Resistant Cells Research Articles

ANGPTL3 diminishes the resistance of ovarian cancer to paclitaxel by blocking the PI3K-AKT-mTOR signaling pathway

Angiopoietin-like protein 3 (ANGPTL3) is key in ovarian cancer (OC) cell growth and metastasis, notably by enhancing natural killer cells' capacity for inducing cell toxicity and apoptosis. However, its role in influencing chemotherapy resistance in OC remains ambiguous. In this study, we discovered a correlation between reduced ANGPTL3 levels and a less favorable outcome in OC patients using the Kaplan-Meier Plotter database. Lower levels of ANGPTL3 were detected in paclitaxel (PTX)-resistant OC tissues and cell lines via western blotting and immunohistochemistry. To investigate ANGPTL3's effects, we established SKOV3/PTX and 2780/PTX as PTX-resistant OC cell lines by incrementally increasing PTX exposure and then transfecting them with overexpress ANGPTL3 (OE-ANGPTL3) lentivirus. We conducted various assays such as CCK-8, colony formation, Edu staining, flow cytometry, and transwell to investigate the impact of ANGPTL3 on PTX resistance. Additionally, this effect was examined in a mouse subcutaneous xenograft model. Both in vitro and in vivo experiments demonstrated that ANGPTL3 overexpression mitigated PTX resistance in OC cells by inactivating the PI3K-AKT-mTOR pathway. In summary, our research reveals that ANGPTL3 enhances PTX sensitivity in OC by downregulating the PI3K-AKT-mTOR pathway. The study of this study suggest that ANGPTL3 could serve as a valuable therapeutic target for OC, signifying its clinical relevance in OC management.

Abstract PR008: Modulating the PPARγ pathway to augment NECTIN4-targeting chimeric antigen receptor (CAR) T cell therapy

Abstract Background: We present the development of NECTIN4-CAR T cells targeting bladder cancer and evaluate the ability of thiazolidinedione, a class of anti-diabetic drugs, to specifically upregulate NECTIN4 expression and expand the therapeutic window of NECTIN4-CAR T cells. In addition, we assess efficacy of NECTIN4-CAR T cell therapy against a model of bladder cancer with acquired resistance to enfortumab vedotin (EV). Methods: NECTIN4-targeting chimeric antigen receptor (CAR) constructs were designed and cloned into a second-generation EQ-28ζ CAR backbone construct. Human primary T cells were transduced with lentiviral NECTIN4-CAR constructs and co-cultured with a panel of bladder cancer cell lines with a range of NECTIN4 expression and against NECTIN4 knockout cells to assess specificity. Tumor cell count over time was monitored in vitro using an IncuCyte S3. Bladder cancer cell lines (RT112, UMUC-1, HT1197) were treated with PPARγ agonists (rosiglitazone) and antagonists (T0070907), and total and surface NECTIN4 protein levels were assessed. Anti-tumor effects of the NECTIN4-CAR T were evaluated in vitro and in vivo, either with or without rosiglitazone pre-treatment. Finally, RT112 bladder cancer cells were exposed to “cycles” of escalating doses of EV to generate EV-resistant cell line models, and anti-tumor efficacy against EV-resistant lines was evaluated. Results: NECTIN4-targeting CAR T cells displayed potent and NECTIN4-specific cytotoxic activity against bladder cancer cell lines. The efficacy of NECTIN4-CAR T cells was strongly correlated with NECTIN4 expression. Treatment with PPARγ agonists and antagonists was shown to upregulate and downregulate NECTIN4 expression, respectively. Treatment with the PPARγ agonist rosiglitazone induced NECTIN4 expression in a PPARγ-dependent and dose responsive manner. NECTIN4 expression was increased in bladder cancer xenografts established in immunodeficient NSG mice that were treated with systemic rosiglitazone. Priming with rosiglitazone enhanced sensitivity to NECTIN4-CAR T cells in vitro, and the combination of rosiglitazone pre-treatment and NECTIN4-CAR T cell therapy showed enhanced tumor control in bladder cancer xenograft models. We generated RT112 cells that exhibited resistance to EV in vitro and in vivo, which retained NECTIN4 expression. Interestingly, EV-resistant RT112 cells remained sensitive to NECTIN4-CAR T cell therapy both in vitro and in vivo. Conclusions: We engineered a NECTIN4-targeting CAR T cell therapy that is highly antigen-specific and demonstrates antitumor activity against multiple bladder cancer models, including EV resistant cells. Modulating the PPARγ pathway increases NECTIN4 expression, which we leveraged by repurposing an FDA-approved anti-diabetes drug rosiglitazone to increase targeting and anti-tumor efficacy of NECTIN4-CAR T cells. These preclinical results lay the groundwork for CAR T therapy development in bladder cancer and suggest rational drug combinations that can expand the therapeutic window of NECTIN4-targeting therapies. Citation Format: Kevin Chang, Henry M. Delavan, Jun Zhu, Corynn Kasap, Elizabeth Yip, Roshan Lodha, Sheng-You Liao, Sima Porten, Terence Friedlander, Vadim Koshkin, Felix Feng, Arun Wiita, John Lee, Carissa E. Chu, Jonathan Chou. Modulating the PPARγ pathway to augment NECTIN4-targeting chimeric antigen receptor (CAR) T cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR008.

