Cisplatin In Non-small Cell Lung Cancer Cells Research Articles

Dihydroartemisinin, a potential PTGS1 inhibitor, potentiated cisplatin-induced cell death in non-small cell lung cancer through activating ROS-mediated multiple signaling pathways

Dihydroartemisinin (DHA) exerts an anti-tumor effect in multiple cancers, however, the molecular mechanism of DHA and whether DHA facilitates the anti-tumor efficacy of cisplatin in non-small cell lung cancer (NSCLC) are unclear. Here, we found that DHA potentiated the anti-tumor effects of cisplatin in NSCLC cells by stimulating reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress, C-Jun-amino-terminal kinase (JNK) and p38 MAPK signaling pathways both in vitro and in vivo. Of note, we demonstrated for the first time that DHA inhibits prostaglandin G/H synthase 1 (PTGS1) expression, resulting in enhanced ROS production. Importantly, silencing PTGS1 sensitized DHA-induced cell death by increasing ROS production and activating ER-stress, JNK and p38 MAPK signaling pathways. In summary, our findings provided new experimental basis and therapeutic prospect for the combined therapy with DHA and cisplatin in some NSCLC patients.

Shikonin inhibited glycolysis and sensitized cisplatin treatment in non-small cell lung cancer cells via the exosomal pyruvate kinase M2 pathway

ABSTRACT The active ingredient of the traditional Chinese medicine comfrey is shikonin, a naphthoquinone compound. The focus of this study was to investigate the effect of shikonin on the proliferation, invasion, migration, and chemoresistance of non-small cell lung cancer (NSCLC) cells, and to explore its underlying molecular biological mechanisms. The results show that shikonin inhibited the viability, proliferation, invasion, and migration of NSCLC cells A549 and PC9, and induced apoptosis. As the inhibitor of pyruvate kinase M2 (PKM2), a key enzyme in glycolysis, shikonin inhibited glucose uptake and the production of lactate, the final metabolite of aerobic glycolysis. In vivo chemotherapeutic assay showed that shikonin reduced the tumor volume and weight in NSCLC mice model and increased the sensitivity to cisplatin chemotherapy. Histoimmunology experiments showed the combination of shikonin and cisplatin downregulated the expression of PKM2 and its transcriptionally regulated downstream gene glucose transporter 1 (Glut1) in tumor tissue. In an assessment of glucose metabolism, micro-PET/CT data showed a combination of shikonin and cisplatin inhibited the fluorodeoxy glucose (18F-FDG) uptake into tumor. Since exosomal PKM2 affected the sensitivity to cisplatin in NSCLC cells, we also demonstrated shikonin could inhibit exosome secretion and exosomal PKM2 through the administration of exosomal inhibitor GW4869. Furthermore, shikonin sensitized cisplatin treatment by reducing the extracellular secretion of exosomal PKM2. In conclusion, we suggest that shikonin not only inhibits PKM2 intracellularly but also reduces glycolytic flux and increases cisplatin sensitivity through the exosomal pathway.

GOLM1 predicts poor prognosis of patients with NSCLC and is associated with the proliferation and chemo-sensitivity of cisplatin in NSCLC cells: bioinformatics analysis and laboratory validation.

