Late Phase Of Apoptosis Research Articles

Gd-GQDs as nanotheranostic platform for the treatment of HPV-positive oropharyngeal cancer.

In this study, we developed new gadolinium-graphene quantum dot nanoparticles (Gd-GQDs) as a theranostic platform for magnetic resonance imaging and improved the efficiency of radiotherapy in HPV-positive oropharyngeal cancer. Based on cell toxicity results, Gd-GQD NPs were nontoxic for both cancer and normal cell lines up to 25µg/ml. These NPs enhance the cytotoxic effect of radiation only on cancer cells but not on normal cells. The flow cytometry analysis indicated that cell death mainly occurred in the late phase of apoptosis. The immunocytochemical analysis was used to evaluate apoptosis pathway proteins. The Bcl-2 and p53 protein levels did not differ statistically significantly between radiation alone group and those that received irradiation in combination with NPs. In contrast, the combination group exhibited a significant increase in Bax protein expression, suggesting that cells could undergo apoptosis independent of the p53 pathway. Magnetic resonance (MR) imaging showed that Gd-GQD NPs, when used at low concentrations, enhanced T1-weighted signal intensity resulting from T1 shortening effects. At higher concentrations, the T2 shortening effect became predominant and was able to decrease the signal intensity. Gd-GQD appears to offer a novel approach for enhancing the effectiveness of radiation treatment and facilitating MR imaging for monitoring HPV-positive tumors.

Human papillomavirus-16 E6-positive cervical cancer attenuated by potent 2-(4-biphenylyl)-N-(1-ethyl-4-piperidinyl) acetamide second-generation analogs with improved binding affinity.

Human papillomavirus (HPV) infection, particularly HPV16, is a major contributor to the development of cervical cancer. Given the urgent need for novel therapeutic strategies targeting HPV-associated cancers, this study focuses on characterizing second-generation analogs of a lead compound, as a potential inhibitor of HPV16-E6. Protein-ligand docking, Gibbs binding free energy estimation, and molecular dynamics simulations were conducted. HPV16-infected SiHa and CaSki cell lines were used. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay for proliferation and flow cytometry for target inhibition and apoptosis were employed. Computational and cell proliferation analyses revealed that modifications to E6-855, particularly in the piperidinyl group, enhanced binding affinities against HPV16-E6, with E6-272 demonstrating superior binding properties. Molecular dynamics simulations confirmed the stable binding of E6-272 to HPV16-E6, supported by favorable binding energy estimates. E6-272 inhibited the proliferation of SiHa and CaSki cells with GI50 values of 32.56 and 62.09nM, respectively. The compound reduced HPV16-E6-positive population, while inducing the early and late phase apoptosis in these cells. Structural alterations at the piperidinyl group of E6-855 identified E6-272 as a promising inhibitor of HPV16-E6 with improved efficacy against HPV16-E6. Further experimental validation of E6-272 and its analogs warrant to advance its clinical utility in combating HPV-associated cancers.

C-5401331 identified as a novel T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) inhibitor to control acute myeloid leukemia (AML) cell proliferation.

T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is a checkpoint protein expressed in exhausted T-cells during cancer scenarios. This exhaustion may end in T-cell effector dysfunction, resulting in suboptimal control of cancers like acute myeloid leukemia (AML). Use of immune checkpoint inhibitors (ICIs) to block checkpoint receptors such as Tim-3 is an emerging, revolutionary concept in the immuno-oncology therapeutic arena; however, ICIs are not effective on myeloid malignancies. Here, a multifaceted approach is utilized to identify novel compounds that target and inhibit Tim-3 with improved efficacy. High-throughput virtual screening of the ChemBridge small molecule library and molecular dynamics simulation yielded a lead molecule C-5401331 predicted to bind with high affinity and inhibit the activity of Tim-3. In vitro evaluations demonstrated the compound to have anti-proliferative effects on Tim-3-positive populations of THP-1 and HC-5401331 AML cells, inducing early and late phase apoptosis. With further development, the lead molecule identified in this work has potential to aid the natural "gatekeeper" functions of the body in immunocompromised AML cancer patients by successfully hampering the binding of Tim-3 to T-cells.

C-71980262, a novel small molecule against human papilloma virus-16 E6 (HPV-16 E6) with anticancer potency against cervical cancer: A computational guided in vitro approach.