Fe-MOF nanoplatform: Specifically overcoming oxaliplatin resistance in colorectal cancer through multifaceted pathways

Oxaliplatin (OXA) is a widely used chemotherapy drug for advanced colorectal cancer (CRC), yet resistance frequently and rapidly develops in patients. Here, we explored a Fe-based metal-organic framework (Fe-MOF) as a nanoplatform to simultaneously deliver OXA and a nitric oxide (NO) donor, specifically l-Arginine (L-Arg). The aim was to overcome OXA resistance in CRC cells, thereby enhancing the anticancer efficacy against drug-resistant CRC. Due to the abundant glutathione (GSH) in tumor tissue, Fe3+ in Fe-MOF undergoes reduction to Fe2+, causing the collapse of Fe-MOF structure and the subsequent release of OXA and L-Arg. This process is accompanied by GSH depletion, amplifying the effectiveness of OXA against drug-resistant CRC. Fe2+ further reacts with overexpressed H2O2 to generate •OH, inducing direct cell apoptosis and subsequently reacting with L-Arg to rapidly produce NO. NO plays multifaceted roles in the anti-CRC effect, reversing drug resistance in CRC cells through multiple pathways, inducing apoptosis of CRC cells to initiate gas therapy, and reacting with •OH to generate highly toxic reactive nitrogen species for cancer cell destruction. This synergistic strategy effectively addresses the challenge of drug resistance in CRC, offering a promising avenue for enhancing the clinical effectiveness of OXA in cases resistant to conventional treatment.

E3 ubiquitin ligase UBR5 promotes gemcitabine resistance in pancreatic cancer by inducing O-GlcNAcylation-mediated EMT via destabilization of OGA.

Pancreatic cancer (PC) is among the deadliest malignancies, with an extremely poor diagnosis and prognosis. Gemcitabine (GEM) remains the first-line drug for treating PC; however, only a small percentage of patients benefit from current immunotherapies or targeted therapies. Resistance to GEM is prevalent and affects long-term survival. We found that ubiquitin-protein ligase E3 module N-recognition 5 (UBR5) is a therapeutic target against GEM resistance. UBR5 was markedly upregulated in clinical GEM-resistant PC samples and GEM-resistant PC cells. UBR5 knockdown markedly increased GEM sensitivity in GEM-resistant PC cell lines. UBR5-mediated GEM resistance was accompanied by activation of epithelial-mesenchymal transition (EMT) and could be mitigated by inhibiting EMT. Further analysis revealed that UBR5 promoted GEM resistance in PC cells by enhancing O-GlcNAcylation-mediated EMT. In addition, UBR5 knockdown resulted in increased O-GlcNAase (OGA) levels, an essential negatively regulated enzyme in the O-GlcNAcylation process. We identified a negative association between OGA and UBR5 levels, which further supported the hypothesis that O-GlcNAcylation-mediated GEM resistance induced by UBR5 is OGA-dependent in PC cells. Mechanistic studies revealed that UBR5 acts as an E3 ubiquitin ligase of OGA and regulates O-GlcNAcylation by binding and modulating OGA, facilitating its degradation and ubiquitination. Additionally, high-throughput compound library screening using three-dimensional protein structure analysis and drug screening identified a Food and Drug Administration drug, Y-39983 dihydrochloride, as a potent GEM sensitiser and UBR5 inhibitor. The combination of Y-39983 dihydrochloride and GEM attenuated tumour growth in a mouse xenograft tumour model. Collectively, these data demonstrated that UBR5 plays a pivotal role in the sensitisation of PC to GEM and provides a potential therapeutic strategy to overcome GEM resistance.

