A549 Tumor Xenografts Research Articles

Targeted Photodynamic Therapy (PDT) of Lung Cancer with Biotinylated Silicon (IV) Phthalocyanine.

Lung cancer is the leading cause of cancer-associated mortality in the world. Traditional cancer therapies prolong the life expectancy of patients but often suffer from adverse reactions. Photodynamic Therapy (PDT) has been recommended as a treatment option for lung cancer in several countries, due to its non-invasive procedures, high selectivity and weak side effects. We have designed and synthesized a biotin receptor-targeted silicon phthalocyanine (IV) (compound 1) which showed a good therapeutic effect on biotin receptor-positive tumors. Since the overexpression of Biotin Receptor (BR) is also present in human lung cancer cells (A549), we explored the therapeutic properties of compound 1 on A549 xenograft tumor models. The selectivity of compound 1 toward A549 cells was studied with a fluorescence microscope and IVIS Spectrum Imaging System. The cytotoxicity was measured using the MTT assay. In vivo anti-tumor activity was investigated on the nude mice bearing A549 xenografts. In vitro assays proved that compound 1 could selectively accumulate in A549 cells via the BR-mediated internalization. In vivo imaging and distribution experiments showed that compound 1 could selectively accumulate in tumor tissues of tumor-bearing mice. After 16 days of the treatment, the volumes of tumor in the PDT group were obviously smaller than that in other groups. This study demonstrates that compound 1 is a promising photosensitizer and has broad application prospects in clinical PDT of lung cancers.

I-BET726 suppresses human skin squamous cell carcinoma cell growth in vitro and in vivo

Bromodomain-containing protein 4 (BRD4) is a potential therapeutic target of skin squamous cell carcinoma (SCC). I-BET726 is a novel BRD4 inhibitor. Its potential effect in skin SCC cells was tested in the present study. We show that I-BET726 potently inhibited survival, proliferation, cell cycle progression, and migration in established (A431/SCC-9/SCC-12/SCC-13 lines) and primary human skin SCC cells. I-BET726 induced significant apoptosis activation in skin SCC cells. It was more efficient in inhibiting skin SCC cells than known BRD4 inhibitors (JQ1, CPI203, and AZD5153). I-BET726 not only downregulated BRD4-regulated proteins (c-Myc, Bcl-2, and cyclin D1), but also inhibited sphingosine kinase 1 (SphK1) and Akt signalings in SCC cells. Restoring Akt activation, by a constitutively active S473D mutant Akt1 (“caAkt1”), partially inhibited I-BET726-induced cytotoxicity in A431 cells. In vivo, I-BET726 oral administration potently inhibited A431 xenograft growth in severe combined immunodeficient mice. Downregulation of BRD4-regulated proteins and inhibition of the SphK1-Akt signaling were detected in I-BET726-treated A431 xenograft tumor tissues. Together, I-BET726 inhibits skin SCC cell growth in vitro and in vivo.

Matrix metalloprotein-triggered, cell penetrating peptide-modified star-shaped nanoparticles for tumor targeting and cancer therapy

BackgroundSpecific targeting ability and good cell penetration are two critical requirements of tumor-targeted delivery systems. In the present work, we developed a novel matrix metalloprotein-triggered, cell-penetrating, peptide-modified, star-shaped nanoparticle (NP) based on a functionalized copolymer (MePEG-Peptide-Tri-CL), with the peptide composed of GPLGIAG (matrix metalloprotein-triggered peptide for targeted delivery) and r9 (cell-penetrating peptide for penetration improvement) to enhance its biological specificity and therapeutic effect.ResultsBased on the in vitro release study, a sustained release profile was achieved for curcumin (Cur) release from the Cur-P-NPs at pH 7.4. Furthermore, the release rate of Cur was accelerated in the enzymatic reaction. MTT assay results indicated that the biocompatibility of polymer NPs (P-NPs) was inversely related to the NP concentration, while the efficiency toward tumor cell inhibition was positively related to the Cur-P-NP concentration. In addition, Cur-P-NPs showed higher fluorescence intensity than Cur-NPs in tumor cells, indicating improved penetration of tumor cells. An in vivo biodistribution study further demonstrated that Cur-P-NPs exhibited stronger targeting to A549 xenografts than to normal tissue. Furthermore, the strongest tumor growth inhibition (76.95%) was observed in Cur-P-NP-treated A549 tumor xenograft nude mice, with slight pulmonary toxicity.ConclusionAll results demonstrated that Cur-P-NP is a promising drug delivery system that possesses specific enzyme responsiveness for use in anti-tumor therapy.

