IC50 Concentration Research Articles

Investigating the Antiproliferative Activity of Novel 4-Chloro-8-Nitro-1,2-Dihydro-3-Quinoline Acylhydrazones on Human Cervical Cancer Cell Lines.

A new series of acyl hydrazones have been synthesized from 4-chloro-8-nitro-1,2-dihydroquinoline-3-carbaldehyde. These compounds were characterized using various spectroscopic techniques. Density functional theoretical (DFT) studies were conducted to understand the correlation between electronic parameters and biological activity. The biological activity of the compounds was theoretically examined through molecular docking and ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analysis. The compounds demonstrated high absorption rates and were found to be non-hepatotoxic. Preliminary cytotoxicity screenings against HeLa cell lines identified compound 7 as the most potent, with an IC50 value of 18.8 μM. This compound was further selected for bioimaging studies. The results indicate that compound 7 induces apoptosis at its IC50 concentration, suggesting its potential as an anticancer agent.

Insight on novel sulfamoylphenyl pyrazole derivatives as anticancer carbonic anhydrase inhibitors.

As another part continue for our previous study, variable substituted pyrazoles bearing sulfamoylphenyl moiety were synthesized and screened against two cancer related human carbonic anhydrase (hCA) isoforms and acetazolamide (AAZ) used as a reference standard. Some compounds as 4e and 6c manifested a promising inhibitory activity against both isoforms (KI = 0.072, 0.081 and 0.073, 0.095 µM), respectively. While others as 4a and 5e showed inhibitory activity against hCA IX only (KI = 0.062, 0.04 µM) or against hCA XII only as compound 5b (KI = 0.106 µM) compared to AAZ (KI = 0.065, 0.046 µM), respectively. Also, the anticancer efficacy against 60 cancer cell lines for the target compounds was assessed, and the most promising ones were 4d and 5a-d. Further investigation of the anticancer activity of 5b on MCF-7 cell line explored (IC50 = 5.21 µM) compared to doxorubicin (IC50 = 11.58 µM). Moreover, compound 5b was exposed to cell cycle analysis and apoptotic assay on MCF-7 breast cancer cell line under both normal and hypoxic conditions at its IC50 concentration with elevation of total apoptotic cells % in MCF-7 relative to the control cells; respectively. Finally, molecular modelling simulations rationalized the in vitro testing results.

DDDR-46. TARGETING TRANSGLUTAMINASE 2 (TGM2) ENHANCES SENSITIVITY OF GLIOBLASTOMA CELL LINES TO RADIOTHERAPY AND TEMOZOLOMIDE

Abstract Glioblastoma (GBM), the most aggressive and common primary brain tumor in adults, remains highly resistant to conventional therapeutics, including radiation therapy (RT), largely due to its molecular heterogeneity. Few biomarkers have been identified that successfully guide or predict treatment response. Transglutaminase 2 (TGM2), a ubiquitous multifunctional enzyme involved in numerous signal transduction pathways related to tumorigenesis, has recently been suggested to drive chemoradioresistance. Here, we investigate the effects of disrupting TGM2 by pharmacologic inhibition or CRISPR knockout (TGM2-KO) on GBM cell’s response to RT and Temozolomide (TMZ) chemotherapy. Using western blotting and clonogenic assays, we show that the allosteric TGM2 inhibitor, GK921, stabilizes p53 expression and impedes reproductive ability at IC50 and IC25 concentrations. TGM2-KO and GK921 (at IC10 and lower) sensitized GBM cell lines to RT with almost equivalent efficacy, resulting in a 2- to 3-fold suppression of clonal expansion compared to scramble controls or irradiation alone. We also demonstrate increased sensitivity of GBM cell lines to TMZ upon inhibition of TGM2 by GK921. Our findings indicate that the use of TGM2 inhibitors, such as GK921, could enhance the efficacy of RT and the potency of TMZ, positioning TGM2 as a promising therapeutic target that may improve standard treatment of GBM cells expressing TGM2.

