Cell Survival Assay Research Articles

Abstract 393: Targeting MCT inhibition to overcome enzalutamide resistance in prostate cancer

Abstract Prostate cancers (PCa) exhibit a unique metabolic profile with reliance on different forms of glucose metabolism at different stages of disease progression. Early stage PCa cells use the more efficient TCA cycle, while metastatic PCa cells switch to glycolysis (Warburg effect), leading to the accumulation of lactate. Lactate is exported out of the cells by monocarboxylate transporters (MCTs) to maintain redox balance. Such metabolic reprogramming can lead to gain-of-function mutations and affect drug sensitivity. Enzalutamide is a second-generation antiandrogen used for the treatment of metastatic castration-resistant PCa. However, resistance to Enzalutamide develops in most patients within 9-15 months. Since Warburg effect is a hallmark of metastatic PCa, we sought to understand whether lactate transport has any effect on Enzalutamide-resistance in PCa. We measured the expression of MCTs in parental and Enzalutamide-resistant C4-2B and 22RV1 cells and found that they are overexpressed in the Enzalutamide resistant subtypes. Based on this observation, we hypothesized that targeting lactate transport might be a potential strategy to overcome Enzalutamide-resistance in PCa cells. Using cell survival and cell proliferation assays, we found that MCT antagonists resensitized Enzalutamide resistant C4-2B and 22Rv1 cells to treatment with Enzalutamide. Using the Seahorse based glycolytic rate assay we found that these combination treatments significantly reduced the extracellular acidification rate by reducing the level of glycolysis in the Enzalutamide-resistant PCa cells. We also found that treatment with these antagonists either singly, or in combination with Enzalutamide suppressed xenograft growth in SCID mice bearing subcutaneously injected parental or Enzalutamide-resistant C4-2B cells. These findings led to the conclusion that targeting MCTs could be an attractive strategy to overcome Enzalutamide resistance in prostate cancer. Citation Format: Sayani Bhattacharjee, Jonathan P. Doan, Rebecca Wynn, Nagalakshmi Nadiminty. Targeting MCT inhibition to overcome enzalutamide resistance in prostate 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 393.

Abstract 5649: Bromodomain inhibition reduces CDK6 expression and reverses CDK4/6 inhibitor resistance in ER+ breast cancer

Abstract Introduction: CDK4/6 inhibitor (CDK4/6i) resistance is a pressing clinical problem for patients with ER+ breast cancer who are treated with CDK4/6 inhibitors combined with hormonal therapy. Our previous work identified CDK6 overexpression as a critical determinant of acquired resistance to CDK4/6i. Bromodomain and extra-terminal domain (BET) family proteins (BRD2, BRD3, BRD4, and BRDT) play important roles in regulating the expression of oncogenes in multiple cancer types. BET inhibitors (BETi), such as JQ1, have been extensively studied for the treatment of cancers. It has been shown that BRD4 is recruited to the CDK6 gene promoter and that BET inhibition reduces CDK6 mRNA and protein expression. In this study, we investigated the potential of BET inhibition to reverse CDK4/6i resistance of ER+ breast cancer. Methods: By long-term exposure to increasing concentrations of a CDK4/6i, we developed MCF7 and T47D ER+ breast cancer cell lines with acquired resistance to several FDA-approved CDK4/6 inhibitors demonstrating marked CDK6 overexpression. Cell viability and survival assays were performed to assess the in vitro efficacy of BETi. Flow cytometry was performed to evaluate cell cycle alterations as well as cell death. Gene expression was analyzed by Western blotting and qPCR to examine cell cycle-related molecular changes. Markers of apoptosis were also evaluated by Western blotting. CDK4/6i-resistant ER+ tumors were established in immunodeficient mouse models to explore the in vivo efficacy of BETi. Results: Our data showed that JQ1 significantly reduced cell viability and survival of CDK4/6i-resistant ER+ breast cancer cells in the presence of palbociclib, a commonly used CDK4/6i for the treatment of metastatic ER+ HER2- breast cancer. JQ1 also induced marked G1 cell cycle arrest and apoptosis in CDK4/6 inhibitor-resistant MCF7 and T47D cell lines in a time and dose-dependent manner. Analyses of key molecular mediators of cell cycle progression identified CDK6 and cyclin D1 as the major targets of JQ1 in CDK4/6i-resistant cells. Furthermore, we demonstrated that JQ1 suppressed CDK6 expression at the mRNA level. Importantly, we determined that growth suppression and killing of CDK4/6i-resistant cells by BETi were largely the consequence of CDK6 downregulation by BET inhibition. We have also established CDK4/6i-resistant xenografts successfully from MCF7 cells to test the in vivo efficacy of BETi in reversing CDK4/6 inhibitor resistance. Conclusions: Our results suggest that BET inhibition has potent efficacy in reversing acquired CDK4/6i resistance of ER+ breast cancer, mainly through its inhibitory regulation of CDK6. BET inhibitors are therefore promising candidate therapeutic agents for the treatment of CDK4/6i-resistant ER+ breast cancer. Citation Format: Renyan Liu, Constantia Pantelidou, Heta Jadhav, Geoffrey Shapiro. Bromodomain inhibition reduces CDK6 expression and reverses CDK4/6 inhibitor resistance in ER+ breast 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 5649.

