A549 Lung Tumor Cells Research Articles

Selective Ru(II)/benzoate complexes against triple-negative breast tumor cells and their interactions with DNA and BSA

New ruthenium(II) complexes bearing benzoate (AB) ligand with the general formula [Ru(AB)(bipy)(PP)]PF6, where 2,2′-bipyridine (bipy) and different diphosphine ligands, (PP) = 1,2′-bis(diphenylphosphine)ethane (dppe, 1), 1,3′-bis(diphenylphosphine)propane (dppp, 2) and 1,2′-bis(diphenylphosphine)ferrocene (dppf, 3), were synthesized. The compounds were characterized by molar conductivity, elemental analysis, cyclic voltammetry, infrared and UV–Vis spectroscopies, NMR, and by single-crystal X-ray diffraction for complexes 1 and 2. The complexes showed a weak interaction to CT-DNA through DNA minor groove, with Kb values at around 103-104 M−1. CT-DNA interaction assays by viscosity and circular dichroism (CD) suggested that the compounds do not significantly alter the secondary DNA structure. The complexes are cytotoxic against MDA-MB-231, MCF-7 (breast) and A549 (lung) tumor cell lines, with IC50 values in the range of 1 to 17 µM. The compounds 1-3 showed high selectivity against triple-negative breast tumor cells. Remarkably, complexes 1 and 3 show greater cytotoxic activity against cells than cisplatin, being promising agents for tumor treatment.

Abstract 5748: Inhibition of tumor growth using exosomes loaded with KRAS siRNA and surface-modified with tumor-targeted proapoptotic peptide

Abstract Exosomes are extracellular vesicles naturally secreted by cells and serve as carriers of RNAs and proteins for cell-to-cell communications. KRAS is frequently mutated and plays a vital role in many tumors. Receptor expressed in lymphoid tissue (RELT), also known as tumor necrosis factor receptor superfamily 19-like, is upregulated in certain tumors such as lung and pancreatic tumors and is a potential tumor target. To selectively deliver cytotoxic agents to tumor, we loaded mesenchymal stem cell (MSC)-derived exosomes with a siRNA against KRAS and surface-modified them with the RELTpep-KLA peptide comprising a RELT-binding peptide (CRQTKN, named RELTpep) and proapoptotic peptide (KLAKLAKKLAKLAK, named KLA). Exosomes were isolated from cell culture medium using ultracentrifugation and transfected with the KRAS siRNA using a lipid-based reagent and then surfaced-modified with the RELTpep-KLA using a phospholipid-based membrane anchor. Compared with control peptide-KLA, RELTpep-KLA-targeted MSC-exosomes were more efficiently internalized into A549 lung tumor and Panc-1 pancreatic tumor cells expressing RELT on their surface and exhibited cytotoxicity to these tumor cells. RELTpep-KLA-targeted and KRAS siRNA-loaded MSC-exosomes reduced the phosphorylation of Erk and exhibited an enhanced cytotoxicity in A549 and Panc-1 tumor cells. Moreover, when intravenously injected, they inhibited tumor growth in Balb/c nude mice bearing subcutaneous Panc-1 human tumor xenografts more efficiently than those loaded with control siRNA or modified with control peptide-KLA. These results demonstrate that RELTpep-KLA-targeted MSC-exosomes carrying KRAS siRNA hold a potential for exosome-based targeted therapy against cancer. Citation Format: Li Kang, Soyeon Jeon, Sri Murugan Vadevoo, Sang-Hyun Kim, Byungheon Lee. Inhibition of tumor growth using exosomes loaded with KRAS siRNA and surface-modified with tumor-targeted proapoptotic peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5748.

Glycocalyx transduces membrane leak in brain tumor cells exposed to sharp magnetic pulsing

