Cisplatin-resistant Lung Cancer Cells Research Articles

Calotropin from Asclepias curasavica induces cell cycle arrest and apoptosis in cisplatin-resistant lung cancer cells

Calotropin (M11), an active compound isolated from Asclepias curasavica L., was found to exert strong inhibitory and pro-apoptotic activity specifically against cisplatin-induced resistant non-small cell lung cancer (NSCLC) cells (A549/CDDP). Molecular mechanism study revealed that M11 induced cell cycle arrest at the G2/M phase through down-regulating cyclins, CDK1, CDK2 and up-regulating p53 and p21. Furthermore, M11 accelerated apoptosis through the mitochondrial apoptotic pathway which was accompanied by increase Bax/Bcl-2 ratio, decrease in mitochondrial membrane potential, increase in reactive oxygen species production, activations of caspases 3 and 9 as well as cleavage of poly ADP-ribose polymerase (PARP). The activation and phosphorylation of JNK was also found to be involved in M11-induced apoptosis, and SP610025 (specific JNK inhibitor) partially prevented apoptosis induced by M11. In contrast, all of the effects that M11 induce cell cycle arrest and apoptosis in A549/CDDP cells were not significant in A549 cells. Drugs with higher sensitivity against resistant tumor cells than the parent cells are rather rare. Results of this study supported the potential application of M11 on the non-small lung cancer (NSCLC) with cisplatin resistance.

Inhibition of disheveled-2 resensitizes cisplatin-resistant lung cancer cells through down-regulating Wnt/β-catenin signaling

Cisplatin (CDDP) is currently recommended as the front-line chemotherapeutic agent for lung cancer. However, the resistance to cisplatin is widespread in patients with advanced lung cancer, and the molecular mechanism of such resistance remains incompletely understood. Disheveled (DVL), a key mediator of Wnt/β-catenin, has been linked to cancer progression, while the role of DVL in cancer drug resistance is not clear. Here, we found that DVL2 was over-expressed in cisplatin-resistant human lung cancer cells A549/CDDP compared to the parental A549 cells. Inhibition of DVL2 by its inhibitor (3289-8625) or shDVL2 resensitized A549/CDDP cells to cisplatin. In addition, over-expression of DVL2 in A549 cells increased the protein levels of BCRP, MRP4, and Survivin, which are known to be associated with chemoresistance, while inhibition of DVL2 in A549/CDDP cells decreased these protein levels, and reduced the accumulation and nuclear translocation of β-catenin. In addition, shβ-catenin abolished the DVL2-induced the expression of BCRP, MRP4, and Survivin. Furthermore, our data showed that GSK3β/β-catenin signals were aberrantly activated by DVL2, and inactivation of GSK3β reversed the shDVL2-induced down-regulation of β-catenin. Taken together, these results suggested that inhibition of DVL2 can sensitize cisplatin-resistant lung cancer cells through down-regulating Wnt/β-catenin signaling and inhibiting BCRP, MRP4, and Survivin expression. It promises a new strategy to chemosensitize cisplatin-induced cytotoxicity in lung cancer.

