Cell NCI-H460 Research Articles

Abstract C168: Developing lung adenocarcinoma cell lines to mitigate lung cancer health disparities

Abstract Lung cancer continues to be the number one cause of cancer-related deaths worldwide and remains a prominent source of cancer health disparities. Black/African American (B/AA) males exhibit a 12% higher lung cancer incidence rate and a 15% higher lung cancer death rate when compared to white males. Lung adenocarcinoma (LUAD) is the most common histological subtype of lung cancer in all ethnic/racial groups, including B/AAs. To further examine this phenomenon, we want to create a collection of lung cancer cell lines that would be useful for molecular studies of lung cancer development, progression, development of new targeted therapies, and pre-clinical drug testing. After reviewing the literature (Leon et al. 2023, Frontiers in Oncology, Vol. 13, p. 1187585, 2023), we identified only 30 unique B/AA cell lines out of ∼800, the majority of which are derived from White and Asian patients. Moreover, only 6 of the 30 cell lines are derived from patients with LUAD. We are in the process of deriving more LUAD cell lines from B/AA patients as well as alveolar epithelial cell lines in order to study the development of LUAD. In addition, we are creating a collection of multiple independent isogenic cell line variants from an existing lung cancer cell line NCI-H23. NCI-H23 was derived from a B/AA male LUAD patient and harbors a KRASG12C heterozygous mutation. G12C is the most common KRAS mutation in LUAD (∼40% of KRAS mutations) followed by G12V (∼20%), G12D (∼15%), G12A (∼10%), and G13C (∼6%). We used CRISPR/Cas-9 genome- based editing to replace the KRASG12C mutation with KRASG12V, KRASG12D, KRASG12A, and KRASG13C. We have derived multiple independent cell lines for most mutations. We are in the process of performing whole genome sequencing of these cell lines to exclude any off-target effects that may have arisen from the CRISPR/Cas-9 engineering. In addition, we will carry out whole genome RNA sequencing to assess the effect of the engineering and/or KRAS mutation switching. As a control, we are also sequencing the genomes and transcriptomes of three monoclonal cell lines we derived from NCI-H23 cells, to determine what variability ensues with the derivation of monoclonal lines, since that is required to obtain the CRISPR/Cas9-edited cells. We will use this collection of cell lines to study the efficacy of polyisoprenylated cysteinyl amide inhibitors (PCAIs) and other drugs on cell lines bearing these KRAS mutations. PCAIs were developed to target cells carrying mutant KRAS. Our efforts will provide new cell lines to study lung adenocarcinoma in B/AA subjects, addressing a key shortcoming and disparity in the lung cancer research toolkit. Citation Format: Sofia A. Lugo, Christopher Leon, Matthew A. Gladstone, Chunli Yan, Daniel J. Mullen, Pablo E. Puente, Kweku Ofosu-Asante, Justin K. Mensah-Mamfo, Kyle R. Phillips, Yong Huang, Nazarius S. Lamango, Ite A. Offringa. Developing lung adenocarcinoma cell lines to mitigate lung cancer health disparities [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C168.

Mucosa‑associated lymphoid tissue lymphoma translocation protein 1 inhibitor, MI‑2, attenuates non‑small cell lung cancer cell proliferation, migration and invasion, and promotes apoptosis by suppressing the JNK/c‑JUN pathway.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) inhibitors are effective in attenuating the progression of several types of cancer. However, their role in lung cancer requires further investigation. Therefore, the present study aimed to explore the effect of the MALT1 inhibitor, MI-2, on the behavior of non-small cell lung cancer (NSCLC) cells and to uncover their possible underlying mechanism of action. The mRNA and protein expression levels of MALT1 were detected in the human normal lung epithelial cell line BEAS-2B, and the NSCLC cell lines, NCI-H1299, NCI-H1650, HCC827, A549 and NCI-H23. Subsequently, NCI-H1650 and A549 cells were treated with MI-2. Additionally, NCI-H1650 and A549 cells were co-treated with anisomycin, a c-JUN N-terminal kinase (JNK) pathway activator, with or without MI-2. The results illustrated that the mRNA and protein expression levels of MALT1 were significantly increased in NCI-H1299, NCI-H1650, A549 and NCI-H23 cells compared with those in BEAS-2B cells. Treatment of NCI-H1650 and A549 cells with MI-2 for 72 h reduced the optical density value as determined using the Cell Counting Kit-8 assay. Consistently, the 5-ethynyl-2'-deoxyuridine assay also showed that proliferation was reduced in MI-2-treated NSCLC cells. In addition, MI-2 downregulated B-cell lymphoma 2 (BCL2), and enhanced BCL2-associated X-protein expression and apoptotic rate in NCI-H1650 and A549 cells. These findings indicated that MI-2 could inhibit NCI-H1650 and A549 cell proliferation and promote apoptosis. Furthermore, treatment of cells with MI-2 only attenuated the migration and invasion of NCI-H1650 cells. Notably, MI-2 decreased the expression levels of phosphorylated (p)-JNK and p-c-JUN in NCI-H1650 and A549 cells, thus suggesting that MI-2 could suppress the JNK/c-JUN signaling pathway. However, NSCLC cell co-treatment with anisomycin (JNK pathway activator) reversed the effect of MI-2 on the proliferation, apoptosis and activation of the JNK/c-JUN pathway in NCI-H1650 and A549 cells. In conclusion, the present study demonstrated that the MALT1 inhibitor, MI-2, could suppress NSCLC cell proliferation, migration and invasion, and induce apoptosis via inactivating the JNK/c-JUN pathway.

