Apoptosis In A549 Cells Research Articles

Ferrocene-modified half-sandwich iridium(III) and ruthenium(II) propionylhydrazone complexes and anticancer application

Ferrocene, ruthenium(II) and iridium(III) organometallic complexes, potential substitutes for platinum-based drugs, have shown good application prospects in the field of cancer therapy. Therefore, in this paper, six ferrocene-modified half-sandwich ruthenium(II) and iridium(III) propionylhydrazone complexes were prepared, and the anticancer potential was evaluated and compared with cisplatin. These complexes showed potential in-vitro anti-proliferative activity against A549 cancer cells, especially for Ir-based complexes, and showing favorable synergistic anticancer effect. Meanwhile, these complexes showed little cytotoxicity and effective anti-migration activity. Ir3, the most active complex (ferrocene-appended iridium(III) complex), could accumulate in the intracellular mitochondria, disturb the cell cycle (S-phase), induce the accumulation of reactive oxygen species, and eventually cause the apoptosis of A549 cells. Then, the design of these complexes provides a good structural basis for the multi-active non‑platinum organometallic anticancer complexes.

A Novel Tetramethylpyrazine Chalcone Hybrid- HCTMPPK, as a Potential Anti-Lung Cancer Agent by Downregulating MELK.

Lung adenocarcinoma currently ranks the leading causes of cancer-related mortality worldwide. Many anti-inflammation herbs, like tetramethylpyrazine, have shown their anti-tumor potentials. Here, we evaluated the role of a novel chalcone derivative of tetramethylpyrazine ((E) -1- (E) -1- (2-hydroxy-5-chlorophenyl) -3- (3,5,6-trimethylpyrazin-2-yl) -2-propen-1, HCTMPPK) in lung adenocarcinoma. The effects of HCTMPPK on cell proliferation, apoptosis, and invasion were investigated by in-vitro assays, including CCK-8, colony formation assay, flow cytometry, transwell assay, and wound-healing assay. The therapeutic potential of HCTMPPK in vivo was evaluated in xenograft mice. To figure out the target molecules of HCTMPPK, a network pharmacology approach and molecular docking studies were employed, and subsequent experiments were conducted to confirm these candidate molecules. HCTMPPK effectively suppressed the proliferative activity and migration, as well as enhanced the apoptosis of A549 cells in a concentration-dependent manner. Consistent with this, tumor growth was inhibited by HCTMPPK significantly in vivo. Regarding the mechanisms, HCTMPPK down-regulated Bcl-2 and MMP-9 and up-regulating Bax and cleaved-caspase-3. Subsequently, we identified 601 overlapping DEGs from LUAD patients in TCGA and GEO database. Then, 15 hub genes were identified by PPI network and CytoHubba. Finally, MELK was verified to be the HCTMPPK targeted site, through the molecular docking studies and validation experiments. Overall, our study indicates HCTMPPK as a potential MELK inhibitor and may be a promising candidate for the therapy of lung cancer.

Globospiramine from Voacanga globosa Exerts Robust Cytotoxic and Antiproliferative Activities on Cancer Cells by Inducing Caspase-Dependent Apoptosis in A549 Cells and Inhibiting MAPK14 (p38α): In Vitro and Computational Investigations.

Bisindole alkaloids are a source of inspiration for the design and discovery of new-generation anticancer agents. In this study, we investigated the cytotoxic and antiproliferative activities of three spirobisindole alkaloids from the traditional anticancer Philippine medicinal plant Voacanga globosa, along with their mechanisms of action. Thus, the alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) showed in vitro cytotoxicity and antiproliferative activities against the tested cell lines (L929, KB3.1, A431, MCF-7, A549, PC-3, and SKOV-3) using MTT and CellTiter-Blue assays. Globospiramine (1) was also screened against a panel of breast cancer cell lines using the sulforhodamine B (SRB) assay and showed moderate cytotoxicity. It also promoted the activation of apoptotic effector caspases 3 and 7 using Caspase-Glo 3/7 and CellEvent-3/7 apoptosis assays. Increased expressions of cleaved caspase 3 and PARP in A549 cells treated with 1 were also observed. Apoptotic activity was also confirmed when globospiramine (1) failed to promote the rapid loss of membrane integrity according to the HeLa cell membrane permeability assay. Network pharmacology analysis, molecular docking, and molecular dynamics simulations identified MAPK14 (p38α), a pharmacological target leading to cancer cell apoptosis, as a putative target. Low toxicity risks and favorable drug-likeness were also predicted for 1. Overall, our study demonstrated the anticancer potentials and apoptotic mechanisms of globospiramine (1), validating the traditional medicinal use of Voacanga globosa.