Human papillomavirus-16 E6 (HPV-16 E6) forms a heterodimer complex to up-regulate the degradation of tumor suppressor protein p53 to promote cervical cancer. This study aims to identify a novel small molecule against E6 with anticancer efficacy against HPV-16, a prime high-risk serotype inducer for cervical cancer. Autodock-vina-based high-throughput virtual screening and atomistic molecular dynamic simulations were used for identification of targeted lead molecules. HPV-16 infected SiHa and CaSki cell lines were used to validate the lead compound in vitro. Proliferation of cancer cells was analyzed by MTT assay and flow cytometry was used to analyze target inhibition, apoptosis, and p53. High throughput virtual screening and molecular dynamic simulation identified C-71980262 as a lead candidate that could bind HPV-E6. Atomistic molecular dynamic simulation of E6 bound C-71980262 for 200 ns showed that the predicted ligand binding was stable with minimal energy expenditure, proposing the viability and veracity of the assessed molecule. C-71980262 inhibited the proliferation of SiHa and CaSki cells with GI50 values of 355.70 nM and 505.90 nM, respectively. The compound reduced HPV-16 E6 while inducing early and late-phase apoptosis in these cells. Treatment with C-71980262 increased the p53-positive populations in SiHa and CaSki cells. C-71980262 was identified as a novel lead molecule that could inhibit the HPV-16 E6 and increase p53 in cervical cancer cells. Further in vitro and in vivo validation is warranted to consolidate and corroborate this lead compound against HPV-induced cancer progression.

Yttrium Oxide nanoparticles induce cytotoxicity, genotoxicity, apoptosis, and ferroptosis in the human triple-negative breast cancer MDA-MB-231 cells

BackgroundTriple-negative breast cancer (TNBC) is a lethal mammary carcinoma subtype that affects females and is associated with a worse prognosis. Chemotherapy is the only conventional therapy available for patients with TNBC due to the lack of therapeutic targets. Yttrium oxide (Y2O3) is a rare earth metal oxide, whose nanoparticle (NPs) formulations are used in various applications, including biological imaging, the material sciences, and the chemical synthesis of inorganic chemicals. However, the biological activity of Y2O3-NPs against TNBC cells has not been fully explored. The current study was conducted to assess Y2O3-NPs’ anticancer activity against the human TNBC MDA-MB-231 cell line.MethodsTransmission electron microscopy (TEM), X-ray diffraction, Zeta potential, and dynamic light scattering (DLS) were used to characterize the Y2O3-NPs. SRB cell viability, reactive oxygen species (ROS) measurement, single-cell gel electrophoresis (comet assay), qPCR, flow cytometry, and Western blot were employed to assess the anticancer activity of the Y2O3-NPs.ResultsOur results indicate favorable physiochemical properties of Y2O3-NPs (with approximately average size 14 nm, Zeta Potential about − 53.2 mV, and polydispersity index = 0.630). Y2O3-NPs showed a potent cytotoxic effect against MDA-MB-231 cells, with IC50 values of 74.4 µg/mL, without cytotoxic effect on the normal retina REP1 and human dermal fibroblast HDF cell lines. Further, treatment of MDA-MB-231 cells with IC50 Y2O3-NPs resulted in increased oxidative stress, accumulation of intracellular ROS levels, and induced DNA damage assessed by Comet assay. Upon Y2O3-NPs treatment, a significant increase in the early and late phases of apoptosis was revealed in MDA-MB-231 cells. qPCR results showed that Y2O3-NPs significantly upregulated the pro-apoptotic genes CASP3 and CASP8 as well as ferroptosis-related gene heme oxygenase-1 (HO-1), whereas the anti-apoptotic gene BCL2 was significantly downregulated.ConclusionThis study suggests that Y2O3-NPs are safe on normal REP1 and HDF cells and exhibited a potent selective cytotoxic effect against the TNBC MDA-MB-231 cells through increasing levels of ROS generation with subsequent DNA damage, and induction of apoptosis and ferroptosis.

Antiproliferative and Apoptotic Effects of Olive Leaf Extract Microcapsules on MCF-7 and A549 Cancer Cells.

