Late Apoptotic Cells Research Articles

Fisetin induces apoptosis in human skin cancer cells through downregulating MTH1

Fisetin, a natural flavonoid molecule, has been shown to have anticancer properties against various malignancies. In this investigation, we discovered that Fisetin decreased cell viability of both the treated skin cancer cell lines A375 and A431 in a dose and time-dependent manner. The IC50 values ranging from 57.60 µM ± 6.59 to 41.70 µM ± 1.25 in A375 and 48.70 µM ± 5.49 to 33.67 µM ± 1.03 for A431 at the observed time ranging between 24 h to 72 h of treatment remained quite enthusiastic when compared with the normal HEK 293 cells. Fisetin significantly decreased colony formation and migratory ability of the cancer cells. Flow cytometry analysis revealed that Fisetin significantly restricted the progression of skin cancer cells in the G 0/G 1 phase of the cell cycle and induced cells to undergo apoptosis by increasing reactive oxygen species, decreasing mitochondrial membrane potential, and elevating the count of early and late apoptotic cells. Our in silico studies of molecular docking followed by molecular dynamics simulation found that the interactions and stability of MTH1 protein with Fisetin further showed a considerable binding affinity for MTH1 (–11.4 kcal/mol) and developed stable complexes maintained throughout 100 ns trajectories. Our western blot analysis endorsed this. We found that Fisetin downregulated the expression levels of MTH1 also in addition, it played a crucial role in regulation of apoptotic events in cancer cells. We therefore, conclude that Fisetin anticancer properties against skin cancer cells are mediated through MTH1 inhibition followed by ATM and P53 upregulation. Communicated by Ramaswamy H. Sarma

P17.10: Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation. This work was carried out with the support of the “Cooperative Research Program for Agriculture Science and Technology Development [Project No. PJ015607 (HK and KO)], Rural Development Administration, Republic of Korea. This research was partly supported by Hallym University Research Fund (HL).

Design, synthesis, and biological screening of a series of 4′-fluoro-benzotriazole-acrylonitrile derivatives as microtubule-destabilising agents (MDAs)

Introduction: Colchicine-binding site inhibitors are some of the most interesting ligands belonging to the wider family of microtubule-destabilising agents. Results: A novel series of 4′-fluoro-substituted ligands (5–13) was synthesised. The antiproliferative activity assays resulted in nM values for the new benzotriazole-acrylonitrile derivatives. Compound 5, the hit compound, showed an evident blockade of HeLa cell cycle in the G2-M phase, but also a pro-apoptotic potential, and an increase of early and late apoptotic cells in HeLa and MCF-7 cell cycle analysis. Confocal microscopy analysis showed a segmented shape and a collapse of the cytoskeleton, as well as a consistent cell shrinkage after administration of 5 at 100 nM. Derivative 5 was also proved to compete with colchicine at colchicine-binding site, lowering its activity against tubulin polymerisation. In addition, co-administration of 5 and doxorubicin in drug-resistant A375 melanoma cell line highlighted a synergic potential in terms of inhibition of cell viability. Discussion: The 4′-fluoro substitution of benzotriazole-acrylonitrile scaffold brought us a step forward in the optimisation process to obtain compound 5 as promising MDA antiproliferative agent at nanomolar concentration.

Amelioration of human acute lymphoblastic leukemia (ALL) cells by ZnO-TiO2-Chitosan-Amygdalin nanocomposites