Anti-NSCLC role of SCN4B by negative regulation of the cGMP-PKG pathway: Integrated utilization of bioinformatics analysis and in vitro assay validation.

Non-small cell lung cancer (NSCLC)is a malignant tumor with low overall cure and survival rates. Uncovering abnormally expressed genes is significantly important for developing novel targeted therapies in NSCLC. This study aimed to discover new differentially expressed genes (DEGs)of NSCLC. The DEGs of NSCLC were identified in eight data sets from Gene Expression Omnibus (GEO)database. The expression profiles and the prognostic significance of SCN4B in LUAD and LUSC were analyzed using GEPIA database. LinkedOmics was used to identify co-expressed genes with SCN4B, which were further subjected to KEGG pathway enrichment analysis. SCN4B-overexpressing plasmid (pcDNA/SCN4B)was transfected into A549 and NCI-H2170 cells to elevate the expression of SCN4B. MTT and TUNEL assays were performed to evaluate cell viability and apoptosis. Relying on the screened DEGs from GEO database, we identified that SCN4B was significantly downregulated in LUAD and LUSC. We confirmed the downregulation of SCN4B in NSCLC tissues using GEPIA database. SCN4B has a prognostic value in LUAD, but not LUSC. KEGG pathway enrichment analysis of SCN4B-related genes showed that cGMP-PKG signaling pathway might be involved in the role of SCN4B in NSCLC. Overexpression of SCN4B in A549 and NCI-H2170 cells inhibited the cell viability. Besides, SCN4B overexpression induced apoptosis of A549 and NCI-H2170 cells. SCN4B inhibited the expression of PKG1 and p-CREBin NSCLC cells. Moreover, the inhibitory effects of SCN4B on tumor malignancy were attenuated by the activator of PKG. In conclusion, integrated bioinformatical analysis proved that SCN4B was downregulated and had a prognostic significance in NSCLC. In vitro experimental studies demonstrated that SCN4B regulated NSCLC cells viability and apoptosis via inhibiting cGMP-PKG signaling pathway.

Synthesis of Flavonols and Assessment of Their Biological Activity as Anticancer Agents.

A series of flavanols were synthesized to assess their biological activity against human non-small cell lung cancer cells (A549). Among the sixteen synthesized compounds, it was observed that compounds 6k (3.14 ± 0.29 µM) and 6l (0.46 ± 0.02 µM) exhibited higher potency compared to 5-fluorouracil (5-Fu, 4.98 ± 0.41 µM), a clinical anticancer drug which was used as a positive control. Moreover, compound 6l (4'-bromoflavonol) markedly induced apoptosis of A549 cells through the mitochondrial- and caspase-3-dependent pathways. Consequently, compound 6l might be developed as a candidate for treating or preventing lung cancer.

VP5 protein of oncolytic herpes simplex virus type 2 induces apoptosis in A549 cells through TP53I3 protein

