Apoptosis In Tumor Cell Lines Research Articles

Effects of α‑solanine on human head and neck squamous cell carcinoma cells and human umbilical vein endothelial cells in vitro.

α-solanine is a glycoalkaloid that is commonly found in nightshades (Solanum) and has a toxic effect on the human organism. Among other things, it is already known to inhibit tumor cell proliferation and induce apoptosis in tumor cell lines. Due to its potential as a tumor therapeutic, the current study investigated the effect of α-solanine on head and neck squamous cell carcinoma (HNSCC). In addition, genotoxic and antiangiogenic effects on human umbilical vein endothelial cells (HUVECs) were evaluated at subtoxic α-solanine concentrations. Cytotoxicity and apoptosis rates were measured in two human HNSCC cell lines (FaDu pharynx carcinoma cells and CAL-33 tongue carcinoma cells), as well as in HUVECs. MTT and Annexin V analyses were performed 24 h after α-solanine treatment at increasing doses up to 30 µM to determine cytotoxic concentrations. Furthermore, genotoxicity at subtoxic concentrations of 1, 2, 4 and 6 µM in HUVECs was analyzed using single-cell gel electrophoresis (comet assay). The antiangiogenic effect on HUVECs was evaluated in the capillary tube formation assay. The MTT assay indicated an induction of concentration-dependent viability loss in FaDu and CAL-33 cancer cell lines, whereas the Annexin V test revealed α-solanine-induced cell death predominantly independent from apoptosis. In HUVECs, the cytotoxic effect occurred at lower concentrations. No genotoxicity or inhibition of angiogenesis were detected at subtoxic doses in HUVECs. In summary, α-solanine had a cytotoxic effect on both malignant and non-malignant cells, but this was only observed at higher concentrations in malignant cells. In contrast to existing data in the literature, tumor cell apoptosis was less evident than necrosis. The lack of genotoxicity and antiangiogenic effects in the subtoxic range in benign cells are promising, as this is favorable for potential therapeutic applications. In conclusion, however, the cytotoxicity in non-malignant cells remains a severe hindrance for the application of α-solanine as a therapeutic tumor agent in humans.

Abstract 5723: Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay

Abstract Kinesin superfamily proteins (KIFs) are a large family of molecular motor proteins, that share a highly conserved motor domain. KIFs play an important role in cell division and transport of vesicles and organelles within cells. Mitotic kinesins hydrolyze ATP through its ATPase activity to move along the spindle microtubules and carry out several functions during mitosis to facilitate precise chromosome segregation. Altered expressions of several kinesins are shown in various cancers, fueling the aggressive nature of cancers leading to genomic instability. KIF11/Eg5 overexpression is shown in various cancers including hepatic carcinoma, lung, prostate, colorectal, gastric, and pancreatic cancers. Small molecule inhibitors of KIF11/Eg5 and CENP-E are shown to result in mitotic arrest leading to apoptosis in tumor cell lines. Despite the poor clinical outcome of several Eg5 and CENP-E inhibitors entered in clinical trials, targeting kinesins remains an attractive anti-cancer therapeutic approach. Recently, an inhibitor of KIF18A has entered clinical trial for treatment of solid tumors with high chromosomal instability. Here we show development of a microplate based biochemical screening assays for a panel of kinesin motor domain proteins. We summarize kinetic characterization of kinesin proteins and study the selectivity of various previously reported KIF18A, Eg5, CENP-E inhibitors across a kinesin selectivity panel. Citation Format: Brenna P. Lee, Sung Won Oh, Kurumi Y. Horiuchi, Safnas F. AbdulSalam. Selectivity profiling of small molecule kinesin inhibitors using microplate-based ATPase activity assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5723.

Synthesis, Characterization, In Vitro Anticancer Potentiality, and Antimicrobial Activities of Novel Peptide-Glycyrrhetinic-Acid-Based Derivatives.

