Human Lung Cancer Cell Lines Research Articles

Abstract LB049: Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway

Abstract Lung cancer is the second most common cancer in both men and women in the United States. Even though treatments including chemotherapy, targeted therapy, and immunotherapy are available, lung cancer has been the leading cause of cancer deaths. The discovery and evaluation of new alternative medications targeting lung cancer are tremendously important for reducing lung cancer mortality. Phytochemicals are very desirable chemotherapeutic agents because of their greater safety profile. Isorhapontigenin (ISO), one of the oral bioavailable dietary stilbenes, can be found in various natural resources. Our result showed that ISO treatment suppressed the invasion and migration of human lung cancer cell lines A549, H23, and H1299 cells. Long non-coding RNA Maternally Expressed Gene 3 (MEG3), a tumor suppressor, was downregulated or lost in various primary human tumor tissues and cancer cell lines. The low expression of MEG3 is correlated with poor prognosis in non-small cell lung cancer (NSCLC). Our results showed that ISO treatment elevated MEG3 levels in these lung cancer cell lines. One of the major genetic causes of lung cancer is mutations in the epidermal growth factor receptor (EGFR) receptor tyrosine kinase, resulting in activation of EGFR which activates several signaling pathways involved in cell proliferation, drug sensitivity, and angiogenesis. Our results showed that ISO decreased the phosphorylation and protein levels of EGFR in A549 cells. Hypoxia-inducible factor-1α (HIF-1α), a transcription factor, regulates the activation of several genes involved in cell proliferation, angiogenesis, invasion, and metastasis. Our results showed that ISO decreased HIF-1α protein levels. Overexpression of MEG3 reduced p-EGFR and HIF-1α, indicating MEG3 negatively regulated EGFR and HIF-1α. Our data also showed that inhibition of EGFR by its tyrosine inhibitor decreased HIF-1α, suggesting that EGFR positively regulates HIF-1α. In cancer cells, Epithelial-Mesenchymal Transition(EMT) endows cells with increased invasiveness and tumor-initiating capacity, contributing to metastasis and resistance to therapeutics. Slug, one of the EMT transcription factors maintains cell plasticity during carcinogenesis. Our results showed that ISO treatment decreased Slugprotein levels in A549 and H23 cells. Transcription factor SOX2 regulates cancer cell proliferation, EMT, migration, invasion, metastasis, tumor initiation, cancer stem cell formation as well as resistance to apoptosis and therapy. Our results showed that ISO decreased SOX2 expression. Knockdown of MEG3 increased the expressions of Slug and SOX2, indicating that MEG3 negatively regulates Slug and SOX2. In conclusion, ISO elevated MEG3, inhibiting EGFR, HIF-1α, Slug, and SOX2, suppressing the migration and invasion of lung cancer cells. Citation Format: Sophia Shi, Zhuo Zhang, Jingxia Li, Huailu Tu, Max Costa. Isoharpontigenin inhibits migration and invasion of lung cancer cells through MEG3/EGFR pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB049.

A facile green synthesis of Ag@MnO2 nanoparticles using Martynia annua plant extract and their biological activity and catalytic reduction of dye

The Ag@MnO2 NPs were characterized by UV, FTIR, HRTEM, SAED, and EDX analysis. UV analysis revealed the characteristic wavelength absorption maxima at 425 nm, which confirmed the synthesis of Ag@MnO2 NPs. Martynia annua plant extract presented phytochemical compounds that act as reducing and capping agents in the synthesis of Ag@MnO2 NPs, as confirmed by FT-IR analysis. HRTEM analysis to determine the Ag@MnO2 NPs morphology images showed the spherical shape of NPs with sizes from 1 to 8 nm. The crystalline nature of Ag@MnO2 NPs was identified using SAED and EDX analysis revealed the composition of these synthesized NPs. In-vitro antioxidant activities of Ag@MnO2 NPs were evaluated using a DPPH assay for the significant radical scavenging ability of these NPs. Our findings revealed that M.annua plant-mediated Ag@MnO2 NPs have been highly active against both bacterial and fungal strains. The BSA and EA methods for inhibition of albumin denaturation were investigated for their anti-inflammatory activity. The Ag@MnO2 NPs showed superior activity as anti-inflammatory agents, reduced the viability of the human lung cancer cell line A549 up to 9.92 % at a concentration of 500 µg/ml, and exhibited a good cytotoxic effect. In the presence of NaBH4, the Ag@MnO2 NPs showed excellent catalytic degradation activity against Fuchsin basic dye.