Host-specific endophytes of Momordica charantia: A promising source for affordable lung cancer therapeutics

Endophyte-host interactions lead to the production of various bioactive compounds. Thus, endophytes from a medicinal plant such as Momordica charantia, can be promising candidates for producing pharmacological compounds. Our study therefore aims to evaluate these endophytes as sustainable sources of potential low-cost lung cancer drugs. We determined the endophytes associated with M. charantia (fruit and leaf) and assessed anti-inflammatory, antioxidant and antiproliferative potential against NCI-H23 lung cancer cells. LC-Q-TOF-MS of the endophyte extracts was conducted to determine the metabolite profile.Leaf endophyte F. circinatum showed anti-inflammatory (114.29 %), antioxidant (IC50=27.39 µg/ml) and antiproliferative activity (IC50=98.62 µl/ml) with decreased Beclin1 expression (autophagy regulator) in NCI-H23 cells. It produced five anticancer compounds namely linamarin, xestoaminol C, phytosphingosine, cucurbitacin E and margarolic acid. Fruit endophyte E. fergusonii also showed significant antiproliferative potential (IC50=170.8 µl/ml) with upregulated apoptosis regulator Bcl2 expression. It produced compounds such as eplerenone, kuguacin C and H. With the production of triterpenoids momordicine I, cucurbitacin E and kuguaglycoside A, leaf endophyte A. faecalis showed antioxidant potential (IC50=29.65 µg/ml) but limited antiproliferative activity. This is the first report of endophytes-mediated biosynthesis of host-specific compounds in M. charantia including momordicine I, cucurbitacin E and kuguacins, which are known to have anti-cancer properties. Thus, these medicinal plant endophytes can be low-cost sustainable candidates with anti-cancerous potential, especially against lung cancer.

Discovery of novel coumarin-based KRAS-G12C inhibitors from virtual screening and Rational structural optimization

KRAS-G12C inhibitors has been made significant progress in the treatment of KRAS-G12C mutant cancers, but their clinical application is limited due to the adaptive resistance, motivating development of novel structural inhibitors. Herein, series of coumarin derivatives as KRAS-G12C inhibitors were found through virtual screening and rational structural optimization. Especially, K45 exhibited strong antiproliferative potency on NCI-H23 and NCI-H358 cancer cells harboring KRAS-G12C with the IC50 values of 0.77 μM and 1.50 μM, which was 15 and 11 times as potent as positive drug ARS1620, respectively. Furthermore, K45 reduced the phosphorylation of KRAS downstream effectors ERK and AKT by reducing the active form of KRAS (KRAS GTP) in NCI-H23 cells. In addition, K45 induced cell apoptosis by increasing the expression of anti-apoptotic protein BAD and BAX in NCI-H23 cells. Docking studies displayed that the 3-naphthylmethoxy moiety of K45 extended into the cryptic pocket formed by the residues Gln99 and Val9, which enhanced the interaction with the KRAS-G12C protein. These results indicated that K45 was a potent KRAS-G12C inhibitor worthy of further study.

Discovery of LLC355 as an Autophagy-Tethering Compound for the Degradation of Discoidin Domain Receptor 1.

Discoidin domain receptor 1 (DDR1) is a potential target for cancer drug discovery. Although several DDR1 kinase inhibitors have been developed, recent studies have revealed the critical roles of the noncatalytic functions of DDR1 in tumor progression, metastasis, and immune exclusion. Degradation of DDR1 presents an opportunity to block its noncatalytic functions. Here, we report the discovery of the DDR1 degrader LLC355 by employing autophagosome-tethering compound technology. Compound LLC355 efficiently degraded DDR1 protein with a DC50 value of 150.8 nM in non-small cell lung cancer NCI-H23 cells. Mechanistic studies revealed compound LLC355 to induce DDR1 degradation via lysosome-mediated autophagy. Importantly, compound LLC355 potently suppressed cancer cell tumorigenicity, migration, and invasion and significantly outperformed the corresponding inhibitor 1. These results underline the therapeutic advantage of targeting the noncatalytic function of DDR1 over inhibition of its kinase activity.