Novel markers of MCL1 inhibitor sensitivity in triple-negative breast cancer cells

Triple negative breast cancer (TNBC) is an aggressive breast cancer sub-type with limited treatment options and poor prognosis. Currently, standard treatments for TNBC includes surgery, chemotherapy, and anti-PDL1 therapy. These therapies have limited efficacy in advanced stages. Myeloid-cell leukemia 1 (MCL1) is an anti-apoptotic BCL2 family protein. High expression of MCL1 contributes to chemotherapy resistance and is associated with worse prognosis in TNBC. MCL1 inhibitors are in clinical trials for TNBC, but response rates to these inhibitors can vary and predictive markers are lacking. Currently we identified a 4-member (AXL, ETS1, IL6, EFEMP1) gene signature (GS) that predicts MCL1 inhibitor sensitivity in TNBC cells. Factors encoded by these genes regulate signaling pathways to promote MCL1 inhibitor resistance. Small molecule inhibitors of the GS factors can overcome resistance and sensitize otherwise resistant TNBC cells to MCL1 inhibitor treatment. These findings offer insights into potential therapeutic strategies and tumor stratification for MCL1 inhibitor use in TNBC.

Fisetin and/or capecitabine causes changes in apoptosis pathways in capecitabine-resistant colorectal cancer cell lines.

Capecitabine is recommended as one of the first-line chemotherapy treatments for advanced or metastatic colorectal cancer. Researches have been conducted on capecitabine's impact on the viability of human colon cancer cells and its potential to induce apoptosis. However, even in cases initially responsive to treatment, the development of acquired resistance significantly limits its efficacy. Challenges still exist in effectively treating patients with chemotherapy, and developing new cytotoxic drugs is hindered by drug resistance. Fisetin alters the cell cycle, inducing apoptosis, inhibiting cancer cell proliferation, and enhancing the therapeutic effectiveness of chemotherapy drugs. This work aims to create a plan for reversing capecitabine resistance. For this purpose, the role of capecitabine and/or fisetin combinations in cell proliferation and apoptosis has been determined in both wild-type and capecitabine-resistant HT29 cells (CR/HT29). We developed capecitabine-resistant cell line from wild-type HT29 cells. This study demonstrated the effects of capecitabine, fisetin, and their combinations on both resistant and wild-type cells through experiments including cell survival skills, cell proliferation, wound healing, colony formation, hoechst staining, and western blot analysis.We established capecitabine-resistant cell lines. P-gp expression increased in CR/HT29 cells. Capecitabine effects on a CR/HT29 cells less than wild-type HT29 cells. The combination of fisetin and capecitabine in cell proliferation caused greater reductions in wild-type HT29 cells than in capecitabine-resistant cells. Fisetin has also additive effects on the apoptotic pathway in CR/HT29 cells.This study provides new perspectives on the combination of capecitabine and/or flavonoid treatment in resistant cells.

Theoretico-experimental analysis of bistability in the oscillatory response of a TaOx ReRAM to pulse train stimuli

Fading memory is the capability of a physical system to approach a unique asymptotic behaviour, irrespective of the initial conditions, when stimulated by an input from a certain class. Standard stimuli from the AC periodic class typically induce fading memory effects in non-volatile memristors, as uncovered for the first time back in 2016. Very recently, a deep investigation of resistance switching phenomena in a TaOx resistive random access memory cell revealed the capability of the nano-device to exhibit one of two possible oscillatory behaviours, depending upon the initial condition, when subject to a particular periodic excitation. This interesting finding was, however, left unexplained. Bistability is the simplest form of local fading memory. In a system, endowed with local fading memory under a given stimulus, the initial condition does not affect the long-term behaviour of the state as long as it is drawn from the basin of attraction of either of the distinct coexisting state-space attractors (two limit cycles for the periodically forced memristor acting as a bistable oscillator). Here, the history of the system, encoded in the initial condition, is, thus, erasable only locally through ad hoc stimulation. Motivated by the discovery of local history erase effects in our resistive random access memory cell, this study applies a powerful system-theoretic tool, enabling the analysis of the response of first-order systems to square pulse train-based periodic stimuli, known as the time-average state dynamic route, to an accurate physics-based mathematical model, earlier fitted to the nano-device, to determine a strategy for specifying the parameters of an excitation signal, consisting of the sequence of two square pulses of opposite polarity per period so as to induce various forms of monostability or multistability in the non-volatile memristor. In particular, as an absolute novelty in the literature, experimental measurements validate the theoretical prediction on the capability of the device to operate as one of two distinct oscillators, depending upon the initial condition, under a specific pulse train excitation signal. The coexistence of multiple oscillatory operating modes in the periodically forced resistive random access memory cell, an example par excellence of their unique non-linear dynamics, may inspire the development and circuit implementation of novel sensing and mem-computing paradigms.