Alginate microcapsules are a talented means for the delivery of broad curative biomacromolecules. In this study, we immobilized olive leaf extract (OLE) by calcium alginate (CA) and chitosan-coated CA (CCA) and characterized the OLE-loaded CA and CCA. The cytotoxic effect, the cell cycle arrest, and the apoptotic effect of OLE and its microcapsules were investigated against breast adenocarcinoma (MCF-7) and lung carcinoma (A549). As a result, the loading capacity of OLE-CA and OLE-CCA was found to be 80 and 99%, respectively, in optimal conditions. Also, OLE-CA and OLE-CCA were characterized by unique FTIR peaks and morphological display relative to the empty CCA microcapsules. The cytotoxicity analysis showed that the IC50 values of OLE-CA and OLE-CCA were determined to be 312 and 0.94 μg mL-1 against A549, respectively, whereas these were found to be 865.4 and 425.5 μg mL-1 for MCF-7 cells. On the other hand, the OLE microcapsules did not possess in any concentration of cytotoxic influence on the BEAS 2B healthy cell line. Also, the exposure of OLE-CCA to MCF-7 and A549 resulted in the arrest of more MCF-7 and A549 cells at the G0/G1 phase compared to the OLE. A549 and MCF-7 cells were predominantly found in the late apoptosis phase and necrosis phase, respectively. Optical microscopy images confirmed that OLE microcapsules were more effective against MCF-7 and A549 than free OLE. The present work suggested that the OLE microcapsules might be administered as nutrition supplements for cancer therapy.

Novel fused imidazotriazines acting as promising top. II inhibitors and apoptotic inducers with greater selectivity against head and neck tumors: Design, synthesis, and biological assessments

Although the great effectiveness of doxorubicin (Dox) in the treatment of many types of tumors, it showed limited effectiveness against the head and neck squamous cell carcinoma (HNSCC) subtype which is attributed to its reported multiple drug resistance (MDR). In the current study, we considered the essential pharmacophoric features of Dox as an effective Top. II inhibitor and sought to develop a novel set of imidazo[1,2-a] [1,3,5]triazin-2-amines (2a-2p) as a suggested anticancer option that could intercalate the DNA base pairs. We evaluated the % inhibition of the newly synthesized compounds on thirteen cancer cell lines and the analysis of structure-activity relationships revealed that the human head and neck cancer cell line (HNO97) was the most sensitive to their growth inhibition effect. Then, the IC50 values were recorded against the most sensitive cancer cell lines (HNO97, MDA-MB-231, and HEPG2), and compared to the normal cell line OEC (human oral epithelial cells). Compounds 2f and 2g showed very strong activities against HNO97 with IC50 values of (4 ± 1 and 3 ± 1.5 μg/mL), respectively, compared to that of Dox (9 ± 1.6 μg/mL). Next, a quantitative determination of human DNA Top. II concentrations in the most sensitive cell line (HNO97) were recorded for the most active anticancer derivatives. Again, compound 2f showed a superior Top. II inhibition with 87.86% compared to that of Dox (86.44%), while compound 2g achieved an inhibition of 81.37% which was close to the effect of Dox. To further investigate their effects on cell cycle progression and apoptosis induction in HNO97 cells, both 2f and 2g were selected for analysis. Both candidates arrested cell cycle progression at both the S and G2-M phases, as well as increased the early and late apoptosis phase ratios. Besides, both 2f and 2g were subjected to protein expression analysis of apoptosis-related genes (p53, BAX, IL-6, and BCL2). Moreover, the antioxidant effect of 2f and 2g was evaluated by measuring GSH, MDA, and NO markers in HNO97 cells. Furthermore, molecular docking for the newly designed tricyclic derivatives against both the Top. II and DNA double helix was carried out.

Auraptene-induced cytotoxic effects in acute myeloid leukemia cell lines.

Acute myeloid leukemia is one of the most commonly identified hematological malignancies with poor prognosis. This research was planned to identify the cytotoxic effects of Auraptene on HL60 and U937 cell lines. The cytotoxic effects of Auraptene were measured by AlamarBlue assay (Resazurin) after 24- and 48-h treatments with different doses of Auraptene. The inductive effects of Auraptene on cellular oxidative stress were investigated by determining cellular ROS levels. The cell cycle progression and cell apoptosis were also evaluated by flow cytometry method. Our findings revealed that Auraptene decreased HL60 and U937 cellular proliferation by downregulation of Cyclin D1. Auraptene also induces cellular oxidative stress by upregulation of cellular ROS levels. Auraptene induces cell cycle arrest the early and late phases of apoptosis by upregulation of Bax and p53 proteins. Our data suggest that the anti-tumor function of Auraptene can be mediated by promoting apoptosis and cell cycle arrest and inducing cellular oxidative stress in HL60 and U937 cell lines. These results support that Auraptene may be used as a potent anti-tumor agent against hematologic malignancies in the further studies.