Oncolytic virotherapy stands out as a burgeoning and promising therapeutic paradigm, harnessing the intrinsic cytotoxicity of oncolytic viruses for selective replication and dissemination within tumors. The primary mode of action revolves around the direct eradication of tumor cells. In our previous investigations, we formulated an oncolytic herpes simplex virus type 2 (OH2) and substantiated its anti-tumor efficacy both in vivo and in vitro. Subsequently, we embarked on a phase I/II clinical trial in China (NMPA, 2018L02743) and the USA (FDA, IND 27137) to assess OH2's safety, biodistribution, and anti-tumor activity as a standalone agent in patients with advanced solid tumors. In this investigation, our primary focus was to comprehend the influence of the major capsid protein VP5 of OH2 on its efficacy as an antitumor agent. Our findings underscore that the VP5 protein significantly amplifies OH2's oncolytic impact on A549 cells. Additionally, we observed that VP5 actively promotes the induction of apoptosis in A549 cells, both in vivo and in vitro. Through comprehensive transcriptional sequencing, we further authenticated that the VP5 protein triggers apoptosis-related signaling pathways and Gene Ontology (GO) terms in A549 cells. Moreover, we scrutinized differentially expressed genes in the p53-dependent apoptosis pathway and conducted meticulous in vitro validation of these genes. Subsequently, we delved deeper into unraveling the functional significance of the TP53I3 gene and conclusively affirmed that the VP5 protein induces apoptosis in A549 cells through the TP53I3 gene. These revelations illuminate the underlying mechanisms of OH2's antitumor activity and underscore the pivotal role played by the VP5 protein. The outcomes of our study harbor promising implications for the formulation of effective oncolytic virotherapy strategies in cancer treatment.

Beta hydroxybutyrate induces lung cancer cell death, mitochondrial impairment and oxidative stress in a long term glucose-restricted condition.

Metabolic plasticity gives cancer cells the ability to shift between signaling pathways to facilitate their growth and survival. This study investigates the role of glucose deprivation in the presence and absence of beta-hydroxybutyrate (BHB) in growth, death, oxidative stress and the stemness features of lung cancer cells. A549 cells were exposed to various glucose conditions, both with and without beta-hydroxybutyrate (BHB), to evaluate their effects on apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) levels using flow cytometry, and the expression of CD133, CD44, SOX-9, and β-Catenin through Quantitative PCR. The activity of superoxide dismutase, glutathione peroxidase, and malondialdehyde was assessed using colorimetric assays. Treatment with therapeutic doses of BHB triggered apoptosis in A549 cells, particularly in cells adapted to glucose deprivation. The elevated ROS levels, combined with reduced levels of SOD and GPx, indicate that oxidative stress contributes to the cell arrest induced by BHB. Notably, BHB treatment under glucose-restricted conditions notably decreased CD133 expression, suggesting a potential inhibition of cell survival through the downregulation of CD133 levels. Additionally, the simultaneous decrease in mitochondrial membrane potential and increase in ROS levels indicate the potential for creating oxidative stress conditions to impede tumor cell growth in such environmental settings. The induced cell death, oxidative stress and mitochondria impairment beside attenuated levels of cancer stem cell markers following BHB administration emphasize on the distinctive role of metabolic plasticity of cancer cells and propose possible therapeutic approaches to control cancer cell growth through metabolic fuels.

Unveiling Nano-Resveratrol induced by oil-in-water binding to human haemoglobin in the presence of holo-transferrin as binary and ternary systems: Spectroscopy, molecular dynamic and hemoglobin oxygenation approaches

Resveratrol (RES) is a member of polyphenols compounds capable of performing antioxidant activities and exhibiting anti-inflammatory and anticancer properties. This study attempted to increase the bioavailability and solubility of RES through an O/W nano-emulsion process. The performance of synthesized Nano-RES with hemoglobin (Hb) and human holo transferrin (HTF) as the binary and ternary systems was investigated by the means of various spectroscopic techniques and cancer cell experiments. Fluorescence quenching measurement configured the static quenching mechanism of Hb-Nano-RES and (Hb-HTF) interaction with Nano-RES complexes. According to the results, the measurements for binding constant of Hb-Nano-RES and (Hb-HTF) Nano-RES complexes were observed to be (6.62 ± 0.08) × 104 and (6.37 ± 0.09) × 104 M−1 at 298 K respectively. Thermodynamic parameters revealed the participation of electrostatic forces throughout the binding of ligands and proteins. Furthermore, the resonance light scattering signal of Hb and Hb-HTF in the presence of Nano-RES displayed the formation and production of complexes in larger sizes. According to the data of far-UV CD and UV/vis, the secondary structure of proteins was altered by the interaction of Nano-RES with both proteins. The results of melting point assessment confirmed the impact of Nano-RES on increasing the thermal stability of Hb-HTF complex as a ternary system. As the different behavior of Hb and Hb-HTF in the presence of Nano-RES concentrations was approved by the conductivity analysis, the distances between donor and acceptor was determined by fluorescence resonance energy transfer measurements (FRET) to be 1.68 nm and 1.77 nm for Hb-Nano-RES and (Hb-HTF) Nano-RES complexes, respectively. Molecular dynamic study was carried out to investigate the binding interactions of RES to Hb and Hb-HTF complex and was followed by MD simulation under the calculation of 100 ns. Lastly, we evaluated the cytotoxicity of Nano-RES in different concentrations on A549 and BEAS-2B cells, which increased the rate of apoptosis in A549 cells.