Curcumin Induces MCF-7 Breast Cancer Cell Apoptosis Through miR-15a Alteration

Background: In recent years, the application of herbal compounds in cancer treatment has shown significant progress. Curcumin (CUR), a natural polyphenol, demonstrates potent anti-cancer effects against various cancers, including breast cancer (BC). Curcumin targets a range of molecular pathways, contributing to the inhibition of cancer cell proliferation, metastasis, and angiogenesis, and promoting apoptosis. Objectives: This study aimed to assess the effect of CUR on BC cells, focusing on alterations in the expression levels of Mir-15a and Bcl-2 through the apoptotic pathway. Methods: The cytotoxicity of CUR was evaluated using an MTT assay. Changes in the expression of Mir-15a and Bcl-2 genes were analyzed by real-time PCR, and cell apoptosis was measured using flow cytometry. Data analysis was conducted using SPSS. Results: The MTT assay results indicated that cell viability decreased as CUR concentration increased (5 – 25 µM). Real-time PCR results showed a significant decrease in the expression of Mir-15a and Bcl-2 (P < 0.05). Flow cytometry findings demonstrated that CUR treatment at the IC50 concentration for 48 hours induced apoptosis in 75.9% of MCF-7 cells. Conclusions: Our study provides a crucial foundation indicating that CUR induces apoptosis in MCF-7 cells by modulating the expression of Mir-15a.

A zinc oxide–tin oxide–nerolidol hybrid nanomaterial: Efficacy against esophageal squamous cell carcinoma

Abstract The sixth most common cancer in the world, esophageal cancer, requires aggressive treatment such as surgery, chemotherapy, radiotherapy, and immunotherapy. Phytochemicals and medicinal plants are being used in the green synthesis of nanoparticles. Our study aimed to synthesize ZnO–SnO2 nerolidol nanocomposite and study its effects on esophageal squamous cell carcinoma cells. UV spectroscopy showed significant absorbance at 288 nm, transmission electron microscopy and DLS showed spherical shapes, and transmission electron microscopy also showed 108 nm average diameters. The ZnO–SnO2 nerolidol nanocomposite was also investigated using energy-dispersive X-ray analysis, Fourier transform infrared spectroscopy, photoluminescence, and field emission scanning electron microscopy. A cytotoxic effect was observed against KYSE-150 cells with an IC50 concentration of 14.9 μg/mL. The ZnO–SnO2 nerolidol nanocomposite inhibited cancer cell proliferation in KYSE-150 cells and enhanced apoptosis by altering its mitochondrial membrane potential. The ZnO–SnO2 nerolidol nanocomposite also enhanced oxidative stress, leading to a decrease in superoxide dismutase, catalase, and glutathione and an increase in lipid peroxidation. Ultimately, ZnO–SnO2 nerolidol nanocomposite enhanced the caspase cascade by inducing caspases 3, 8, and 9 in KYSE-150 cells. On the whole, we suggest that the ZnO–SnO2 nerolidol nanocomposite can be an effective treatment strategy against esophageal squamous cell carcinoma in KYSE-150 cells. However, understanding molecular circuits is still warranted.

Efficient one-pot green synthesis of carboxymethyl cellulose/folic acid embedded ultrafine CeO2 nanocomposite and its superior multi-drug resistant antibacterial activity and anticancer activity.

Due to the prevalence of drug-resistant bacteria and the ongoing shortage of novel antibiotics as well as the challenge of treating breast cancer, the therapeutic and clinical sectors are consistently seeking effective nanomedicines. The incorporation of metal oxide nanoparticles with biological macromolecules and an organic compound emerges as a promising strategy to enhance breast cancer treatment and antibacterial activity against drug-resistant bacteria in various biomedical applications. This study aims to synthesize a unique nanocomposite consisting of CeO2 embedded with folic acid and carboxymethyl cellulose (CFC NC) via a green precipitation method using Moringa oleifera. Various spectroscopic and microscopic analyses are utilized to decipher the physicochemical characteristics of CFC NC and active phytocompounds of Moringa oleifera. Antibacterial study against MRSA (Methicillin-resistant Staphylococcus aureus) demonstrated a higher activity (95.6%) for CFC NC compared to its counterparts. The impact is attributed to reactive oxygen species (ROS), which induces a strong photo-oxidative stress, leading to the destruction of bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CFC NC are determined as 600µg/mL and 1000µg/mL, respectively. The anticancer activity against breast cancer cell resulted in the IC50 concentration of 10.8μg/mL and 8.2μg/mL for CeO2 and CFC NC respectively.The biocompatibility test was conducted against fibroblast cells and found 85% of the cells viable, with less toxicity. Therefore, the newly synthesized CFC NC has potential applications in healthcare and industry, enhancing human health conditions.