Abstract 1125: TNIK induced chemoradiation resistance in small cell lung cancer

Abstract Chemoradiation (CRT) is the standard first-line therapy for limited stage small cell lung cancer (LS-SCLC), which is characterized by early metastasis, intrinsic-acquired CRT resistance and tumor recurrence. CRT confers a 20-25% 5-year overall survival in patients with LS-SCLC. In this study, we utilized patient-derived xenograft (PDX) models to demonstrate CRT resistance of SCLC and identified CRT resistance candidate genes from various SCLC PDX that represent the majority of molecular subtypes (n=4 that include SCLC-A and SCLC-N subtypes). RNA-seq data identified TNIK (Traf2- and Nck-interacting kinase) as one such gene that is consistently upregulated in SCLC PDXs exposed to CRT compared to single modality treatments. Copy number gains of TNIK were also present in human SCLC samples. Overcoming this CRT resistance is crucial in improving treatment outcomes and patient survival. Genetic depletion or pharmacological inhibition of TNIK reduced the in vitro clonogenic survival of TNIKhigh SCLC cells, NCI-H446 and makes them increasingly sensitive to CRT. In vivo, pharmacological inhibition of TNIK with the inhibitor NCB-0846, enhances the CRT sensitivity of NCI-H446 cell line-derived xenografts (CDX) in NOD SCID immunodeficient mice. Furthermore, pharmacological inhibition of TNIK in vivo demonstrated sensitivity to CRT in LX33 PDX. In conclusion, our results indicate that TNIK plays a role in conferring resistance to CRT in vitro in SCLC cell lines and in vivo in SCLC CDX and PDX models and therefore, can be a potential therapeutic target in limited stage SCLC. Citation Format: Dipanwita Dutta Chowdhury, Eddie Imada, Nick Connis, Triet Nguyen, Jinhee Chang, Danielle Council, Audrey Lafargue, Mohammad Rezaee, Phuoc T. Tran, Luigi Marchionni, Christine L. Hann. TNIK induced chemoradiation resistance in small cell lung cancer [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 1125.

Abstract A163: Exoribonuclease XRN1 is a therapeutic vulnerability in tumors with intrinsically elevated type I interferon signaling

Abstract Background: 5′→3′ exoribonuclease 1 (XRN1) degrades single stranded mRNA from the 5′→3′ direction and plays a key role in endogenous cellular mRNA turnover. In addition, XRN1 can degrade double-stranded RNA (dsRNA), and as such plays a role in innate immunity by prevention of dsRNA accumulation and pPKR induction in virus-infected cells. We identified XRN1 as a putative selective vulnerability in tumor cells with intrinsic elevation of a Type I Interferon Stimulated Gene (TISG) signature through analysis of publicly available CRISPR screen data across 483 tumor cell lines. We further explored the role of XRN1 in a set of lung cancer cell lines with differential TISG expression in order to validate XRN1 as a compelling target for oncology drug development. Materials and Methods: NCI-H1650, NCI-H1703, NCI-H1944, and NCI-H838 lung adenocarcinoma cell lines were infected with lentiviruses containing Cas9 and sgRNAs targeting XRN1. Non-targeting sgRNAs were used as negative controls. XRN1 knockout (KO) cells and controls were evaluated in proliferation, apoptosis, qPCR and western blot assays. Results: Consistent with a selective dependency on XRN1 in cells with high intrinsic interferon signaling, XRN1 KO via CRISPR leads to robust anti-proliferative effects in TISG high NCI-H1650 and NCI-H1703 cells, but not in TISG low NCI-H838 and NCI-H1944 cells. Annexin V staining of NCI-H1650 cells upon XRN1 KO demonstrates that apoptosis is induced in TISG high cells. XRN1 KO in the TISG high setting leads to robust upregulation of Interferon-β mRNA consistent with activation of the MDA5 pathway by elevation of cytoplasmic dsRNA. Similarly, XRN1 loss induces pPKR activation in TISG high, but not TISG low cells. As pPKR activation triggers translational shutdown, TISG high selective pPKR elevation is consistent with the cell death observed. Exogenous treatment with Interferon-β or dsRNA mimetic Poly I:C to induce elevated Type I Interferon expression sensitizes NCI-H838 cells to XRN1 KO, with effects on cell proliferation. Conclusions: Taken together, these results demonstrate a critical role of XRN1 in cancer cells with intrinsically elevated Type I Interferon signaling. Cell death is triggered via accumulation of dsRNA and downstream activation of the MDA5 and PKR innate immune pathways. These data suggest that XRN1, through its regulation of dsRNA burden, is a compelling oncology target for TISG high tumors. Conflict of Interests: All authors are current or former employees and shareholders of Accent Therapeutics, Inc. Citation Format: Maureen M Lynes, Sophie A Shen, Sunaina P Nayak, Brian A Sparling, Scott Ribich, Stephen J Blakemore, Serena J Silver. Exoribonuclease XRN1 is a therapeutic vulnerability in tumors with intrinsically elevated type I interferon signaling [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A163.

