Drug-sensitive Cell Lines Research Articles

TROP2 expression and SN38 antitumor activity in malignant pleural mesothelioma cells provide a rationale for antibody-drug conjugate therapy

ObjectivesMalignant pleural mesothelioma (MPM) is an aggressive cancer which at large is not amenable to curative surgery. Despite the recent approval of immune checkpoint inhibitor therapy, the response rates and survival following systemic therapy is still limited. Sacituzumab govitecan is an antibody-drug conjugate targeting the topoisomerase I inhibitor SN38 to trophoblast cell-surface antigen 2 (TROP-2)-positive cells. Here we have explored the therapeutic potential of sacituzumab govitecan in MPM models. Materials and methodsTROP2 expression was analyzed in a panel of two well established and 15 pleural effusion derived novel lines by RT-QPCR and immunoblotting, TROP2 membrane-localization was studied by flow cytometry and immunohistochemistry. Cultured mesothelial cells and pneumothorax pleura served as controls. The sensitivity of MPM cell lines to irinotecan and SN38 was studied using cell viability, cell cycle, apoptosis and DNA damage assays. Drug sensitivity of cell lines was correlated with RNA expression of DNA repair genes. Drug sensitivity was defined as an IC50 below 5 nM in the cell viability assay. ResultsTROP2 expression was detected at RNA and protein level in 6 of the 17 MPM cell lines, but not in in cultured mesothelial control cells or in the mesothelial layer of the pleura. TROP2 was detectable on the cell membrane in 5 MPM lines and was present in the nucleus in 6 cell models. Ten of 17 MPM cell lines showed sensitivity to SN38 treatment, among those 4 expressed TROP2. High AURKA RNA expression and high proliferation rate correlated with sensitivity to SN38-induced cell death, DNA damage response, cell cycle arrest and cell death. Sacituzumab govitecan treatment effectively induced cell cycle arrest and cell death in TROP2-positive MPM cells. ConclusionTROP2 expression and sensitivity to SN38 in MPM cell lines support biomarker-selected clinical exploration of sacituzumab govitecan in patients with MPM.

Ingenol mebutate inhibits the growth of pancreatic cancer cells in vitro via STING with an efficacy comparable to that of clinically used anticancer agents.

Pancreatic cancer is associated with a poor prognosis; thus, there is an urgent need to develop new and effective treatments. Ingenol mebutate (IM), which is isolated from the latex of Euphorbia peplus, was recently shown to be effective against pancreatic cancer cell lines; however, its mechanism of action has not been fully elucidated. In this study, we focused on the less drug-sensitive pancreatic cancer cell line Panc-1 and compared IM to commercially available anticancer drugs using cell survival assays. In addition, we aimed to identify novel biomolecules that may be involved in the mechanism of action of IM using RNA sequencing, western blotting, and inhibition assays. The IC50 values after 72h of exposure to IM and SN-38, drugs to which the Panc-1 cells are most sensitive among the tested anticancer agents, were 43.1 ± 16.8nM and 165 ± 37nM, respectively. IM showed a cytostatic effect equal to or greater than that of the clinically used pancreatic cancer therapeutic drugs. RNA sequencing and protein expression analysis revealed that expression of stimulator of interferon genes (STING) increased at low IM concentration, whereas cell viability decreased. Co-exposure of IM and STING inhibitor, H-151, to Panc-1 or MIA PaCa-2 cell lines canceled the growth-inhibitory effects of IM alone. In conclusion, IM may have an efficacy comparable to that of existing pancreatic cancer therapeutic agents on the less drug-sensitive Panc-1 cell line and the immune-related molecule STING plays a role in the mechanism of action of IM.

Half-Sandwich Rhodium Complexes with Releasable N-Donor Monodentate Ligands: Solution Chemical Properties and the Possibility for Acidosis Activation.

Cancer chemotherapeutics usually have serious side effects. Targeting the special properties of cancer and activation of the anticancer drug in the tumor microenvironment in situ may decrease the intensity of the side effects and improve the efficacy of therapy. In this study, half-sandwich Rh complexes are introduced, which may be activated at the acidic, extracellular pH of the tumor tissue. The synthesis and aqueous stability of mixed-ligand complexes with a general formula of [Rh(η5-Cp*)(N,N/O)(N)]2+/+ are reported, where (N,N/O) indicates bidentate 8-quinolate, ethylenediamine and 1,10-phenanthroline and (N) represents the releasable monodentate ligand with a nitrogen donor atom. UV-visible spectrophotometry, 1H NMR, and pH-potentiometry were used to determine the protonation constants of the monodentate ligands, the proton dissociation constants of the coordinated water molecules in the aqua complexes, and the formation constants of the mixed-ligand complexes. The obtained data were compared to those of the analogous Ru(η6-p-cymene) complexes. The developed mixed-ligand complexes were tested in drug-sensitive and resistant colon cancer cell lines (Colo205 and Colo320, respectively) and in four bacterial strains (Gram-positive and Gram-negative, drug-sensitive, and resistant) at different pH values (5-8). The mixed-ligand complexes with 1-methylimidazole displayed sufficient stability at pH 7.4, and their activation was found in cancer cells with decreasing pH; moreover, the mixed-ligand complexes demonstrated antimicrobial activity in Gram-positive and Gram-negative bacteria, including the resistant MRSA strain. This study proved the viability of incorporating releasable monodentate ligands into mixed-ligand half-sandwich complexes, which is supported by the biological assays.

