Chemoresistance Of Non-small Cell Lung Cancer Cells Research Articles

Hsa_circ_0000190 Promotes NSCLC Cell Resistance to Cisplatin via the Modulation of the miR-1253/IL-6 Axis.

This study explored the mechanistic basis for nonsmall cell lung cancer (NSCLC) cisplatin (DDP) treatment resistance in an effort to define effective approaches to abrogating the emergence of such chemoresistance. Analyses of NSCLC expression of hsa_circ_0000190, miR-1253, and interleukin 6 (IL-6) were conducted via a quantitative real-time polymerase chain reaction (qPCR) approach, while the ability of these tumor cells to resist DDP treatment was evaluated with a CCK-8 assay. Interactions between different RNA molecules were assessed using both RNA immunoprecipitation and dual-luciferase reporter assays. NSCLC cell lines and tissues resistant to DDP were found to express higher levels of hsa_circ_0000190, and knocking down this circRNA in NSCLC cells was associated with greater sensitivity to DDP exposure. Further research identified miR-1253 as a hsa_circ_0000190 target, with the ability of hsa_circ_0000190 knockdown to restore DDP sensitivity being largely attributable to the ability of this circRNA to suppress miR-1253 activity. IL-6 was identified as a major miR-1253 target in this context, with miR-1253 regulating chemoresistance in NSCLC cells in part by preventing IL-6 upregulation. Together, these data suggest that hsa_circ_0000190 can promote DDP chemoresistance in NSCLC cells through its ability to modulate miR-1253/IL-6 axis activity, highlighting a novel pathway that can be targeted in an effort to guide the more effective diagnosis and management of DDP-resistant tumors.

Survivin degradation by bergenin overcomes pemetrexed resistance.

Chemoresistance is a primary factor for treatment failure and tumor recurrence in non-small cell lung cancer (NSCLC) patients. The oncoprotein survivinis commonly upregulated in human malignancies and is associated with poor prognosis, but its effect on carcinogenesis and chemoresistance in NSCLC is not yet evident, and to explore an effective inhibitor targeting survivin expression is urgently needed. The protumor characteristics of survivin and antitumor activities of bergenin in NSCLC cells were examined by MTS, colony formation assays, immunoblot, immunohistochemistry, and in vivo xenograft development. Survivin was upregulated in non-small cell lung cancer (NSCLC) tissues, while its depletion inhibited NSCLC tumorigenesis. The current study focused on bergenin, identifying its effective antitumor effect on NSCLC cells both in vivo and in vitro. The results showed that bergenin could inhibit cell proliferation and induce the intrinsic pathway of apoptosis via downregulating survivin. Mechanistically, bergenin reduced the phosphorylation of survivin via inhibiting the Akt/Wee1/CDK1 signaling pathway, thus resulting in enhanced interaction between survivin and E3 ligase Fbxl7 to promote survivin ubiquitination and degradation. Furthermore, bergenin promoted chemoresistance in NSCLC cells re-sensitized to pemetrexed treatment. Survivin overexpression is required for maintaining multiple malignant phenotypes of NSCLC cells. Bergenin exerts a potent antitumor effect on NSCLC via targeting survivin, rendering it a promising agent for the treatment of NSCLC.

MiR-466 Contributes to the Enhanced Antitumor Effect of Bortezomib on Non-Small-Cell Lung Cancer by Inhibiting CCND1

Introduction: Changes in microRNAs (miRs) contribute to the alternative chemo-resistance of cancers. Bortezomib (BTZ) is a well-characterized anticancer agent that inhibits proteasome, and its effect is associated with the function of miRs. Based on the data of microarray assay and comprehensive bioinformatics analyses, in the current study, we explored the role of miR-466 and its downstream effector CCND1 in the BTZ-resistance of non-small-cell lung cancer (NSCLC) cells. Methods: miR expression profiles in NSCLC tissues and paratumor tissues were determined with microarray assay. The potential miR involved in the chemo-resistance of NSCLC cells was explored via a series of bioinformatics analyses, and miR-466 was selected. Afterward, levels of miR-466 and CCND1 were investigated in NSCLC samples and analyzed by clinicopathologic parameters, including age, sex, stage of NSCLC, tumor size, tumor differentiation status, and lymphocytic infiltration status. The expression of CCND1 and miR-466 was then modulated in vitro to explore the influence on cell phenotypes, which was then verified with mouse models. Results: Based on microarray detection, 287 miRs were dysexpressed between NSCLC tissues and paratumor tissues, including 90 upregulated members and 197 downregulated members. After bioinformatics analyses and reverse transcription quantitative PCR validation, miR-466 and CCND1 were selected. Following clinical investigations, miR-466 was downregulated, while CCND1 was upregulated in NSCLC samples, contributing to the advanced cancer progression. The overexpression of CCND1 increased cell viability, suppressed cell apoptosis, decreased p21 and induced N-cadherin, CCND2, and CDK4 under BTZ treatment. The induced expression of miR-466 re-sensitized NSCLC cells to BTZ treatment. In the animal model, the overexpression of CCND1 impaired the inhibitory effect of BTZ on the growth and metastasis of solid tumor, which was restored by miR-466 induction. Conclusion: The findings showed that the interaction between BTZ, miR-466, and CCND1 determined the antitumor effect of BTZ on NSCLC.