Abstract 1289: The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells

Abstract Lung cancer is the leading cause of cancer deaths worldwide and despite significant progress, current therapies are limited in efficacy. The CK2 Ser/Thr kinase has been historically linked with cancer. It is involved in cell proliferation, survival and apoptosis by modulating diverse signaling pathways, including Wnt and NF-κB among the most relevant. CIGB-300 is an antitumor peptide with a novel mechanism of action, capable of binding to CK2 substrates thus preventing the enzyme activity. Previously, we have determined that CIGB-300 induces apoptosis through caspase-3 activation in different lung cancer cell lines. Moreover, CIGB-300 strongly inhibited RelA/NF-κB (p65) nuclear translocation, even in the presence of a phorbol-ester activating stimulus. NF-κB activation is known to reduce chemotherapy efficiency in different malignancies, including lung cancer. Based on this evidence, we hypothesize that supplementing cisplatin with CIGB-300 would improve the treatment efficiency. Indeed, we observed by Western blot that nuclear p65 levels were highly increased after treating human NCI-H125 cells with cisplatin. Moreover, when cells were treated with cisplatin plus CIGB-300, NF-κB activation was completely abolished. Therefore, the CIGB-300 effect on NF-κB signaling pathway prevails over cisplatin. These promising results on NF-κB inhibition led us to evaluate the combined treatment in chemoresistant setting. For this purpose we developed a cisplatin resistant A549 lung cancer cell line (A549-Rcisp) by the chronic administration of cisplatin during six months. A549-Rcisp viability was 40% higher than parental cells, confirming the cisplatin-acquired resistance. Remarkably, cisplatin resistant cells showed a significant increase in CIGB-300 sensitivity as compared to the parental cell line (p<0.01; Student's t-test). More on, only A549-Rcisp showed an increased p65 nuclear level after cisplatin treatment, suggesting that both cisplatin resistance and CIGB-300 sensitization might be linked to the NF-kB transcription factor. Given that NF-κB dimer stability is regulated by the proteasome-selective proteolysis of its inhibitory proteins, we studied the effect of CIGB-300 on this process. Surprisingly, we observed a significant increase on protease activities associated with the proteasome after 30 minutes of CIGB-300 treatment. Thus, proteasome complex is a newly identified target of CIGB-300 that could be relevant for its mechanism of action and deserves further exploration in order to determine the association with the observed perturbation of different signaling pathways. Citation Format: Stéfano M. Cirigliano, María Inés Díaz Bessone, Carolina Flumian, Damián E. Berardi, Silvio Perea, Elisa Bal De Kier Joffé, Hernán Farina, Laura Todaro, Alejandro Urtreger. The synthetic peptide CIGB-300 inhibits nuclear factor κB (NF-κB) affecting the survival and chemoresistance of human lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1289.

Abstract 1038: Exploiting ROS and metabolic differences to selectively killing cisplatin resistant lung cancer

Abstract Cisplatin resistance remains a major problem in the treatment of lung cancer. We have discovered that cisplatin resistant (CR) lung cancer cells, regardless of the KRAS mutation status, share one common parameter which is increased reactive oxygen species (ROS) when compared to their parental cells counterpart. Importantly, we found that CR cells were no longer addicted to the glycolytic pathway, but rather relied on oxidative metabolism (OXMET) for energy and biosynthesis. Thus, alteration in mitochondria which resulted in metabolic changes and higher basal levels of ROS may be one of the major factors contributing to cisplatin resistance. We have investigated mitochondrial morphology in parental vs. CR cells using transmission electron microscopy (TEM). Our data clearly showed that CR cells possessed significantly higher number of mitochondria per total cell area (n = 4; p = 0.0006). CR cells also consumed 2-4 fold more oxygen when compared to their parental cells. To further confirm that oxidative phosphorylation was utilized more in CR cells, we assayed mitochondrial membrane potential (MMP) using TMRE staining. MMP was significantly higher in CR cells suggesting that CR cells possess highly active mitochondria. Key glycolytic enzymes hexokinase II (HKII) and lactate dehydrogenase A (LDHA), as well as lactate production were decreased in all CR cell lines which corresponded to the resistance to glycolytic inhibitor, 2DG (n = 8; p<0.03). Moreover, CR cells were highly sensitive to glutamine deprivation. Glutamine withdrawal for 72h resulted in 75-80% cell death in CR cells while only 10-15% cell death occurred in parental cells. CR also take up twice as much L-[G-3H] glutamine when compared to its parental counterpart (n = 4, p<0.05). Here, using Riluzole (the FDA approved drug that interferes with cystine/glutamate pump and results in reduced intracellular glutathione (GSH) levels), can selectively kill CR cells. ROS levels were increased (2-3X) more in resistant cells after Riluzole treatment while no significant change occurred in parental cells. The ID50 dosage of parental cells were 3-4 fold more than theirs CR cell counterparts and no cytotoxicity were found in normal lung fibroblast (n = 8; p<0.05). However, antioxidant, N-acetylcysteine (NAC, 0.1mM), cannot completely abort cell death by Riluzole suggesting other mechanisms are involved. Importantly, Riluzole is also effective in 2 mouse xenograft models (H460CR (KRAS mutant) and SC (wt)). Tumors completely disappeared in wild type mice and were significant reduced in KRAS mutant mice (n = 5 per treatment group; p<0.002). We conclude that addiction to glutamine instead of glucose and higher basal ROS levels seen in CR cells can make them hypersensitive to Riluzole. Repurpose this drug should be considered for future treatment of CR lung cancer patients. Supported by Department of Veterans Affairs, CDA2 award (1K2BX001289) and Woman Cancer Association Fund. Citation Format: Medhi Wangpaichitr, Chunjing Wu, Ying Ying Li, Sumedh Shah, Shu-Mei Chen, Vy Dinh, Macus T. Kuo, Lynn G. Feun, Niramol Savaraj. Exploiting ROS and metabolic differences to selectively killing cisplatin resistant lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1038.