The present study aims to study the cytotoxicity of ZnO-TiO 2 -Chitosan-Amygdalin nanocomposites (ZnO-TiO 2 -Chitosan-Amygdalin) on T lymphoblast cancer cells (MOLT-4). In a study, nanocomposites containing 2.5 to 15 µg/ml MTT were screened for their anticancer activity. Its anticancer properties were significantly higher than those of other nanocomposites with an IC 50 value of 10.34 µg/ml. We studied the mechanism of action for cytotoxic cell death by fluorescence microscopy using Acridine Orange/EtBr (AO/EtBr) and Rhodamine 123 staining procedures. Using DCFH-DA, ZnO-TiO 2 -Chitosan-Amygdalin nanocomposites were analyzed to determine ROS production. The change in apoptotic protein expression for the 24 h following treatment with MOLT-4 cells for Caspase-3, 8, and 9. Nanocomposites containing ZnO-TiO 2 -Chitosan-Amygdalin increased the number of early and late apoptotic cells in MOLT-4 cells. ZnO-TiO 2 -Chitosan-Amygdalin nanocomposites also enhanced mitochondrial apoptosis through Caspase cascade signaling. MOLT-4 cells phosphorylated Caspase cascade in response to ZnO-TiO 2 -Chitosan-Amygdalin nanocomposites. Compared to the control group, the cancer cells treated with ZnO-TiO 2 -Chitosan-Amygdalin nanocomposites significantly arrest the proliferation and induces cleavage of pro-apoptotic proteins which leads to apoptotic cell death. Accordingly, ZnO-TiO 2 -Chitosan-Amygdalin nanocomposites might be effective against T lymphoblast cancer.

Norepinephrine promotes anoikis resistance in fallopian tube secretory cells through beta adrenergic receptor signaling (247)

<b>Objectives:</b> High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecologic malignancy with a 5-year survival rate approaching 40%. Unfortunately, most women present at an advanced stage due to subtle symptomatology at disease onset and inadequate screening protocols. Thus, understanding the cellular events that lead to the development and malignant transformation of fallopian tube (FT) precursor cells into metastatic disease may lead to actionable targets to improve outcomes. Fallopian tube secretory epithelium is considered the cell of origin of HGSOC, where <i>TP53</i> and other genetic defects accrue to permit exfoliation and seeding of the ovary and peritoneum, leading to widespread metastasis. Follicular fluid bathes the fallopian tube monthly during the reproductive years and contains several factors contributing to carcinogenesis. Recently, neurotransmitters such as norepinephrine (NE) have been implicated in ovarian cancer growth and metastasis through multiple mechanisms and are also present within the follicular fluid. Thus, the objective of the current study was to evaluate the effects of NE on FT epithelial cell function under 3D conditions. <b>Methods:</b> FT cell lines (FT189, FT190, FT194, FT237, FT240, FT246, FT282), previously immortalized through several mechanisms, were plated into round bottom ultralow adhesion plates with 10µM NE or vehicle control (VHC), in the presence or absence of adrenergic antagonist propranolol (10µM). Spheroids were serially imaged over 24h. For FT237 and FT246, live/dead cell staining was performed using ReadyProbe cell viability assay, and apoptotic activity was assessed via annexin staining analyzed by flow cytometry. qPCR and western blotting were used to assess receptor expression. <b>Results:</b> All FT lines examined expressed <i>ADRB1</i> and <i>ADRB2</i> by both western blot and qPCR. Treatment with NE resulted in significant compaction of spheroid formation by 33% (+/- 5%, p <0.001) at 24h compared to VHC. This response was completely abrogated by preexposure to propranolol (p <0.05). NE nor propranolol had a significant impact on cell viability under immunofluorescence. However, annexin staining revealed a significant 5% increase in cell survival with a concomitant decrease in early and late apoptotic cell populations exposed to NE in 3D culture (p<0.05), compared to VHC. FT exposure to NE resulted in significantly increased expression of EMT genes, <i>SNAI1</i> and <i>SNAI2</i>, which was completely reversed by pretreatment with propranolol (p<0.05). <b>Conclusions:</b> Taken together, these findings demonstrate that NE exposure promotes FT cell spheroid formation and EMT gene transcription through activation of ADRB-mediated signaling. Thus, NE may potentiate metastasis of early ovarian cancer precursor lesions through stimulation of EMT genes and enhancing anoikis resistance, two factors that are critical to the coelomic spread of ovarian cancer. Future studies examining NE-induced carcinogenesis in FT cells are warranted.