Lentinan mitigates pemetrexed chemoresistance by the PI3K/Akt pathway in non-small cell lung cancer.

Pemetrexed is a folate analog metabolic inhibitor that is given for therapy of non-small cell lung cancer (NSCLC). Drug resistance affects the efficacy of pemetrexed in NSCLC. Lentinan is a polysaccharide extracted from Shiitake mushrooms which has antitumor roles in multiple cancers, including lung cancer. However, the effects of lentinan on pemetrexed resistance in NSCLC remain unclear. In present study, The pemetrexed-resistant NSCLC cells were established and exposed to pemetrexed and lentinan. Oxidative stress was investigated via mitochondrial membrane potential (JC-1 staining), levels of MDA and SOD.The phosphorylation and total of PI3K and Akt levels were actuated using specific activator 740Y-P and measured through western blot. We observed that Lentinan decreased IC50 of pemetrexed in resistant NSCLC cells. Lentinan aggravated pemetrexed-induced proliferation inhibition of resistant NSCLC cells via reducing PCNA levels. Lentinan exacerbated pemetrexed-triggered oxidative stress through increasing ROS and MDA levels, and reducing mitochondrial membrane potential and SOD levels. Lentinan inhibited PI3K/Akt signaling activation in pemetrexed-treated cells. Activated PI3K/Akt pathway using activator 740Y-P reversed the effects of lentinan on pemetrexed-mediated proliferation inhibition and oxidative stress. Our findings uncover that Lentinan mitigates pemetrexed resistance in NSCLC through inhibiting cell proliferation and inducing oxidative stress by suppressing PI3K/Akt signaling.

Coordinative Compounds Based on Unsaturated Carboxylate with Versatile Biological Applications.

This review presents an overview of the biological applications of coordinative compounds based on unsaturated carboxylates accompanied by other ligands, usually N-based heterocyclic species. The interest in these compounds arises from the valuable antimicrobial and antitumor activities evidenced by some species, as well as from their ability to generate metal-containing polymers suitable for various medical purposes. Therefore, we describe the recently discovered aspects related to the synthesis, structure, and biological activity of a wide range of unsaturated carboxylate-containing species and metal ions, originating mostly from 3d series. The unsaturated carboxylates encountered in coordinative compounds are acrylate, methacrylate, fumarate, maleate, cinnamate, ferulate, coumarate, and itaconate. Regarding the properties of the investigated compounds, it is worth mentioning the good ability of some to inhibit the development of resistant strains or microbial biofilms on inert surfaces or, even more, exert antitumor activity against resistant cells. The ability of some species to intercalate into DNA strands as well as to scavenge ROS species is also addressed.

Effects of KEAP1 Silencing on NRF2 and NOTCH Pathways in SCLC Cell Lines.

The KEAP1/NRF2 pathway is a master regulator of several redox-sensitive genes implicated in the resistance of tumor cells against therapeutic drugs. The dysfunction of the KEAP1/NRF2 system has been correlated with neoplastic patients' outcomes and responses to conventional therapies. In lung tumors, the growth and the progression of cancer cells may also involve the intersection between the molecular NRF2/KEAP1 axis and other pathways, including NOTCH, with implications for antioxidant protection, survival of cancer cells, and drug resistance to therapies. At present, the data concerning the mechanism of aberrant NRF2/NOTCH crosstalk as well as its genetic and epigenetic basis in SCLC are incomplete. To better clarify this point and elucidate the contribution of NRF2/NOTCH crosstalk deregulation in tumorigenesis of SCLC, we investigated genetic and epigenetic dysfunctions of the KEAP1 gene in a subset of SCLC cell lines. Moreover, we assessed its impact on SCLC cells' response to conventional chemotherapies (etoposide, cisplatin, and their combination) and NOTCH inhibitor treatments using DAPT, a γ-secretase inhibitor (GSI). We demonstrated that the KEAP1/NRF2 axis is epigenetically controlled in SCLC cell lines and that silencing of KEAP1 by siRNA induced the upregulation of NRF2 with a consequent increase in SCLC cells' chemoresistance under cisplatin and etoposide treatment. Moreover, KEAP1 modulation also interfered with NOTCH1, HES1, and DLL3 transcription. Our preliminary data provide new insights about the downstream effects of KEAP1 dysfunction on NRF2 and NOTCH deregulation in this type of tumor and corroborate the hypothesis of a cooperation of these two pathways in the tumorigenesis of SCLC.

