Adenocarcinoma Cell Line Research Articles

Abstract 2962: Molecular mechanisms underlying the sensitizing effect of mutant p53 protein expression for Auranofin treatment of NSCLC and PDAC cells

Abstract Introduction: Auranofin (AF) is an orally available, organogold compound that is FDA-approved for its use in rheumatoid arthritis. AF gained interest as an anti-cancer agent since it acts as a thioredoxin reductase (TrxR) inhibitor. Data extracted from The Cancer Genome Atlas show that TXNRD1 is abundantly expressed in lung adenocarcinoma patients and negatively affects their outcome, making TrxR1 an attractable target in non-small cell lung cancer (NSCLC) patients. The goal of this study was to determine if mutant p53 expression can be used as a predictive biomarker for the treatment of AF in NSCLC. In addition, we aimed to obtain a profound understanding of the molecular mechanisms that govern the sensitizing effect of mutant p53 towards AF-treated NSCLC cells by investigating the type of cell death. Material and methods: We determined IC50-values using the sulforhodamine-B assay for AF in a panel of NSCLC and pancreatic ductal adenocarcinoma (PDAC) cell lines with different p53 mutations and expression levels. In addition, we correlated these IC50-values to baseline expression levels of different antioxidant related proteins. Results were confirmed in the NSCLC cell line NCI-H1299 (p53 deletion, null) and its two isogenic derivates (mutant p53 R175H or R273H knock-in). mRNA-Seq of the isogenic cell line panel was performed after exposure to a medium dose of AF. Finally, the IncuCyte ZOOM system was used to determine the percentage of AF-induced cell death by the means of the Cytotox Green reagent after pretreatment of the tumor cells with a panel of apoptosis, necroptosis and ferroptosis inhibitors. Results: A strong inverse correlation was observed between mutant p53 protein expression and AF IC50-values and baseline Trx levels in the NSCLC and PDAC cells, indicating that mutant p53 can sensitize cancer cells for AF. This sensitizing effect was confirmed in the p53Mutant isogenic NSCLC cells. Transcriptome analysis revealed the up- and downregulation of specific genes involved in ferroptosis. More specifically, AF induced cell death through various mechanisms, including apoptosis and ferroptosis, which were dependent on the expression levels of p53 rather than the type of p53 mutation. In cells with high expression levels of mutant p53, AF induced ferroptosis and lipid peroxidation. At lower expression levels of p53, the apoptosis pathway was triggered by AF with an increase in caspase-3/7 positive cells and inhibition of cell death by the caspase inhibitor Z-VAD-FMK. Conclusion: Mutant p53 protein expression renders NSCLC and PDAC cells more susceptible to AF. In an isogenic setting, medium levels of mutant p53 sensitize NSCLC cells for apoptosis while cells with higher levels are effectively killed through ferroptosis. Altogether, AF presents a potential novel therapeutic strategy to efficiently kill p53 mutated NSCLC tumor cells by various types of cell death. Citation Format: Laurie Freire Boullosa, Filip Lardon, Evelien Smits, Christophe Deben. Molecular mechanisms underlying the sensitizing effect of mutant p53 protein expression for Auranofin treatment of NSCLC and PDAC cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2962.

Abstract 4717: Ly6-neurotoxin1 knockout in PDAC cells inhibits their growth in vitro and in vivo