Abstract A62: Inhibition of tumor growth by novel CART redirected against cells expressing high levels of fibroblast activation protein

Abstract Cancer-associated fibroblasts (CAFs) support tumor growth and metastasis of virtually all solid tumors, making them a potential “universal therapeutic target.” However, there are challenges to designing CAF targeted therapies. First, CAFs are heterogeneous, consisting of tumor-promoting and tumor-restraining populations. Second, molecular targets expressed strictly by tumor-promoting CAFs have yet to be defined. Nonetheless, Fibroblast Activation Protein (FAP) is selectively overexpressed on tumor-promoting CAFs. Moreover, deleting FAP+ cells inhibits tumor growth in preclinical models due to reductions in stroma-dependent signals that support tumor cell survival, growth, and progression and in stroma-derived immunosuppressive factors and can enhance efficacy of other therapies when given in combination. Prior studies in preclinical models suggest, however, that selective elimination of FAPhigh cells while sparing normal FAP+ cells that typically express lower levels of FAP may be necessary to avoid toxicity due to on-target, off-tumor effects. Thus, the aim of this study was to generate a FAP-chimeric antigen receptor (CAR) that selectively targets FAPhigh cells but that exhibits minimal activity against FAPlow cells for use in future clinical trials. We generated a FAP-CAR containing a scFv based on a monoclonal antibody (mAb) we generated that reacts with human and mouse FAP, the CD8 transmembrane region, and the CD3z and 41BB cytoplasmic domains. We show that primary human T cells transduced with this novel FAP-CAR exhibit strong reactivity against cells expressing high levels of human or mouse FAP but not FAPlow or FAP-negative cells. Moreover, these FAP-CART cells inhibited the growth of human FAP-negative A549 tumor xenografts infiltrated by FAP+ murine CAFs in NSG mice. Based on these characteristics, this novel FAP-CAR will be developed for use in clinical trials for human cancer patients. Citation Format: Estela Noguera-Ortega, Leslie Todd, James Monslow, Jing Sun, Steven M. Albelda, Ellen Pure. Inhibition of tumor growth by novel CART redirected against cells expressing high levels of fibroblast activation protein [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A62.

RBBP6 induces non-small cell lung cancer cell proliferation and high expression is associated with poor prognosis.

Lung cancer is the most common cause of cancer-associated mortality in China with 85% of patients having non-small cell lung cancer (NSCLC). Identifying NSCLC driver genes and prognostic markers is critical to reducing these numbers. The studies of retinoblastoma binding protein 6 (RBBP6) performed on NSCLC is limited. The present study aimed to investigate the molecular function and the prognostic potential of RBBP6 in NSCLC using the A549 cell line and patient samples, respectively. The functional effect on cancer cell proliferation and prognostic value of RBBP6 were examined in vitro and in vivo using reverse transcription-quantitative PCR, immunofluorescence, immunohistochemistry (IHC) and xenograft implantation. The results demonstrated that RBBP6 mRNA expression was significantly higher in NSCLC tissues compared with in adjacent normal samples. When RBBP6 mRNA expression was interfered with using short hairpin RNA, A549 cell proliferation and xenograft tumor growth were reduced. Additionally, IHC and survival analysis demonstrated that patients with NSCLC with high expression levels of RBBP6 had a shorter median overall survival time compared with patients with low RBBP6 expression (31 vs. 51.5 months), and this was more prominent in stage I–II patients (43 vs. >67 months). High expression levels of RBBP6 indicated poor prognosis in patients with NSCLC. This may be due to the ability of RBBP6 to promote cancer cell proliferation. RBBP6 may be a potential prognostic biomarker and a therapeutic target for NSCLC.