Mechanisms by which electric (E) or magnetic (B) fields might be harnessed to affect tumor cell behavior remain poorly defined, presenting a barrier to translation. We hypothesized in early studies that the glycocalyx of lung cancer cells might play a role in mediating plasma membrane leak by low-frequency pulsed magnetic fields (Lf-PMF) generated on a low-energy solenoid platform. In testing glioblastoma and neuroblastoma cells known to overexpress glycoproteins rich in modifications by the anionic glycan sialic acid (Sia), exposure of brain tumor cells on the same platform to a pulse train that included a 5 min 50Hz Lf-PMF (dB/dt ∼ 2 T/s at 10 ms pulse widths) induced a very modest but significant protease leak above that of control nonexposed cells (with modest but significant reductions in long-term tumor cell viability after the 5 min exposure). Using a markedly higher dB/dt system (80 T/s pulses, 70 μs pulse-width at 5.9 cm from a MagVenture coil source) induced markedly greater leak by the same cells, and eliminating Sia by treating cells with AUS sialidase immediately preexposure abrogated the effect entirely in SH-SY5Y neuroblastoma cells, and partially in T98G glioblastoma cells. The system demonstrated significant leak (including inward leak of propidium iodide), with reduced leak at lower dB/dt in a variety of tumor cells. The ability to abrogate Lf-PMF protease leak by pretreatment with sialidase in SH-SY5Y brain tumor cells or with heparin lyase in A549 lung tumor cells indicated the importance of heavy Sia or heparan sulfate glycosaminoglycan glycocalyx modifications as dominant glycan species mediating Lf-PMF membrane leak in respective tumor cells. This “first-physical” Lf-PMF tumor glycocalyx event, with downstream cell stress, may represent a critical and “tunable” transduction mechanism that depends on characteristic anionic glycans overexpressed by distinct malignant tumors.

New Nanostructured Materials Based on Mesoporous Silica Loaded with Ru(II)/Ru(III) Complexes with Anticancer and Antimicrobial Properties.

A new series of nanostructured materials was obtained by functionalization of SBA-15 mesoporous silica with Ru(II) and Ru(III) complexes bearing Schiff base ligands derived from salicylaldehyde and various amines (1,2-diaminocyclohexane, 1,2-phenylenediamine, ethylenediamine, 1,3-diamino-2-propanol, N,N-dimethylethylenediamine, 2-aminomethyl-pyridine, and 2-(2-aminoethyl)-pyridine). The incorporation of ruthenium complexes into the porous structure of SBA-15 and the structural, morphological, and textural features of the resulting nanostructured materials were investigated by FTIR, XPS, TG/DTA, zeta potential, SEM, and N2 physisorption. The ruthenium complex-loaded SBA-15 silica samples were tested against A549 lung tumor cells and MRC-5 normal lung fibroblasts. A dose-dependent effect was observed, with the highest antitumoral efficiency being recorded for the material containing [Ru(Salen)(PPh3)Cl] (50%/90% decrease in the A549 cells' viability at a concentration of 70 μg/mL/200 μg/mL after 24 h incubation). The other hybrid materials have also shown good cytotoxicity against cancer cells, depending on the ligand included in the ruthenium complex. The antibacterial assay revealed an inhibitory effect for all samples, the most active being those containing [Ru(Salen)(PPh3)Cl], [Ru(Saldiam)(PPh3)Cl], and [Ru(Salaepy)(PPh3)Cl], especially against Staphylococcus aureus and Enterococcus faecalis Gram-positive strains. In conclusion, these nanostructured hybrid materials could represent valuable tools for the development of multi-pharmacologically active compounds with antiproliferative, antibacterial, and antibiofilm activity.

“Half-Sandwich” Ruthenium Complexes with Alizarin as Anticancer Agents: In Vitro and In Vivo Studies

Upon exploration of the chemistry of the combination of ruthenium/arene with anthraquinone alizarin (L), three new complexes with the general formulas [Ru(L)Cl(η6-p-cymene)] (C1), [Ru(L)(η6-p-cymene)(PPh3)]PF6 (C2), and [Ru(L)(η6-p-cymene)(PEt3)]PF6 (C3) were synthesized and characterized using spectroscopic techniques (mass, IR, and 1D and 2D NMR), molar conductivity, elemental analysis, and X-ray diffraction. Complex C1 exhibited fluorescence, such as free alizarin, while in C2 and C3, the emission was probably quenched by monophosphines and the crystallographic data showed that hydrophobic interactions are predominant in intermolecular contacts. The cytotoxicity of the complexes was evaluated in the MDA-MB-231 (triple-negative breast cancer), MCF-7 (breast cancer), and A549 (lung) tumor cell lines and MCF-10A (breast) and MRC-5 (lung) nontumor cell lines. Complexes C1 and C2 were more selective to the breast tumor cell lines, and C2 was the most cytotoxic (IC50 = 6.5 μM for MDA-MB-231). In addition, compound C1 performs a covalent interaction with DNA, while C2 and C3 present only weak interactions; however, internalization studies by flow cytometry and confocal microscopy showed that complex C1 does not accumulate in viable MDA-MB-231 cells and is detected in the cytoplasm only after cell permeabilization. Investigations of the mechanism of action of the complexes indicate that C2 promotes cell cycle arrest in the Sub-G1 phase in MDA-MB-231, inhibits its colony formation, and has a possible antimetastatic action, impeding cell migration in the wound-healing experiment (13% of wound healing in 24 h). The in vivo toxicological experiments with zebrafish indicate that C1 and C3 exhibit the most zebrafish embryo developmental toxicity (inhibition of spontaneous movements and heartbeats), while C2, the most promising anticancer drug in the in vitro preclinical tests, revealed the lowest toxicity in in vivo preclinical screening.

