Cell Carcinoma Of Cervix Research Articles

From 2D to 3D In Vitro World: Sonodynamically-Induced Prooxidant Proapoptotic Effects of C60-Berberine Nanocomplex on Cancer Cells.

The primary objective of this research targeted the biochemical effects of SDT on human cervix carcinoma (HeLa) and mouse Lewis lung carcinoma (LLC) cells grown in 2D monolayer and 3D spheroid cell culture. HeLa and LLC monolayers and spheroids were treated with a 20 µM C60-Ber for 24 h, followed by irradiation with 1 MHz, 1 W/cm2 US. To evaluate the efficacy of the proposed treatment on cancer cells, assessments of cell viability, caspase 3/7 activity, ATP levels, and ROS levels were conducted. Our results revealed that US irradiation alone had negligible effects on LLC and HeLa cancer cells. However, both monolayers and spheroids irradiated with US in the presence of the C60-Ber exhibited a significant decrease in viability (32% and 37%) and ATP levels (42% and 64%), along with a notable increase in ROS levels (398% and 396%) and caspase 3/7 activity (437% and 246%), for HeLa monolayers and spheroids, respectively. Similar tendencies were observed with LLC cells. In addition, the anticancer effects of C60-Ber surpassed those of C60, Ber, or their mixture (C60 + Ber) in both cell lines. The detected intensified ROS generation and ATP level drop point to mitochondria dysfunction, while increased caspase 3/7 activity points on the apoptotic pathway induction. The combination of 1 W/cm2 US with C60-Ber showcased a promising platform for synergistic sonodynamic chemotherapy for cancer treatment.

Synthesis, molecular docking, dynamics study and preliminary cytotoxicity study of some coumarin linked imidazothiadiazoles

A new series of coumarin-linked imidazo[2,1-b][1,3,4]thiadiazoles were synthesized and evaluated for their cytotoxicity inhuman T-lymphocyte (CEM), human cervix carcinoma (HeLa) and murine leukemia cells (L1210). Among the tested compounds, 3-[5‑bromo-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazol-6-yl]-2H-chromen-2-one 9b exhibited cytotoxicity at 0.77 μM against CEM, 1.6 μM against L1210 and 0.38 μM against HeLa cells. Compound 6-(2-oxo-2H-chromen-3-yl)-2-(thiophen-2-ylmethyl)imidazo[2,1-b][1,3,4]thiadiazol-5-carbaldehyde 10b showed cytotoxicity at 3.5 μM against CEM and at 9.5 μM against HeLa cells. Further, molecular docking simulations performed to evaluate the possibility of these synthesized analogs as inhibitors of heat shock protein 90 (Hsp90), Pdb ID: 1UYL revealing that docking predictions are in good agreement with in-vitro results (-10.7 for 9b and -10.5 for 10b). The in-silico ADME parameters, pharmacokinetics and drug-likeness of all derivatives were examined in detail using the Swiss ADME webserver. By applying molecular dynamics simulation experiments a better stability was observed of compounds 8b, 9a, 10b and 11b.

Plakevulin A induces apoptosis and suppresses IL-6-induced STAT3 activation in HL60 cells

(+)-Plakevulin A (1), an oxylipin isolated from an Okinawan sponge Plakortis sp. inhibits enzymatic inhibition of DNA polymerases (pols) α and δ and exhibits cytotoxicity against murine leukemia (L1210) and human cervix carcinoma (KB) cell lines. However, the half-maximal inhibitory concentration (IC50) value for cytotoxicity significantly differed from those observed for the enzymatic inhibition of pols α and β, indicating the presence of target protein(s) other than pols. This study demonstrated cytotoxicity against human promyelocytic leukemia (HL60), human cervix epithelioid carcinoma (HeLa), mouse calvaria-derived pre-osteoblast (MC3T3-E1), and human normal lung fibroblast (MRC-5) cell lines. This compound had selectivity to cancer cells over normal ones. Among these cell lines, HL60 exhibited the highest sensitivity to (+)-plakevulin A. (+)-Plakevulin A induced DNA fragmentation and caspase-3 activation in HL60 cells, indicating its role in apoptosis induction. Additionally, hydroxysteroid 17-β dehydrogenase 4 (HSD17B4) was isolated from the HL60 lysate as one of its binding proteins through pull-down experiments using its biotinylated derivative and neutravidin-coated beads. Moreover, (+)-plakevulin A suppressed the activation of interleukin 6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3). Because the knockdown or inhibition of STAT3 induces apoptosis and HSD17B4 regulates STAT3 activation, (+)-plakevulin A may induce apoptosis in HL60 cell lines by suppressing STAT3 activation, potentially by binding to HSD17B4. The present findings provide valuable information for the mechanism of its action.