Suppression of asparagine synthetase enhances the antitumor potency of ART and artemalogue SOMCL-14-221 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality. Artemisinin (ART) and SOMCL-14-221 (221), a spirobicyclic analogue of ART, have been reported to inhibit the proliferation of A549cells with unclear underlying mechanism. In the present study, we validated that both ART and 221 inhibited the proliferation and migration of NSCLC cells and the growth of A549 xenograft tumors without appreciable toxicity. The proteomic data revealed proteins upregulated in ART and 221 groups were involved in "response to endoplasmic reticulum stress" and "amino acid metabolism". Asparagine synthetase (ASNS) was identified as a key node protein in these processes. Interestingly, knockdown of ASNS improved the antitumor potency of ART and 221 in vitro and in vivo, and treatments with ART and 221 disordered the amino acid metabolism of A549cells. Moreover, ART and 221 activated ER stress, and inhibition of ER stress abolished the anti-proliferative effects of ART and 221. In conclusion, this study demonstrates that ART and 221 suppress tumor growth by triggering ER stress, and the inhibition of ASNS enhances the antitumor activity of ART and 221, which provides new strategy for drug combination therapy.

Structural analysis of a novel sulfated galacto-fuco-xylo-glucurono-mannan from Sargassum fusiforme and its anti-lung cancer activity

Polysaccharide (HFSGF) was purified from Sargassum fusiforme. Autohydrolysis and gel column chromatography were performed to fractionate HFSGF into three components (HFSGF-S, HFSGF-L and HFSGF-H). Compositional analysis, mass spectrometry and nuclear magnetic resonance spectroscopy were used to elucidate the structural features of HFSGF. HFSGF-S was a mixture of sulfated galacto-fuco-oligomers, from the branches terminal ends; in HFSGF-L, the branches of HFSGF, was a sulfated galactofucan, containing a backbone of 1,3-linked α-L-fucan sulfated at C2/4 and/or C4 and interspersed with galactose (Gal); and in HFSGF-H, the backbone of HFSGF, was composed of alternating 1,2-linked α-D-mannose (Man) and 1,4-linked β-D-glucuronic acid (GlcA), branched with sulfated galactofucan or sulfated fucan, 1,3-linked α-L-fucan sulfated at C2/4 and/or C4 and partly interspersed with Gal. Some fucose (Fuc) residues were also partially branched with xylose (Xyl). The anti-lung cancer activities of HFSGF-L and HFSGF-H against human lung cancer A549 cells in vitro and A549 xenograft tumor growth in vivo were determined. HFSGF-H had higher activity in vitro (IC50 ~12 mg/mL for 24 h) and in vivo (tumor inhibition ~51%.) than HFSGF-L, indicating that HFSGF-H might be a leading compound for a potential new therapeutics for the treatment of lung cancer.

Multi-functional self-assembled nanoparticles for pVEGF-shRNA loading and anti-tumor targeted therapy.

Although RNA interference (RNAi) technology shows great potential in cancer treatment, the tumor target delivery and sufficient cytosolic transport of RNAi agents are still the main obstacles for its clinical applications. Herein, we report a functional supramolecular self-assembled nanoparticle vector for RNAi agent loading and tumor target therapy. Molecular block adamantane-grafted poly(ethylene glycol) (Ad-PEG) was modified with epidermal growth factor receptor (EGFR)-specific binding ligand GE11 or pH-sensitive fusogenic peptide GALA and then used for self-assembly with cyclodextrin-grafted branched polyethylenimine (CD-PEI), adamantane-grafted polyamidoamine dendrimer (Ad-PAMAM), and plasmid DNA containing a small hairpin RNA expression cassette against vascular endothelial growth factor (VEGF) into functional DNA-loaded supramolecular nanoparticles (GE11&GALA-pshVEGF@SNPs) based on molecular recognition and charge interaction. These functional peptides facilitated the target cell binding, internalization, and endosomal escape of GE11&GALA-pshVEGF@SNPs, resulting in increased reporter gene expression and efficient targeted gene silencing. The systemic delivery of the GE11&GALA-pshVEGF@SNPs can efficiently downregulate the intratumoral VEGF protein levels, reduce blood vessel formation, and significantly inhibit A549 xenograft tumor growth. These results reveal the potential of these multifunctional self-assembled nanoparticles as a nucleic acid drug delivery system for the treatment of lung cancer.