Abstract Ly6/neurotoxin1 (Lynx1) functions as a brake for nicotinic receptors and was defined as a tumor suppressor in lung cancer. As pancreatic cancer development may be slowed down by cholinergic signaling, we investigated the role of Lynx1 in pancreatic ductal adenocarcinoma (PDAC) cell lines, both in vitro and in vivo. Lynx1 knockout cell clones were generated by transfecting the CRISPRCas9 plasmid - (pSpCas9 (BB)-2A-Puro) into Miapacaluci and BXPC3luci PDAC cells using jet Pei and jet Prime as transfecting agents, respectively. The annealed gRNA was directed towards exon 3 of the Lynx1 gene. Control clones were transfected with the plasmid alone. Clones from both cell lines were tested by qRT-PCR and Western blot for knockout efficiency as well as by proliferation assay. In addition, the expression levels of p-MEK, p-MAPK, p-mTOR and p-Rictor were verified in two BXPC3 cell clones in relation to Lynx1 expression. Furthermore, 4x106 cells of four BXPC3 cell clones (one control, 3 knockout clones) were injected, respectively, into the portal vein of nude rats to control for a possibly reduced tumor growth in the liver. The knockdown of Lynx1 was incomplete at mRNA level and ranged from 20 to 60% in BXPC3 clones and from 0 to 30% in Miapaca clones. At protein level, the respective values ranged from 0 to 90% in BXPC3 and from 22 to 50% in Miapaca clones. However, Lynx1 protein levels increased at later time points. All BXPC3 clones proliferated less quickly than the respective control when tested by MTT assay. Miapaca clones, however, didn't show a significant difference from the respective control, although their growth was clearly disturbed shortly after transfection. Concomitantly with reduced Lynx1 protein levels, there was reduction of p-mTOR (90%), p-Rictor (30%) and p-MEK (40%) in BXPC3 cells. In vivo, the BXPC3 clones showed a lag period of 1 to 2 weeks till the appearance of a first bioluminescence signal indicating tumor growth. Rats injected with cells from the control clone showed a steady increase in the bioluminescence signal (n=7 of 10) as compared to the most sensitive knockdown clone, which didn't show any signal in any of 4 injected rats (p=0.05). Cells of 2 other BXPC3 clones showed a reduced growth rate at best in 2 of 2 rats used for each clone, respectively. In conclusion, knockout of Lynx1 was incomplete at both mRNA and protein levels. Nevertheless, the respective BXPC3 clones exhibited reduced proliferation in vitro, which was associated with diminished p-mTOR, p-Rictor and p-MEK levels. In addition, they failed to establish a tumor in vivo or showed a reduced tumor growth rate. These findings suggest that Lynx1 is a vital gene and may play an important role in the growth and establishment of PDAC cells. Citation Format: Doaa Ali, Michael Zepp, Matthias Bozza, Maria Nikolova, Richard Harbottle, Martin R. Berger. Ly6-neurotoxin1 knockout in PDAC cells inhibits their growth in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4717.

Abstract 5091: TIMP-1 in tumor-associated fibroblasts drives tumor progression in lung adenocarcinoma through CD63 interaction

Abstract Tumor associated fibroblasts (TAFs) are important regulators of tumor growth and resistance to therapies. We have recently shown that lung TAFs in vitro respond to the antifibrotic drug nintedanib in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). We also showed that the tumor-promoting effects of TAFs are driven by different mechanisms in ADC and SCC, which remain to be elucidated. Tissue inhibitor of metalloproteinases 1 (TIMP-1) has been associated with poor prognosis in lung cancer, its expression is downregulated by nintedanib, and our preliminary results reveal that its putative receptor, CD63, is overexpressed in ADC patients compared to SCC, supporting a selective crosstalk between TAFs and cancer cells in ADC through TIMP-1 and CD63. The aim of this study was to test this hypothesis using in vitro preclinical models. Primary fibroblasts were obtained with the patient informed consent, and using protocols approved by the Ethics Committee of the Hospital Clinic. ADC-TAFs and SCC-TAFs were stimulated with TGF-β1 in the presence or absence of nintedanib, and the TIMP-1 content in their conditioned medium was determined by ELISA. TIMP-1 was knocked-down in ADC-TAFs by siRNA, and the corresponding conditioned medium was used to stimulate growth and invasion of the high-CD63 ADC cell line, H1437. Likewise, CD63 expression in H1437 cells was reduced by siRNA. Our in vitro results showed that TIMP-1 secretion induced by TGF-β1 is significantly larger in ADC-TAFs compared to SCC-TAFs. Likewise, nintedanib elicited a higher downregulation of secreted TIMP-1 in ADC-TAFs compared to SCC-TAFs. Of note, TIMP-1 from ADC-TAFs was necessary to induce growth and invasion of H1437 cells. Likewise, knocking-down CD63 in H1437 ADC cells was sufficient to reduce the growth and invasion elicited by the conditioned medium of TGF-β1 activated ADC-TAFs. Collectively, our results unveil a novel stroma-carcinoma crosstalk driven by TIMP-1 and CD63 selectively in lung ADC, and support that such heterotypic crosstalk may underlie the aberrant tumor-promoting effects of ADC-TAFs that are selectively downregulated by nintedanib. Citation Format: Paula Duch, Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Frank Hillberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. TIMP-1 in tumor-associated fibroblasts drives tumor progression in lung adenocarcinoma through CD63 interaction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5091.

Proteins from Lignosus tigris with selective apoptotic cytotoxicity towards MCF7 cell line and suppresses MCF7-xenograft tumor growth.

