs were obtained. Then miRNA-200c, downregulated in both MaSCs and BCSCs, were verified as anti-oncogene, and played essential role in regulating self-renewal of both kinds of stem-like cells. These findings reveal a novel insights of breast tumorigenesis. CONCLUSIONS: PDCD10 is a target gene of miR-200c and also a possible mechanism by which miR-200c plays a role in regulating the stemness of BCSCs and MaSCs.

Regulation of miRNAs affects radiobiological response of lung cancer stem cells.

Radiotherapy (RT) is a key therapeutic strategy for lung cancer, the most common cause of cancer-related deaths worldwide, but radioresistance often occurs and leads to failure of RT. It is therefore important to clarify the mechanism underlying radioresistance in lung cancer. Cancer stem cells (CSCs) are considered the fundamental reason for radioresistance. MicroRNAs (miRNAs) have been regarded as important regulatory molecules of CSCs, carcinogenesis, and treatment response of cancers. It is crucial to clarify how regulation of miRNAs affects repair of DNA damage, redistribution, repopulation, reoxygenation, and radiosensitivity (5R) of lung cancer stem cells (LCSCs). A thorough understanding of the regulation of miRNAs affecting 5R of LCSCs has potential impact on identifying novel targets and thus may improve the efficacy of lung cancer radiotherapy.

Molecular mechanism of microRNA involvement in genesis of myelodysplastic syndrome and its transformation to acute myeloid leukemia.

Myelodysplastic syndrome (MDS) is a group of blood-related malignancies caused by disorders of hematopoietic stem cells (HSCs). It is mainly characterized by the ineffective hematopoiesis. Many MDS patients will transform into acute myeloid leukemia (AML) with poor prognosis, often due to infection, hemorrhage and death. However, the molecular mechanism underlying the transformation remains unknown. MicroRNAs (miRNAs), a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression, are considered playing an important role in regulating gene translation during development. Aberrant expression of miRNAs plays an important role in the mutation of HSCs and their transformation to MDS stem cells, suggesting an important mechanism of MDS pathogenesis and its transformation to leukemia. So far, few studies have focused on molecular mechanisms of the transformation of MDS to AML. The role of miRNAs in the transformation needs to be fully understood. Here, we reviewed the molecular mechanism of miRNA involvement in the damage of HSCs, the pathogenesis of MDS and its transformation to leukemia. These results will be of theoretical importance for understanding the mechanism underlying the pathogenesis of MDS, and have potential applications in the prevention and treatment of MDS and in preventing its transformation to leukemia.

Role of inhibitor of apoptosis protein Livin in radiation resistance in nonsmall cell lung cancer.

OBJECTIVE: The objective of the present study was to explore the role of the inhibitor of apoptosis protein (IAP) Livin in radioresistance in nonsmall cell lung cancer (NSCLC). METHODS: Lung adenocarcinoma cell lines A549 and SPC-A1 were used for this study. Using the technique of molecular cloning and gene transfection, two Livin isoforms, Livinα and ß, respectively, were expressed in A549 cells with the purpose of exploring the role of Livin in radiation resistance of A549 cells. Moreover, a Livin-specific gene-silencing system was developed using SPC-A1 cell line with the purpose of increasing radiosensitivity of SPC-A1 cells. RESULTS: A549 cells were induced by radiation to express Livin isoforms, Livinα and ß. A549 cells expressed Livin isoforms stably after gene transfection and the transfected cells demonstrated characteristics of antiradiation. However, Livin gene-silenced SPC-A1 cells exhibited remarkably enhanced radiation sensitivity. CONCLUSION: The IAP Livin is an important molecule in antiradiotherapy of NSCLC. Livin-specific gene silencing is likely to be an effective means to enhance radiation sensitivity of lung cancer.

Microarray-based analysis of microRNA expression in breast cancer stem cells.