A Potential Anti-Glioblastoma Compound LH20 Induces Apoptosis and Arrest of Human Glioblastoma Cells via CDK4/6 Inhibition.

Glioblastoma (GBM) is a deadly brain tumor characterized by signaling dysregulation and aberrant cell cycle control. The CDK4/6-Rb axis is dysregulated in approximately 80% of all GBM cases. In this study, the anti-GBM effect of a novel pyrimidin-2-amine, LH20 was evaluated in vitro using the primary GBM cell lines U87MG and U251. GBM cells were administered LH20 at concentrations of 0.1, 1, 4, 8, 10, 20, 100, and 200 µM for 24 and 48 h, and the proliferation rate was evaluated using a CCK8 assay. Migration, apoptosis, and cell cycle were also assessed using a wound healing assay, Annexin V-FITC/PI apoptosis assay, and cell cycle staining, respectively. The targets of LH20 were predicted using SwissTargetPrediction and molecular docking. Western blotting analysis was performed to confirm the anti-GBM mechanism of LH20. We found that at concentrations of 4, 8, and 10 µM, LH20 significantly inhibited the proliferation and migration of U87MG and U251 cells, induced late phase apoptosis, promoted tumor cell necrosis, and arrested the G2/M phase of the cell cycle. LH20 also inhibited CDK4 and CDK6 activities by decreasing the phosphorylation of Rb. Our results suggest LH20 as a potential treatment strategy against GBM.

Phase 1B/2A safety, pharmacokinetics and pharmacodynamics study of fosciclopirox alone and in combination with cytarabine in patients with relapsed/refractory acute myeloid leukemia.

TPS7078 Background: Fosciclopirox (CPX-POM) is a broad-spectrum anticancer agent being developed through a public-private partnership between CicloMed LLC and KU Cancer Center (KUCC). Following intravenous administration, fosciclopirox is rapidly and completely metabolized to its active metabolite ciclopirox (CPX). Given fosciclopirox is a prodrug lacking pharmacologic activity, in vitro mechanistic studies were conducted utilizing CPX as the test article. In collaboration with Notable Labs (Foster City CA), we characterized ex vivo sensitivity to CPX in bone marrow and peripheral blood samples from ten newly diagnosed and relapsed acute myeloid leukemia (AML) patients. Reductions in blast counts were seen in each of the 10 patients studied at clinically relevant concentrations. In vitro experiments in AML cell lines (HL-60 and MV4-11) characterized the mechanism(s) of the anti-leukemia activity of CPX. CPX inhibited proliferation of AML cell lines in a dose- and time-dependent manner with IC50 values ranging from 2.5-4 µM. CPX induced cell cycle arrest in the subG0 phase and CPX-treated cells lost their ability to enter to G0-G1 phase. Moreover, CPX induced early and late phase apoptosis. CPX also activated Caspase 3/7 activity. This preclinical data led to the development of a multi-center Phase 1B/2A clinical proof of concept trial (NCT04956042) in AML patients. Methods: Eligible AML patients are adults with primary refractory (two prior regimens) or relapsed AML; ECOG PS 0-2; adequate end organ function. Prior allogeneic stem cell transplant is allowed if patient is more than 100 days post-transplant with no active graft-versus-host disease. CPX-POM is administered as 900 mg/m2 once daily as an IV infusion on Days 1 to 5 of each 21-day cycle. Day 8 bone marrow sample is collected for correlative studies. Ex vivo Drug Sensitivity Screening (DSS) will be performed on samples obtained prior to treatment and C1 Days 8 and 21. 14 evaluable patients will be enrolled in Cohort 1a. If disease response is observed in at least 4 of 14 patients, an additional 14 patients will be enrolled in Cohort 1b. During Cohort 1a, patients who do not respond to CPX-POM alone may be switched to the combination of CPX-POM + cytarabine. Patients who respond to the combination treatment may continue until evidence of disease progression. If disease response to CPX-POM alone is not observed in at least 4 of 14 patients in Cohort 1a, the study may be terminated or a Cohort 2a may be initiated using the combination of CPX-POM and cytarabine. The trial is currently open at two sites with 14 patients enrolled and 8 evaluable for the primary endpoint. Clinical trial information: NCT04956042 .

