Induced Cell Shrinkage Research Articles

Levofloxacin induces erythrocyte contraction leading to red cell death.

Levofloxacin, a fluoroquinolone, is an extensively used antibiotic effective against both positively and negatively staining bacteria. It works by inhibiting bacterial topoisomerase type II and topoisomerase type IV, resulting in impaired DNA synthesis and bacterial cell death. Eryptosis is another term for apoptotic cell death of erythrocyte marked by cell shrinkage, phosphatidylserine (PS) flipping, and membrane blebbing. The intent of the present research was to look at the eryptotic effect of levofloxacin by exposing erythrocytes to therapeutical doses (7, 14 µM) of levofloxacin for 48 hours. Cell size evaluation, PS subjection to outside, and calcium channel inhibition were carried out to investigate eryptosis. Oxidative stress generated by levofloxacin was measured as a putative mechanism of eryptosis using glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase activities. Similarly, hemolysis measurements demonstrated levofloxacin's cytotoxic effect. Our findings showed that therapeutic doses of levofloxacin can cause a considerable decline in antioxidant enzymes activities, as well as induce cell shrinkage, PS externalization, and hemolysis in erythrocytes. The role of calcium in triggering erythrocyte shrinkage was also confirmed. In conclusion, our findings showed that the indicated levofloxacin doses caused oxidative stress, which leads to erythrocyte death via eryptosis and hemolysis. These findings emphasize the importance of using levofloxacin with caution and the need for additional research to mitigate these side effects.

OR03-06 Transmembrane Water Channels Within The Tsh Receptor

Abstract Disclosure: R. Latif: None. T.F. Davies: Advisory Board Member; Self; Board of Kronus Inc (Starr, ID, USA);. M. Mezei: None. Using our recently reported hydrated model of the TSH receptor transmembrane (TM) region (Endocrinology 2021,162:7;1-12), we have now identified a network of putative TM water channels within this structure. By analyzing the molecular simulation trajectory for water molecules crossing the plasma membrane embedded TSHR we found evidence of putative water channels suggestive of active transport. To ascertain the biological activity of such TSHR channels we examined the movement of water down osmotic gradients using calcein aceto-oxymethyl ester (Calcein-AM) fluorescence in rat thyrocyte cells (FRTL5). Calcein-AM is a membrane permeant fluorescent dye that is quenched in a concentration-dependent manner and is, therefore, an effective probe of cell volume changes that would result from water movement across the cell membrane. Since thyroid cells are known to contain aquaporin (AQP) channels, principally AQP4, we first blocked these channels (using 3μM of TGN020) prior to examining the effects of TSHR activation using TSH (10-103 µU/ml), stimulating TSHR monoclonal antibodies (mAbs MS1 and M22 at 10μg/ml and 1μg/ml respectively) and TSHR-specific small molecule allosteric agonists (MS437 and MS438 at 10µM) and measured the changes in fluorescence quenching. D-Mannitol at 400mM was used as the hypertonic medium to induce cell shrinkage measured by continuous fluorescence measurements with read intervals of 50msec. Analyzing the normalized fluorescence of cells treated with TSH in a dose-dependent manner we observed ∼25% quenching with 103 µU/ml of TSH. On blocking the binding of TSH using a TSHR blocking mAb K1-70 (1μg/ml), we found marked de-quenching. Furthermore, using equimolar concentrations of stimulating TSHR antibodies the quenching was even more significantly enhanced to 54-60%. These data suggested that ligands that activate the TSHR by binding to the TSHR ectodomain could influence the TM water transport. However, using the allosteric activators, which act at the TM, we also showed similar changes indicating this was not ectodomain dependent. These data confirm the presence of the modeled putative TSHR water channels indicating that TSH and TSHR autoantibodies have an important role in regulating TM water passage. Presentation: Thursday, June 15, 2023

UVA/UVB Irradiation Exerts a Distinct Phototoxic Effect on Human Keratinocytes Compared to Human Malignant Melanoma Cells.

