Noninvasive Evaluation of Multidrug Resistance via Imaging of ABCG2/BCRP Multidrug Transporter in Lung Cancer Xenograft Models.

Chemotherapy is an important method for the treatment of lung cancer, but multidrug resistance (MDR) greatly reduces the efficacy. The superfamily of ATP-binding cassette (ABC) transport proteins is related to MDR. As a subfamily of ABC proteins, ABCG2/BCRP (breast cancer resistance protein, BCRP) is considered a major player in the development of cancer MDR. For the stratification of chemotherapeutic choices, we constructed Cy5.5- or 89Zr-labeled ABCG2-targeted monoclonal antibody (mAb) ABCG2-PKU1 for noninvasive evaluation of ABCG2 expression in lung cancer xenograft models. ABCG2 expression was screened in H460/MX (mitoxantrone resistant), H460, and H1299 human lung cancer cell lines using Western blotting. ELISA, flow cytometry, and cell immunofluorescent staining were used to evaluate the binding ability of ABCG2-PKU1 to ABCG2 antigen. Lung cancer murine xenograft models were built for in vivo experiments. ABCG2-PKU1 was labeled with Cy5.5 (Cy5.5-ABCG2) for fluorescent imaging and radiolabeled with 89Zr (89Zr-DFO-ABCG2) for immunoPET imaging following the conjugation with p-SCN-deferoxamine (DFO). In vivo imaging was performed in lung cancer models at 2, 24, 48, 72, 96, 120, 144, and 168 h postinjection. Ex vivo biodistribution was conducted after the terminal time point of imaging. Finally, tissue immunohistochemical staining was used to evaluate the tumor expression of ABCG2. Western blotting showed that the H460/MX cells had a high ABCG2 expression level whereas H460 and H1299 had moderate and low levels. ELISA, flow cytometry, and cell immunofluorescent staining results validated the good binding affinity between ABCG2-PKU1 and ABCG2. The H460/MX and H460 cells were used to build positive lung cancer models, and H1299 cells were used to build negative models. The fluorescent imaging showed that the tumor average radiant efficiency of Cy5.5-ABCG2 reached the maximum at 72 and 120 h in H460/MX and H460 respectively (n = 3, P < 0.01). The tumor uptake of Cy5.5-ABCG2 in H1299 (n = 3) was significantly lower than H460/MX and H460 (P < 0.01). ImmunoPET imaging showed that the tumor uptake of 89Zr-DFO-ABCG2 in H460/MX was significantly higher than H460, with a maximum of 4.15 ± 0.41 %ID/g and 2.81 ± 0.24 %ID/g at 168 and 144 h, respectively (n = 5, P < 0.01). The H1299 tumors showed significantly lower uptake than H460/MX and H460 (n = 5, P < 0.01). The radioactive uptake of 89Zr-DFO-ABCG2 among three groups in the heart, liver, and kidney gradually decreased over time. Ex vivo biodistribution verified the differential tumor uptake among the three groups (P < 0.01). Immunohistochemical staining revealed that the H460/MX tumor had the highest expression of ABCG2, whereas H460 and H1299 had the moderate and lowest expression, respectively. Therefore, in this study, fluorescent and immunoPET imaging of lung cancer MDR models using Cy5.5-ABCG2 and 89Zr-DFO-ABCG2 noninvasively evaluated the differential expression of ABCG2, which are expected to be used for the diagnosis and the selection for clinical treatment options for lung cancer MDR patients in future applications.

Lidamycin regulates p53 expression by repressing Oct4 transcription.

Antitumor antibiotic lidamycin (LDM) is widely used in the treatment of a variety of cancers. Here we demonstrated that LDM up-regulates the expression of the tumor suppressor p53 gene by repressing Oct4 transcription. We showed that low dose LDM-induced increase of p53 expression and decrease of Oct4 expression in P19 and HCT116-p53(+/+) cells. Knockdown of Oct4 expression by siRNA led to activation of p53 in both cell lines, whereas ectopical expression of Oct4 significantly inhibited p53 expression in P19 cells. LDM-induced p53 activation was blocked by ectopical expression of Oct4.

Clinical value of the Patient Global Assessment with Ankylosing Spondylitis: A cross-sectional study.

