Mean IC50 Values Research Articles

Combination treatment effect of an immunotherapeutic agent with microtubule inhibitors.

e14619 Background: The microtubule (MT) is a polymer formed by self-assembly of αβ-tubulin heterodimers. MT inhibitors either stabilize or destabilize microtubule assembly and halt cell proliferation during the mitotic phase of the cell cycle. However, due to non-specificity, high resistance, and adverse reactions observed with MT-targeting drugs, various approaches to novel combination chemotherapies are being used in the clinic. The specific goal of this study is to investigate the ability of an immunotherapeutic agent to alter the activity of MT-targeting chemotherapeutics when used in a combination treatment approach. Methods: The combination treatment effect of the immunotherapeutic agent with microtubule inhibitors was investigated using two methods: 1) Tubulin polymerization studies using purified tubulin and microtubule inhibitors, paclitaxel (a microtubule stabilizing agent) and dolastatin 10 (a microtubule destabilizing agent), studied alone or in combination with the immunotherapeutic agent. (2) Co-culture system expressing the stably expressing Nuclear Factor of Activated T cells (NFAT)- Renilla luciferase (NFAT- Rluc) reporter gene to study anti-tumor immune response of MT inhibitors alone or in combination with the immunotherapeutic agent. Results: Spectrophotometric-based tubulin polymerization studies demonstrated that the immunotherapeutic agent (0.01 - 0.1 mg/mL) altered tubulin polymerization when studied in combination with either paclitaxel (10 µM) or dolastatin 10 (0.5 mM). Luminescence-based reporter gene expression studies in a co-culture system, consisting of CD3− antigen presenting Chinese hamster ovary and CD3+ effector Jurkat T cells, demonstrated the combination treatment effectively induced NFAT- Rluc activity when compared to the controls, the immunotherapeutic agent alone or to the MT-inhibitor alone. Statistical analysis was done using one-way analysis of variance (ANOVA). A significant difference ( p < 0.05) in mean IC50 values (expressed as ng/mL ± SEM, N=4) was observed between the immunotherapeutic plus dolastatin 10 versus the immunotherapeutic agent alone, and a 6.5-fold decrease (85% reduction) in the IC50 value was observed for induction of NFAT- Rluc activity in the presence of immunotherapeutic plus dolastatin 10 versus the immunotherapeutic alone. There was a notable decrease (1.9-fold or 46% reduction) in the IC50 value observed for induction of NFAT- Rluc activity in the presence of the immunotherapeutic plus paclitaxel versus the immunotherapeutic alone. Conclusions: These results suggest a boost in T-cell stimulation by the immunotherapeutic agent in the presence of MT inhibitors. The implications of enhanced T-cell activation on antitumor immunity and modulation of tubulin polymerization by a combination treatment of the immunotherapeutic agent and microtubule inhibitors are currently under investigation.

Abstract 5957: Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer

Abstract Introduction Transcription regulators, such as cyclin-dependent kinase 9 (CDK9), have been implicated in the super-enhancer driven transcriptional control of oncogenes in multiple cancers including pancreatic cancer. CDK9 has been shown to be employed by cancer cells for the constant production of short-lived oncoproteins such as c-Myc to maintain their survival. Due to its critical role in regulating transcription in cancer cells, CDK9 has emerged as a potential therapeutic target. Here, we report the antitumor activity of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, in preclinical models for pancreatic cancer. Experimental Procedures Fragment-based MolecuLern driven AI workflow methods were used to design a series of ATP-competitive CDK9 specific kinase inhibitors. BLX-3030 was selected as one of the most potent leads with an IC50 of 1.1 nM in a cell-free CDK9 kinase assay. A Sulforhodamine B (SRB) assay was used to evaluate the cell growth inhibitory activity of BLX-3030 in a panel of 10 pancreatic ductal adenocarcinoma (PDAC) and 2 pancreatic neuroendocrine tumor (pNET) cell lines. Western blotting was used to determine the effects of BLX-3030 treatment on c-Myc expression in cancer cells. The combination effects between BLX-3030 and standard of care regimen, gemcitabine (GEM) and nab-paclitaxel (NP), was assessed using the SRB assay. Finally, the in vivo antitumor activity of BLX-3030 alone or in combination with GEM and NP was evaluated in a mouse cell line xenograft model for pancreatic cancer. Results BLX-3030 exhibited potent cell growth inhibitory activity in the panel of pancreatic cancer cell lines with IC50 values ranging from 133.5 nM to 508.1 nM. The potency of BLX-3030 in the cell lines appears to correlate with the expression level of c-Myc in the cell lines. BLX-3030 showed similar activities in PDAC and pNET cell lines with mean IC50 values of 244.5 nM and 194.6 nM, respectively. However, treatment with BLX-3030 for 48 hours did not seem to significantly reduce c-Myc expression in PSN-1 PDAC cells. When combined with GEM and NP, BLX-3030 demonstrated mostly additive effects in PSN-1 cells. Preliminary data from the in vivo study show that BLX-3030 improves the antitumor activity of GEM and NP. Conclusion In summary, BLX-3030 potently inhibited the growth of both PDAC and pNET cells in vitro. The growth inhibitory activity of BLX-3030 in PDAC cell lines correlates with their c-Myc expression levels. BLX-3030 showed an additive effect when combined with standard of care drugs (GEM and NP) in PDAC cell lines. Overall, the novel CDK9 inhibitor, BLX-3030, demonstrated potent activity in pancreatic cancer cell line models and presents a promising preclinical lead for pancreatic cancer. (This work was supported in part by Biolexis Therapeutics and the Seena Magowitz Foundation). Citation Format: Yesenia Barrera-Millan, Zhaoliang Li, Hariprasad Vankayalapati, David J. Bearss, Daniel D. Von Hoff, Haiyong Han. Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5957.