Influence of temperature, solvent and extraction procedure on the content of phenolic compounds and antioxidant activity of <i>Aloysia citriodora</i> Palau leaves

Introduction: The leaves of Aloysia citriodora Palau are a source of phenolic compounds that have antioxidant properties. Objective: The objective of this study was to compare the effects of temperature, type of solvent, maceration, and ultrasound processes in the extraction of phenolic compounds from A. citriodora Palau leaves that allow for maintaining the highest antioxidant capacity. Methods: In this study, phenolic compounds were extracted from A. citriodora by two methods, maceration, and ultrasonic treatment, using water and 80% ethanol as solvents and extraction temperatures of 25 °C, 45 °C, and 65 °C. Total phenolic compounds (TPhen), total flavonoid compounds (TFlav), and antioxidant capacity, evaluated by DPPH and ABTS assay, were characterized and reported as inhibition concentration (IC50). Results: The highest CPhen content was obtained with ethanolic extract using the ultrasonic method at 65 °C, with a value of 15.32 mg EAG/mL at 65 °C. For TFlav, the highest value was obtained with the ultrasonic treatment at 45 °C using ethanol as solvent, obtaining a value of 6.52 mg EQ/mL. The best antioxidant capacity was shown by the sonication with ethanol at 65 and 45 °C, with IC50 values of 27.10 µg/mL and 20.40 µg/mL, respectively. Conclusion: Ultrasound extraction at higher temperatures (45°C and 65°C) proved more effective than maceration, yielding greater amounts of phenolic and flavonoid compounds and exhibiting better antioxidant capacity.”. Keywords: Aloysia citrodora, phenolic compounds, antioxidant activity, inhibitory concentration IC50.

Exploring the therapeutic potential of Pongamia pinnata plant extract against skin cancer: In-silico and in-vitro study

Ethnopharmacological relevancePongamia pinnata Linn belonging to the Fabaceae family, holds significance as a crucial remedy in herbal medicine. Aim of the studyThe present study aims to determine the anticancer and antioxidant properties of the plant extract. Material and methodsThe methodology includes extraction of the leaf sample by soxhlet method followed by the phytochemical analysis of leaf extract. Through in-silico approach three anti-cancer receptors were analyzed with 10 ligands which were studied using molecular docking and molecular simulation approach. The prediction outcome of in-silico tests on the petroleum ether plant extract served as a foundation for the subsequent in-vitro studies. Phytochemical profiling of plant extracts using GC-MS analysis, and cytotoxicity testing for A431 skin cell line using MTT Assay. ResultsThe binding affinity of Pongamia pinnata as an anti-cancer agent with respect to the targets EGF, EGFR, ERBB2 was evident. In the in-silico studies, the highest binding affinity with respect to the docked complexes were -8.2 kcal/mol for EGF with Pazopanib, -8.3 kcal/mol for EGFR with pongachromene, -9.5 kcal/mol for ERBB2 with Vitexin. Further these complexes were assessed by molecular simulations and it confirms the stability of these complexes. In in-vitro studies the HPLC results indicated the presence of 0.176 ± 0.001 mg/mL of phenols and 0.159 ± 0.001 mg/mL of flavonoids. The IC50 value was 1.051 which revealed the antioxidant potential. The cytotoxicity studies against the A431 skin cancer cell resulted in an IC50 concentration of 89.59 μg/ml. ConclusionsThese studies show that P. pinnata has the properties to serve as a therapeutic agent for the treatment of skin tumors. The molecular simulation studies approach is a predictor for drug discovery, acting as a basis for testing anti-cancer activity against the skin tumor. As a result, it can be a useful source of crude drug for the treatment of melanomas.