Abstract 2803: Distinct roles of intracellular ROS in cisplatin and evofosfamide cytotoxicity

Abstract Cisplatin is one of the most widely used anticancer agents for the treatment of an array of malignancies. Although multiple mechanisms are involved in cisplatin efficacy, the most prominent and best understood modes of action are the generation of DNA lesions and induction of apoptosis and mitochondrial reactive oxygen species (ROS)-dependent effects on mitochondrial integrity. However, intrinsic or acquired cisplatin resistance can become a limiting factor in cisplatin-based therapy. Evofosfamide is a hypoxia-activated prodrug that is reduced at its nitroimidazole group and preferentially under hypoxic conditions releases Br-IPM, a DNA cross-linker. Evofosfamide is currently being investigated in multiple clinical trials for the treatment of cancer including indications and settings where resistance to cisplatin is observed (e.g. second-line non-small cell lung cancer; NCT01403610). We have previously demonstrated both shared and distinct DNA repair pathways involved with evofosfamide and cisplatin DNA damage. In the current study, we have conducted comparative studies to identify any distinctions in the relative role of ROS underlying cell sensitivity to cisplatin and evofosfamide to help evaluate the potential of evofosfamide to treat cisplatin-resistant tumors. The cisplatin-sensitive cervical cancer cell line ME180, the intermediate cisplatin-resistant human ovarian cancer cell line SK-OV-3, and cisplatin-resistant human lung cancer cell line A549 were investigated with respect to their chemosensitivity to evofosfamide. Unlike cisplatin, evofosfamide exhibited similar potency across all three cell lines. Addition of the ROS scavenger N-acetyl-L-cysteine (NAC) reduced the potency of cisplatin- but not evofosfamide-mediated cytotoxicity, indicating ROS plays a role in cytotoxicity mediated by cisplatin, but not evofosfamide. ROS levels were investigated using the dye H2DCFDA. Cisplatin induced a concentration-dependent and NAC-inhibitable increase of cellular ROS in cisplatin-sensitive ME-180 cells but not in cisplatin-resistant A549 cells. In contrast, ROS levels were not affected by the addition of evofosfamide under normoxia and hypoxia in both ME180 and A549 cells. In order to ascertain whether mitochondrial pathway-mediated apoptosis was involved in cytotoxicity mediated by evofosfamide and cisplatin, mitochondrial membrane depolarization was investigated with the dye JC-1. Cisplatin induced a concentration-dependent JC-1 polarization and these effects were abolished by NAC. In contrast, evofosfamide did not affect mitochondrial membrane potential. In conclusion, this data demonstrates that ROS is an essential factor in cisplatin-mediated cytotoxicity, but not in evofosfamide-mediated cytotoxicity. Citation Format: Fanying Meng, Deepthi Bhupathi, Geraldine Chan, Charles P. Hart. Distinct roles of intracellular ROS in cisplatin and evofosfamide cytotoxicity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2803.