Abstract 1132: A systems biology approach combining ProLiFiler and Cancer Data Miner for an enhanced preclinical characterization of the WEE-1 inhibitor Adavosertib

Abstract Adavosertib (AZD1775, MK-1775), a clinical stage inhibitor of the tyrosine kinase WEE1, was investigated in a cell proliferation and survival assay with 140 human cancer cell lines (CLs) representing all major tumor types (ProLiFiler platform of Reaction Biology) followed by mechanism of action (MoA) and biomarker analyses using 4HF Biotec’s Cancer Data Miner in silico platform. Adavosertib exhibited a broad anti-cancer activity across all hematological and solid tumor types with IC50 values ranging from 0.06 to 10 µM (median: 0.38 µM), matching the consistently high expression of the WEE1 gene. Among 900 reference compounds, the activity profile of adavosertib correlated best with the profiles of compounds targeting the replication stress response including other WEE1 inhibitors but also inhibitors of checkpoint kinase 1 and 2 (CHK1/2) or ataxia telangiectasia-mutated (ATM). Significant correlations were also seen with compounds blocking mitosis, DNA replication and DNA repair. Interestingly, we observed a subset of cell lines that were resistant to both DNA synthesis and PARP inhibitors but were sensitive to WEE1 inhibition. Moreover, by using multiple datasets of WEE1 inhibitors connected to the molecular annotations of CLs for a data driven biomarker screening, we identified MYC mutations as a predictive marker of sensitivity and PIK3CA or ERBB2 gene amplifications as predictors of resistance. Transcriptome analysis identified up to 900 genes for which higher expression in CLs was associated with CL sensitivity to the compound. Preliminary pathway analysis indicated that these genes are well represented among nuclear factor and Myc-regulated genes. In conclusion, our studies demonstrate broad anticancer activity of adavosertib and confirm its proposed MoA. The biomarkers we identified will facilitate the selection of pre-clinical in vivo tumor models and, if confirmed, even patient selection for clinical trials. The combined use of the ProLiFiler and Cancer Data Miner Platforms has the potential to accelerate and de-risk the development of anti-cancer agents. Citation Format: Vincent Vuaroqueaux, Daniel Feger, Anne-Lise Peille, Oliver Siedentopf, Sadhana Panzade, Sarah Ulrich, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Erik Ehlert. A systems biology approach combining ProLiFiler and Cancer Data Miner for an enhanced preclinical characterization of the WEE-1 inhibitor Adavosertib [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 1132.

Abstract 204: Notch2NL is a novel regulator of radiation sensitivity in non-small cell lung cancer and a positive regulator of MYC activity