Glycyrrhetinic acid (GA) is one of many interesting pentacyclic triterpenoids showing significant anticancer activity by triggering apoptosis in tumor cell lines. This study deals with the design and synthesis of new glycyrrhetinic acid (GA)–amino acid peptides and peptide ester derivatives. The structures of the new derivatives were established through various spectral and microanalytical data. The novel compounds were screened for their in vitro cytotoxic activity. The evaluation results showed that the new peptides produced promising cytotoxic activity against the human breast MCF-7 cancer cell line while comparing to doxorubicin. On the other hand, only compounds 3, 5, and 7 produced potent activity against human colon HCT-116 cancer cell line. The human liver cancer (HepG-2) cell line represented a higher sensitivity to peptide 7 (IC50; 3.30 μg/mL), while it appeared insensitive to the rest of the tested peptides. Furthermore, compounds 1, 3, and 5 exhibited a promising safety profile against human normal skin fibroblasts cell line BJ-1. In order to investigate the mode of action, compound 5 was selected as a representative example to study its in vitro effect against the apoptotic parameters and Bax/BCL-2/p53/caspase-7/caspase-3/tubulin, and DNA fragmentation to investigate beta (TUBb). Additionally, all the new analogues were subjected to antimicrobial assay against a panel of Gram-positive and Gram-negative bacteria and the yeast candida Albicans. All the tested GA analogues 1–8 exhibited more antibacterial effect against Micrococcus Luteus than gentamicin, but they exhibited moderate antimicrobial activity against the tested bacterial and yeast strains. Molecular docking studies were also simulated for compound 5 to give better rationalization and put insight to the features of its structure.

Derivatives of Deoxypodophyllotoxin Induce Apoptosis through Bcl-2/Bax Proteins Expression.

Deoxypodophyllotoxin, isolated from the Traditional Chinese Medicine Anthriscus sylvestris, is well-known because of its significant anti-tumor activity with strong toxicity in vitro and in vivo. In this article, a series of deoxypodophyllotoxin derivatives were synthesized and their anti-tumor effectiveness was evaluated. The anti-tumor activity of deoxypodophyllotoxin derivatives was investigated by the MTT assay method. Apoptosis percentage was measured by flow cytometer analysis using Annexin-V-FITC. The derivatives revealed obvious cytotoxicity in the MTT assay by decreasing the number of late cancer cells. The decrease of Bcl-2/Bax could be observed in MCF-7, HepG2, HT-29, and MG-63 using Annexin V-FITC. The ratio of Bcl-2/Bax in the administration group was decreased, which was determined by the ELISA kit. The derivatives of deoxypodophyllotoxin could induce apoptosis in tumor cell lines by influencing Bcl-2/Bax.

Effect of microvesicles from Moringa oleifera containing miRNA on proliferation and apoptosis in tumor cell lines

Human microvesicles are key mediators of cell–cell communication. Exosomes function as microRNA transporters, playing a crucial role in physiological and pathological processes. Plant microvesicles (MVs) display similar features to mammalian exosomes, and these MVs might enhance plant microRNA delivery in mammals. Considering that plant microRNAs have been newly identified as bioactive constituents in medicinal plants, and that their potential role as regulators in mammals has been underlined, in this study, we characterized MVs purified from Moringa oleifera seeds aqueous extract (MOES MVs) and used flow cytometry methods to quantify the ability to deliver their content to host cells. The microRNAs present in MOES MVs were characterized, and through a bioinformatic analysis, specific human apoptosis-related target genes of plant miRNAs were identified. In tumor cell lines, MOES MVs treatment reduced viability, increased apoptosis levels associated with a decrease in B-cell lymphoma 2 protein expression and reduced mitochondrial membrane potential. Interestingly, the effects observed with MOES MVs treatment were comparable to those observed with MOES treatment and transfection with the pool of small RNAs isolated from MOES, used as a control. These results highlight the role of microRNAs transported by MOES MVs as natural bioactive plant compounds that counteract tumorigenesis.