A comprehensive study on synthesis and crystal structures of Cu(II) and Ni(II) complexes: In vitro and in silico evaluation of biomolecular interactions, antiproliferative activities and molecular docking

Cu(II) and Ni(II) complexes, [Cu(NNN)2](NO3)2 (1) and [Ni(NNN)2](NO3)2 (2) (NNN-donor ligand: 2,2′:6′,2″-terpyridine), have been synthesized and characterized by electronic absorption spectroscopy, CHN analysis, FTIR, ESI-MS and X-ray crystallography techniques. Interaction of the complexes 1 and 2 with biomolecules (calf thymus DNA (CT-DNA) and bovine serum albumin (BSA)) has been investigated by electronic absorption and fluorescence spectroscopy. The results show that the complexes 1 and 2 can bind to CT-DNA via a moderate intercalation mode. Moreover, the fluorescence quenching mechanism between the complexes 1 and 2 and BSA is a static quenching process. The antiproliferative activities of the complexes 1 and 2 against human breast cancer (MCF-7) and lung cancer (A549 and H1299) cell lines were investigated. The complex 1 was found to have promising antiproliferative activity in selected cell line, with lower IC50 values than cisplatin. Molecular docking studies conducted across the complexes 1 and 2 have provided their potential as prospective chemotherapeutic agents against tumors. The complexes 1 and 2 demonstrated spontaneous binding with targets including anaplastic lymphoma kinase (ALK), heat shock protein 90 (Hsp90), epidermal growth factor receptor (EGFR), bovine hemoglobin (BHb), human epidermal growth factor receptor 2 (HER2), and B-DNA. These interactions are primarily driven by van der Waals forces and π-π interactions. Determining binding constants and the multiplicity of binding sites enhances understanding of these molecular interactions.

Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolite Regulated in Treated Cancer Cell Lines

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints, for cancer prevention. Rice callus suspension cultures (RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluated cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilution of A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity, and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced effects.

Exploring the Synthesis, Structure and Bioactivity of Pyrimidine Carboxylic Acid-Derived Organic Antimony (V) Complexes: Cytostatic and Antimicrobial Evaluations

Eight new organoantimony complexes, R3SbL2 (R = Ph, p-MePh; L = L1, L2) and (R3Sb)2OL2 (R = Ph, m-MePh; L = L1, L2), [Ph3Sb(C5H3N2O2)2] (1), [(Ph3Sb)2O(C5H3N2O2)2] (2), [(p-tol)3Sb(C5H3N2O2)2] (3), [(m-tol)6Sb2O(C5H3N2O2)2] (4), [Ph3Sb(C5H3N2O2)2] (5), [(Ph3Sb)2O(C5H3N2O2)2] (6), [(p-tol)3Sb(C5H3N2O2)2] (7) and [(m-tol)6Sb2O(C5H3N2O2)2] (8), were designed and synthesized by the reaction of 4-pyrimidine carboxylic acid (HL1) or 5-pyrimidine carboxylic acid (HL2) with organoantimony precursors. All the complexes were characterized by elemental analysis, FT-IR, NMR (1H and 13C), and single crystal X-ray diffraction analysis. Crystal structure results reveal that the central antimony atom of all the complexes displays a triangular biconical geometry. Among them, complexes 1, 3, 5 and 7 show a monomeric structure, while complexes 2, 4, 6 and 8 have a dimeric structure with oxygen bridges. The in vitro cytostatic activities of the eight complexes against a human alveolar basal epithelial lung cancer cell lines (A549) and hepatocellular carcinoma cell lines (HepG-2) were investigated, and the results showed that the organoantimony derivatives with the tolyl groups (3, 4, 7, 8) exhibited higher in vitro cytostatic activities compared with those with the phenyl groups (1, 2, 5, 6). At the same time, bacteriostatic tests were conducted to assess the inhibitory effects of complexes 1-8 against three prevalent plant pathogenic fungi and one bacterium. The results revealed that complex 4 exhibited significant fungicidal activities, on the other hand, complex 3 exhibited higher antibacterial activity against MRSA. These results will provide significant guidance for the further exploration of potential efficient biological activity complexes.

Nano-Titanium Dioxide: Antioxidant Activity and Cytotoxicity Against Human Lung Cancer Cell Line

The scope of this study is to synthesize titanium dioxide nanoparticles by chemical method and to assess their antioxidant and anticancer properties. Titanium isopropoxide was employed as a precursor in the production of the nanoparticles of titanium dioxide. The sample was analyzed using a variety of characterization techniques, including X-ray diffraction, Ultraviolet-Visible Spectroscopy, Fourier Transform Infrared Spectroscopy, Field Emission Scanning Electron Microscopy, Energy Dispersive X-ray analysis. Further, their antioxidant and anticancer activities have been investigated. The crystallite size of titanium dioxide nanoparticles was revealed by X-ray diffraction, and the functional groups were confirmed by Fourier Transform Infrared Spectroscopy. Field emission scanning electron microscopy and energy dispersive X-ray analysis reveal the structure of the nanoparticles and the presence of titanium, carbon, and oxygen molecules, respectively. The antioxidant activity indicates that as the concentration of the TiO2  nanoparticles increases, it reaches the activity of ascorbic acid (standard). The sample shows good cytotoxic effect against A549 human lung cancer cell lines.