Abstract Background: A gain-of-function genetic screen identified NOTCH2 N-terminal like (NOTCH2NL) as a candidate novel gene involved in radiation resistance in non-small cell lung cancer (NSCLC). NOTCH2NL is located on chromosome position 1q21.1 which was incorrectly aligned in previous builds of the reference human genome sequence. NOTCH2NL was derived from a segmental duplication event in NOTCH2 during the evolutionary divergence between humans and non-human primates, forming three Notch2NL proteins: Notch2NL-A, Notch2NL-B and Notch2NL-C. Consequently, the only species whose genomes contain NOTCH2NL are modern and ancient humans. Notch2NL-B is a secreted protein and has been shown to promote Notch signalling in cortical stem cell progenitors via inhibition of inhibitory cis-DLL1/NOTCH signalling. Methods: Stable overexpression of NOTCH2NLB and secreted Notch2NL-B protein were validated by RT-qPCR and Western Blotting. The effects of NOTCH2NLB overexpression; transfer of Notch2NL-B protein from conditioned media onto wild-type cell lines; and siRNA knockdown of NOTCH1-4 transmembrane receptors on Notch signalling were quantified by a Notch luciferase reporter assay, RT-qPCR of classical NOTCH target genes and cancer stem cell marker (CSC) and RNA-Seq. Clonogenic cell survival assays determined the impact of NOTCH2NLB overexpression on radiation response. Cell proliferation was measured using the IncuCyte Zoom system. shRNA stable cell lines to knockdown NOTCH2NL were generated to mitigate the NOTCH2NLB overexpression phenotype we observed. RNA-Seq data from the first 100 patients from the TRACERx lung cancer study was used to quantify hallmark gene expression signatures and NOTCH2NLB expression. Results: NOTCH2NLB overexpression upregulates CSL-dependent Notch reporter luciferase activity; confers a radiation-resistant phenotype and impacts proliferation rate in NSCLC cell lines. Furthermore, we demonstrate that NOTCH2NLB overexpression and secreted Notch2NL-B protein transfer increase expression of classical NOTCH target genes (c-MYC, HES1 and HEY1) and CSC marker, ALDH1A1. Analysis of RNA-Seq data from the Lung TRACERx cohort study shows that NOTCH2NL is commonly expressed in NSCLC. NOTCH2NL expression positively correlates with notch hallmark gene expression signature in squamous (R=0.59, p=5.6e-9) and non-squamous carcinoma (R=0.51, p=4.7e-10). Interestingly, NOTCH2NL expression also positively correlated with the myc hallmark gene expression signature in squamous cell carcinoma (R=0.45, p=2.4e-5). Conclusions: Notch2NL is a novel determinant of radiation therapy response and impacts canonical Notch signalling in lung cancer. It is commonly expressed in NSCLC and can act in a paracrine manner as a secreted protein. Notch2NL, through canonical notch signalling, can regulate MYC expression and increase activity in the MYC pathway. Citation Format: Simranpreet Kaur Summan, Barbara Chow, Rhona Millar, Su Kit Chew, Philip East, Robert Hynds, Carlos Martinez Ruiz, Eva Gronroos, Mariam Jamal-Hanjani, Kevin Litchfield, Nicholas McGranahan, Nnennaya Kanu, Charles Swanton, Crispin Thomas Hiley. Notch2NL is a novel regulator of radiation sensitivity in non-small cell lung cancer and a positive regulator of MYC activity [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 204.

Abstract 5685: Recognition of internal mRNA N7-methylguanosine by QKI shuttles transcripts into stress granules and modulates drug resistance

Abstract Introduction: Albeit that over 170 types of chemical modifications have been identified in RNAs, the biological functions of most of those decorations are still elusive. N7-methylguanosine (m7G), routinely occurring at the 5’ cap of mRNA or within tRNA and rRNA, also exists in internal mRNA. However, the “reader” proteins that recognize internal mRNA m7G and regulate the metabolism and fate of target mRNAs have yet to be identified. Here, we aim to identify the reader(s) of internal mRNA m7G and elucidate the biological function. Methods: To identify internal m7G reader(s), we utilized RNA-pull down, mass spectrometry (MS), RNA immunoprecipitation (RIP)-seq, and m7G-seq. To evaluate the interaction between QKI and G3BP1, we used Co-IP, immunofluorescence (IF) and proximity ligation assay (PLA). To investigate the function of QKI in stress granule (SG), we performed polyA & m7G RNA fluorescence in situ hybridization (FISH), triple quadrupole MS (QQQ-MS), and Ribo-seq. To assess the role of QKI in drug resistance, we carried out both in vitro cell survival assays and in vivo xenograft mouse model. Results: Our QQQ-MS results suggest cytoplasmic mRNA, in contrast to nuclei mRNA, are highly enriched with internal m7G modification. Quaking proteins (QKI), especially QKI7, can selectively recognize the internal mRNA m7G decoration in the cytosol of various cell types. Our high-throughput sequence data revealed over 1000 confident m7G-modified and QKI-binding RNA targets with a conserved motif, “GANGAN (N=A/U/G)”. Those transcripts are functionally enriched in the “pathways in cancer” and “Hippo signaling pathway”. More strikingly, internal m7G reader QKI7 directly interacts with the SG core protein G3BP1 and can shuttle a subset of m7G-modified transcripts into SG mRNA pool under oxidative stress condition. Additionally, we identified 314 confident m7G-modified & QKI7-binding & SG-enriched target genes in U2OS cells. Ribo-seq indicated that QKI7 modulates the translation efficiency of a set of m7G-modified transcripts via sequestering and silencing them in SGs. Moreover, in line with the observation that many chemotherapy drugs could trigger the assembly of SGs, QKI7 increases the sensitivity of cancer cells to drug treatment (e.g., doxorubicin) in vitro and in vivo in a m7G-dependent manner. Consistently, by analyzing the hepatocellular carcinoma (HCC) pharmacogenomic landscape study dataset, we found that HCC cell lines with higher QKI expression levels are more sensitive to a variety of chemotherapy or targeted therapeutic drugs. Conclusion: Our study identifies QKI as the first internal mRNA m7G reader, provides insights into the biological function of internal mRNA m7G modification in remodeling stress granules transcriptome, and highlights the therapeutic potential of targeting the QKI7/m7G axis in overcoming drug resistance. Citation Format: Zhicong Zhao, Lei Dong, Li Han, Yangchan Li, Jianhuang Xue, Keren Zhou, Wei Li, Xiaolan Deng, Ying Qing, Brandon Tan, Zhenhua Chen, Chao Shen, Andrew Small, Kitty Wang, Keith Leung, Zheng Zhang, Xi Qin, Qiang Xia, Rui Su, Jianjun Chen. Recognition of internal mRNA N7-methylguanosine by QKI shuttles transcripts into stress granules and modulates drug resistance [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 5685.