Abstract 3511: Chloroquine-induced lysosomal membrane permeabilization restores sensitivity to cisplatin in refractory lung cancer cells

Abstract Introduction: Lung cancer is the leading cause of cancer-related death. Resistance to treatment contributes to poor outcome. A form of resistance to antineoplastic agents involves the sequestration of chemotherapy inside lysosomes followed by its delivery outside the cell through exocytosis. Lysosomal membrane permeabilization (LMP) not only releases the trapped drug, but also allows the translocation of lysosomal proteases into the cytosol, triggering cell demise. Since lysosomes in cancer cells are more susceptible to LMP than normal cells, we hypothesize that targeting LMP in cisplatin-resistant cells may provide a successful strategy to restore sensitivity to chemotherapy. Materials and Methods: Cisplatin-resistant (cisR) A549 lung cancer cells, obtained by treating parental A549 cells with incremental concentrations of cisplatin, were kindly provided by Dr. Martin Barr. To detect LMP, lysosomes were loaded with FITC-dextran 40KDa and immunofluorescence images were obtained by confocal microscopy. To confirm LMP, cells were homogenized, cytosolic and lysosomal fractions were separated by ultracentrifugation, and cell lysates were immunoblotted with specific antibodies against cathepsin D, α-tubulin and LAMP-1. Cell viability was evaluated using CellTiter blue assay. To measure apoptosis, cells were stained for Annexin-V/Propidium iodide, or FITC-conjugated caspase 3 substrate, and mounted on the Cellometer vision CBA platform, an automated image-based fluorescence microscope equipped with bio-analysis system. Results: cisR A549 cells loaded with FITC-dextran, and incubated with different concentrations of chloroquine (25-100μM) for 24 hours demonstrated increased green haziness in the cytosol and loss of the punctate pattern of the dextran-loaded lysosomes, indicative of dextran leakage following LMP. Release of cathepsins into the cytosol due to LMP was confirmed by the increase in cathepsin D signal in the cytosolic extract on western blot. Next, cisR A549 cells were treated with chloroquine, cisplatin or the drug mix. Cell viability assay revealed a synergistic effect of the combination, with a combination index of 0.70 for cisplatin 25μM and chloroquine 100μM at 48 hours. Notably, there is significant increase in Annexin V stained apoptotic cells, accompanied by a corresponding increase in caspase 3 activity, in the combination. Interestingly, inhibition of lysosomal proteases using E64 was cytoprotective for cells treated with cisplatin and chloroquine, suggesting that chloroquine-induced cell death is LMP mediated. Conclusion: Chloroquine re-sensitizes cisR A549 cells to cisplatin in an LMP-mediated manner. Our preliminary results support the concept that targeting LMP may provide a viable therapeutic strategy to restore sensitivity to chemotherapy in refractory lung cancer. Screening for other repurposed LMP inducer drugs is ongoing. Citation Format: Magdalena Circu, James Cardelli, Glenn Mills, Martin Barr, Hazem E. El-Osta. Chloroquine-induced lysosomal membrane permeabilization restores sensitivity to cisplatin in refractory lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3511.

Abstract 3537: Cisplatin refractory lung cancer cells exhibit an increased autophagic flux at baseline and following exposure to cisplatin

