Cisplatin-resistant Non-small Cell Lung Cancer Cells Research Articles

MCL-1 is the key target of adjuvant chemotherapy to reverse the cisplatin-resistance in NSCLC

Cisplatin is one of the most effective chemotherapeutic agents for the treatment of lung cancer. However, the acquired resistance occurred in cancer cells limits the clinical application of cisplatin. MCL-1, which is an important member in the pro-survival Bcl-2 family, plays a critical role in multidrug resistance (MDR). The aim of the present study is to investigate the value of Pan-Bcl-2 inhibitor as sensitizer for the chemotherapy of cisplatin-resistant non-small cell lung cancer (NSCLC) cells. We found the obatoclax but not the ABT-737 significantly decreased the IC50 (half maximal inhibitory concentration) of cisplatin in cisplatin-resistant NSCLC cells. Furthermore, we demonstrated that the mechanism of obatoclax-promoted cell death induced by cisplatin was dependent on the inhibition of MCL-1, which couldn't be inhibited by ABT-737 but is the target of obatoclax. Moreover, inhibition of MCL-1 recovered the function of NOXA and BAK in cisplatin-resistant NSCLC cells, leading to the promotion of mitochondrial apoptosis induced by cisplatin. Interestingly, our date indicated the obatoclax also reversed the cross-resistance in cisplatin-resistant NSCLC cells. Therefore, we demonstrated that the targeted therapy with MCL-1 inhibitors, such as obatoclax, may represent a novel strategy for cancer therapy.

Downregulation of miR-101 contributes to epithelial-mesenchymal transition in cisplatin resistance of NSCLC cells by targeting ROCK2.

Chemoresistance and epithelial-mesenchymal transition (EMT) in cancer are linked phenomena. EMT contributes to chemoresistance, however, little is known about whether chemotherapy can induce EMT in cancer cells. Here, we found that miR-101 expression was downregulated in cisplatin-resistant non-small cell lung cancer (NSCLC) cells. Restoration of miR-101 expression inhibited EMT and increased the sensitivity of cisplatin-resistant NSCLC cells to cisplatin in vitro by targeting ROCK2. Furthermore, ROCK2 protein level was inversely correlated with miR-101 level in NSCLC tissue samples. Kaplan-Meier analysis revealed that low miR-101 expression in NSCLC was correlated with poor survival time. In summary, our results provide novel mechanistic insights into the role of miR-101/ROCK2 signaling in the cisplatin resistance of NSCLC cells. Targeting of miR-101 is a potential therapeutic approach for NSCLC.

Differential expression of inflammasomes in lung cancer cell lines and tissues.

As pivotal elements involved in inflammation, inflammasomes represent a group of multiprotein complexes triggering the maturation of proinflammatory cytokine interleukin (IL)-1β and IL-18. Although the importance of the inflammasomes in inflammatory diseases is well appreciated, a precise characterization of their expressions in lung cancer remains obscure. This study aimed to determine the expressions of inflammasomes in various lung cancer cell lines and tissues to understand their potential roles in lung cancer. Our findings showed that inflammasome components were markedly upregulated in lung cancer and elicited the maturation of IL-1β and IL-18. In addition, enormous variations in subtypes and levels of inflammasomes were detected in lung cancers depending on their histological type and grading, invasion ability, as well as chemoresistance. Generally, AIM2 inflammasome was overexpressed in nonsmall cell lung cancer (NSCLC), while NLRP3 inflammasome was upregulated in lung adenocarcinoma (ADC) and small cell lung cancer (SCLC). The high-metastatic or cisplatin-sensitive NSCLC cells expressed more inflammasome components and products than their counterpart low-metastatic or cisplatin-resistant NSCLC cells, respectively. In resected lung cancer tissues, high-grade ADC expressed more inflammasome components and products than low-grade ADC. Together, these findings suggest that inflammasomes may be crucial biomarkers for lung cancer as well as potential modulators of the biological behaviors of lung cancer. Further, pharmacotherapeutics targeting inflammasomes might be novel adjuvant therapy strategies for lung cancer.

Matrine induces mitochondrial apoptosis in cisplatin-resistant non-small cell lung cancer cells via suppression of β-catenin/survivin signaling.