Lung cancer is one of the deadliest malignant tumors with non-small cell lung cancer (NSCLC) being the most prevalent type. Patients with NSCLC usually were diagnosed at the advance clinical stages, and these patients often had high rate of tumor-recurrence, thus leading to poor prognosis. Yet, the molecular mechanisms underlying NSCLC progression and recurrence are largely unknown. This study aimed to identify potential hub genes associated with the pathophysiology of NSCLC by bioinformatics analysis and laboratory validation. The GSE51852, GSE52248 and GSE75037 datasets were downloaded from the Gene Expression Omnibus database. The overlapping differentially expressed genes (DEGs) were analyzed by GEO2R tool. Gene Ontology (GO) and KEGG pathway enrichment analysis were performed on these overlapping DEGs. The protein-protein interaction network was constructed to identify hub genes from DEGs. The expression and survival analysis of these hub genes were performed by using the integrated bioinformatics tools. Finally, the effects of GOLM1 on the proliferation and chemo-sensitivity of NSCLC cells were determined by in vitro functional assays. A total of 197 overlapping DEGs (37 up-regulated and 160 down-regulated) were identified from the microarray datasets. Furthermore, the PPI network with 89 nodes and 768 edges was constructed and 17 hub genes were identified from PPI network by using MCODE analysis. The survival analysis revealed that the expression of 5 hub genes (FGF2, GOLM1, GPC3, IL6 and SPP1) were significantly correlated with overall survival of patients with lung cancer. Furthermore, the in vitro functional studies showed that GOLM1 overexpression promoted the NSCLC cell proliferation and colony formation; while GOLM1 knockdown exerted the opposite effects. Importantly, GOLM1 overexpression reduced the chemo-sensitivity of cisplatin in NSCLC cells by attenuating the inhibitory effects of cisplatin on the cell proliferation and colony formation. In conclusion, the present study showed that 5 hub genes including FGF2, GOLM1, GPC3, IL6 and SPP1 were deregulated in NSCLC tissues and may predict the prognosis of patients with NSCLC. GOLM1 may play an important role in regulating the cell proliferation and chemo-sensitivity of cisplatin in NSCLC.

Induction of Apoptosis and Inhibition of EGFR/NF-κB Signaling Are Associated With Regorafenib-sensitized Non-small Cell Lung Cancer to Cisplatin.

The combination of regorafenib with cisplatin/pemetrexed has indicated controllable safety and encouraging antitumor activity in non-small cell lung cancer (NSCLC) patients. However, the anti-NSCLC effects and action mechanisms of regorafenib combined with cisplatin is ambiguous. The major goal of the study was to study the inhibitory effects and action mechanisms of regorafenib combined with cisplatin in NSCLC cells. Cell viability, flow cytometry, immunofluorescence staining, western blotting, migration, and invasion assays were employed to verify the anti-NSCLC effects and mechanisms of regorafenib in combination with cisplatin. Cisplatin-induced epidermal growth factor receptor (EGFR)/nuclear factor κB (NF-κB) signaling was effectively inhibited by regorafenib treatment. Regorafenib, erlotinib (EGFR inhibitor) and QNZ (NF-κB inhibitor) may all enhance the cytotoxicity effect of cisplatin. The invasion ability was effectively decreased by combination treatment. Caspase-dependent and -independent apoptosis was activated by cisplatin combined with regorafenib. Apoptosis induction and EGFR/NF-κB inactivation correlate with regorafenib-enhanced anti-NSCLC efficacy of cisplatin. This study provides evidence of the therapeutic efficacy of regorafenib in combination with cisplatin on NSCLC.

RING finger protein 38 induces the drug resistance of cisplatin in non-small-cell lung cancer.

Cisplatin resistance of non-small-cell lung cancer (NSCLC) needs to be well elucidated. RING finger protein (RNF38) has been proposed as a biomarker of NSCLC poor prognosis. However, its role in drug resistance in NSCLC is poorly understood. RNF38 expression was detected in normal lung epithelial cell and four NSCLC cell lines. RNF38 was stably overexpressed in A549 and H460 cells or silenced in H1975 and cisplatin-resistant A549 cells (A549-CDDP resistant) using lentiviral vectors. RNF38 expression levels were determined using quantitative real-time polymerase chain reaction and western blotting analysis. Cell viability in response to different concentrations of cisplatin was evaluated by Cell Counting Kit-8 assay. RNF38 expression levels were markedly elevated in NSCLC cells and cells harboring high RNF38 were less sensitive to cisplatin. Overexpression of RNF38 reduced, while RNF38 silencing increased the drug sensitivity of cisplatin in NSCLC cells. Cisplatin-resistant cells expressed high RNF38 level. RNF38 silencing promoted cell apoptosis and enhanced the drug sensitivity of cisplatin in cisplatin-resistant NSCLC cells. These findings indicate that RNF38 might induce cisplatin resistance of NSCLC cells via promoting cell apoptosis and RNF38 could be a novel target for rectify cisplatin resistance in NSCLC cases.