Exploring Performance Parameters of Artificial Allosteric Protein Switches

Biological information processing networks rely on allosteric protein switches that dynamically interconvert biological signals. Construction of their artificial analogues is a central goal of synthetic biology and bioengineering. Receptor domain insertion is one of the leading methods for constructing chimeric protein switches. Here we present an in vitro expression-based platform for the analysis of chimeric protein libraries for which traditional cell survival or cytometric high throughput assays are not applicable. We utilise this platform to screen a focused library of chimeras between PQQ-glucose dehydrogenase and calmodulin. Using this approach, we identified 50 chimeras (approximately 23% of the library) that were activated by calmodulin-binding peptides. We analysed performance parameters of the active chimeras and demonstrated that their dynamic range and response times are anticorrelated, pointing to the existence of an inherent thermodynamic trade-off. We show that the structure of the ligand peptide affects both the response and activation kinetics of the biosensors suggesting that the structure of a ligand:receptor complex can influence the chimera’s activation pathway. In order to understand the extent of structural changes in the reporter protein induced by the receptor domains, we have analysed one of the chimeric molecules by CD spectroscopy and hydrogen–deuterium exchange mass spectrometry. We concluded that subtle ligand-induced changes in the receptor domain propagated into the GDH domain and affected residues important for substrate and cofactor binding. Finally, we used one of the identified chimeras to construct a two-component rapamycin biosensor and demonstrated that core switch optimisation translated into improved biosensor performance.

S-allylmercapto-N-acetylcysteine protects bone cells from oxidation and improves femur microarchitecture in healthy and diabetic mice.

Oxidative stress is involved in the deterioration of bone quality and mechanical strength in both diabetic and aging adults. Therefore, we studied the ability of the antioxidant compound, S-allylmercapto-N-acetylcysteine (ASSNAC) to protect bone marrow stromal cells (BMSCs) from advanced glycation end-products (AGEs) cytotoxicity and improve bone microarchitecture of adult healthy and obese/diabetic (db/db) female mice. ASSNAC effect on AGEs-treated cultured rat BMSCs was evaluated by Neutral Red and XTT cell survival and reactive oxygen species (ROS) level assays. Its effect on healthy (C57BL/6) and obese/diabetic (C57BLKS/J Leprdb+/+; db/db) female mice femur parameters, such as (1) number of adherent BMSCs, (2) percentage of CD73+/CD45- cells in bone marrow (BM), (3) glutathione level in BM cells, and (4) femur microarchitecture parameters by microcomputed tomography, was studied. ASSNAC treatment protected BMSCs by significantly decreasing AGEs-induced ROS production and increasing their cellular resistance to the cytotoxic effect of AGEs. ASSNAC treatment of healthy female mice (50 mg/kg/day; i.p.; age 12-20 weeks) significantly increased the number of BMSCs (+60%), CD73+/CD45- cells (+134%), and glutathione level (+110%) in the femur bone marrow. Furthermore, it increased the femur length (+3%), cortical diameter (+3%), and cortical areal moment of inertia (Ct.MOI; +10%) a surrogate for biomechanical strength. In db/db mice that demonstrated a compromised trabecular bone and growth plate microarchitecture, ASSNAC treatment restored the trabecular number (Tb.N, +29%), bone volume fraction (Tb.BV/TV, +130%), and growth plate primary spongiosa volumetric bone mineral density (PS-vBMD, +7%) and thickness (PS-Th, +18%). In conclusion, this study demonstrates that ASSNAC protects bone marrow cells from oxidative stress and may improve bone microarchitecture in adult healthy and diabetic female mice.