Targeted Overexpression of NDRG2 using Survivin Promoter Reduces Viability and Invasiveness of A549 Cell Line

Anti-tumor effects of N-myc Downstream Regulated Gene2 (NDRG2) have been demonstrated in many tumors. In the present study, NDRG2 was specifically overexpressed in lung cancer cell line using Survivin Promoter (Sur-P). Then, the effects of NDRG2 overexpression on viability, apoptosis, migration, and invasion of A549 cells were evaluated. Recombinant pAdenoVator-Sur-P-NDRG2-IRES-GFP plasmid harboring NDRG2 gene under transcriptional control of Sur-P and mock plasmid were constructed. A549 lung tumor cells and LX-2 cells (non-tumor cell line) were transfected with pAdenoVator-Sur-P-NDRG2-IRES-GFP, pAdenoVator-CMV-NDRG2-IRES-GFP, or mock plasmids. Tumor specificity of Sur-P was evaluated using fluorescent microscopy for GFP expression. The effects of NDRG2 overexpression on cell viability, apoptosis, and migration of A549 cells were measured using MTT, annexinV/7-AAD flow cytometry, and transwell migration assay, respectively. NDRG2 and matrix metalloproteinase-2 (MMP-2) expression were measured using real time-PCR. pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection resulted in a huge GFP expression in A549 cells, but not in LX-2 cells. The results of real time-PCR analysis also showed that pAdenoVator-Sur-P-NDRG2-IRES-GFP transfection led to an abundant NDRG2 expression in A549 cells. NDRG2 overexpression decreased A549 cell viability through increasing cell apoptosis. In addition, migration, invasion, and MMP-2 expression decreased following NDRG2 overexpression in A549 cells. The findings indicate that the targeted overexpression of NDRG2 using Sur-P can reduce the viability and invasiveness of A549 cells, suggesting possible benefits of this approach in lung cancer therapy.

Smurf2 inhibition enhances chemotherapy and radiation sensitivity in non-small-cell lung cancer

Lung cancer has been the most common cancer worldwide for several decades. The outcomes of patients with locally advanced lung cancer remain dismal, and only a minority of patients survive more than 5 years. However, tumor therapeutic resistance mechanisms are poorly studied. Identification of therapeutic resistance pathways in lung cancer in order to increase the sensitivity of lung tumor cells to therapeutic agents is a crucial but challenging need. To identify novel genes that modulate the response to platinum-based therapy, we performed a genome-wide high-throughput ribonucleic acid interference (RNAi) screen via transfection of human lung cancer (PC9) cells with a viral short hairpin RNA (shRNA) library. We further validated a potential target via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic survival assays on PC9 and A549 lung tumor cells transfected with small interfering RNAs (siRNAs) to successfully downregulate protein expression and then treated with increasing doses of cisplatin or X-ray radiation. We determined protein expression by immunohistochemistry (IHC) after chemoradiotherapy and analyzed gene expression-based survival outcomes in two cohorts of human non-small-cell lung cancer (NSCLC) patients. The screen identified several targets involved in epithelial-to-mesenchymal transition (EMT), including Smurf1, Smurf2, YAP1, and CEBPZ, and glycolytic pathway proteins, including PFKFB3. Furthermore, we found that the small molecule proteasome inhibitor bortezomib significantly downregulated Smurf2 in lung cancer cells. The addition of bortezomib in combination with cisplatin and radiation therapy in PC9 and A549 cells led to an increase in deoxyribonucleic acid (DNA) double-strand breaks with increased numbers of γ-H2AX-positive cells and upregulation of apoptosis. Finally, we found that Smurf2 protein expression was upregulated in situ after treatment with cisplatin and radiation therapy in a relevant cohort of patients with stage III NSCLC. Additionally, Smurf2 gene expression was the strongest predictor of survival in patients with squamous NSCLC after chemotherapy or chemoradiotherapy. We successfully identified and validated Smurf2 as both a common modulator of resistance and an actionable target in lung cancer. These results suggest the urgent need to investigate clinical Smurf2 inhibition via bortezomib in combination with cisplatin and radiation for patients with locally advanced NSCLC.