Microtubule inhibitors containing immunostimulatory agents promote cancer immunochemotherapy by inhibiting tubulin polymerization and tryptophan-2,3-dioxygenase

A combination therapeutic regimen via introducing tryptophan 2,3-dioxygenase inhibitors into microtubule inhibitors was performed and evaluated for their antitumor activity. Thereinto, HT2, composed of combretastatin A-4 (CA-4) and tryptophan-2,3-dioxygenase (TDO) inhibitor by a linker, displayed the most potent activity with 10-fold higher than its parent CA-4 against HepG2, A549 and HCT-116 cancer cell lines. Mechanism studies suggested that HT2 inhibited tubulin polymerization and cell migration, caused G2 phase arrest, induced apoptosis by mitochondrial mediated apoptotic pathway, concurrent depolarized the mitochondria membrane potentials and caused reactive oxygen species (ROS) production in HepG2 cells. Moreover, HT2 could enhance T-cell immune responses in vitro by releasing a TDO inhibitor to suppress TDO expression and blockade kynurenine production. As expected, HT2 could remarkably promote the antitumor activity of CA-4 in either immunocompetent H22 or immunodeficient A549 tumor xenograft models without observable toxic effects. More importantly, HT2 increased the level of splenic and tumor-infiltrated T cells and in turn effectively boosted the inhibition effect in H22 xenografted tumor growth. Collectively, this immunochemotherapeutic strategy can be applied to promote chemotherapeutic effect.

Water-Soluble Chlorin/Arylaminoquinazoline Conjugate for Photodynamic and Targeted Therapy.

A new water-soluble conjugate, consisting of a chlorin-e6 photosensitizer part, a 4-arylaminoquinazoline moiety with affinity to epidermal growth factor receptors, and a hydrophilic β-d-maltose fragment, was synthesized starting from methylpheophorbide-a in seven steps. The prepared conjugate exhibited low levels of dark cytotoxicity and pronounced photoinduced cytotoxicity at submicromolar concentrations in vitro, with an IC50(dark)/IC50(light) ratio of ∼368 and a singlet oxygen quantum yield of about 20%. In tumor-bearing Balb/c nude mice, conjugate 1 preferentially accumulates in the tumor tissue. Irradiation of the nude mice bearing A431 xenograft tumors after intravenous administration of the prepared conjugate with a relatively low light dose (50 J/cm2) produced an excellent therapeutic effect with profound tumor regression and low systemic toxicity.

BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo

USE1 has been demonstrated to play crucial roles in the development and progression of human lung cancer. However, the antitumor efficacy of RNA interference (RNAi) targeting of USE1 has not yet been evaluated as a possible clinical application. We here synthesized USE1 targeting bubbled RNA-based cargo (BRC) composed of densely packed multimeric pre-siRNAs with specific Dicer cleavage sites to enable efficient siRNA release upon entry to target cells. The physical entanglement and continuous networking of RNAs via hybridization during enzymatic replication serve as a driving force for the self-assembly of BRCs. These molecules effectively suppressed the transcription of their target genes, leading to tumor growth suppression in vitro and in vivo. Moreover, their repeated intravenous administration efficiently inhibited the growth of A549 tumor xenografts. Based on these findings of a reduced cancer cell viability following a USE1 knockdown, we further explored cell cycle arrest and apoptosis pathways. The observed tumor cell growth suppression was found to be controlled by cell cycle arrest and apoptosis signals induced by the USE1 reduction. These results suggest that USE1 BRCs may have future clinical applications as an RNAi-based cancer therapy.