Solar ultraviolet radiation (UVR) is responsible for the development of many skin diseases, including malignant melanoma (MM). This study assessed the phototoxic effects of UVA, and UVB radiations on healthy and pathologic skin cells by evaluating the behavior of human keratinocytes (HaCaT) and MM cells (A375) at 24 h post-irradiation. The main results showed that UVA 10 J/cm2 exerted no cytotoxicity on HaCaT and A375 cells, while UVB 0.5 J/cm2 significantly reduced cell viability and confluence, induced cell shrinkage and rounding, generated nuclear and F-actin condensation, and induced apoptosis by modulating the expressions of Bax and Bcl-2. The association of UVA 10 J/cm2 with UVB 0.5 J/cm2 (UVA/UVB) induced the highest cytotoxicity in both cell lines (viability < 40%). However, the morphological changes were different-HaCaT cells showed signs of necrosis, while in A375 nuclear polarization and expulsion from the cells were observed, features that indicate enucleation. By unraveling the impact of different UVR treatments on the behavior of normal and cancer skin cells and describing enucleation as a novel process involved in the cytotoxicity of UVA/UVB irradiation, these findings bridge the gap between the current and the future status of research in the field.

Impact of Quorum Sensing Signal N-3-Oxododecanoyl Homoserine Lactone on Intestinal Porcine Enterocyte Cell

Quorum sensing (QS) is an autoinducer (AI) molecules mediated signaling system in bacteria. It is generally believed that bacteria can regulate their own characteristics or virulence through OS. This study investigated the direct effects of a quorum sensing signal molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL, synonyms: OdDHL) on host cells, to further reveal the function of QS system in the interaction between bacteria and host cells. Intestinal porcine enterocyte cell line (IPEC-J2) were used as model cells, and the AHL signal molecule OdDHL was used to stimulate the cells to observe the effect of quorum sensing signals on host cells. The results showed that OdDHL had an obvious effect on the morphology of IPEC-J2 cells, and a lower concentration and shorter time of stimulation could induce cell shrinkage and rounding. MTT assay revealed that OdDHL had no effect on IPEC-J2 cell viability, suggesting that the cell morphological changes were not from signaling molecule toxicity. Using different concentrations and different exposure times of OdDHL to stimulate IPEC-J2 cells, we demonstrated that changes in cell morphology were positively correlated with the concentration of AHL and exposure time. Bacterial adhesion assay showed that OdDHL could enhance the adhesion of enteropathogenic E. coli to host cell IPEC-J2, while reducing its invasion into host cell. Fluorescent quantitative PCR and Western blot revealed that the expression of tight junction protein ZO-1 was significantly increased. The expression levels of multiple genes that related to cytoskeletons and cell junction were all regulated by OdDHL. In conclusion, OdDHL could directly affect the biological characteristics of IPEC-J2 cells such as morphology and tight junction, reduce the intestinal defences of hosts, thereby promoting the adhesion and injury of pathogenic bacteria to the intestine of piglets. This study provides a basic reference for further understanding of the pathogenic role of regulating pathogenic bacterial populations and their prevention against diseases.

Uncovering the physiological impacts of soybean meal replacement by Narbonne vetch (Vicia narbonensis) meal in rainbow trout (Oncorhynchus mykiss) diets: Towards the future and sustainable European aquaculture.

The identification and implementation of raw materials to replace fish meal (FM) or its major alternative, soybean meal (SBM), among other factors, is crucial for the sustainable growth of aquaculture. Narbonne vetch (Vicia narbonensis) meal (NVM) has previously been identified as a promising alternative raw material locally produced in Europe to replace SBM. Previous work has showed that 33% replacement of SBM by NVM treated with a commercially available phytase (Rovabio® PHY) does not compromise fish growth performance in a 63-day trial. Here, a deeper characterization of the potential use of NVM to replace SBM was performed to unveil any potential undesired physiological impact in rainbow trout (Oncorhynchus mykiss) juveniles with an initial weight of 38.04 ± 0.07 g. After a 63-day feeding trial, high (66%) SBM replacement by NVM (even when treated with Rovabio® PHY) had a negative impact on rainbow trout: decreasing the activity of alkaline proteases, trypsin, chymotrypsin, and α-amylase; inducing cell shrinkage in hepatocytes; and reducing plasma triglycerides and hepatic vitamin E (VE) levels. No biochemical alterations were observed in blood plasma glucose, cholesterol or redox enzymes, regardless of the % of SBM replacement and/or Rovabio® PHY treatment. In fish fed diets with 33% replacement of SBM by NVM (regardless of being treated with Rovabio® PHY or not), only a reduced content of plasma triglycerides, and a slight modification in the position of the nucleus in enterocytes (more intermediate) from the proximal intestine, were reported. Altered metabolism of both carbohydrates and VE was also confirmed at transcriptional level, possibly being related to the presence of non-starch polysaccharides (NSPs) in NVM. The present study identifies future strategies to improve the use of NVM as an alternative raw material to replace SBM, allowing greater sustainability, and a lower carbon footprint in European aquafeeds to be achieved.