BackgroundLignosus tigris, a recently discovered species of the unique Lignosus family, has been traditionally used by the indigenous communities in Peninsular Malaysia to treat various ailments and as an alternative medicine for cancer treatment. The L. tigris cultivar sclerotia (Ligno TG-K) was found to contain numerous bioactive compounds with beneficial biomedicinal properties and the sclerotial extract exhibited potent antioxidant activity. However, the anticancer property of the Ligno TG-K including in vitro and in vivo antitumor effects as well as its anticancer active compounds and the mechanisms has yet to be investigated.MethodsThe cytotoxicity of the Ligno TG-K against human breast (MCF7), prostate (PC3) and lung (A549) adenocarcinoma cell lines was evaluated using MTT cytotoxicity assay. The cytotoxic mechanisms of the active high molecular weight proteins (HMWp) fraction were investigated through detection of caspases activity and apoptotic-related proteins expression by Western blotting. The in vivo antitumor activity of the isolated HMWp was examined using MCF7 mouse xenograft model. Shotgun LC-MS/MS analysis was performed to identify the proteins in the HMWp.Results and DiscussionCold water extract of the sclerotia inhibited proliferation of MCF7, A549 and PC3 cells with IC50 ranged from 28.9 to 95.0 µg/mL. Bioassay guided fractionation of the extract revealed that HMWp exhibited selective cytotoxicity against MCF7 cells via induction of cellular apoptosis by the activation of extrinsic and intrinsic signaling pathways. HMWp activated expression of caspase-8 and -9 enzymes, and pro-apoptotic Bax protein whilst inhibiting expression of tumor survivor protein, Bcl-2. HMWp induced tumor-cell apoptosis and suppressed growth of tumor in MCF-7 xenograft mice. Lectins, serine proteases, RNase Gf29 and a 230NA deoxyribonuclease are the major cytotoxic proteins that accounted for 55.93% of the HMWp.ConclusionThe findings from this study provided scientific evidences to support the traditional use of the L. tigris sclerotia for treatment of breast cancer. Several cytotoxic proteins with high abundance have been identified in the HMWp of the sclerotial extract and these proteins have potential to be developed into new anticancer agents or as adjunct cancer therapy.

Doxorubicin Imprinted Photoluminescent Polymer as a pH-Responsive Nanocarrier.

In this work, an innovative doxorubicin (DOX) imprinted photoluminescent polymer via the precipitation free-radical polymerization strategy was developed based on graphene quantum dots as a pH-responsive nanocarrier. The prepared materials were characterized by Fourier transform infrared, scanning electron microscopy, photoluminescence, and dynamic light scattering techniques. Binding kinetics of DOX established specific recognition binding sites in the photoluminescent nanoscale molecularly imprinted polymer (PLMIP) structure. In vitro drug release behaviors exhibited a pH-controlled release in a sustained manner for the prepared photoluminescent nanocarriers. Due to the presence of pseudopeptide skeletons in the nanocarrier and a positively charged structure, the cytotoxicity study indicated that a DOX-loaded nanocarrier against human lung adenocarcinoma A549 cell lines has notable cytotoxicity. According to the obtained results, the prepared pH-responsive PLMIP has the potential to be employed as an anticancer and biodetection platform.

Hairy Garlic (Allium subhirsutum) from Sicily (Italy): LC-DAD-MSn Analysis of Secondary Metabolites and In Vitro Biological Properties.

Allium subhirsutum, known as hairy garlic, is a bulbous plant widespread in the Mediterranean area and locally used as a food and spice. In the present study, the chemical profile of the ethanolic extracts from bulbs (BE) and aerial parts (APE) were analyzed by HPLC-ESI-MSn, and antioxidant properties were evaluated by DPPH, ABTS and TEAC assays. The traditional use in the diet, and the well documented biological activity of Allium species suggest a potential as a new nutraceutical. For this reason, the potential usefulness of this food can be considered in the treatment and prevention of degenerative Alzheimer disease. For this reason, acetylcholinesterase inhibitory property was investigated. Furthermore, due to the observed presence of sulfur-containing and phenolic constituents, the cytotoxicity on tumor cells line was investigated. Results revealed significant AChE inhibitory activity for BE and APE. Both extracts exhibited also moderate antioxidant properties in the in vitro assays. Finally, limited cytotoxic activity was observed towards Human colon carcinoma and adenocarcinoma cell line, with differences between the individual parts tested. HPLC-ESI-MSn analysis showed that hairy garlic is a good source of sulphur compounds, flavonoids and phenylpropanoids derivatives, thus being a valid alternative to the common garlic (A. sativum). This work opens new opportunities for the application of A. subhirsutum as a health-promoting food.

Targeting platelets for improved outcome in KRAS-driven lung adenocarcinoma.