Molecular profiles of predictive biomarkers for platinum-based chemotherapy in Non-Small Cell Lung Cancer (NSCLC)

BackgroundNon-small cell lung cancer (NSCLC) is the principal subtype of lung cancer. Among all therapeutic options, platinum-based chemotherapy agents, especially Cisplatin, are still commonly used treatment for NSCLC patients. However, developing chemoresistance in NSCLC cells often gives rise to chemotherapy failure. Therefore, more studies are required to shed light on gene interaction and cellular pathways involved in initiating and developing resistance to platinum-based chemotherapy in NSCLC. Hence, it is urgent to find the key genes, microRNA (miRNAs), and potential molecular mechanisms implicated in chemoresistance and present markers to predict response to platinum-based chemotherapy in NSCLC patients. MethodsThe microarray datasets GSE6410, GSE7035, GSE14814, GSE26704, GSE73302 were downloaded from the Gene Expression Omnibus (GEO) database and were analyzed using R software. Functional and pathway enrichment analyses were performed using the Enrich R site. Then, the protein-protein interaction (PPI) network and hub genes were obtained using the Cytoscape software. Further, the miRSystem database was performed to predict the miRNAs regulating the hub genes. Moreover, Cytoscape software and the CytoHubba plugin were used to construct the miRNA-target interaction network and hub modules. Finally, the Kaplan–Meier curve was used to demonstrate the survival curves and assess the association of the genes signature with clinical outcomes. ResultsA total of 142 differentially expressed genes (DEGs) were found. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses present the p53 signaling pathway as the most significant pathway.Besides, from the top ten terms obtained of Biological Process, Molecular Function, and Cellular Component, the first ones, including cholesterol biosynthetic process, the extrinsic component of external side of plasma membrane, cytokine activity, were selected respectively. Based on the PPI network, the ten nodes with the highest degree were screened as hub genes. In addition, from the miRNA–target regulatory network in Cytoscape, ten hub nodes were found. Ultimately, according to Kaplan–Meier curve, BTG2 and TP53I3 with p-value <0.05 were associated with a better prognosis. ConclusionsIn the present study, DEGs, candidate miRNAs, and underlying mechanisms involved in chemoresistance were identified to suggest potential biomarkers to provide new clues for the prediction of response to platinum-based chemotherapy.

CAFs-Derived Exosomal miRNA-130a Confers Cisplatin Resistance of NSCLC Cells Through PUM2-Dependent Packaging.

PurposeChemoresistance is a significant barrier to the treatment and management of non-small cell lung cancer (NSCLC). Exosomes play an essential role in intercellular communication. Understanding the mechanism underlying the role of tumor stroma, especially cancer-associated fibroblasts (CAFs), during chemoresistance would significantly contribute to the clinical application of chemotherapy agents.ResultsIn this study, we demonstrated that NSCLC-derived CAFs were innately resistant to cisplatin treatment and CAFs-conditioned medium significantly promoted the survival rate of NSCLC cells after cisplatin treatment. Additionally, CAFs-derived exosomes were taken up by NSCLC cells. Moreover, exosomal miRNA-130a was transferred from CAFs to recipient NSCLC cells and knockdown of miRNA-130a reversed the effect of CAFs-derived exosomes during chemoresistance of NSCLC cells. Furthermore, pumilio homolog 2 (PUM2), a RNA-binding protein, mediated the packaging of miRNA-130a into exosomes. The overexpression and knockdown of PUM2 promoted and inhibited tumor growth of xenograft mice, respectively.ConclusionTaken together, these results suggest that CAFs-derived exosomes confer cisplatin resistance of NSCLC cells through transferring miRNA-130a and that PUM2 is a critical factor for packaging miRNA-130a into exosomes. This study indicates that CAFs-derived exosomal miRNA-130a may be a potential therapeutic target for cisplatin resistance in NSCLC.