In vitro evaluation of pediatric restorative materials on human dental pulp stem cells

This in vitro study aimed to compare the cellular viability of four different restorative materials on human dental pulp stem cells (hDPSCs). The necessary tissues of isolated hDPSCs were obtained from 10 impacted third molars of healthy individuals 19–25 years old undergoing surgical extraction. The effects of GC Fuji IX GP, GC EQUIA Forte, APA Glass Carbomer, and APA Esthetic Fill eluates on hDPSCs’ viability on the first, third, and seventh days were examined by flow cytometry using an Annexin V binding assay. The cell viability ratios differed significantly between the restorative material groups (p < 0.001). When the restorative materials and the control group were compared, the control group had the highest cell viability on all experiment days, while the Glass Carbomer group had the highest percentage of viable cells of all the restorative material groups. A significant difference between days in terms of cellular viability (p < 0.001) was found. Considering the early apoptotic cell ratios, no significant difference was found between the groups and days (p > 0.05). Furthermore, based on time, except for the first day of the GC Fuji IX GP group, no statistically significant difference was observed in the early apoptotic cell ratios in each group (p > 0.05). When the rates of late apoptotic cells were compared between the groups, no significant difference was observed, except for with the control group, which had the lowest rate of late apoptotic cells (p < 0.001). However, there was a significant difference in necrotic cell ratios between groups and days (p < 0.001; p = 0.001). The lowest rates of necrotic cells were found in the control group and Glass Carbomer groups, while the rates of necrotic cells were similar among the other groups. Therefore, we concluded that APA Glass Carbomer presents the highest cellular viability among the materials.

Diverse actions of sirtuin-1 on ovulatory genes and cell death pathways in human granulosa cells

BackgroundHuman granulosa-lutein cells (hGLCs) amply express sirtuin-1 (SIRT1), a NAD + -dependent deacetylase that is associated with various cellular functions. SIRT1 was shown to elevate cAMP on its own and additively with human chorionic gonadotropin (hCG), it is therefore interesting to examine if SIRT1 affects other essential hGLC functions.MethodsPrimary hGLCs, obtained from the follicular aspirates of women undergoing IVF and SV40-transfected, immortalized hGLCs (SVOG cells), were used. Primary cells were treated with SIRT1 specific activator SRT2104, as well as hCG or their combination. Additionally, siRNA-targeting SIRT1 construct was used to silence endogenous SIRT1 in SVOG cells. PTGS2, EREG, VEGFA and FGF2 expression was determined using quantitative polymerase chain reaction (qPCR). Apoptotic and necroptotic proteins were determined by specific antibodies in western blotting. Cell viability/apoptosis was determined by the XTT and flow cytometry analyses. Data were analyzed using student t-test or Mann–Whitney U test or one-way ANOVA followed by Tukey HSD post hoc test.ResultsIn primary and immortalized hGLCs, SRT2104 significantly upregulated key ovulatory and angiogenic genes: PTGS2, EREG, FGF2 and VEGFA, these effects tended to be further augmented in the presence of hCG. Additionally, SRT2104 dose and time-dependently decreased viable cell numbers. Flow cytometry of Annexin V stained cells confirmed that SIRT1 reduced live cell numbers and increased late apoptotic and necrotic cells. Moreover, we found that SIRT1 markedly reduced anti-apoptotic BCL-XL and MCL1 protein levels and increased cleaved forms of pro-apoptotic proteins caspase-3 and PARP. SIRT1 also significantly induced necroptotic proteins RIPK1 and MLKL. RIPK1 inhibitor, necrostatin-1 mitigated SIRT1 actions on RIPK1 and MLKL but also on cleaved caspase-3 and PARP and in accordance on live and apoptotic cells, implying a role for RIPK1 in SIRT1-induced cell death. SIRT1 silencing produced inverse effects on sorted cell populations, anti-apoptotic, pro-apoptotic and necroptotic proteins, corroborating SIRT1 activation.ConclusionsThese findings reveal that in hGLCs, SIRT1 enhances the expression of ovulatory and angiogenic genes while eventually advancing cell death pathways. Interestingly, these seemingly contradictory events may have occurred in a cAMP-dependent manner.