Multiple Beneficial Effects of Aloesone from Aloe vera on LPS-Induced RAW264.7 Cells, Including the Inhibition of Oxidative Stress, Inflammation, M1 Polarization, and Apoptosis

Aloesone is a major metabolic compound in Aloe vera, which has been widely used as a food source and therapeutic agent in several countries. Our recent study demonstrated that aloesone has anti-epileptic effects on glutamate-induced neuronal injury by suppressing the production of reactive oxygen species (ROS). Unless ROS are naturally neutralized by the endogenous antioxidant system, they lead to the activation of inflammation, polarization, and apoptosis. This study aimed to identify the multiple beneficial effects of aloesone and explore its molecular mechanism in macrophages. Hence, the murine macrophage cell line RAW264.7 was pretreated with aloesone and then exposed to lipopolysaccharides (LPS). The results demonstrated that aloesone, within a dosage range of 0.1-100 µM, dramatically decreased the LPS-induced elevation of ROS production, reduced nitric oxide (NO) release, inhibited the M1 polarization of RAW264.7 cells, and prevented cells from entering the LPS-induced early and late phases of apoptosis in a dose-dependent manner. Simultaneously, aloesone significantly decreased the mRNA expression of inflammation-related genes (iNOS, IL-1ꞵ, TNF-α) and increased the expression of antioxidant enzymes (Gpx-1 and SOD-1). The core genes HSP90AA1, Stat3, Mapk1, mTOR, Fyn, Ptk2b, and Lck were closely related to these beneficial effects of aloesone. Furthermore, immunofluorescence staining and flow cytometry data confirmed that aloesone significantly repressed the activation of mTOR, p-mTOR, and HIF-1α induced by LPS and inhibited the protein expression of TLR4, which is the target of LPS. In conclusion, aloesone demonstrated multiple protective effects against LPS-induced oxidative stress, inflammation, M1 polarization, and apoptosis in macrophages, suggesting its potential as a prodrug.

Theranostics platform of Abemaciclib using magnetite@silica@chitosan nanocomposite

The current study was designed to synthesize a nanoformula comprising of magnetite nanoparticles (MN) with mesoporous silica (MS), which was in turn coated with chitosan (CS) and further loaded with a chemotherapeutic agent, Abemaciclib (ABE). The prepared formula, MN@MS@CS@ABE, was characterized by XRD, FTIR, HRTEM, FESEM, DLS, VSM, BET, and BJH. The ABE loading capacity and entrapment efficiency were calculated, and an in vitro drug release experiment was conducted. Cytoxicity was studied by the MTT assay. The formula was investigated as an anticancer agent versus MCF-7 cells by performing Annexin V-FITC flow cytometry and cell cycle analysis. Furthermore, we examine the formula as a contrast agent in magnetic resonance imaging (MRI). ABE loading and encapsulation efficiency were 24.2 % and 63.4 %, respectively. The formula demonstrated sustained drug release behavior for 72 h. The MTT assay revealed a higher cytotoxicity of free ABE in MCF-7 cells compared to MN@MS@CS@ABE. Flow cytometry revealed early and late phases of apoptosis and necrosis with different percentages. The formula stimulated a reduction in signal intensity in the MR T2-weighted imaging technique. In conclusion, the current study developed a nanoformula which could be a promising theranostic agent in cancer therapy and diagnosis.

Apoptosis Detection Assays.

Many apoptosis assays are available since there are many proteins regulated at multiple points and involved in apoptosis signaling cascade. To detect apoptosis accurately, two or more assays should be used since there are many overlapped features between apoptosis and necrosis. There are six major groups of available assays to detect apoptosis: membrane alteration, mitochondrial assays, cytomorphological alterations, DNA fragmentation, detection of caspase, cleaved substrate, inhibitors and regulators, and detection of apoptosis in whole mounts. Among those assay, early apoptosis could be detected through annexin V, which is based on the loss of the cellular membrane integrity. Also, there are many assays that can detect midphase of apoptosis using caspase activation and molecular processing including PARP degradation. Late phase of apoptosis could be detected with DNA fragmentation assays. Combinations of these assays allow us to identify the mechanisms of apoptosis induction after specific stimulus. This chapter will introduce three apoptosis detection assays including annexin assay, DNA/chromatin condensation assays, and TUNEL assay.