Influence of low-dose X-ray on plasma membrane properties of erythroleukemia cell lines (K562, K562/adr)

Background: Low-dose X-ray in medical use for diagnosis and therapy can result in cellular biology either directly or indirectly. In cell biology, the interaction of low-dose radiation generates many radical molecules that interact with cellular organelles, such as the plasma membrane. Objectives: This study aimed to evaluate the effect of low-dose X-ray on both drug-sensitive (K562) and drug-resistant (K562/adr) erythroleukemic cell lines. Materials and methods: Cells were exposed by using an X-ray at 135 kVp to obtain the absorbed dose of 0.05, 0.1, and 0.2 Gy. The intracellular reactive oxidant species (RS), malondialdehyde, membrane fluidity, drug uptake, and drug accumulation were instantly observed after radiation. Results: The result showed a significant increase in RS in both cell lines as a function of radiation dose. In K562, the malondialdehyde (MDA) value increased in a radiation dose manner, while membrane fluidity was significantly modified at 0.1 and 0.2 Gy. In K562/adr, the uptake rate of pirarubicin (THP) and IC20 were altered but not significantly different from sham control. Conclusion: Low-dose X-ray significantly increased the intracellular RS in both cell lines and decreased the membrane fluidity at 0.1 Gy of K562. There are alterations of anticancer drug uptake rate in both cell lines, but they are not significant.

A Novel Protocol for the Synthesis of 1,2,4-Oxadiazoles Active against Trypanosomatids and Drug-Resistant Leukemia Cell Lines.

Cancer and parasitic diseases, such as leishmaniasis and Chagas disease, share similarities that allow the co-development of new antiproliferative agents as a strategy to quickly track the discovery of new drugs. This strategy is especially interesting regarding tropical neglected diseases, for which chemotherapeutic alternatives are extremely outdated. We designed a series of (E)-3-aryl-5-(2-aryl-vinyl)-1,2,4-oxadiazoles based on the reported antiparasitic and anticancer activities of structurally related compounds. The synthesis of such compounds led to the development of a new, fast, and efficient strategy for the construction of a 1,2,4-oxadiazole ring on a silica-supported system under microwave irradiation. One hit compound (23) was identified during the in vitro evaluation against drug-sensitive and drug-resistant chronic myeloid leukemia cell lines (EC50 values ranging from 5.5 to 13.2 µM), Trypanosoma cruzi amastigotes (EC50 = 2.9 µM) and Leishmania amazonensis promastigotes (EC50 = 12.2 µM) and amastigotes (EC50 = 13.5 µM). In silico studies indicate a correlation between the in vitro activity and the interaction with tubulin at the colchicine binding site. Furthermore, ADMET in silico predictions indicate that the compounds possess a high druggability potential due to their physicochemical, pharmacokinetic, and toxicity profiles, and for hit 23, it was identified by multiple spectroscopic approaches that this compound binds with human serum albumin (HSA) via a spontaneous ground-state association with a moderate affinity driven by entropically and enthalpically energies into subdomain IIA (site I) without significantly perturbing the secondary content of the protein.

The Anti-Breast Cancer Activity of Dihydroartemisinin-5-methylisatin Hybrids Tethered via Different Carbon Spacers.

Sixteen dihydroartemisinin-5-methylisatin hybrids 6a-c and 7a-m tethered via different carbon spacers were assessed for their antiproliferative activity against MCF-7, MDA-MB-231, MCF-7/ADR and MDA-MB-231/ADR breast cancer cell lines as well as cytotoxicity towards MCF-10A cells to investigate the influence of the length of carbon spacers on the activity. The preliminary results illustrated that the length of the carbon spacer was the main parameter which affected the activity, and hybrids tethered via the two-carbon linker showed the highest activity. Amongst the synthesized hybrids, the representative hybrid 7a (IC50: 15.3-20.1 µM) not only demonstrated profound activity against both drug-sensitive and drug-resistant breast cancer cell lines, but also possessed excellent safety and selectivity profile. Collectivity, hybrid 7a was a promising candidate for the treatment of both drug-sensitive and drug-resistant breast cancers and worthy of further preclinical evaluations.