Does Low-Level Laser Therapy affect Bcl-2 Gene Expression in Oral Squamous Cell Carcinoma; An invitro study

Objectives: Oral squamous cell carcinoma, which is known as the most common cancer of the oral cavity, is associated with high morbidity and mortality rate. To date, many efforts have been made to find effective methods to stop the growth of cancer cells. One of the emerging methods in this field is the application of low-level lasers.
 Material and Method: Human head and neck carcinoma cell lines were prepared and irradiated with four different wavelengths of low-level laser with a density of 1 j/cm2 and a power of 100 mW. The expression of the Bcl-2 and Bax genes and the Bax / Bcl-2 ratio were investigated by real-time PCR.
 Results: The highest percentage of Bcl-2 gene expression was related to 660 nm wavelength and the highest percentage of Bax gene expression was related to 810 nm infrared wavelength. The lowest level of expression of the Bcl-2 gene related to the 810 nm infrared wavelength and the Bax gene related to the 532 nm wavelength was obtained. The lowest Bax/Bcl-2 ratio was obtained at the wavelength of 660 nm and the highest ratio was obtained at the wavelength of 810 nm.
 Conclusion: Despite the extensive studies conducted in the field of low-level laser application in oncology, more studies are needed to investigate the effect of this technology on cancer cells.

Anti-cancer drug-mediated increase in mitochondrial mass limits the application of metabolic viability-based MTT assay in cytotoxicity screening.

The high-throughput metabolic viability-based colorimetric MTT test is commonly employed to screen the cytotoxicity of different chemotherapeutic drugs. The assay assumes a cell density-dependent linear correlation with the MTT spectral absorbance. Therefore, the present study aimed to compare the cytotoxicity assessment between the MTT assay and gold standard cell number enumeration. The cytotoxicity was induced by Cisplatin, Etoposide, and Doxorubicin in human lung epithelial adenocarcinoma cells (A549) and cervix carcinoma (HeLa) cell lines. The mitochondrial mass was estimated, and immunoblotting of succinate dehydrogenase (SDH-A) was performed following drug treatment in both cell lines. Student's t-test paired analysis was employed to calculate the significance of the results, where the value p < 0.05 was considered statistically significant. The drug-induced cytotoxic response estimated by MTT absorbance did not show any significant difference with respect to control, and no correlation was observed with the enumerated cell number in both A549 and HeLa cells. Interestingly, per-cell metabolic viability was found to be increased by 1.18 to 3.26-fold (p < 0.05) following drug treatment. Further, mechanistic investigation revealed a drug concentration-dependent significant increase in mitochondrial mass (1.21 to 4.2-fold) and upregulation of SDH protein (50-70%) as well as enzymatic activity with respect to control in both A549 and Hela cells. The limitation of the MTT assay for drug-induced cytotoxicity assessment is due to increased mitochondrial mass and SDH upregulation in surviving cells, leading to enhanced formazan formation. This leads to a lack of correlation between cell number and MTT spectral absorbance, suggesting that the MTT assay may provide an erroneous conclusion for cytotoxicity assessment.

Concomitant [18F]F-FAZA and [18F]F-FDG Imaging of Gynecological Cancer Xenografts: Insight into Tumor Hypoxia.