Mitochondrial ncRNA targeting induces cell cycle arrest and tumor growth inhibition of MDA-MB-231 breast cancer cells through reduction of key cell cycle progression factors

The family of long noncoding mitochondrial RNAs (ncmtRNAs), comprising sense (SncmtRNA), and antisense (ASncmtRNA-1 and ASncmtRNA-2) members, are differentially expressed according to cell proliferative status; SncmtRNA is expressed in all proliferating cells, while ASncmtRNAs are expressed in normal proliferating cells, but is downregulated in tumor cells. ASncmtRNA knockdown with an antisense oligonucleotide induces massive apoptosis in tumor cell lines, without affecting healthy cells. Apoptotic death is preceded by proliferation blockage, suggesting that these transcripts are involved in cell cycle regulation. Here, we show that ASncmtRNA knockdown induces cell death preceded by proliferative blockage in three different human breast cancer cell lines. This effect is mediated by downregulation of the key cell cycle progression factors cyclin B1, cyclin D1, CDK1, CDK4, and survivin, the latter also constituting an essential inhibitor of apoptosis, underlying additionally the onset of apoptosis. The treatment also induces an increase in the microRNA hsa-miR-4485-3p, whose sequence maps to ASncmtRNA-2 and transfection of MDA-MB-231 cells with a mimic of this miRNA induces cyclin B1 and D1 downregulation. Other miRNAs that are upregulated include nuclear-encoded hsa-miR-5096 and hsa-miR-3609, whose mimics downregulate CDK1. Our results suggest that ASncmtRNA targeting blocks tumor cell proliferation through reduction of essential cell cycle proteins, mediated by mitochondrial and nuclear miRNAs. This work adds to the elucidation of the molecular mechanisms behind cell cycle arrest preceding tumor cell apoptosis induced by ASncmtRNA knockdown. As proof-of-concept, we show that in vivo knockdown of ASncmtRNAs results in drastic inhibition of tumor growth in a xenograft model of MDA-MB-231 subcutaneous tumors, further supporting this approach for the development of new therapeutic strategies against breast cancer.

Oxysterols selectively promote short-term apoptosis in tumor cell lines

Oxysterols are 27-carbon oxidation products of cholesterol metabolism. Oxysterols possess several biological actions, including the promotion of cell death. Here, we examined the ability of several oxysterols to induce short-term death in cancerous (human breast cancer and mouse skin melanoma cells) and non-cancerous (human endothelial cells and lung fibroblasts) cell lines. We determined cell viability, Ki67 expression, cell cycle regulation, and apoptosis after 24-h incubations with oxysterols. We found that different oxysterols had different effects on the studied parameters. Moreover, the effects depended on cell type and oxysterol concentration. Three cytotoxic oxysterols (7-ketocholesterol, cholestane-3β-5α-6β-triol, and 5α-cholestane-3β,6β-diol) inhibited the S phase and stimulated the G0/G1 or G2/M phases. These oxysterols promoted apoptosis, determined with Annexin V and propidium iodide assays. These results showed that different oxysterols have cytotoxic effects depending on the cell line. The findings suggest a potential pharmacological utility of cytotoxic oxysterols.

Phase I study of procaspase activating compound-1 (PAC-1) for treatment of advanced malignancies.

TPS2621 Background: Dysregulation in apoptotic pathways is a hallmark feature of cancer, offering opportunities for pharmacologic intervention in its treatment. Caspases, a family of proteases, play several roles in apoptosis. Caspase-3 catalyzes the intra-cellular proteolysis that characterizes part of the apoptotic process. Caspase-3 levels are low in many tumors including: glioblastoma; breast, colon, lung, and liver cancers: lymphoma: neuroblastoma; and melanoma. In contrast, procaspase-3, the precursor of caspase-3 is elevated in these tumors. We describe a phase I study of PAC-1, a drug that catalyzes the conversion of procaspase -3 to caspase-3. It induces apoptosis in tumor cell lines in vitro. In vivo activity has also been shown, when combined with alkylating chemotherapy, in rodent glioma models and in canines diagnosed with malignant glioma. Methods: This Phase I dose escalation study has two components: the first (C1) to determine the maximum tolerated dose (MTD) of PAC-1 in advanced malignancies; the second (C2) to determine the MTD of PAC-1 when combined with temozolomide (TMZ) in patients with recurrent anaplastic astrocytoma (AA) or glioblastoma (GBM). A modified Fibonacci 3 + 3 design is used, expanding to nine subjects at the MTD level in each component. PAC-1 pharmacokinetics is assessed in all subjects during the first cycle. Secondary objectives include pharmacodynamics and correlations of PAC-1 activity with procaspase-3 expression in tumor tissue. Neurologic toxicity is closely monitored throughout the study. Inclusion criteria: diagnoses of advanced malignancies (C1) and recurrent AA or GBM (C2), ECOG PS 0-2, adequate organ function. Exclusion criteria: prior cytotoxic therapy in the last 3-6 weeks (varying with drug class) or uncontrolled chronic illness. Administration and design: For C1, PAC-1 (orally administered) is dosed at 75-1,000 mg daily on days 1-21 of each 28 day cycle. For C 2, the first PAC-1 dose is 375 mg daily with potential for escalation to 1,000 mg daily. TMZ, PO, is dosed at 150 mg/m2 daily, days 8-12, of each cycle. Enrollment to date for C1 is 27, (dose level 6); and for C2 is 4 (dose level 1). The study is open to accrual. Clinical trial information: NCT02355535.