Pharmacogenomic Profiling of Cisplatin-Resistant and -Sensitive Human Osteosarcoma Cell Lines by Multimodal Targeted Next Generation Sequencing.

Cisplatin (CDDP) is a drug for high-grade osteosarcoma (HGOS) treatment. Several germline pharmacogenetic studies have revealed associations between single nucleotide polymorphisms (SNPs) and CDDP-based therapy response or CDDP-related toxicity in patients with HGOS. Whether these variants could play a biological role in HGOS cells has not been studied so far. The aim of this study was to explore 28 SNPs of 14 genes in 6 CDDP-resistant and 12 drug-sensitive human HGOS cell lines. An innovative multimodal targeted next generation sequencing (mmNGS) approach with custom primers designed for the most commonly reported SNPs of genes belonging to DNA repair, CDDP transport or detoxification, or associated with CDPP-related toxicity was applied. The mmNGS approach was validated by TaqMan genotyping assays and emerged to be an innovative, reliable tool to detect genetic polymorphisms at both the DNA and RNA level. Allele changes in three SNPs (ERCC2 rs13181 and rs1799793, ERCC1 rs11615) were identified on both DNA and RNA derived libraries in association with CDDP resistance. A change of the GSTP1 rs1695 polymorphism from AA to AG genotype was observed in the RNA of all six CDDP-resistant variants. These SNPs emerged to be causally associated with CDDP resistance in HGOS cells.

Plant-Derived Sulforaphane Suppresses Growth and Proliferation of Drug-Sensitive and Drug-Resistant Bladder Cancer Cell Lines In Vitro.

Simple SummaryThe natural compound sulforaphane is highly popular among tumor patients, since it is suggested to prevent oncogenesis and cancer progression. However, knowledge about its precise mode of action, particularly when drug resistance has been established, remains poor. The present study demonstrates the proliferation-blocking effects of SFN on a panel of drug-resistant bladder cancer cell lines.Combined cisplatin–gemcitabine (GC) application is standard for treating muscle-invasive bladder cancer. However, since rapid resistance to treatment often develops, many patients turn to supplements in the form of plant-based compounds. Sulforaphane (SFN), derived from cruciferous vegetables, is one such compound, and the present study was designed to investigate its influence on growth and proliferation in a panel of drug-sensitive bladder cancer cell lines, as well as their gemcitabine- and cisplatin-resistant counterparts. Chemo-sensitive and -resistant RT4, RT112, T24, and TCCSUP cell lines were exposed to SFN in different concentrations, and tumor growth, proliferation, and clone formation were evaluated, in addition to apoptosis and cell cycle progression. Means of action were investigated by assaying cell-cycle-regulating proteins and the mechanistic target of rapamycin (mTOR)/AKT signaling cascade. SFN significantly inhibited growth, proliferation, and clone formation in all four tumor cell lines. Cells were arrested in the G2/M and/or S phase, and alteration of the CDK–cyclin axis was closely associated with cell growth inhibition. The AKT/mTOR signaling pathway was deactivated in three of the cell lines. Acetylation of histone H3 was up-regulated. SFN, therefore, does exert tumor-suppressive properties in cisplatin- and gemcitabine-resistant bladder cancer cells and could be beneficial in optimizing bladder cancer therapy.

The EMT-activator ZEB1 is unrelated to platinum drug resistance in ovarian cancer but is predictive of survival

The IGROVCDDP cisplatin-resistant ovarian cancer cell line is an unusual model, as it is also cross-resistant to paclitaxel. IGROVCDDP, therefore, models the resistance phenotype of serous ovarian cancer patients who have failed frontline platinum/taxane chemotherapy. IGROVCDDP has also undergone epithelial-mesenchymal transition (EMT). We aim to determine if alterations in EMT-related genes are related to or independent from the drug-resistance phenotypes. EMT gene and protein markers, invasion, motility and morphology were investigated in IGROVCDDP and its parent drug-sensitive cell line IGROV-1. ZEB1 was investigated by qPCR, Western blotting and siRNA knockdown. ZEB1 was also investigated in publicly available ovarian cancer gene-expression datasets. IGROVCDDP cells have decreased protein levels of epithelial marker E-cadherin (6.18-fold, p = 1.58e−04) and higher levels of mesenchymal markers vimentin (2.47-fold, p = 4.43e−03), N-cadherin (4.35-fold, p = 4.76e−03) and ZEB1 (3.43-fold, p = 0.04). IGROVCDDP have a spindle-like morphology consistent with EMT. Knockdown of ZEB1 in IGROVCDDP does not lead to cisplatin sensitivity but shows a reversal of EMT-gene signalling and an increase in cell circularity. High ZEB1 gene expression (HR = 1.31, n = 2051, p = 1.31e−05) is a marker of poor overall survival in high-grade serous ovarian-cancer patients. In contrast, ZEB1 is not predictive of overall survival in high-grade serous ovarian-cancer patients known to be treated with platinum chemotherapy. The increased expression of ZEB1 in IGROVCDDP appears to be independent of the drug-resistance phenotypes. ZEB1 has the potential to be used as biomarker of overall prognosis in ovarian-cancer patients but not of platinum/taxane chemoresistance.