Assessment of antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone in subcutaneous xenografts of A-549 tumor cell culture.

e20520 Background: The aim of the study was an assessment of antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone for subcutaneous xenografts of A-549 lung tumor cells in immunodeficient Balb/c Nude mice. Methods: 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone is a new compound in the series of 2-quinoline-2-yl-1,3-tropolone derivatives. The study included 50 immunodeficient Balb/c Nude mice divided into 5 groups depending on the dosage of the studied substance (0.0055; 0.055; 0.55 and 2.75 mg/g). The A-549 cells of lung cancer were used as the xenograft. Antitumor effect of tropolone was evaluated by the inhibition of tumor growth (ITG%) and the index of tumor growth (I). The experiment lasted for 36 days starting from the first administration of substances. Results: Indicators of the average tumor volume on day 36 of the experiment in experimental groups 1, 2, 3 and 4 and in the control group were 678.9; 952.3; 1746.4; 2150.8 and 2729.5 mm3, respectively. The indices of tumor growth in groups 1, 2, 3 and 4 were statistically significantly lower than in group 5 (controls) starting from days 15, 21, 21 and 24, respectively, and until the end of the experiment. Maximal differences between groups 1 and 5 were observed on days 33 and 36 – by 3.7 (p = 0.01) and 4.1 (p = 0.003) times, respectively. Antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone demonstrated by the study could be associated with various mechanisms. For example, its closely related compound – hinoktiol, according to numerous studies, has a cytotoxic effect associated with stopping the cell cycle, induction of apoptosis, DNA damage, and autophagic death of tumor cells. Conclusions: The study demonstrated statistically significant differences in xenograft volumes between all experimental groups and the control group. 2.75 mg/g of mouse weight was the most effective dosage of the studied compound leading to a slow decrease in tumor growth rates and a decrease in the volumes of subcutaneous xenografts.

Sulfated galactoglucan impedes xenografted lung cancer cell growth by blocking angiogenesis via binding BMPRs

Evidences propound tumor growth may be impeded by blocking angiogenesis. Before we showed that sulfated glucan or arabinogalactan might bind to BMP2 or its receptors to inhibit angiogenesis. Whether sulfated galactoglucan can target both BMPRIA and BMPRII to impede angiogenesis and tumor cells growth is still vague. Here, we prepare galactoglucan and its sulfated derivatives Sul-CDA-0.05. The sulfate groups substituted are at the C-6 of 1, 4-linked α-Glcp and 1, 4-linked α-Galp backbone and at the C-6 of branch chain T-linked α-Glcp. Sul-CDA-0.05 can inhibit angiogenesis in vitro and in vivo. Indeed, Sul-CDA-0.05 impedes xenografted A549 lung tumor cells growth. Mechanism study demonstrates that this polysaccharide may target both BMPRIA and BMPRII to block BMP/Smad/Id1 signaling and attenuate VEGF and its transcription factor. Our evidences suggest that Sul-CDA-0.05 may be a new drug candidate for anti-lung cancer therapy by targeting both BMPRIA and BMPRII.

Assessment of an antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone in A-549 tumor cell subcutaneous xenografts

Rationale: Chemotherapy is one of the lung cancer treatment methods. The search for new substances with antitumor effect against malignant lung neoplasms is relevant because of low efficacy and side effects of cytotoxic agents. A promising substance class with various biological activities, including antitumor, includes alkaloids of the tropolone family, such as heptamerous non-benzoid aromatic compounds. 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)- 5,6,7-trichloro-1,3-tropolone has been synthesized in Institute of Physical and Organic Chemistry; it is a new compound belonging to 2-quinoline-2-yl derivatives of 1,3-tropolone.Aim: To assess the antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)- 5,6,7-trichloro-1,3-tropolone on subcutaneous xenografts of A-549 lung tumor cells in immunodeficient Balb/c Nude mice.Materials and methods: The study included 50 immunodeficient Balb/c Nude mice divided into 4 experimental groups depending on the dosage of the study substance (0.0055, 0.055, 0.55, and 2.75 mg/g); group 5 was the control group. A-549 cells of lung cancer were used as a xenograft. The antitumor effect of tropolone was evaluated by the inhibition of tumor growth and the index of tumor growth. The experiment lasted for 36 days starting from the first administration of the substances.Results: The mean tumor volumes on day 36 of the experiment in the control group and four experimental groups were 2729.5; 2150.8; 1746.4; 952.3 and 678.9 mm3 , respectively. The indices of tumor growth in groups 1, 2, 3 and 4 were significantly lower than in group 5 (control) starting from days 24, 21, 21 and 15, respectively, and till the end of the experiment. Maximal differences between groups 4 and 5 were observed at days 33 and 36 (by 3.7, p=0.01 and 4.1, p=0.003 times, respectively).Discussion: The anti-tumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)- 5,6,7-trichloro-1,3-tropolone demonstrated in the study could be related to various mechanisms. For example, numerous studies have shown that its related compound hinokitiol exerts a cytotoxic effect associated with cessation of the cell cycle, apoptosis induction, DNA damage, and autophagic death of tumor cells.Conclusion: The study demonstrated significant differences in xenograft volumes in all experimental groups, compared to the control group. In mice, 2.75 mg/g bodyweight was the most effective dosage of the studied compound leading to a slow decrease in tumor growth rates and a decrease in the volumes of subcutaneous xenografts.

