Effective Lung Cancer Therapy Research Articles

Abstract 4571: Antitumor effects of an antibody (cetuximab)-targeted nanoparticle containing siRNA against EGFR

Abstract Objective: Effective therapies for lung cancer that have mutated EGFR remain elusive. Because of the success of antibody-drug-conjugates, we are exploring the use of antibody targeted nanoparticles (NP) containing siRNA to produce multifunctional therapeutics. Here, we investigate the use of cetuximab (CTX) (commercial antibody to EGFR) targeted NPs containing siRNA against EGFR (sequence is for a region of the mRNA conserved in all types of mutations) in H1975 NSCLC xenografts. Methods: We synthesized a cationic mucic acid polymer by polymerizing a derivative of mucic acid, mucic acid ethylenediamine, with dimethyl suberimidate. This polycation can self-assemble with siRNA to form the NP. A methoxy polyethylene glycol (PEG) was modified to have a 5-nitrophenyl boronic acid (5-nPBA) moiety at one end to attach to diol groups on mucic acid and stabilize the NP. 5-nPBA-PEG with a carboxylic acid end group was conjugated to CTX to target the NP to EGFR receptors on the surface of cancer cells. These polymers were mixed with siRNA to form NPs, characterized by dynamic light scattering and cryo-transmission electron microscopy for size, and zeta potential measurements for surface charge. The NP was tested in vivo against tumors grown with H1975 NSCLC cells that contain the L858R and T790M mutations. NPs dosed iv (tail vein) at 9.3 mg/kg CTX targeting agent and 5 mg/kg siEGFR were injected twice a week for two weeks into nude mice with H1975 NSCLC xenografts and the anti-tumor effects compared to 9.3 mg/kg CTX (dosed iv tail vein) alone. Results: The CTX-targeted cMAP-siRNA NPs are ca. 40 nm in diameter as determined by both dynamic light scattering and cryo-TEM. The NPs' surface charge is slightly negative at -3.5 mV in 10 mM phosphate buffered at pH 7.4 (surrogate for extracellular conditions in vivo). When the NPs are in 1 mM KCl at pH 5.5 (surrogate for intracellular, endocytosis localization), the NP zeta potential is +1 mV indicating detachment of 5-nPBA-PEG from the NP at acidic pH. Thus, the NP is stabilized and targeted until it is endocytosed, where it disassembles to release the siRNA payload. We confirmed delivery of siEGFR to the tumor following two NP doses via a real-time PCR assay. In contrast to CTX treated mice that showed tumor growth delay but ultimately had tumors that grew to over 1000 mm3, NP treated mice had tumors that remained below 200 mm3 53 days following completion of 4 doses. Conclusions: NPs containing full antibodies as targeting agents and siRNA payloads can be formulated into well defined, stable experimental therapeutics. The NP system used here has a pH-tunable 5-nPBA that allows for targeting and stabilizing the NP at a physiologic pH of 7.4. The targeting agent and PEG coating is able to detach from the NP at acidic pH like those found in endosomes, enabling the siRNA payload to escape and reach its site of action within the cell. These NPs produce significant tumor regression in vivo that is superior to CTX alone. Citation Format: Dorothy W. Pan, Mark E. Davis. Antitumor effects of an antibody (cetuximab)-targeted nanoparticle containing siRNA against EGFR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4571. doi:10.1158/1538-7445.AM2014-4571

Ligand-conjugated mesoporous silica nanorattles based on enzyme targeted prodrug delivery system for effective lung cancer therapy