Abstract Introduction: Lung cancer is the leading cause of cancer-related death. Platinum-based therapy is a standard treatment for advanced lung cancer. However, most patients develop resistance to treatment within few months. Autophagy is a catabolic degradation process whereby cellular proteins and organelles are engulfed by autophagosomes, degraded in lysosomes, and recycled to sustain cellular metabolism. Activation of autophagy in response to cellular stress enables cancer cell survival, which can lead to tumor growth and therapeutic resistance. Materials and Methods: Cisplatin-resistant (cisR A549) lung cancer cells, obtained by treating parental A549 cells with incremental concentrations of cisplatin, were kindly provided by Dr. Martin Barr. To measure autophagy, cells were stained for CytoID and mounted on the Cellometer vision CBA platform, an automated image-based fluorescence microscope equipped with bio-analysis system. Autophagy was confirmed by immunoblotting for LC3II/I and p62 in cell lysates. Lysosomal membrane permeabilization (LMP) was detected after loading lysosomes with FITC-dextran 40KDa and detecting dextran leakage into the cytosol on confocal microscopy. Cell viability was evaluated using CellTiter blue assay. Results: We observed an increase in baseline autophagic flux in cisR A549 lung cancer cells as compared to parental cells in full media condition. This was demonstrated by an increase in LC3II/I ratio, and reduction in p62 level in untreated cells. Compared with untreated cells, cisplatin induced a time-dependent increase in autophagosome formation in cisR A549 cells as manifested by the enhanced cytoID signal. To examine whether the increase in CytoID signal was due to autophagic flux stimulation or autophagosome accumulation secondary to blockage of the last step of autophagy, cells were treated with cisplatin in combination with chloroquine, a known lysosomal inhibitor. This had led to a significant increase in cytoID signal as compared to each drug alone, suggesting that autophagy was induced following exposure to cisplatin in cisR cells. Interestingly, increased CytoID signal following exposure to cisplatin was less pronounced in cisplatin-sensitive cells, suggesting that autophagy stimulation following exposure to the drug is inherent for resistant cells. Inhibition of autophagy using chloroquine sensitized resistant cells to cisplatin. Chloroquine was found to induce LMP. Indeed, neutralization of leaked lysosomal proteases with E64, a pan-protease inhibitor, attenuated the synergistic effect of chloroquine. Further works evaluating whether specific autophagy inhibition employing siRNA ATG3 or 3-MA can restore susceptibility to cisplatin are ongoing. Conclusion: Our findings suggest that increased autophagy may be involved in acquired resistance to cisplatin in lung cancer. Targeting autophagy is a viable strategy to reverse chemoresistance. Citation Format: Magdalena Circu, James Cardelli, Martin Barr, Hazem E. El-Osta. Cisplatin refractory lung cancer cells exhibit an increased autophagic flux at baseline and following exposure to cisplatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3537.

Biotin-targeted Pluronic® P123/F127 mixed micelles delivering niclosamide: A repositioning strategy to treat drug-resistant lung cancer cells

With the aim to develop alternative therapeutic tools for the treatment of resistant cancers, here we propose targeted Pluronic® P123/F127 mixed micelles (PMM) delivering niclosamide (NCL) as a repositioning strategy to treat multidrug resistant non-small lung cancer cell lines. To build multifunctional PMM for targeting and imaging, Pluronic® F127 was conjugated with biotin, while Pluronic® P123 was fluorescently tagged with rhodamine B, in both cases at one of the two hydroxyl end groups. This design intended to avoid any interference of rhodamine B on biotin exposition on PMM surface, which is a key fundamental for cell trafficking studies. Biotin-decorated PMM were internalized more efficiently than non-targeted PMM in A549 lung cancer cells, while very low internalization was found in NHI3T3 normal fibroblasts. Biotin-decorated PMM entrapped NCL with good efficiency, displayed sustained drug release in protein-rich media and improved cytotoxicity in A549 cells as compared to free NCL (P<0.01). To go in depth into the actual therapeutic potential of NCL-loaded PMM, a cisplatin-resistant A549 lung cancer cell line (CPr-A549) was developed and its multidrug resistance tested against common chemotherapeutics. Free NCL was able to overcome chemoresistance showing cytotoxic effects in this cell line ascribable to nucleolar stress, which was associated to a significant increase of the ribosomal protein rpL3 and consequent up-regulation of p21. It is noteworthy that biotin-decorated PMM carrying NCL at low doses demonstrated a significantly higher cytotoxicity than free NCL in CPr-A549. These results point at NCL-based regimen with targeted PMM as a possible second-line chemotherapy for lung cancer showing cisplatin or multidrug resistance.

Rejection of adenovirus infection is independent of coxsackie and adenovirus receptor expression in cisplatin-resistant human lung cancer cells.

The adenovirus vector-based cancer gene therapy is controversial. Low transduction efficacy is believed to be one of the main barriers for the decreased expression of coxsackie and adenovirus receptor (CAR) on tumor cells. However, the expression of CAR on primary tumor tissue and tumor tissue survived from treatment has still been not extensively studied. The present study analyzed the adenovirus infection rates and CAR expression in human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP. The results showed that although the CAR expression in A549 and A549/DDP was not different, compared with the A549, A549/DDP appeared obviously to reject adenovirus infection. Moreover, we modified CAR expression in the two cell lines with proteasome inhibitor MG-132 and histone deacetylase inhibitor trichostatin A (TSA), and analyzed the adenovirus infection rates after modifying agent treatments. Both TSA and MG-132 pretreatments could increase the CAR expression in the two cell lines, but the drug pretreatments could only make A549 cells more susceptible to adenovirus infectivity.