Elemental Analysis, Phytochemical Screening and Evaluation of Antioxidant, Antibacterial and Anticancer Activity of Pleurotus ostreatus through In Vitro and In Silico Approaches.

Oyster mushrooms form an integral part of many diets owing to their characteristic aroma, delicious taste and nutraceutical value. In this study, we examined oyster mushrooms by direct arc optical emission spectroscopy for the presence of various biologically important elements. Furthermore, we screened phytochemicals present in Pleurotus ostreatus by applying GC-MS. Additionally, the antioxidant, antibacterial and anticancer activities of the ethanolic extract of Pleurotus ostreatus were studied. Moreover, we docked the phytochemicals and examined their binding affinities with EGFR, PR and NF-κB proteins, which are overexpressed in breast cancer. The elemental analysis showed the presence of Fe, K, Na, Ca, Mg, Cr and Sr in the spectrum. Moreover, GC-MS data revealed the presence of 32 biologically active compounds in oyster mushrooms. The ethanolic extract displayed remarkable free radical scavenging activity (~50%) against DPPH. The mushroom has shown promising antibacterial activity against both Gram-positive (S. aureus) and Gram-negative bacteria (Pseudomonas aeruginosa, Proteus vulgaris and Proteus mirabilis). The present study also revealed that oyster mushrooms possess significant anticancer activity. The ethanolic extract inhibited the growth and proliferation of MCF-7 cells. It also induced cell shrinkage, membrane blebbing and nuclear fragmentation, resulting in apoptosis of malignant cells. The molecular docking analysis showed that ligand 15 (Linoleic acid ethyl ester), ligand 27 (Ergosta-5,7,9(11),22-tetraen-3-ol, (3. beta.,22E), ligand 28 (Stigmasta-5,22-dien-3-ol, acetate, (3. beta.,22Z), ligand 30 (Ergosta-5,7,22-Trien-3-Ol, (3. Beta.,22E) and ligand 32 (gamma. Sitosterol) exhibited better binding affinities with EGFR, PR and NF-κB proteins. This result provides a strong ground for confirmation of the in vitro anticancer effect of Pleurotus ostreatus. From the present in vitro and in silico studies, it can be concluded that Pleurotus ostreatus is a useful source of essential elements and reservoir of bioactive compounds which confer its significant antioxidant, antibacterial and anticancer properties.

Inhibition of Neurotoxicity/Anticancer Activity of Bortezomib by Caffeic Acid and Chlorogenic Acid.

Bortezomib, used for the treatment of multiple myeloma, has been reported to induce potent neurotoxicity. The present study investigated whether eight popular polyphenols inhibit bortezomib-induced neurotoxicity without affecting its anticancer activity. Viable cell number was determined with the MTT method. Tumor-specificity was determined by the relative cytotoxicity in human oral squamous cell carcinoma vs. normal oral cells. Neurotoxicity was determined by the relative cytotoxicity in differentiated rat neuronal PC12 cells vs. normal cells. Apoptotic cells were quantified by cell cycle analysis. Bortezomib induced cell shrinkage, disruption of neurites, and accumulation of PC-12 cells in subG1. Only chlorogenic acid and caffeic acid protected PC-12 cells from bortezomib-induced neurotoxicity. Ferulic acid that has one of the two hydroxyl groups replaced by a methoxy group showed a significantly reduced neuroprotective effect. Caffeic acid and the chlorogenic acid also neutralized the anticancer potential of bortezomib. Caffeic acid and the chlorogenic acid may reduce the biological activity of bortezomib by forming a conjugate.