MHY2251, a New SIRT1 Inhibitor, Induces Apoptosis via JNK/p53 Pathway in HCT116 Human Colorectal Cancer Cells.

Sirtuins (SIRTs) belong to the nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase family. They are key regulators of cellular and physiological processes, such as cell survival, senescence, differentiation, DNA damage and stress response, cellular metabolism, and aging. SIRTs also influence carcinogenesis, making them potential targets for anticancer therapeutic strategies. In this study, we investigated the anticancer properties and underlying molecular mechanisms of a novel SIRT1 inhibitor, MHY2251, in human colorectal cancer (CRC) cells. MHY2251 reduced the viability of various human CRC cell lines, especially those with wild-type TP53. MHY2251 inhibited SIRT1 activity and SIRT1/2 protein expression, while promoting p53 acetylation, which is a target of SIRT1 in HCT116 cells. MHY2251 treatment triggered apoptosis in HCT116 cells. It increased the percentage of late apoptotic cells and the sub-G1 fraction (as detected by flow cytometric analysis) and induced DNA fragmentation. In addition, MHY2251 upregulated the expression of FasL and Fas, altered the ratio of Bax/Bcl-2, downregulated the levels of pro-caspase-8, -9, and -3 proteins, and induced subsequent poly(ADP-ribose) polymerase cleavage. The induction of apoptosis by MHY2251 was related to the activation of the caspase cascade, which was significantly attenuated by pre-treatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, MHY2251 stimulated the phosphorylation of c-Jun N-terminal kinase (JNK), and MHY2251-triggered apoptosis was blocked by pre-treatment with SP600125, a JNK inhibitor. This finding indicated the specific involvement of JNK in MHY2251-induced apoptosis. MHY2251 shows considerable potential as a therapeutic agent for targeting human CRC via the inhibition of SIRT1 and activation of JNK/p53 pathway.

Radiation Response of Human Leukemia/Lymphoma Cells was Improved by 7-Geranyloxycoumarin.

ObjectivesAdult T-cell leukemia/lymphoma (ATLL) is a blood neoplasm with specific geographic distribution. Although radiotherapy is a palliative treatment that provides long-term local control, single use of radiation leads to complications for patients. To introduce a novel multimodal approach against ATLL, we investigated combinatorial effects of 7-geranyloxycoumarin and radiation in vitro.MethodsViability of MT-2 cells was determined by resazurin assay upon administration of 7-geranyloxycoumarin alone and followed by radiation. Then, apoptosis was detected by annexin V and propidium iodide, and the expression of candidate genes was analyzed by qPCR.ResultsFindings revealed significant (P<.0001) improvement in radiation effects upon 7-geranyloxycoumarin pretreatment, most notably when cells were pretreated with 5 µg/ml 7-geranyloxycoumarin for 96 h, exposed to 6 Gy radiation and recovered for 48 h. These results were confirmed by flow cytometry, as the percentage of early and late apoptotic cells was increased after combinatorial treatment. In addition, significant (P< .0001) changes in CD44, c-MYC, cFLIPL, BMI-1, NF-κB (Rel A), and P53 expression was induced by 7-geranyloxycoumarin and radiation.ConclusionsCurrent research indicated, for the first time, that combinatorial use of 7-geranyloxycoumarin and ionizing radiation could be considered as an effective therapeutic modality for ATLL.

Induction of apoptosis and suppression of Ras gene expression in MCF human breast cancer cells.

Breast cancer is the leading invasive cancer in women globally. This study aimed at evaluating the anti-apoptotic activity of p-Coumaric acid (PCA) on MCF-7 breast cancer cell line. Experiments were conducted in which the MCF-7 cell line was treated with PCA. which showed decreased cell viability, increased lactate dehydrogenase activity, and caspase-3 activation. The results were evaluated with real-time polymerase chain reaction which revealed that PCA reduced the amount of H-Ras and K-Ras transcript in MCF-7 breast cancer cells. In the presence of PCA there was a significant increase in the levels of mRNA gene Bax and late apoptotic cells which was dose dependent. It also retarded the relative expression of antiapoptotic gene, Bcl2 in treated cells. The results suggest that PCA exhibits anti-cancer properties against MCF-7 cells. PCA inhibited the growth of MCF7 cell. The optimum concentration of PCA was 75-150 mM. PCA can inhibit the growth of MCF-7 cells by reducing Ras expression and inducing cell apoptosis. Our results suggest that PCA could prove valuable in the search for possible inhibitors of Ras oncogene functionality and gain further support for its potential utilization in the treatment of patients with breast cancer. PCA is safe and could complement current treatments employed for the disease.