Anthocyanins prevent the development and progression of urethane-induced lung cancer by regulating energy metabolism in mice.

Anthocyanin (ACN) is a natural antioxidant with multiple biological activities, and the aim of this study was to evaluate the protective effect of ACN on the development and progression of lung cancer and to further explore its possible mechanism of action. In vivo, we fed C57BL/6J mice a 0.5%ACN diet or a control diet to observe their effects on the development and progression of urethane-induced lung cancer. In vitro, multiple lung cancer cell lines were used to investigate the effects of C3G on cell viability. The results showed a reduction in lung tumor burden and downregulation of oxidative phosphorylation and fatty acid degradation pathways in lung tissue of urethane-administrated ACN-fed mice compared with control diet-fed mice. In vitro, cyanidin-3-O-glucoside chloride (C3G) intervention treatment significantly inhibited proliferation and apoptosis of A549 cells. This process is likely due to the modulation of AMPK/mTOR signaling pathway by C3G to regulate cellular fatty acid metabolism and reduce intracellular lipid accumulation which affects the growth of lung cancer cells. These results suggest that ACN can inhibit the development and progression of urethane-induced lung tumors and alter the lipid metabolism of tumors in C57BL/6J mice.

Cold atmospheric plasma attenuates skin cancer via ROS induced apoptosis.

Cold atmospheric plasma (CAP) has been widely used in biomedical research, especially in vitro cancer therapy. Cutaneous squamous cell carcinoma (CSCC) is a malignant tumor originating from epidermal keratinocytes. However, the mechanism of CAP therapy on CSCC remains unclear. The animal models of CSCC induced by 7,12-dimethylbenz(a) anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) were constructed. For the CAP treatment group, after each TPA application, CAP was administered for 3min twice weekly after drying. HE staining were used to detect the pathological status of tumor tissue in each group. The levels of PCNA, Bcl-2, Bax, MMP2 and MMP9 were evaluated by western blot and qPCR. TUNEL staining were used to detect apoptosis in tumor tissues. In vivo, serum samples were used for ELISA of total ROS. MTT assay was used to detect the viability of A431 cells. Western blot and qPCR were used to detect the levels of PCNA, Bcl-2, Bax, MMP2 and MMP9 in A431 cells. A431 cell proliferation was examined by colony formation assay. The proportions of apoptosis of A431 cells were detected by flow cytometry. Transwell assessed the ability of A431 cells migration and proliferation. We found that CAP could induce skin cancer cells apoptosis and inhibit the progress of skin cancer. Through experiments in vitro, reactive oxygen species (ROS) generated by N-acetylcysteine (NAC) and CAP inhibited the proliferation and migration of A431 skin cancer cells while promoting apoptosis. These evidences suggest the protective effect of CAP in CSCC, and CAP has the potential clinical application of CSCC.

Mechanisms underlying reversed TRAIL sensitivity in acquired bortezomib-resistant non-small cell lung cancer cells.

Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

Hibiscus manihot L. flower extract induces anticancer activity through modulation of apoptosis and autophagy in A549 cells

Lung cancer is a major public health issue and heavy burden in China and worldwide due to its high incidence and mortality without effective treatment. It’s imperative to develop new treatments to overcome drug resistance. Natural products from food source, given their wide-ranging and long-term benefits, have been increasingly used in tumor prevention and treatment. This study revealed that Hibiscus manihot L. flower extract (HML) suppressed the proliferation and migration of A549 cells in a dose and time dependent manner and disrupting cell cycle progression. HML markedly enhanced the accumulation of ROS, stimulated the dissipation of mitochondrial membrane potential (MMP) and that facilitated mitophagy through the loss of mitochondrial function. In addition, HML induced apoptosis by activation of the PTEN-P53 pathway and inhibition of ATG5/7-dependent autophagy induced by PINK1-mediated mitophagy in A549 cells. Moreover, HML exert anticancer effects together with 5-FU through synergistic effect. Taken together, HML may serve as a potential tumor prevention and adjuvant treatment for its functional attributes.

Design, synthesis and activity evaluation of quinolinone derivatives as EZH2 inhibitors

The enhancer of zeste homologue 2 (EZH2) is the core catalytic subunit of polycomb repressive complex 2, which catalyzes lysine 27 methylation of histone H3.Herein, a series of quinolinone derivatives were designed and synthesized based on the structure of Tazemetostat as the lead compound. Compound 9l (EZH2WT IC50 = 0.94 nM) showed stronger antiproliferative activity in HeLa cells than the lead compound. Moreover, compound 9e (EZH2WT IC50 = 1.01 nM) significantly inhibited the proliferation and induced apoptosis in A549 cells.

Synergistic Effects of Chemotherapy and Phototherapy on Ovarian Cancer Using Follicle-Stimulating Hormone Receptor-Mediated Liposomes Co-Loaded with SN38 and IR820.

We have developed an ovarian cancer-targeted drug delivery system based on a follicle-stimulating hormone receptor (FSHR) peptide. The lipophilic chemotherapeutic drug SN38 and the photosensitizer IR820 were loaded into the phospholipid bilayer of liposomes. The combination of chemotherapy and phototherapy has become a promising strategy to improve the therapeutic effect of chemotherapy drugs on solid tumors. IR820 can be used for photodynamic therapy (PDT), effectively converting near-infrared light (NIR) into heat and producing reactive oxygen species (ROS), causing damage to intracellular components and leading to cell death. In addition, PDT generates heat in near-infrared, thereby enhancing the sensitivity of tumors to chemotherapy drugs. FSH liposomes loaded with SN38 and IR820 (SN38/IR820-Lipo@FSH) were prepared using thin-film hydration-sonication. FSH peptide binding was analyzed using 1H NMR spectrum and Maldi-Tof. The average size and zeta potential of SN38/IR820-Lipo@FSH were 105.1 ± 1.15 nm (PDI: 0.204 ± 0.03) and -27.8 ± 0.42 mV, respectively. The encapsulation efficiency of SN38 and IR820 in SN38/IR820-Lipo@FSH liposomes were 90.2% and 91.5%, respectively, and their release was slow in vitro. FSH significantly increased the uptake of liposomes, inhibited cell proliferation, and induced apoptosis in A2780 cells. Moreover, SN38/IR820-Lipo@FSH exhibited better tumor-targeting ability and anti-ovarian cancer activity in vivo when compared with non-targeted SN38/IR820-Lipo. The combination of chemotherapy and photodynamic treatment based on an FSH peptide-targeted delivery system may be an effective approach to treating ovarian cancer.

Gefitinib Induces Apoptosis in NSCLC Cells by Promoting Glutaminolysis and Inhibiting the MEK/ERK Signaling Pathway.