To analyze the factors associated with the overall patient condition and explore the clinical value of the Patient Global Assessment (PGA) index for assessing the disease state in patients with Ankylosing Spondylitis (AS). This cross-sectional study used a standardized questionnaire to record the basic information of patients with AS. The collected data included the Ankylosing Spondylitis Disease Activity Score (ASDAS)-C-reactive protein (CRP), ASDAS-erythrocyte sedimentation rate (ESR), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), PGA, and other clinical indicators. Statistical analysis was performed using SPSS 25.0 software, and the scale was assessed for retest reliability and structural validity. The Kruskal-Wallis H test and Spearman or Pearson correlation analysis were used to analyze the factors influencing PGA scores. The receiver operator characteristic (ROC) curve was used to identify the cutoff value of the PGA for predicting disease activity in AS. The patient age, disease duration, family history, and history of ocular inflammation significantly differed between PGA groups (P < .05). The median PGA was significantly lower in patients with disease remission than in those with disease activity (P < .01). The various clinical indexes significantly differed between PGA groups (P < .01). The PGA was significantly correlated with various clinical indicators (P < .01). The area under the ROC curve (AUC) for disease activity based on the ASDAS-CRP was 0.743 (P < .01) with a PGA cutoff value of 1.38; the AUC for disease activity based on the BASDAI was 0.715 (P < .01) with a PGA cutoff value of 1.63. The PGA was significantly correlated with patient-reported outcomes, disease activity, function, and psychological status, and may indicate the level of inflammation in patients with AS. A PGA of around 1.5 indicates disease activity.

The Role of H3K27me3-Mediated Th17 Differentiation in Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a common chronic progressive inflammatory autoimmune disease. T helper 17 (Th17) cells are the major effector cells mediating AS inflammation. Histone 3 Lys 27 trimethylation (H3K27me3) is an inhibitory histone modification that silences gene transcription and plays an important role in Th17 differentiation. The objective of this study was to investigate the expression of H3K27me3 in patients with AS and to explore its epigenetic regulation mechanism of Th17 differentiation during AS inflammation. We collected serum samples from 45 patients with AS at various stages and 10 healthy controls to measure their Interleukin-17 (IL-17) levels using ELISA. A quantitative polymerase chain reaction was used to quantify the mRNA levels of RORc and the signaling molecules of the JAK2/STAT3 pathway, JMJD3, and EZH2. Additionally, Western blot analysis was performed to quantify the protein levels of H3K27me3, RORγt, JAK2, STAT3, JMJD3, and EZH2 in cell protein extracts. The results showed that H3K27me3 expression in peripheral blood mononuclear cells (PBMCs) was significantly lower in patients with active AS compared to both the normal control groups and those with stable AS. Moreover, a significant negative correlation was observed between H3K27me3 expression and the characteristic transcription factor of Th17 differentiation, RORγt. We also discovered that patients with active AS exhibited significantly higher levels of JMJD3, an inhibitor of H3K27 demethylase, compared to the normal control group and patients with stable AS, while the expression of H3K27 methyltransferase (EZH2) was significantly lower. These findings suggest that H3K27me3 may be a dynamic and important epigenetic modification in AS inflammation, and JMJD3/EZH2 regulates the methylation level of H3K27me3, which may be one of the key regulatory factors in the pathogenesis of AS. These findings contribute to our understanding of the role of epigenetics in AS and may have implications for the development of novel therapeutic strategies for AS.

Lidamycin induces neural differentiation of mouse embryonic carcinoma cells through down-regulation of transcription factor Oct4.

Lidamycin is a potential anti-cancer drug, which is widely used in a variety of human cancer types. It has been reported that lidamycin inhibited mouse embryonic carcinoma (EC) cells growth through down-regulation of embryonic stem (ES) cell-like genes. In this study, whether 0.01 nM lidamycin induces neuronal differentiation of mouse EC cells was investigated. It was observed that lidamycin decreased transcription factor Oct4, and increased both p21 mRNA and protein expression in P19 EC cells. Furthermore, luciferase assay showed that lidamycin activated p21 promoter activity through suppression of Oct4, and Chromatin immunoprecipitation (ChIP) assay confirmed that binding of transcription factor Oct4 to the p21 promoter decreased in lidamycin-exposed cells. Knockdown of Oct4 resulted in neuron-like differentiation and up-regulation of p21 expression. In accordance, overexpression of Oct4 blocked neural differentiation and down-regulated p21 in lidamycin-treated P19 cells. Taken together, these results suggested that neuronal differentiation of EC cells induced by lidamycin was associated with the inhibition of Oct4 expression and the activation of p21 transcription. Our results have provided a novel mechanism, in which lidamycin led to cancer cell differentiation.

Inhibition of mouse embryonic carcinoma cell growth by lidamycin through down-regulation of embryonic stem cell-like genes Oct4, Sox2 and Myc.