Apigenin promotes cell death in NCI-H23 cells by upregulation of PTEN: potential involvement of the binding of apigenin with WWP2 protein

The tumour suppressor protein PTEN is often down-regulated in non-small cell lung cancer. A major protein promoting the lowering of the PTEN protein is WWP2. Polyphenols have been shown to promote the expression of tumour suppressor genes like PTEN. We carry out the study to check for the ability of apigenin to bind with the WWP2 protein using in-silico investigation comprising docking and simulation. We checked for the cytotoxic effect of apigenin upon the non-small cell lung cancer cell line NCI-H23. We checked the PTEN expression status at the gene and protein levels. The expression levels of the apoptotic regulators BCL2, BAX and CASPASE3 genes along with the activity levels of the caspase-3 protein were checked. The ultrastructure of the cells was analysed. Our Autodock analysis showed that apigenin bound favourably with the WWP2 protein. Molecular dynamics simulation revealed that apigenin increased the parameters of RMSD, Rg and SASA when bound with the WWP2 protein. The protein-ligand complex had hydrogen bonding and majorly van der Wal’s interactions. PCA analysis revealed greater fluctuations in the apigenin-bound state of the protein. The mutant form of the WWP2 revealed similar results in the presence of apigenin. Apigenin showed efficacy against the NCI-H23 cell line and promoted PTEN protein levels, lowered BCL2 gene expression and up-regulated BAX and CASPASE3 gene expression. Increased caspase-3 activity and ultra-structural analysis revealed the occurrence of apoptosis. Thus the binding of apigenin with WWP2 could promote PTEN protein levels and lead to apoptotic activity in NCI-H23 cells. Communicated by Ramaswamy H. Sarma

Different RONS Generation in MTC-SK and NSCL Cells Lead to Varying Antitumoral Effects of Alpha-Ketoglutarate + 5-HMF.

Carbonylated proteins (CPs) serve as specific indicators of increased reactive oxygen and nitrogen species (RONS) production in cancer cells, attributed to the dysregulated mitochondrial energy metabolism known as the Warburg effect. The aim of this study was to investigate the potential of alpha-ketoglutarate (aKG), 5-hydroxymethylfurfural (5-HMF), and their combination as mitochondrial-targeting antioxidants in MTC-SK or NCI-H23 cancer cells. MTC-SK and NCI-H23 cells were cultured in the absence or presence of varying concentrations (0-500 µg/mL) of aKG, 5-HMF, and the combined aKG + 5-HMF solutions. After 0, 24, 48, and 72 h, mitochondrial activity, cancer cell membrane CP levels, cell growth, and caspase-3 activity were assessed in aliquots of MTC-SK and NCI-H23 cells. The mitochondrial activity of MTC-SK cells exhibited a concentration- and time-dependent reduction upon treatment with aKG, 5-HMF, or the combined aKG + 5-HMF. The half-maximal inhibitory concentration (IC50%) for mitochondrial activity was achieved at 500 µg/mL aKG, 200 µg/mL 5-HMF, and 200 µg/mL aKG + 66.7 µg/mL 5-HMF after 72 h. In contrast, NCI-H23 cells showed a minimal reduction (10%) in mitochondrial activity even at the highest combined concentration of aKG + 5-HMF. The CP levels in MTC-SK cells were measured at 8.7 nmol/mg protein, while NCI-H23 cells exhibited CP levels of 1.4 nmol/mg protein. The combination of aKG + 5-HMF led to a decrease in CP levels specifically in MTC-SK cells. The correlation between mitochondrial activity and CP levels in the presence of different concentrations of combined aKG + 5-HMF in MTC-SK cells demonstrated a linear and concentration-dependent decline in CP levels and mitochondrial activity. Conversely, the effect was less pronounced in NCI-H23 cells. Cell growth of MTC-CK cells was reduced to 60% after 48 h and maintained at 50% after 72 h incubation when treated with 500 µg/mL aKG (IC50%). Addition of 500 µg/mL 5-HMF inhibited cell growth completely regardless of the incubation time. The IC50% for 5-HMF on MTC-CK cell growth was calculated at 375 µg/mL after 24 h incubation and 200 µg/mL 5-HMF after 72 h. MTC-SK cells treated with 500 µg/mL aKG + 167 µg/mL 5-HMF showed no cell growth. The calculated IC50% for the combined substances was 250 µg/mL aKG + 83.3 µg/mL 5-HMF (48 h incubation) and 200 µg/mL aKG + 66.7 µg/mL 5-HMF (72 h incubation). None of the tested concentrations of aKG, 5-HMF, or the combined solution had any effect on NCI-H23 cell growth at any incubation time. Caspase-3 activity increased to 21% in MTC-CK cells in the presence of 500 µg/mL aKG, while an increase to 59.6% was observed using 500 µg/mL 5-HMF. The combination of 500 µg/mL aKG + 167.7 µg/mL 5-HMF resulted in a caspase-3 activity of 55.2%. No caspase-3 activation was observed in NCI-H23 cells when treated with aKG, 5-HMF, or the combined solutions. CPs may serve as potential markers for distinguishing between cancer cells regulated by RONS. The combination of aKG + 5-HMF showed induced cell death in high-RONS-generating cancer cells compared to low-RONS-generating cancer cells.