Design and Characterization of Spray-Dried Proliposomes for the Pulmonary Delivery of Curcumin.

PurposeThe goal was to directly deliver curcumin, a natural polyphenolic anticancer and anti-inflammatory compound, to the lung tissues with minimal systemic exposure through the fabrication of proliposomes, overcoming its poor aqueous solubility and oral bioavailability.MethodsNano-spray drying was employed to prepare proliposomes using hydroxypropyl beta-cyclodextrin as a carrier. Lecithin and cholesterol were used as lipids, stearylamine and Poloxamer 188 were added as positive charge inducer and a surfactant, respectively. Different characterization parameters were evaluated like percentage yield, entrapment efficiency, drug loading, aerodynamic particle size, in vitro release besides morphological examination. Cytotoxicity studies on cell line A549 lung tumor cells as well as in vivo lung pharmacokinetic studies were also carried.ResultsThe optimized formulations showed superior aerosolization properties coupled their enhanced ability to reach deep lung tissues with a high % of fine particle fraction. Cytotoxicity studies using MTT assay demonstrated enhanced growth inhibitory effect on lung tumor cells A549 and significant reduction of proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6 and interleukin-10 compared to the pure drug. Results of lung pharmacokinetic tests confirmed the superiority of proliposomal curcumin over curcumin powder in both, the rate and extent of lung tissue absorption, as well as the mean residence time within the lung tissues.ConclusionThe pulmonary delivery of curcumin-loaded proliposomes as dry powder provides a direct approach to lung tissues targeting while avoiding the limitations of the oral route and offering a non-invasive alternative to the parenteral one.

Sensitization of A-549 lung cancer cells to Cisplatin by Quinacrine-loaded lipidic nanoparticles via suppressing Nrf2 mediated defense mechanism.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is believed to be responsible for the control mechanisms of cellular defense response and master regulator of antioxidant system by adjustment of endogenous antioxidants, phase II detoxifying enzymes and transporters, so inhibition of Nrf2 could be considered molecule target to overcome drug resistance and cancer progression. By harnessing liposome as an advanced nanoparticles transporter, we formulated Quinacrine known as nrf2 inhibitor into nano-carrier, and sensitized A-549 lung tumor cells to Cisplatin. The aim of this work was to prepare liposome nano-carriers to enhance the bioavailability of Quinacrine and to improve passive targeting in A549 cells. Quinacrine formulation into liposome exposed a mean particle size of 80±5 nm in passive targeting and 110±3 after decoration with chitosan oligosaccharides (COS), respectively. The highest amount of cell death (p<0.05) occurred with the co-incubation of the A549 cells with new formulation and Cisplatin. Additionally, Quinacrine-loaded liposomes declined Nrf2 expression more than Quinacrine alone (p<0.05). Correspondingly, the expression of Nrf2 downstream genes, MRP1, Trx, and bcl2 decreased significantly. Taking all the data into consideration, liposomes containing Quinacrine could ameliorate the effectiveness of Cisplatin by raising the permeability of cancer cells to the abovementioned chemical treatment and might be then given as a candidate to boost the therapeutic protocols in cancer patients.

Interpretation of the absorbed constituents and pharmacological effect of Spica Schizonepetae extract on non-small cell lung cancer.