Effects of Cultivation Stress on the Glaucophyte Cyanophora paradoxa and Bioactive Potential in Human Cancer Cell Lines

ABSTRACT The propensity to accumulate high-value metabolites under specific conditions makes microalgae promising candidates for commercial applications. Here, the freshwater microalga Cyanophora paradoxa CCAP 981/1 was cultivated under varying conditions (control, nitrogen limitation, NaCl supplementation and blue light exposure) and the biological activity of the corresponding biomass extracts was assessed against human breast (MCF-7) and lung (A549) cancer cell lines. The stress treatments hindered the growth of C. paradoxa, the control displaying the highest specific growth rate (0.16 d−1 ± 0.01). Biomass screening by GCMS and HPLC identified diverse bioactive compounds including pigments (chlorophylls, xanthophylls, carotenes) and fatty acids (e.g., EPA). The control cultivation conditions returned the highest yields of biomass (724 mg), zeaxanthin (6.8 mg g−1), EPA (1.1 mg g−1) and phycocyanin (113.9 mg g−1), while the blue light treatment enhanced the proportions of unsaturated forms of C18 (34.3%). Extracts of dried biomass obtained by cold maceration using solvents of increasing polarity were used in the treatment of MCF-7 and A549 cancer cells. Ethyl acetate extracts from nitrogen-limited cultures reduced MCF-7 viability (~70%) after 72 h. However, the diethyl ether and methanol extracts showed no impact on MCF-7 and A549 cell viability. Furthermore, acridine orange staining revealed no significant DNA damage in these cell lines upon exposure to C. paradoxa extracts. Overall, the minimal impacts on cell viability observed here supports considering C. paradoxa as a promising source of natural bioactive compounds with applications in various biotechnological sectors.

Trichostatin C Synergistically Interacts with DNMT Inhibitor to Induce Antineoplastic Effect via Inhibition of Axl in Bladder and Lung Cancer Cells

Aberrant epigenetic modifications are fundamental contributors to the pathogenesis of various cancers. Consequently, targeting these aberrations with small molecules, such as histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors, presents a viable strategy for cancer therapy. The objective of this study is to assess the anti-cancer efficacy of trichostatin C (TSC), an analogue of trichostatin A sourced from the fermentation of Streptomyces sp. CPCC 203909. Our investigations reveal that TSC demonstrates potent activity against both human lung cancer and urothelial bladder cancer cell lines, with IC50 values in the low micromolar range. Moreover, TSC induces apoptosis mediated by caspase 3/7 and arrests the cell cycle at the G2/M phase. When combined with the DNMT inhibitor decitabine, TSC exhibits a synergistic anti-cancer effect. Additionally, protein analysis elucidates a significant reduction in the expression of the tyrosine kinase receptor Axl. Notably, elevated concentrations of TSC correlate with the up-regulation of the transcription factor forkhead box class O1 (FoxO1) and increased levels of the proapoptotic proteins Bim and p21. In conclusion, our findings suggest TSC as a promising anti-cancer agent with HDAC inhibitory activity. Furthermore, our results highlight the potential utility of TSC in combination with DNMT inhibitors for cancer treatment.