Matrine is an alkaloid isolated from Sophora flavescens and shows anticancer activities. The present study was carried out to determine the cytotoxic effects of matrine on cisplatin-resistant non-small cell lung cancer (NSCLC) cells and the associated molecular mechanisms. Parental and cisplatin-resistant A549 and H460 NSCLC cells were treated with 1 or 2 g/l of matrine for 48 h, and cell viability and apoptosis were assessed. β-catenin-mediated transcriptional activity, mitochondrial membrane potential (ΔΨm) changes, activation of caspases, and survivin expression were examined. The effect of overexpression of survivin on the anticancer activity of matrine was investigated. Compared to the parental cells, cisplatin-resistant NSCLC cells showed increased β-catenin transcriptional activity. Matrine treatment resulted in a significant reduction in β-catenin activation and survivin expression in the cisplatin-resistant cells. Matrine caused apoptotic death in the cisplatin-resistant NSCLC cells, coupled with loss of ΔΨm and activation of caspase-9 and -3. Matrine-induced apoptosis of the cisplatin-resistant NSCLC cells was significantly reversed by overexpression of survivin. In conclusion, matrine exposure induces mitochondrial apoptosis in cisplatin-resistant NSCLC cells, which is largely mediated through inactivation of β-catenin/survivin signaling. Further investigation of the therapeutic benefit of matrine in overcoming cisplatin resistance in NSCLC is warranted.

Aurora-A contributes to cisplatin resistance and lymphatic metastasis in non-small cell lung cancer and predicts poor prognosis.

BackgroundPlatinum-based chemotherapy improves survival among patients with non-small cell lung cancer (NSCLC), but the efficiency is limited due to resistance. In this study, we aimed to identify the expression of Aurora-A and its correlation with cisplatin resistance and prognosis in NSCLC.MethodsWe used immunohistochemical analysis to determine the expression of Aurora-A protein in 102 NSCLC patients treated by surgery and adjuvant cisplatin-based chemotherapy. The prognostic significances were assessed by Kaplan-Meier survival estimates and Cox models. The potential role of Aurora-A in the regulation of cisplatin resistance in NSCLC cells was examined by transfections using expression vector and small interfering RNA or using small-molecule inhibitors.ResultsAurora-A expression was significantly associated with clinical stage (p = 0.018), lymph node metastasis (p = 0.038) and recurrence (p = 0.005), and was an independent prognostic parameter in multivariate analysis. High level of Aurora-A expression predicted poorer overall survival (OS) and progression-free survival (PFS). In vitro data showed that Aurora-A expression was elevated in cisplatin-resistant lung cancer cells, and overexpression or knockdown of Aurora-A resulted in increased or decreased cellular resistance to cisplatin. Furthermore, inhibition of Aurora-A reversed the migration ability of cisplatin-resistant cells.ConclusionsThe current findings suggest that high Aurora-A expression is correlated with cisplatin-based chemotherapeutic resistance and predicts poor patient survival in NSCLC. Aurora-A might serve as a predictive biomarker of drug response and therapeutic target to reverse chemotherapy resistance.

FHIT loss confers cisplatin resistance in lung cancer via the AKT/NF-κB/Slug-mediated PUMA reduction.

Fragile histidine triad (FHIT) loss by the two-hit mechanism of loss of heterozygosity and promoter hypermethylation commonly occurrs in non-small cell lung cancer (NSCLC) and may confer cisplatin resistance in NSCLC cells. However, the underlying mechanisms of FHIT loss in cisplatin resistance and the response to cisplatin-based chemotherapy in NSCLC patients have not yet been reported. In the present study, inhibition concentration of 50% cell viability induced by cisplatin (IC50) and soft agar growth and invasion capability were increased and decreased in FHIT-knockdown and -overexpressing cells, respectively. Mechanistically, Slug transcription is upregulated by AKT/NF-κB activation due to FHIT loss and, in turn, Slug suppresses PUMA expression; this decrease of PUMA by FHIT loss is responsible for cisplatin resistance. In addition, cisplatin resistance due to FHIT loss can be conquered by AKT inhibitor-perifosine in xenograft tumors. Among NSCLC patients, low FHIT, high p-AKT, high Slug and low PUMA were correlated with shorter overall survival, relapse-free survival and poorer response to cisplatin-based chemotherapy. Therefore, the AKT inhibitor perifosine might potentially overcome the resistance to cisplatin-based chemotherapy in NSCLC patients with low-FHIT tumors, and consequently improve the outcome.