Antioxidant radical scavenging capacity and total carotenoid content of narrow-clawed crayfish (Pontastacus leptodactylus, Eschscholtz, 1823) in Atikhisar Reservoir (Çanakkale, Türkiye)

In this study, antioxidant radical scavenging capacity and total carotenoid content in the meat and shells of Pontastacus leptodactylus were investigated. Concerning the antioxidant scavenging effect, the highest IC50 values were found to be 388.77 mg g-1 and 155.53 mg g-1 for females and males in July and March, respectively. The mean IC50 values of the meat were calculated as 239.83 mg g-1 and 105.21 mg g-1 for females and males, respectively. The mean total carotenoid content in the meat was found to be 14.35 and 12.78 µg g-1 for females and males, respectively. The results indicated that crayfish meat had antioxidant radical scavenging capacity and was rich in carotenoid content.

Apoptosis and oxidative stress of mouse breast carcinoma 4T1 and human intestinal epithelial Caco-2 cell lines caused by the phycotoxin gymnodimine-A

Gymnodimine-A (GYM-A) is a cyclic imine phycotoxin produced by some marine dinoflagellates. It can cause rapid death of mice via intraperitoneal administration and frequently accumulate in shellfish potentially threatening human health. In this study, four different cell lines were exposed to GYM-A for the viability assessment. Results showed that GYM-A was cytotoxic with concentration-dependent pattern to each cell type, with mean IC50 values ranging from 1.39 to 2.79 μmol L−1. Results suggested that the loss of cell viability of 4T1 and Caco-2 cells was attributed to apoptosis. Furthermore, the collapse of mitochondrial membrane potential and caspases activation were observed in the GYM-A-treated cells. Reactive oxygen species (ROS) and lipid peroxides (LPO) levels were markedly increased in 4T1 and Caco-2 cells exposed to GYM-A at 2 μmol L−1, and the oxidative stress in 4T1 cells was more obvious than that in Caco-2 cells. Additionally, unusual ultrastructure impairment on mitochondria and mitophagosomes occurred in the GYM-A-treated cells. These results suggested that an ROS-mediated mitochondrial pathway for apoptosis and mitophagy was implicated in the cytotoxic effects induced by GYM-A. This is the first report to explore the cytotoxic mechanisms of GYM-A through apoptosis and oxidative stress, and it will provide theoretical foundations for the potential therapeutic applications of GYM-A.

Antiproliferative activity and apoptosis-inducing mechanism of Amaryllidaceae alkaloid montanine on A549 and MOLT-4 human cancer cells

The isoquinoline alkaloids found in Amaryllidaceae are attracting attention due to attributes that can be harnessed for the development of new drugs. The possible molecular mechanisms by which montanine exerts its inhibitory effects against cancer cells have not been documented. In the present study, montanine, manthine and a series of 15 semisynthetic montanine analogues originating from the parent alkaloid montanine were screened at a single test dose of 10 μM to explore their cytotoxic activities against a panel of eight cancer cell lines and one non-cancer cell line. Among montanine and its analogues, montanine and its derivatives 12 and 14 showed the highest cytostatic activity in the initial single-dose screening. However, the native montanine exhibited the greatest antiproliferative activity against cancer cells, with a lower mean IC50 value of 1.39 µM, compared to the displayed mean IC50 values of 2.08 µM for 12 and 3.57 µM for 14. Montanine exhibited the most potent antiproliferative activity with IC50 values of 1.04 µM and 1.09 µM against Jurkat and A549 cell lines, respectively. We also evaluated montanine’s cytotoxicity and cell death mechanisms. Our results revealed that montanine triggered apoptosis of MOLT-4 cells via caspase activation, mitochondrial depolarisation and Annexin V/PI double staining. The Western blot results of MOLT-4 cells showed that the protein levels of phosphorylated Chk1 Ser345 were upregulated with increased montanine concentrations. Our findings provide new insights into the mechanisms underlying the cytostatic, cytotoxic and pro-apoptotic activities of montanine alkaloids in lung adenocarcinoma A549 and leukemic MOLT-4 cancer cell types.