Lidamycin (LDM, also known as C-1027) as an anti-cancer agent inhibits growth in a variety of cancer cells by inducing apoptosis and cell cycle arrest. In this study we demonstrated that inhibition of mouse embryonic carcinoma (EC) cell growth using LDM at low concentrations can be attributed to a loss of the cell's self-renewal capability but not to apoptosis or cell death, which can be correlated to the down-regulation of embryonic stem (ES) cell-like genes Oct4, Sox2 and c-Myc. MTT assays showed that LDM inhibited the growth of mouse P19 EC cells in a time- and dose-dependent manner. The EC cells exposed to a low dose (0.01 nM) of LDM lost their capability to generate colonies, as evidenced by the colony forming assay. Flow cytometer analyses demonstrated that LDM induced G1 arrest in exposed EC cells without apoptosis. Real-time qPCR, Western blotting and immunocytochemistry revealed that Oct4, Sox2 and c-Myc were down-regulated in LDM-exposed EC cells, but not adriamycin (ADM)-exposed cells. Furthermore, a combination of the low dose of LDM and ADM significantly reduced the proliferation of the cancer cells than single-agent treatment. This suggested that synergy of ADM and LDM improved chemotherapy. Taking together, our results indicate that LDM can reduce the capability for self-renewal that mouse EC cells possess through the repression of ES cell-like genes, thereby inhibiting carcinoma cell growth. This data also suggests that LDM might have potential for application in CSC-based therapy and be a useful tool for studying ES cell pluripotency and differentiation.

RNF8-mediated regulation of Akt promotes lung cancer cell survival and resistance to DNA damage.

Despite the tremendous success of targeted and conventional therapies for lung cancer, therapeutic resistance is a common and major clinical challenge. RNF8 is a ubiquitin E3 ligase that plays essential roles in the DNA damage response; however, its role in the pathogenesis of lung cancer is unclear. Here, we report that RNF8 is overexpressed in lung cancer and positively correlates with the expression of p-Akt and poor survival of patients with non-small-cell lung cancer. In addition, we identify RNF8 as the E3 ligase for regulating the activation of Akt by K63-linked ubiquitination under physiological and genotoxic conditions, which leads to lung cancer cell proliferation and resistance to chemotherapy. Together, our study suggests that RNF8 could be a very promising target in precision medicine for lung cancer.

LyGDI is a promising biomarker for ovarian cancer.

INTRODUCTION: LyGDI is an inhibitor of Rho protein activation by blocking its transformation between guanosine 5'-diphosphate- and guanosine 5'-triphosphate-bound states. The aim of this study was to investigate the usefulness of LyGDI as a biomarker for the detection of ovarian cancer, and its specificity and sensitivity were compared with those of cancer antigen 125 (CA125). METHODS: The serum levels of LyGDI were determined by enzyme-linked immunosorbent assay in 42 patients with ovarian disease, including 30 ovarian cancers and 12 benign ovarian lesions, and 76 healthy controls. The expression of LyGDI was also evaluated by immunohistochemical staining in resected ovarian tissues of these patients. RESULTS: The serum LyGDI level of cancers was significantly greater than those of the benign and healthy groups (P = 0.002 and P < 0.0001, respectively), whereas no difference was observed between the benign and control groups (P = 0.889). Based upon receiver operating characteristic curve analysis, LyGDI levels were able to distinguish ovarian cancer from benign ovarian disease (P = 0.0001) and healthy control (P < 0.0001; areas under the receiver operating characteristic curves, 0.876 and 0.833, respectively). For ovarian cancers, 83.3% (25/30) or 80.0% (24/30) was identified by serum LyGDI (> or = 1.5 ng/mL) alone or by CA125 (>35 U/mL) alone. It is of particular importance to note that all cancer patients were identified by use of both markers, and the specificity was 83.3% for the benign group. Moreover, in early-stage cancers, 88.9% (8/9) had elevated serum LyGDI levels as compared with 44.4% (4/9) elevation of CA125 levels (P = 0.125). Immunohistochemical staining confirmed the expression of LyGDI on cancerous epithelial cells other than benign ovarian epithelium. CONCLUSIONS: These results suggest that LyGDI has significant potential as a marker for detection of ovarian cancer in the patients with ovarian enlargement, including detection of early-stage cancers.

Identification of novel proteins in chemoresistant lung cancer cells by quantitative proteomics.