Phlorizin from Lithocarpus litseifolius [Hance] Chun ameliorates FFA-induced insulin resistance by regulating AMPK/PI3K/AKT signaling pathway

BackgroundInsulin resistance (IR) is the central pathophysiological feature in the pathogenesis of metabolic syndrome, obesity, type 2 diabetes mellitus (T2DM), hypertension, and dyslipidemia. As the main active ingredient in Lithocarpus litseifolius [Hance] Chun, previous studies have shown that phlorizin (PHZ) can reduce insulin resistance in the liver. However, the effect of phlorizin on attenuating hepatic insulin resistance has not been fully investigated, and whether this effect is related to AMPK remains unclear. PurposeThe present study aimed to further investigate the effect of phlorizin on attenuating insulin resistance and the potential action mechanism. MethodsFree fatty acids (FFA) were used to induce insulin resistance in HepG2 cells. The effects of phlorizin and FFA on cell viability were detected by MTT analysis. Glucose consumption, glycogen synthesis, intracellular malondialdehyde (MDA), superoxide dismutase (SOD), total cholesterol (TC), and triglyceride (TG) contents were quantified after phlorizin treatment. Glucose uptake and reactive oxygen species (ROS) levels in HepG2 cells were assayed by flow cytometry. Potential targets and signaling pathways for attenuating insulin resistance by phlorizin were predicted by network pharmacological analysis. Moreover, the expression levels of proteins related to the AMPK/PI3K/AKT signaling pathway were detected by western blot. ResultsInsulin resistance was successfully induced in HepG2 cells by co-treatment of 1 mM sodium oleate (OA) and 0.5 mM sodium palmitate (PA) for 24 h. Treatment with phlorizin promoted glucose consumption, glucose uptake, and glycogen synthesis and inhibited gluconeogenesis in IR-HepG2 cells. In addition, phlorizin inhibited oxidative stress and lipid accumulation in IR-HepG2 cells. Network pharmacological analysis showed that AKT1 was the active target of phlorizin, and the PI3K/AKT signaling pathway may be the potential action mechanism of phlorizin. Furthermore, western blot results showed that phlorizin ameliorated FFA-induced insulin resistance by activating the AMPK/PI3K/AKT signaling pathway. ConclusionPhlorizin inhibited oxidative stress and lipid accumulation in IR-HepG2 cells and ameliorated hepatic insulin resistance by activating the AMPK/PI3K/AKT signaling pathway. Our study proved that phlorizin played a role in alleviating hepatic insulin resistance by activating AMPK, which provided experimental evidence for the use of phlorizin as a potential drug to improve insulin resistance.

O8G Site-Specifically Modified tRF-1-AspGTC: A Novel Therapeutic Target and Biomarker for Pulmonary Hypertension.

Hypoxia and oxidative stress contribute to the development of pulmonary hypertension (PH). tRNA-derived fragments play important roles in RNA interference and cell proliferation, but their epitranscriptional roles in PH development have not been investigated. We aimed to gain insight into the mechanistic contribution of oxidative stress-induced 8-oxoguanine in pulmonary vascular remodeling. Through small RNA modification array analysis and quantitative polymerase chain reaction, a significant upregulation of the 8-oxoguanine -modified tRF-1-AspGTC was found in the lung tissues and the serum of patients with PH. This modification occurs at the position 5 of the tRF-1-AspGTC (5o8G tRF). Inhibition of the 5o8G tRF reversed hypoxia-induced proliferation and apoptosis resistance in pulmonary artery smooth muscle cells. Further investigation unveiled that the 5o8G tRF retargeted mRNA of WNT5A (Wingless-type MMTV integration site family, member 5A) and CASP3 (Caspase3) and inhibited their expression. Ultimately, BMPR2 (Bone morphogenetic protein receptor 2) -reactive oxygen species/5o8G tRF/WNT5A signaling pathway exacerbated the progression of PH. Our study highlights the role of site-specific 8-oxoguanine-modified tRF in promoting the development of PH. Our findings present a promising therapeutic avenue for managing PH and propose 5o8G tRF as a potential innovative marker for diagnosing this disease.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKβ independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Targeting the glutamine metabolism to suppress cell proliferation in mesenchymal docetaxel-resistant prostate cancer