The Anticancer Poperties of Diosgenin on Colorectal Cancer Cells Induced by Wnt/Β Catenin Signaling Pathway

Background: Colorectal cancer (CC) is one of the most prevalent cancers globally. Due to the severe side effects and the development of drug resistance in CC treatments, current therapeutic strategies often prove ineffective. As a result, there is a growing interest in exploring traditional medicinal plants as alternative treatment options for various human diseases. Diosgenin, a bioactive compound derived from plants, has shown potential in inhibiting the growth of human CC cells. The Wnt/β-catenin signaling pathway plays a critical role in colorectal tumorigenesis. Objectives: This study investigates the effects of Diosgenin on the Wnt/β-catenin pathway in CC cells. Methods: Colorectal cancer cells were treated with Diosgenin, and cell viability and plasma membrane integrity were assessed using the MTT assay and lactate dehydrogenase (LDH) activity assay, respectively. Apoptosis was evaluated through Annexin V/PI staining. The expression of Wnt/β-catenin-related genes was analyzed by quantitative PCR (qPCR). Results: Diosgenin significantly inhibited CC cell viability in a time- and concentration-dependent manner after 24, 48, 72, and 96 hours of exposure (P < 0.05). The IC50 values were determined to be 203.55, 122.95, 70.11, and 7.34 µM at 24, 48, 72, and 96 hours, respectively. Diosgenin at its IC50 concentration induced a significant increase in cell apoptosis after 24 hours of treatment (P < 0.05). Additionally, it caused a significant reduction in the expression of β-catenin, cyclin D1, and Pin1 (βCP). Conclusions: Diosgenin exhibits anti-CC effects by inhibiting the expression of βCP genes involved in the Wnt/β-catenin pathway, suggesting its potential as a therapeutic agent for CC.

In vitro cytotoxic evaluation of Hypericum perforatum and molecular docking and dynamic analysis of PINK-1 inhibitors on model organism Tribolium castaneum and Homo sapiens

Hypericum species are especially known for their pharmacological characteristic. One of the major component of the plant is hypericin that can be used in tumor inhibition with its potent cytotoxic effects. In this study, the anticancer effect of H. perforatum essential oil on the MCF-7 breast cancer cell line was examined, and the binding potential of the compounds of Hypericum perforatum L., hypericin, hyperoside and hyperforin, to the PINK1 protein of both human and model organism, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) was investigated in silico. Recently, many insect species have been proposed as model organisms, including T. castaneum, that the first species with whole genome sequenced. Worlwide distribution of insects, their environmental importance and realtively inexpensive cultivation have increased the interest in them Therefore, in in silico studies, T. castaneum was used as a model to compare binding similarities with humans and model organisms. In the study, the IC50 concentration of H. perforatum L. on MCF-7 cells was determined to be 98.765 μg/ml. Based on in silico findings, the most favorable binding affinity of -12.5 kcal/mol was observed between the Hypericin molecule and the insect PINK1 protein. The fact that these plant components bind with high energy to the PINK1 protein, which is believed to guard cells from mitochondrial dysfunction triggered by stress, is promising for the development of plant-based medical drugs and biopesticides.

Synthesis, characterization, in silico and in vitro assessment of 2-aminopyridine nicotinamide cocrystal as a new agent for breast cancer therapy