Elevated platelet count is associated with poor survival in certain solid cancers, including lung cancer. In addition, experimental transplantation of cancer cell lines has uncovered a role for platelets in blood-borne metastasis. These studies, however, do not account for heterogeneity between lung cancer subtypes. Subsequently, the role of platelets in the major subtypes of non-small cell lung cancer (adenocarcinoma (ADC) and squamous cell carcinoma (SqCC)) is not fully understood. We utilised an autochthonous KrasLSL-G12D/+;p53flox/flox mouse model of lung ADC together with genetic models of thrombocytopenia to interrogate the role of platelets in lung cancer growth and progression. While thrombocytopenia failed to impact primary tumour growth, in experimental metastatic models however, thrombocytopenic mice displayed significantly extended survival. Utilising a novel thrombocytopenic immunocompromised mouse, the importance of platelets in metastatic dissemination was confirmed with human KRAS-mutant ADC cell lines. Finally, retrospective analysis of a NSCLC patient cohort revealed thrombocytosis was predictive of poor survival in ADC patients with metastatic disease. Interestingly, this association was not apparent in SqCC patients. Overall, these data highlight the possibility of patient stratification using thrombocytosis as a biomarker, and indicates opportunities for potential novel treatment strategies that combine anti-platelet and lung cancer therapies.

Bipyrazole Based Novel Bimetallic µ-oxo Bridged Au(III) Complexes as Potent DNA Interacalative, Genotoxic, Anticancer, Antibacterial and Cytotoxic Agents

Six bipyrazole based Au(III) complexes were synthesized and characterized by 1H-NMR, 13C (APT) NMR, FT-IR, Mass spectrometry, elemental analysis and conductance measurement. The analysis show that complexes are ionic in nature and bimetallic with oxygen atom as bridging ligand. The DNA binding ability of compounds were checked as change in absorption band in UV-Vis spectra of HS-DNA and change in relative viscosity of DNA in presence of compounds. The results support intercalation mode of binding with good affinity of compounds toward DNA as evaluated by estimation of binding constant values. The molecular docking study also supports the experimental data. The anticancer activity of complexes were evaluated against A549 (Lung adenocarcinoma) cell line by MTT assay in terms of IC50 value. The cell viability assay showed significant potency of complexes as staining of death cells with trypan blue dye. The genotoxicity of compounds were checked by smearing observed in DNA of S. pombe cell under the influence of complexes. The compounds showed significant cytotoxicity against brine shrimp.

YES1 amplification as a primary driver of lung tumorigenesis and YES1/YAP1 amplifications as mediators of acquired resistance (AR) to ALK and EGFR tyrosine kinase inhibitors (TKIs).

e21591 Background: Our previous work identified YES1 amplification as a mechanism of AR to EGFR TKIs. The current study investigates the potential role of YES1 amplification as a primary driver of tumorigenesis and of YES1/YAP1 amplifications as mediators of AR to ALK and EGFR TKIs. Methods: Models of ectopic expression were established and characterized for YES1 and YAP1 in human bronchial epithelial cells (HBECs) and ALK fusion-positive ( ALK+) and EGFR-mutant ( EGFRm) lung adenocarcinoma (LUAD) cell lines. MSK-IMPACT data for all LUAD cases and for ALK and EGFR TKI AR cases were surveyed for YES1 and YAP1 amplification. Results: YES1 was found to be amplified in 0.5% of all LUAD cases currently in the MSK-IMPACT database. To demonstrate its potential as a primary driver of tumorigenesis, YES1 was inducibly expressed in HBECs and shown to promote EGF-independent growth, focus formation, and increases in proliferation rate and sensitivity to Src family kinase (SFK) TKIs. Consistent with these findings, a partial response by RECIST criteria to third-line dasatinib was observed for a patient with stage IV LUAD in which MSK-IMPACT detected YES1 amplification and no established primary driver alteration. In models of AR, overexpression of either YES1 or YAP1 in multiple ALK+ and EGFRm cell lines conferred resistance to ALK and EGFR TKIs. Sensitivity to ALK or EGFR TKIs was partially recovered in YES1-overexpressing cells lines by siRNA-mediated gene knockdown of YAP1, and was restored in YAP1-overexpressing cells lines by knockdown of YES1 or mutation of the YES1-phosphorylation site on YAP1. ALK + or EGFR m cells overexpressing either YES1 or YAP1 were sensitive to dual inhibition of the primary driver and SFKs with single-agent repotrectinib or dasatinib, respectively. In contrast, treatment of YAP1-overexpressing ALK+ H3122 cells with alectinib induced changes in morphology and gene expression consistent with epithelial-to-mesenchymal transition (EMT) and rendered the cells insensitive to repotrectinib. Similar resistance to dual inhibition was seen in other YAP1-overexpressing cell lines after treatment with only ALK or EGFR blockade. To date, one LUAD case each of EGFR and ALK TKI AR has been found through MSK-IMPACT™ to be associated with YAP1 amplification. Conclusions: SFK inhibition can potentially be exploited to therapeutically target YES1 amplification as a primary driver in tumorigenesis and YES1/YAP1 amplifications as mediators of AR to ALK and EGFR TKIs.