Impact of the APE1 Redox Function Inhibitor E3330 in Non-small Cell Lung Cancer Cells Exposed to Cisplatin: Increased Cytotoxicity and Impairment of Cell Migration and Invasion.

Elevated expression levels of the apurinic/apyrimidinic endonuclease 1 (APE1) have been correlated with the more aggressive phenotypes and poor prognosis of non-small cell lung cancer (NSCLC). This study aimed to assess the impact of the inhibition of the redox function of APE1 with E3330 either alone or in combination with cisplatin in NSCLC cells. For this purpose, complementary endpoints focusing on cell viability, apoptosis, cell cycle distribution, and migration/invasion were studied. Cisplatin decreased the viability of H1975 cells in a time- and concentration-dependent manner, with IC50 values of 9.6 µM for crystal violet assay and 15.9 µM for 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. E3330 was clearly cytotoxic for concentrations above 30 µM. The co-incubation of E3330 and cisplatin significantly decreased cell viability compared to cisplatin alone. Regarding cell cycle distribution, cisplatin led to an increase in sub-G1, whereas the co-treatment with E3330 did not change this profile, which was then confirmed in terms of % apoptotic cells. In addition, the combination of E3330 and cisplatin at low concentrations decreased collective and chemotactic migration, and also chemoinvasion, by reducing these capabilities up to 20%. Overall, these results point to E3330 as a promising compound to boost cisplatin therapy that warrants further investigation in NSCLC.

YBX1 mediates autophagy by targeting p110\u03b2 and decreasing the sensitivity to cisplatin in NSCLC

Y-box binding protein 1 (YBX1) is involved in the development of multiple types of tumors. However, the relationship between YBX1 and autophagy in non-small cell lung cancer (NSCLC) remains unclear. In this study, we analyzed the expression and clinical significance of YBX1 and markers of autophagy (LC3I/II) in NSCLC and examined their roles in regulating sensitivity to cisplatin in NSCLC. The retrospective analysis of patients with NSCLC indicated that YBX1 was positively correlated with autophagy. Increased levels of YBX1 or autophagy also observed in NSCLC cells compared with those in 16HBE cells. Compared to the controls, the knockdown of YBX1 expression suppressed autophagy, increased drug sensitivity and promoted apoptosis in response to cisplatin in NSCLC cells by targeting the p110β promoter and inhibiting p110β/Vps34/beclin1 signaling pathways. We also demonstrated in an in vivo study that the overexpressed YBX1 effectively increased NSCLC growth and progression and decreased the sensitivity to cisplatin by inducing autophagy in a xenograft tumor model, and these effects were concomitant with the increasing of p110β and beclin1 expression. Collectively, these results show that YBX1 plays an essential role in autophagy in NSCLC.

Exosomal Transfer Of Cisplatin-Induced miR-425-3p Confers Cisplatin Resistance In NSCLC Through Activating Autophagy.

IntroductionExosomes are important mediators of intercellular communication. Previously, we characterized circulating exosomal miR-425-3p as a non-invasive prognostic marker for predicting clinical response to platinum-based chemotherapy in patients with non-small cell lung cancer (NSCLC).MethodsCirculating exosomal miR-425-3p was validated by qRT-PCR in paired serum samples from NSCLC patients during the course of platinum-based chemotherapy. Cell coculture was performed to examine the effects of exosomal miR-425-3p on the sensitivity of recipient A549 cells to cisplatin. Using bioinformatics, ChIP and luciferase reporter assays, the transcription factor essential for miR-425-3p expression was identified. Autophagic activity in the recipient cells was determined by Western blot and fluorescence microscopy.ResultsHigher levels of exosomal miR-425-3p were found in serum samples from the patients in tolerance versus those at baseline. An upward trend in the expression of circulating exosomal miR-425-3p was revealed during chemotherapy. Furthermore, the expression of exosomal miR-425-3p could be induced by cisplatin in NSCLC cells. Exosomes isolated from either cisplatin-treated or cisplatin-resistant NSCLC cells conferred chemoresistance to sensitive A549 cells in a miR-425-3p-dependent manner. Cisplatin-induced c-Myc was found to directly bind the miR-425-3p promoter and transactivated its expression. Exosomal miR-425-3p facilitated autophagic activation in the recipient cells by targeting AKT1, eventually leading to chemoresistance.DiscussionOur results suggest that apart from a prognostic marker of treatment response, exosomal miR-425-3p might be a potential dynamic biomarker to tailor cisplatin resistance in NSCLC patients during the treatment and represent a promising therapeutic target for therapy-resistant NSCLC.