Breast cancer is the leading cause of cancer-related death among women globally, in spite of major advances in diagnosis and treatment. 2-Aminopyridine Nicotinamide (2APNA) cocrystal was synthesized and cocrystal formation was confirmed through powder XRD. Computational investigations in structural fields and spectroscopic parameters were conducted and compared with the experimental results. Comparing the optimized geometrical characteristics, it was observed that molecular properties of 2APNA closely match the previously reported experimental data. FT-IR and FT-Raman spectroscopic analysis revealed the theoretical wavenumbers agree better with recorded data and in UV–visible absorption a single peak was recorded at 247 nm showing n → π* transition. The 2APNA's stability, reactive nature, charge relocations were studied through quantum chemical calculation such as FMO, MEP, NBO, NLO and Mulliken charge analysis. The topological features of cocrystal 2APNA was examined through ELF, LOL and RDG studies. The anti-apoptotic protein BCL-2 was selected as a target in molecular docking analysis and the results expressed 2APNA's binding score as -8.2 kcal/mol. In vitro studies confirmed that 2APNA has 2APNA demonstrated an enhanced cell death in MCF-7 cells with the IC50 concentration of 56.58 µg/mL and decreased the expression of BCL-2, thus resulting in apoptotic cell death. These results indicated that 2APNA possess a potent anticancer property for the treatment of breast cancer.

Assessment of cytotoxicity of 5-arylaminouracil derivatives

We have previously shown that 5-arylaminouracil derivatives can inhibit HIV-1, herpesviruses, mycobacteria and other pathogens through various mechanisms. The purpose of this study was to evaluate the potential of 5-arylaminouracils and their derivatives against leukemia, neuroblastoma and glial brain tumors. The cytotoxicity of 5-aminouracils with various substituents, as well as their 5’-norcabocyclic and ribo derivatives, was screened against two neuroblastoma cell lines (SH-SY5Y and IMR-32), lymphoblastic cells K-562, promyeoloblastic cells HL-60 and low-passage variants of well-differentiated glioblastoma multiforme (GBM5522 and GBM6138). As a result of assessing the cytotoxicity of the resulting compounds on the above cell lines using the standard MTT test, it was revealed that most of the compounds do not have significant toxicity. However, in the GBM-6138 cell line, 5-(4-isopropylphenylamine)uracil and 5-(4-tert-butylphenylamine)uracil exhibited a dose-dependent toxic effect, with half-maximal inhibition concentrations IC50 of 9 μM and 2.3 μM, respectively. The antitumor activity of compounds of this type has been demonstrated for the first time and can serve as a starting point for further research.

Design, Development of Pyrazole-Linked Spirocyclopropyl Oxindole-Carboxamides as Potential Cytotoxic Agents and Type III Allosteric VEGFR-2 Inhibitors.

Tumor progression depends on angiogenesis, which is stimulated by growth factors like VEGF, targeting VEGFR kinase with small molecules is an effective anti-angiogenic therapeutic approach. The rational modification of sunitinib (VEGFR-2 inhibitor) to spirocyclopropyloxindoline carboxamides have been performed and their in vitro cytotoxic profiling was evaluated. The molecular modelling studies enabled the screening of designed analogues and identifying the possible interactions within the type III allosteric inhibitor binding site of VEGFR-2. The biological screening of synthesized compounds 15 a-y, revealed the ability of compound 15 w to inhibit the cell growth in MCF-7 cell line with IC50 value of 3.87±0.19 μM and alongside inhibition of VEGFR-2 kinase at a IC50 concentration of 4.34±0.13 μM was observed. Also, VEGFR-2 inhibition was validated through HUVEC tube formation inhibition assay. The qualitative assessment of apoptosis induction by 15 w in MCF-7 cells was evaluated through staining studies such as AO/EB and DAPI staining, whereas quantification of apoptosis and cell cycle analysis were performed through FACS analysis. The metastatic ability of the cancer cells was evaluated through inhibition of cell migration by a scratch wound healing assay. The current study strives to sequentially optimize the structural attributes of the 3-alkenyl oxindole core to surpass the existing challenges of well-known VEGFR-2 inhibitors. The findings observed from this study highlights that compound 15 w to be a prominent lead towards the development of clinical drug candidates.