Protective effects of ADAM8 against cisplatin‐mediated apoptosis in non‐small‐cell lung cancer

A disintegrin, metalloproteinase 8 (ADAM8), is overexpressed in the vast majority of lung cancers and can be a diagnostic marker of lung cancer. We have investigated the effect of ADAM8 on the cisplatin resistance in non-small-cell lung cancer (NSCLC) cell lines. Stable cell lines overexpressing ADAM8 in A549 and H460 cells were generated, both of which have low endogenous ADAM8. Ectopic expression of ADAM8 rendered cells more resistant to cisplatin-induced toxicity, increasing the half maximal inhibitory concentration (IC(50) ) values by 1.85-fold in A549 cells and 3.91-fold in H460 cells relative to mock-transfected cells. Moreover, silencing of ADAM8 in H647 cells with high endogenous level of ADAM8 sensitised them to cisplatin-induced toxicity, with a lower IC(50) value of 11.2 µM relative to an IC(50) of 25.3 µM in mock-transfected cells. Moreover, knockdown of ADAM8 caused a significant increase in cisplatin-induced apoptosis assessed by annexin-V/propidium iodide double staining, accompanying with enhanced cleavage of caspase-3 and poly(ADP-ribose) polymerase. Western blot analysis showed that a greater amount of phosphorylated signal transducer and activator of transcription 3 (STAT3) in ADAM8-overexpressing A549 cells compared to parental or mock-transfected cells. STAT3 silencing increased the susceptibility of ADAM8-overexpressing A549 cells to cisplatin. Both Bcl-2 and Mcl-1 in ADAM8-overexpressing A549 cells were profoundly diminished by STAT3 knockdown. Thus, ADAM8 is implicated in cisplatin resistance of NSCLC cells through activation of the STAT3 signalling pathway, and thus represents a potential therapeutic target in this malignancy.

148P - Targeting NFKB in Cisplatin Resistant NSCLC

ABSTRACT The most effective systemic chemotherapy for Non Small Cell Lung Cancer (NSCLC) is cisplatin-based combination treatment. However, chemoresistance is a major therapeutic problem with the patients' tumours either de novo resistant to therapy or ultimately developing resistance to cisplatin. Understanding the mechanisms underpinning cisplatin resistance (CR) may lead to the development of predictive biomarkers and novel therapies for NSCLC and other platinum treated tumours in the future. The NF-κB pathway has been implicated in tumour initiation, progression, and resistance to chemotherapy. Although small-molecule inhibitors of NF-κB have been proposed as single-agent therapies for cancers with aberrant NF-κB activity, most classic NF-κB inhibitors are poorly selective and result in off-target effects. Dehydroxymethyl-epoxyquinomicin (DHMEQ) is an inhibitor of low molecular weight designed from the structure of the antibiotic epoxyquinomicin C. The aim of this study is to investigate the role of the PI3K-NFκB pathway in resistance to cisplatin in NSCLC and assess the effect of NF-κB inhibition by DHMEQ in cisplatin sensitive and resistant cell lines. A panel of cisplatin resistant NSCLC cell lines (H460, SKMES1, A549) were developed in our laboratory. H460 parent & resistant cell lines were screened for changes in mRNA expression using the PI3K Profiler array (SABiosciences). Changes in gene expression were validated by QPCR and protein expression by Western blot and HCA. IkBa exons 3-5 were screened for mutations by sequencing. The effect of DHMEQ (inhibitor of NF-κB translocation) was assessed by HCA assays. An 11.99 fold increase in IkBa mRNA expression was identified in the cisplatin resistant H460 cells compared to parent cells. QPCR, Western blot and HCA confirmed this overexpression of IkBa. No mutations were identified in exons 3-5 of the IkBa gene. Inhibition of NF-kB by DHMEQ led to increased apoptosis and decreased proliferation in cisplatin resistant cells versus parent cells implying NF-kB plays a significant role in cisplatin resistance. We are evaluating the synergistic effects of DHMEQ and cisplatin both in vitro and in vivo on tumour growth and spontaneous lung metastasis in athymic nude mice. DHMEQ may provide a beneficial treatment strategy for NSCLC patients who progress on cisplatin. Disclosure All authors have declared no conflicts of interest.