Inducing apoptosis using chemical treatment and acidic pH, and detecting it using the Annexin V flow cytometric assay.

Cell death is important in physiology, and can happen as a result of structural damage, or as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies, and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V/PI apoptosis assay.

Withanolide modulates the potential crosstalk between apoptosis and autophagy in different colorectal cancer cell lines

Withaferin A (WFA), a withanolide, is isolated from plants of Withania somnifera (L.) Dual (Solanaceae), known as Indian ginseng, Indian winter cherry or Ashwagandha. It has been reported to exert multifaceted anti-neoplastic effects. Here, we analyzed the impact of WFA on apoptosis and autophagy activation in different human colorectal cancer cell lines. We observed that WFA exposure caused an increased aggregation of cells in the subG1 arrest in cell cycle, and increased the number of late apoptotic cells. WFA also induced the apoptosis via PARP and caspase-3 cleavage accompanied with suppression of levels of anti-apoptotic proteins like Bcl-2 and Bcl-xl. The influence of WFA on autophagy was validated by acridine orange, MDC staining, and immunocytochemistry of LC3. It was found that 24 h treatment of WFA increased the acridine and MDC stained autophagosome with induced the LC3 and other autophagy markers Atg7 and beclin-1 activation. We used Z-DEVD-FMK, a caspase-3 blocker, and 3-MA, an autophagy inhibitor, to confirm whether these effects were specific to apoptosis and autophagy, and observed the recovery of both these processes upon exposure to WFA. Moreover, the activation of β-catenin protein was attenuated by WFA. Interestingly, small interfering RNA (siRNA)-promoted β-catenin knockdown augmented the WFA-induced active form of p-GSK-3β, and stimulated autophagy and apoptosis through PARP and LC3 activation. These findings suggested that WFA could stimulate activation of both apoptosis and autophagy process via modulating β-catenin pathway.

Myrcene exerts anti-tumor effects on oral cancer cells in vitro via induction of apoptosis

Purpose: To investigate the anticancer potential of myrcene against oral cancer cells.Methods: The effect of myrcene on oral cancer cell growth were studied using CCK-8 proliferation andclonogenic assays, while Annexin V-FITC/PI and DAPI staining methods were used for the determination of its effect on cell apoptosis. Protein expression levels were assayed by western blotting technique. Wound healing and Transwell chamber assays were used to determine the effect of myrcene on oral cancer cell migration and invasion, respectively.Results: Myrcene significantly and dose-dependently inhibited oral cancer cell growth (p &lt; 0.05). The anti-proliferative effect of myrcene was comparatively lower on Hs27 human fibroblast cells than on SCC9 oral cancer cells. Myrcene stimulated apoptosis in human oral cancer cells via enhancement of Bax expression and repression of Bcl-2 expression. The percentage of early and late apoptotic cells increased from 4.03 in untreated control to 27.77 when treated with 20 μM myrcene. Moreover, myrcene in vitro significantly suppressed the migration and invasion of oral cancer cells (p &lt; 0.05).Conclusion: These results indicate that myrcene exerts potent anticancer effects on oral cancer cells via induction of apoptosis, and should be further investigated in vivo for this purpose.

Modulatory Impact of Lamiaceae Metabolites on Apoptosis of Human Leukemia Cells.