Abstract 2391: Alterations in metformin mediated metabolism in chemoresistant ovarian cancer cells

Abstract Ovarian cancer (OVCA) is the fifth leading cause of death in post-menopausal women in comparison to all other gynecologic malignancies. Late detection of OVCA is common as screening is done mostly in women over 63. This delay in screening coupled with poor prognosis presents the need for novel treatment measures. Repurposing of Metformin (Met), a biguanide used to increase insulin responses in the body, has been identified as a promising approach for therapeutic treatment in OVCA patients. The purpose of this study is to isolate metabolic pathways used by Met to alter OVCA onset. Preliminary results show that Met inhibits cell proliferation and clonogenic growth in both HeyA8 and HeyA8MDR which are drug-sensitive and drug-resistant cell lines, respectively. In combination with carboplatin, Met treatment enhanced chemosensitivity in both cell lines. In addition, lipidomic analysis was done to monitor the impact of lipid metabolites on OVCA cells. Our analysis showed differences in neutral lipid composition in drug-sensitive and resistant cells which indicates the role of various lipid species play in mediating drug-resistance. Furthermore, Met treatment triggered anti-cancerous pathways through syntheses of lipid metabolites. This data indicates that cell proliferation, along with clonogenic growth are inhibited dose dependently by Met treatment in drug-resistant and drug-sensitive cell lines. Chemosensitivity is enhanced by Met treatment in combination with carboplatin in HeyA8 and HeyA8MDR cell lines. Together, the data suggests that autophagy and lipid biogenesis can be altered through metabolic pathways by Met treatment. Through the use of repurposed Metformin, this approach shows promising results of a treatment regimen and potential prevention of OVCA. Citation Format: Brenita Colette Jenkins, Debarshi Roy, Kristal Maner-Smith, Viji Shridhar. Alterations in metformin mediated metabolism in chemoresistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2391.

Abstract 2710: Associations between structural variant signatures and drug sensitivity in cell lines

Abstract Structural variants (SVs) within the cancer genome reflect defects in DNA damage repair, impact gene regulation, and alter the genomic landscape. SVs impact a larger fraction of the cancer genome than any other genetic aberration, yet their effects on modulating therapeutic response have been largely uncharacterized. Patterns of rearrangements, otherwise known as signatures, vary across tumor and tissue type, and can indicate underlying biological mechanisms responsible for cancer etiology. We have extracted SV signatures from whole genome sequencing data from hundreds of cancer cell lines. Using datasets indicating the cell lines’ response to over a thousand small molecule inhibitors, we have identified associations between SV signatures and response to therapeutics. These results will enhance our understanding of mechanisms underlying rearrangement signatures and nominate new biomarkers of drug response. Citation Format: Simona Dalin, Frank Dubois, Shu Zhang, Alexander Crane, Jeremie Kalfon, Javad Noorbakhsh, Rameen Beroukhim. Associations between structural variant signatures and drug sensitivity in cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2710.

Abstract 5356: RIDR-PI-103, ROS-activated PI3K inhibitor prodrug inhibits cell growth and impairs the PI3K/Akt pathway in BRAF and MEK inhibitor resistant BRAF-mutant melanoma cells