RETRACTED: Exploring cytotoxicity of cordycepin loaded nanovesicles against (HCT116) colon cancer cells: Optimization and cellular evaluation

Numerous researchers have investigated cordycepin (COR) as an anti-tumor compound. COR has been documented to have cytotoxic effects on several cancer cells. The current work used a Box–Behnken mathematical design to minimize COR's size. The design incorporated COR concentration, phospholipid concentration and sonication time as variables to minimize the vesicles of COR emulsomes (COR-EMLs). To evaluate degree of improvement of COR cytotoxicity against colorectal cancer (HCT116) cells, cell viability, cell cycle analysis and apoptosis have been assessed. In addition, wound scratching and mitochondrial membrane potential were evaluated. Results of Box–Behnken design achieved COR-EMLs sizes in range from 91.54 ± 2.3–343.83 ± 3.7 nm. Moreover, the optimized formula morphology's was evaluated using transmission electron microscope and showed nanospheres in range of 100 nm. COR released from COR-EMLs exceeded 80% after 12 h.The half-maximal inhibitory concentration (IC50) of the refined COR-EML formula was about four times lower than that of COR-raw. The cell cycle study revealed that administration of COR-EML considerably hindered HCT116 cellular propagation in contrast to plain emulsomes (EMLs) and COR-raw with a denser cell compilation in G2/M. Moreover, the optimized formula notably enhanced the proportion of cells in both the initial and late phases of apoptosis. The augmentation of COR cytotoxicity was confirmed by its inhibition of cancer cell wound healing by approximately 40%. The mitochondrial membrane potential was significantly lower than in cells treated with COR-raw and EMLs. Finally, loading COR into the EMLs increased COR’s capacity to lower mitochondrial membrane functionality and significantly improved its cytotoxicity.

Abstract 1848: Ultra-high concentrations of gaseous nitric oxide show rapid cytotoxic capabilities against colon, breast, pancreatic and other cancer cells in vitro

Abstract Background: Nitric oxide (NO) acts as a signaling molecule in various pathological conditions, including cancer. High concentrations of NO are known to have an anti-tumor effect. It is anticipated that NO induces cancer cell cytotoxicity through DNA damage induction, leading to apoptotic cell death. To examine the cytotoxic effect of gaseous NO (gNO) on various cancer types, we exposed 6 cancer cell lines to gNO at ultra-high concentrations, of 10,000-100,000 ppm. Methods: Four mouse cancer cell lines, colon (CT26), breast (4T1), melanoma (B16F10) and lung (LLC1), and 2 human cancer cell lines, pancreatic (Panc02.03) and ovarian (OVCAR-3), were exposed directly to gNO at 10,000 - 100,000 ppm for 10 - 600 seconds (n=3). Air and nitrogen controls were used to assess whether the effect is mediated by gas flow or lack of oxygen (n=3). Cell viability was measured 24 hours after exposure by an XTT-based cell proliferation method. Additionally, cells were examined after exposure using Annexin V and Propidium Iodide cell apoptosis assays to determine cell death mechanisms. Results: gNO resulted in significant death of cancer cells in a dose and time dependent manner as revealed by XTT assay: 1. At a concentration of 100,000 ppm gNO, cell viability of CT26, 4T1, B16F10, LLC1 and Panc02.03 was reduced by ~100% after 10 seconds of exposure compared to non-treated (NT) cells (p<0.001) and OVCAR-3 was reduced by 84%. 2. Exposure of cells to 25,000-50,000 ppm gNO resulted in complete eradication of CT26, 4T1, B16F10, LLC1, Panc02.03 and OVCAR-3 cell viability after 1 minute of exposure compared to NT cells (p<0.001). 3. Exposure to 10,000 ppm gNO resulted in elimination of CT26, 4T1, B16F10 LLC1 and Panc02.03 after 2.5 minutes compared to NT cells (p<0.001) with OVCAR-3 viability reduced by 94%. 4. Viability of cells exposed to both nitrogen and air was comparable to NT cells. Additionally, a longer exposure time to 50,000 ppm gNO resulted in an increase in late apoptosis phase for LLC1 (from ~16% for 10 seconds to ~85% for 1 minute). Conclusion: gNO treatment resulted in a dose dependent cytotoxic response on various cancer cell lines, with higher concentrations leading to near instant cell death and lower concentrations requiring a longer exposure period to elicit cell death. Together with the known ability of NO to activate and recruit the immune system, these results suggest that gNO may be a potent therapeutic agent for tumor treatment across a range of tumor types. Citation Format: Hila Confino, Matan Goldshtein, Shani Puyesky, Shay Yarkoni, Amir Avniel, Steve Lisi, Ido Wolf. Ultra-high concentrations of gaseous nitric oxide show rapid cytotoxic capabilities against colon, breast, pancreatic and other cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1848.