The influence of simulated microgravity on proliferation and apoptosis in U251 glioma cells.

Several studies have indicated that microgravity can influence cellular progression, proliferation, and apoptosis in tumor cell lines. In this study, we observed that simulated microgravity inhibited proliferation and induced apoptosis in U251 malignant glioma (U251MG) cells. Furthermore, expression of the apoptosis-associated proteins, p21 and insulin-like growth factor binding protein-2 (IGFBP-2), was upregulated and downregulated, respectively, following exposure to simulated microgravity. These findings indicate that simulated microgravity inhibits proliferation while inducing apoptosis of U251MG cells. The associated effects appear to be mediated by inhibition of IGFBP-2 expression and stimulation of p21 expression. This suggests that simulated microgravity might represent a promising method to discover new targets for glioma therapeutic strategy.

Abstract 3025: Preclinical characterization of a novel monoclonal antibody targeting a neo-antigen expressed in ovarian and GI malignancies

Abstract Objectives: We have developed a novel humanized IgG1 monocolonal antibody, NEO-201, which targets a neoantigen expressed on ovarian and GI malignancies. This antigen has homology to CEACAM; however, it has specificity to tumor tissues and not normal tissues. Our aims were as follows: (1) to evaluate the expression of this antigen on tumor cell lines derived from various ovarian and GI cancers; and (2) to determine the functionality of NEO-201. Methods: Expression of the antigen was identified by western blot analysis and flow cytometry. Functionality was determined by an antibody dependent cell-mediated cytotoxicity (ADCC) assay using the antigen-expressing cell lines identified by western blot analysis. Cell lines not expressing the antigen were used as negative controls. The effector cells used in the ADCC assay were natural killer (NK) cells isolated from PBMCs of normal donors and high-affinity activated NK (haNK) cells. haNK cells are NK-92 cells genetically engineered to express the high-affinity variant of human CD16 (FcgRIIIA-V158) and the human IL-2 gene. haNK cells have little or no inhibitory expression of killer cell immunoglobulin-like receptors, a unique feature that may optimize their highly cytotoxic activity against a broad range of malignancies. Results: Using western blot analysis, 2 of 5 ovarian cancer cell lines (OV90 and PE01) and 1 of 2 colorectal cancer cell lines (LS174T) stained positively for NEO-201. Flow cytometry analysis on ovarian, colorectal, and pancreatic cancer cell lines confirmed the expression of the NEO-201 neoantigen on 1 of 2 ovarian cancer cell lines (OV90), 1 of 2 colorectal cancer cell lines (LS174T), and 2 of 2 pancreatic cancer cell lines (ASPC-1 and CFPAC-1). ADCC assay was performed using OV90 and ASPC-1 tumor cell lines. Cell lines not expressing the neoantigen were used as negative controls. Killing of tumor cells expressing the neoantigen was also observed with NEO-201 alone; however, the lysis was significantly augmented when NK cells from the PBMCs of normal donors or haNK cells were included in the ADCC assay. Conclusions: These finding demonstrate that the NEO-201 neoantigen is expressed on several tumor cell lines representing ovarian and GI malignancies. NEO-201 can mediate ADCC activity in the presence of NK cells and NK cell lines. In addition, NEO-201 can induce apoptosis in tumor cell lines expressing the NEO-201 neoantigen. Furthermore, preliminary studies demonstrated that this neoantigen is expressed in numerous cancer types (not shown). In vivo studies as well as IND-related studies are planned. Citation Format: Kristen Zeligs, Philip M. Arlen, Kwong Tsang, Lidia Hernandez, Massimo Fantini, Christina M. Annunziata. Preclinical characterization of a novel monoclonal antibody targeting a neo-antigen expressed in ovarian and GI malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3025. doi:10.1158/1538-7445.AM2017-3025