Black tea bioactive phytoconstituents realign NRF2 for anticancer activity in lung adenocarcinoma.

Constitutive activation of nuclear factor erythroid 2-related factor 2 (NRF2) is pivotal in bestowing therapy resistance in cancer cells. Several phytochemicals have been reported with the potential of modulating NRF2. Therefore, it was hypothesized that NRF2-deregulated chemoresistance in lung adenocarcinoma (LUAD) may be counteracted by theaflavin-rich black tea (BT). A non-responsive LUAD cell line, A549, was the best sensitized towards cisplatin upon pre-treatment with BT. BT-mediated NRF2 reorientation was observed to be dependent on concentration and duration of treatment as well as on the mutational profile of NRF2 in A549 cells. Transient exposure of low-concentration BT hormetically downregulated NRF2, its downstream antioxidants, and drug transporter. BT also influenced the Kelch-like ECH-associated protein (KEAP1)-dependent cullin 3 (Cul3) and KEAP-1-independent signaling through epidermal growth factor receptor (EGFR) - rat sarcoma virus (RAS) - rapidly accelerated fibrosarcoma (RAF) - extracellular signal-regulated kinase 1/2 (ERK) - matrix metalloproteinase (MMP)-2 and MMP-9. The realignment of NRF2 in KEAP1-suppressed A549 cells enhanced the chemotherapeutic outcome. But a higher concentration of the same BT surprisingly upregulated NRF2 and its transcriptional targets with a subsequent decrease in the NRF2-regulatory machinery in NCI-H23 cells (a KEAP1-overexpressed LUAD cell line), ultimately resulting in a better anticancer response. The BT-mediated bidirectional NRF2 modulation was reconfirmed upon comparison with the action of a pharmacological NRF2 inhibitor, ML-385, in A549 and a known NRF2 activator, tertiary-butylhydroquinone, in NCI-H23 respectively. BT-mediated regulation of NRF2-KEAP1 and their upstream networks (EGFR/RAS/RAF/ERK) sufficed as a better anticancer agent than synthetic NRF2 modulators. Therefore, BT may be indicated as a potent multi-modal small molecule for increasing drug responsiveness in LUAD cells by maintaining NRF2/KEAP1 axis at an optimum level.

Simultaneous activation of CXC chemokine receptor 4 and histamine receptor H1 enhances calcium signaling and cancer cell migration

C-X-C chemokine receptor 4 (CXCR4) is widely overexpressed in various types of cancer and is involved in several cancer phenotypes including tumor growth, survival, and metastasis. The roles of histamine and histamine receptor H1 (HRH1) in cancer pathogenesis remain controversial. Here, we show that HRH1 is widely expressed in various cancer cell lines and cancer tissues and that coexpression of CXCR4 and HRH1 is associated with poor prognosis in breast cancer. Using bimolecular fluorescence complementation and bioluminescence resonance energy transfer donor saturation assays, we demonstrate that CXCR4 and HRH1 can assemble into a heteromeric complex. Simultaneous activation of CXCR4 and HRH1 synergistically increases calcium flux in MDA-MB-231 cells that endogenously express CXCR4 and HRH1 but not in cells deficient in CXCR4 or HRH1. Costimulation of CXCR4 and HRH1 also significantly enhances CXCL12-induced MDA-MB-231 cell migration, while histamine alone does not induce cell migration. Synergistic effects on calcium flux and cell migration are inhibited by the Gαi inhibitor pertussis toxin and the Gαq inhibitor YM254890, suggesting that the Gαi and Gαq pathways are involved in the synergy. Enhanced calcium signaling and cell migration are also observed in NCI-H23 and HeLa cells, which coexpress CXCR4 and HRH1. Taken together, our findings demonstrate an interplay between CXCR4 and HRH1, and suggest the possibility of the CXCR4-HRH1 heteromer as a potential therapeutic target for anticancer therapy.