Ex vivo and In vitro antiplasmodial activities of approved drugs predicted to have antimalarial activities using chemogenomics and drug repositioning approach

High malaria mortality coupled with increased emergence of resistant multi-drug resistant strains of Plasmodium parasite, warrants the development of new and effective antimalarial drugs. However, drug design and discovery are costly and time-consuming with many active antimalarial compounds failing to get approved due to safety reasons. To address these challenges, the current study aimed at testing the antiplasmodial activities of approved drugs that were predicted using a target-similarity approach. This approach is based on the fact that if an approved drug used to treat another disease targets a protein similar to Plasmodium falciparum protein, then the drug will have a comparable effect on P. falciparum. In a previous study, in vitro antiplasmodial activities of 10 approved drugs was reported of the total 28 approved drugs. In this study, six out of 18 drugs that were previously not tested, namely epirubicin, irinotecan, venlafaxine, palbociclib, pelitinib, and PD153035 were tested for antiplasmodial activity. The drug susceptibility in vitro assays against five P. falciparum reference strains (D6, 3D7, W2, DD2, and F32 ART) and ex vivo assays against fresh clinical isolates were done using the malaria SYBR Green I assay. Standard antimalarial drugs were included as controls. Epirubicin and irinotecan showed excellent antiplasmodial ex vivo activity against field isolates with mean IC50 values of 0.044 ± 0.033 μM and 0.085 ± 0.055 μM, respectively. Similar activity was observed against W2 strain where epirubicin had an IC50 value of 0.004 ± 0.0009 μM, palbociclib 0.056 ± 0.006 μM, and pelinitib 0.057 ± 0.013 μM. For the DD2 strain, epirubicin, irinotecan and PD 153035 displayed potent antiplasmodial activity (IC50 < 1 μM). Epirubicin and irinotecan showed potent antiplasmodial activities (IC50 < 1 μM) against DD2, D6, 3D7, and F32 ART strains and field isolates. This shows the potential use of these drugs as antimalarials. All the tested drugs showed antiplasmodial activities with IC50 values below 20 μM, which suggests that our target similarity-based strategy is successful at predicting antiplasmodial activity of compounds thereby circumventing challenges in antimalarial drug discovery.

Anticancer potential of Carica papaya Linn black seed extract against human colon cancer cell line: in vitro study

BackgroundSince cancer is one of the most prevalent diseases in the world, further studies are needed to identify the effective therapeutic modalities. The second deadliest and third most common cancer is colorectal cancer (CRC). Papaya (Carica papaya Linn) seeds offer anti-cancer properties that can cure various types of cancer, such as liver and prostate cancer.MethodsThe study aimed to evaluate the anti-cancer activity of Carica papaya seed extract on colorectal cancer cell lines (Caco-2) and used techniques to assess the anti-cancer potential. The effectiveness of SE on cell proliferation and the viability of HTB-37 Caco-2 and C-166 cells were assessed using the MTT test. Real-time RT-PCR was used to measure gene expression levels and evaluate the activity of genes involved in apoptosis, including caspase-3, p53, Cycs, and Bcl-2. Finally, flow cytometry was used to analyze apoptosis induction by detecting changes in cell morphology and DNA content.ResultsThe study showed that the MTT reduction assay was dependent on cancer cell type and concentration of SE compared to the control cells and C-166, with a mean IC50 value of 9.734 ug/ml. The cytotoxicity was accompanied by some morphological alterations in the colorectal cancer cell line (Caco-2). The expression of the genes for p53, Cycs, and caspase-3 was substantially up-regulated, while Bcl-2 was dramatically down-regulated compared to control cells. The cell cycle arrested at the G2-M phase and the presence of early and late apoptotic characteristics post-treatment increased the apoptotic profile.ConclusionIt concluded that papaya seeds aqueous extract could act as a novel therapeutic option for colorectal cancer (CRC).

Cellular Uptake of Catharanthus roseus-Silver Nanoparticles in Human Hepatocellular Carcinoma HepG2 Cells

Introduction: Nanoparticles exhibit unique features and currently at the forefront of cutting-edge research. Silver nanoparticles (AgNPs) are among the most promising and widely commercialised nanoproducts in various fields. The interaction of these AgNPs with cells remain unclear to connect with its toxicological endpoints. The aim of this study was to investigate the cellular uptake of C. roseus-AgNPs in hepatocellular carcinoma cells HepG2. Methods: The HepG2 cells were treated with the mean IC50 value of C. roseus-AgNPs which was 4.95±0.26 µg/mL for 24, 48 and 72 hours. The effects were compared with the untreated cells and other treatments which include camptothecin, C. roseus-aqueous extract, and AgNO3. Inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to quantify the intracellular Ag+ and Ca2+, while transmission electron microscopy (TEM) imaging was used to visualise the nanoparticle distribution. Results: The HepG2 cells have significantly taken up Ag+ from C. roseus-AgNPs with at least six times higher compared to Ag+ from AgNO3. The intracellular Ca2+ detected in HepG2 cells for all treatments were significantly higher than the untreated cells, in time-dependent manner. TEM images indicated the endocytosis of C. roseus-AgNPs with the presence of endosomes and exocytic vesicles. Conclusion: The significant accumulation of intracellular Ag+ demonstrated the efficiency of the C. roseus-AgNPs uptake while the increased Ca2+ indicated the early sign of cell injury. The cellular uptake was mainly through endocytosis. These findings are crucial to correlate the physicochemical properties of C. roseus-AgNPs with the anticancer mechanisms towards the development of liver cancer therapy.