Epidermal growth factor receptor antibody (EGFRAb) conjugated silica nanorattles (SNs) were synthesized and used to develop receptor mediated endocytosis for targeted drug delivery strategies for cancer therapy. The present study determined that the rate of internalization of silica nanorattles was found to be high in lung cancer cells when compared with the normal lung cells. EGFRAb can specifically bind to EGFR, a receptor that is highly expressed in lung cancer cells, but is expressed at low levels in other normal cells. Furthermore, in vitro studies clearly substantiated that the cPLA2α activity, arachidonic acid release and cell proliferation were considerably reduced by pyrrolidine-2 loaded EGFRAb-SN in H460 cells. The cytotoxicity, cell cycle arrest and apoptosis were significantly induced by the treatment of pyrrolidine-2 loaded EGFRAb-SN when compared with free pyrrolidine-2 and pyrrolidine-2 loaded SNs in human non-small cell lung cancer cells. An in vivo toxicity assessment showed that silica nanorattles and EGFRAb-SN-pyrrolidine-2 exhibited low systemic toxicity in healthy Balb/c mice. The EGFRAb-SN-pyrrolidine-2 showed a much better antitumor activity (38%) with enhanced tumor inhibition rate than the pyrrolidine-2 on the non-small cell lung carcinoma subcutaneous model. Thus, the present findings validated the low toxicity and high therapeutic potentials of EGFRAb-SN-pyrrolidine-2, which may provide a convincing evidence of the silica nanorattles as new potential carriers for targeted drug delivery systems.

EGFR-targeted hybrid plasmonic magnetic nanoparticles synergistically induce autophagy and apoptosis in non-small cell lung cancer cells.

BackgroundThe epidermal growth factor receptor (EGFR) is overexpressed in 80% of non-small cell lung cancer (NSCLC) and is associated with poor survival. In recent years, EGFR-targeted inhibitors have been tested in the clinic for NSCLC. Despite the emergence of novel therapeutics and their application in cancer therapy, the overall survival rate of lung cancer patients remains 15%. To develop more effective therapies for lung cancer we have combined the anti-EGFR antibody (Clone 225) as a molecular therapeutic with hybrid plasmonic magnetic nanoparticles (NP) and tested on non-small cell lung cancer (NSCLC) cells.Methodology/Principal FindingsCell viability was determined by trypan-blue assay. Cellular protein expression was determined by Western blotting. C225-NPs were detected by electron microscopy and confocal microscopy, and EGFR expression using immunocytochemistry. C225-NP exhibited a strong and selective antitumor effect on EGFR-expressing NSCLC cells by inhibiting EGFR-mediated signal transduction and induced autophagy and apoptosis in tumor cells. Optical images showed specificity of interactions between C225-NP and EGFR-expressing NSCLC cells. No binding of C225-NP was observed for EGFR-null NSCLC cells. C225-NP exhibited higher efficiency in induction of cell killing in comparison with the same amount of free C225 antibody in tumor cells with different levels of EGFR expression. Furthermore, in contrast to C225-NP, free C225 antibody did not induce autophagy in cells. However, the therapeutic efficacy of C225-NP gradually approached the level of free antibodies as the amount of C225 antibody conjugated per nanoparticle was decreased. Finally, attaching C225 to NP was important for producing the enhanced tumor cell killing as addition of mixture of free C225 and NP did not demonstrate the same degree of cell killing activity.Conclusions/SignificanceWe demonstrated for the first time the molecular mechanism of C225-NP induced cytotoxic effects in lung cancer cells that are not characteristic for free molecular therapeutics thus increasing efficacy of therapy against NSCLC.

Downregulation of transcription factor SOX2 in cancer stem cells suppresses growth and metastasis of lung cancer

Background:The cancer stem cell hypothesis suggests that neoplastic clones are maintained exclusively by a small subpopulation of cells, which have indefinite proliferation and differentiation potentials and give rise to phenotypically diverse cancer cells. Cancer stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the ‘side population’ (SP).Methods and results:The Hoechst efflux assay was used to isolate and characterize the SP from murine D121 lung carcinoma cells. Here, we demonstrated that D121-SP cells contain cancer stem cell characteristics, that is, upregulation of the transcription factors SOX2 and Oct 4 in D121-SP cells. In addition, the migration of D121-SP was decreased, and apoptosis of D121-SP was upregulated following knocking down of SOX2 in D121 cells. Importantly, downregulation of SOX2 in D121 cells markedly suppressed their metastatic potential in syngeneic mice.Conclusions:These results suggest that the SP is an enriched source of lung tumour cells with stem cell properties and that SOX2 has an important role in maintaining stem cell properties and functions that may be a potential target for effective lung cancer therapy.