CircRNA CDR1as/miR-641/HOXA9 pathway regulated stemness contributes to cisplatin resistance in non-small cell lung cancer (NSCLC)

BackgroundCisplatin (DDP) is the first-line chemotherapeutic drug for non-small cell lung cancer (NSCLC), and long-term DDP stimulation increased resistance of NSCLC cells to this drug by enriching cancer stem cells (CSCs), which contributed to recurrence and worse prognosis of NSCLC, but the molecular mechanisms are still not fully delineated.MethodsReal-Time qPCR and Western Blot analysis were conducted to examine gene expressions at mRNA and protein levels, respectively. Dual-luciferase reporter gene system was used to validate the targeting sites among circRNA CDR1as, miR-641 and HOXA9 mRNA. Cell growth was evaluated by CCK-8 assay, trypan blue staining assay and colony formation assay. The Annexin V-FITC/PI double staining method was employed to measure cell apoptosis ratio. Spheroid formation and flow cytometer assay was used to evaluate cell stemness. Xenograft mice models were established to measure tumorgenicity in vivo, and Ki67 expressions in mice tumor tissues were examined by immunohistochemistry (IHC).ResultsHere we identified a novel circRNA CDR1as/miR-641/Homeobox protein Hox-A9 (HOXA9) pathway regulated stemness and DDP chemoresistance in NSCLC. Mechanistically, circRNA CDR1as and HOXA9 were high-expressed, while miR-641 was low-expressed in DDP-resistant NSCLC cells, instead of their corresponding parental DDP-sensitive NSCLC cells. Additionally, we validated that circRNA CDR1as positively regulated HOXA9 in NSCLC cells by serving as an RNA sponge for miR-641, and knock-down of circRNA CDR1as increased the sensitivity of DDP-resistant NSCLC cells, which were reversed by downregulating miR-641 and upregulating HOXA9. Consistently, overexpression of circRNA CDR1as increased drug resistance of DDP-sensitive NSCLC cells by regulating miR-641/HOXA9 axis. In addition, the expression levels of stemness signatures (SOX2, OCT4 and Nanog) were higher in DDP-resistant NSCLC cells, which also tended to form spheres and enrich CD44+CD166+ population compared to their parental DDP-sensitive NSCLC cells, suggesting that CSCs were enriched in DDP-resistant NSCLC cells. Notably, knock-down of circRNA CDR1as inhibited stemness of DDP-resistant NSCLC cells by inhibiting HOXA9 through upregulating miR-641.ConclusionsTaken together, this study identified that circRNA CDR1as regulated stemness and DDP chemoresistance in NSCLC cells by targeting miR-641/HOXA9 axis.

MiR-199a-5p is involved in doxorubicin resistance of non-small cell lung cancer (NSCLC) cells

Non-small cell lung cancer (NSCLC) is one of the prevalent and deadly cancers worldwide. Chemotherapy resistance is one of the most challenging problems for NSCLC and other cancer treatment. Recent study suggested that miRNAs are involved in therapeutic functions of chemotherapy during cancer treatment. Our present study established doxorubicin (Dox) resistant NSCLC A549 and H460 cells (named A549Dox/R and H460 Dox/R). We found that miR-199a-5p was significantly down regulated in Dox resistant cells. Over expression of miR-199a-5p can increase the Dox sensitivity of resistant cells. Among various targets of miR-199a-5p, chemoresistance can increase the expression of ABCC1 and HIF-1α. Gain and loss of function studies confirmed that both ABCC1 and HIF-1α were involved in the chemoresistance of NSCLC cells. Collectively, our data showed that miR-199a-5p regulated expression of ABCC1 and HIF-1α were involved in Dox resistance of NSCLC.

The miRNA-149-5p/MyD88 axis is responsible for ursolic acid-mediated attenuation of the stemness and chemoresistance of non-small cell lung cancer cells.