Herein we assessed the feasibility of imaging protocols using both hypoxia-specific [18F]F-FAZA and [18F]F-FDG in bypassing the limitations derived from the non-specific findings of [18F]F-FDG PET imaging of tumor-related hypoxia. CoCl2-generated hypoxia was induced in multidrug resistant (Pgp+) or sensitive (Pgp-) human ovarian (Pgp- A2780, Pgp+ A2780AD), and cervix carcinoma (Pgp- KB-3-1, Pgp+ KB-V-1) cell lines to establish corresponding tumor-bearing mouse models. Prior to [18F]F-FDG/[18F]F-FAZA-based MiniPET imaging, in vitro [18F]F-FDG uptake measurements and western blotting were used to verify the presence of hypoxia. Elevated GLUT-1, and hexokinase enzyme-II expression driven by CoCl2-induced activation of hypoxia-inducible factor-1α explains enhanced cellular [18F]F-FDG accumulation. No difference was observed in the [18F]F-FAZA accretion of Pgp+ and Pgp- tumors. Tumor-to-muscle ratios for [18F]F-FAZA measured at 110-120 min postinjection (6.2±0.1) provided the best contrasted images for the delineation of PET-oxic and PET-hypoxic intratumor regions. Although all tumors exhibited heterogenous uptake of both radiopharmaceuticals, greater differences for [18F]F-FAZA between the tracer avid and non-accumulating regions indicate its superiority over [18F]F-FDG. Spatial correlation between [18F]F-FGD and [18F]F-FAZA scans confirms that hypoxia mostly occurs in regions with highly active glucose metabolism. The addition of [18F]F-FAZA PET to [18F]F-FGD imaging may add clinical value in determining hypoxic sub-regions.

N-SALICYLOYL AMIDES AND THEIR SALTS: SYNTHESIS, BIOLOGICAL ACTIVITY AND TOXICITY

The Schotten-Baumann reaction was used to synthesize N-salicyloyl derivatives of Morpholine, Glycine and gamma-aminobutyric acid in the process between salicyloyl chloride with morpholine, glycine or gamma-aminobutyric acid with high yield. Sodium and lithium salts of the salicylamides were synthesized by the reaction of an amide with sodium ethylate or lithium hydroxide in inert solvent. The synthesized compounds were charachterized by 1H nuclear magnetic resonance spectra and elemental analysis. Purity was checked by thin layer chromatography. Quantitative analysis of metal ions was done by potentiometry. To select the most promising compounds exhibiting psychotropic (Pa &gt; 0.8) and antiviral (Pa about 0.5) activities, computer analysis was carried out through the PASS program. In the present study, psychotropic (the open-field exploratory test, the forced swim test, the elevated plus-maze test, the passive avoidance test), analgesic (current vocalization threshold) activities and acute toxicity were evaluated. The results of psychotropic activity revealed that amides possess significant psychotropic activity and low acute toxicity. Disodium salicylurate showed a significant antiamnesic action. The results suggested a significant antidepressant activity and antiamnesic action for dilithium salicylurate and sodium salt of salycyloyl morphlide, a psychostimulant action for lithium salt of salicyloyl morphlide. Lithium gamma-(N-salycylamino)butyrate demonstrated psychostimulant and antiamnesic activities. Our present investigation demonstrated that all compounds are very safe for consumption with high LD50 value. All obtained compounds were evaluated for their antiviral activities against a large number of DNA and RNA viruses including herpes simplex viruses 1 and 2, vaccinia virus, vesicular stomatitis virus, HIV-1 and HIV-2. These compounds were evaluated against human cervix carcinoma cells (HeLa) and CEM T-lymphocytes as well as murine leukemia cells (L1210). The antiviral activity studies depicted that none of the tested compounds were active against DNA or RNA viruses.

Cytotoxic clerodane diterpenoids from the roots of Casearia barteri Mast.

A study of diterpenoids as active ingredients against cancer from the active roots extract of Casearia barteri Mast. (IC50 = 1.57 μg mL-1) led to the isolation of six new clerodane diterpenoids, named as barterins A-F (1-6) alongside seven known compounds, caseamembrin A, caseamembrin E, casearlucin A, graveospene G, N-trans-feruloyltyramine, N-cis-feruloytyramine and sitosterol-3-O-β-D-(6-O-palmitoyl)-glucopyranoside. Their structures were elucidated based on NMR spectroscopic data and mass spectrometry. The absolute configurations of 1-6 were established by the time-dependent density functional theory (TDDFT), electronic circular dichroism (ECD) calculations and experimental data analysis. The cytotoxic effects of compounds 1-6 were evaluated against a human cervix carcinoma cell line KB-3-1. Barterins A-D (1-4) showed cytotoxic effects against the KB-3-1 cell line with IC50 values ranging from 1.34-4.73 μM.