Abstract A64: NFκB-IκBα Interaction: A Mechanism of Resistance to Cisplatin in Non Small Cell Lung Cancer (NSCLC)

Abstract Introduction: The purpose of this study was to investigate the role of the PI3K-NFκB pathway in resistance to cisplatin in Non-small cell lung cancer (NSCLC). This form of malignancy is the leading cause of cancer morbidity and mortality in the western world with a poor overall 5 year survival of <15%. The most effective systemic chemotherapy for NSCLC is cisplatin-based combination treatment. However, chemoresistance is a major therapeutic problem and understanding the mechanisms involved is critical to the development of new therapeutic intervention strategies. The PI3K/AKT/mTOR pathway signals through NFκB and plays an important role in NSCLC, representing a novel therapeutic target for overcoming cisplatin resistance. Methods: A panel of cisplatin resistant NSCLC cell lines (H460, SKMES1, A549) were developed in our laboratory. H460 parent & resistant cell lines were screened for changes in mRNA expression using the PI3K Profiler array (SABiosciences). Changes in gene expression were validated by QPCR and protein expression by Western blot and HCA. IκBα exons 3–5 were screened for mutations by sequencing. DHMEQ is an inhibitor of NFκB translocation. The effects of DHMEQ on NFκB translocation as well as on cell apoptosis and proliferation were assessed by HCA and BrdU assays. Results: An 11.99 fold increase in IκBα mRNA expression was identified in the cisplatin resistant H460 cells compared to parent cells. QPCR, Western blot and HCA confirmed this overexpression of IκBα. No mutations were identified in exons 3-5 of the IκBα gene. Inhibition of NFκB by DHMEQ led to significantly increased apoptosis and significantly decreased proliferation in cisplatin resistant cells versus parent cells. Conclusions: The transcription factor NFκB is involved in cell survival and regulates the transcription of its own inhibitor, IκBα. Both IκBα and NFκB were found to be upregulated in cisplatin resistant cells compared to cisplatin sensitive cells. Inhibiton of NFκB by the inhibitor DHMEQ succeeded in reducing resistant cell survival and proliferation. NFκB inhibition could represent a novel therapeutic strategy to overcome NSCLC resistance to cisplatin.

IGFBP-3 hypermethylation-derived deficiency mediates cisplatin resistance in non-small-cell lung cancer

Cisplatin-based chemotherapy is the paradigm of non-small-cell lung cancer (NSCLC) treatment; however, it also induces de novo DNA-hypermethylation, a process that may be involved in the development of drug-resistant phenotypes by inactivating genes required for drug-cytotoxicity. By using an expression microarray analysis, we aimed to identify those genes reactivated in a set of two cisplatin (CDDP) resistant and sensitive NSCLC cell lines after epigenetic treatment. Gene expression, promoter methylation and CDDP-chemoresponse were further analyzed in three matched sets of sensitive/resistant cell lines, 23 human cancer cell lines and 36 NSCLC specimens. Results revealed specific silencing by promoter hypermethylation of IGFBP-3 in CDDP resistant cells, whereas IGFBP-3 siRNA interference, induced resistance to CDDP in sensitive cells (P<0.001). In addition, we found a strong correlation between methylation status and CDDP response in tumor specimens (P<0.001). Thus, stage I patients, whose tumors harbor an unmethylated promoter, had a trend towards increased disease-free survival (DFS). We report that a loss of IGFBP-3 expression, mediated by promoter-hypermethylation, results in a reduction of tumor cell sensitivity to cisplatin in NSCLC. Basal methylation status of IGFBP-3 before treatment may be a clinical biomarker and a predictor of the chemotherapy outcome, helping to identify patients who are most likely to benefit from CDDP therapy alone or in combination with epigenetic treatment.