Therapeutic potential of dual CDK12 and 13 inhibition in colorectal cancer.

e15601 Background: To date, studies on cyclin-dependent kinase inhibitors in colorectal cancer (CRC), such as targeting CDK4/6 with Palbociclib, have shown little to no clinical benefit. However, tumor profiling of CRC has shown that mutations and amplifications in CDK 12 and 13 are prevalent in 5 to 10% of CRC patients, suggesting that CDK12/13 inhibition may be a potential target for patients with CRC. Methods: To study the effects of inhibiting CDK12 and 13 in CRC, a total of 11 patient-derived organoids (PDO) were first established from patients undergoing biopsy or resection of their primary or metastatic CRC. In vitro drug sensitivity assays were performed by treating each PDO with THZ531, a selective dual CDK12/13 inhibitor; 5-flurouracil, oxaliplatin and SN38 as a standard of care; or Palbociclib (CDK 4/6 inhibitor) at seven different drug concentrations (0.0064 μM - 100 μM) with five replicates at each concentration. Viability was assessed 72 hours following the addition of each drug using CellTiter-Glo (Promega) and IC50s were calculated. Results: Targeting CDK12/13 with THZ531 demonstrated dose dependent activity in the majority of CRC PDOs, with IC50 values ranging from 0.06 μM to 4.2 μM. In comparison, targeting CDK4/6 with Palbociclib was ineffective, with IC50 values ranging from 2.5 μM to 17 μM (p = 0.0003, 95% CI 3.7 to 10.4). Inhibition of CDK12/13 was also more effective than standard of care chemotherapy with either 5- FU or oxaliplatin (mean IC50 values were 4.2 μM and 21.4 μM for 5-fluorouracil and oxaliplatin, p &lt; 0.05) and was comparable to irinotecan (mean IC50 was 0.07 μM for SN38). Finally, no significant correlation was found between sensitivity to THZ531 and microsatellite status, mutations in RAS and BRAF, or grade of the original patient tumor. Conclusions: We identify dual CDK12/13 inhibition as a promising therapy to treat CRC. Further studies will focus on characterizing mechanisms of action, evaluating efficacy and toxicity in vivo, and determining biomarkers of response based on actionable genomic alterations.

A Novel and Highly Selective Epidermal Growth Factor Receptor Inhibitor, SMUZ106, for the Treatment of Glioblastoma.

Targeting the epidermal growth factor receptor (EGFR) is one of the potential ways to treat glioblastoma (GBM). In this study, we investigate the anti-GBM tumor effects of the EGFR inhibitor SMUZ106 in both in vitro and in vivo conditions. The effects of SMUZ106 on the growth and proliferation of GBM cells were explored through MTT and clone formation experiments. Additionally, flow cytometry experiments were conducted to study the effects of SMUZ106 on the cell cycle and apoptosis of GBM cells. The inhibitory activity and selectivity of SMUZ106 to the EGFR protein were proved by Western blotting, molecular docking, and kinase spectrum screening methods. We also conducted a pharmacokinetic analysis of SMUZ106 hydrochloride following i.v. or p.o. administration to mice and assessed the acute toxicity level of SMUZ106 hydrochloride following p.o. administration to mice. Subcutaneous and orthotopic xenograft models of U87MG-EGFRvIII cells were established to assess the antitumor activity of SMUZ106 hydrochloride in vivo. SMUZ106 could inhibit the growth and proliferation of GBM cells, especially for the U87MG-EGFRvIII cells with a mean IC50 value of 4.36 μM. Western blotting analyses showed that compound SMUZ106 inhibits the level of EGFR phosphorylation in GBM cells. It was also shown that SMUZ106 targets EGFR and presents high selectivity. In vivo, the absolute bioavailability of SMUZ106 hydrochloride was 51.97%, and its LD50 exceeded 5000 mg/kg. SMUZ106 hydrochloride significantly inhibited GBM growth in vivo. Furthermore, SMUZ106 inhibited the activity of U87MG-resistant cells induced by temozolomide (TMZ) (IC50: 7.86 μM). These results suggest that SMUZ106 hydrochloride has the potential to be used as a treatment method for GBM as an EGFR inhibitor.

Synthesis, anticancer evaluation, and molecular docking study of some arylidenehydrazono analogues

A series of 2-(arylidenehydrazono)-5-(2-oxo-2-arylethyl)thiazolidin-4-one derivatives was synthesized, characterized by spectral analyses and evaluated for their in vitro antitumor activity. The IC50 determination of compounds was investigated on the human breast cancer cell line MCF-7. Among the series, compounds 3, 6, 10, 16, 17, and 24 showed remarkable anticancer activity with mean IC50 values 2.34, 0.73, 2.69, 3.40, 1.18, and 1.76 µg/mL, respectively, against MCF-7 cancer cells. Compound 16 enhanced the concentration of caspase-9, inhibited the concentration of Ki67 and showed a profound reduction in the amount of MMP9 secreted into the medium of MCF-7-treated cells. Furthermore, compound 16 revealed anti-angiogenic activity through downregulation of the concentration of VEGF in the medium of MCF-7-treated cells. Compound 16 exerted cytotoxic effects on MCF-7 tumor cells via antiproliferative, apoptotic, anti-angiogenic, and antimetastatic activities. Molecular docking methodology was performed for the most effective anticancer compounds to rationalize the possible interactions with active site of VEGFR-2 enzyme.