Abstract 2089: A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications

Abstract The majority of RAS oncogenic activity is thought to be mediated by its canonical signaling, mainly via RAS-GTP interactions with its effectors RAF and PI3K at the plasma membrane, which respectively activate MEK/ERK and AKT/mTOR pathways. However, in this study, we have identified a new, fundamental, pro-oncogenic, non-canonical activity of RAS-GTP that increases the Exportin 1 (XPO1)-dependent export of nuclear proteins into the cytoplasm and is independent of RAF/MEK and PI3K/AKT signaling. The nuclear protein export machinery is known to depend on a trimeric protein complex composed of RAN-GTP:XPO1:nuclear protein cargo. Unexpectedly, we have found that RAS-GTP forms a stable complex with RAN GTPase-Activating Protein 1 (RAN-GAP) at the nuclear envelope, which facilitates the release of nuclear protein cargo by increasing the hydrolysis of RAN-GTP to RAN-GDP. We have determined that RAS-GTP and RAN-GAP form this complex in primary human lung tumors, patient derived xenografts, and human lung cancer cell lines. Interestingly, the RAS-GTP:RAN-GAP complex is found at the nuclear envelope and in the cytoplasm, but not at the plasma membrane. The RAS-dependent step mediated by the RAS-GTP:RAN-GAP complex acts downstream of XPO1 by increasing RAN-GTP hydrolysis, which promotes protein cargo release. Our fundamental findings have important clinical implications. For example, in human lung cancer models that harbor a KRAS-G12C mutation, the combined inhibition of MEK and PI3K (MEKi + PI3Ki) does not inhibit tumor growth to the same degree as a KRAS-G12C inhibitor. Remarkably, adding an XPO1 inhibitor to MEKi + PI3Ki inhibits tumor growth to the same degree as the KRAS-G12C inhibitor. One critical pro-oncogenic target of the RAS-GTP:RAN-GAP complex is nuclear export of the EZH2 methyltransferase, whose increase in the cytoplasm leads to a direct methylation of the cytoplasmic DLC1 tumor suppressor protein, leading to ubiquitin-dependent proteasomal degradation of DLC1 protein. Analysis of a KRAS mutant cell line from which the DLC1 gene has been disrupted by CRISPR-Cas9 technology indicates that DLC1 makes an important contribution to the growth inhibition by inhibitors that reduce nuclear protein export. These findings establish nuclear protein export as a critical, pro-oncogenic, non-canonical RAS function that is mediated by the RAS-GTP:RAN-GAP complex and provide a mechanistic explanation for poor clinical responses to combined inhibition of MEK and PI3K, which do not regulate the nuclear protein activity identified in this study. Citation Format: Brajendra K. Tripathi, Nicole H. Hirsh, David Hansen, Xiaolan Qian, Marian E. Durkin, Dunrui Wang, Alex G. Papageorge, Ross Lake, Yvonne A. Evrard, Adam Marcus, Suresh S. Ramalingam, Mary Dasso, Karen Vousden, James H. Doroshow, Kylie J. Walters, Douglas R. Lowy. A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications [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 2089.

Abstract 3384: Rapid and automated tumor cell phenotyping based on annexin-mediated enforce blebbing response

Abstract Introduction: Accumulating evidence supports the tight correlation between the physical characteristics of cells (including morphology, mechanical, and electrical properties) with the progression of cancer. Cellular blebbing, a distinctive feature observed during apoptosis and cell migration, has gained significant recognition. However, existing tools such as atomic force microscopy and optical tweezers have limitations in terms of throughput and complexity. Microfluidics has emerged as a promising technology to overcome these limitations. Methods: In this study, we developed a novel microfluidic device with a double-layer compression mechanism to induce controlled blebbing of cells. We implemented a convolution-based tracing program to enhance the accuracy and efficiency of cell area measurement. The device was used to examine the enforced blebbing response of two human breast cancer cell lines (MDA-MB-231 and MCF-7) and investigate the potential of blebbing as a marker for drug resistance using a human lung cancer cell line (HCC4006). Additionally, we elucidated the role of annexin-6 (A6) in enforced blebbing and its underlying mechanism. Results: Our microfluidic device successfully implemented a double-layered design that induced cell deformation through compression from the second pressurized polydimethylsiloxane layer. The findings revealed that highly invasive cell lines, such as MDA-MB-231 and osimertinib-resistant HCC4006, required significantly higher critical compression levels compared to their non-invasive counterparts (MCF-7 cell line) or osimertinib-sensitive cells. Moreover, the drug-resistant groups exhibited a greater compression strain requirement for blebbing compared to the control group, indicating their enhanced resilience against disruptive forces and superior survivability. Prior studies have demonstrated that A6 protein is recruited to the site of membrane injury and facilitates the formation of blebs, thereby contributing to cellular repair processes. Our further investigation into the role of A6 protein demonstrated its involvement in cell blebbing. Knockdown of A6 resulted in a notable 27% reduction in cellular volume, reflecting reduced intracellular pressure, and a corresponding 1.7-fold increase in the critical compressive strain necessary for inducing blebbing. Conclusion: Our microfluidic device allows for rapid and simultaneous testing and automated analysis of hundreds of cells, making it suitable for applications such as cell sorting, highly invasive and drug-resistant cancer cell detection, without the need for labeling. Furthermore, it serves as a powerful tool for fundamental studies of membrane and cytoskeleton dynamics. The label-free nature of the device, coupled with its high throughput capabilities, highlights its potential in future applications, including cancer monitoring and drug-resistance screening. Citation Format: Wei Huang, Zheng Wang, Dennis Wai-Yin Au, Bingxian Tang, Baiqian Qi, William C. Cho, Yuan Lin. Rapid and automated tumor cell phenotyping based on annexin-mediated enforce blebbing response [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 3384.