As a traditional Chinese medicine (TCM) with a usage history of over 2,000 years in China, Spica Schizonepetae possesses definite clinical activity in the treatment of non-small cell lung cancer (NSCLC). However, its active ingredients and mechanism of action remain unclear at present. The further exploration of its active components and underlying mechanism will provide a basis for the development of candidate anti-tumor drugs. Our previous study explored the chemical constituents of Spica Schizonepetae extract (SSE). On this basis, molecular networking technology was applied in analyzing the QTOF-MS/MS data of rat plasma after intragastric administration of SSE using the GNPS database platform. A total of 26 components were found, including 9 proterotype components and 17 metabolites, which revealed the potential active ingredients of SSE. Later, the Lewis lung cancer mouse model was established, and the inhibition rate and histopathological sections were used as the indicators to investigate the anti-tumor effect of SSE, whereas the body weight, survival rate, thymus index and spleen index served as the indicators to explore the pharmacological effects of SSE on improving mouse immunity. The results showed that SSE had comparable anti-tumor efficacy to cisplatin, which enhanced the immunity, improved the quality of life, and extended the survival time of lung cancer mice. Furthermore, human A549 lung tumor cells were selected to explore the mechanism of SSE in treating NSCLC based on cell metabonomics. After data mining by the MPP software, 23 differential endogenous metabolites were identified between SSE and tumor groups. Moreover, results of pathway enrichment analysis using the MetaboAnalyst 4.0 software indicated that these metabolites were mainly enriched in four metabolic pathways (p < 0.1). By adopting the network pharmacology method, the metabolic pathways discovered by cell metabolomics were verified against the ChEMBL, STITCH, UniProt and TCGA databases, and differences in the underlying mechanism between cells and humans were found. It was proved that SSE affected the metabolism of purine, arachidonic acid and histidine to exert the anti-tumor efficacy. Furthermore, the multi-target, multi-pathway, and immunoenhancement mechanism of SSE in anti-tumor treatment was revealed, which provided a scientific basis for new drug development and the rational application of Spica Schizonepetae in clinic.

Synthesis and evaluation of novel fluorinated hematoporphyrin ether derivatives for photodynamic therapy

A photosensitizer with high phototoxicity, suitable amphipathy and low dark toxicity could play a pivotal role in photodynamic therapy (PDT). In this study, a facile and versatile approach was adopted to synthesize a series of novel fluorinated hematoporphyrin ether derivatives (I1-I5 and II1-II4), and the photodynamic activities of these compounds were studied. Compared to hematoporphyrin monomethyl ether (HMME), all PSs showed preferable photodynamic activity against A549 lung tumor cells. The longest visible absorption wavelength of these compounds was approximately 622 nm. Among them, II3 revealed the highest singlet oxygen yield (0.0957 min−1), the strongest phototoxicity (IC50 = 1.24 μM), the lowest dark toxicity in vitro, and exhibited excellent anti-tumor effects in vivo. So compound II3 could act as new drug candidate for photodynamic therapy.

Ruthenium (II) complex cis-[RuII(\u014b2-O2CC7H7O2)(dppm)2]PF6-hmxbato induces ROS-mediated apoptosis in lung tumor cells producing selective cytotoxicity

Ruthenium complexes have been extensively explored as potential molecules for cancer treatment. Considering our previous findings on the remarkable cytotoxic activity exhibited by the ruthenium (II) complex 3-hydroxy-4-methoxybenzoate (hmxbato)-cis-[RuII(ŋ2-O2CC7H7O2)(dppm)2]PF6 against Leishmania promastigotes and also the similar metabolic characteristics between trypanosomatids and tumor cells, the present study aimed to analyze the anticancer potential of hmxbato against lung tumor cells, as well as the partial death mechanisms involved. Hmxbato demonstrated selective cytotoxicity against A549 lung tumor cells. In addition, this complex at a concentration of 3.8 µM was able to expressively increase the generation of reactive oxygen species (ROS) in tumor cells, causing an oxidative stress that may culminate in: (1) reduction in cellular proliferation; (2) changes in cell morphology and organization patterns of the actin cytoskeleton; (3) cell arrest in the G2/M phase of the cell cycle; (4) apoptosis; (5) changes in the mitochondrial membrane potential and (6) initial DNA damage. Furthermore, we demonstrated that the induction of programmed cell death can occur by the intrinsic apoptotic pathway through the activation of caspases. It is also worth highlighting that hmxbato exhibited predominant actions on A549 tumor cells in comparison to BEAS-2B normal bronchial epithelium cells, which makes this complex an interesting candidate for the design of new drugs against lung cancer.

Lung Tumor Cell-Derived Exosomes Promote M2 Macrophage Polarization.