Synthesis, characterization and antitumor activity of Ln(III) complexes with hydrazone Schiff base derived from 2-acetylpyridine and isonicotinohydrazone.

In the present study, two isostructural lanthanide (Ln)(III) complexes, namely Ln(HL)2(NO3)(CH3OH)2)·CH3OH, where Ln = La in complex 1 and Ce in complex 2, and hydrogen ligand (HL) = (E)-N'-[1-(2-pyridinyl)ethylidene]isonicotinohydrazone, have been isolated and characterized by elemental analysis, infrared spectra and single-crystal X-ray diffraction analysis. The results revealed that the acylhydrazone ligand HL in each complex was deprotonated as an anionic ligand and coordinated to the central La(III) ion via enolization of oxygen and nitrogen atoms. Furthermore, the antitumor effects and potential mechanisms of the two complexes were explored in the human lung cancer cell line A549 and in the human gastric cancer cell lines BGC823 and SGC7901. In the present study, the roles the two complexes on the proliferation and apoptosis of the above tumor cell lines were determined by MTT assay and Annexin V/propidium iodide flow cytometry, respectively. Furthermore, various apoptosis-associated key genes, including caspase 3, B cell lymphoma (Bcl)-2-associated X protein (Bax) and Bcl-2, were detected by western blotting to explore the possible antitumor mechanisms of the two complexes. The results revealed that the two complexes had comparable antitumor activities in terms of inhibiting proliferation and inducing apoptosis in tumor cell lines. The changes in the protein expression levels of caspase 3, Bax and Bcl-2 further verified the apoptosis-promoting mechanisms of the two complexes in tumor cell lines. These findings have a great potential in biomedical applications of novel Ln(III) complexes.

Mechanism of the cytotoxic effect of l-amino acid oxidase isolated from Bothrops alternatus snake venom

BaltLAAO-I, an L-amino acid oxidase isolated from Bothrops alternatus, is a glycoprotein enzyme with a pI⿿5.3, 15% sugar and a related molecular mass of 66,000Da in its monomeric form, and 123,000Da in its dimeric form. The objective of this study is to describe the cytotoxicity activity induced by BaltLAAO-I isolated from Bothrops alternatus venom and its possible mechanism of action on tumor cells. Our results clearly depict that BaltLAAO-I has a strong selective cytotoxic activity on tumor cell lines (JURKAT, SK-BR-3 and B16F10). On the other hand, the results show low cytotoxicity on human peripheral blood mononuclear cells. Furthermore, our findings demonstrate that BaltLAAO-I induces the apoptosis of tumor cell lines through a cytotoxic activity exerted by a generation of reactive oxygen intermediates. All in all, the data indicate that LAAOs exert a selective cytotoxic role on tumor cells, demonstrating a great potential for future use in clinical therapy.

Chelidonium majus crude extract inhibits migration and induces cell cycle arrest and apoptosis in tumor cell lines