Evaluation of antibacterial and anticancer properties of secondary metabolites isolated from soil Bacillus spp focusing on two strains of Bacillus licheniformis and Bacillus siamensis

BackgroundBacillus strains are well recognized for their inherent production of bioactive compounds that exhibit antibacterial and anticancer properties. This study aims to evaluate the antimicrobial and anticancer effects of the secondary metabolite isolated from Bacillus licheniformis and Bacillus siamensis strain.Material and methodWe developed and purified a new soil-derived Bacillus strain to study its metabolites on cancer cells and bacteria. After evaluating the antimicrobial effects of the selected strains’ secondary metabolites by well diffusion, growth conditions and temperature optimised using liquid-liquid extraction, secondary metabolites isolated, and active compounds identified using GC-MS. Evaluation of PC-3 and HPrEpC cytotoxicity. AV/PI staining and comet assay assessed necrosis and apoptosis. Real-time PCR measured apoptotic gene expression. Finally, the scratch test measured cell movement.ResultsBacillus strain metabolites exhibit dual-purpose antimicrobial and anticancer properties. Bacillus licheniformis isolate 56 and S2-G12 isolate 60 demonstrated the greatest antibacterial activity. Among all Bacillus isolates, isolates 56 (Bacillus licheniformis) and 60 (Bacillus siamensis strain) had the highest antibacterial activity. Crude extracts obtained from strains 56 and 60 decreased PC-3 cell viability in a dose-dependent manner. At 200 µg/mL, the survival rate of cells treated with strain 56 and 60 crude extract was 23% and 25%, respectively (p < 0.001). The treatment of PC-3 cells with strains 56 and 60 crude extract led to considerable apoptosis (46.2% and 50.09%, respectively) compared to the control group. After treatment with the crude extract from strains 56 and 60 at an IC50 concentration, a significant number of PC-3 cells showed comet formation, indicating DNA fragmentation. Metabolites extracted from strain 56 and 60 enhanced caspase 3, caspase 8, and Bax genes expression and reduced Bcl-2 expression (p < 0.001). Cell migration was also prevented.ConclusionOur findings show that the secondary metabolites of B. licheniformis and B. siamensis have antibiotic and anticancer properties. However in vivo studies are necessary to confirm these findings.

Anti-viral and Apoptotic Induction of m-TOR Inhibitor Drugs against Hepatitis C Virus Activity and Hepatocellular Carcinoma Cell Line: In vitro and in silico.

This study investigated the potential of m-TOR inhibitor drugs (sirolimus, everolimus, and tacrolimus) in combating both hepatocellular carcinoma (HCC) and hepatitis C virus (HCV) replication. After treating HepG2 and PBMCs with the mammalian target of Rapamycin (m-TOR) inhibitors drugs; sirolimus, everolimus, and tacrolimus at different concentrations (1, 5, and 10 µM/µl), cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Antioxidant activities (total antioxidant, glutathione S-transferase, and glutathione reductase), Fas-ligand level, tumor necrosis factor-α (TNF-α) level, caspase-3, -8, and -9 activities, and cell cycle analysis were measured. quantitative Real-time PCR, colony forming assay, molecular docking studies after infection of PBMCs with 1 ml (1.5 × 106 HCV) serum then incubated with m-TOR inhibitor drugs at their respective IC50 concentrations. In HepG2 cells, treatment with these inhibitors resulted in suppressed cell viability, increased dead cell accumulation, and enhanced apoptotic signaling through elevated Fas-ligand and caspase activities. Additionally, cell cycle analysis revealed arrest in G0/G1 and G2/M phases, further hindering HCC progression. Furthermore, m-TOR inhibitor drugs significantly reduced HCV viral load and colony formation in infected PBMCs. This antiviral effect was accompanied by decreased TNF-α activity, suggesting potential modulation of the inflammatory response associated with HCV infection. Molecular docking studies provided theoretical support for these findings, with Sovaldi demonstrating the highest binding affinity towards key HCV targets compared to other m-TOR inhibitors. This suggests its potential as a potent HCV inhibitor, while also highlighting the potential of exploring m-TOR inhibitors for future HCV treatment development. Overall, this study provides encouraging evidence for the potential of m-TOR inhibitor drugs as promising therapeutic agents for both HCC and HCV, warranting further investigation and optimization for clinical applications.

Exploring the 1-(4-Nitrophenyl)-3-arylprop-2-en-1-one Scaffold for the Selective Inhibition of Monoamine Oxidase B.