Effects of Prebiotic Fructooligosaccharides on Cytokine and Tight Junction Protein Expression in Cultured Intestinal Epithelial Cells

Prebiotic molecules such as fructooligosaccharides (FOS) can promote the growth and metabolism of beneficial bacteria in the host intestine. There are several published studies suggesting that prebiotic supplementation can alleviate symptoms of colitis in animal models. In addition, a recent study showed evidence for direct effects of FOS on tight junction protein expression in cultured intestinal epithelial cells. This study aimed to assess effects of FOS on pro‐inflammatory cytokine and tight junction protein expression in CMT‐93 and Caco‐2 colonic epithelial cells. Cells were treated with 0.1 mg/ml, 1 mg/ml or 10 mg/ml FOS and expression of cyclooxygenase‐2 (COX‐2), pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐6, and tight junction proteins claudin and ZO‐1 were evaluated by real‐time qPCR. The number of viable cells was analyzed after overnight exposure to FOS using a cell survival assay. Expression of ZO‐1 and claudin and localization of nuclear factor kappa‐B (NFkB) were evaluated by immunofluorescent staining. Surprisingly, FOS‐treated CMT‐93 cells showed a slight reduction in claudin mRNA expression compared to untreated cells. ZO‐1 and COX‐2 mRNA expression were not altered by FOS treatment compared to untreated controls. Immunofluorescent staining showed no translocation of NFkB to the nucleus after 2 hours of exposure to FOS. In conclusion, prebiotic fructooligosaccharides had minimal to no effects on the mRNA expression of claudin, ZO‐1, and COX‐2. In addition, FOS did not activate the NFkB pathway at the time points studied. These data suggest that FOS may not cause inflammatory responses in intestinal epithelial cells.

A Novel Curcumin Isomer, cis‐transCurcumin, Activates Adenosine Receptor Subtypes A1 And A3: Potential for Pain Therapy

A wide variety of plants produce compounds that contain the 4‐hydroxy‐3‐methoxybenzyl or “vanillyl” group. These vanilloid compounds include such well‐known food molecules as vanillin, capsaicin, gingerol, and curcumin. Vanilloid compounds have been widely studied for their medicinal properties, and one property that most of them have in common is the ability to induce analgesia. Studies have indicated multiple potential cellular mechanisms for this analgesic activity, including TRPV1 receptors, opioid receptors, and adenosine receptors (ARs). There are only a few studies on the interaction of vanilloid compounds with ARs, and to our knowledge no previous study has measured the binding affinity of any vanilloid compound with either of the two AR subtypes that are linked to the inhibitory Gαsubunit (subtypes A1and A3). Our study objective was to assess the binding affinity and receptor activation of a novel curcumin isomer, cis‐transcurcumin (CTCUR), at AR subtypes A1and A3. Since agonism of these subtypes is associated with analgesia, we hypothesized that CTCUR would be an agonist at AR subtypes A1 and A3. Two lines of Chinese hamster ovary cells were used, each transfected to overexpress one of the AR subtypes. Cell survival assays were performed to assess toxicity. Competitive binding assays and confocal microscopy were performed to measure binding affinity. Computer docking was also performed, to assess location of binding. Assays of intracellular cAMP levels were performed to measure receptor activation. Survival results indicate that CTCUR is not toxic to CHO cells at any of the concentrations tested (the highest being 100 μM). Competition assay results indicate that CTCUR binds to subtype A1 with a Ki of 306 nM and to subtype A3 with a Ki of 400 nM. Microscopy results confirm sub‐micromolar affinity for A1, but not for A3. Docking results confirm sub‐micromolar affinity for both subtypes, and furthermore indicate that CTCUR binds to the “toggle switch” domain of α helix 6 that is important for receptor activation. Intracellular cAMP results indicate that CTCUR acts as an agonist of ARs. Thus, we fail to reject our hypothesis. These results are of interest because they provide the first in vitro confirmation of the hypothesis that vanilloid compounds can activate ARs, in accordance with the results of previous studies in vivo. Vanilloid compounds that activate ARs are therefore worthy of further study, as a potential class of analgesic therapeutics.

Transcriptome Profiling Analysis Identifies LCP1 as a Contributor for Chidamide Resistance in Gastric Cancer.