Anti-tumor effect of chitin oligosaccharide plus cisplatin in vitro and in vivo.

BackgroundLung cancer is one of the most common malignant tumors in human beings, and cisplatin is a widely used chemotherapy drug, but its clinical application is limited because of its dose-dependent toxicity and drug resistance. Chitin is known to have various biological activities including anti-tumor, but the insoluble feature in common solvents greatly restricts its application. Chitin oligosaccharide is a small water-soluble molecule degraded from chitin without any toxic effect.MethodsChitin oligosaccharide was adopted to investigate the effects on lung adenocarcinoma A549 cells and tumor xenografts of nude mice. The experiments were divided into control group, chitin oligosaccharide group, cisplatin group and combination group. MTS assay, cell scratch test and migration assay were used to observe the proliferation and migration of A549 cells, and Western blot was used to detect the expression levels of caspase8, caspase3 and BAK. Ki67 and P53 expressions of tumor xenografts were detected to explore the effects of drugs on tumor prognosis.ResultsThe results in vitro showed that chitin oligosaccharides could inhibit the proliferation and migration of A549 cells, and the effect was superior to chitin oligosaccharide or cisplatin when combined with cisplatin. Chitin oligosaccharide plus cisplatin up-regulated the expression level of caspase8 and caspase3, while had minor influence on the expression level of BAK. In vivo experiments showed that chitin oligosaccharide plus cisplatin could down-regulate the expression level of Ki67, while had minor influence on the expression level of P53.ConclusionThe study demonstrated that chitin oligosaccharide plus cisplatin had positive synergistic effects, and it is possible to improve the prognosis of lung adenocarcinoma patients by up-regulating the expression level of caspase8, caspase3 and down-regulating the expression level of Ki67.

The effects and mechanisms of a biosynthetic ginsenoside 3β,12β-Di-O-Glc-PPD on non-small cell lung cancer.

BackgroundA biosynthetic ginsenoside, 3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-dammar-24-ene-3β, 12β, 20S-triol (C3C12PPD), showed antitumor activity against many tumor cells in vitro, especially had better anti-lung cancer activity than Rg3 in vitro and in vivo. However, the effects and molecular mechanisms of C3C12PPD on non-small cell lung cancer (NSCLC) remain unclear. According to previous studies, we hypothesized ginsenoside C3C12PPD could inhibit the tumor growth of NSCLC by targeting proliferation, migration and angiogenesis.MethodsA thiazolyl blue tetrazolium bromide assay (MTT) was performed to evaluate cell viability. Additionally, Transwell and tube formation assays were conducted to analyze cell migration and angiogenesis. The Lewis and A549 tumor xenograft experiments were also performed to investigate the effects of C3C12PPD on tumor growth in vivo, Western blotting and IHC assay were performed to analyze protein expression.ResultsC3C12PPD could effectively inhibit the proliferation and migration of lung cancer cells, and tube formation of EA.hy926 cell. Ginsenoside C3C12PPD suppressed Lewis and A549 tumor growth in vivo without obvious side effects on body weight and the hematology index. In addition, the Western blot analysis revealed that the effects of C3C12PPD on lung cancer were mediated by inhibiting Raf/MEK/ERK, AKT/mTOR and AKT/GSK-3β/β-Catenin signaling pathways. Finally, C3C12PPD could significantly inhibit the proliferation index and vessel number in Lewis xenograft tumors analyzed by IHC.ConclusionThe results of the present study suggest that ginsenoside C3C12PPD may serve as a potential therapeutic candidate compound against NSCLC.