Although the inhibitory roles of ursolic acid (UA) have been established in various tumors, its effects on the stemness of non-small cell lung cancer (NSCLC) cells are still unclear. Here, we constructed NSCLC cells with paclitaxel resistance (A549-PR) and showed that A549-PR exhibited a remarkably stronger stemness than the parental A549 cells, which is evident by the increase of spheroid formation capacity, stemness marker expression, and ALDH1 activity. Additionally, UA significantly reduced the stemness and paclitaxel resistance of A549-PR cells. Mechanistic investigations revealed that UA inhibited the miR-149-5p/MyD88 signaling, which is responsible for UA-mediated effects on the stemness of A549-PR cells. Notably, miR-149-5p/MyD88 axis promoted the stemness of A549 cells, while inhibition of this axis attenuated the stemness of A549-PR cells. Therefore, these results suggest that UA could attenuate the stemness and chemoresistance of NSCLC cells through targeting miR-149-5p/MyD88 axis.

PIDD interaction with KEAP1 as a new mutation-independent mechanism to promote NRF2 stabilization and chemoresistance in NSCLC

Chemotherapy resistance is a major problem in non-small cell lung cancer (NSCLC) treatment. A major mechanism of chemoresistance involves stabilization of the NRF2 transcription factor. NRF2 levels are normally tightly regulated through interaction with KEAP1, an adaptor that targets NRF2 to the CUL3 E3 ubiquitin ligase for proteolysis. In NSCLC, aberrant NRF2 stabilization is best understood through mutations in NRF2, KEAP1, or CUL3 that disrupt their interaction. Biochemical studies, however, have revealed that NRF2 can also be stabilized through expression of KEAP1-interacting proteins that competitively sequester KEAP1 away from NRF2. Here, we have identified PIDD, as a novel KEAP1-interactor in NSCLC that regulates NRF2. We show that this interaction allows PIDD to reduce NRF2 ubiquitination and increase its stability. We also demonstrate that PIDD promotes chemoresistance in NSCLC cells both in vitro and in vivo, and that this effect is dependent on NRF2. Finally, we report that NRF2 protein expression in a NSCLC cohort exceeds the typical incidence of combined NRF2, KEAP1, and CUL3 mutations, and that NRF2 expression in this cohort is correlated with PIDD levels. Our data identify PIDD as a new NRF2 regulator, and suggest that variations in PIDD levels contribute to differential chemosensitivities among NSCLC patients.

LncRNA MALAT1 Depressed Chemo-Sensitivity of NSCLC Cells through Directly Functioning on miR-197-3p/p120 Catenin Axis.

This study was aimed to explore if lncRNA MALAT1 would modify chemo-resistance of non-small cell lung cancer (NSCLC) cells by regulating miR-197-3p and p120 catenin (p120-ctn). Within this investigation, we totally recruited 326 lung cancer patients, and purchased 4 NSCLC cell lines of A549, H1299, SPC-A-1 and H460. Moreover, cisplatin, adriamycin, gefitinib and paclitaxel were arranged as chemotherapies, and half maximal inhibitory concentration (IC50) values were calculated to evaluate the chemo-resistance of the cells. Furthermore, mice models of NSCLC were also established to assess the impacts of MALAT1, miR-197-3p and p120-ctn on tumor growth. Our results indicated that MALAT1 and miR-197-3p were both over-expressed within NSCLC tissues and cells, when compared with normal tissues and cells (P < 0.05). The A549, H460, SPC-A-1 and SPC-A-1 displayed maximum resistances to cisplatin (IC50 = 15.70 μg/ml), adriamycin (IC50 = 5.58 μg/ml), gefitinib (96.82 μmol/L) and paclitaxel (141.97 nmol/L). Over-expression of MALAT1 and miR-197-3p, or under-expression of p120-ctn were associated with promoted viability and growth of the cancer cells (P < 0.05), and they could significantly strengthen the chemo-resistance of cancer cells (P < 0.05). MALAT1 Wt or p120-ctn Wt co-transfected with miR-197-3p mimic was observed with significantly reduced luciferase activity within NSCLC cells (P < 0.05). Finally, the NSCLC mice models were observed with larger tumor size and weight under circumstances of over-expressed MALAT1 and miR-197-3p, or under-expressed p120-ctn (P < 0.05). In conclusion, MALAT1 could alter chemo-resistance of NSCLC cells by targeting miR-197-3p and regulating p120-ctn expression, which might assist in improvement of chemo-therapies for NSCLC.