Abstract 1899: Novel combinations of aplitabart, a DR5 agonist IgM antibody, with ADCs or chemotherapeutic agents lead to robust anti-tumor responses in solid tumor models

Abstract Agonism of death receptor 5 (DR5), a tumor necrosis factor receptor superfamily member, induces the killing of tumor cells via the extrinsic apoptotic pathway upon receptor multimerization. We have engineered a multivalent IgM DR5 agonist antibody, aplitabart (IGM-8444), that induces robust anti-tumor activity in both in vitro and in vivo preclinical models. We also demonstrated potent synergistic in vitro activity of aplitabart with different classes of chemotherapeutic agents including microtubule inhibitors, nucleoside analogs and topoisomerase inhibitors in a range of solid cancer cell lines. Preliminary results from an ongoing phase 1a/ab clinical study show that aplitabart has an encouraging safety profile and promising activity in combination with FOLFIRI ± bevacizumab in heavily pretreated metastatic colorectal cancer patients. To further explore the combination therapies for aplitabart in lung and breast cancers, we evaluated standard of care chemotherapies, such as paclitaxel, carboplatin, and antibody drug conjugates (ADCs) in vitro on a range of human lung and breast cancer cell lines. We observed dose-dependent synergistic cytotoxic effects of aplitabart on tumor cells when combined with these agents. Additionally, these agents increased the expression of DR5 on tumor cells and could explain their synergistic combinatorial activity with aplitabart. Representative cancer cell lines were selected to evaluate these combinations in vivo using xenograft mouse models where robust anti-tumor responses were demonstrated in the xenograft lung and breast cancer models, resulting in enhanced tumor growth inhibition and extended survival. Our findings demonstrate potent in vitro synergistic cytotoxicity and enhanced anti-tumor response in preclinical models in vivo by combining aplitabart with ADCs, or carboplatin and paclitaxel. These results support the combination of DR5 agonist IgM antibodies with agents known to upregulate DR5 expression on solid tumors. Aplitabart is currently under evaluation in a randomized clinical study in combination with FOLFIRI + bevacizumab in comparison with FOLFIRI + bevacizumab in patients with colorectal cancer (NCT04553692). Citation Format: Mélanie Desbois, Susan E. Calhoun, Kevin C. Hart, Carolyn R. Denson, Poonam Yakkundi, Thomas J. Matthew, Maya K. Leabman, Sanela Bilic, Genevive Hernandez, Eric W. Humke, Albert F. Candia, Bruce Keyt, Angus M. Sinclair, Maya F. Kotturi, Beatrice T. Wang. Novel combinations of aplitabart, a DR5 agonist IgM antibody, with ADCs or chemotherapeutic agents lead to robust anti-tumor responses in solid tumor models [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 1899.

Abstract 1220: Deep mutational scanning of EGFR reveals potential domain-specific TKI sensitivities in lung cancer and glioblastoma

Abstract The epidermal growth factor receptor, EGFR, is frequently activated in lung cancer and glioblastoma by genomic alterations including missense mutations. The different mutation spectra in these diseases are reflected in divergent responses to EGFR inhibition: significant patient benefit in lung cancer, but limited in glioblastoma. Here, we report a comprehensive mutational analysis of EGFR function. We performed saturation mutagenesis of EGFR and assessed function of ~22,500 variants in a human EGFR-dependent lung cancer cell line. This approach revealed enrichment of erlotinib-insensitive variants of known and unknown significance in the dimerization, transmembrane, and kinase domains. Multiple EGFR extracellular domain variants, lacking approved targeted therapies, were sensitive to dacomitinib. Strikingly, at least two glioblastoma patients with G598V dimerization domain mutations showed responses to dacomitinib treatment together with subsequent off-target resistance in one case. In summary, this comprehensive screen reveals novel functional EGFR variants and suggests broader clinical investigation of EGFR inhibition for cancers harboring extracellular domain mutations. Citation Format: Tikvah Hayes, Elisa Aquilanti, Erica Kim, Nicole Persky, Lisa Brenan, Amy Goodale, Ted Sharpe, Doug Alan, Robert Shue, Lior Golomb, Brianna Silverman, Yvonne Li, Lindsay Westlake, Federica Piccioni, Andrew Cherniack, Xiaoping Yang Yang, David Root, J. Kevin Hicks, Andrew Chi, Jorg Dietrich, Daniel Cahill, Cory Johannessen, Tracy Batchelor, Matthew Meyerson. Deep mutational scanning of EGFR reveals potential domain-specific TKI sensitivities in lung cancer and glioblastoma [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 1220.