Cytotoxic Activity, Cell Cycle Inhibition, and Apoptosis-Inducing Potential of Athyrium hohenackerianum (Lady Fern) with Its Phytochemical Profiling.

In the present study, we investigated the cytotoxic effects of Athyrium hohenackerianum ethanolic extract (AHEE) on the proliferation of breast, lung, and colon cancer cells. The AHEE was tested for its effect on the progression of the cell cycle, followed by induction of apoptosis determination by flow cytometry. Real-time qRT-PCR was also utilized to observe the initiation of apoptosis. In addition, GC-MS was performed in order to identify the active phytochemicals present in the AHEE. A cytotoxic activity with an IC50 value of 123.90 μg/mL against HCT-116 colon cancer cells was exhibited by AHEE. Following propidium iodide staining, annexin-V/PI, and clonogenic assays, AHEE treatment results in cell arrest in the S phase, causing an increase in the early and late phases of apoptosis and displaying antiproliferative potential, respectively. The morphological alterations were further monitored using acridine orange/ethidium bromide (AO/EB) staining. When compared with the control cells, features of apoptotic cell death, including nuclear fragmentation, in the AHEE-treated cells were noticed. The apoptosis was also confirmed by detecting the increased expression of p53 and caspase-3 along with the downregulation of Bcl-2. GC-MS analysis revealed that trans-linalool oxide, loliolide, phytol, 4,8,12,16-tetramethylheptadecan-4-olide, and gamma-sitosterol were the major phytochemical constituents. Based on these findings, it can be suggested that AHEE causes cellular death via apoptosis, which should be further explored for the identification of active compounds responsible for these observed effects. Therefore, AHEE can be used in the pharmaceutical development of anticancer agents for cancer therapeutics.

Delivery of miRNA-126 through folic acid-targeted biocompatible polymeric nanoparticles for effective lung cancer therapy

Objective: Nanoparticle-based drug delivery systems (DDSs) have been playing a considerable role in the eradication of cancer. In this experimental study, we designed and synthesized folic acid (FA)-decorated chitosan (CS) nanocarrier for targeted delivery of miR-126 (as a therapeutic agent) to lung cancer A549 cells. Materials and methods: Therefore, the FA-CS-miR-126 nano-complex was perfectly developed and characterized by various analytical devices such as Fourier transform infrared (FT-IR) and dynamic light scattering (DLS) spectroscopies and as well as transmission electron microscopy (TEM). The size was determined lower than 100 nm for synthetics. Then, a gel retardation assay was performed to investigate the entrapment efficiency of nano-complex. Afterward, the sort of in vitro assays was implemented on A549 (FA receptor-positive lung cancer cell line) and MRC5 (normal human diploid cell line) to evaluate the therapeutic efficiency of FA-CS-miR-126. Results: As the cell viability (40.7 ± 2.98% cell viability after 72 h treatment with 500 nM), migration assay (weaker migration after 24 h and 48 h), apoptotic and autophagy genes expression level (Caspse9: sixfolds; BAX: 17 folds; ATG5: fourfolds; and BECLIN1: threefolds more than the control group), the reduced expression level of EGF-L7, as a target gene for miR-126 was evaluated by Real-Time PCR too, then, cell cycle arrest (8.66% of cells in sub-G1 phase), and cell apoptosis assay (21.0% of cancer cell in late apoptosis phase) were scrutinized. Conclusion: These results are remarkably approved the biocompatible and efficient performance of FA-CS-miR-126 as a promising DDS.

Slow sulfide donor GYY4137 potentiates effect of paclitaxel on colorectal carcinoma cells