Knockdown of KLF5 suppresses hypoxia-induced resistance to cisplatin in NSCLC cells by regulating HIF-1\u03b1-dependent glycolysis through inactivation of the PI3K/Akt/mTOR pathway

BackgroundHypoxia-mediated chemoresistance has been regarded as an important obstacle in the development of cancer treatment. Knockdown of krüppel-like factor 5 (KLF5) was reported to inhibit hypoxia-induced cell survival and promote cell apoptosis in non-small cell lung cancer (NSCLC) cells via direct regulation of hypoxia inducible factor-1α (HIF-1α) expression. However, the roles of KLF5 in the development of hypoxia-induced cisplatin (DDP) resistance and its underlying mechanism in NSCLC cells remain to be further elucidated.MethodsWestern blot was performed to determine the protein levels of KLF5, P-glycoprotein (P-gp) and HIF-1α in treated NSCLC cells. Cell survival was examined by MTT assay. The effect of KLF5 knockdown on hypoxia-induced glycolysis was assessed by measuring glucose consumption and lactate production. The effect of KLF5 knockdown on the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway was analyzed by western blot.ResultsHypoxia upregulated the expression of KLF5 in NSCLC cells. KLF5 knockdown suppressed hypoxia-induced DDP resistance in NSCLC cells, as demonstrated by the increased cytotoxic effects of DDP and reduced P-gp expression in NSCLC cells in hypoxia. Moreover, KLF5 knockdown inhibited hypoxia-induced HIF-1α expression and glycolysis, and KLF5 knockdown suppressed hypoxia-induced DDP resistance by inhibiting HIF-1α-dependent glycolysis in NSCLC cells. Furthermore, KLF5 knockdown suppressed hypoxia-induced activation of the PI3K/Akt/mTOR pathway in NSCLC cells and KLF5 overexpression promoted hypoxia-induced DDP resistance in NSCLC cells through activation of the PI3K/Akt/mTOR pathway.ConclusionsKLF5 knockdown could suppress hypoxia-induced DDP resistance, and its mechanism may be due to the inhibition of HIF-1α-dependent glycolysis via inactivation of the PI3K/Akt/mTOR pathway.

MiR-202 Enhances the Anti-Tumor Effect of Cisplatin on Non-Small Cell Lung Cancer by Targeting the Ras/MAPK Pathway

Background/Aims: KRas is usually mutated in non-small cell lung cancer (NSCLC). The mutated KRas gene is a negative prognostic indicator that promotes tumor proliferation, metastasis, and drug resistance in NSCLC, and thus has become a target for cancer therapy. This study is focused on the effects of the microRNA (miR)-202/KRas axis in regulating chemosensitivity in NSCLC. Methods: Quantitative reverse transcriptase real-time PCR analysis was performed to examine the expression of miR-202. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays were performed to evaluate the sensitivity of cisplatin against NSCLC cells. The miR-202/KRas axis was confirmed by western blot and luciferase reporter assays. Cell apoptosis was measured by flow cytometry. KRas expression, MEK1/2 and ERK1/2 phosphorylation, and activation of caspase-9 and caspase-3 were detected by western blot. Results: A significant decrease in miR-202 expression was observed in NSCLC cells both in vivo and in vitro. In addition, miR-202 expression was associated with drug resistance. Recovery of miR-202 expression levels was found to increase the sensitivity of both NCI-H441 and A549 NSCLC cells to cisplatin treatment. Mechanically, as the Ras/mitogen-activated protein kinase (MAPK) pathway was aberrantly activated in NCI-H441 and A549 NSCLC cells, the overexpression of miR-202 was found to inhibit the Ras/MAPK pathway by targeting the KRas gene. As a result, increased miR-202 expression expanded apoptosis signaling induced by cisplatin in NSCLC cells. Conclusion: The miR-202/KRas axis controlled the chemosensitivity of NSCLC by mediating the Ras/MAPK pathway. Thus, the combination of platinum-based drugs with miR-202 may represent a novel strategy to enhance the anti-tumor effect against NSCLC.