Bioactive constituents of the stem bark of Bersama abyssinica Fresen subsp. abyssinica Verdc. (Melianthaceae)

A new xanthone glycoside (1) and a new bufadienolide (2) named bersamaxanthoside and bersamabufadienolide, respectively, together with seventeen known compounds (3–19) were isolated from the stem bark of Bersama abyssinica Fresen subsp. abyssinica Verdc. Their structures were elucidated based on their spectroscopic data. The extract, fractions and compounds were assessed for their cytotoxicity on the cervix carcinoma cell line KB-3–1. The MeOH extract and the EtOAc and n-BuOH soluble fractions exhibited weak activity with IC50 values of 0.085, 0.084 and 0.075 mg/mL, respectively. The other tested samples were inactive. In addition, some of the isolates were tested for their DPPH radical scavenging activity and only compounds 1, 11–13, 16, and 17 displayed antioxidant properties with IC50 values ranging from 2.9 to 38.1 µg/mL.

Integrated High-Throughput Screening and Large-Scale Isobolographic Analysis to Accelerate the Discovery of Radiosensitizers With Greater Selectivity for Cancer Cells

High-throughput screening (HTS) platforms have been widely used to identify candidate anticancer drugs and drug-drug combinations; however, HTS-based identification of new drug-ionizing radiation (IR) combinations has rarely been reported. Herein, we developed an integrated approach including cell-based HTS and computational large-scale isobolographic analysis to accelerate the identification of radiosensitizing compounds acting strongly and more specifically on cancer cells. In a 384-well plate format, 160 compounds likely to interfere with the cell response to radiation were screened on human glioblastoma (U251-MG) and cervix carcinoma (ME-180) cell lines, as well as on normal fibroblasts (CCD-19Lu). After drug exposure, cells were irradiated or not and short-term cell survival was assessed by high-throughput cell microscopy. Computational large-scale dose-response and isobolographic approach were used to identify promising synergistic drugs radiosensitizing cancer cells rather than normal cells. Synergy of a promising compound was confirmed on ME-180 cells by an independent 96-well assay protocol, and finally, by the gold-standard colony forming assay. We retained 4 compounds synergistic at 2 isoeffects in U251-MG and ME-180 cell lines and 11 compounds synergistically effective in only one cancer cell line. Among these 15 promising radiosensitizers, 5 compounds showed limited toxicity combined or not with IR on normal fibroblasts. Overall, this study demonstrated that HTS chemoradiation screening together with large-scale computational analysis is an efficient tool to identify synergistic drug-IR combinations, with concomitant assessment of unwanted toxicity on normal fibroblasts. It sparks expectations to accelerate the discovery of highly desired agents improving the therapeutic index of radiation therapy.

5′-O-methyl-14-hydroxyarmillane, a new armillane-type sesquiterpene from cultures of Guyanagaster necrorhiza

Protoilludene-type sesquiterpene aryl esters are a unique and very diverse compound class that were exclusively isolated from members of the genus Armillaria (Agaricomycetes, Physalacriaceae) up to this point. Herein, we describe the isolation and structural characterization of 5′-O-methyl-14-hydroxyarmillane (1), a new armillane-type derivative, that was obtained from submerged cultures of Guyanagaster necrorhiza (CBS 138623) together with the known congeners melleolide G (2), melleolide B (3), and 10-dehydroxy-melleolide B (4). ROESY data and coupling constants assigned the relative configurations of 1, while common absolute configurations were confirmed from comparison of its ECD spectrum to the one of 10-hydroxy-5′-methoxy-6′-chloroarmillane (5). Additionally, the configuration of melleolide G (2) was revised based on observed ROESY correlations. It is the first time that protoilludene-type sesquiterpene aryl esters were isolated from another genus, namely, Guyanagaster, that is closely related to Armillaria. 1–4 were evaluated for their biological activities in a serial dilution assay against several yeast, fungi, and bacteria, as well as in a cytotoxicity assay against different cell lines. Compound 4 was moderately active against Bacillus subtilis, Staphylococcus aureus, and Mucor hiemalis. Furthermore, 1, 3, and 4 showed weak cytotoxic effects against the mouse fibroblast cell line L929 and the cervix carcinoma cell line KB3.1.