Abstract 6247: Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response

Abstract Background - SCLC is the most aggressive lung cancer type and with the worst prognosis. There are four molecular subtypes based on the high expression of distinct transcription factors and with different therapeutic vulnerabilities. However, all share transcriptional addiction as pathogenic mechanism. Lurbinectedin is a novel oncogenic transcription inhibitor, approved in the United States and other countries for the treatment of adult patients with metastatic SCLC with disease progression on or after platinum-based chemotherapy. The aim of this study was to analyze the activity of lurbinectedin in the different SCLC molecular subtypes and to investigate new biomarkers of response. Methods and Results - We have characterized a panel of 20 SCLC human cell lines based on the expression of ASCL1, NEUROD1, YAP1 and POU2F3, where lurbinectedin yielded a mean IC50 value of 6.52 nM, which is considerable greater than the activity exerted by topotecan, irinotecan, carboplatin, etoposide, olaparib, alisertib and navitoclax (IC50 100 µM-100 nM). Lurbinectedin potency is high in the four molecular subtypes, with IC50 values of 4.1, 14.9, 7.2, and 0.2 nM for SCLC-A, -N, -I and -P, respectively. However, high expression of POU2F3 correlated with better responses. In fact, mean IC50 was 0.28 nM for POU2F3high cells versus 12.1 nM for all POU2F3low cells (p=0.0443). Additionally, basal SLFN11 levels were evaluated, observing that SLFN11high cells responded better to lurbinectedin, with IC50 values of 1.1 nM versus 11.8 nM for SLFN11low (p=0.05). Likewise, lurbinectedin treatment induced greater antitumor activity in SLFN11high tumor-bearing mice (H526: T/C, 18% on day 11) than in SLFN11low tumor-bearing mice (H82: T/C, 65% on day 7) after intravenous treatment at 0.18 mg/kg (on days 0, 7 and 14). Finally, SLFN11 expression was evaluated by IHC in FFPE tumor samples from SCLC patients participating in a multicenter phase II clinical trial in advanced solid tumors (NCT02454972), which allowed lurbinectedin accelerated approval in this indication by FDA. Patients with higher expression of SLFN11 had a slightly better overall survival (OS at 6 months: 68.4% (&amp;lt;15%, N=20) vs. 98.7% (≥15%, N=20) p=0.0261)), especially in the refractory/resistant subgroup (OS at 6 months: 33.3% (&amp;lt;15%, N=9) vs. 100.0% (≥15%, N=6) p&amp;lt;0.0001). Conclusions - Lurbinectedin is highly effective in all molecular subtypes of SCLC in vitro and in vivo, with IC50 values at least two logs more potent than for other antitumoral agents and its activity is even greater in tumors with high POU2F3 expression and/or high SLFN11 expression. Citation Format: Marta Martínez Diez, Gema Santamaría Nuñez, María José Guillén, Daniel Rueda, Eva Maria Garrido-Martin, Pablo Avilés, Carmen Cuevas. Lurbinectedin shows potent activity in all four molecular subtypes of small cell lung cancer (SCLC) and POU2F3 and SLFN11 are biomarkers for a better response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6247.

MRNA transcriptome profiling of human hepatocellular carcinoma cells HepG2 treated with Catharanthus roseus-silver nanoparticles.

The demand for the development of cancer nanomedicine has increased due to its great therapeutic value that can overcome the limitations of conventional cancer therapy. However, the presence of various bioactive compounds in crude plant extracts used for the synthesis of silver nanoparticles (AgNPs) makes its precise mechanisms of action unclear. To assessed the mRNA transcriptome profiling of human HepG2 cells exposed to Catharanthus roseus G. Don (C. roseus)-AgNPs. The proliferative activity of hepatocellular carcinoma (HepG2) and normal human liver (THLE3) cells treated with C. roseusAgNPs were measured using MTT assay. The RNA samples were extracted and sequenced using BGIseq500 platform. This is followed by data filtering, mapping, gene expression analysis, differentially expression genes analysis, Gene Ontology analysis, and pathway analysis. The mean IC50 values of C. roseusAgNPs on HepG2 was 4.38 ± 1.59 μg/mL while on THLE3 cells was 800 ± 1.55 μg/mL. Transcriptome profiling revealed an alteration of 296 genes. C. roseusAgNPs induced the expression of stress-associated genes such as MT, HSP and HMOX-1. Cellular signalling pathways were potentially activated through MAPK, TNF and TGF pathways that are responsible for apoptosis and cell cycle arrest. The alteration of ARF6, EHD2, FGFR3, RhoA, EEA1, VPS28, VPS25, and TSG101 indicated the uptake of C. roseus-AgNPs via both clathrin-dependent and clathrin-independent endocytosis. This study provides new insights into gene expression study of biosynthesised AgNPs on cancer cells. The cytotoxicity effect is mediated by the aberrant gene alteration, and more interestingly the unique selective antiproliferative properties indicate the C. roseusAgNPs as an ideal anticancer candidate.

Synthesis of 3-(2-Alkylthio-4-chloro-5-methylbenzenesulfonyl)-2-(1-phenyl-3-arylprop-2-enylideneamino)guanidine Derivatives with Pro-Apoptotic Activity against Cancer Cells.