Abstract The five-year survival rate for patients diagnosed with metastatic melanoma is only 27%. About half of melanomas have the presence of the BRAFV600E oncoprotein, which leads to hyperactivation of the mitogen activated protein kinase (MAPK) pathway. The MAPK pathway is known to crosstalk with the PI3K/Akt pathway. Treatment with the BRAF inhibitor, Dabrafenib and the MEK inhibitor, Trametinib is limited as patient responses begin to drop due to acquired resistance to these kinase inhibitors. High concentrations of reactive oxygen species (ROS) accompany this resistance. ROS-induced drug release (RIDR)-PI-103 is a novel prodrug with a self-cyclizing moiety linked to PI-103, a PI3K inhibitor. Under high ROS conditions, RIDR-PI-103 releases PI-103, which inhibits conversion of PIP2 to PIP3. We have generated Trametinib and Dabrafenib resistant (TDR) cells for 3 BRAF-mutant cell lines: A375, WM115 and WM983B from parental (drug sensitive) cell lines. Studies in our lab demonstrate that TDR cells maintain p-Akt levels compared to parental counterparts and have significantly higher ROS. This is a rationale to explore the efficacy RIDR-PI-103 in TDR cells. We tested the effect of RIDR-PI-103 on cell viability for 1789C melanocytes, 1788B melanocytes, A375 TDR, WM983B TDR, and WM115 TDR using an MTT proliferation assay. RIDR-PI-103 exhibited less toxicity as compared to PI-103 at 5 µM in normal melanocytes. While RIDR-PI-103 significantly inhibited TDR cell proliferation at 5 and 10 µM compared to cells treated with vehicle DMSO and melanocytes. This effect was consistent when TDR cells were treated with RIDR-PI-103 in long-term crystal violet cell proliferation assays. WM115 TDR and WM983B TDR cells were highly sensitive to RIDR-PI-103 at 5 µM, while A375 TDR cells had only ~50% inhibition at 5 µM in both cell proliferation assays. 24 hour treatment with RIDR-PI-103 inhibited p-Akt (S473) along with downstream p-S6 (S240/244) and p-S6 (235/236). To examine the mechanism of action of RIDR-PI-103 and to test that the drug release is ROS induced, we assessed the effect of glutathione (GSH), an antioxidant on the TDR cells in the presence or absence of RIDR-PI-103. GSH alone did not affect proliferation of TDR cells. Addition of GSH to RIDR-PI-103 significantly rescued the cell proliferation in all three TDR cell lines. We next tested the effect of t-butyl hydrogen peroxide (TBHP), a ROS inducer on TDR cells with lower concentrations of RIDR-PI-103. Addition of TBHP to RIDR-PI-103 significantly induced proliferation in TDR cells. Ongoing experiments are focused on assessing the effect of RIDR-PI-103 on the mTOR pathway. Examining the efficacy of RIDR-PI-103 on BRAF and MEK inhibitor resistant cells will expand possible treatment options and open up avenues for the development of new treatment therapies for BRAF-mutant melanoma patients. Citation Format: Hima Patel, Rosalin Mishra, Adam Wier, Nazanin Mokhtarpour, Edward J. Merino, Joan T. Garrett. RIDR-PI-103, ROS-activated PI3K inhibitor prodrug inhibits cell growth and impairs the PI3K/Akt pathway in BRAF and MEK inhibitor resistant BRAF-mutant melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5356.

Abstract 1088: Therapeutic targeting of sphingosine kinase 2 in tyrosine kinase inhibitor resistance and non-small cell lung cancer tumorigenicity

Abstract It is anticipated that there will be 235,760 cases and 131,880 deaths due to lung cancer in the US in 2021. Tyrosine Kinase Inhibitors (TKIs) such as Erlotinib (ER) and Osimertinib (OR) are currently being used to treat lung cancer patients with EGFR mutations; however, patients develop resistance to these drugs within 10-18 months. Sphingosine kinase 2 (SK2), which is upregulated in Non-Small Cell Lung Cancer (NSCLC), phosphorylates sphingosine to sphingosine-1-phosphate (S1P), promoting cancer cell proliferation. The sphingolipid rheostat controls the cell fate by maintaining a balance between pro-survival and pro-apoptotic sphingolipids, which is regulated by SK2. However, the role of SK2 in TKI resistance is not established. To determine if SK2 causes TKI resistance, we have studied its role in NSCLC and TKI resistance. We hypothesize that SK2 may be involved in inducing TKI- resistance in NSCLC cells and is positively correlated with tumor progression. We studied SK2 in both drug resistant (OR and ER) and drug sensitive (parental) H358, H2170, H3255 and PC9 NSCLC cell lines. Our study investigated SK2 expression in drug resistant cells versus parental cells using qPCR and Western Blotting. The effects of SK2 inhibitor, Opaganib, on NSCLC cell proliferation was studied through an MTT assay. We have also studied its expression in early- and late-stage tumors using Immunohistochemistry (IHC). In drug resistant cells, qPCR studies showed that SK2 was upregulated in H2170 by 1.30-fold (ER) and 1.5-fold (OR); in H358 by 1.36-fold (ER) and 1.17-fold (OR); in PC9 by 3.3-fold (ER) and 1.8-fold (OR) and in H3255 by 4.3-fold (OR) compared to parental cells. A western blotting experiment demonstrated that SK2 was upregulated significantly by 1.7-fold and 1.5-fold in H358OR and H2170OR cells, respectively, compared to parental cells. These results suggest that SK2 may play a role in TKI resistance, as displayed by SK2 upregulation in TKI-resistant cells. MTT assay data showed 71% inhibition of cell growth by Osimertinib and SK2 inhibitor in H2170OR. The inhibition due to the combination of the drug and inhibitor was more than additive by 9% compared to the drug and inhibitor individually. This indicates SK2s potential to overcome TKI- Resistance. To analyze SK2 expression in cancer progression, IHC experiments were performed on 63 early and 48 late stage lung cancer patient sections. We observed upregulation of SK2 in late- stage lung cancer (p<0.05), indicating its role in cancer progression. In conclusion, SK2 may be a key biomarker in combating TKI-resistance and play a role in cancer progression in NSCLC. Thus, SK2 may be a promising candidate for molecular targeted therapy against NSCLC. Citation Format: Namrata Umeshchandra Dube, Katie Lam, Neelu Puri. Therapeutic targeting of sphingosine kinase 2 in tyrosine kinase inhibitor resistance and non-small cell lung cancer tumorigenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1088.