Krüppel-Like Factor 4 Enhances Sensitivity of Cisplatin to Lung Cancer Cells and Inhibits Regulating Epithelial-to-Mesenchymal Transition.

In order to improve therapeutic efficacy, it is a current emergency to better know the mechanisms underlying cisplatin resistance in lung cancer cells. In this study, we aim to investigate the role of Krüppel-like factor 4 (KLF4) in cisplatin-resistant lung cancer cells. We developed cisplatin-resistant lung cancer cell line A549/DDP, and then a battery of experiments was used to analyze the effects of KLF4 in cisplatin resistance of lung cancer. We found that KLF4 was significantly downregulated in cisplatin-resistant A549 cells and forced KLF4 expression inhibited cell growth and induced apoptosis. Further, we found that overexpression of KLF4 was able to inhibit cell migration and invasion, to inhibit the expression of Slug, Twist, and vimentin, and to increase the expression of E-cadherin and subsequent inhibition of the EMT process. Thus, overexpression of KLF4 may be a potential strategy for lung cancer treatment, especially for cisplatin-resistant cases.

Combination of gambogic acid with cisplatin enhances the antitumor effects on cisplatin-resistant lung cancer cells by downregulating MRP2 and LRP expression.

Cisplatin resistance is a main clinical problem of lung cancer therapy. Gambogic acid (GA) could prohibit the proliferation of a variety of human cancer cells. However, the effects of GA on cisplatin-resistant lung cancer are still unclear. The objective of the present study was to find out the antitumor effects of GA on cisplatin-resistant human lung cancer A549/DDP cells and further explore its underlying mechanisms. Cell Counting Kit-8 assay was used to observe the impacts of GA and/or cisplatin on the proliferation of lung cancer cells; flow cytometry was used to detect the effects of GA on cell cycle and apoptosis; Western blot was used to examine the effects of GA on the expression of lung resistance protein (LRP) and multidrug resistance-associated protein 2 (MRP2) protein in A549/DDP cells. Our results showed that GA dose- and time-dependently prohibited the proliferation and induced significant cell apoptosis in A549 and A549/DDP cells. GA also induced G0/G1 arrest in both A549/DDP and A549 cells. Moreover, GA upregulated protein expression level of cleaved caspase-3 and Bax and downregulated protein expression level of pro-caspase-9 and Bcl-2 in time- and dose-dependent way in A549/DDP cells. GA combined with cisplatin enhanced the cells apoptotic rate and reduced the cisplatin resistance index in A549/DDP cells. In addition, GA reduced the MRP2 and LRP protein expression level in A549/DDP cells. GA inhibits the proliferation, induces cell cycle arrest and apoptosis in A549/DDP cells. Combination of GA with cisplatin enhances the antitumor effects on cisplatin-resistant lung cancer cells by downregulating MRP2 and LRP expression.

Co-Delivery of Cisplatin Prodrug and Chlorin e6 by Mesoporous Silica Nanoparticles for Chemo-Photodynamic Combination Therapy to Combat Drug Resistance.