Lamiaceae species are rich sources of biologically active compounds which have been applied in medicine since ancient times. Especially their antineoplastic properties have been thoroughly studied with respect to their putative application in chemoprevention and adjuvant therapy of cancer. However, the most known biological effects of Lamiaceae have been ascribed to their essential oil fractions, whereas their (poly)phenolic metabolites being also abundant in these plants, are much less recognized, nevertheless contributing to their beneficial properties, such as anti-cancer actions. The aim of this study was to evaluate the impact of dried aqueous extracts from common thyme (Thymus vulgaris L.) (ExTv), wild thyme (Thymus serpyllum L.) (ExTs), sweet marjoram (Origanum majorana L.) (ExOm), and peppermint (Mentha × piperita L.) (ExMp), as well as (poly)phenolic compounds: caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), luteolin-7-O-β-glucuronide (Lgr), luteolin-7-O-rutinoside (Lr), eriodictyol-7-O-rutinoside (Er), and arbutin (Ab), on unstimulated Jurkat cells, in comparison with their effect on staurosporine-stimulated Jurkat cells. Jurkat T cells were incubated with different concentrations of ExTv, ExTs, ExOm, ExMp, Lgr, LA, Er, Lr, RA, CA, or Ab. Subsequently, staurosporine was added to half of the samples and flow cytometry combined with fluorescence-activated cell sorting analysis was conducted, which allowed for the selection of early and late apoptotic cells. Both ExTs and ExOm stimulated apoptosis of Jurkat cells and enhanced the proapoptotic effect of staurosporine. Conversely, ExTv and ExMp demonstrated no clear effect on apoptosis. CA and RA raised the staurosporine-induced apoptotic effect. The impact of Er and Lgr on Jurkat cells showed fluctuations depending on the compound concentration. Neither Er nor Ab altered staurosporine-induced apoptosis in Jurkat cells, whereas Lgr seemed to weaken the proapoptotic action of staurosporine. The most evident observation in this study was the pro-apoptotic action of ExTs and ExOm observed both in staurosporine-unstimulated and stimulated Jurkat cells. Additionally, an enhancement of staurosporine-induced apoptosis by caffeic and rosmarinic acids was reported. Therefore, it might be concluded that these are the mixtures of biologically active polyphenols which often exert more pronounced beneficial effects than purified molecules.

Crocus sativus L. Tepal Extract Induces Apoptosis in Human U87 Glioblastoma Cells.

Crocus sativus (C. sativus) is considered as the costliest spice and an important medicinal plant. Herein, we investigated the effects of tepal extract (TE) of C. sativus on the viability of the human glioblastoma cells. Results revealed that TE significantly (P < 0.05) inhibited the proliferation of U87 glioblastoma cells in a dose-dependent manner with comparatively lower toxic effects against normal astrocytes. The IC50 of TE against U87 glioblastoma cells was found to be 130 μg/mL as compared to 600 μg/mL against normal astrocytes. TE also inhibited the colony formation of U87 cells significantly (P < 0.05). The AO/EB and Annexin V/PI staining assays indicated that TE stimulated apoptosis in U87 cells dose dependently. The early and late apoptotic U87 cells increased from 0.66% and 2.3% at control to 14.2% and 21.4% at 260 μg/mL of TE. Moreover, TE caused upregulation of Bax and suppression of Bcl-2. Wound healing assay showed that migration of the U87 cells was suppressed significantly (P < 0.05) at 80 μg/mL of TE. Taken together, these results suggest that TE exhibits antiproliferative effects against U87 glioma cells and may prove to be an important source of natural anticancer agents.

Artesunate protects pancreatic β-cells from streptozotocin-induced diabetes via inhibition of the NLRP3/caspase-1/GSDMD pathway