Abstract 4047: Targeted liposomes against ovarian cancer

Abstract Purpose: Cisplatin (CPT) is a first-line chemotherapeutic agent for the treatment of ovarian cancer. However, CPT has severe adverse effects. The purpose of this study is to develop targeted liposomes that selectively target the xCT transporter which is upregulated in ovarian cancer and carry CPT to ovarian cancer only, thereby avoiding the adverse effects of CPT. We evaluated the stability, the cytotoxicity and the cellular uptake of targeted liposomes in ovarian cancer cell lines. Methods: CPT was encapsulated by the remote loading method in liposomes. The stability of liposomes was evaluated by measuring the drug-leakage from liposomes and the growth of liposome size stored at 4°C and room temperature over a 30-day period. For the drug-leakage study, liposomes were placed into a dialysis tubing and dialysate aliquots were removed and analyzed twice weekly for CPT content. The liposome size was evaluated using Dynamic Light Scattering (Nano Brook Omni, Brookhaven Instruments). The cellular uptake of targeted liposomes (encapsulating the fluorescent dye sulforhodamine B, SRB) was evaluated in drug-resistant (A2870cis, OVCAR3, OVCAR4, IGROV1, and NCI/ADR-RES) and drug sensitive (A2870 and OVCAR8) ovarian cancer cell lines (purchased from the NIH), were evaluated using flow cytometry (BD Accuri C6). The cytotoxicity of CPT in targeted liposomes was evaluated in the various ovarian cell lines using the MTS assay (Novus Biologicals). Results: The targeted liposomes over the 30-day period, show that 6.8% of the total loaded CPT leaked out of the liposomes. During that period the growth of the liposomes was <5% for the liposomes stored at 4°C and <1% of their initial size, stored at 25°C. The cellular uptake of ovarian cancer cell lines, A2870, A2870cis, OVCAR3, OVCAR4, OVCAR8, and NCI/ADR-Res the targeted SRB loaded liposomes showed significantly more fluorescence intensity when compared to free SRB and non-targeted liposomes (p<0.05). In the cell line IGROV1, there is a similar trend seen, where there was a higher fluorescence intensity compared to free SRB and non-targeted. The fluorescence intensity was found to be higher compared to non-targeted liposomes (p<0.0001) but not statistically significant higher compared to free SRB. For cytotoxicity, a similar trend is seen in cell lines A2870, A2870cis, IGROV1, OVCAR3, OVCAR4, OVCAR8, and NCI/ADR-res, where the cisplatin loaded targeted liposomes had the lowest IC50 compared to free drug and non-targeted liposomes. Non-targeted liposomes had the highest IC50 values compared to free drug and targeted liposomes. In cell lines A2870, A2870cis, IGROV1, OVCAR3, OVCAR4, OVCAR8, and NCI/ADR-res the IC50 values show a 2.4, 5.3, 4.1, 3.3, 1.7, 1.6, and 2.5-fold lower values compared to free cisplatin, respectively. Conclusion: Targeted liposomes encapsulating CPT show stability, enhanced in-vitro cellular uptake and anti-cancer activity in resistant and sensitive ovarian cancer cell lines. Citation Format: Antonia Marashio, Maria (Mary) P. Lambros, Adenike Oyegbesan. Targeted liposomes against ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4047.