Cellular cross-talk within the tumor microenvironment (TME) by exosomes is known to promote tumor progression. Tumor promoting macrophages with an M2 phenotype are suppressors of anti-tumor immunity. However, the impact of tumor-derived exosomes in modulating macrophage polarization in the lung TME is largely unknown. Herein, we investigated if lung tumor-derived exosomes alter transcriptional and bioenergetic signatures of M0 macrophages and polarize them to an M2 phenotype. The concentration of exosomes produced by p53 null H358 lung tumor cells was significantly reduced compared to A549 (p53 wild-type) lung tumor cells, consistent with p53-mediated regulation of exosome production. In co-culture studies, M0 macrophages internalized tumor-derived exosomes, and differentiated into M2 phenotype. Importantly, we demonstrate that tumor-derived exosomes enhance the oxygen consumption rate of macrophages, altering their bioenergetic state consistent with that of M2 macrophages. In vitro co-cultures of M0 macrophages with H358 exosomes demonstrated that exosome-induced M2 polarization may be p53 independent. Murine bone marrow cells and bone marrow-derived myeloid-derived suppressor cells (MDSCs) co-cultured with lewis lung carcinoma (LLC)-derived exosomes differentiated to M2 macrophages. Collectively, these studies provide evidence for a novel role for lung tumor-exosomes in M2 macrophage polarization, which then offers new therapeutic targets for immunotherapy of lung cancer.

Antioxidant Enzymes Level Detection in Cisplatin Treated Iraqi Lung Cancer Patients and In vitro Estimating the Cytotoxic and Reactive Oxygen Species Generation in A549 Cell Line

Cisplatin is one of the most important and effective antitumor drug used as treatment against different types of tumors. Cisplatin directly affects DNA integrity as well as the generation of reactive oxygen species (ROS) and cell death induction. Changes in the activities of antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were investigated in healthy controls and lung tumor patients before and after cisplatin treatment. Untreated patients showed a significant reduction in CAT, GPx, and SOD levels, while cisplatin-treated patients retained the normal levels of antioxidant enzymes compared with healthy controls. Cisplatin exhibited cytotoxic activity against A549 lung tumor cells in a dose-dependent manner with an IC50 of 10.33 μg mL-1. Cisplatin was found to induce the generation of ROS in cisplatin-treated A549 cells compared with a negative control. Such an increase in ROS production was significantly correlated with increasing cisplatin concentration.

An Improved Patient-Derived Xenograft Humanized Mouse Model for Evaluation of Lung Cancer Immune Responses.

Human tumor xenograft models do not replicate the human immune system and tumor microenvironment. We developed an improved humanized mouse model, derived from fresh cord blood CD34+ stem cells (CD34+ HSC), and combined it with lung cancer cell line-derived human xenografts or patient-derived xenografts (Hu-PDX). Fresh CD34+ HSCs could reconstitute detectable mature human leukocytes (hCD45+) in mice at four weeks without the onset of graft-versus-host disease (GVHD). Repopulated human T cells, B cells, natural killer (NK) cells, dendritic cells (DC), and myeloid-derived suppressor cells (MDSC) increased in peripheral blood, spleen, and bone marrow over time. Although cultured CD34+ HSCs labeled with luciferase could be detected in mice, the cultured HSCs did not develop into mature human immune cells by four weeks, unlike fresh CD34+ HSCs. Ex vivo, reconstituted T cells, obtained from the tumor-bearing humanized mice, secreted IFNγ upon treatment with phorbol myristate acetate (PMA) or exposure to human A549 lung tumor cells and mediated antigen-specific CTL responses, indicating functional activity. Growth of engrafted PDXs and tumor xenografts was not dependent on the human leukocyte antigen status of the donor. Treatment with the anti-PD-1 checkpoint inhibitors pembrolizumab or nivolumab inhibited tumor growth in humanized mice significantly, and correlated with an increased number of CTLs and decreased MDSCs, regardless of the donor HLA type. In conclusion, fresh CD34+HSCs are more effective than their expanded counterparts in humanizing mice, and do so in a shorter time. The Hu-PDX model provides an improved platform for evaluation of immunotherapy.

Abstract 2731: A novel PDE10/beta-catenin inhibitor, MCI-048, suppresses lung tumorigenesis to block metastasis