Over 80% of lung cancer cases constitute non-small cell lung cancer (NSCLC), making it the most prevalent type of lung cancer globally and the leading cause of cancer-related deaths. The treatment of NSCLC patients with gefitinib has demonstrated promising initial efficacy. However, the underlying mechanism remains unclear. This study aims to investigate how gefitinib affects the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway-mediated growth and death of NSCLC cells. In this study, the NSCLC cell line A549 was cultured in vitro and divided into a control group and a gefitinib group. The viability of the A549 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect apoptosis in A549 cells, and the expression of glutamate dehydrogenase (GDH1) mRNA in these cells was determined using real-time quantitative PCR (RT-PCR). Western blotting was utilized to evaluate the protein expression levels of key components in the MEK/ERK signaling pathway, including phospho-MEK1/2, MEK1/2, phospho-ERK1/2, and ERK1/2. Additionally, intracellular glutamine content in A549 cells was measured using a colorimetric method. In contrast to the control group, the proliferation of A549 cells, the transcription level of glutamate dehydrogenase (GDH1), the intracellular glutamine content, and the protein expression levels of phospho-MEK1/2 and phospho-ERK1/2 were significantly lower in the gefitinib group. Moreover, apoptosis markedly increased. Gefitinib expedites apoptosis and diminishes proliferation in the NSCLC cell line A549 by downregulating the epidermal growth factor receptor (EGFR)/MEK/ERK signaling pathway. This effect is accomplished by fostering the expression of GDH1 to augment glutaminolysis in A549 cells.

Apoptin Inhibits Glycolysis and Regulates Autophagy by Targeting Pyruvate Kinase M2 (PKM2) in Lung Cancer A549 Cells.

Pyruvate kinase M2 (PKM2) is a key enzyme in aerobic glycolysis and plays an important role in tumor energy metabolism and tumor growth. Ad-apoptin, a recombinant oncolytic adenovirus, can stably express apoptin in tumor cells and selectively causes cell death in tumor cells. The relationship between the anti-tumor function of apoptin, including apoptosis and autophagy activation, and the energy metabolism of tumor cells has not been clarified. In this study, we used the A549 lung cancer cell line to analyze the mechanism of PKM2 involvement in apoptin-mediated cell death in tumor cells. PKM2 expression in lung cancer cells was detected by Western blot and qRT-PCR. In the PKM2 knockdown and over-expression experiments, A549 lung cancer cells were treated with Ad-apoptin, and cell viability was determined by the CCK-8 assay and crystal violet staining. Glycolysis was investigated using glucose consumption and lactate production experiments. Moreover, the effects of Ad-apoptin on autophagy and apoptosis were analyzed by immunofluorescence using the Annexin v-mCherry staining and by western blot for c-PARP, p62, and LC3-II proteins. Immunoprecipitation analysis was used to investigate the interaction between apoptin and PKM2. In addition, following PKM2 knockdown and overexpression, the expression levels of p-AMPK, p-mTOR, p-ULK1, and p-4E-BP1 proteins in Ad-apoptin treated tumor cells were analyzed by western blot to investigate the mechanism of apoptin effect on the energy metabolism of tumor cells. The in vivo antitumor mechanism of apoptin was analyzed by xenograft tumor inhibition experiment in nude mice and immunohistochemistry of tumors' tissue. As a result, apoptin could target PKM2, inhibit glycolysis and cell proliferation in A549 cells, and promote autophagy and apoptosis in A549 cells by regulating the PKM2/AMPK/mTOR pathway. This study confirmed the necessary role of Ad-apoptin in the energy metabolism of A549 cells.

492 Novel BH3 Mimetic Bcl-2 Inhibitor Promotes Autophagic Cell Death and Reduces in Vivo Glioblastoma Tumor Growth

INTRODUCTION: Anti-apoptotic members of the Bcl-2 family proteins play central roles in the regulation of cell death in glioblastoma, the most malignant type of brain tumor. Despite the advances in GBM treatment, there is still an urgent need for new therapeutic approaches. METHODS: In silico studies resulted in 500,000 molecules being screened with computer-aided drug design methods and the molecule with the highest binding affinity was selected and named BAU-243. Cell viability, IC50 value, tumor formation potential, tumor spheroid formation potential, tumor invasion studies were investigated following BAU-243 application. The molecular changes caused by Bcl-2 inhibition were demonstrated by quantitative PCR, western blot and immunofluorescence methods. In in vivo studies, glioma tumors were transplanted intracranially into Nod Scid Gamma(NSG) mice using the mouse xenograft model. RESULTS: Here, we report a novel 4-thiazolidinone derivative BH3 mimetic, BAU-243 that binds to Bcl-2 with a high affinity. BAU-243 effectively reduced overall glioblastoma cell proliferation including a subpopulation of cancer-initiating cells in contrast to the selective Bcl-2 inhibitor ABT-199. While ABT-199 successfully induces apoptosis in high BCL2-expressing neuroblastoma SHSY-5Y cells, BAU-243 triggered autophagic cell death rather than apoptosis in A172 cells, indicated by the upregulation of BECN1, ATG5, and MAP1LC3B expression. Lc3b-II, a potent autophagy marker, was significantly upregulated following BAU-243 treatment. Moreover, BAU-243 significantly reduced tumor growth in vivo in orthotopic brain tumor models when compared to the vehicle group, and ABT-199 treated animals. To elucidate the molecular mechanisms of action of BAU-243, we performed computational modeling simulations that were consistent with in vitro results. CONCLUSIONS: Results indicated that BAU-243 activates autophagic cell death by disrupting Beclin 1:Bcl-2 complex, and may serve as a potential small molecule for the treatment of glioblastoma.