Abstract 4493: Protein structure inspired drug discovery

Abstract Introduction: Drug action is mediated by a small molecule therapeutic binding to a specific structural motif on a protein. As structure determines function, it follows that unique structural motifs are enriched for sites of unique function and high therapeutic target value. We hypothesized that protein structure constitutes high value primary information that can drive the discovery of novel therapeutic agents. Method: We developed a unique physics-based protein structure analysis platform scaled to run on a supercomputer, allowing us to consider dynamic protein flexibility and the influence of solvent upon protein structure, and to probe large protein libraries as a primary source of structure information that can support up-front structure-based screens. We developed and then probed a high resolution protein x-ray crystallographic library, created and curated by us. From a 60 million compound library, we conducted virtual screens for docking to identified sites. 8-day colony formation assays were conducted on a panel of 8 human breast, prostate, lung and colon cancer cell lines, as well as 8- and 14-day trilineage hematopoietic colony formation assays on human cord blood stem cells. Findings: From the first 180 deposited protein structures, we screened for unique protein surface structure. Structures were deemed unique if they were not otherwise known to be associated with any particular functional roles and were not the binding site of known therapeutics. Further selection included those that were clefts and possessed physical characteristics compatible with binding small chemicals whose properties have been linked to effective therapeutics. For each of 8 sites on 6 different proteins, a suite of analytics probed docking solutions from the compound library, generating a ranked list of 100 compounds for each site. Based on factors inclusive of availability and low potential for toxicity, compounds were acquired and used in in-parallel selection and de-selection functional assays. Cancer cell growth inhibition was a positive selection criterion, with additional ranking based on greater potency and broader efficacy. Bone marrow toxicity is limiting for most drugs, and inhibition of bone marrow stem cell growth was as a deselection criterion. Multiple compounds clustered around 2 sites on 2 different proteins. One compound, Dxr2-017, exhibited very high relative efficacy against colon and breast cancer cells. Evaluation of Dxr2-017 in the NCI-60 panel corroborated these findings, and showed even higher efficacy against melanoma. We demonstrated that Dxr2-017 inhibited melanoma growth with low nanomolar efficacy, while concentrations of over 2300-fold higher had only limited inhibitory effects on bone marrow stem cells. Conclusions: This study provides proof-of-principle that protein structure constitutes high value primary information that can support identification of novel therapeutics. This approach is widely applicable and expandable. Citation Format: Fangfang Qiao, T. Andrew Binkowski, Wayne Anderson, Weining Chen, Gray Schiltz, Karl Scheidt, Amarnath Natarajan, Raymond Bergan. Protein structure inspired drug discovery [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 4493.

Synthesis and biological evaluation of novel benzothiazole derivatives as potential anticancer and antiinflammatory agents.

Introduction: Cancer, a significant global health concern, necessitates innovative treatments. The pivotal role of chronic inflammation in cancer development underscores the urgency for novel therapeutic strategies. Benzothiazole derivatives exhibit promise due to their distinctive structures and broad spectrum of biological effects. This study aims to explore new anti-tumor small molecule drugs that simultaneously anti-inflammatory and anticancer based on the advantages of benzothiazole frameworks. Methods: The compounds were characterized by nuclear magnetic resonance (NMR), liquid chromatograph-mass spectrometer (LC-MS) and high performance liquid chromatography (HPLC) for structure as well as purity and other related physicochemical properties. The effects of the compounds on the proliferation of human epidermoid carcinoma cell line (A431) and human non-small cell lung cancer cell lines (A549, H1299) were evaluated by MTT method. The effect of compounds on the expression levels of inflammatory factors IL-6 and TNF-α in mouse monocyte macrophages (RAW264.7) was assessed using enzyme-linked immunosorbent assay (ELISA). The effect of compounds on apoptosis and cell cycle of A431 and A549 cells was evaluated by flow cytometry. The effect of compounds on A431 and A549 cell migration was evaluated by scratch wound healing assay. The effect of compounds on protein expression levels in A431 and A549 cells was assessed by Western Blot assay. The physicochemical parameters, pharmacokinetic properties, toxicity and drug similarity of the active compound were predicted using Swiss ADME and admetSAR web servers. Results: Twenty-five novel benzothiazole compounds were designed and synthesized, with their structures confirmed through spectrogram verification. The active compound 6-chloro-N-(4-nitrobenzyl) benzo[d] thiazol-2-amine (compound B7) was screened through a series of bioactivity assessments, which significantly inhibited the proliferation of A431, A549 and H1299 cancer cells, decreased the activity of IL-6 and TNF-α, and hindered cell migration. In addition, at concentrations of 1, 2, and 4μM, B7 exhibited apoptosis-promoting and cell cycle-arresting effects similar to those of the lead compound 7-chloro-N-(2, 6-dichlorophenyl) benzo[d] thiazole-2-amine (compound 4i). Western blot analysis confirmed that B7 inhibited both AKT and ERK signaling pathways in A431 and A549 cells. The prediction results of ADMET indicated that B7 had good drug properties. Discussion: This study has innovatively developed a series of benzothiazole derivatives, with a focus on compound B7 due to its notable dual anticancer and anti-inflammatory activities. B7 stands out for its ability to significantly reduce cancer cell proliferation in A431, A549, and H1299 cell lines and lower the levels of inflammatory cytokines IL-6 and TNF-α. These results position B7B7 as a promising candidate for dual-action cancer therapy. The study's mechanistic exploration, highlighting B7's simultaneous inhibition of the AKT and ERK pathways, offers a novel strategy for addressing both the survival mechanisms of tumor cells and the inflammatory milieu facilitating cancer progression.