Chloroquine Enhances Death in Lung Adenocarcinoma A549 Cells Exposed to Cold Atmospheric Plasma Jet.

Cold atmospheric plasma (CAP) is an intensively-studied approach for the treatment of malignant neoplasms. Various active oxygen and nitrogen compounds are believed to be the main cytotoxic effectors on biotargets; however, the comprehensive mechanism of CAP interaction with living cells and tissues remains elusive. In this study, we experimentally determined the optimal discharge regime (or semi-selective regime) for the direct CAP jet treatment of cancer cells, under which lung adenocarcinoma A549, A427 and NCI-H23 cells demonstrated substantial suppression of viability, coupled with a weak viability decrease of healthy lung fibroblasts Wi-38 and MRC-5. The death of CAP-exposed cancer and healthy cells under semi-selective conditions was caspase-dependent. We showed that there was an accumulation of lysosomes in the treated cells. The increased activity of lysosomal protease Cathepsin D, the transcriptional upregulation of autophagy-related MAPLC3B gene in cancer cells and the changes in autophagy-related proteins may have indicated the activation of autophagy. The addition of the autophagy inhibitor chloroquine (CQ) after the CAP jet treatment increased the death of A549 cancer cells in a synergistic manner and showed a low effect on the viability of CAP-treated Wi-38 cells. Downregulation of Drp1 mitochondrial protein and upregulation of PINK1 protein in CAP + CQ treated cells indicated that CQ increased the CAP-dependent destabilization of mitochondria. We concluded that CAP weakly activated pro-survival autophagy in irradiated cells, and CQ promoted CAP-dependent cell death due to the destabilization of autophagosomes formation and mitochondria homeostasis. To summarize, the combination of CAP treatment with CQ could be useful for the development of cold plasma-based antitumor approaches for clinical application.

Prognostic prediction and expression validation of NSD3 in pan-cancer analyses

<b>Background:</b> Nuclear receptor binding SET domain protein-3 (NSD3) is a histone lysine methyltransferase and a crucial regulator of carcinogenesis in several cancers. We aimed to investigate the prognostic value and potential function of NSD3 in 33 types of human cancer. <b>Methods:</b> The data were obtained from The Cancer Genome Atlas. Kaplan-Meier analysis, CIBERSORT, gene set enrichment analysis, and gene set variation analysis were performed. The expression of NSD3 was measured using quantitative real-time polymerase chain reaction and western blot. <b>Results:</b> The expression of NSD3 was altered in pan-cancer samples. Patients with higher levels of NDS3 generally had shorter overall survival and disease-specific survival. Levels of NSD3 were positively correlated with DNA copy number variation (CNV) in pan-cancer. NSD3 expression was also associated with tumor mutation burden and microsatellite instability. The levels of immune-cell infiltration differed significantly between high and low NSD3 expression. NSD3 negatively correlated with levels of CD8+ T cells. Functional enrichment analysis showed that while NSD3 expression was positively associated with several immune cell-related and histone methylation-related pathways, it was negatively correlated with cell metabolism-related, drug transport-related, and drug metabolism-related pathways. NSD3 levels in the cell lines tested were significantly different. In U251 and NCI-H23 cells, silencing NSD3 inhibited cell proliferation and promoted apoptosis. <b>Conclusions:</b> NSD3 expression was changed in pan-cancer samples that was also verified in cell lines. NSD3 was associated with CNV and immune-cell infiltration. A poor prognosis was predicted in patients with high expression of NSD3. NSD3 might hence be a potential marker for predicting tumor prognosis.