Abstract 5285: The role of HIV-1 TAT interactive protein 2 in modulating tumor response to hypoxia

Abstract While there is ample evidence that HIV-1 TAT interactive protein 2 (HTATIP2) is a tumor suppressor exerting its pro-apoptotic and anti-metastatic activities by regulating the expression of a subset of pro-apoptotic and anti-apoptotic genes and metastasis suppressor genes, it remains unclear if HTATIP2 has a role in tumor response to hypoxia. The objective of this study was to define the role of HTATIP2 in mediating certain molecular and cellular changes in response to hypoxia in tumors. We employed a series of isogenic A549 human lung adenocarcinoma sublines that were genetically deficient for HTATIP2 and/or HIF-1α to examine the potential modulatory effect of HTATIP2 on oncogenic signaling pathways mediated by HIF-1α and HIF-2α, and established the HTATIP2-deficient A549 xenograft model to explore the impact of deletion of HTATIP2 on tumor response to hypoxia. Results from the human HIF-regulated cDNA plate array study revealed that under hypoxic condition (0.5% O2), several genes promoting tumor metastasis and resistance to apoptosis and anticancer treatment, including HIF1α, CYSD, MMP1, MXI1, NDRG2, PAI1 and NDRG1, were upregulated to a greater extent in HTATIP2 knockdown A549 cells than in the control A549 cells. In contrast, genes involved in angiogenesis, glucose metabolism and protection of apoptosis under hypoxia, such as PDGF, PDK1, CA9, and SAG, were upregulated to a greater extent in control A549 cells than in HTATIP2-knockdown A549 cells. Results from the in vivo study demonstrated that the mean growth rate of HTATIP2-deficient A549 xenograft tumors was significantly increased as compared with that of parental A549 xenograft tumors irrespective of the presence or absence of sorafenib treatment. Moreover, the data of photoacoustic imaging (PAI) of tumor hypoxia revealed that the parental A549 tumors were better oxygenated than the counterpart HTATIP2-deficient A549 tumors. After the 10-day sorafenib treatment, the oxygenated regions in sorafenib-treated parental A549 tumors were significantly reduced as compared with the vehicle-treated parental A549 tumors, whereas no significant change in the oxygenated regions was found between vehicle- and sorafenib-treated HTATIP2-deficient A549 tumors. The overall finding of our study suggests that downregulation of HTATIP2 affords growth advantage to tumor cells by fine-tuning tumor adaptation to hypoxia, leading to tumor progression and aggression. Citation Format: Qingyu Stephanie Zhou, Xiaofang Guo, Minghua Li, Hua Pan. The role of HIV-1 TAT interactive protein 2 in modulating tumor response to hypoxia [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 5285.

A chimeric poxvirus with J2R (thymidine kinase) deletion shows safety and anti-tumor activity in lung cancer models

Oncolytic viruses have shown excellent safety profiles in preclinical and clinical studies; however, in most cases therapeutic benefits have been modest. We have previously reported the generation of a chimeric poxvirus (CF33), with significantly improved oncolytic characteristics, through chimerization among different poxviruses. Here we report the sequence analysis of CF33 and oncolytic potential of a GFP-encoding CF33 virus (CF33-GFP) with a J2R deletion in lung cancer models. Replication of CF33-GFP and the resulting cytotoxicity were higher in cancer cell lines compared to a normal cell line, in vitro. After infection with virus, cancer cells expressed markers for immunogenic cell death in vitro. Furthermore, CF33-GFP was safe and exerted potent anti-tumor effects at a dose as low as 1000 plaque forming units in both virus-injected and un-injected distant tumors in A549 tumor xenograft model in mice. Likewise, in a syngeneic model of lung cancer in mice, the virus showed significant anti-tumor effect and was found to increase tumor infiltration by CD8+ T cells. Collectively, these data warrant further investigation of this novel chimeric poxvirus for its potential use as a cancer bio-therapeutic.