Highly efficient Pyrococcus furiosus recombinant L-asparaginase with no glutaminase activity: Expression, purification, functional characterization, and cytotoxicity on THP-1, A549 and Caco-2 cell lines

L-Asparaginase (L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study, Pyrococcus furiosus L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS, and purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 5.7 purification fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of ~33,660 Da on SDS-PAGE and showed maximal activity at 50 °C and pH 8.0. It retained 98.3% and 60.7% initial activity after 60 min at 37 °C and 50 °C, respectively. The recombinant enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for l-glutamine, urea, and acrylamide at 10 mM concentration. The Km and Vmax of the purified recombinant enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623 mM and 105 μmol min−1 mg−1, respectively. Human leukemia cell line THP-1 treated with recombinant L-ASNase showed significant morphological changes, and the IC50 of the purified enzyme was found to be 0.8 IU. Moreover, the purified recombinant L-ASNase induced cytotoxic effects on lung adenocarcinoma A549 and colorectal adenocarcinoma Caco-2 cell lines with IC50 of 1.78 IU and 30 IU, respectively.

Hydroxychloroquine enhances anticancer effect of DOX/folate-phytosterol-carboxymethyl cellulose nanoparticles in A549 lung cancer cells

Purpose: To study the in vitro anticancer effect of doxorubicin-loaded folate-phytosterol-carboxymethyl cellulose nanoparticles (DOX/FPCMC NPs), alone and in combination with the antimalarial drug hydroxychloroquine (HCQ) on human lung cancer cells (A549 cells).
 Methods: Human lung adenocarcinoma A549 cell line was treated with blank FPCMC NPs, HCQ, free DOX, DOX/FPCMC NPs, free DOX + HCQ or DOX/FPCMC NPs + HCQ. The concentrations of HCQ, DOX and FPCMC NPs varied within the ranges of 20-120 μmol/L, 2-12 mg/L and 50-500 mg/L, respectively. Cell viability and free folate competitive inhibition were determined using MTT assay. Cell proliferation and migration were investigated with wound healing assay, while confocal laser scanning microscopy (CLSM) was used to determine cellular uptake of drugs.
 Results: In all formulations, the DOX/FPCMC NPs + HCQ produced the highest cytotoxicity in A549 cells due to high cytotoxicity arising from folate-receptor-mediated endocytosis and HCQ-induced inhibition of autophagy. Free folate competitively inhibited the cytotoxicity of DOX/FPCMC NPs on A549 cells. Wound healing assay showed that A549 cells treated with DOX/FPCMC NPs + HCQ had the lowest cell levels of proliferation and migration capacity. The cellular uptake of DOX/FPCMC NPs by A549 cells was higher than that of free DOX.
 Conclusion: The combination of DOX/FPCMC NPs and HCQ produced the best antitumor effect and had a promising potential for reversal of MDR
 Keywords: Folate-phytosterol-carboxymethyl cellulose, Doxorubicin, Hydroxychloroquine, Anticancer, Lung cancer

Quantitative SWATH-Based Proteomic Profiling for Identification of Mechanism-Driven Diagnostic Biomarkers Conferring in the Progression of Metastatic Prostate Cancer.

Prostate cancer (PCa), the most frequently diagnosed malignancy in men is associated with significant mortality and morbidity. Therefore, demand exists for the identification of potential biomarkers for patient stratification according to prognostic risks and the mechanisms involved in cancer development and progression to avoid over/under treatment of patients and prevent relapse. Quantitative proteomic mass spectrometry profiling and gene enrichment analysis of TGF-β induced-EMT in human Prostate androgen-dependent (LNCaP) and androgen-independent (PC-3) adenocarcinoma cell lines was performed to investigate proteomics involved in Prostate carcinogenesis and their effect onto the survival of PCa patients. Amongst 1,795 proteins, which were analyzed, 474 proteins were significantly deregulated. These proteins contributed to apoptosis, gluconeogenesis, transcriptional regulation, RNA splicing, cell cycle, and MAPK cascade and hence indicating the crucial roles of these proteins in PCa initiation and progression. We have identified a panel of six proteins viz., GOT1, HNRNPA2B1, MAPK1, PAK2, UBE2N, and YWHAB, which contribute to cancer development, and the transition of PCa from androgen dependent to independent stages. The prognostic values of identified proteins were evaluated using UALCAN, GEPIA, and HPA datasets. The results demonstrate the utility of SWATH-LC-MS/MS for understanding the proteomics involved in EMT transition of PCa and identification of clinically relevant proteomic biomarkers.