Lactiplantibacillus plantarum K8 lysates regulate hypoxia-induced gene expression

Hypoxic responses have been implicated in critical pathologies, including inflammation, immunity, and tumorigenesis. Recently, efforts to identify effective natural remedies and health supplements are increasing. Previous studies have reported that the cell lysates and the cell wall-bound lipoteichoic acids of Lactiplantibacillus plantarum K8 (K8) exert anti-inflammatory and immunomodulative effects. However, the effect of K8 on cellular hypoxic responses remains unknown. In this study, we found that K8 lysates had a potent suppressive effect on gene expression under hypoxia. K8 lysates markedly downregulated hypoxia-induced HIF1α accumulation in the human bone marrow and lung cancer cell lines, SH-SY5Y and H460. Consequently, the transcription of known HIF1α target genes, such as p21, GLUT1, and ALDOC, was notably suppressed in the K8 lysate supplement and purified lipoteichoic acids of K8, upon hypoxic induction. Intriguingly, K8 lysates decreased the expression of PHD2 and VHL proteins, which are responsible for HIF1α destabilization under normoxic conditions, suggesting that K8 may regulate HIF1α stability in a non-canonical pathway. Overall, our results suggest that K8 lysates desensitize the cells to hypoxic stresses and suppress HIF1α-mediated hypoxic gene activation.

Biofabricated Green Synthesized Hibiscus Silver Nanoparticles Potentiate Antibacterial Activity and Cytotoxicity in Human Lung Cancer Cells.

Traditional medicine has long employed the shrub Hibiscus sabdariffa to treat a variety of illnesses.The biochemical characteristics of silver nanoparticles made using the plant extract of Hibiscus sabdariffa were examined in this work. According to the results, the plant extract of Hibiscus sabdariffa had a total phenolic quantity of 84.9mg/gm and a total quantity of flavonoids of 41.50mg/gm. The extract also showed antibacterial action against Escherichia coli and Staphylococcus aureus (75.15% scavenging activity). The silver nanoparticles of plant extracts were stable in PBS solution for at least 30days and had a mean size of particles of 21.22nm. Silver nanoparticles were shown to both be cytotoxic on human lung cancer cell line A-549 and have anti-inflammatory action. Overall, the research's findings demonstrate the fascinating biological activity of the silver nanoparticles made from the extract of the Hibiscus sabdariffa plant. To assess these compounds' potential as medicines, more research is required.

Trilobolide-6-O-isobutyrate from Sphagneticola trilobata acts by inducing oxidative stress, metabolic changes and apoptosis-like processes by caspase 3/7 activation of human lung cancer cell lines