Gastric cancer (GC) remains a significant health problem and carries with it substantial morbidity and mortality. Chidamide is a novel and orally administered histone deacetylase (HDAC) inhibitor and has been demonstrated its anti-tumor efficacy on different kinds of hematological and solid tumors. However, the underlying mechanism of chidamide resistance is still poorly characterized. We established chidamide resistant GC cell lines, AGS ChiR and MGC803 ChiR and investigated the toxicologic effects through cell survival, colony formation and flow cytometry assays in vitro, and a subcutaneous xenograft model in vivo. RNA-sequence was then performed to screen chidamide resistance-associated genes between AGS and AGS ChiR cells. The role of Lymphocyte cytosolic protein 1 (LCP1) in chidamide resistance was explored by gain- and loss-of-function analyses. We found that chidamide significantly inhibited cell proliferation and induced the apoptosis in a concentration-dependent manner in wild-type GC cell lines as compared to chidamide resistant cell lines. The transcriptomic profiling, quantitative RT-PCR, and western blot data revealed that LCP1 was upregulated in AGS ChiR cells compared with parental cells. Overexpression of LCP1 conferred and knockdown of LCP1 attenuated the chidamide resistance of GC cells. Epigenetic derepression of LCP1 by chidamide may be a possible reason for the contribution of LCP1 to chidamide resistance. These findings illustrated that LCP1 may play a chidamide resistance role in GC, suggesting that LCP1 could be a potential target for the therapy of GC combined with chidamide.

A New Generation of Ultrasmall Nanoparticles Inducing Sensitization to Irradiation and Copper Depletion to Overcome Radioresistant and Invasive Cancers

An emerging target to overcome cancer resistance to treatments is copper, which is upregulated in a wide variety of tumors and may be associated with cancer progression and metastases. The aim of this study was to develop a multimodal ultrasmall nanoparticle, CuPRiX, based on the clinical AGuIX nanoparticle made of the polysiloxane matrix on which gadolinium chelates are grafted. Such hybrid nanoparticles allow: (i) a localized depletion of copper in tumors to prevent tumor cell dissemination and metastasis formation and (ii) an increased sensitivity of the tumor to radiotherapy (RT) due to the presence of high Z gadolinium (Gd) atoms. CuPRiX nanoparticles are obtained by controlled acidification of AGuIX nanoparticles. They were evaluated in vitro on two cancer cell lines (lung and head and neck) using the scratch-wound assay and clonogenic cell survival assay. They were able to reduce cell migration and invasion and displayed radiosensitizing properties.

A Boronated Derivative of Temozolomide Showing Enhanced Efficacy in Boron Neutron Capture Therapy of Glioblastoma.

There is an incontestable need for improved treatment modality for glioblastoma due to its extraordinary resistance to traditional chemoradiation therapy. Boron neutron capture therapy (BNCT) may play a role in the future. We designed and synthesized a 10B-boronated derivative of temozolomide, TMZB. BNCT was carried out with a total neutron radiation fluence of 2.4 ± 0.3 × 1011 n/cm2. The effects of TMZB in BNCT were measured with a clonogenic cell survival assay in vitro and PET/CT imaging in vivo. Then, 10B-boronated phenylalanine (BPA) was tested in parallel with TMZB for comparison. The IC50 of TMZB for the cytotoxicity of clonogenic cells in HS683 was 0.208 mM, which is comparable to the IC50 of temozolomide at 0.213 mM. In BNCT treatment, 0.243 mM TMZB caused 91.2% ± 6.4% of clonogenic cell death, while 0.239 mM BPA eliminated 63.7% ± 6.3% of clonogenic cells. TMZB had a tumor-to-normal brain ratio of 2.9 ± 1.1 and a tumor-to-blood ratio of 3.8 ± 0.2 in a mouse glioblastoma model. BNCT with TMZB in this model caused 58.2% tumor shrinkage at 31 days after neutron irradiation, while the number for BPA was 35.2%. Therefore, by combining the effects of chemotherapy from temozolomide and radiotherapy with heavy charged particles from BNCT, TMZB-based BNCT exhibited promising potential for therapeutic applications in glioblastoma treatment.

Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA4 and LPA6 knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA2 knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2S)-OMPT (LPA3 agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA4 and LPA6 knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.

Radiosensitization Effect of Gold Nanoparticles on Plasmid DNA Damage Induced by Therapeutic MV X-rays.