A small library of 1-(4-nitrophenyl)-3-arylprop-2-en-1-one derivatives was synthesized to identify new human monoamine oxidase B selective inhibitors. Their inhibitory activity toward MAO-A and MAO-B isoforms was evaluated to determine their potency and selectivity. All newly synthesized compounds were nanomolar inhibitors of the B isoform with IC50 concentrations ranging from 120 to 2.2 nM. Conversely, their activity toward the A isozyme was only observed at micromolar concentrations. Our results bear out the hypothesis that the 1,3-diarylpropenone scaffold could represent a valuable starting point for designing efficient and selective MAO-B inhibitors.

Disulfiram Upgrades the Radiosensitivity of Osteosarcoma by Enhancing Apoptosis and P53-Induced Cell Cycle Arrest.

The prognosis of osteosarcoma has not been improved for decades. As radioresistance is one of the major reasons, effective radiotherapy sensitization drugs need to be discovered. HOS and K7M2 osteosarcoma cell lines were treated with disulfiram (DSF) and radiation to assess cell viability, proliferation, migration ability, apoptosis level, ROS and Ca2+ level, and cell cycle in vitro. A HOS-derived subcutaneous tumor mouse model was constructed to evaluate tumor growth after DSF combined with radiation, and the Tunel assay and immunohistochemistry of Ki67 were conducted. Western blot was used to evaluate the protein expression level. The IC50 and working concentration of DSF in osteosarcoma cell lines were ascertained. When combined with radiation, DSF effectively suppressed cell viability, proliferation, and migration, while enhancing apoptosis in osteosarcoma cells. The cell cycle postirradiation exhibited a downward shift in the G1 phase, but the addition of DSF counteracted this trend. The combination of DSF and radiation exhibited inhibitory effects on tumor growth invivo, which was corroborated by Ki67 staining and Tunel assay. Western blot analysis revealed that DSF upregulated the expression of P53, P21, CDKN2C, BAX, and cleaved Caspase-3 while downregulating BCL2, CDK4/6, and CyclinD1 after irradiation. Our results document that DSF exerts its radiosensitization effects invivo and in vitro, and is a valuable radiosensitizing drug option for osteosarcoma. The radiosensitization effect is mainly achieved by activating the apoptotic pathway and promoting cell cycle arrest induced by P53/P21 and CDKN2C after irradiation.

Anti-Inflammatory Activity and Wound Healing Ability of Coconut Oil Mouthwash on Gingival Fibroblast Cell In Vitro

Coconut oil-pulling therapy is used for maintaining oral health. The procedure has benefits for the prevention of oral disease, including dental caries, oral malodor, bleeding gums. However, virgin coconut oil (VCO) has unsatisfying oily taste. Therefore, coconut oil mouthwash (CoMW) was recently developed. This in vitro study aimed to evaluate the anti-inflammatory activity of coconut oil mouthwash consisting of 60% v/v virgin coconut oil (VCO), 30% v/v propylene glycol (PG), and 10% v/v distilled water on human gingival fibroblast (HGF) cells compared with the activity on murine macrophage (Raw 264.7) cells. The cytotoxicity of CoMW, 0.12% chlorhexidine gluconate (CHX), VCO, and PG was assessed. IC50 concentration of CoMW, CHX, and PG were 1:8 (v/v), 1:32 (v/v), and 1:16 (v/v), respectively. All tested concentrations of VCO had no impact on cell viability. Their anti-inflammatory effects of each IC50 concentration were further studied. Notably, the IC50 concentration of CHX also significantly inhibited nitric oxide production in lipopolysaccharide (LPS)- activated Raw 264.7 cells. Moreover, the IC50 concentrations of CoMW, VCO, PG, and CHX could suppress the interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) gene expressions in LPS-activated HGF cells, while also enhancing the cell migration of HGF cells as likely to the effect observed with the IC50 concentration of CHX. Wound healing ability of CoMW was also demonstrated after testing with a scratch assay. These findings indicated promising potential for coconut oil mouthwash as an effective agent in reducing inflammation and facilitating wound healing.

