Purpose: For treatment decision and prognostic applications, we evaluated p53/epidermal growth factor receptor (EGFR) somatic aberrations in multiple primary lung cancers to differentiate multifocal tumors from intrapulmonary metastasis. Experimental Design: Fifty-eight multiple primary lung cancers of 1,037 patients in a 10-year period were identified to investigate somatic mutations and altered expression of p53 and EGFR for clonality assessment. Genomic DNA was extracted from microdissected cells of paraffinembedded multiple primary lung cancer tissues. Overexpression and somatic mutations in exons of p53 (exons 5-8) and tyrosine kinase domain of EGFR (exons 18-22) were examined by immunohistochemical staining and DNA sequencing, respectively. Results: High frequency of somatic mutations in p53 (33 of 58, 56.9%) and/or EGFR (44 of 58, 75.9%) resulted in high discrimination rate of tumor clonality (50 of 58, 86.2%) of multiple primary lung cancers. Twenty-two cases (37.9%) were assessed as having the same clonality and 28 cases (48.3%) were determined as having different clonality, which further supported the carcinogenic theory of field cancerization. Notably, the occurrence of lymph node metastasis was more commonly observed in tumors with the same clonality (P = 0.045) and was associated with poor patient 5-year survival rate (P = 0.001). However, no correlation was found between tumor clonality and patient survival (P = 0.630). The EGFR somatic aberrations in 58 multiple primary lung cancers, including vascular invasion associated with EGFR overexpression (P = 0.012) and mutation (P = 0.025), further suggested the potential benefits of target therapy of inoperable multiple primary lung cancers. Conclusions: Our results suggest that analysis of somatic alterations in p53 and EGFR can significantly improve the clonality assessment and impact management of multiple primary lung cancer patients.
Luteolin is a falvonoid compound derived from Lonicera japonica Thunb. Numerous reports have demonstrated that luteolin has anticancer effects on many kinds of tumors. This study investigated the effects of luteolin on prostate cancer (PCa), assessing the PC3 and LNCaP cells. The cell viability and apoptosis were assessed by performing Cell Counting Kit-8 assay and Annexin V–fluorescein isothiocyanate/propidium iodide double staining. Luteolin was found to inhibit androgen-sensitive and androgen-independent PCa cell lines’ growth and induced apoptosis. To uncover the exact mechanisms and molecular targets, microRNA (miR) array analysis was performed. miR-301 was found to be markedly downregulated. Then, the expression of miR-301 was retrospectively analyzed in the primary PCa tissues by quantitative reverse transcription polymerase chain reaction and in situ hybridization methods. According to the quantitative reverse transcription polymerase chain reaction results of miR-301, the 54 PCa patients were divided into two groups: high and low miR-301 groups. The division indicator is a relative expression ≥5. Compared to the low-expression group, high miR-301 expression was associated with a significantly shorter overall survival (P=0.029). The proapoptotic gene, DEDD2, was predicted to be the direct target of miR-301. It was clarified in accordance with bioinformatics and luciferase activity analyses. The overexpression of miR-301 by plasmid decreased the luteolin effect. Taken together, these results suggest that luteolin inhibits PCa cell proliferation through miR-301, the poor predictive factor of PCa.
Aim: Multi-drug resistance poses a critical bottleneck in chemotherapy. given the up-regulation of mToR pathway in many chemoresistant cancers, we examined whether sirolimus (rapamycin), a first generation mTOR inhibitor, might induce human osteosarcoma (os) cell apoptosis and increase the sensitivity of os cells to anticancer drugs in vitro. Methods: human os cell line Mg63/aDM was treated with sirolimus alone or in combination with doxorubicin (aDM), gemcitabine (GEM) or methotrexate (MTX). Cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry, respectively. MiRnas in the cells were analyzed with miRna microarray. The targets of miR-34b were determined based on Targetscan analysis and luciferase reporter assays. The expression of relevant mRna and proteins was measured using qRT-PCR and Western blotting. MiR-34, PaK1 and aBCB1 levels in 40 tissue samples of os patients were analyzed using qRT-PCR and in situ hybridization assays. Results: Sirolimus (1−100 nmol/L) dose-dependently suppressed the cell proliferation (IC 50 =23.97 nmol/L) and induced apoptosis. Sirolimus (10 nmol/L) significantly sensitized the cells to anticancer drugs, leading to decreased IC 50 values of aDM, geM and MTX (from 25.48, 621.41 and 21.72 μmol/L to 4.93, 73.92 and 6.77 μmol/L, respectively). Treatment of with sirolimus increased miR-34b levels by a factor of 7.5 in the cells. upregulation of miR-34b also induced apoptosis and increased the sensitivity of the cells to the anticancer drugs, whereas transfection with miR-34b-aMo, an inhibitor of miR-34b, reversed the anti-proliferation effect of sirolimus. Two key regulators of cell cycle, apoptosis and multiple drug resistance, PaK1 and aBCB1, were demonstrated to be the direct targets of miR-34b. In 40 tissue samples of OS patients, significantly higher miR-34 ISH score and lower PAK5 and ABCB1 scores were detected in the chemo-sensitive group. Conclusion: sirolimus increases the sensitivity of human os cells to anticancer drugs in vitro by up-regulating miR-34b interacting with PaK1 and aBCB1. a low miR-34 level is an indicator of poor prognosis in os patients.
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