Gold nanoparticles (AuNPs) can be used with megavolt (MV) X-rays to exert radiosensitization effects, as demonstrated in cell survival assays and mouse experiments. However, the detailed mechanisms are not clear; besides physical dose enhancement, several chemical and biological processes have been proposed. Reducing the AuNP concentration while achieving sufficient enhancement is necessary for the clinical application of AuNPs. Here, we used positively charged (+) AuNPs to determine the radiosensitization effects of AuNPs combined with MV X-rays on DNA damage in vitro. We examined the effect of low concentrations of AuNPs on DNA damage and reactive oxygen species (ROS) generation. DNA damage was promoted by 1.4 nm +AuNP with dose enhancement factors of 1.4 ± 0.2 for single-strand breaks and 1.2 ± 0.1 for double-strand breaks. +AuNPs combined with MV X-rays induced radiosensitization at the DNA level, indicating that the effects were physical and/or chemical. Although −AuNPs induced similar ROS levels, they did not cause considerable DNA damage. Thus, dose enhancement by low concentrations of +AuNPs may have occurred with the increase in the local +AuNP concentration around DNA or via DNA binding. +AuNPs showed stronger radiosensitization effects than −AuNPs. Combining +AuNPs with MV X-rays in radiation therapy may improve clinical outcomes.

Cellular Cytotoxicity and Oxidative Potential of Recurrent Molds of the Genus Aspergillus Series Versicolores.

Molds are ubiquitous biological pollutants in bioaerosols. Among these molds, the genus Aspergillus is found in the majority of indoor air samples, and includes several species with pathogenic and toxigenic properties. Aspergillus species in the series Versicolores remain little known despite recurrence in bioaerosols. In order to investigate their toxicity, we studied 22 isolates of clinical and environmental origin, corresponding to seven different species of the series Versicolores. Spore suspensions and ethyl acetate extracts prepared from fungal isolates were subjected to oxidative potential measurement using the dithiothreitol (DTT) test and cell survival measurement. The DTT tests showed that all species of the series Versicolores had an oxidative potential, either by their spores (especially for Aspergillus jensenii) or by the extracts (especially from Aspergillus amoenus). Measurements of cell survival of A549 and HaCaT cell lines showed that only the spore suspension containing 105 spores/mL of Aspergillus jensenii caused a significant decrease in survival after 72 h of exposure. The same tests performed with mixtures of 105 spores/mL showed a potentiation of the cytotoxic effect, with a significant decrease in cell survival for mixtures containing spores of two species (on A549 cells, p = 0.05 and HaCaT cells, p = 0.001) or three different species (on HaCaT cells, p = 0.05). Cell survival assays after 72 h of exposure to the fungal extracts showed that Aspergillus puulaauensis extract was the most cytotoxic (IC50 < 25 µg/mL), while Aspergillus fructus caused no significant decrease in cell survival.

SARS-CoV-2 spike protein inhibits growth of prostate cancer: a potential role of the COVID-19 vaccine killing two birds with one stone

To investigate the effects of isolated SARS-CoV-2 spike protein on prostate cancer cell survival. The effects of SARS-CoV-2 spike protein on LNCaP prostate cancer cell survival were assessed using clonogenic cell survival assay, quick cell proliferation assay, and caspase-3 activity kits. RT-PCR and immunohistochemistry were performed to investigate underlying molecular mechanisms. SARS-CoV-2 spike protein was found to inhibit prostate cancer cell proliferation as well as promote apoptosis. Further investigation revealed that anti-proliferative effects were associated with downregulation of the pro-proliferative molecule cyclin-dependent kinase 4 (CDK4). The increased rate of apoptosis was associated with the upregulation of pro-apoptotic molecule Fas ligand (FasL). SARS-CoV-2 spike protein inhibits the growth of LNCaP prostate cancer cells in vitro by a two-pronged approach of downregulating the expression of CDK4 and upregulating FasL. The introduction of SARS-CoV-2 spike protein into the body via COVID-19 vaccination may have the potential to inhibit prostate cancer in patients. This potential beneficial association between COVID-19 vaccines and prostate cancer inhibition will require more extensive studies before any conclusions can be drawn about any in vivo effects in a human model.

Big dynorphin is a neuroprotector scaffold against amyloid β-peptide aggregation and cell toxicity

Amyloid β-peptide (Aβ) misfolding into β-sheet structures triggers neurotoxicity inducing Alzheimer’s disease (AD). Molecules able to reduce or to impair Aβ aggregation are highly relevant as possible AD treatments since they should protect against Aβ neurotoxicity. We have studied the effects of the interaction of dynorphins, a family of opioid neuropeptides, with Aβ40 the most abundant species of Aβ. Biophysical measurements indicate that Aβ40 interacts with Big Dynorphin (BigDyn), lowering the amount of hydrophobic aggregates, and slowing down the aggregation kinetics. As expected, we found that BigDyn protects against Aβ40 aggregates when studied in human neuroblastoma cells by cell survival assays. The cross-interaction between BigDyn and Aβ40 provides insight into the mechanism of amyloid pathophysiology and may open up new therapy possibilities.