An Oncolytic Adenovirus Vector Expressing p14 FAST Protein Induces Widespread Syncytium Formation and Reduces Tumor Growth Rate In Vivo

Intratumoral injection of oncolytic viruses provides a direct means of tumor cell destruction for inoperable tumors. Unfortunately, oncolytic vectors based on human adenovirus (HAdV) typically do not spread efficiently throughout the tumor mass, reducing the efficacy of treatment. In this study, we explore the efficacy of a conditionally replicating HAdV vector expressing the p14 Fusion-Associated Small Transmembrane (FAST) protein (CRAdFAST) in both immunocompetent and immunodeficient mouse models of cancer. The p14 FAST protein mediates cell-cell fusion, which may enhance spread of the virus-mediated, tumor cell-killing effect. In the murine 4T1 model of cancer, treatment with CRAdFAST resulted in enhanced cell death compared to vector lacking the p14 FAST gene, but it did not reduce the tumor growth rate in vivo. In the human A549 lung adenocarcinoma model of cancer, CRAdFAST showed significantly improved oncolytic efficacy in vitro and in vivo. In an A549 xenograft tumor model in vivo, CRAdFAST induced tumor cell fusion, which led to the formation of large acellular regions within the tumor and significantly reduced the tumor growth rate compared to control vector. Our results indicate that expression of p14 FAST from an oncolytic HAdV can improve vector efficacy for the treatment of cancer.

Glucocorticoid modulatory element-binding protein 1 (GMEB1) interacts with the de-ubiquitinase USP40 to stabilize CFLARL and inhibit apoptosis in human non-small cell lung cancer cells

BackgroundGMEB1 was originally identified via its interaction with GMEB2, which binds to the promoter region of the tyrosine aminotransferase (TAT) gene and modulates transactivation of the glucocorticoid receptor gene. In the cytosol, GMEB1 interacts with and inhibits CASP8, but the molecular mechanism is currently unknown.MethodsHuman non-small cell lung cancer cells and 293FT cells were used to investigate the function of GMEB1/USP40/CFLARL complex by WB, GST Pull-Down Assay, Immunoprecipitation, Immunofluorescence and Flow cytometry analysis. A549 cells overexpressing green fluorescent protein and GMEB1 shRNA were used for tumor xenograft using female athymic nu/nu 4-week-old mice.ResultsWe found GMEB1 interacted with CFLARL (also known as c-FLIPL) in the cytosol and promoted its stability. USP40 targeted CFLARL for K48-linked de-ubiquitination. GMEB1 promoted the binding of USP40 to CFLARL. USP40 knockdown did not increase CFLARL protein level despite GMEB1 overexpression, suggesting GMEB1 promotes CFLARL stability via USP40. Additionally, GMEB1 inhibited the activation of pro-caspase 8 and apoptosis in non-small cell lung cancer (NSCLC) cell via CFLARL stabilization. Also, GMEB1 inhibited the formation of DISC upon TRAIL activation. CFLARL enhanced the binding of GMEB1 and CASP8. Downregulation of GMEB1 inhibited A549 xenograft tumor growth in vivo.ConclusionsOur findings show the de-ubiquitinase USP40 regulates the ubiquitination and degradation of CFLARL; and GMEB1 acts as a bridge protein for USP40 and CFLARL. Mechanistically, we found GMEB1 inhibits the activation of CASP8 by modulating ubiquitination and degradation of CFLARL. These findings suggest a novel strategy to induce apoptosis through CFLARL targeting in human NSCLC cells.

Polymorphs, co-crystal structure and pharmacodynamics study of MBRI-001, a deuterium-substituted plinabulin derivative as a tubulin polymerization inhibitor.

MBRI-001, a deuterium-substituted plinabulin derivative, has been reported to have better pharmacokinetic and similar antitumor effects in comparison with plinabulin. In this approach, we further carried out its polymorphs, co-crystal structure of MBRI-001-tubulin and tubulin inhibition study. Among the different polymorphs, Form F (MBRI-001/H2O) was prepared and evaluated, which had better physical stability and suitable process for scale-up production. Co-crystal structure of MBRI-001-tubulin (PDB:5XI5) was prepared and analyzed. The result of tubulin polymerization assay demonstrated that MBRI-001 could inhibit tubulin polymerization which was similar as plinabulin. Subsequently, the anti-proliferative activities of plinabulin and MBRI-001 were evaluated against two different human lung cancer cell lines. In vivo study, MBRI-001 revealed similar antitumor inhibition in comparison with plinabulin in A549 xenograft tumor model. Therefore, we suggested that MBRI-001 could be developed as a promising anti-cancer agent in near future.