Transcription Factors and MiRNAs Regulate the Mechanism of Drug Resistance in Oesophageal Cancer

The presence of drug resistance can lead to differences in treatment outcomes among patients using the same drug. Therefore, identification of key biological markers associated with drug resistance can help clinicians to quickly select the appropriate drug to prolong the survival time of patients, and it is important for drug development and detailed study of the drug’s mechanism of action. Firstly, we screened different drug resistance and sensitive cell lines in esophageal cancer cell lines to find different drug resistance related genes, and annotated these calculated drug resistance related genes into the transcriptional regulatory network we constructed. The regulatory relationships within this transcriptional regulatory network were all experimentally confirmed and further filtered by real esophageal cancer data to identify drug resistance related modules and key regulators. Thirteen drug resistance-associated modules were identified, each containing 1-6 drug resistance-associated key regulators. Among them, transcription factors SP1, has-miR-21-5p and hsa-miR-1-5p play key regulatory roles in the resistance modules of various drugs, and they regulate drug resistance-associated differentially expressed genes through one-step or multi-step regulatory relationships. Key transcription factors and miRNA in the transcriptional regulatory network that regulate drug resistance-associated genes can be used as potential biomarkers to identify drug resistance to the corresponding drugs in tumor patients.

The Profile of MicroRNA Expression and Potential Role in the Regulation of Drug-Resistant Genes in Doxorubicin and Topotecan Resistant Ovarian Cancer Cell Lines.

Epithelial ovarian cancer has the highest mortality among all gynecological malignancies. The main reasons for high mortality are late diagnosis and development of resistance to chemotherapy. Resistance to chemotherapeutic drugs can result from altered expression of drug-resistance genes regulated by miRNA. The main goal of our study was to detect differences in miRNA expression levels in two doxorubicin (DOX)- and two topotecan (TOP)-resistant variants of the A2780 drug-sensitive ovarian cancer cell line by miRNA microarray. The next aim was to recognize miRNAs as factors responsible for the regulation of drug-resistance genes. We observed altered expression of 28 miRNA that may be related to drug resistance. The upregulation of miR-125b-5p and miR-935 and downregulation of miR-218-5p was observed in both DOX-resistant cell lines. In both TOP-resistant cell lines, we noted the overexpression of miR-99a-5p, miR-100-5p, miR-125b-5p, and miR-125b-2-3p and decreased expression of miR-551b-3p, miR-551b-5p, and miR-383-5p. Analysis of the targets suggested that expression of important drug-resistant genes such as the collagen type I alpha 2 chain (COL1A2), protein Tyrosine Phosphatase Receptor Type K (PTPRK), receptor tyrosine kinase—EPHA7, Roundabout Guidance Receptor 2 (ROBO2), myristoylated alanine-rich C-kinase substrate (MARCK), and the ATP-binding cassette subfamily G member 2 (ABCG2) can be regulated by miRNA.

The anti-lung cancer activity of propylene tethered dihydroartemisinin-isatin hybrids

Nineteen propylene tethered dihydroartemisinin-isatin hybrids 5a-g and 6a-l were designed, synthesized, and screened for their in vitro antiproliferative activity against three lung cancer cell lines, inclusive of drug-sensitive (A549), doxorubicin-resistant A549 (A549/DOX) and cisplatin-resistant A549 (A549/DDP) cell lines. The cytotoxicity of the synthesized hybrids towards normal lung epithelial cell line (BEAS-2B) was also assessed to evaluate the selectivity. The structure-activity relationship (SAR) elucidated that (1) alkyloxylimino fragment at C-3 position of isatin moiety were more favorable than the carbonyl and benzoxylimino, and the relative contricution order was methoxylimino > ethoxylimino > carbonyl > benzoxylimino; halogen atom at C-5 or C-6 position of isatin fragment could enhance the activity. Among them, hybrid 6f (IC 50 : 21.7–28.9 μM) showed promising activity against the three tested lung cancer cell lines, and the activity was not inferior to that of cisplatin (IC 50 : 19.7 and 66.9 μM) and doxorubicin (IC 50 : 54.3 and 15.1 μM) against multidrug-resistant A549/DOX and A549/DDP lung cancer cell lines. In addition, hybrid 6f (IC 50 : >100 μM) was non-cytotoxic towards normal lung epithelial cell line (BEAS-2B), and the RI values of hybrid 6f were 1.12 and 1.33. Further, hybrid 6f also possessed acceptable stability in mouse and human microsomes. Accordingly, hybrid 6a was a promising anti-lung cancer chemotherapeutic candidate and merited further evaluations.

High RAD51 gene expression is associated with aggressive biology and with poor survival in breast cancer.