Antioxidant, anti-inflammatory and healing potential of ethyl acetate fraction of Bauhinia ungulata L. (Fabaceae) on in vitro and in vivo wound model.

The present work aimed to investigate the antioxidant, anti-inflammatory and wound healing potential of ethyl acetate fraction from Bauhinia ungulata L. (FABU) on in vitro and in vivo models. Wound healing assay using human lung adenocarcinoma A549 cell line was employed to evaluate the ability of FABU in modulating cell migration. In addition, a surgical wound model in C57BL/6 mice was used to study the healing potential of FABU incorporated into gel carbomer 940 (Carbopol®). Evaluation of lipid peroxidation, inflammatory and anti-inflammatory mediator gene expression, rate of wound closure, and histological analysis were done. FABU significantly reduced the gap area in in vitro wound healing assay, 24h after treatment. In the animal model, FABU at 0.5% topically applied once-daily for 5days to the surgical wounds significantly reduced the lesion area. Moreover, it significantly decreased the levels of lipid peroxidation in the lesions and decreased the relative gene expression levels of IL-1β and TNF-α in the injured region. In conclusion, our study suggests that Bauhinia ungulata can effectively promote the wound healing, probably by regulating the inflammatory environment during the early stages of the process.

Expression of large tumour suppressor (LATS) kinases modulates chemotherapy response in advanced non-small cell lung cancer.

BackgroundThe Hippo signalling pathway plays an important role in regulating organ size and cell proliferation. Down-regulation of large tumour suppressor (LATS) protein homologs LATS1 or LATS2 has been found in lung cancer. LATS1 and LATS2 are the core components of the Hippo signalling pathway. LATS1 and LATS2 share some conserved structural features and exhibit redundant biological functions. The aim of this study was to dissect the interaction between these two homologs.MethodsIn lung adenocarcinoma (AD) cells, protein expression of LATS1 and LATS2 were determined by western blotting; cell viability and apoptosis were measured by MTT and annexin V staining after treatment with cisplatin; subcellular distributions of LATS proteins were determined by immunofluorescence microscopy; LATS2 expression was modulated by shRNA-mediated knockdown or ectopic expression in cancer cell lines.ResultsManipulation of the expression of these two LATS kinases influenced cisplatin response in advanced lung AD cell lines. High LATS2-to-LATS1 ratio in H2023 cells was associated with cisplatin resistance, while low LATS2-to-LATS1 ratio in CL1-0 and CL83 cells was associated with sensitivity to cisplatin. Manipulating the LATS2-to-LATS1 ratio by LATS2 over-expression in CL1-0 and CL83 rendered them resistant to cisplatin treatment, whereas LATS2 knockdown in H2023 alleviated the LATS2-to-LATS1 ratio and sensitized cancer cells to cisplatin exposure.ConclusionsOur data suggested that the ratio of expression of LATS kinases played a role in the modulation of cisplatin sensitivity in advanced lung AD, and targeting of LATS proteins as a novel therapeutic strategy for lung AD deserves further investigation.

Significance of achaete-scute complex homologue 1 (ASCL1) in pulmonary neuroendocrine carcinomas; RNA sequence analyses using small cell lung cancer cells and Ascl1-induced pulmonary neuroendocrine carcinoma cells.

ASCL1 is one of the master transcription factors of small cell lung carcinoma (SCLC). To investigate the significance of ASCL1 in pulmonary neuroendocrine carcinoma, we performed 2 comparative RNA-seq studies between H69 (ASCL1-positive, classical type SCLC) and H69AR (ASCL1-negative, variant type SCLC) and between ASCL1-transfected A549 adenocarcinoma cell lines (A549(ASCL1+) cell lines) and A549(control) cell lines. RNA-seq analyses revealed that 940 genes were significantly different between the H69 and H69AR cell lines, and 728 between the A549(ASCL1+) and A549(control) cell lines. In total, 120 common genes between these analyses were selected as candidate ASCL1-related genes, and included genes with various cellular functions, such as neural development, secretion, growth, and morphology. Their expression degrees in three classical and two variant SCLC cell lines, two A549(ASCL1+) and two A549(control) cell lines were subjected to quantitative PCR analyses. Since the candidate ASCL1-related genes were strongly expressed in the classical SCLC and A549(ASCL1+) cell lines and more weakly expressed in the variant SCLC and A549(control) cell lines, the ASCL1-related 7 molecules INSM1, ISL1, SYT4, KCTD16, SEZ6, MS4A8, and COBL were further selected. These molecules suggested diverse functions for A549(ASCL1+): INSM1 and ISL1 are transcription factors associated with neuroendocrine differentiation, while SYT4, KTCD16, and SEZ6 may be related to neurosecretory functions and MS4A8 and COBL to cell growth and morphology. An immunohistochemistry of these seven molecules was performed on lung carcinoma tissues and the xenotransplanted tumors of A549(ASCL1+), and they were preferentially and positively stained in ASCL1-postive tumor tissues.