Docetaxel (DX) serves as a palliative treatment option for metastatic prostate cancer (PCa). Despite initial remission, acquired DX resistance is inevitable. The mechanisms behind DX resistance have not yet been deciphered, but a mesenchymal phenotype is associated with DX resistance. Mesenchymal phenotypes have been linked to metabolic rewiring, obtaining most ATP production by oxidative phosphorylation (OXPHOS) powered substantially by glutamine (Gln). Likewise, Gln is known to play an essential role in modulating bioenergetic, redox homeostasis and autophagy. Herein, investigations of Gln deprivation on DX-sensitive and -resistant (DR) PCa cells revealed that the DR cell sub-lines were susceptible to Gln deprivation. Mechanistically, Gln deprivation reduced OXPHOS and ATP levels, causing a disturbance in cell cycle progression. Genetic and chemical inhibition of the Gln-metabolism key protein GLS1 could validate the Gln deprivation results, thereby representing a valid therapeutic target. Moreover, immunohistological investigation of GLS1 revealed a high-expressing GLS1 subgroup post-docetaxel failure, exhibiting low overall survival. This subgroup presents an intriguing opportunity for targeted therapy focusing on glutamine metabolism. Thus, these findings highlight a possible clinical rationale for the chemical inhibition of GLS1 as a therapeutic strategy to target mesenchymal DR PCa cells, thereby delaying accelerated tumour progression.

Global DNA Methylation analysis of imatinib resistant and sensitive K562 cells

OBJECTIVE: Chronic myeloid leukemia (CML) is a hematological disease which is known by the presence of Philadelphia chromosome (Ph+). BCR-ABL protein is expressed by Ph+ chromosome, represents constant increased tyrosine kinase activity. Imatinib (IMA) is a tyrosine kinase inhibitor (TKI) which is utilized as a first line treatment in CML. Emergence of IMA resistance at some point of therapy leads to treatment failure. DNA methylation is considered to be the most stable epigenetic change and several studies have shown that epigenetic changes may play a role in drug resistance. We investigated the global methylation profile of IMA-sensitive K562S, IMA-resistant K562R and IMA-resistant and adherent K562R (K562R-adh) cells to determine whether epigenetic reprogramming is involved in the resistance to IMA and the change in phenotype due to this resistance. MATERIAL AND METHODS: In this study, morphologically distinct, IMA-sensitive K562S and 5µM IMA-resistant K562R and K562R-adh in-vitro CML cell models were used to analyze the global DNA methylation profile. After DNA was isolated from the cells, global 5mC DNA methylation profiles were investigated by ELISA using equal amounts of DNA. RESULTS: Compared to K562S, the global methylation of K562R showed an increase in DNA methylation profile, but this increase in methylation was not statistically significant. Whereas, a slight hypermethylation was observed in the DNA of the K562R-adh vs K562S and K562R-adh vs K562R which is statistically significant. We observed slight hypermethylation in IMA-resistant cells lines versus to the IMA-sensitive cell line. CONCLUSION: Our observed differences in 5methyl-Cytosine on CpG islands (5mC) in K562S versus K562R and K562R-adh cell lines suggest that the DNA methylation alteration in resistant cells may partly contributed in phenotype switching.