Although paclitaxel (PTX) is potent chemotherapeutic agent commonly used in variety of cancers, in colorectal carcinoma its usage is excluded because of low effectivity. Up to now, some experimental attempts were utilized to improve sensitivity of colorectal carcinoma to PTX. We used a slow sulfide donor GYY4137 to increase sensitivity of colorectal carcinoma cells to PTX. As a model of colorectal carcinoma, we utilized three different cell lines – HCT116, SW620 and DLD1. We compared IC50 for PTX and PTX/GYY4137, cell cycle, apoptosis, ATP levels and changes in intracellular pH. We observed significant decrease in IC50 levels in PTX/GYY4137 groups compared to PTX in all three cell lines. PTX arrested cell cycle in G2/M phase. Differences in S phase were observed in HCT116 and DLD1 cells treated with 20 nM PTX/GYY4137, but not in SW620 cell. GYY4137 increased early, but not late phase of apoptosis. This increase was not detected in non-cancer EAHy926 cells. Upregulation of IP3R1 suggested involvement of these receptors in PTX and/or GYY4137 induced apoptosis. We also observed partial ATP depletion and intracellular acidification in PTX treated groups. In PTX/GYY4137 groups of all three cell lines no ATP depletion was detectable and intracellular acidification was lower than in PTX treated groups. Slight differences in all measured parameters were determined among HCT116, SW620 and DLD1 cells, which is probably due to physiological variations in these cells. Taking together, sensitivity of PTX to colorectal carcinoma cell lines could be increased by slow sulfide donor GYY4137, probably through potentiation of apoptosis.

Development of a New Combination Compound (Cyclopamine) Isolated from Veratrum californicum and 5-FU Acting as Anti-invasive and Initiator of Intrinsic Apoptosis Mechanism against Aggressive Type Colorectal Cancer Cells

Characteristics of programmed cell death are generally regarded as defined by cell outgrowth or cellular aging and distinct morphological characteristics featuring nuclear condensation and formation of apoptotic bodies. Apoptosis is considered to have a function at cellular level and is essential to long-lived cell differentiation undergoing senescence and apoptosis. Inappropriate apoptosis is a factor that results in serious conditions, causing multiple disease progression as well as cancer. The ability to modulate the life or death of a cancer cell is recognized for its immense therapeutic potential. Therefore, the research findings that are focused on the elucidation of a newly synthesized drug or drug derived from a natural plant to stop the cell cycle mechanism and signaling pathways that control cell cycle arrest and apoptosis are considered necessary. The field research of apoptosis is moving forward at a rapid rate. The goal of this paper is to investigate a natural active constituent, which is simply identified as “a drug for near future co-chemotherapy”, as well as to explore the ability to activate the apoptotic pathway. Our results demonstrated that cyclopamine provides an excellent anti-tumor activity and changing of the morphology of cancerous cells in the early and late phases of apoptosis, indicating that cyclopamine has the potential to chemosensitize SW-620 cancer cells to 5-FU based chemotherapy, which may be useful for the treatment of colorectal cancer and the fight to overcome drug resistance in advanced colorectal cancer.

Niclosamide suppresses T‑cell acute lymphoblastic leukemia growth through activation of apoptosis and autophagy.

T-cell acute lymphoblastic leukemia (T-ALL) is a common pediatric malignancy, characterized by the abnormal presence of immature T-cell progenitors. Conventional treatments for T-ALL fail to prevent or cure the disease, with a high-risk of recurrence after the first remission. Thus, medical options are in demand to develop novel therapies for patients suffering with T-ALL. Niclosamide, a traditional oral anti-helminthic drug, has been reported to be a potential anticancer agent that regulates intracellular signaling pathways. Few studies have yet investigated the effects of niclosamide on the development of T-ALL. Here, the present study aimed to investigate the anti-leukemia effects of niclosamide on T-ALL. We first hypothesized that the suppressive effects of niclosamide on the tumor growth of T-ALL are exerted by regulating autophagy and apoptosis. Following niclosamide treatment, T-ALL cell viability was evaluated using MTT assay, and apoptosis with Annexin V/propidium iodide staining. In T-ALL cells treated with niclosamide, changes in apoptosis- and autophagy-related proteins were analyzed by western blotting. In addition, in an in vivo model, T-ALL xenograft mice were used to study the anti-leukemia effects of niclosamide. The results showed that niclosamide significantly reduced the viability of Jurkat and CCRF-CEM T-ALL cells in both a dose- and time-dependent manner. Niclosamide significantly activated the early and late phases of apoptosis in Jurkat (at 2 µM) and CCRF-CEM cells (at 1 µM). Furthermore, niclosamide upregulated protein expression of cleaved caspase-3 and LC3B, while downregulated those of Bcl-2 and p62, in a dose-dependent manner in both Jurkat and CCRF-CEM cells. The in vivo results showed that niclosamide treatment significantly suppressed tumor growth and the disease progression in T-ALL xenograft mice by activating cleaved caspase-3 and LC3B. We conclude that niclosamide plays an anti-leukemia role, and that it represents a novel approach for the treatment of T-ALL.