5ʹ-Ectonucleotidase CD73/NT5E supports EGFR-mediated invasion of HPV-negative head and neck carcinoma cells

BackgroundEpithelial-to-mesenchymal transition (EMT) of malignant cells is a driving force of disease progression in human papillomavirus-negative (HPV-negative) head and neck squamous cell carcinomas (HNSCC). Sustained hyper-activation of epidermal growth factor receptor (EGFR) induces an invasion-promoting subtype of EMT (EGFR-EMT) characterized by a gene signature (“‘EGFR-EMT_Signature’”) comprising 5´-ectonucleotidase CD73. Generally, CD73 promotes immune evasion via adenosine (ADO) formation and associates with EMT and metastases. However, CD73 regulation through EGFR signaling remains under-explored and targeting options are amiss.MethodsCD73 functions in EGFR-mediated tumor cell dissemination were addressed in 2D and 3D cellular models of migration and invasion. The novel antagonizing antibody 22E6 and therapeutic antibody Cetuximab served as inhibitors of CD73 and EGFR, respectively, in combinatorial treatment. Specificity for CD73 and its role as effector or regulator of EGFR-EMT were assessed upon CD73 knock-down and over-expression. CD73 correlation to tumor budding was studied in an in-house primary HNSCC cohort. Expression correlations, and prognostic and predictive values were analyzed using machine learning-based algorithms and Kaplan–Meier survival curves in single cell and bulk RNA sequencing datasets.ResultsCD73/NT5E is induced by the EGF/EGFR-EMT-axis and blocked by Cetuximab and MEK inhibitor. Inhibition of CD73 with the novel antagonizing antibody 22E6 specifically repressed EGFR-dependent migration and invasion of HNSCC cells in 2D. Cetuximab and 22E6 alone reduced local invasion in a 3D-model. Interestingly, combining inefficient low-dose concentrations of Cetuximab and 22E6 revealed highly potent in invasion inhibition, substantially reducing the functional IC50 of Cetuximab regarding local invasion. A role for CD73 as an effector of EGFR-EMT in local invasion was further supported by knock-down and over-expression experiments in vitro and by high expression in malignant cells budding from primary tumors. CD73 expression correlated with EGFR pathway activity, EMT, and partial EMT (p-EMT) in malignant single HNSCC cells and in large patient cohorts. Contrary to published data, CD73 was not a prognostic marker of overall survival (OS) in the TCGA-HNSCC cohort when patients were stratified for HPV-status. However, CD73 prognosticated OS of oral cavity carcinomas. Furthermore, CD73 expression levels correlated with response to Cetuximab in HPV-negative advanced, metastasized HNSCC patients.ConclusionsIn sum, CD73 is an effector of EGF/EGFR-mediated local invasion and a potential therapeutic target and candidate predictive marker for advanced HPV-negative HNSCC.

The effect of matrices on the gene expression profile of patient-derived head and neck carcinoma cells for in vitro therapy testing

ObjectiveHead and neck squamous cell carcinoma (HNSCC) is a highly aggressive tumor with a 5-year mortality rate of ~ 50%. New in vitro methods are needed for testing patients’ cancer cell response to anti-cancer treatments. We aimed to investigate how the gene expression of fresh carcinoma tissue samples and freshly digested single cancer cells change after short-term cell culturing on plastic, Matrigel or Myogel. Additionally, we studied the effect of these changes on the cancer cells’ response to anti-cancer treatments.Materials/methodsFresh tissue samples from HNSCC patients were obtained perioperatively and single cells were enzymatically isolated and cultured on either plastic, Matrigel or Myogel. We treated the cultured cells with cisplatin, cetuximab, and irradiation; and performed cell viability measurement. RNA was isolated from fresh tissue samples, freshly isolated single cells and cultured cells, and RNA sequencing transcriptome profiling and gene set enrichment analysis were performed.ResultsCancer cells obtained from fresh tissue samples changed their gene expression regardless of the culturing conditions, which may be due to the enzymatic digestion of the tissue. Myogel was more effective than Matrigel at supporting the upregulation of pathways related to cancer cell proliferation and invasion. The impacts of anti-cancer treatments varied between culturing conditions.ConclusionsOur study showed the challenge of in vitro cancer drug testing using enzymatic cell digestion. The upregulation of many targeted pathways in the cultured cells may partially explain the common clinical failure of the targeted cancer drugs that pass the in vitro testing.