BackgroundReports in recent years have shown that pancreatic β-cell pyroptosis represents a critical mechanism involved with the progressive failure of pancreatic function. Previous research from our laboratory has indicated that artemether can increase the number of cells in pancreatic islets of db/db mice. In this study, we further examined whether artesunate (ART) protects pancreatic β-cells from the damage of streptozotocin (STZ) by inhibiting pyroptosis. Materials and methodsIn vitro, MIN6 cells exposed to 1 mM STZ were treated with ART (0.8 or 1.6 μM). The effects of ART on STZ-treated cells were evaluated through CCK-8 assay, flow cytometry and western blot, and further compared the effects of ART with the NLRP3 inhibitor, Mcc950 upon pyroptosis pathway proteins using western blot. In vivo, Male C57 mice were administered with a single intraperitoneal injection of STZ, and those with confirmed diabetes mellitus were given ART (0.5 or 1.0 mg/ml in drinking water) for 18 days. The effects of ART on STZ-induced diabetes were assessed by the observation of the general situation, glucose tolerance test, hematoxylin-eosin (HE) staining and immunohistochemistry. ResultsIn MIN6 cells treated with STZ, we found that ART increased cell viability, decreased the number of late apoptotic cells (including pyroptosis cells) and inhibited the expression of proteins associated with the pyroptosis pathway. In STZ-induced animal model, the administration of ART reduced blood glucose levels, improved the consumption status within this diabetic mouse model and inhibited the expression of proteins include in the pyroptosis pathway in mice pancreats. ConclusionsInhibition of pyroptosis may be a critical mechanism through which artesunate exerts protective effects upon pancreatic β cells.

Green route synthesis of copper oxide nanoparticles using Vitex altissima [L] leaves extract and their potential anticancer activity against A549 cell lines and its apoptosis induction

Green route synthesis of copper oxide nanoparticles (CuO NPs) using Vitex altissima leaves extract has gained a greater interest and it is simple and ecofriendly. It has been confirmed by using different techniques such as X-ray diffraction, UV-visible spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, Zeta potential, energy dispersive X-ray analysis, scanning electron microscope, and transmission electron microscopy. In this study, we examined the cytotoxic effect of CuO NPs on human lung cancer cells (A549). Cytotoxicity was evaluated using MTT assay, which results in IC50 values. We found that CuO NPs exerted a cytotoxic effect on A549 cells. Evaluation of anticancer potential through induction of apoptosis by flow cytometry was confirmed by annexin V and propidium iodide staining. The present result indicates a substantial rise in early and late apoptotic cell percentage, caspase-3 expression, and cell cycle arrest in G2/M phase in A549 cells. Overall, A549 cells showed that an increase in apoptotic activity due to increased caspase-3 expression revealed that CuO NPs has anticancer property. Highlights Green route synthesis of copper oxide nanoparticles (CuO NPs) using Vitex altissima [L.] leaves extract by combustion method. Green route synthesis of CuO NPs possessed a crystalline nature, which perfectly matched the monoclinic structure. Highly remarkable cytotoxicity effect of CuO NPs on human lung cancer cells (A549). The cytotoxicity of CuO NPs has potential anticancer activity against A549 cell line via induction of apoptosis and cell cycle arrest.

Protective Effect of Agmatine Against Cisplatin- Induced Cellular Apoptosis in an Auditory Cell Line.

The aims of this study were to evaluate the protective effects of agmatine against cisplatin-induced cellular apoptosis in an audi- tory cell line and to prove the protective mechanism of agmatine. The House Ear Institute-Organ of Corti 1 cells were co-treated with agmatine at different concentrations and 15 μM of cisplatin for 48 hours. Cell viability and proliferation were measured. Annexin V-fluorescein isothiocyanate /propidium iodide staining was performed to analyze apoptosis. The levels of intracellular reactive oxygen species were measured using flow cytometry. The expression of BCL2-associated X protein and the enzymatic activity of caspase-3 was measured to examine the pathway of apoptosis induction. In normal conditions, the maximal protective effect occurred with 10 mM of agmatine. However, in the presence of cisplatin, the maximal protective effect was observed from 8 mM of agmatine. Thus, 8 mM was chosen as the ideal agmatine concentration for the analysis of protective effects against cisplatin-induced cytotoxicity. Agmatine exerted a significant protective effect against 15 μM of cisplatin when applied for 48 hours and reduced the proportion of necrotic and late apoptotic cells. Agmatine did not significantly reduce the cisplatin-induced increase in reactive oxygen species but decreased the expression of BCL2-associated X protein and the activity of caspase-3. Agmatine protected against cisplatin-induced cellular apoptosis in an auditory cell line. These effects were mediated by the pro- tection of mitochondrial function and inhibition of apoptosis.