The untypical course of reaction between chalcones and benzenesulfonylaminoguanidines led to the new 3-(2-alkylthio-4-chloro-5-methylbenzenesulfonyl)-2-(1-phenyl-3-arylprop-2-enylideneamino)guanidine derivatives 8-33. The new compounds were tested in vitro for their impact on the growth of breast cancer cells MCF-7, cervical cancer cells HeLa and colon cancer cells HCT-116 by MTT assay. The results revealed that the activity of derivatives is strongly related to the presence of hydroxy group in the benzene ring at the 3-arylpropylidene fragment. The most cytotoxic compounds 20 and 24 displayed mean IC50 values of 12.8 and 12.7 μM, respectively, against three tested cell lines and were almost 3- and 4-fold more active toward MCF-7 and HCT-116 when compared with non-malignant HaCaT cells. Furthermore, compound 24 induced apoptosis in cancer cells and caused a decrease of mitochondrial membrane potential as well as an increase of cells in sub-G1 phase in contrast to its inactive analog 31. The strongest activity against the most sensitive HCT-116 cell line was found for compound 30 (IC50 = 8 μM), which was 11-fold more effective in the growth inhibition of HCT-116 cells than those of HaCaT cells. Based on this fact, the new derivatives may be promising leading structures for the search for agents for the treatment of colon cancer.

Mechanistic in vitro studies indicate that the clinical drug-drug interactions between protease inhibitors and rosuvastatin are driven by inhibition of intestinal BCRP and hepatic OATP1B1 with minimal contribution from OATP1B3, NTCP and OAT3.

Previous use of a mechanistic static model to accurately quantify the increased rosuvastatin exposure due to drug-drug interaction (DDI) with coadministered atazanavir underpredicted the magnitude of area under the plasma concentration-time curve ratio (AUCR) based on inhibition of breast cancer resistance protein (BCRP) and organic anion transporting polypeptide (OATP) 1B1. To reconcile the disconnect between predicted and clinical AUCR, atazanavir and other protease inhibitors (darunavir, lopinavir and ritonavir) were evaluated as inhibitors of BCRP, OATP1B1, OATP1B3, sodium taurocholate cotransporting polypeptide (NTCP) and organic anion transporter (OAT) 3. None of the drugs inhibited OAT3, nor did darunavir and ritonavir inhibit OATP1B3 or NTCP. All drugs inhibited BCRP-mediated estrone 3-sulfate transport or OATP1B1-mediated estradiol 17β-D-glucuronide transport with the same rank order of inhibitory potency (lopinavir>ritonavir>atazanavir>>darunavir) and mean IC50 values ranging from 15.5 ± 2.80 μM to 143 ± 14.7 μM or 0.220 ± 0.0655 μM to 9.53 ± 2.50 μM, respectively. Atazanavir and lopinavir also inhibited OATP1B3- or NTCP-mediated transport with a mean IC50 of 1.86 ± 0.500 μM or 65.6 ± 10.7 μM and 5.04 ± 0.0950 μM or 20.3 ± 2.13 μM, respectively. Following integration of a combined hepatic transport component into the previous mechanistic static model using the in vitro inhibitory kinetic parameters determined above for atazanavir, the newly predicted rosuvastatin AUCR reconciled with the clinically observed AUCR confirming additional minor involvement of OATP1B3 and NTCP inhibition in its DDI. The predictions for the other protease inhibitors confirmed inhibition of intestinal BCRP and hepatic OATP1B1 as the principal pathways involved in their clinical DDI with rosuvastatin.

Significance of logistic regression scoring model based on natural killer cell-mediated cytotoxic pathway in the diagnosis of colon cancer.

The poor clinical accuracy to predict the survival of colon cancer patients is associated with a high incidence rate and a poor 3-year survival rate. This study aimed to identify the poor prognostic biomarkers of colon cancer from natural killer cell-mediated cytotoxic pathway (NKCP), and establish a logistical regression scoring model to predict its prognosis. Based on the expressions and methylations of NKCP-related genes (NRGs) and the clinical information, dimensionality reduction screening was performed to establish a logistic regression scoring model to predict survival and prognosis. Risk score, clinical stage, and ULBP2 were used to establish a logistic regression scoring model to classify the 3-year survival period and compare with each other. Comparison of survival, tumor mutation burden (TMB), estimation of immune invasion, and prediction of chemotherapeutic drug IC50 were performed between low- and high-risk score groups. This study found that ULBP2 was significantly overexpressed in colon cancer tissues and colon cancer cell lines. The logistic regression scoring model was established to include six statistically significant features: S = 1.70 × stage - 9.32 × cg06543087 + 6.19 × cg25848557 + 1.29 × IFNA1 + 0.048 × age + 4.37 × cg21370856 - 8.93, which was used to calculate risk score of each sample. The risk scores, clinical stage, and ULBP2 were classified into three-year survival, the 3-year prediction accuracy based on 10-fold cross-validation was 80.17%, 67.24, and 59.48%, respectively. The survival time of low-risk score group was better than that of the high-risk score group. Moreover, compared to high-risk score group, low-risk score group had lower TMB [2.20/MB (log10) vs. 2.34/MB (log10)], higher infiltration score of M0 macrophages (0.17 vs. 0.14), and lower mean IC50 value of oxaliplatin (3.65 vs 3.78) (p < 0.05). The significantly upregulated ULBP2 was a poor prognostic biomarker of colon cancer. The risk score based on the six-feature logistic regression model can effectively predict the 3-year survival time. High-risk score group demonstrated a poorer prognosis, higher TMB, lower M0 macrophage infiltration score, and higher IC50 value of oxaliplatin. The six-feature logistic scoring model has certain clinical significance in colon cancer.