BACKGROUND: This study aimed to determine the miRNA profile in breast cancer stem cells (BCSCs) and to explore the functions of characteristic BCSC miRNAs. METHODS: We isolated ESA+CD44+CD24-/low BCSCs from MCF-7 cells using fluorescence-activated cell sorting (FACS). A human breast cancer xenograft assay was performed to validate the stem cell properties of the isolated cells, and microarray analysis was performed to screen for BCSC-related miRNAs. These BCSC-related miRNAs were selected for bioinformatic analysis and target prediction using online software programs. RESULTS: The ESA+CD44+CD24-/low cells had up to 100- to 1000-fold greater tumor-initiating capability than the MCF-7 cells. Tumors initiated from the ESA+CD44+CD24-/low cells were included of luminal epithelial and myoepithelial cells, indicating stem cell properties. We also obtained miRNA profiles of ESA+CD44+CD24-/low BCSCs. Most of the possible targets of potential tumorigenesis-related miRNAs were oncogenes, anti-oncogenes or regulatory genes. CONCLUSIONS: We identified a subset of miRNAs that were differentially expressed in BCSCs, providing a starting point to explore the functions of these miRNAs. Evaluating characteristic BCSC miRNAs represents a new method for studying breast cancer-initiating cells and developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation of cells in breast cancer.

Detection of lymphangiogenesis in non-small cell lung cancer and its prognostic value.

BACKGROUND: Our aim was to detect lymphatic endothelial marker podoplanin, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and vascular endothelial growth factor receptor-3 (VEGFR)-3 and study the prognostic relevance of lymphangiogenesis in non-small cell lung cancer (NSCLC). MATERIALS: 82 paraffin-embedded tissues and 40 fresh frozen tissues from patients with NSCLC were studied. Tumor samples were immunostained for the lymphatic endothelial markers. Lymphangiogenesis was assessed by immunohistochemical double stains for Podoplanin and Ki-67. The prognostic relevance of lymphangiogenesis-related clinicopathological parameters in NSCLC was evaluated. RESULTS: We found that the number of podoplanin positive vessels was correlated positively with the number of LYVE-1 positive vessels. Most of VEGFR-3 positive, few of LYVE-1 positive and none of podoplanin positive vessels were blood vessels. Peritumoral lymphatic vessel density (ptLVD), pathologic stage, lymph node status, lymphatic vessel invasion (LVI), vascular endothelial growth factor-C (VEGF-C) expression and Ki-67 index of the endothelium cells of the micro lymphatic vessels (Ki67%) were associated significantly with a higher risk of tumor progress. ptLVD, pathologic stage, lymph-node metastasis and Ki67% were independent prognostic parameters for overall survival. CONCLUSION: Podoplanin positive ptLVD might play important roles in the lymphangiogenesis and progression of NSCLC. Patients with high podoplanin+ ptLVD have a poor prognosis.

Tumor targeting of vincristine by mBAFF-modified PEG liposomes in B lymphoma cells.

Malignant hematologic diseases are highly malignant and refractory to conventional therapies. Ligand-mediated targeting of liposomal anticancer drugs to surface receptors expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. BAFF plays an important role in the maintenance of normal B-cell development and homeostasis and the expression of its receptors is significantly increased in numerous B-cell malignancies. mBAFF (a soluble BAFF mutant with amino acid 217-224 being replaced by two glycine residues) may be used as a competitive inhibitor for BAFF to treat relevant malignant hematologic diseases. It may also hold promise as a novel ligand for targeted anticancer therapy. In this study, we show that liposomes that are sterically stabilized by PEG and surface decorated with mBAFF exhibited strong affinity and specificity to cultured human Raji B lymphoma cells. Vincristine formulated in the targeted liposomes showed significantly higher levels of cytotoxicity towards Raji cells than the nontargeted liposomal drug. Therapeutic experiments in SCID mice implanted with Raji cells showed significantly prolonged survival time with targeted liposomal vincristine compared to either free VCR or vincristine formulated in nontargeted liposomes. These studies suggest the potential of the mBAFF-modified liposomal drugs in targeted therapy of B-cell malignancies.

MicroRNAs play a role in the development of human hematopoietic stem cells.

MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene translation during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. We isolated CD34(+)CD38(-) hematopoietic stem cells (HSCs) from human umbilical cord blood, on the basis of cell-surface markers using fluorescence-activated cell sorting (FACS). Also, CD34(+) subpopulation was FACS isolated as the control. Next, using microarray containing oligonucleotides corresponding to 517 miRNAs from human, mouse, and rat genomes, we obtained miRNA gene expression profiles of both subpopulations. We focused on the HSCs correlative miRNAs with comparison to the control. The miRNAs of particular interest were confirmed by real-time RT-PCR. HSCs-overexpressed hsa-miR-520h and underexpressed hsa-miR-129 were selected for target prediction and analysis. The result showed that EIF2C3 and CAMTA1, genes related to miRNAs processing or transcription regulation, were proved to be real targets for hsa-miR-129. And ABCG2, involved in stemness maintaining, a real target for hsa-miR-520h. Finally, we chose hsa-miR-520h, enriched in HSCs but low in CD34(+) cells, for functional characterization, because of its possible role in differentiation of HSCs by regulating ABCG2. As a result, hsa-miR-520h transduction into CD34(+) cells greatly increased number of different progenitor colonies in Colony-Forming-Cell assays, suggesting that hsa-miR-520h may promote differentiation of HSCs into progenitor cells by inhibiting ABCG2 expression. This study paves the way for identifying HSC-specific miRNAs and their roles in HSC development.

Functional analysis on the 5'-flanking region of human FXR gene in HepG2 cells.

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Although modulation of FXR expression has been reported, the mechanisms underlying the regulation of human FXR are yet unclear. Functional assays showed that the -150/+29 nucleotides region from the first nucleotide at the Exon I is the minimal promoter of the human FXR gene by the technique of serial deletion and point mutants of the 5'-flanking region. Chromatin immunoprecipitation analysis and electrophoretic mobility shift assay revealed that hepatic nuclear factor 1alpha (HNF1alpha) interacted with the region. Co-transfection of the promoter with HNF1alpha expression vectors enhanced promoter activity of FXR gene. Over-expression of HNF1alpha up-regulated FXR expression in HepG2 cells. These data indicate that (a) the identified HNF1alpha binding site serves as a positive regulatory sequence, (b) the binding site is functionally active both in vivo and in vitro, and (c) the transcription factor HNF1alpha that binds to this site plays an important role in the regulation of human FXR promoter activity.

[Study on enhancing sensitivity of SPC-A1 cells to chemotherapy by Livin isoform-specific gene silencing].

BACKGROUND: As a new member of inhibitor of apoptosis protein(IAP) family,Livin,especially Livin α,is known to be involved in occurrence and development of lung cancer.Livin is an important mechanism of chemotherapy resistance of lung cancer cell.The aim of this study is to set up Livin isoform(α & ß)-specific gene silencing system in SPC-A1 cells by gene transfection and RNA interference(RNAi),and to explore the different functions and value of the isoforms in enhancing chemosensitivity of SPC-A1 cells. METHODS: Livinα+ß,Livinα and Livinß specific siRNA were expressed stably in SPC-A1 cells,respectively.MTT was performed to study sensitivity of the cells to chemotherapy drugs.In vivo experiment was performed to test sensitivity of mouse bearing tumor to cisplatin after gene silencing of Livin. RESULTS: After silencing of Livinα+ß,Livinα and Livinß genes,sensitivity of SPC-A1 cells to many chemotherapy drugs(including cisplatin,carboplatin,cyclophosphamide and adriblastine) was markedly increased(P < 0.05).Among them,gene silencing of Livinα+ß showed the strongest enhancement effect on chemosensitivity of SPC-A1 cells(P < 0.01).Animal experiment showed that tumor inhibition rate of pSilencer-Livinα+ß,pSilencer-Livinα and pSilencer-Livinß groups was 146.1%,130.7% and 110.5%,respectively. CONCLUSIONS: The results suggest that Livin isoform,especially Livinα+ß is hopeful to be a molecular target for increasing sensitivity of lung cancer cell to chemotherapy.Gene silencing may be a new means of gene therapy for non-small cell lung cancer.