Overexpression of Flap Endonuclease 1 Correlates with Enhanced Proliferation and Poor Prognosis of Non–Small-Cell Lung Cancer

Flap endonuclease 1 (FEN1) plays a crucial role in both DNA replication and damage repair. In this study, FEN1 expression and its clinical-pathologic significance in non-small-cell lung cancer (NSCLC) was investigated. Quantitative RT-PCR and immunohistochemistry analysis identified that both FEN1 mRNA and protein were highly overexpressed in about 36% of 136 cancer tissues compared to adjacent tissues, in which FEN1 was generally undetectable. Notably, patients with FEN1-overexpressed cancers were prone to have poor differentiation and poor prognosis. A strong positive correlation between the levels of FEN1 and Ki-67 staining was identified in these NSCLC tissues (r=0.485), suggesting overexpressed FEN1 conferred a proliferative advantage to NSCLC. Furthermore, knockdown of FEN1 resulted in G1/S or G2/M phase cell cycle arrest and suppressed invitro cellular proliferation in NSCLC cancer cells. Consistently, a selective FEN1 inhibitor was shown to effectively inhibit cellular proliferation of NSCLC cells in a dose-dependent manner. Additionally, knockdown of FEN1 significantly attenuated homologous DNA repair efficiency and enhanced cytotoxic effects of cisplatin in NSCLC cells. Taken together, these findings have indicated that overexpressed FEN1 represents a prognostic biomarker and potential therapeutic target for NSCLC treatment, which warrants further study.

ABT-737 Synergizes with Cisplatin Bypassing Aberration of Apoptotic Pathway in Non-small Cell Lung Cancer

A subset of non-small cell lung cancer (NSCLC), which does not have a druggable driver mutation, is treated with platinum-based cytotoxic chemotherapy, but it develops resistance triggered by DNA damage responses. Here, we investigated the effect of activation of STAT3 by cisplatin on anti-apoptotic proteins and the effectiveness of a co-treatment with cisplatin and a BH3 mimetic, ABT-737. We analyzed the relationship between cisplatin and STAT3 pathway and effect of ABT-737, when combined with cisplatin in NSCLC cells and K-ras mutant mouse models. The synergism of this combination was evaluated by the Chou-Talalay Combination Index method. In vivo activity was evaluated by micro-CT. In NSCLC cells, there was a time and dose-dependent phosphorylation of SRC-JAK2-STAT3 by cisplatin, followed by increased expression of anti-apoptotic molecules. When the expression of the BCL-2 protein family members was evaluated in clinical samples, BCL-xL was most frequently overexpressed. Dominant negative STAT3 suppressed their expression, suggesting that STAT3 mediates cisplatin mediated overexpression of the anti-apoptotic molecules. ABT-737 displaced BCL-xL from mitochondria and induced oligomerization of BAK. ABT-737 itself showed cytotoxic effects and a combination of ABT-737 with cisplatin showed strong synergistic cytotoxicity. In a murine lung cancer model, co-treatment with ABT-737 and cisplatin induced significant tumor regression. These findings reveal a synergistic cytotoxic and anti-tumor activity of ABT-737 and cisplatin co-treatment in preclinical models, and suggest that clinical trials using this strategy may be beneficial in advanced NSCLC.

Knockdown of REV3 synergizes with ATR inhibition to promote apoptosis induced by cisplatin in lung cancer cells.