Ethnopharmacological relevanceChelidonium majus L (Papaveraceae) is widely used in alternative medicine for treatment of various disorders. Antitumor activities of alkaloids isolated from this plant have been reviewed, while there are only a few studies that examine properties of the whole extract. Aim of the studyThe aim of the present study was to investigate direct cytotoxic effects, as well as indirect antitumor effects of Chelidonium majus ethanolic extract against different tumor cell lines,. Materials and methodsMTT and SRB assays were performed to estimate cytotoxic effects of Chelidonium majus extract against human tumor cell lines A549, H460, HCT 116, SW480, MDA-MB 231 and MCF-7 and peripheral blood mononuclear cells from healthy individuals. Cell cycle analysis was performed by flow cytometry. Type of cell death induced by extract was determined by flow cytometry and cell morphology assessment. Inhibitory effect on migration of cancer cells was assessed by wound healing assay. ResultsChelidonium majus extract showed selective time- and dose-dependent increase of cytotoxicity in all six cell lines, with individual cell line sensitivities. Extract promoted cell cycle arrest and induced apoptosis. Cotreatment with doxorubicin enhanced cytotoxicity of the drug. Also, inhibitory effect on migration was shown with non-toxic extract concentration. ConclusionsThese results indicate possible usefulness of Chelidonium majus crude extract in antitumor therapy, whether through its direct cytotoxic effect, by prevention of metastasis, or as adjuvant therapy.

Down-regulation of anti-apoptotic genes in tumor cell lines is facilitated by suppression of OCT4B1

PurposeThe OCT4B1 as a variant of OCT4 is expressed in both cancer cells and tissues. The anti-apoptotic property of this variant aid cancer cells to escape from apoptosis. Therefore, the aim of the present study was to determine the effects of OCT4B1 suppression on regulation of 25 genes involved in anti-apoptotic pathway in tumor cell lines. Material and methodsAGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cells were transfected with specific OCT4B1 siRNA and a scramble siRNA by siRNA silencing gene technology, using Lipofectamine 2000 commercial kit. The real-time PCR technique was employed to examine and calculate fold changes of evaluated genes using the 2−ΔΔCT formula. ResultsPresent results demonstrated that 22 (88%) of interested genes were similarly down-regulated in all three examined cell lines. Our results also indicated that three genes (CASP2, IGF1R,TNF) were up-regulated. The CFLAR gene was down-regulated in AGS, while it was inversely up-regulated in 5637 and U87MG cells. ConclusionsIt may possibly be concluded that suppression of OCT4B1 can lead to apoptosis in tumor cell lines and this is at least facilitated via down-regulation of examined anti-apoptotic genes. Accordingly, suppression of OCT4B1 may probably be considered as useful tool in cancer therapy and research.

SOX17 increases the cisplatin sensitivity of an endometrial cancer cell line.

BackgroundEndometrial cancer (EC) is the most common form of malignant gynecological tumor. Treatment with cisplatin (CDDP) is the mainstay of EC chemotherapy. The apoptotic machinery is regarded as an important etiological factor in chemoresistance. Recent evidence has suggested that overexpression of the transcription factor SOX17 prevented apoptosis in tumor cell lines. The effect of SOX17 on apoptosis in EC cisplatin chemoresistance remains unclear.MethodsImmunohistochemistry and the reverse transcription-polymerase chain reaction were employed to detect gene expression in paraffin-embedded EC tissues and blood samples. The anti-proliferative ability of SOX17 on EC cells was assessed by MTT. Flow cytometric analysis was used to detect cell apoptosis by annexin V/PI double-staining. The expression of apoptosis-related proteins was analyzed by western blot. In the in vivo study, nude mice were subcutaneously injected with EC cells, and received cisplatin treatment through intraperitoneal chemotherapy. Apoptosis of in vivo samples was analyzed by TUNEL assay.ResultsSOX17 expression decreased the chemical resistance of EC cells to CDDP. HEC-1B cells with an elevated expression of SOX17 had a lower cell viability and higher apoptosis rate after cisplatin exposure. Overexpression SOX17 up-regulated wild type p53 after being exposed to cisplatin, while the expression of BCL2-associated X protein and cleaved caspase-3 simultaneously increased. Caspase-9 inhibitor reduced the efficacy of SOX17 in HEC-1B cells after cisplatin treatment. In the in vivo study, SOX17 overexpression clearly restrained the tumor growth and increased the cisplatin toxicity and apoptosis of tumor cells.ConclusionsSOX17 is involved in the p53-mediated apoptosis pathway, and increases the sensitivity of HEC-1B cells to cisplatin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0304-7) contains supplementary material, which is available to authorized users.

The Nitric Oxide Prodrug JS-K Induces Ca(2+)-Mediated Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells.