Fusobacterium nucleatum-triggered purine metabolic reprogramming drives tumorigenesis in head and neck carcinoma

BackgroundFusobacterium nucleatum (F. nucleatum) is a vital pro-oncogenic bacterium. Our previous study revealed that a high abundance of F. nucleatum in head and neck squamous cell carcinoma (HNSCC) is correlated with poor patient prognosis. However, the impact of F. nucleatum on metabolic reprogramming and tumor progression in HNSCC awaits more exploration.MethodsLiquid chromatography‒mass spectrometry (LC‒MS) was applied to analyze the altered metabolites in a head and neck carcinoma cell line (AMC-HN-8) after coculture with F. nucleatum for 24 hrs and 48 hrs. Both univariate and multivariate analyses were used to screen for differential metabolites. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway enrichment analysis was further used to explore the metabolic changes.ResultsWe observed a significantly altered metabolic profile in AMC-HN-8 cells over time after coculture with F. nucleatum. Among the several enriched pathways, the purine metabolic pathway was the most significantly enriched (P = 0.0005), with downregulation of purine degradation. Furthermore, uric acid, the end product of purine metabolism, significantly reversed F. nucleatum-triggered tumor progression and altered the intracellular reactive oxygen species (ROS) level. Moreover, the negative correlation between the serum uric acid level and the abundance of F. nucleatum was verified in 113 HNSCC patients (P = 0.0412, R = − 0.1924).ConclusionsOur study revealed obviously aberrant purine metabolism driven by F. nucleatum in HNSCC, which was closely related to tumor progression and patient prognosis. These findings indicate the possibility of targeting F. nucleatum-induced purine metabolism reprogramming in the future treatment of HNSCC.

An insight into hypothesized biological mechanisms contributing to the Flash effect

In recent years, FLASH radiotherapy (FLASH RT) has gained attention in preclinical studies as a potential advancement in cancer treatment. The great advantage of FLASH RT is the ultra-fast, high doses delivery that have a similar or greater effect on cancer cells while sparing normal, healthy tissue surrounding the tumor site. This is known as the FLASH effect. However, currently, there are not enough in vitro and in vivo data to transpose FLASH RT to human trials. This mini review summarizes the available in vitro data on electron beam FLASH, focusing on possible mechanisms of the FLASH effect. Current studies have focused on various types of cancer, including lung cancer, glioblastoma, uterus adenocarcinoma, cervix carcinoma, prostate carcinoma, melanoma, breast cancer, head and neck squamous cell carcinoma, and colon adenocarcinoma. Several hypotheses have been proposed to explain the biological mechanisms contributing to the to the selective FLASH effect, including differences between healthy and cancer cells in production of reactive oxygen species and free radicals, limitation of Fenton reaction caused by high Fe2+/3+ levels in tumor cells, and impaired DNA damage repair mechanisms occurring in cancer.

Biological Activity of Naphthoquinones Derivatives in the Search of Anticancer Lead Compounds.

Naphthoquinones are a valuable source of secondary metabolites that are well known for their dye properties since ancient times. A wide range of biological activities have been described highlighting their cytotoxic activity, gaining the attention of researchers in recent years. In addition, it is also worth mentioning that many anticancer drugs possess a naphthoquinone backbone in their structure. Considering this background, the work described herein reports the evaluation of the cytotoxicity of different acyl and alkyl derivatives from juglone and lawsone that showed the best activity results from a etiolated wheat coleoptile bioassay. This bioassay is rapid, highly sensitive to a wide spectrum of activities, and is a powerful tool for detecting biologically active natural products. A preliminary cell viability bioassay was performed on cervix carcinoma (HeLa) cells for 24 h. The most promising compounds were further tested for apoptosis on different tumoral (IGROV-1 and SK-MEL-28) and non-tumoral (HEK-293) cell lines by flow cytometry. Results reveal that derivatives from lawsone (particularly derivative 4) were more cytotoxic on tumoral than in non-tumoral cells, showing similar results to those obtained with of etoposide, which is used as a positive control for apoptotic cell death. These findings encourage further studies on the development of new anticancer drugs for more directed therapies and reduced side effects with naphthoquinone skeleton.