LncRNA BLACAT1 is involved in chemoresistance of non‑small cell lung cancer cells by regulating autophagy.

The aim of the present study was to determine the effect of the long non‑coding RNA (lncRNA) bladder cancer‑associated transcript1 (BLACAT1) in chemoresistance of non‑small cell lung cancer (NSCLC) cells. Expression of lncRNA BLACAT1, microRNA (miR)‑17, autophagy‑related protein7 (ATG7), multidrug‑resistance protein1 (MRP1), and the autophagy‑associated proteins light chain3 (LC3)‑II/LC3‑I and Beclin1 were detected using the reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Cell viability was determined using an MTT assay. The interaction between BLACAT1 and miR‑17 was determined using RNA immunoprecipitation and RNA pull‑down assays. A cisplatin (DDP)‑resistant NSCLC cell A549/DDP xenograft model in nude mice was established to investigate the effect of BLACAT1 on the chemoresistance of NSCLC cells. Compared with in DDP‑sensitive NSCLC cells, expression of BLACAT1, ATG7, MRP1, LC3‑II/LC3‑I and Beclin1 was significantly upregulated in DDP‑resistant NSCLC cells, whereas miR‑17 was downregulated in DDP‑resistant NSCLC cells. Short interfering RNA against BLACAT1 decreased the viability of DDP‑resistant NSCLC cells. In addition, BLACAT1 interacted with miR‑17, and negatively regulated miR‑17. BLACAT1 promoted ATG7 expression through miR‑17, and facilitated autophagy and promoted chemoresistance of NSCLC cells through miR‑17/ATG7. Finally, in vivo experiments indicated that inhibition of BLACAT1 ameliorated the chemoresistance of NSCLC. BLACAT1 was upregulated in DDP‑resistant NSCLC cells, and promoted autophagy and chemoresistance of NSCLC cells through the miR‑17/ATG7 signaling pathway.

MiR-181c contributes to cisplatin resistance in non-small cell lung cancer cells by targeting Wnt inhibition factor 1.

miRNAs are implicated in drug resistance of multiple cancers including non-small cell lung cancer (NSCLC), highlighting the potential of miRNAs as chemoresistance regulators in cancer treatment. This study aims to explore the relationship between miR-181c and chemoresistance of NSCLC cells. qRT-PCR was conducted to examine the expression of miR-181c in NSCLC tissues, and parental and cisplatin (DDP)-resistant NSCLC cells. MTT assay and flow cytometry were performed to detect the survival rate and apoptosis in NSCLC cells. Luciferase reporter assay was performed to confirm the potential target of miR-181c. Xenograft tumor experiment was applied to confirm the effect of miR-181c on DDP sensitivity of DDP-resistant NSCLC cells in vivo. miR-181c was upregulated in NSCLC tissues, and parental and DDP-resistant NSCLC cells. miR-181c downregulation or WIF1 overexpression increased DDP sensitivity of DDP-resistant NSCLC cells by decreasing survival rate and promoting DDP-induced apoptosis. miR-181c was demonstrated to be able to bind to WIF1 and negatively regulate the expression of WIF1. WIF1 knockdown abolished anti-miR-181c-induced DDP sensitivity. Moreover, anti-miR-181c suppressed the Wnt/β-catenin pathway by regulating WIF1. XAV939 treatment reversed miR-181c-induced increase in IC50 value and miR-181c-triggered decrease in apoptosis. Finally, anti-miR-181c improved DDP sensitivity of DDP-resistant NSCLC cells in vivo. miR-181c contributed to DDP resistance in NSCLC cells through activation of the Wnt/β-catenin pathway by targeting WIF1, providing a potential therapeutic application for the treatment of patients with DDP-resistant NSCLC in the future.

Synergistic Effect of Notch-3-Specific Inhibition and Paclitaxel in Non-Small Cell Lung Cancer (NSCLC) Cells Via Activation of The Intrinsic Apoptosis Pathway.