It has been demonstrated that REV3, the catalytic subunit of the translesion synthesis (TLS) polymerase ζ, play an important role in DNA damage response (DDR) induced by cisplatin, and Ataxia-telangietasia mutated and Rad-3-related (ATR) knase is a central player in activating cell cycle checkpoint, stabilizing replication forks, regulating DDR, and promoting repair of DNA damage caused by cisplatin. Cancer cells deficient in either one of REV3 and ATR are more sensitive to cisplatin. However, whether co-inhibition of REV3 and ATR can further increase sensitivity of non-small cell lung cancer (NSCLC) cells to cisplatin is not clear. In this study, we show that REV3 knockdown combined with ATR inhibition further enhance cytotoxicity of cisplatin in NSCLC cells, including cisplatin-sensitive and -resistant cell lines, compared to individual knockdown of REV3 or ATR, which are accompanied by markedly caspase-dependent apoptosis response, pronounced DNA damage accumulation and severe impediment of interstrand crosslink (ICL), and double strand break (DSB) repair. Our results suggest that REV3 knockdown synergize strongly with ATR inhibition to significantly increase sensitivity of cisplatin in NSCLC cells by inhibiting ICL and DSB repair. Thus simultaneously targeting REV3 and ATR may represent one approach to overcome cisplatin resistance and improve chemotherapeutic efficacy in NSCLC treatment.

Curcumin downregulates p38 MAPK-dependent X-ray repair cross-complement group 1 (XRCC1) expression to enhance cisplatin-induced cytotoxicity in human lung cancer cells.

Cisplatin is a well-studied and widely used chemotherapeutic agent and is effective in the treatment of the advanced human non-small cell lung cancer (NSCLC). Curcumin is a yellow pigment derived from the rhizome of Curcuma longa and has been proved to have antioxidant and antitumor properties. XRCC1 is an important scaffold protein involved in base excision repair and plays an important role in the development of lung cancer. In this study, we characterize the role of curcumin in the cytotoxicity, p38 MAPK activation, and XRCC1 expression affected by cisplatin in NSCLC cells. We show that curcumin enhanced the cytotoxicity induced by cisplatin in two NSCLC cells, A549 and H1703. Treatment with cisplatin alone increased XRCC1 mRNA and protein expression through p38 MAPK activation. Moreover, SB2023580 (p38 inhibitor) decreased the XRCC1 mRNA and protein stability upon cisplatin treatment. Knockdown of XRCC1 in NSCLC cells by transfection of XRCC1 siRNA or inactivation of p38 MAPK resulted in enhancing the cytotoxicity and cell growth inhibition induced by cisplatin. Curcumin inhibited the expression of XRCC1 in cisplatin-exposed NSCLC cells. Furthermore, transfection with constitutive active MKK6 or HA-p38 MAPK vectors rescued the XRCC1 protein level and also the cell survival suppressed by cisplatin and curcumin combination in A549 and H1703 cells. These findings suggested that the downregulation of XRCC1 expression by curcumin can enhance the chemosensitivity of cisplatin in NSCLC cells.

Astragaloside IV Enhances Cisplatin Chemosensitivity in Non-Small Cell Lung Cancer Cells Through Inhibition of B7-H3

Background: Chemoresistance is a major obstacle to successful chemotherapy for human non-small cell lung cancer (NSCLC). Astragaloside IV, the component of Astragalus membranaceus, has been reported to exhibit anti-inflammation, anti-cancer and immunoregulatory properties. In the present study, we investigated the role of astragaloside IV in the chemoresistance to cisplatin in NSCLC cells. Methods: We established astragaloside IV-suppressed NSCLC cell lines including A549, HCC827, and NCI-H1299 and evaluated their sensitivity to cisplatin in vitro. In addition, we examined the mRNA and protein levels of B7-H3 in response to cisplatin-based chemotherapy. Results: We showed that high doses of astragaloside IV (10, 20, 40 ng/ml) inhibited NSCLC cell growth, whereas low concentrations of astragaloside IV (1, 2.5, 5 ng/ml) had no obvious cytotoxicity on cell viability. Moreover, combined treatment with astragaloside IV significantly increased chemosensitivity to cisplatin in NSCLC cells. On the molecular level, astragaloside IV co-treatment significantly inhibited the mRNA and protein levels of B7-H3 in the presence of cisplatin. In addition, ectopic expression of B7-H3 diminished the sensitization role of astragaloside IV in cellular responses to cisplatin in NSCLC cells. Conclusion: These results demonstrate that astragaloside IV enhances chemosensitivity to cisplatin via inhibition of B7-H3 and that treatment with astragaloside IV and inhibition of B7-H3 serve as potential therapeutic approach for lung cancer patients.