Apigenin targets protein and lipids of HeLa cells and exhibits protective efficacy against oxidative stress

Apigenin, non-toxic and non-mutagenic flavonoid, is an alternative to classical drugs that interact with membranes. Hence, the objects of the study were to examine its effects on liposomes made of egg yolk phosphatidylcholine (EYPC) with the use of 1H NMR, four-electrode BLM and EPR techniques, and determine its activity on lipids and proteins of human cervix carcinoma (HeLa) cells. In addition its protective efficacy against H2O2- induced oxidative shock was investigated. FTIR spectroscopy was applied to study molecular interactions with membrane lipids and proteins of HeLa cells. Microscopic techniques (SEM, light, and fluorescence), flow cytometer analysis, and NR assays were employed to reveal apigenin involvement in apoptosis in different cell conditions. Apigenin affected mainly the region of choline head groups and the hydrophobic core below this area. Simultaneously, the ordering effect was shown. The fingerprint region of lipids in the HeLa cells was found as a target for apigenin. Furthermore, in the amide I and amide II regions, a decrease in β-sheets and an increase in turns, loops, and unordered structures were noted. Apigenin reduced viability of cells and induced apoptosis. SEM observations revealed characteristic changes in morphology of the examined cells. Pretreatment of HeLa cells with apigenin protected them against H2O2-induced oxidative stress by the increase in glutathione content as well as superoxide dismutase and catalase levels. The consequences of molecular changes related to membranes and cancer cells make apigenin a unique and very interesting bio-compound with great significance for medical and biological applications.

Synthesis and Preliminary Evaluations of [18F]fluorinated Pyridine-2- carboxamide Derivatives for Targeting PD-L1 in Cancer.

Treatment with immune checkpoint inhibitors has improved both progressionfree survival and overall survival in a subset of patients with tumors. However, the selection of patients who benefit from immune checkpoint inhibitor treatment remains challenging. Positron Emission Tomography (PET) is a non-invasive molecular imaging tool that offers a promising alternative to the current IHC for detecting the PD-L1 expression in malignant cells in vivo, enabling patient selection and predicting the response to individual patient immunotherapy treatment. Herein, we report the development of novel [18F]labeled pyridine-2-carboxamide derivatives [18F]2 and [18F]3 as small-molecule probes for imaging immune checkpoint (PD-1/PD-L1) in cancer using PET. [18F]2 and [18F]3 were prepared by a one-step radiofluorination in 44 ± 5% and 30 ± 4% radiochemical yield and > 98% radiochemical purity for a potential clinical translation. The total synthesis time, including HPLC purification, was less than 45 min. [18F]2 and [18F]3 showed excellent stability in injection solution and a significant accumulation and retention in PD-1/PD-L1 expressing MDA-MB-231 breast cancer and in HeLa cervix carcinoma cells (2- 5 cpm/1000 cells). In addition, autoradiographic analysis and inhibition experiments on tumor slices confirm the potential of both compounds as specific imaging probes for the PD-1/PD-L1 axis in tumors. The in vitro evaluation in PD-L1 expressing cells together with results from autoradiographic analysis in PD-L1 positive tumor sections, suggest that [18F]2 and [18F]3 could be potential imaging probes for assessing PD-L1 expression in tumors and warrant further biological evaluations in vivo.

Abstract 327: Knockdown of VRK1 inhibits the proliferation of small cell neuroendocrine carcinoma of the uterine cervix