Cytotoxic activity of non-specific lipid transfer protein (nsLTP1) from Ajwain (Trachyspermum ammi) seeds

BackgroundTrachyspermum ammi, commonly known as Ajwain, is a member of the Apiaceae family. It is a therapeutic herbal spice with diverse pharmacological properties, used in traditional medicine for various ailments. However, all previous studies were conducted using small molecule extracts, leaving the protein’s bioactivity undiscovered.AimThe current study aimed to demonstrate the cytotoxic activity of Ajwain non-specific lipid transfer protein (nsLTP1) in normal breast (MCF10A), breast cancer (MCF-7), and pancreatic cancer (AsPC-1) cell lines. Also, to evaluate its structural stability in human serum as well as at high temperature conditions.MethodsThe cytotoxic activity of Ajwain nsLTP1 was evaluated in MCF-7 and AsPC-1 cell lines using MTT assay. Annexin V-FITC and PI staining were used to detect the early apoptotic and late apoptotic cells. The role of nsLTP1 in inducing apoptosis was further studied by quantifying Bcl-2, Bax, Caspase-3, Survivin, EGFR, and VEGF genes expression using RT-PCR. CD spectroscopy analyzed the nsLTP1 conformational changes after thermal treatment for structure stability determination. The RP-HPLC was used to analyze the nsLTP1 degradation rate in human serum at different time intervals incubated at 37 °C.ResultsAjwain nsLTP1 showed a potent cytotoxic effect in MCF-7 and AsPC-1. The IC50 value obtained in MCF-7 was 8.21 μM, while for AsPC-1 4.17 μM. The effect of nsLTP1 on stimulating apoptosis revealed that the proportions of apoptotic cells in both cell lines were relatively increased depending on the concentration. The apoptotic cells percentage at 20 μM was in MCF-7 71% (***P < 0.001) and AsPC-1 88% (***P < 0.001). These results indicate that nsLTP1 might efficaciously induce apoptosis in multiple types of cancerous cells. Genes expression in MCF-7 and AsPC-1 showed significant upregulation in Bax and Caspase-3 and downregulation in Bcl-2, Survivin, EGFR, and VEGF protein. The CD analysis of nsLTP1 showed a significant thermostable property. In serum, nsLTP1 showed a slow degradation rate, indicating high stability with a half-life of ~ 8.4 h.ConclusionOur results revealed the potential anticancer activity of Ajwain nsLTP1 and its mechanism in inducing apoptosis. It further exhibited thermostable properties at high temperatures and in human serum, which suggested this protein as a promising anticancer agent.

Transfer of IGF2BP3 Through Ara-C-Induced Apoptotic Bodies Promotes Survival of Recipient Cells.

Cytosine arabinoside (Ara-C) has been the standard therapeutic agent for myelodysplastic syndromes (MDS) and adult acute myeloid leukemia (AML) patients for decades. Considerable progress has been made in development of new treatments for MDS/AML patients, but drug resistance remains a major clinical problem. Apoptotic bodies (ABs), produced by late apoptotic cells, can enclose bioactive components that affect cell-cell interactions and disease progression. We isolated and identified drug-induced ABs from Ara-C-tolerance cells. Treatment of sensitive cells with Ara-C-induced ABs resulted in Ara-C-resistant phenotype. We further investigated components and functions of Ara-C-induced ABs. Proteomics analysis in combination with mass spectrometry revealed that Ara-C-induced ABs carried numerous RNA-binding proteins, notably including insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). Delivery of AB-encapsulated IGF2BP3 promoted survival of recipient cells by activating PI3K-AKT and p42-44 MAPK pathways. High IGF2BP3 level in ABs from MDS/AML patient plasma was correlated with poor overall survival. Our findings demonstrate that AB-derived IGF2BP3 plays an essential role in acquired Ara-C resistance in MDS/AML patients, and is a potential therapeutic target for suppression of Ara-C resistance.