Promotion of p53 expression and reactive oxidative stress production is involved in zerumbone-induced cisplatin sensitization of non-small cell lung cancer cells.

p53 signaling plays an important role in cell death. Zerumbone, a natural cyclic sesquiterpene, has shown cytotoxic activity against many cancers. This study was done to investigate the anticancer effects of zerumbone on non-small cell lung cancer (NSCLC) cells and explored the involvement of p53 signaling. Cell viability was assessed by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium assay. Apoptosis was confirmed by annexin-V/propidium iodide staining and caspase activity assay. Mitochondrial membrane potential (Δφm) and reactive oxygen species (ROS) production were measured by flow cytometry. Depletion of p53 was achieved by transfection of specific small interfering RNA. Gene expression changes were determined by Western blot analysis. Zerumbone treatment caused a dose-dependent inhibition of A549 and H460 NSCLC cell viability. Zerumbone-induced mitochondrial apoptosis of NSCLC cells, evidenced loss of Δφm, release of mitochondrial cytochrome c, and activation of caspase-9 and -3. There was increased p53 and Bax expression and ROS production in zerumbone-treated cells. Downregulation of p53 or scavenging ROS interfered with the pro-apoptotic action of zerumbone. Combinational treatment with zerumbone and cisplatin significantly accelerated apoptosis and promoted p53 expression and ROS production in NSCLC cells, compared with each alone. These findings demonstrate that zerumbone induces mitochondrial apoptosis and enhances the susceptibility to cisplatin in NSCLC cells, which are, at least partially, mediated through activation of p53 signaling and promotion of ROS generation. This study may provide a rationale for the potential clinical application of zerumbone as a chemotherapeutic agent against NSCLC.

Inhibitory effect of Disulfiram/copper complex on non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in both men and women worldwide. Recently, Disulfiram has been reported to be able to inhibit glioblastoma, prostate, or breast cancer cell proliferation. In this study, the synergistic effect of Disulfiram and copper on NSCLC cell growth was investigated. Inhibition of cancer cell proliferation was detected by 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and cell cycle analysis. Liquid colony formation and tumor spheroid formation assays were used to evaluate their effect on cancer cell clonogenicity. Real-time PCR was performed to test the mRNA level of cancer stem cell related genes. We found that Disulfiram or copper alone did not potently inhibit NSCLC cell proliferation in vitro. However, the presence of copper significantly enhanced inhibitory effect of Disulfiram on NSCLC cell growth, indicating a synergistic effect between Disulfiram and copper. Cell cycle analysis showed that Disulfiram/copper complex caused NSCLC cell cycle arrest in G2/M phase. Furthermore, Disulfiram/copper significantly increased the sensitivity of cisplatin in NSCLC cells tested by MTT assay. Liquid colony formation assay revealed that copper dramatically increased the inhibitory effect of Disulfiram on NSCLC cell colony forming ability. Disulfiram combined with copper significantly attenuated NSCLC cell spheroid formation and recuded the mRNA expression of lung cancer stem cell related genes. Our data suggest that Disulfiram/copper complex alone or combined with other chemotherapy is a potential therapeutic strategy for NSCLC patients.

USP1 deubiquitinase: cellular functions, regulatory mechanisms and emerging potential as target in cancer therapy