Abstract B31: Viagenpumatucel-L (HS-110) plus nivolumab in previously treated patients with advanced non-small cell lung cancer (NSCLC)

Abstract Background: Viagenpumatucel-L (HS-110) is an allogeneic cellular vaccine derived from a human lung adenocarcinoma (Ad) cell line transfected with the gp96-Ig fusion protein that functions as an antigen chaperone for cross-presentation and dendritic cell activation. In this context, gp96 serves as a potent immune adjuvant via Toll-like receptor 4/2 signaling, which serves to activate APCs to specialized dendritic cells as wells as cross-presentation of the gp96-chaperoned cancer testis peptide antigens (up to 725 represented) directly to MHC class I molecules for direct activation and expansion of CD8+ T-cells. DURGA is a multicohort study evaluating the combination of HS-110 and anti-PD-1 monoclonal antibodies in patients with advanced NSCLC. We report on Cohort A, which enrolled previously treated patients who have not received a checkpoint inhibitor prior to study entry. Methods: Patients (pts) with previously treated NSCLC received 1 X 107 HS-110 cells intradermally every week for 18 weeks, and standard-of-care nivolumab IV until intolerable toxicity or tumor progression. Tissue was tested at baseline for PD-L1 expression (≥ 1% or < 1%) and tumor-infiltrating lymphocytes (TILs). TIL high was defined by more than 10% CD8+ lymphocytes in the tumor stroma. The primary objectives were safety and objective response rate (ORR). Results: As of the March 2019 enrollment cut-off, there were 46 pts enrolled into cohort A (43 Ad and 3 squamous cell carcinoma), 6 (13%) of whom were EGFR positive. 9 pts (20%) were TIL high, 13 (28%) TIL low, and 24 (52%) TIL unknown. 8 pts (17%) were PD-L1 ≥ 1%, 21 (46%) were PD-L1 < 1%, and 17 (37%) were PD-L1 unknown. ORR was 22% and clinical benefit rate (objective response plus stable disease) was 48%. Median progression-free and overall survival were 1.9 and 16.9 months, respectively, with a median follow-up of 17 months. One- and 2-year survival were 48% and 30%, respectively. There were no statistically significant differences in subgroup analyses of PFS or OS based on TIL or PD-L1. In a prospectively defined secondary analysis, the presence of at least one injection site reaction (ISR) during treatment was associated with improved progression-free and overall survival: mPFS 6.1 vs. 1.6 months (HR 0.51 [95% CI 0.26, 0.97] p = 0.04), and mOS 42.1 vs. 5.9 months (HR 0.14 [95% CI 0.05, 0.36] p < 0.0001). 46 (100%) pts experienced at least one adverse event (AE), of which 29 (63%) were grade 1 or 2. The most common AEs were fatigue (26%), cough and arthralgia (17% each), and decreased appetite, constipation, and diarrhea (15% each). There were 2 grade 5 AEs (pulmonary embolism and acute MI) and 1 grade 4 AE (hyponatremia), none of which were considered to be treatment related. Conclusions: The combination of HS-110 and nivolumab appears safe and well tolerated. Efficacy results are independent of TIL and PD-L1 levels, whereas the occurrence of ISR is associated with improved progression-free and overall survival. Citation Format: Daniel Morgensztern, Lyudmila Bazhenova, Saiama N. Waqar, Lori McDermott, Jeff Hutchins, Wael Harb, Nathan Pennell, Roger B. Cohen. Viagenpumatucel-L (HS-110) plus nivolumab in previously treated patients with advanced non-small cell lung cancer (NSCLC) [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 B31.

Unveiling the cytotoxicity of phytosynthesised silver nanoparticles using Tinospora cordifolia leaves against human lung adenocarcinoma A549 cell line.

Biosynthesis of silver nanoparticles (AgNPs) using plant extract is a cheap, easily accessible and natural process in which the phyto-constituents of the plants act as capping, stabilising and reducing agent. The present study explored the biosynthesis of AgNPs using aqueous leaf extract of Tinospora cordifolia and characterised via various techniques such as Fourier transform infrared, scanning electron microscopy, transmission electron microscopy (TEM), energy dispersive X-ray analysis and X-ray diffraction. Here, TEM confirmed the spherical morphology with 25-50 nm size of synthesised AgNPs. Further, anticancer efficiency of AgNPs synthesised using T. cordifolia leaves were evaluated against human lung adenocarcinoma cell line A549 by MTT, trypan blue assay, apoptotic morphological changes using Annexin V-FITC and Propidium iodide (PI), nuclear morphological changes by DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) staining, reactive oxygen species generation and mitochondrial membrane potential determination. Results confirmed the AgNPs synthesised using T. cordifolia leaves are found to be highly toxic against human lung adenocarcinoma cell line A549.