Hepatocellular carcinoma is one of the most common and deadly forms of human malignancies. JS-K, O(2)-(2, 4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1, 2-diolate, has the ability to induce apoptosis of tumor cell lines. In the present study, JS-K inhibited the proliferation of HepG2 cells in a time- and concentration-dependent manner and significantly induced apoptosis. JS-K enhanced the ratio of Bax-to-Bcl-2, released of cytochrome c (Cyt c) from mitochondria and the activated caspase-9/3. JS-K caused an increasing cytosolic Ca(2+) and the loss of mitochondrial membrane potential. Carboxy-PTIO (a NO scavenger) and BAPTA-AM (an intracellular Ca(2+) chelator) significantly blocked an increasing cytosolic Ca(2+) in JS-K-induced HepG2 cells apoptosis, especially Carboxy-PTIO. Meanwhile, Carboxy-PTIO and BAPTA-AM treatment both attenuate JS-K-induced apoptosis through upregulation of Bcl-2, downregulation of Bax, reduction of Cyt c release from mitochondria to cytoplasm and inactivation of caspase-9/3. In summary, JS-K induced HepG2 cells apoptosis via Ca(2+)/caspase-3-mediated mitochondrial pathway.

439. Oncolytic Newcastle Disease Virus Iraqi Virulent Strain Induce Apoptosis In Vitro through intrinsic Pathway and Association of Both Intrinsic and Extrinsic Pathways In Vivo

Newcastle disease virus is very promising antitumor agent. Iraqi virulent strain showed pronounced oncolytic activity. This study aimed to investigate the specific pathway of apoptosis induced by Iraqi strain of Newcastle disease virus (NDV) on tumor cells In vitro and In vivo. Several cell lines were tested (Human Rhabdomyosarcoma cell line (RD), Human Glioblastoma multiforme culture (AMGM) and Murine mammary adenocarcinoma cell line (AMN3) for ability of Iraqi NDV strain to induce apoptosis and to give deep look about the main pathway that induce apoptosis in tumor cell lines by the virus. The cell lines were infected with Newcastle disease virus and investigated for apoptotic pathway by using immunocytochemistry for caspase 3, 8, and 9. The result of this in vitro experiment revealed that NDV induce apoptosis in tumor cell lines when compared to control group with marked increase in the percentage of cells expressing caspase 9 with low percentage of cells expressing caspase 8 in NDV treated group at 24,48hr. These results indicated the role of NDV Iraqi strain in inducing apoptosis through intrinsic mitochondrial pathway in vitro. The in vivo experiment included female mice transplanted with mammary adenocarcinoma transplantable tumor line (AM3), after tumor growth; tumor bearing mice randomly divided into groups, first group treated by NDV by intratumoral injection, second group treated by NDV intrapretonially. Caspase 3, caspase 8, and caspase 9 expression in tumor sections were checked after 24hrs, 48hrs, 72hrs, 7 and 14 days of treatment with NDV. The results revealed that NDV induce apoptosis significantly in treated tumors by both ways IT and IP when compared with control group. There is significant (p<0.05) marked increase in the mean percentage of cells expressing caspase 9 in NDV treated groups compared with mean percentage of cells expressing caspase 8 and untreated control group at 24,48,72, 1week, 2 weeks post single injection treatment. Moreover, Caspase-8 expression in IT and IP groups was significantly higher than control untreated group. These results revealed that NDV had a powerful effect on inducing mitochondrial (intrinsic) apoptotic pathway in vivo in association of extrinsic pathway. This association can explained by NDV induction for antitumor immune response and this immune response can trigger tumor killing by extrinsic pathway. The current work suggest that Iraqi oncolytic virulent strain of NDV induce apoptosis through intrinsic pathway directly as the results of in vitro study and induce extrinsic pathway indirectly through immune stimulation properties of NDV.

SMK-17, a MEK1/2-specific inhibitor, selectively induces apoptosis in β-catenin-mutated tumors.