Proto-oncogene c-Myb potentiates cisplatin resistance of ovarian cancer cells by downregulating lncRNA NKILA and modulating cancer stemness and LIN28A-let7 axis

Ovarian cancer is a major gynecological cancer that has poor prognosis associated mainly to its late diagnosis. Cisplatin is an FDA approved ovarian cancer therapy and even though the therapy is initially promising, the patients mostly progress to resistance against cisplatin. The underlying mechanisms are complex and not very clearly understood. Using two different paired cell lines representing cisplatin-sensitive and the cisplatin-resistant ovarian cancer cells, the ES2 and the A2780 parental and cisplatin-resistant cells, we show an elevated proto-oncogene c-Myb in resistant cells. We further show down-regulated lncRNA NKILA in resistant cells with its de-repression in resistant cells when c-Myb is silenced. NKILA negatively correlates with cancer cell and invasion but has no effect on cellular proliferation or cell cycle. C-Myb activates NF-κB signaling which is inhibited by NKILA. The cisplatin resistant cells are also marked by upregulated stem cell markers, particularly LIN28A and OCT4, and downregulated LIN28A-targeted let-7 family miRNAs. Whereas LIN28A and downregulated let-7s individually de-repress c-Myb-mediated cisplatin resistance, the ectopic expression of let-7s attenuates LIN28A effects, thus underlying a c-Myb-NKILA-LIN28A-let-7 axis in cisplatin resistance of ovarian cancer cells that needs to be further explored for therapeutic intervention.

β-Elemene Reverses Gefitinib Resistance in NSCLC Cells by Inhibiting lncRNA H19-Mediated Autophagy.

Lung cancer is a leading cause of mortality worldwide, especially among Asian patients with non-small cell lung cancer (NSCLC) who have epidermal growth factor receptor (EGFR) mutations. Initially, first-generation EGFR tyrosine kinase inhibitors (TKIs) are commonly administered as the primary treatment option; however, encountering resistance to these medications poses a significant obstacle. Hence, it has become crucial to address initial resistance and ensure continued effectiveness. Recent research has focused on the role of long noncoding RNAs (lncRNAs) in tumor drug resistance, especially lncRNA H19. β-elemene, derived from Curcuma aromatic Salisb., has shown strong anti-tumor effects. However, the relationship between β-elemene, lncRNA H19, and gefitinib resistance in NSCLC is unclear. This study aims to investigate whether β-elemene can enhance the sensitivity of gefitinib-resistant NSCLC cells to gefitinib and to elucidate its mechanism of action. The impact of gefitinib and β-elemene on cell viability was evaluated using the cell counting kit-8 (CCK8) assay. Furthermore, western blotting and qRT-PCR analysis were employed to determine the expression levels of autophagy-related proteins and genes, respectively. The influence on cellular proliferation was gauged through a colony-formation assay, and apoptosis induction was quantified via flow cytometry. Additionally, the tumorigenic potential in vivo was assessed using a xenograft model in nude mice. The expression levels of LC3B, EGFR, and Rab7 proteins were examined through immunofluorescence. Our findings elucidate that the resistance to gefitinib is intricately linked with the dysregulation of autophagy and the overexpression of lncRNA H19. The synergistic administration of β-elemene and gefitinib markedly attenuated the proliferative capacity of resistant cells, expedited apoptotic processes, and inhibited the in vivo proliferation of lung cancer. Notably, β-elemene profoundly diminished the expression of lncRNA H19 and curtailed autophagic activity in resistant cells, thereby bolstering their responsiveness to gefitinib. Moreover, β-elemene disrupted the Rab7-facilitated degradation pathway of EGFR, facilitating its repositioning to the plasma membrane. β-elemene emerges as a promising auxiliary therapeutic for circumventing gefitinib resistance in NSCLC, potentially through the regulation of lncRNA H19-mediated autophagy. The participation of Rab7 in this dynamic unveils novel insights into the resistance mechanisms operative in lung cancer, paving the way for future therapeutic innovations.

Shikonin reduces M2 macrophage population in ovarian cancer by repressing exosome production and the exosomal galectin 3-mediated β-catenin activation