Although the DNA repair mechanism is important in preventing carcinogenesis, its activation in established cancer cells may support their proliferation and aggravate cancer progression. RAD51 cooperates with BRCA2 and is essential in the homologous recombination of DNA repair. To this end, we hypothesized that RAD51 gene expression is associated with cancer cell proliferation and poor prognosis of breast cancer (BC) patients. A total of 8515 primary BC patients with transcriptome and clinical data from 17 independent cohorts were analyzed. The median value was used to divide each cohort into high and low RAD51 expression groups. High RAD51 expression enriched the DNA repair gene set and was correlated with DNA repair-related genes. Nottingham histological grade, Ki67 expression and cell proliferation-related gene sets (E2F Targets, G2M Checkpoint and Myc Targets) were all significantly associated with the high RAD51 BC group. RAD51 expression was positively correlated with Homologous Recombination Deficiency, as well as both mutational burden and neoantigens that accompanied a higher infiltration of immune cells. Primary BC with lymph node metastases was associated with high expression of RAD51 in two cohorts. There was no strong correlation between RAD51 expression and drug sensitivity in cell lines, and RAD51 expression was lower after the neoadjuvant chemotherapy compared to before the treatment. High RAD51 BC was associated with poor prognosis consistently in three independent cohorts. RAD51 gene expression is associated with aggressive cancer biology, cancer cell proliferation, and poor survival in breast cancer.

Artemisinin-isatin hybrids with potential antiproliferative activity against breast cancer

Three series of artemisinin-isatin hybrids 8a-i , 10a-c and 11a-e were designed, synthesized and evaluated for their antiproliferative activity against breast cancer cells (MCF-7, MDA-MB-231 and doxorubicin-resistant MCF-7 (MCF-7/DOX)), as well as the cytotoxicity towards normal MCF-10A breast cells. The preliminary results showed that a significant part of the synthesized hybrids (IC 50 : 20.7–99.9 µM) were active against both drug-sensitive and doxorubicin-resistant breast cancer cell lines. The structure–activity relationship illustrated that the linker between artemisinin and isatin moieties as well as the substituents on C-3 and C-5 position of isatin motif had great influence on the activity. In particular, hybrids 11c,d were found to be most active against all tested breast cancer cell lines, and their activity was not inferior to that of doxorubicin. Therefore, hybrids 11c,d could serve as useful templates for the development of novel anti-breast cancer agents.

Abstract P4-01-14: The RXR agonist, IRX4204, increases the anti-tumor activity of HER2-targeted therapies in HER2-amplified breast cancer

Abstract Background: HER2 targeted monoclonal antibodies and tyrosine kinase inhibitors (TKI) are effective treatments for HER2-amplified primary tumors. However, the challenge persists that primary and acquired resistance to anti-HER2 therapy is common, and that anti-HER2 targeted therapies fail to achieve a cure in the metastatic setting. There is a critical need for safe and effective therapies that can overcome anti-HER2 drug resistance and eliminate breast cancer metastases. Here, we present data demonstrating the activity of IRX4204, a highly specific agonist of the nuclear retinoid X receptor (RXR), to inhibit the growth of HER2-amplified breast cancer cell lines alone and in combination with current anti-HER2 therapies. Methods: In vitro cell growth assays were conducted to evaluate the efficacy of IRX4204 alone and in combination with trastuzumab, tucatinib, neratinib or T-DM1, using a panel of breast cancer cell lines expressing various levels of HER2. A calculated coefficient of drug interaction (CDI) and isobologram analysis was used to assess the additive, synergistic or antagonistic effects of IRX4204 with each anti-HER2 targeted therapy. Results: IRX4204 treatment alone inhibited the growth of HER2-overexpressing cell lines, SkBr3, AU565, HCC1419 and ZR75-1 in vitro, but not the growth of HER2-normal cell lines MCF7, MDA-MB-231, or HCC1143. When combined with trastuzumab, tucatinib, neratinib or T-DM1, additive or synergistic effects were observed in all of the drug-sensitive cell lines. Moreover, IRX4204 in combination with neratinib or tucatinib inhibited the growth of a TKI-resistant breast cancer cell line (HCC1954). Conclusion: These data demonstrate a novel use of an RXR agonist, IRX4204, to inhibit the growth of HER2-amplified breast cancer cell lines and to enhance the activity of anti-HER2 therapy in vitro. These results also demonstrate that IRX4204 can overcome anti-HER2 resistance to inhibit the growth of HER2-positive cells. These data demonstrate that IRX4204 can enhance the anti-cancer activity of anti-HER2 therapies even in TKI-resistant cells. In vivo experiments and mechanistic studies are ongoing. Citation Format: Cassandra L Moyer, Abhijit Mazumdar, Jamal Hill, Martin E Sanders, Powel Brown. The RXR agonist, IRX4204, increases the anti-tumor activity of HER2-targeted therapies in HER2-amplified breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-14.