Phytochemical analysis and anticancer effect of Camellia oleifera bud ethanol extract in non-small cell lung cancer A549 cells.

Camellia oleifera is a medicine food homology plant widely cultivated in the Yangtze River Basin and southern China due to its camellia oil. Camellia oleifera bud and fruit exist simultaneously, and its bud is largely discarded as waste. However, C. oleifera bud has been used in traditional Chinese medicine to treat a variety of ailments. Thus, the purpose of this study was to identify the chemical components of C. oleifera bud ethanol extract (EE) and first evaluate its anticancer effects in non-small cell lung cancer A549 cells. Based on UHPLC-Q-Orbitrap-MS analysis, seventy components were identified. For anticancer activity, C. oleifera bud EE had remarkable cytotoxic effect on non-small cell lung cancer A549 (IC50: 57.53 ± 1.54μg/mL) and NCI-H1299 (IC50: 131.67 ± 4.32μg/mL) cells, while showed lower cytotoxicity on non-cancerous MRC-5 (IC50 > 320μg/mL) and L929 (IC50: 179.84 ± 1.08μg/mL) cells. It dramatically inhibited the proliferation of A549 cells by inducing cell cycle arrest at the G1 phase. Additionally, it induced apoptosis in A549 cells through a mitochondria-mediated pathway, which decreased mitochondrial membrane potential, upregulated Bax, activated caspase 9 and caspase 3, and resulted in PARP cleavage. Wound healing and transwell invasion assays demonstrated that C. oleifera bud EE inhibited the migration and invasion of A549 cells in a dose-dependent manner. The above findings indicated that C. oleifera bud EE revealed notable anticancer effects by inhibiting proliferation, inducing apoptosis, and suppressing migration and invasion of A549 cells. Hence, C. oleifera bud ethanol extract could serve as a new source of natural anticancer drugs.

Synthesis and biological evaluation of novel benzothiazole derivatives as potential anticancer and antiinflammatory agents.