Proteasome inhibition enhances the anti-leukemic efficacy of chimeric antigen receptor (CAR) expressing NK cells against acute myeloid leukemia

BackgroundRelapsed and refractory acute myeloid leukemia (AML) carries a dismal prognosis. CAR T cells have shown limited efficacy in AML, partially due to dysfunctional autologous T cells and the extended time for generation of patient specific CAR T cells. Allogeneic NK cell therapy is a promising alternative, but strategies to enhance efficacy and persistence may be necessary. Proteasome inhibitors (PI) induce changes in the surface proteome which may render malignant cells more vulnerable to NK mediated cytotoxicity. Here, we investigated the potential benefit of combining PIs with CAR-expressing allogeneic NK cells against AML.MethodsWe established the IC50 concentrations for Bortezomib and Carfilzomib against several AML cell lines. Surface expression of class-I HLA molecules and stress-associated proteins upon treatment with proteasome inhibitors was determined by multiparameter flow cytometry. Using functional in vitro assays, we explored the therapeutic synergy between pre-treatment with PIs and the anti-leukemic efficacy of NK cells with or without expression of AML-specific CAR constructs against AML cell lines and primary patient samples. Also, we investigated the tolerability and efficacy of a single PI application strategy followed by (CAR-) NK cell infusion in two different murine xenograft models of AML.ResultsAML cell lines and primary AML patient samples were susceptible to Bortezomib and Carfilzomib mediated cytotoxicity. Conditioned resistance to Azacitidine/Venetoclax did not confer primary resistance to PIs. Treating AML cells with PIs reduced the surface expression of class-I HLA molecules on AML cells in a time-and-dose dependent manner. Stress-associated proteins were upregulated on the transcriptional level and on the cell surface. NK cell mediated killing of AML cells was enhanced in a synergistic manner. PI pre-treatment increased effector-target cell conjugate formation and Interferon-γ secretion, resulting in enhanced NK cell activity against AML cell lines and primary samples in vitro. Expression of CD33- and CD70-specific CARs further improved the antileukemic efficacy. In vivo, Bortezomib pre-treatment followed by CAR-NK cell infusion reduced AML growth, leading to prolonged overall survival.ConclusionsPIs enhance the anti-leukemic efficacy of CAR-expressing allogeneic NK cells against AML in vitro and in vivo, warranting further exploration of this combinatorial treatment within early phase clinical trials.

Evaluation of the effects of vitamin D analogs, bevacizumab, and radiotherapy in uveal melanoma cells

Due to the lack of a definitive effective treatment method that provides a complete cure and increases survival rates in uveal melanoma, the search for alternative treatments at the molecular level continues. In this context, we aimed to comparatively analyze the therapeutic effects of 25-hydroxyvitamin D3 (D2), 1a, 25-dihydroxyvitamin D3 (D3), bevacizumab and radiotherapy (RT) in a uveal melanoma cell line (MP41). Cytotoxicity was evaluated using XTT cell proliferation kit and Xcelligence cell analyzer system. RT dose was determined after a clonogenic assay. Annexin V/PI staining and Western blot analyses for caspase-3, -8, and -9 were performed to analyze apoptosis. Additionally, cell cycle analyses were also conducted. As a result, we found that D2 and D3 did not show cytotoxic effects, while bevacizumab and RT showed time and dose-dependent cytotoxicity. IC50 concentration of bevacizumab was 6.945 mg/mL. Radiotherapy and bevacizumab significantly reduced cell survival and induced apoptosis when administered both as monotherapy and in combination. A significant increase in caspase proteins was detected at high bevacizumab concentrations. However, the combination of bevacizumab and radiotherapy caused a substantial decrease in caspase-3, -8 and -9 expressions. No significant difference in cell cycle distribution was detected in any treatment. Our results showed that bevacizumab inhibited MP41 cell proliferation and had an additive effect when administered with RT. In conclusion, our study offers a different perspective on the treatment of uveal melanoma, and these results, when supported by animal experiments and clinical studies in the future, might be a new step in the treatment of this challenging ocular tumor.