Soil Bacterial Community Tolerance to Three Tetracycline Antibiotics Induced by Ni and Zn

A laboratory work has been carried out to determine the tolerance of soil bacterial communities to Ni and Zn and co-tolerance to tetracycline antibiotics (chlortetracycline (CTC), oxytetracycline (OTC) and tetracycline (TC)) in soils individually spiked with five different concentrations of Ni or Zn (1,000, 750, 500, 250, and 125 mg kg−1), and an uncontaminated (0 mg kg−1) control soil. The PICT parameter (pollution-induced community tolerance) was estimated for the bacterial community using the tritium (3H)-labeled leucine incorporation technique, and the values corresponding to log IC50 were used as toxicity index. The mean log IC50 values observed in the uncontaminated soil samples indicate that Zn (with log IC50 = −2.83) was more toxic than Ni (log IC50 = −2.73). In addition, for the soil with the lowest carbon content (C = 1.9%), Ni-contaminated samples showed increased tolerance when the Ni concentrations added were ≥500 mg kg−1, while for the soils with higher carbon content (between 5.3% and 10.9%) tolerance increased when Ni concentrations added were ≥1,000 mg kg−1. Regarding the soils contaminated with Zn, tolerance increased in all the soils studied when the Zn concentrations added were ≥125 mg kg−1, regardless of the soil carbon content. The co-tolerance increases obtained after exposure of the bacterial suspension to TC, OTC and CTC showed an identical behavior within these tetracycline antibiotics. However, it was dependent on the heavy metal tested (Ni or Zn). In the case of soils 1 (C = 1.1%) and 2 (C = 5.3%), the soil bacterial communities showed increases in co-tolerance to TC, OTC and CTC for Ni concentrations added of ≥125 mg kg−1, while for soil 3 (with C = 10.9%) co-tolerance took place when Ni was added at ≥1,000 mg kg−1. However, in soils contaminated with Zn, increases in co-tolerance to CTC, OTC and TC occurred at Zn concentrations added of ≥125 mg kg−1 for the 3 soils tested. These results can be considered relevant when anticipating possible environmental repercussions related to the simultaneous presence of various types of pollutants, specifically certain heavy metals and antibiotics.

Novel fluorinated amino acid derivatives as potent antitumor agents against MCF-7 and HepG2 cells: Synthesis, characterization, in vitro assays and molecular docking studies

To tackle healthcare challenges including neoplastic, cardiac, neurological disease and infectious disease faced by the world community, it is highly demanding to design and synthesize innovative molecules. Herein using a variety of amino acids and fluorinated aromatic amines, we have designed and synthesized novel fluorinated amino acid derivatives (1a–6b) as potential anticancer agents. Among synthesized fluorinated compounds 1a, 1b, and 3b showed potent anticancer activity against MCF-7 cancer cell lines. Similarly, compounds 1b and 2a showed moderate activity against HepG2 cancer cell lines. Compound 1b was found to be the most effective cytotoxic compound against MCF-7 breast cancer cells with a mean IC50 value of 11.63 µM and also effective against HepG2 liver cancer cells with a mean IC50 value of 34.10 µM. Additionally, a molecular docking study was carried out to determine the molecular binding affinity of the synthesized fluorinated derivatives. The compound 1b showed the highest binding affinity (binding energy: −12.6 kcal/mol) which interacted more effectively with PRO 385 (4.75 Å) than standard drug doxorubicin binding affinity (binding energy: −10.4 kcal/mol).

Dual BCL-XL/2 Protac 753B Not Only Potently Induces Apoptosis in Venetoclax-Resistant Primary AML Cells but Also Effectively Eliminates Chemotherapy-Induced Senescent AML Cells