Cladribine Enhances Apoptotic Cell Death in Lung Carcinoma Cells Over-Expressing DNase .GAMMA.

Worldwide, lung cancer is the most common form of cancer and often has a poor prognosis. Establishment of effective therapies for lung cancer is a major concern in clinical cancer research. We compared the cytotoxic effects of chemotherapeutic agents including cisplatin, 5-fluorouracil, vinorelbine and cladribine, on a human lung cancer cell line, A549, and its derivative transfected with the DNase γ gene. We observed selective cytotoxicity of cladribine on the DNase γ-expressing sub-cell line of A549. Cladribine induces selective apoptosis in DNase γ-expressing A549 cells, which depends on activation of caspases. These results suggest that a combination therapy that includes cladribine along with the introduction of DNase γ has potential as a new therapeutic strategy for lung cancer.

The influence of arsenic trioxide combined with cisplatin on the growth and expression of X-linked inhibitor of apoptosis protein, XIAP of human non-small cell lung cancer cells

Lung cancer is the leading cancer of malignant tumors in China. It is the direction that people make effect to seek effective therapy of lung cancer. The aim of this study is to investigate the influence of arsenic trioxide (As₂O₃) injection combined with cisplatin (DDP) on the growth, apoptosis and XIAP mRNA of human non-small cell lung cancer cell lines. The influence of growth of human non-small cell lung cancer cell lines H460 induced by As₂O₃ combined with DDP or without DDP was analysed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The apoptosis rate of H460 was measured by flow cytometry. XIAP mRNA level of the cells was detected by RT-PCR before and after treatment of As₂O₃ combined with DDP or without DDP. Compared to individual drug, As₂O₃ injection combined with DDP may obviously inhibit the proliferation of H460 cells, increase the apotosis rate of cells and strengthen inhibition of XIAP mRNA expression in cells. As₂O₃ injection combined with DDP can significantly increase the chemosensitivity on human non-small cell lung cancer, and the mechanism may be related to its inhibition on the expression of XIAP.

Neuroendocrine-like differentiation of non-small cell lung carcinoma cells: regulation by cAMP and the interaction of mac25/IGFBP-rP1 and 25.1

The need to develop more effective therapies for lung cancer has led to investigations in understanding the molecular mechanisms of the differentiation process, in particular neuroendocrine (NE) differentiation. Recent studies have demonstrated that NE differentiation in non-small cell lung carcinoma (NSCLC) is not uncommon. Those NSCLCs with NE differentiation are considered a form of in transition NE carcinoma and show a more aggressive clinical course compared with NSCLC without NE differentiation. 25.1, a novel protein interacting with mac25/insulin-like growth factor-binding protein-related protein 1 (mac25/IGFBP-rP1), induced NE-like differentiation when collectively overexpressed in M12 prostate cancer cells. We have examined mac25/IGFBP-rP1 and 25.1 as potential molecular regulators in vitro of the NE-differentiation process in lung cancer. In a panel of SCLC and NSCLC cell lines, mac25/IGFBP-rP1 and 25.1 were expressed at higher levels in SCLC. An increase and sustained activation of adenosine 3',5'-cyclic monophosphate (cAMP) levels induced NE-like differentiation in NSCLC cell lines, and a concomitant increase in the expression of mac25/IGFBP-rP1 and 25.1 was observed during the cAMP-regulated differentiation of NCI-H157 cells, suggesting the involvement of these proteins. Furthermore, the collective overexpression of mac25/IGFBP-rP1 and 25.1 in NSCLC cells induced NE-like differentiation as early as 6 h postinfection. The present data suggest that mac25/IGFBP-rP1 and 25.1 may play a functional role in the NE differentiation of NSCLC cell lines and may provide a novel therapeutic target for treating lung cancers, in particular NSCLC with NE differentiation.