BackgroundShikonin (SK), a naphthoquinone with anti-tumor effects, has been found to decrease production of tumor-associated exosomes (exo). This study aims to verify the treatment effect of SK on ovarian cancer (OC) cells, especially on the production of exo and their subsequent effect on macrophage polarization.MethodsOC cells SKOV3 and A2780 were treated with SK. The exo were isolated from OC cells with or without SK treatment, termed OC exo and SK OC exo, respectively. These exo were used to treat PMA-induced THP-1 cells (M0 macrophages). M2 polarization of macrophages was determined by measuring the M2 specific cell surface markers CD163 and CD206 as well as the secretion of M2 cytokine IL-10. The functions of galectin 3 (LGALS3/GAL3) and β-catenin in macrophage polarization were determined by gain- or loss-of-function assays. CB-17 SCID mice were subcutaneously injected with SKOV3 cells to generate xenograft tumors, followed by OC exo or SK OC exo treatment for in vivo experiments.ResultsSK suppressed viability, migration and invasion, and apoptosis resistance of OC cells in vitro. Compared to OC exo, SK OC exo reduced the M2 polarization of macrophages. Regarding the mechanism, SK reduced exo production in cancer cells, and it decreased the protein level of GAL3 in exo and recipient macrophages, leading to decreased β-catenin activation. M2 polarization of macrophages was restored by LGALS3 overexpression but decreased again by the β-catenin inhibitor FH535. Compared to OC exo, the SK OC exo treatment reduced the xenograft tumor growth in mice, and it decreased the M2 macrophage infiltration within tumor tissues.ConclusionThis study suggests that SK reduces M2 macrophage population in OC by repressing exo production and blocking exosomal GAL3-mediated β-catenin activation.

The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in p53 proficient colon cancer cells

YAP and TAZ, the Hippo pathway terminal transcriptional activators, are frequently upregulated in cancers. In tumor cells, they have been mainly associated with increased tumorigenesis controlling different aspects from cell cycle regulation, stemness, or resistance to chemotherapies. In fewer cases, they have also been shown to oppose cancer progression, including by promoting cell death through the action of the p73/YAP transcriptional complex, in particular after chemotherapeutic drug exposure. Using HCT116 cells, we show here that oxaliplatin treatment led to core Hippo pathway down-regulation and nuclear accumulation of TAZ. We further show that TAZ was required for the increased sensitivity of HCT116 cells to oxaliplatin, an effect that appeared independent of p73, but which required the nuclear relocalization of TAZ. Accordingly, Verteporfin and CA3, two drugs affecting the activity of YAP and TAZ, showed antagonistic effects with oxaliplatin in co-treatments. Importantly, using several colorectal cell lines, we show that the sensitizing action of TAZ to oxaliplatin is dependent on the p53 status of the cells. Our results support thus an early action of TAZ to sensitize cells to oxaliplatin, consistent with a model in which nuclear TAZ in the context of DNA damage and p53 activity pushes cells towards apoptosis.

M6A RNA Methylation-Mediated Dysregulation of AGAP2-AS1 Promotes Trastuzumab Resistance of Breast Cancer

Introduction: Trastuzumab is commonly used to treat human epidermal growth factor receptor-2-positive (HER2+) breast cancer, but its efficacy is often limited by chemotherapy resistance. Recent studies have indicated that long non-coding RNAs (lncRNAs) play important roles in tumor progression and response to therapy. However, the regulatory mechanisms associating lncRNAs and trastuzumab resistance remain unknown. Methods: Quantitative polymerase chain reaction was performed to detect the expression of related genes. Western blot and immunofluorescence assays were used to evaluate protein expression levels. A series of gain- or loss-of-function assays confirmed the function of AGAP2-AS1 in trastuzumab resistance, both in vitro and in vivo. RNA immunoprecipitation and pull-down analyses were conducted to verify the interaction between METTL3/YTHDF2 and lncRNA AGAP2-AS1. Results: AGAP2-AS1 was upregulated in trastuzumab-resistant cells and SKBR-3R-generated xenografts in nude mice. Silencing AGAP2-AS1 significantly decreased trastuzumab-induced cytotoxicity both in vitro and in vivo. Furthermore, m6A methylation of AGAP2-AS1 was reduced in trastuzumab-resistant cells compared to that in parental cells. In addition, METTL3 increased m6A methylation of AGAP2-AS1, which finally induced the suppressed AGAP2-AS1 expression. Moreover, YTHDF2 was essential for METTL3-mediated m6A methylation of AGAP2-AS1. Functionally, AGAP2-AS1 regulated trastuzumab resistance by inducing autophagy and increasing ATG5 expression. Conclusion: we demonstrated that METTL3/YTHDF2-mediated m6A methylation increased the expression of AGAP2-AS1, which could promote trastuzumab resistance in breast cancer. AGAP2-AS1 regulates trastuzumab resistance by inducing autophagy. Therefore, AGAP2-AS1 may be a promising predictive biomarker and therapeutic target in patients with breast cancer.