BCL-XL and BCL-2 (BCL-XL/2 ) are key antiapoptotic proteins and validated cancer targets. 753B is a novel BCL-XL/2 proteolysis targeting chimera (PROTAC) that targets both BCL-XL and BCL-2 to the Von Hippel-Lindau (VHL) E3 ligase, leading to BCL-XL/2 ubiquitination and degradation (Lv et al. Nat. Comm. 2021). It exhibits an improved potency against BCL-XL/2 leukemia cells while reduced toxicity to platelets compared to the dual BCL-XL/2 inhibitor navitoclax or ABT263 in vitro. Here we examined the potential therapeutic efficacy of 753B against venetoclax-resistant primary AML cells and chemotherapy-induced senescent AML cells in vitro and in vivo. We first evaluated the sensitivity of 24 genetically diverse leukemia cell lines (17 AML, 5 T-ALL, and 2 AML secondary to myeloproliferative neoplasms (MPN-AML) cell lines) to ABT263, DT2216 (a BCL-XL selective PROTAC) (Khan et.al Nat. Med, 2019) and 753B. 753B induced time and dose-dependent BCL-XL degradation in all lines with DC50 (the concentrations with 50% protein degradation) ranging from 0.01 μM to 0.54 μM. BCL-2 was degraded in all except Loucy cells, with DC50 ranging from 0.02 μM to >1 μM. 753B caused dose-dependent reduction of viability in AML cell lines by CellTiter-Glo (CTG) assay at 24hr (IC50 range 0.01μM - 27.35 μM). 753B was more potent than ABT-263 in 12/17 AML and 3/5 T-ALL lines, with the mean IC50 values of 0.10 μM and 0.48 μM, respectively. The degradation of BCL-XL/2 by 753B was accompanied with MCL-1 upregulation in 10/17 cell lines tested, which was reversely correlated with the IC50. Recent findings indicate that chemotherapy (Ara-C)-induced senescence is associated with chemoresistance (Duy et al., Cancer Discovery 2021), and BCL-XL was previously reported to be the primary survival factor of senescent cells (Chang et al.,Nat. Med.2016). As previously reported, Ara-C indeed induced cellular senescence (SnCs) in MOLM-14 and Kasumi-1 AML cells, with increased cell size, higher senescence-associated β-galactosidase activity, upregulation of cell cycle regulator proteins (p16, p21, p53), and expression of senescence-associated secretory phenotype (IL-8 and CCR5). 753B cleared chemotherapy-induced SnCs by induction of apoptosis. To explore the senolytic mechanism of 753B, we FACS-sorted senescence-high or -low populations based on C12-FDG positivity (fluorogenic substrate di-β-D-galactopyranoside). C12-FDG high senescent AML population expressed higher levels of BCL-XL/2, priming the cells to 753B-induced apoptosis. We next isolated blasts from 16 primary AML samples and tested their sensitivity to 753B. 753B potently reduced cells viability with median IC50 value of 0.23 mM (range 0.02 - 2.29 mM). Similar to cell lines, 753B was more potent in degrading BCL-XL and inducing apoptosis than DT2216 in primary AML cells. 753B showed potency comparable to ABT-263 in all tested AML samples, including seven venetoclax-resistant samples (median IC50, 753B 0.22 mM; ABT-263 0.13 mM). 753B degraded both BCL-XL and BCL-2 in primary myeloblasts in association with the induction of apoptosis. BCL-2 degradation was observed in 4 of 6 samples tested. To investigate the antileukemia activity of 753B in vivo, we developed a patient-derived xenograft (PDX) model using NSG mice injected with AML PDX #8550 (harboring FLT3-ITD, DNMT3A, IDH1, KIT, and NPM1 mutations). Mice were randomized into 2 groups to receive either vehicle or 753B (5 mg/kg intraperitoneally every 4 days) for 3 weeks after leukemia engraftment was confirmed (day 33). Mice tolerated 753B therapy well with no significant changes in body weight and no significant platelet toxicity ascertained by blood counts. 753B as a single agent reduced the circulating leukemia cell burden, serially measured by flow cytometry, and extended median survival time from 61 days to 71 days (P = 0.0023)(Fig. 1A). In summary, the novel BCL-XL/2 PROTAC 753B potently reduced cell viability in leukemia cell lines with diverse genetic background, in primary AML cells including venetoclax-resistant AML blasts, and extended survival of mice harboring human AML PDX in vivo without hematologic toxicity. Our data indicate that co-targeting of BCL-2 and BCL-XL may eliminate senescent AML cells surviving Ara-C chemotherapy, supporting the utility of future combination studies in leukemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pharmacological Landscape of FDA-Approved Anticancer Drugs Reveals Sensitivities to Ixabepilone, Romidepsin, Omacetaxine, and Carfilzomib in Aggressive Meningiomas.

To date, there are no systemic treatment options for patients with recurrent or refractory meningioma. To identify effective drugs, we performed a large-scale drug screening using FDA-approved drugs on several meningioma cell lines. The impact of the top four compounds was assessed on cell viability, proliferation, colony formation, migration, and apoptosis. In addition, the antineoplastic effects of the selected drugs were validated in a heterotopic xenograft mouse model. Analyses of the viability of meningioma cells treated with 119 antineoplastic FDA-approved drugs resulted in categorization into sensitive and resistant drug-response groups based on the mean IC50 values and peak serum concentrations (Cmax) in patients. Eighty drugs, including 15 alkylating agents, 14 antimetabolites, and 13 tyrosine kinase inhibitors, were classified as resistant (IC50 > Cmax). The sensitive drug-response group (n = 29, IC50 < Cmax) included RNA/protein synthesis inhibitors, proteasome inhibitors, topoisomerase, tyrosine-kinase, and partial histone deacetylase and microtubule inhibitors. The IC50 value of the four most effective compounds (carfilzomib, omacetaxine, ixabepilone, and romidepsin) ranged from 0.12 to 9.5 nmol/L. Most of them caused cell-cycle arrest in the G2-M-phase and induced apoptosis. Furthermore, all drugs except romidepsin significantly inhibited tumor growth in vivo. The strongest antineoplastic effect was observed for ixabepilone, which reduced tumor volume by 86%. In summary, a large-scale drug screening provides a comprehensive insight into the anti-meningioma activities of FDA-approved drugs, and identified carfilzomib, omacetaxine, ixabepilone, and romidepsin as novel potent antineoplastic agents for the treatment of aggressive meningiomas. The most pronounced effects were observed with ixabepilone mandating for further clinical investigation.