Abstract C024: Study of therapeutic effects of polyisoprenylated cysteinyl amide inhibitors on lung cancer cells of Black American patients using 3D-printed alveolar models

Abstract Lung cancer is one of the top five deadliest cancers, and Black Americans have a higher mortality rate when compared to White Americans. Lung adenocarcinoma (LUAD) is the most common histological lung cancer subtype in all population groups including Blacks. Unfortunately, there are no in vitro model systems to study lung cancer and its treatments for Black subjects. Herein we aim to utilize three-dimensional (3D) printing to develop an in vitro three-dimensional (3D) alveolar model using a lung adenocarcinoma cell line from a Black patient (NCI-H23). The developed model is further utilized to study the therapeutic effects of potential LUAD therapeutic agents, polyisoprenylated cysteinyl amide inhibitors (PCAIs), which target and disrupt the RAS-mediated signaling pathway. KRAS is the most commonly mutated oncogene in LUAD in Blacks, and is mutated in NCI-H23 cells. For comparison, hAEC-FT-C7 cells, which are immortalized human alveolar epithelial cells and do not carry the KRAS mutation, will also be used separately to print the alveolar models and test the effects of PCAIs. The experimental results will be further compared to those using two-dimensional (2D) models to better understand the therapeutic effects of PCAIs on Black LUAD. The resulting knowledge will help design better treatments for ethnically/racially diverse patients in whom the RAS signaling pathway is frequently altered. Citation Format: Nadine L. Belony, Bing Ren, Phuc Pham, Matthew Gregory, Pablo E. Puente, Nazarius S. Lamango, Ite A. Offringa, Yong Huang. Study of therapeutic effects of polyisoprenylated cysteinyl amide inhibitors on lung cancer cells of Black American patients using 3D-printed alveolar models [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C024.

BV6 enhances apoptosis in Lung cancer cells by ameliorating caspase expressions through attenuation of XIAP, cIAP-1, and cIAP-2 proteins

The present study aimed to investigate the inhibitory role of second mitochondria determined activator of caspases mimetic on inhibitor of apoptosis proteins (IAPs) and regulation of caspases in nonsmall cell lung cancer cell line. Dimethyl sulfoxide and 3-(4, 5-dimethyl thizol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was done to determine the IC50 of BV6 using NCI-H23 cell line. The levels of mRNA of X-linked IAP (XIAP), cellular IAP (cIAP-1), cIAP-2, caspase-6, and caspase-7 in H23 cell line were evaluated by a quantitative real-time polymerase chain reaction, while their protein expressions were tested using western blotting. Two doses of BV6 dependently downregulated the expression of mRNA of XIAP (P = 0.002, P= 0.0003 vs. untreated), cIAP-1 (P = 0.05, P = 0.005 vs. untreated), and cIAP-2 (P = 0.001, P = 0.0002 vs. untreated), respectively, while the compound upregulated the mRNA expression of caspase-6 (P = 0.001, P < 0.0001 vs. untreated) and caspase-7 (P = 0.001, P = 0.0004 vs. untreated), respectively. Dose dependent of BV6 treatment significantly decreased the protein level of XIAP (P = 0.003, P = 0.007 vs. untreated), cIAP-1 (P = 0.02, P = 0.01 vs. untreated), and cIAP-2 (P = 0.008,P = 0.008 vs. untreated), respectively. However, the compound increased the protein level of caspase-6 and caspase-7 when compared to untreated control (P = 0.006,P = 0.001) and (P = 0.01, P = 0.001), respectively. The result showed that BV6 treatment reduced the level of mRNA of XIAP, cIAP-1, and cIAP-2 and increased the gene expression of caspase-6 and caspase-7 in NCI-H23 cell line. Therefore, the study revealed that BV6 could be used in future as additional therapeutics in lung cancer.

Identification of the novel natural product inhibitors of SHP2 from the plant Toona sinensis: In vitro and in silico study

In this study, we tested the inhibitory activity of 45 natural products extracted from the plant Toona sinensis on SHP2 protein, and identified four natural product inhibitors. The natural product 1,2,3,6-Tetragalloylglucose (A-1) was first reported as a competitive inhibitor of SHP2, with an IC50 value of 0.20 ± 0.029 μM and the selectivity of 1.8-fold and 4.35-fold to high homologous proteins SHP1 and PTP1B, respectively. Compound A-1 also showed high inhibitory activity on SHP2-E76K and SHP2-E76A mutants, with IC50 values of 0.95 ± 0.21 μM and 0.29 ± 0.045 μM, respectively. Cell viability assay showed that compound A-1 could inhibit the proliferation of a variety of cancer cells. Apoptosis assay showed that compound A-1 could effectively induce apoptosis of KRASG12C-mut NCI-H23 and KRASG12S-mut A549 cells. Western blot assay showed that compound A-1 could down regulate the phosphorylation levels of Erk1/2 and Akt in NCI-H23 and A549 cells. Molecular docking showed that compound A-1 could effectively dock to the catalytic active region of SHP2. Molecular dynamics simulation explored the effect of compound A-1 on SHP2, revealing the deep-seated binding mechanism. This study would provide valuable clues for the development of SHP2 and its mutant inhibitors.