BackgroundLung cancers are resistant to conventional chemotherapeutic interventions such as paclitaxel. Notch signaling is crucial in the chemoresistance of lung cancer cells. The Notch inhibitor gamma-secretase inhibitor (GSI) inhibits the Notch signaling pathway.Material/MethodsHere, we evaluated how Notch-3 inhibition by GSI can enhance the sensitivity of lung cancer cells to paclitaxel. To study how Notch-3-specific inhibition affects non-small cell lung cancer (NSCLC), we compared the cell viability, apoptosis, and colony formation of A549 and H1299 cells treated with Notch-3 siRNA and GSI.ResultsThe expression levels of Notch-3 or Notch intracellular domain 3 (NICD3) and apoptosis-related proteins were measured and compared between different groups. Notch-3 was significantly overexpressed in both cell lines, and Notch-3 expression was elevated after paclitaxel treatment, indicating activation of the Notch signaling pathway. Inhibition of the Notch signaling pathway by GSI and Notch-3 siRNA reduced cell proliferation and induced apoptosis in A549 and H1299 cells, thereby boosting sensitivity of the cell lines to paclitaxel. Concomitant treatment with paclitaxel and GSI or siRNA downregulated Bcl-2 expression and upregulated Bax expression levels.ConclusionsThese results indicate a synergistic effect of Notch-3-specific inhibition and paclitaxel through alteration of the intrinsic apoptosis pathway, which was involved in Notch-3-induced chemoresistance in NSCLC cells, and GSI inhibited Notch-3-induced chemoresistance in a concentration-dependent manner. This approach that combines Notch-3-specific inhibition and paclitaxel would be likely to apply in NSCLC.

Disturbance of the let-7/LIN28 double-negative feedback loop is associated with radio- and chemo-resistance in non-small cell lung cancer.

Radio- and chemo-resistance represent major obstacles in the therapy of non-small-cell lung cancer (NSCLC) and the underlying molecular mechanisms are not known. In the present study, during induction of radio- or chemo-resistance in NSCLC cells, dynamic analyses revealed that decreased expression of let-7 induced by irradiation or cisplatin resulted in increased expression of its target gene LIN28, and increased expression of LIN28 then contributed to further decreased expression of let-7 by inhibiting its maturation and biogenesis. Moreover, we showed that down-regulation of let-7 and up-regulation of LIN28 expression promoted resistance to irradiation or cisplatin by regulating the single-cell proliferative capability of NSCLC cells. Consequently, in NSCLC cells, let-7 and LIN28 can form a double-negative feedback loop through mutual inhibition, and disturbance of the let-7/LIN28 double-negative feedback loop induced by irradiation or chemotherapeutic drugs can result in radio- and chemo-resistance. In addition, low expression of let-7 and high expression of LIN28 in NSCLC patients was associated significantly with resistance to radiotherapy or chemotherapy. Therefore, our study demonstrated that disturbance of the let-7/LIN28 double-negative feedback loop is involved in the regulation of radio- and chemo-resistance, and that let-7 and LIN28 could be employed as predictive biomarkers of response to radiotherapy or chemotherapy in NSCLC patients.

Anticancer effect of luteolin is mediated by downregulation of TAM receptor tyrosine kinases, but not interleukin-8, in non-small cell lung cancer cells.

TAM receptor tyrosine kinases (RTKs), Tyro3, Axl and MerTK, transduce diverse signals responsible for cell survival, growth, proliferation and anti-apoptosis. In the present study, we demonstrated the effect of luteolin, a flavonoid with antioxidant, anti-inflammatory and anticancer activities, on the expression and activation of TAM RTKs and the association with its cytotoxicity in non-small cell lung cancer (NSCLC) cells. We observed the cytotoxic effect of luteolin in parental A549 and H460 cells as well as in cisplatin-resistant A549/CisR and H460/CisR cells. Exposure of these cells to luteolin also resulted in a dose‑dependent decrease in clonogenic ability. Next, luteolin was found to decrease the protein levels of all three TAM RTKs in the A549 and A549/CisR cells in a dose‑dependent manner. In a similar manner, in H460 and H460/CisR cells, the protein levels of Axl and Tyro3 were decreased following luteolin treatment. In addition, Axl promoter activity was decreased by luteolin, indicating that luteolin suppresses Axl expression at the transcriptional level. We next found that luteolin abrogated Axl phosphorylation in response to growth arrest-specific6 (Gas6), its ligand, implying the inhibitory effect of luteolin on Gas6-induced Axl activation. Ectopic expression of Axl was observed to attenuate the antiproliferative effect of luteolin, while knockdown of the Axl protein level using a gold nanoparticle-assisted gene delivery system increased its cytotoxicity. In contrast to the inhibitory effect of luteolin on the expression of TAM RTKs, interleukin-8 (IL-8) production was not decreased by luteolin in H460 and H460/CisR cells, while IL-8 production/cell was increased. Collectively, our data suggest that TAM RTKs, but not IL-8, are promising therapeutic targets of luteolin to abrogate cell proliferation and to overcome chemoresistance in NSCLC cells.