Induction of p53-mediated senescence is essential for the eventual anticancer therapeutic effect of RH1.

RH1 (2, 5-diaziridinyl-3-(hydroxymethy)-6-methyl-1, 4-benzoquinone) is a bioreductive anticancer drug. The mechanism underlying its therapeutic properties has not yet been elucidated. In this study, we aimed to determine whether RH1 exerts its anticancer effect via p53-mediated apoptosis and senescence in vitro and in vivo. RH1 displayed dose-dependent biphasic effects in vitro, i.e., it induced apoptosis at higher dose and senescence at lower dose accompanied by marked activation of p53. Thus, RH1 primarily induced cell death by apoptosis. The cytotoxicity of RH1 was inhibited in A549 cells treated with the p53-inhibitor pifithrin-α or transfected p53 siRNA and in human colon cancer HCT116 isogenic (p53-/-) cells. At sub-lethal doses of RH1, the cells survived and underwent senescence. The senescent cells showed flattened and enlarged morphology, and exhibited blue color in senescence-associated β-galactosidase staining. These changes were found to be related to p53. RH1-induced senescence decreased in A549-E6 cells (suppressed p53 level) and HCT 116 p53-/- cells. The growth of A549 xenograft tumors in nude mice was significantly delayed by intraperitoneal injection of RH1, and senescent cells were observed in these xenograft tumors. These results suggest that the in vivo anticancer therapeutic effect of RH1 is mediated by senescence via p53 activation.

Imaging of Tumor Spheroids, Dual-Isotope SPECT, and Autoradiographic Analysis to Assess the Tumor Uptake and Distribution of Different Nanobodies

PurposeRecent studies have shown rapid accumulation of nanobodies (NBs) in tumors and fast clearance of the unbound fraction, making NBs exceptional tracers for cancer imaging. In this study, we investigate the combination of in vitro imaging of tumor spheroids, in vivo dual-isotope single-photon emission computed tomography (SPECT), and ex vivo autoradiographic analysis of tumors to efficiently, and with few mice, assess the tumor uptake and distribution of different NBs.ProceduresThe irrelevant NB R2 (16 kDa) and the EGFR-targeted NBs 7D12 (16 kDa) and 7D12-R2 (32 kDa) were investigated. Confocal microscopy was used to study the penetration of the NBs into A431 tumor spheroids over time, using the anti-EGFR monoclonal antibody (mAb) cetuximab (150 kDa) as a reference. Dual-isotope [111In]DOTA-NB/[177Lu]DOTA-NB SPECT was used for longitudinal imaging of multiple tracers in the same animal bearing A431 tumor xenografts. Tumor sections were analyzed using autoradiography.ResultsNo binding of the irrelevant NB was observed in spheroids, whereas for the specific tracers an increase in the spheroid’s covered area was observed over time. The NB 7D12 saturated the spheroid earlier than the larger, 7D12-R2. Even slower penetration was observed for the large mAb. In vivo, the tumor uptake of 7D12 was 19-fold higher than R2 after co-injection in the same animal, and 2.5-fold higher than 7D12-R2 when co-injected. 7D12-R2 was mainly localized at the rim of tumors, while 7D12 was found to be more evenly distributed.ConclusionsThis study demonstrates that the combination of imaging of tumor spheroids, dual-isotope SPECT, and autoradiography of tumors is effective in comparing tumor uptake and distribution of different NBs. Results were in agreement with published data, highlighting the value of monomeric NBs for tumor imaging, and re-enforcing the value of these techniques to accurately assess the most optimal format for tumor imaging. This combination of techniques requires a lower number of animals to obtain significant data and can accelerate the design of novel tracers.