Combination therapy shows great promise in circumventing cisplatin resistance. We report herein the development of a novel nanoscale drug delivery system (nDDS) based nanotherapeutic that combines chemotherapy and photodynamic therapy (PDT) into one single platform to achieve synergistic anticancer capacity to conquer cisplatin resistance. Mesoporous silica nanoparticle (MSNs) was used as the drug delivery vector to conjugate cisplatin prodrug and to load photosensitizer chlorin e6 (Ce6) to afford the dual drug loaded delivery system MSNs/Ce6/Pt. The hybrid nanoparticles have an average diameter of about 100 nm and slightly positive surface charge of about 18.2 mV. The MSNs/Ce6/Pt nanoparticles can be efficiently internalized by cells through endocytosis, thereby achieving much higher cellular Pt uptake than cisplatin in cisplatin-resistant A549R lung cancer cells. After 660 nm light irradiation (10 mW/cm(2)), the cellular reactive oxygen species (ROS) level in MSNs/Ce6/Pt treated cells was elevated dramatically. As a result of these properties, MSNs/Ce6/Pt exhibited very potent anticancer activity against A549R cells, giving a half-maximal inhibitory concentration (IC50) value for the combination therapy of 0.53 μM, much lower than that of cisplatin (25.1 μM). This study suggests the great potential of nDDS-based nanotherapeutic for combined chemo-photodynamic therapy to circumvent cisplatin resistance.

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.

The molecular and clinical verification of therapeutic resistance via the p38 MAPK-Hsp27 axis in lung cancer.

Treatment failure followed by relapse and metastasis in patients with non-small cell lung cancer is often the result of acquired resistance to cisplatin-based chemotherapy. A cancer stem cell (CSC)-mediated anti-apoptotic phenomenon is responsible for the development of drug resistance. The underlying molecular mechanism related to cisplatin resistance is still controversial, and a new strategy is needed to counteract cisplatin resistance. We used a nonadhesive culture system to generate drug-resistant spheres (DRSPs) derived from cisplatin-resistant H23 lung cancer cells. The expressions of drug-resistance genes, properties of CSCs, and markers of anti-apoptotic proteins were compared between control cells and DRSPs. DRSPs exhibited upregulation of cisplatin resistance-related genes. Gradual morphological alterations showing epithelial-to-mesenchymal transition phenomenon and increased invasion and migration abilities were seen during induction of DRSPs. Compared with control cells, DRSPs displayed increased CSC and anti-apoptotic properties, greater resistance to cisplatin, and overexpression of p-Hsp27 via activation of p38 MAPK signaling. Knockdown of Hsp27 or p38 decreased cisplatin resistance and increased apoptosis in DRSPs. Clinical studies confirmed that the expression of p-Hsp27 was closely associated with prognosis. Overexpression of p-Hsp27 was usually detected in advanced-stage patients with lung cancer and indicated short survival.SummaryDRSPs were useful for investigating drug resistance and may provide a practical model for studying the crucial role of p-Hsp27 in the p38 MAPK–Hsp27 axis in CSC-mediated cisplatin resistance. Targeting this axis using siRNA Hsp27 may provide a treatment strategy to improve prognosis and prolong survival in lung cancer patients.

Highlights for ESMO 40: celebration review for lifetime achievement awards

My work, my crucial achievement, has been in the management of the transition of the conduct of cancer research/management from basic researchers/surgeons to medical oncologists (figure 1). Figure 1 The Flower of Hope—the ESMO Lifetime Achievement Award. This transition can be divided into four categories: 1. The development, progression and consolidation of global Medical Oncology in Asian countries, especially in Japan. 2. Building a close collaboration with European Society of Medical Oncology (ESMO) and receiving strong support from ESMO, through (A) Adaptation of the ESMO/ASCO Core Curriculum in Medical Oncology,1 (B) Mutual ESMO/ Japanese Society of Medical Oncology (JSMO) joint symposia during their respective meetings, (C) making Annals of Oncology JSMO's official scientific publication,2 along with (D) my own work as an Associate Editor of Annals of Oncology (2011–2014), (E) my work as ESMO's National/Regional Representative and Membership Committee member (2005–2011). 3. Leadership of clinical research in Japan as a key opinion leader and primary investigator in IND (investigational new drug) trials and a chairman of the Japanese Clinical Oncology Group (JCOG; 2000–2009), organising a governmental clinical trial group; and the development of new gold standards in the treatment of malignant diseases, especially in thoracic malignancy by investigator-initiated randomised controlled clinical trials. 4. Encouraging translational research and the publication of numerous articles, especially in the area of the identification of molecular targets for anticancer drugs, as Chief of the Pharmacology Division in the National Cancer Center Research Institute. When I began my training in Medical Oncology, cancer research, including translational research, was conducted by basic researchers, and cancer treatment, including chemotherapy, was conducted by surgeons. There was no place for medical oncologists to play an active part in cancer research and treatment. ESMO was established 40 years ago and now has more than 10 000 professionals as members, and medical oncology has been recognised as …

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.