MiR-96 induces cisplatin chemoresistance in non-small cell lung cancer cells by downregulating SAMD9.

Cisplatin is effective as a single agent or in combination with other drugs for the treatment of non-small cell lung cancer (NSCLC). A concerning clinical challenge with cisplatin-based NSCLC chemotherapy is the intrinsic and acquired chemoresistance to cisplatin. The sterile α motif domain-containing (SAMD9) gene has been reported as a potent tumor suppressor gene that inhibits tumorigenesis and progression of NSCLC. microRNAs (miRNA) have been revealed to play important roles in the regulation of cancer chemoresistance. To the best of our knowledge the present study explored the role of miRNA/SAMD9 signaling in regulating cisplatin chemoresistance in NSCLC for the first time. Out of the several candidate miRNAs predicted to bind the 3′-untranslated region (UTR) of the SAMD9 gene, miRNA-96 (miR-96) demonstrated significant target-sequence-specific inhibition of the SAMD9 3′-UTR luciferase reporter activity in NSCLC cells. In addition, while NSCLC tumor samples exhibited significantly higher expression levels of miR-96 compared with adjacent normal tissues, the expression levels of SAMD9 were significantly lower than those in adjacent normal tissues. miR-96 and SAMD9 were overexpressed and knocked down in the human NSCLC H358 and H23 cell lines and the half maximal inhibitory concentration (IC50) of cisplatin and cell apoptosis rate under cisplatin treatment were used as measures of cisplatin chemoresistance. The present results identified that overexpression of miR-96 in NSCLC cells markedly decreased SAMD9 expression and cisplatin-induced apoptosis, and increased the cisplatin IC50, which could be eliminated by overexpression of SAMD9. By contrast, knocking down miR-96 in NSCLC cells using antagomir-96 significantly increased SAMD9 expression and the cisplatin-induced apoptosis and decreased cisplatin IC50, which could be completely reversed by a knockdown of SAMD9. In conclusion, the current study demonstrates that miR-96 targets and downregulates SAMD9 in NSCLC, which decreases cisplatin-induced apoptosis and induces cisplatin chemoresistance in NSCLC cells. The findings of the present study add novel insights into the function of miR-96 and SAMD9 in cancer, as well as into the molecular mechanisms underlying NSCLC chemoresistance.

Overexpression of Sphk2 is associated with gefitinib resistance in non-small cell lung cancer

Lung cancer is the major cause of cancer-related death worldwide, and 80% of them are non-small cell lung cancer (NSCLC) cases. Recent studies have shown that sphingosine kinase 2 (SphK2) could promote tumor progression; however, whether SphK2 could affect the chemoresistance of NSCLC to chemotherapy remains unclear. To determine whether SphK2 serves as a potential therapeutic target of NSCLC, we utilized small interference RNA (siRNA) to knock down SphK2 expression in human NSCLC cells and analyzed their phenotypic changes. The data demonstrated that knockdown of SphK2 led to decreased proliferation and enhanced chemosensitivity and apoptosis to gefitinib in NSCLC cells. In this study, we describe the findings that overexpression of SphK2 promotes chemoresistance in NSCLC cells. Inhibition of SphK2 might be considered as a strategy in NSCLC treatment with gefitinib.

MicroRNA-92a promotes growth, metastasis, and chemoresistance in non-small cell lung cancer cells by targeting PTEN.

MicroRNA-92a (miR-92a) has been reported to play important roles in tumorigenesis of human various cancers. However, the roles and underlying molecular mechanism of miR-92a in non-small cell lung cancer (NSCLC) have not been totally elucidated. Therefore, the aims of this study were to determine the role of miR-92a and to elucidate its regulatory mechanism in NSCLC. We found that miR-92a was significantly upregulated in NSCLC tissues compared to matched adjacent normal lung tissues, and its expression is significantly associated with clinical characteristics of patients, including tumor, node, and metastasis (TNM) stage; tumor size; and lymph node metastasis (all P < 0.01). Function assays demonstrated that upregulation of miR-92a in NSCLC cells promoted cell proliferation, migration, and invasion, decreased apoptosis and caspase-3 activity, and enhanced chemoresistance of NSCLC cells, whereas downregulation of miR-92a showed the opposite effects. Moreover, phosphatase and tensin homolog (PTEN), a unique tumor suppressor gene, was confirmed as a direct target of miR-92a, and PTEN messenger RNA (mRNA) expression was decreased in NSCLC tissues and was inversely correlated with miR-92a. Downregulation of PTEN could mimic the same effects of miR-92a mimic in NSCLC cells and rescue the effects on NSCLC cells induced by miR-92a inhibitor. Taken together, these findings suggested that miR-92a could promote growth, metastasis, and chemoresistance in NSCLC cells at least partially by targeting PTEN.