Recycled Pequi (Caryocar brasiliense, Camb.) Shell Ethanolic Extract Induces Apoptosis in Canine Osteosarcoma Cells

Abstract Osteosarcoma is a mesenchymal cancer associated with high mortality in dogs and in humans. The biodiversity-rich Cerrado, the predominant biome in the Midwest region of Brazil, is native to locally appreciated fruits such as pequi (Caryocar brasiliense, Camb). Although this plant has been frequently used in folk medicine, the pharmacological properties of pequi fruit shells have not been fully evaluated. Thus, this study aimed to determine the cytotoxic activity of ethanol extract of recycled pequi shells on canine osteosarcoma cells in vitro. Cells were cultured and treated with final extract concentrations of 0, 0.029 µg/µL, 0.29 µg/µL, and 2.91 µg/µL for 24, 48, or 72 hours. Cell viability assay using trypan blue exclusion method and tetrazolium reduction method, cell survival assay, and double labeling with annexin V and propidium iodide were performed in the treated osteosarcoma cells. These allowed the determination of IC50, survival fraction, and type of cell death, respectively. Pequi shell ethanol extract at a concentration of 2.91 µg/µL showed the greatest inhibition of osteosarcoma cell growth in vitro, resulting in a 71.80% decrease in growth compared to the control. The mean IC50 was 155.2 μg/mL at 72 hours. The calculated survival fractions showed that cell growth at 72 hours was 3.33% lower in cells treated with 2.91 µg/µL extract. Results from the double labeling experiment suggest that apoptosis was the predominant type of cell death in cells treated with 2.91 µg/µL extract. These results demonstrate that ethanol extract of recycled pequi shells promotes apoptosis in canine osteosarcoma cells.

PI3 kinase isoform p110δ is more important than p110α in KIT signaling in hematopoietic cells

PI3 kinases are important for KIT signaling and KIT mutants mediated cell transformation. In order to know the difference of PI3 kinase isoforms p110α and p110δ in the signaling of wild-type KIT and the often occurred KIT mutation D816V in hematopoietic malignancy mastocytosis, the predominant PI3 kinase isoform p110δ in hematopoietic tissues was knocked out in hematopoietic cells. We found that loss of p110δ expression dramatically inhibits PI3 kinase activation mediated by both wild-type KIT and KIT/D816V. By over expression of p110α in p110δ knock out cells, wild-type KIT mediated PI3 kinase activation was not changed while over expression of p110δ increased PI3 kinase activation. Similarly, in KIT/D816V expressing cells without p110δ expression, over expression of p110δ but not p110α restored PI3 kinase activation. In agreement with the signaling results, cell proliferation, cell survival and cell cycle assay further showed that over expression of p110δ but not p110α in p110δ knock out cells increases both wild-type KIT and KIT/ D816V mediated cell survival and proliferation. These results suggested that p110δ plays a more important role than p110α in KIT signaling and KIT mutant mediated cell transformation in hematopoietic cells.

RTKN2 Enhances Radioresistance in Gastric Cancer through Regulating the Wnt/β-Catenin Signalling Pathway

Adjuvant therapy and radiotherapy improves the survival of patients with metastatic and locally advanced gastric cancer (GC). However, the resistance to radiotherapy limits its clinical usage. Rhotekin 2 (RTKN2) functions as an oncogene and confers resistance to ultraviolet B-radiation and apoptosis-inducing agents. Here, the role of RTKN2 in radiosensitivity of GC cell lines was investigated. RTKN2 was found to be elevated in GC tissues and cells. A series of functional assays revealed that over-expression of RTKN2 induced GC cell proliferation, promoted GC cell migration and invasion, while inhi­biting GC cell apoptosis. However, silence of RTKN2 promoted GC cell apoptosis, while repressing GC cell proliferation, invasion and migration. GC cells were exposed to irradiation, and data from cell survival and apoptotic assays showed that knock-down of RTKN2 enhanced radiosensitivity of GC through up-regulation of apoptosis and down-regulation of proliferation in irradiation-exposed GC cells. More­over, the protein expression of β-catenin and c-Myc in GC cells was enhanced by RTKN2 over-expression, but reduced by RTKN2 silence. Interference of RTKN2 down-regulated nuclear β-catenin expression, while up-regulating cytoplasmic β-catenin in GC. In conclusion, RTKN2 contributed to cell growth and radioresistance in GC through activation of Wnt/β-catenin signalling.