Negative prognostic value of high levels of intracellular poly(ADP-ribose) in non-small cell lung cancer

Cisplatin-resistant non-small cell lung cancer (NSCLC) cells are often characterized by alterations in vitamin B-related metabolic processes, including the overexpression and hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1) and the downregulation of pyridoxal kinase (PDXK), correlating with elevated apoptosis resistance. Low PDXK expression is an established negative prognostic factor in NSCLC. We determined by immunohistochemistry the expression of PARP1 and the level of its product, poly(ADP-ribose) (PAR), in two independent cohorts of patients with resected NSCLC. Intratumoral high levels (above median) of PAR (but not PARP1 protein levels) had a negative prognostic impact in both the training (92 stage I subjects) and validation (133 stage I and II subjects) cohorts, as determined by univariate and multivariate analyses. The simultaneous assessment of PAR and PDXK protein levels improved risk stratification. NSCLC patients with high intratumoral PARP1 activity (i.e. elevated PAR levels above median) and low PDXK expression (below median) had a dismal prognosis, while patients with low PARP1 activity and high PDXK expression had a favorable outcome. Altogether, these results underscore the clinical potential and possible therapeutic relevance of these biomarkers.

RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells.

BackgroundCisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance.In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin.ResultUsing small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage.ConclusionKnockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.

Impact of epidermal growth factor receptor (EGFR) inhibitors on cisplatin-resistant non-small cell lung cancer (NSCLC) cell lines in parallel to naïve cells at therapeutic doses

Introduction: Chemotherapy represents the backbone of treatment of advanced NSCLC; a platinum-based doublet is standard in first-line treatment. But resistance limits the effectiveness of anticancer drugs and is the major cause of relapse and cancer mortality. Cisplatin activates membrane-integrated proteins, like the EGFR. As in more than 60% of NSCLC s EGFR is overexpressed and in about 10% activated by a driver mutation, EGFR is a further target. We tested whether EGFR TKIs influence cisplatin resistance (CR).

Sodium/iodide symporter gene transfection increases radionuclide uptake in human cisplatin-resistant lung cancer cells.

The sodium/iodide symporter (NIS) is involved in iodide uptake and has been used for the diagnosis and treatment of thyroid cancer. Transfection of the NIS gene in A549 human lung cancer cells can induce radioactive iodine ((131)I) and radioactive technetium ((99m)Tc) uptake. The aim of the present study was to assess the role of NIS in (99m)Tc and (131)I uptake by the A549/DDP human cisplatin-resistant lung cancer cell line. To do so, recombinant adenovirus, adenovirus-enhanced green fluorescent protein-human NIS (Ad-eGFP-hNIS) and Ad-eGFP-rat NIS (Ad-eGFP-rNIS) vectors were established. These vectors were transfected into A549/DDP cells and xenograft tumors in nude mice. Assessment of (99m)Tc and (131)I uptake was performed. Results showed that the transfection efficiency of Ad-eGFP-hNIS and Ad-eGFP-rNIS in A549/DDP cells was at least 90 % in all experiments, and that the uptake ability of (99m)Tc and (131)I was highly enhanced (14-18 folds for (99m)Tc, and 12-16 folds for (131)I). However, the radionuclide concentration in transfected NIS genes' A549/DDP cells reached a plateau within 30-60 min, indicating that NIS transport led rapidly to (99m)Tc and (131)I saturation in cells. In xenograft tumor models, uptake of (99m)TcO4 (-) was obviously higher in the hNIS and rNIS groups compared with controls. In conclusion, these results support the hypothesis that A549/DDP cells can effectively uptake (99m)Tc and (131)I when transfected with the hNIS and rNIS gene. The rNIS or hNIS gene could be used as an effective method for the effective delivery of radioactive products to specific tissues for imagery and/or treatment.