Modulatory role of BV6 and chloroquine on the regulation of apoptosis and autophagy in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is one of the aggressive tumors mostly diagnosed in the advanced stage. Therapeutic failure and drug resistance pose a major problem in NSCLC treatment primarily due to alterations in autophagy and loss of apoptosis. Therefore, the present study aimed to investigate the importance of the second mitochondria-derived activator of caspase mimetic BV6 and autophagy inhibitor chloroquine (CQ) on the regulation of apoptosis and autophagy, respectively. Study was conducted on NCI-H23 and NCI-H522 cell lines to evaluate the effect of BV6 and CQ on the transcription and translation level of LC3-II, caspase-3, and caspase-9 genes by quantitative real-time-polymerase chain reaction and western blotting techniques. In NCI-H23 cell line, BV6 and CQ treatments showed increased mRNA and protein expression of caspase-3, and caspase-9 compared to its untreated counterpart. BV6 and CQ treatments also caused downregulation of LC3-II protein expression compared to its counterpart. In NCI-H522 cell line, BV6 treatment showed a significantly increased expression of caspase-3 and caspase-9 mRNA and protein expression levels whereas BV6 treatment downregulated the expression level of LC3-II protein. A similar pattern was also observed in CQ treatment when compared with the respective controls. Both BV6 and CQ modulated in vitro expression of caspases and LC3-II which have critical regulatory roles in apoptosis and autophagy, respectively. Our findings suggest that BV6 and CQ could be promising candidates in NSCLC treatment and there is a need to explore them in vivo and in clinical applications.

Capsaicin induces ferroptosis of NSCLC by regulating SLC7A11/GPX4 signaling in vitro

NSCLC is the first cause of cancer-related deaths in China and threatens life expectancy of the people. Novel drugs and treatment strategies are urgently required. Capsaicin is noticed as a potential new drug for lots of tumors due to its anti-proliferative effect on cancer cells. Our study evaluated the roles of capsaicin in NSCLC cells (A549 and NCI-H23) and further explored its underlying mechanisms. Effect of capsaicin treatment on cell viability was determined by MTT assay and IC50 values for A549 and NCI-H23 cells were ascertained. The iron kit detected the total iron levels and the ferric divalent ions levels in A549 and NCI-H23 cells. GSH kit was used to detect the expression of GSH in A549 and NCI-H23 cells. Additionally, mRNA and protein levels of SLC7A11 and GPX4 were analyzed by real-time PCR and western blot analysis. Through MTT assay, we found that 200 μM capsaicin in cultured A549 cells for 48 h could reach the IC50 value, and the condition was 100 μM and 48 h for NCI-H23 cells. Capsaicin increased total iron levels and ferrous ion levels in A549 and NCI-H23 cells in contrast with the control group, whereas the levels of GSH was reduced in contrast with the control group. Besides, mRNA and protein levels of SLC7A11 and GPX4 were decreased significantly in A549 and NCI-H23 cells treated with capsaicin in contrast with the control group. Our study indicated that capsaicin inhibited the proliferation of A549 and NCI-H23 cells and induced ferroptosis by inactivating SLC7A11/GPX4 signaling. Capsaicin could be used as a potential anticancer agent in the treatment of NSCLC.

KRAS G12C as a Target of Naringenin for Inducing Cell Death in NCI-H23 Cells

KRAS G12C as a Target of Naringenin for Inducing Cell Death in NCI-H23 Cells

Chip-DSF: A rapid screening strategy for drug protein targets

Identification of the drug target of lead compounds is an important means for rapid and efficient drug discovery. Protein chips are a high-throughput protein function analysis technology that has been widely used in screening drug protein targets in recent years. However, the verification of the results after high-throughput protein chip screening is still cumbersome. Based on our mature protein chip preparation platform, we prepared a protein chip containing 150 important high-frequency protein targets and used antibodies to prove the availability of the protein chip. To improve the accuracy of target screening, we combined the label-free differential scanning fluorimetry (DSF) with the protein chip, proposing the Chip–DSF strategy. Subsequently, we tested the method with small molecular ginsenoside-Rg2 (Rg2). The Chip–DSF strategy was used to successfully screen the potential target protein KRAS(G12C) of Rg2. Consistently, we found that Rg2 could inhibit NCI-H23 cell proliferation by inducing cell cycle arrest. Also, we found that Rg2 could reduce the amount of KRAS protein and inhibit the phosphorylation of KRAS downstream key signaling protein ERK1, RPS6, and P70S6K in NCI-H23 cells. Collectively, our Chip-DSF strategy could achieve rapid target verification which improved the accuracy and efficiency of target screening of protein chips.