XIAP-mediated protection of H460 lung cancer cells against cisplatin

Molecular mechanism(s) responsible for drug resistance of non-small cell lung cancer (NSCLC) cells to cisplatin was investigated. Results showed that cisplatin (50muM)-induced cell death (apoptosis) was more significant in CH27 and A549 cell lines than in H460. The high protein levels of X-linked inhibitor-of-apoptosis protein (XIAP) observed in H460 cells appeared to play a key role in the regulation of cisplatin resistance of H460 cells. XIAP can bind to and suppress the activities of caspase 3 in H460 cells and lead to apoptosis inhibition of these cells. Blockade of XIAP activity by Embelin (XIAP inhibitor) or siRNA has increased caspase 3 activities and promoted cisplatin-induced cell death of H460 cells. The results indicate a therapeutic value of Embelin and/or XIAP siRNA in the control of cisplatin-resistant NSCLC cells (H460).

Interrelationships between cellular nucleotide excision repair, cisplatin cytotoxicity, HER-2/neu gene expression, and epidermal growth factor receptor level in non-small cell lung cancer cells.

Nucleotide excision repair (NER) is a major repair mechanism for DNA lesions induced by cisplatin. Overexpressions of epidermal growth factor receptor (EGFR) and HER‐2/neu have been reported to affect the sensitivity of certain human cancer cells to cisplatin, presumably by modification of DNA repair activity through interference with NER. Using an in vitro repair assay, we investigated NER activity of cisplatin‐induced DNA lesions in a panel of 16 non‐small cell lung cancer (NSCLC) cell lines. The interrelationships between NER activity, cisplatin sensitivity, HER‐2/neu expression and EGFR level, were also analyzed. The results showed that high NER activity was closely correlated with cisplatin resistance and high levels of HER‐2/neu expression (P < 0.05). Analysis of the relationships between EGFR level and each of the other three parameters revealed no statistically significant correlations (all P values were > 0.05 by Spearman rank correlation), but a trend of association (all the values of proportion of accordance were ≥62.5% by using a 2x2 contingency table). These results suggest that NER activity may play an important role in the cisplatin resistance of NSCLC cells and there may be an association between enhanced NER activity and high levels of p185neu and probably EGFR in NSCLC cells. The finding that high levels of EGFR showed very little influence on the relationship between p185neu and cisplatin resistance suggests that EGFR may be a less crucial factor in modulating the chemoresistance of NSCLC cells when compared with HER‐2/neu.

Determinants of response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in human lung cancer cell lines.

Small-cell lung cancer (SCLC) is more sensitive to anticancer agents than non-small-cell lung cancer (NSCLC), but few studies have analyzed the mechanisms of natural drug resistance responsible for this difference. To elucidate these mechanisms, we determined drug sensitivity and evaluated the biochemical parameters affecting response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in both types of cancer cell lines, in particular the activity and content of DNA topoisomerase II, as well as etoposide uptake and cell doubling time. Drug sensitivity and cellular uptake of etoposide were determined by clonogenic assay and accumulation of radiolabeled drug, respectively. The topoisomerase II activity was assayed by decatenation of kinetoplast DNA to minicircle DNA using nuclear protein, and the content was determined by immunoblot analysis of nuclear extracts. We also compared the topoisomerase II content in parent cell lines with that in lines with cisplatin resistance acquired in vitro. Sensitivities to doxorubicin and etoposide were higher in SCLC cell lines than in NSCLC lines, and the difference was statistically significant. Etoposide uptake in SCLC cells was higher than in NSCLC cells; the difference was statistically significant, but this difference may not be sufficient to account for the variation in sensitivities of the cell lines. Topoisomerase II activities of nuclear protein from SCLC cell lines were reproducibly twofold higher than those for NSCLC cell lines. The topoisomerase II content in nuclear protein appeared to be higher in SCLC cell lines than in NSCLC cell lines and corresponded to the sensitivities to doxorubicin and etoposide. In the cisplatin-resistant NSCLC cell lines PC-7/CDDP and PC-14/CDDP, the topoisomerase II content was increased compared with that in the parent lines, but the topoisomerase II content in other cisplatin-sensitive parent lines was similar to that in resistant sublines. These findings suggest that the topoisomerase II activity and content may be major factors in determining sensitivity to topoisomerase II inhibitors.