Prognostic significance of STAT3 expression and its correlation with chemoresistance of non-small cell lung cancer cells

Activation of signal transducer and activator of transcription 3 (STAT3) plays important roles in tumorigenesis and tumor development. Previously, we have reported that overexpression of STAT3 potentiates growth, survival and radioresistance of non-small cell lung cancer (NSCLC) cells. The aim of this study was to investigate the prognostic significance of STAT3 expression and its correlation with chemoresistance of NSCLC cells. Semi-quantitative RT-PCR was performed to detect the expression of STAT3 mRNA in 12 NSCLC and corresponding adjacent lung tissues. Immunohistochemistry was performed to detect the expression of STAT3 protein in 76 NSCLC tissue samples. Additionally, the correlation between STAT3 expression and prognosis of NSCLC patients was statistically analyzed. The role of STAT3 in chemoresistance of NSCLC cells was also assessed by the vector-based small interfering RNA. The expression level of STAT3 mRNA in NSCLC tissues was significantly higher than that in corresponding adjacent lung tissues (P<0.05). Positive immunostaining of STAT3 protein was mainly located in the cytoplasm of tumor cells. The expression of STAT3 protein was significantly correlated with tumor differentiation, clinical stage and lymph node metastasis of NSCLC patients. Moreover, the 5-year overall survival rate of patients with high STAT3 expression (42.3%) was significantly lower than that of patients with low STAT3 expression (58.8%; P<0.001). Multivariate analysis using the Cox proportional hazard model showed that high STAT3 protein expression was an independent prognostic factor for NSCLC patients (P=0.021). Furthermore, two stably transfected cell lines (A549/shSTAT3 and SPC-A1/shSTAT3) were successfully established, and RNAi-mediated STAT3 inhibition could significantly increase the sensitivity of NSCLC cells to cisplatin by enhancing caspase-3-dependent apoptosis. Together, the expression of STAT3 might be an independent prognostic marker for NSCLC patients and RNAi-mediated STAT3 inhibition would be a potential strategy for chemosensitization of NSCLC cells.

RB protein status and chemosensitivity in non-small cell lung cancers.

The retinoblastoma gene product (RB protein), one of the tumor suppressor proteins has been found as wild-type in most non-small cell lung cancer (NSCLC) cells, usually showing multidrug chemoresistance. Recently, carboplatin (CBDCA) and etoposide (VP-16) have been introduced for the treatment of NSCLC as well as small cell lung cancer (SCLC). We examined the correlation of RB protein expression levels and chemosensitivities of a panel of NSCLC cell lines with wild-type RB for CBDCA and VP-16 in order to clarify the role of RB protein in chemoresistance of NSCLC cells. There was significant correlation between chemosensitivity and high level of RB protein expression (P=0.049 for VP-16, P=0.016 for CBDCA). We next examined the effect of VP-16 on RB protein status and cell cycle phases of two NSCLC cell lines (Ma-12, the most chemosensitive cell line; Ma-31, the most chemoresistant cell line). VP-16 suppressed RB protein expression level of Ma-12 cells but did not suppress that of Ma-31 cells, following 24 h exposure at 0.1-10 microM. And the alteration of phosphorylation status of Rb protein expression were quite different in these cells. After the treatment of VP-16, dephosphorylated RB protein became dominant in Ma-12 cells, but phosphorylated RB protein became dominant in Ma-31. High sensitive non-small lung cancer Ma-12 cells were accumulated in G2/M phases of the cell cycle. But no major changes were found in low sensitive non-small lung cancer Ma-31 cells. This shows that phosphorylation status of RB protein might be useful for the assessment for the sensitivity to VP-16 and for the cell cycle inhibition.