Although clinical studies have evaluated several MEK1/2 inhibitors, it is unlikely that MEK1/2 inhibitors will be studied clinically. BRAF mutations have been proposed as a responder marker of MEK1/2 inhibitors in a preclinical study. However, current clinical approaches focusing on BRAF mutations have shown only moderate sensitivity of MEK1/2 inhibitors. This has led to insufficient support for their promoted clinical adoption. Further characterization of tumors sensitive to MEK inhibitors holds great promise for optimizing drug therapy for patients with these tumors. Here, we report that β-catenin mutations accelerate apoptosis induced by MEK1/2 inhibitor. SMK-17, a selective MEK1/2 inhibitor, induced apoptosis in tumor cell lines harboring β-catenin mutations at its effective concentration. To confirm that β-catenin mutations and mutant β-catenin-mediated TCF7L2 (also known as TCF4) transcriptional activity is a predictive marker of MEK inhibitors, we evaluated the effects of dominant-negative TCF7L2 and of active, mutated β-catenin on apoptosis induced by MEK inhibitor. Indeed, dominant-negative TCF7L2 reduced apoptosis induced by MEK inhibitor, whereas active, mutated β-catenin accelerated it. Our findings show that β-catenin mutations are an important responder biomarker for MEK1/2 inhibitors.

Characterization of Haemanthus coccineus extract and its bioactive component: A promising and novel anti-inflammatory approach

Haemanthus coccineus (Amaryllidaceae) extracts (HCEs) have been used in traditional African medicine against febrile colds and asthma. The main ingredient of HCE, the non-basic alkaloid narciclasine, was recently reported to induce apoptosis in tumor cell lines [1]. Beyond this anti-cancer action, we hypothesized that HCE and narciclasine could exhibit an anti-inflammatory potential. Dried bulbs of H. coccineus were extracted using 60% (w/w) ethanol. The ethanol was largely removed and the remaining solution was partitioned with ethyl acetate. The organic phase was separated and dried (DER 50:1). The resulting HCE contained 2.2% narciclasine. In vitro, HCE (3 ng/ml to 10 µg/ml) concentration-dependently inhibited the proliferation of lymphocytes and the synthesis of pro-inflammatory cytokines (TNF-α, IL-6, IL-1 β) in murine macrophages without inducing cytotoxicity. Moreover, HCE decreased the TNF-α-induced adhesion of leukocytes to human endothelial cells (ECs) and the surface expression of EC adhesion molecules (ICAM-1, VCAM-1, E-selectin) without affecting EC viability. Extract fractions containing basic alkaloids did not display any activity, whereas the non-basic main alkaloid narciclasine (1 nM to 10µM) clearly reduced adhesion molecule expression. HCE as well as narciclasine attenuated the TNF-α-triggered expression of NF-κB-dependent genes (reporter gene assay) without influencing NF-κB DNA-binding activity (gel shift assay), IκB-α degradation (Western blot), or p65 nuclear translocation (microscopy). In vivo, HCE (450 mg/kg, orally) was found to clearly reduce edema formation and leukocyte infiltration in a dermal ear edema model by croton oil or arachidonic acid (AA). Also in a renal injury model we could demonstrate that HCE (50 µg/animal, i.p.) strongly attenuates leukocyte infiltration and cytokine expression. In conclusion, our study highlights that the use of HCE/narciclasine could represent a novel interesting anti-inflammatory approach.

Synthesis and Biological Activity of New Resveratrol Derivative and Molecular Docking: Dynamics Studies on NFkB

Resveratrol (RVS) is a naturally occurring antioxidant, able to display an array of biological activities. In the present investigation, a new derivative of RVS, RVS(a), was synthesized, and its biological activity was determined on U937 cells. It was observed that RVS(a) showed pronounced activity on U937 cells than RVS. RVS(a) is able to induce apoptosis in tumor cell lines through subsequent DNA fragmentation. From the EMSA results, it was evident that RVS(a) was able to suppress the activity of NFkB by interfering its DNA binding ability. Furthermore, the molecular interaction analysis (docking and dynamics) stated that RVS(a) has strong association with the IkB-alpha site of NFkB compared with RVS; this binding nature of RVS(a) might be prevent the NFkB binding ability with DNA. The present findings represent the potential activity of propynyl RVS on U937 cells and signifying it as a one of putative chemotherapeutic drugs against cancer.