Cytotoxic and Antioxidant Effects of Antimalarial Herbal Mixtures.

Many developing countries depend on herbal mixtures as the first line and cost-effective therapy for malaria. These mixtures with such curative tendencies may also be a source of toxicity to host cells. On the other hand, these mixtures may have anticancer potential activity characterized by cytotoxicity to cancer cells. The aim of the study was to determine the cytotoxic and antioxidant effects of five different antimalarial herbal mixtures. Five antimalarial herbal mixtures commonly used in Ghana (coded as STF, SMH, SMM, SGM, and STT) were purchased and freeze-dried. The dried samples were tested on human acute T-cell leukemia (Jurkat) and breast adenocarcinoma (MCF-7) cell lines. Cytotoxicity was assessed using the tetrazolium-based colorimetric (MTT) assay while antioxidant activity was determined using DPPH free-radical scavenging assay. Among the mixtures, SMM and SGM exhibited the strongest cytotoxicity towards Jurkat cells (IC50 values 59.17 μg/ml and 49.57 μg/ml, respectively), whereas STT showed the weakest cytotoxicity (IC50 = 244.94 μg/ml). Cytotoxic effect of SMM was also strongest towards MCF-7 cells whilst the least cytotoxic sample was SGM (IC50 > 1000 μg/ml). SMM had the highest antioxidant percentage (EC50 = 1.05 mg/ml). The increasing order of antioxidant percentage among the five herbal mixtures is SMM > SMH > STT > STF > SGM. The herbal mixtures may be potential sources of toxic agents to host cells. Therefore, further toxicity studies must be performed to safeguard health of the public. Interestingly, cytotoxicities exhibited by SMM and SGM suggest the presence of anticancer constituents in them which warrant further studies.

Elevated SRC3 expression predicts pemetrexed resistance in lung adenocarcinoma

Lung cancer has been the leading cause of cancer-related death for many years worldwide. Pemetrexed, either as monotherapy or combined with other agents, is the preferred chemotherapy regimen for lung adenocarcinoma. However, both de novo and acquired resistance against pemetrexed frequently occur and lead to poor prognosis of patients. The underlying mechanisms remain poorly characterized. Here, RNA-seq analysis is utilized to compare gene expression levels in an adenocarcinoma cell line A549 with those in its pemetrexed-resistant counterpart, A549/PEM. We show that SRC3 is one of the most significantly upregulated genes in pemetrexed-resistant cells. SRC3 specifically enhances pemetrexed resistance in cultured adenocarcinoma cells. In addition, SRC3 increases pemetrexed resistance by decreasing chemotherapy-induced apoptosis via downregulating ROS level. Mechanistically, SRC3 enhances pemetrexed resistance via regulating Nrf2 and AKT signaling pathway. High SRC3 expression is positively correlated with decreased responsiveness to pemetrexed rather than other chemotherapeutic agents and predicts a poorer clinical outcome in lung adenocarcinoma patients. These data indicate that knockdown of SRC3 may be useful to treat pemetrexed-resistant lung cancer and may also provide a specific biomarker to predict pemetrexed responsiveness in lung cancer.

Cytotoxicity of a new tirucallane derivative isolated from Stereospermum acuminatissimum K. Schum stem bark

One new tirucallan derivative, leutcharic acid (1) was isolated from Stereospermum acuminatissimum stem bark together with the known compounds 3-oxo-22-hydroxyhopane (2), 3,4-secotirucalla-4(28),7,24-trien-3,21-dioic acid (3), 3-oxotirucalla-7,24-dien-21-oic acid (7), lupeol (4), β-sitosterol (5) and stigmasterol (6). The structures of the isolated compounds were elucidated using 1 D and 2 D NMR spectroscopy in combination with literature data. To the best of our knowledge, this is the first report on the cytotoxic properties’ constituents of S. acuminatissimum. Cytotoxicity of compounds 1 and 2 was assessed in vitro with the WST-1 assay on human lung adenocarcinoma A549 and THP-1 human monocytic leukaemia cell lines. Both compounds showed antiproliferative activity on the cancer cells. Compound 2 was more active against THP-1 with an IC50 value of 26.83 µM. The sensitivity of THP-1 cells to compounds 1 and 2 indicated that these compounds might be potential leads for anticancer agent development against leukaemia.