Introduction: Cancer, a significant global health concern, necessitates innovative treatments. The pivotal role of chronic inflammation in cancer development underscores the urgency for novel therapeutic strategies. Benzothiazole derivatives exhibit promise due to their distinctive structures and broad spectrum of biological effects. This study aims to explore new anti-tumor small molecule drugs that simultaneously anti-inflammatory and anticancer based on the advantages of benzothiazole frameworks. Methods: The compounds were characterized by nuclear magnetic resonance (NMR), liquid chromatograph-mass spectrometer (LC-MS) and high performance liquid chromatography (HPLC) for structure as well as purity and other related physicochemical properties. The effects of the compounds on the proliferation of human epidermoid carcinoma cell line (A431) and human non-small cell lung cancer cell lines (A549, H1299) were evaluated by MTT method. The effect of compounds on the expression levels of inflammatory factors IL-6 and TNF-α in mouse monocyte macrophages (RAW264.7) was assessed using enzyme-linked immunosorbent assay (ELISA). The effect of compounds on apoptosis and cell cycle of A431 and A549 cells was evaluated by flow cytometry. The effect of compounds on A431 and A549 cell migration was evaluated by scratch wound healing assay. The effect of compounds on protein expression levels in A431 and A549 cells was assessed by Western Blot assay. The physicochemical parameters, pharmacokinetic properties, toxicity and drug similarity of the active compound were predicted using Swiss ADME and admetSAR web servers. Results: Twenty-five novel benzothiazole compounds were designed and synthesized, with their structures confirmed through spectrogram verification. The active compound 6-chloro-N-(4-nitrobenzyl) benzo[d] thiazol-2-amine (compound B7) was screened through a series of bioactivity assessments, which significantly inhibited the proliferation of A431, A549 and H1299 cancer cells, decreased the activity of IL-6 and TNF-α, and hindered cell migration. In addition, at concentrations of 1, 2, and 4μM, B7 exhibited apoptosis-promoting and cell cycle-arresting effects similar to those of the lead compound 7-chloro-N-(2, 6-dichlorophenyl) benzo[d] thiazole-2-amine (compound 4i). Western blot analysis confirmed that B7 inhibited both AKT and ERK signaling pathways in A431 and A549 cells. The prediction results of ADMET indicated that B7 had good drug properties. Discussion: This study has innovatively developed a series of benzothiazole derivatives, with a focus on compound B7 due to its notable dual anticancer and anti-inflammatory activities. B7 stands out for its ability to significantly reduce cancer cell proliferation in A431, A549, and H1299 cell lines and lower the levels of inflammatory cytokines IL-6 and TNF-α. These results position B7B7 as a promising candidate for dual-action cancer therapy. The study's mechanistic exploration, highlighting B7's simultaneous inhibition of the AKT and ERK pathways, offers a novel strategy for addressing both the survival mechanisms of tumor cells and the inflammatory milieu facilitating cancer progression.

The molecular mechanism of action for the potent antitumor component extracted using supercritical fluid extraction from Croton crassifolius root

Ethnopharmacological relevanceThe root of Croton crassifolius has been used as a traditional Chinese medicine (TCM), called Radix Croton Crassifolius, and commonly known as “Ji Gu Xiang” in Chinese. Its medicinal value has been recorded in several medical books or handbooks, such as “Sheng Cao Yao Xing Bei Yao”, “Ben Cao Qiu Yuan” and “Zhong Hua Ben Cao”. It has been traditional employed for treating sore throat, stomach-ache, rheumatism and cancer. Aim of the studyAt present, there are limited studies on the evaluation of low-polarity extracts of roots in C. crassifolius. Consequently, the aim of this study was to evaluate the antitumor effect of the low-polarity extract of C. crassifolius root. Materials and methodsExtracts were obtained by supercritical fluid extraction. The extracts were tested for antitumor effects in vitro on several cancer cell lines. A CCK-8 kit was used for further analysis of cell viability. A flow cytometer and propidium iodide staining were used to evaluate the cell cycle and apoptosis. Hoechst staining, JC-1 staining and the fluorescence probe DCFH-DA were used to evaluate apoptotic cells. Molecular mechanisms of action were analyzed by quantitative RT‒PCR and Western blotting. Immunohistochemistry was used for the evaluation of xenograft tumors in male BALB/c mice. Finally, molecular docking was employed to predict the bond between the desired bioactive compound and molecular targets. ResultsEleven diterpenoids were isolated from low-polarity C. crassifolius root extracts. Among the compounds, chettaphanin II showed the strongest activity (IC50 = 8.58 μM) against A549 cells. Evaluation of cell viability and the cell cycle showed that Chettaphanin II reduced A549 cell proliferation and induced G2/M-phase arrest. Chttaphanin II significantly induced apoptosis in A549 cells, which was related to the level of apoptosis-related proteins. The growth of tumor tissue was significantly inhibited by chettaphanin II in experiments performed on naked mice. The antitumor mechanism of chettaphanin II is that it can obstruct the mTOR/PI3K/Akt signaling pathway in A549 cells. Molecular docking established that chettaphanin II could bind to the active sites of Bcl-2 and Bax. ConclusionsTaken together, the natural diterpenoid chettaphanin II was identified as the major antitumor active component, and its potential for developing anticancer therapies was demonstrated for the first time by antiproliferation evaluation in vitro and in vivo.