Abstract We previously reported that phosphodiesterase 10A (PDE10) is overexpressed during early stages of lung cancer and is essential for lung tumor cell growth. Here we characterize a novel PDE10 inhibitor, MCI-048, that was identified by screening a library of indene compounds. MCI-048 potently inhibited the growth and induced apoptosis of multiple human lung cancer cell lines expressing PDE10, while normal human airway epithelial cells lacking PDE10 expression were appreciably less sensitive. The mechanism of action of MCI-048 involves PDE10 inhibition, cGMP elevation, PKG activation, and phosphorylation of β-catenin at key residues that induce ubiquitination and proteosomal degradation of the oncogenic pool of β-catenin in cytoplasm to suppress the translocation of active β-catenin to the nucleus and Lef/Tcf-mediated transcription of genes encoding for proteins such as c-myc, cyclin D, and survivin, which are essential for tumor cell proliferation and survival. Pharmacokinetic and tissue distribution studies revealed a unique feature of MCI-048 to accumulate at high concentrations in lungs relative to plasma and other tissues. To assess the potential of MCI-048 for the treatment of lung cancer and blocking metastasis, we tested the drug in two mouse models of lung cancer involving either orthotopic implantation of KRAS mutant A549 lung tumor cells or the chemical carcinogen, urethane. Oral administration of MCI-048 significantly inhibited tumor growth and extended survival in the orthotopic model using two different protocols to either treat the primary tumor or metastasis. Similarly, MCI-048 significantly reduced tumor burden as measured by both surface counting and histopathological analysis in the urethane-induced model of lung tumorigenesis. Biochemical analysis showed that MCI-048 reduced levels of urethane-induced active Ras-GTP and PDE10 levels, as well as other oncogenic markers, including pEGFR and c-Myc. Both mouse models revealed that MCI-048 was well tolerated with no discernable toxicity, thus supporting preclinical development for the treatment of lung cancer. Funding provided by NCI grants R21CA182941, R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Bing Zhu, Veronica Ramirez-Alcantara, Antonio Ward, Kristy Berry, Adam B. Keeton, Michael R. Boyd, Yulia Maxuitenko, Xi Chen, Gary A. Piazza. A novel PDE10/beta-catenin inhibitor, MCI-048, suppresses lung tumorigenesis to block metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2731.

Abstract 1543: Discovery of antibodies for ADC therapy from cancer patients who show durable clinical response to checkpoint inhibitors

Abstract Successful anti-tumor therapy using antibody-drug conjugates (ADCs) depends, in part, on antibodies that bind tumor selective targets that are subsequently internalized at a high rate into tumor cells. Only a select number of tumor-selective internalizing antibodies have been identified, and this has limited the scope of ADC therapeutic strategies in the clinic. Here we demonstrate, using Atreca’s Immune Repertoire Capture (IRC™) technology, that we can identify tumor selective antibodies that have internalization activity suitable for ADC therapeutics directly from cancer patients showing durable clinical response to checkpoint inhibitors. To identify anti-tumor antibodies with hallmarks suitable for ADC applications, we used IRC™ to analyze blood plasmablasts, a type of antibody secreting activated B cell collected from patients with non-progressing metastatic cancer. Antibodies representing expanded clonal families were expressed recombinantly and analyzed for binding to human tumor and non-tumor tissues, as well as for internalization activity using a pH-sensitive dye and cancer cell lines. Those antibodies with a high internalization rate were subsequently tested for in vitro ADC activity using a secondary antibody conjugated with the auristatin MMAE. We identified multiple patient-derived antibodies, from several cancer types, that bound to human tumor tissue, but not adjacent normal tissue. Of these, several also showed internalization into human A549 lung tumor cells when measured through the detection of a pH-sensitive dye. When used in combination with a secondary antibody conjugated to the MMAE cytotoxic agent, internalizing antibodies were able to induce target cell death in vitro. In this study, we used Atreca’s IRC™ platform to identify patient-derived antibodies that bind to tumor-selective antigens and a subset that also internalize into cancer cells. Furthermore, we demonstrate that the internalizing antibodies were able to deliver a cytotoxic payload to induce tumor cell death. Overall, we show that the antibody repertoires of cancer patients contain tumor-selective antibodies with potent internalization properties and strong ADC therapeutic potential. Citation Format: Alexander Scholz, Jerald Aurellano, Michael Harbell, Danhui Zhang, Samantha O'Connor, Beatriz Millare, Felix Chu, Sheila Fernandez, Cathrin J. Czupalla, Iraz Aydin, Amy Manning-Bog, Yvonne Leung, Kevin Williamson, Chantia Carroll, Dongkyoon Kim, Xiaomu Chen, Sean M. Carroll, Ish Dhawan, Ngan Nguyen, Shweta Thyagarajan, Mark Whidden, Gregg Espiritu Santo, Nicole Haaser, Guy Cavet, Lawrence Steinman, Tito A. Serafini, Wayne Volkmuth, Jonathan Benjamin, William H. Robinson, Norman M. Greenberg, Daniel Emerling, Jeff DeFalco. Discovery of antibodies for ADC therapy from cancer patients who show durable clinical response to checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1543.