Abstract Background: The prognosis of small cell neuroendocrine carcinoma of the uterine cervix (SCNECC) is extremely poor due to the aggressive natural history. Since it is a rare cancer, there are few studies describing its molecular biology. Small cell lung cancer (SCLC) accounts for 95% of small cell carcinomas, therefore, clinically the chemotherapy regimens used for SCNECC have been based on regimens used for SCLC. This study aims to detect therapeutic targets for SCNECC by taking clues from the molecular biological commonalities between SCNECC and SCLC. Methods: We analyzed protein expression of cancer tissue-originated spheroids from lung and cervical cancers using isobaric tags for relative and absolute quantitation (iTRAQ). In vitro, protein expression levels of VRK1 in SCNECC cell lines (TC-YIK and HCSC-1), SCLC cell lines (SBC3 and OS2RA) and cell lines of mucinous adenocarcinoma and squamous cell carcinoma of the uterine cervix (HeLa and ME180) were assessed by western blotting (WB), and the change of cell proliferation induced by siRNA/shRNA-mediated VRK1 knockdown was assessed by WST-8 assay. In vivo, in mice xenograft models of SCNEC, the change of tumor size due to downregulation of VRK1 by shRNA were assessed. Furthermore, to explore the possible role of VRK1 in small carcinoma, pathway enrichment and ontology analysis between VRK1-high and -low-SCLC was performed by Gene Set Enrichment Analysis (GSEA), and it was compared with RNA-seq results of between shRNA-mediated VRK1 knockdown and control SCNECC cell lines. Results: iTRAQ showed that SCNECC and SCLC revealed similar protein expression profiles, rather than organ of origin-specific patterns. In addition, higher expression of VRK1 was detected in small cell carcinomas compared to the others. (SCLC/mucinous adenocarcinoma of the lung=2.86, SCNECC/mucinous adenocarcinoma of the cervix=2.56, SCNECC/squamous cell carcinoma of the cervix=2.17). WB showed that all SCNECC and SCLC cell lines expressed more highly VRK1 than other cervical cancer cell lines. In vitro, siRNA/shRNA-mediated VRK1 knockdown significantly suppressed the cell proliferation of SCNECC and SCLC (-29.0±0.3% in SBC-3 and -33.1±6.7% in TC-YIK, p&amp;lt;0.05, other cell lines were comparable). In vivo, the downregulation of VRK1 significantly suppressed tumor size in mice xenograft models of SCNEC (1726±442mm3 vs 544±233mm3 in TC-YIK, p&amp;lt;0.05, HCSC-1 was comparable). GSEA showed that VRK1 might participate in DNA repair to affect the biological process of SCLC, and the genes contained in this gene set was also detected in RNA-seq of SCNEC, suggesting involvement with VRK1. Conclusion: These findings suggest that VRK1 is involved in the proliferation of SCNECC in relation to DNA repair pathways. Our ongoing research is investigating the functional relationship between VRK1 and genes related to DNA repair and its effect on proliferation of SCNECC in vitro. Citation Format: Mariya Kobayashi, Satoshi Nakagawa, Yuji Kamei, Tatsuo Masuda, Mamoru Kakuda, Kosuke Hiramatsu, Ai Miyoshi, Tomomi Takata, Toshihiro Kimura, Eiji Kobayashi, Yutaka Ueda, Tadashi Kimura. Knockdown of VRK1 inhibits the proliferation of small cell neuroendocrine carcinoma of the uterine cervix [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 327.

Anticancer evaluation and molecular docking of new pyridopyrazolo-triazine and pyridopyrazolo-triazole derivatives

3-Amino-4,6-dimethylpyrazolopyridine was applied as a precursor for the synthesis of some new pyridopyrazolo-triazine and pyridopyrazolo-triazole derivatives through diazotization, followed by coupling with many 2-cyanoacetamide compounds, ethyl 3-(phenylamino)-3-thioxopropanoate, 3-oxo-N-phenylbutanethioamide, and α-bromo-ketone reagents [namely; 2-bromo-1-(4-fluorophenyl)ethan-1-one, 5-bromo-2-(bromoacetyl)thiophene, 3-(2-bromoacetyl)-2H-chromen-2-one and/or 3-chloroacetylacetone]. The prepared compounds were identified by spectroscopic analyses as IR, 1H NMR, and mass data. The anticancer activity of these pyrazolopyridine analogues was investigated in colon, hepatocellular, breast, and cervix carcinoma cell lines. The pyridopyrazolo-triazine compound 5a substituted with a carboxylate group gave a distinguished value of IC50 = 3.89 µM against the MCF-7 cell line compared to doxorubicin as a reference drug. Also, the pyridopyrazolo-triazine compound 6a substituted with the carbothioamide function gave good activity toward HCT-116 and MCF-7 cell lines with IC50 values of 12.58 and 11.71 µM, respectively. The discovered pyrazolopyridine derivatives were studied theoretically by molecular docking, and this study exhibited suitable binding between the active sides of pyrazolopyridine ligands and proteins (PDB ID: 5IVE). The pyridopyrazolo-triazine compound 6a showed the highest free binding energy (− 7.8182 kcal/mol) when docked inside the active site of selected proteins.