BackgroundLung cancer, a chronic and heterogeneous disease, is the leading cause of cancer-related death on a global scale. Presently, despite a variety of available treatments, their effectiveness is limited, often resulting in considerable toxicity and adverse effects. Additionally, the development of chemoresistance in cancer cells poses a challenge. Trilobolide-6-O-isobutyrate (TBB), a natural sesquiterpene lactone extracted from Sphagneticola trilobata, has exhibited antitumor effects. Its pharmacological properties in NSCLC lung cancer, however, have not been explored. PurposeThis study evaluated the impact of TBB on the A549 and NCI-H460 tumor cell lines in vitro, examining its antiproliferative properties and initial mechanisms of cell death. MethodsTBB, obtained at 98 % purity from S. trilobata leaves, was characterized using chromatographic techniques. Subsequently, its impact on inhibiting tumor cell proliferation in vitro, TBB-induced cytotoxicity in LLC-MK2, THP-1, AMJ2-C11 cells, as well as its effects on sheep erythrocytes, and the underlying mechanisms of cell death, were assessed. ResultsIn silico predictions have shown promising drug-likeness potential for TBB, indicating high oral bioavailability and intestinal absorption. Treatment of A549 and NCI-H460 human tumor cells with TBB demonstrated a direct impact, inducing significant morphological and structural alterations. TBB also reduced migratory capacity without causing toxicity at lower concentrations to LLC-MK2, THP-1 and AMJ2-C11 cell lines. This antiproliferative effect correlated with elevated oxidative stress, characterized by increased levels of ROS, superoxide anion radicals and NO, accompanied by a decrease in antioxidant markers: SOD and GSH. TBB-stress-induced led to changes in cell metabolism, fostering the accumulation of lipid droplets and autophagic vacuoles. Stress also resulted in compromised mitochondrial integrity, a crucial aspect of cellular function. Additionally, TBB prompted apoptosis-like cell death through activation of caspase 3/7 stressors. ConclusionThese findings underscore the potential of TBB as a promising candidate for future studies and suggest its viability as an additional component in the development of novel anticancer drugs prototypes.

Immuno-oncological effects of standard anticancer agents and commonly used concomitant drugs: an in vitro assessment

BackgroundIt has become evident in the field of oncology that the outcome of medical treatment is influenced by the combined effect exerted on both cancer- and immune cells. Therefore, we evaluated potential immunological effects of 46 standard anticancer agents and 22 commonly administered concomitant non-cancer drugs.MethodsWe utilized a miniaturized in vitro model system comprised of fluorescently labeled human colon and lung cancer cell lines grown as monocultures and co-cultured with activated peripheral blood mononuclear cells (PBMCs). The Bliss Independence Model was then applied to detect antagonism and synergy between the drugs and activated immune cells.ResultsAmong the standard anticancer agents, tyrosine kinase inhibitors (TKIs) stood out as the top inducers of both antagonism and synergy. Ruxolitinib and dasatinib emerged as the most notably antagonistic substances, exhibiting the lowest Bliss scores, whereas sorafenib was shown to synergize with activated PBMCs. Most concomitant drugs did not induce neither antagonism nor synergy. However, the statins mevastatin and simvastatin were uniquely shown to synergize with activated PBMC at all tested drug concentrations in the colon cancer model.ConclusionWe utilized a miniaturized tumor-immune model to enable time and cost-effective evaluation of a broad panel of drugs in an immuno-oncology setting in vitro. Using this approach, immunomodulatory effects exerted by TKIs and statins were identified.

Chemical constituents of Penicillium ferraniaense GE-7 and their cytotoxicities

Phytochemical investigation on the plant endophytic fungus Penicillium ferraniaense GE-7 led to the isolation of 18 compounds including one new α-pyrone derivative, peniferranige A (1). The structure including the absolute configuration of compound 1 was elucidated by NMR, HRMS, and ECD data. Demethoxyfumitremorgin C (16) and meleagrin (17) possessed moderate activities against the human lung cancer cell line H1975 with IC50 values of 28.52 ± 1.07 and 13.94 ± 1.92 μM, respectively.

Production and characterization of novel marine black yeast’s exopolysaccharide with potential antiradical and anticancer prospects

The marine black yeasts are characterized by the production of many novel protective substances. These compounds increase their physiological adaptation to multi-extreme environmental stress. Hence, the exopolysaccharide (EPS) producing marine black yeast SAHE was isolated in this study. It was molecularly identified as Hortaea werneckii (identity 98.5%) through ITS1 and ITS4 gene sequencing analysis. The physicochemical properties of the novel SAHE-EPS were investigated through FTIR, GC-MS, TGA, ESM, and EDX analysis, revealing its heteropolysaccharide nature. SAHE-EPS was found to be thermostable and mainly consists of sucrose, maltose, cellobiose, lactose, and galactose. Furthermore, it exhibited an amorphous texture and irregular porous surface structure. SAHE-EPS showed significant antiradical activity, as demonstrated by the DPPH radical scavenging assay, and the IC50 was recorded to be 984.9 μg/mL. In addition, SAHE-EPS exhibited outstanding anticancer activity toward the A549 human lung cancer cell line (IC50 = 22.9 μg/mL). Conversely, it demonstrates minimal cytotoxicity toward the WI-38 normal lung cell line (IC50 = 203 μg/mL), which implies its safety. This study represents the initial attempt to isolate and characterize the chemical properties of an EPS produced by the marine black yeast H. werneckii as a promising antiradical and anticancer agent.