Lung cancer is the leading cause of cancer-related deaths worldwide, with a five-year survival rate of only 18%. Non-small cell lung cancer (NSCLC) in addition to large cell lung cancer, comprise 85%-90% of all lung cancer diagnoses. Chemoresistance of the cancer cells is one of the reasons for the poor survival rate. In this research, we used mitoxantrone-induced resistant (MXR) NCl-H460 cells to find the mechanism of chemoresistance. We found that the MXR-resistant cells had high single-cell clonogenic ability like cancer stem cells. From the quantitative proteomics study, we found that the MXR-resistant cells high upregulated many metabolism and stem cell-related proteins, such as STAT3 and ALDH. The high level expression of histone 3.1 showed the possibility of genetic changing of resistant cells. Using Western blot assays, we confirmed enhancement of EZH2 in MXR-resistant NCl-H460 cells. Therefore, the EZH2-STAT3 pathway has an important role in the MXR-resistant NCI-H460 cancer cells. Both EZH2 and STAT3 can be used as new target proteins for chemotherapy in the treatment of large cell lung cancer.

Quantitative proteomic study of mitoxantrone-resistant NCI-H460 cell-xenograft tumors.

Mitoxantrone is one kind of chemical therapy medicine for cancer but certain kinds of cancer cells are chemical-resistant to it. In this research, we analyzed the quantitative proteomic difference between tumors in vivo xenograft by mitoxantrone-resistant (M group) and wild NCI-H460 cells (N group). Protein expression profiling in combination with pathway analysis was deployed to investigate molecular events associated with the tumors using a label-free quantitative proteomic approach. A total of 173 proteins were significantly differentially expressed in mitoxantrone-resistant tumors. Bioinformatics analysis using the cytoscape platform indicated that biological processes, including actin-mediated cell contraction, muscle system process, muscle filament sliding, and muscle contraction, are involved in mitoxantrone-resistance. As KEGG pathway enrichment analysis has shown, systemic lupus erythematosus, alcoholism, viral carcinogenesis, and tight junction are strongly regulated with chemical-resistance. By protein-protein interaction analysis, three protein clusters were found using k-means clustering algorithm. Dysregulation results can be verified by Western blotting. Further studies into the molecular functions of dysregulated proteins will help to provide new perspectives regarding chemoresistance for non-small cell lung cancers.

Lidamycin inhibits the cancer cell PKC activity induced by basic fibroblast growth factor.

AIM: To study the mechanism of inhibition of basic fibroblast growth factor (bFGF) related signal transduction by lidamycin in cancer cells. METHODS: MTT assay was used to determine the growth inhibitory effect of lidamycin (LDM) and adriamycin (ADR) in several cancer cell lines. The inhibition of bFGF bound to its receptor by LDM was measured with [125I]-bFGF binding assay. Intracellular Ca2+ stimulated by bFGF was measured by Fura-3. The formation of bFGF-receptor immune complex and the inhibitory effect of LDM on the activity of PKC isoenzymes induced by bFGF in cancer cells were identified by Western blotting analysis. RESULTS: LDM displayed extremely potent growth inhibitory effect on several cancer cell lines in a dose-dependent manner. A comparison of the IC50 values showed that the effect of LDM was 1000-fold more potent than that of ADR. LDM blocked the specific binding of [125I]-bFGF to rat lung membranes with an IC50 value of 2.0 x 10(-4) nmol x L(-1). As detected by anti-FGFR specific antibody, LDM inhibited the formation of bFGF-receptor immune complex. bFGF induced cytosolic Ca2+ response was obstructed by pretreatment with 10 nmol x L(-1) LDM. Immunoblotting demonstrated that LDM inhibited the activity of PKC isoenzymes in cancer cells stimulated with bFGF. CONCLUSION: The blocking of bFGF receptors in the signal transduction pathway may be involved in the effect of LDM on cancer cells.

Long-term cultured human neural stem cells undergo spontaneous transformation to tumor-initiating cells.

In this report, we describe the spontaneous malignant transformation of long-term cultured human fetal striatum neural stem cells (hsNSCs, passage 17). After subcutaneous transplantation of long-term cultured hsNSCs into immunodeficient nude mice, 2 out of 15 mice formed xenografts which expressed neuroendocrine tumor markers CgA and NSE. T1 cells, a cell line that we derived from one of the two subcutaneous xenografts, have undergone continuous expansion in vitro. These T1 cells showed stem cell-like features and expressed neural stem cell markers nestin and CD133. The T1 cells were involved in abnormal karyotype, genomic instability and fast proliferation. Importantly, after long-term in vitro culture, the T1 cells did not result in subcutaneous xenografts, but induced intracranial tumor formation, indicating that they adjusted themselves to the intracranial microenvironment. We further found that the T1 cells exhibited an overexpressed level of EGFR, and the CD133 positive T1 cells showed a truncation mutation in the exons 2-7 of the EGFR (EGFRvIII) gene. These results suggest that continuous expansion of neural stem cells in culture may lead to malignant spontaneous transformation. This phenomenon may be functionally related to EGFR by EGFRvIII gene mutation.