Reversible protein ubiquitination is emerging as a key process for maintaining cell homeostasis, and the enzymes that participate in this process, in particular E3 ubiquitin ligases and deubiquitinases (DUBs), are increasingly being regarded as candidates for drug discovery. Human DUBs are a group of approximately 100 proteins, whose cellular functions and regulatory mechanisms remain, with some exceptions, poorly characterized. One of the best-characterized human DUBs is ubiquitin-specific protease 1 (USP1), which plays an important role in the cellular response to DNA damage. USP1 levels, localization and activity are modulated through several mechanisms, including protein-protein interactions, autocleavage/degradation and phosphorylation, ensuring that USP1 function is carried out in a properly regulated spatio-temporal manner. Importantly, USP1 expression is deregulated in certain types of human cancer, suggesting that USP1 could represent a valid target in cancer therapy. This view has gained recent support with the finding that USP1 inhibition may contribute to revert cisplatin resistance in an in vitro model of non-small cell lung cancer (NSCLC). Here, we describe the current knowledge on the cellular functions and regulatory mechanisms of USP1. We also summarize USP1 alterations found in cancer, combining data from the literature and public databases with our own data. Finally, we discuss the emerging potential of USP1 as a target, integrating published data with our novel findings on the effects of the USP1 inhibitor pimozide in combination with cisplatin in NSCLC cells.

Inhibition of thymidine phosphorylase expression by using an HSP90 inhibitor potentiates the cytotoxic effect of cisplatin in non-small-cell lung cancer cells

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. In this study, we examined the role of TP expression in relation to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1650. Treatment with 17-AAG (0.1–1μM) resulted in a decrease in cellular TP protein and mRNA levels, which was accompanied by a downregulation of phosphorylated MKK1/2-ERK1/2 and AKT protein levels. The 17-AAG treatment disrupted the interaction between HSP90 and TP and triggered TP protein degradation through the ubiquitin-26S proteasome pathway. Specific inhibition of TP expression by siRNA further enhanced the cell death and growth inhibition that had been induced by 17-AAG. An enhancement of ERK1/2 or AKT activation by transfecting the cancer cells with constitutively active MKK1/2 or AKT expression vectors significantly restored the 17-AAG-reduced TP protein levels as well as cell viability. In contrast, a combination of U0126 (MKK1/2 inhibitors) or LY294002 (PI3K inhibitor) further decreased the TP expression and cell viability induced by 17-AAG. Moreover, 17-AAG enhanced the cisplatin-induced cytotoxic effect through downregulation of the cisplatin-induced TP expression and ERK1/2 and AKT activation. Taken together, our results suggest that the down-modulation of TP protein induced by 17-AAG represents a key factor in enhancing the cytotoxic effects of cisplatin in NSCLC cells.

MKP1 repression is required for the chemosensitizing effects of NF-κB and PI3K inhibitors to cisplatin in non-small cell lung cancer

Treatment of non-small cell lung cancer (NSCLC) with cisplatin has a level of antitumor activity still modest. We have shown previously that MKP1/DUSP1 inhibits cisplatin-induced apoptosis in NSCLC cells and is overexpressed in tumors from most patients with stage I–II NSCLC. Here, using different NSCLC cell lines we found that MKP1 and NF-κB are differentially expressed. We studied whether targeting MKP1, NF-κB or both affects cisplatin-induced cell death. MKP1 is expressed in H460 and H727 cells. H727 and H1299 cells showed constitutive phosphorylation of Akt and increased NF-κB activity than did H460 cells. H460-MKP1-siRNA-expressing cells (but not H727-MKP1-siRNA or H1299-MKP1-siRNA cells) exhibit a marked increase in cisplatin response compared with parental cells. Treatment with the PI3K inhibitor LY294002 or the NF-κB inhibitor BAY11-7082 enhanced cisplatin antitumor activity in parental H1299 cells but only weakly affected responses of H727 and H460 cells. MKP1-siRNA expression enhanced the chemosensitization effect of LY294002 and BAY11-7082 on H727 and H460 cells. Additionally, NSCLC cell lines with higher NF-κB-constitutive activation were the most sensitive to PS-341 (Bortezomib), a non-specific NF-κB inhibitor. This finding suggests the proteasome as a suitable strategy in treating NSCLC tumors with high constitutive NF-κB activity. Altogether, these results showed that either an activated PI3K/Akt/NF-κB pathway and/or high MKP1 was linked to reduced sensitivity to cisplatin in NSCLC cells. Inhibition of NF-κB or PI3K potently enhanced cisplatin cytotoxicity in cells with endogenous or genetically induced low MKP1 levels. These findings support the potential improvement in cisplatin responses by co-targeting NF-κB or Akt and MKP1.