Protective effect and mechanism of betaine against hyperosmotic stress in porcine intestinal epithelium

• Betaine exerts osmoprotective role in small intestine. • Betaine ameliorated hyperosmotic stress induced cell shrinkage and elevation ROS of intestinal epithelium. • Betaine attenuated mitochondria-related apoptosis caused by hyperosmolarity via maintaining mitochondrial homeostasis. • Betaine alleviated hyperosmolarity-induced autophagy with the decrease of autophagosomes and autolysosomes. Hyperosmolarity under physiological conditions resulting from lumen contents is a big challenge to the normal function of intestinal epithelium. Betaine is a potent osmolyte which is mostly studied in kidney. This study was aimed to gain insight into the osmoprotectant role of betaine in intestinal epithelium under hyperosmotic condition with intestinal porcine epithelial cells (IPEC-J2) and piglets. The result showed that betaine could attenuate the hyperosmolarity-induced reduction of cell viability after treating 12 h. Betaine ameliorates hyperosmotic stress in intestinal epithelium via decreasing reactive oxygen species (ROS) with the alleviation of cell shrinkage. Moreover, betaine prevented mitochondria from membrane collapse and alleviated mitochondrial-related apoptosis induced by hyperosmolarity in vivo and vitro . In addition, betaine ameliorated autophagy caused by hyperosmolarity by up-regulation LC3 II and down-regulation p62. In summary, betaine protects porcine small intestinal epithelium from hyperosmotic stress by alleviating cell shrinkage, ROS accumulation as well as apoptosis and autophagy.

An In Vitro Anti-Cancer Activity of Ocimum tenuiflorum Essential Oil by Inducing Apoptosis in Human Gastric Cancer Cell Line

Background and Objectives: The effects of Ocimum tenuiflorum essential oil (OTEO) against gastric cancer remain unknown and merit investigation. Materials and Methods: In the present study, the anti-cancer activity of OTEO was examined in a human gastric cancer cell line (AGS). After OTEO treatment, AGS cell viability was determined by an MTT assay, and inhibition of metastasis was determined by cell migration and invasion assays. The expression of apoptosis-related genes in treated AGS cells was determined by qRT-PCR. Results: OTEO significantly decreased AGS cell viability in a dose-dependent manner (IC50 163.42 µg/mL) and effectively inhibited cell migration and invasion. Morphological examination demonstrated that OTEO induced cell shrinkage, chromatin condensation, and fragmentation, which are considered typical morphologies of apoptotic cell death. Pro-apoptotic genes (TP53, BAX, and BAK) were significantly up-regulated, while anti-apoptotic genes (BCL-2 and BCL-xL) were significantly down-regulated after treatment with OTEO. In addition, significantly increased gene expression was detected for CASP8, CASP9, and CASP3 in AGS cells exposed to OTEO. GC-MS analysis demonstrated that the major compound of OTEO was caryophyllene (25.85%) and α-pinene (11.66%). Conclusions: This in vitro study demonstrates for the first time that OTEO has potential anti-gastric cancer activity and may induce apoptosis in AGS cells through extrinsic and intrinsic pathways.

Photodynamic therapy of multidrug resistant leukemic murine cells by 3,6-bis(alkylthiourea)acridine hydrochlorides.

Efforts to overcome multidrug resistance in cancer have led to the development of several novel strategies including photodynamic therapy (PDT). PDT is based on the use of photosensitizers (PSs) photoactivation, which causes the formation of reactive oxygen species that can induce cell death. In the last decade, the development of new PSs has been significantly accelerated. Recently, acridine-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been investigated as a new group of PSs and we have shown that PDT/AcrDTUs caused cell death of mouse leukemic cells L1210. In this study, we investigated the efficacy of PDT/AcrDTUs for the treatment of L1210/VCR cells as a model of chemoresistant cells (overexpressing P-glycoprotein, P-gp). The photoactivation (365 nm, 1.05 J/cm2) increased the cytotoxicity of AcrDTUs 10-15 times. Inhibition of P-gp (verapamil) has been shown to have no significant effect on the accumulation of propyl-AcrDTU (the most potent derivative) in L1210/VCR cells. The intracellular distribution of this acridine derivative has been studied. Prior to irradiation of the resistant cells, propyl-AcrDTU was sequestered mainly in the cytosol, partly in the mitochondria, and, unlike in the sensitive cells, the AcrDTU was not found in the lysosomes. PDT with 1 µM propyl-AcrDTU induced cell shrinkage and "ladder DNA" formation, and although a drastic decrease of the intracellular ATP level was observed at the same time, there was no increase in extracellular LDH activity. AIF in the nucleus can induce DNA fragmentation and we have actually observed a mitochondrio-nuclear translocation of AIF. We concluded that AcrDTUs are photocytotoxic against L1210/VCR cells and that mitochondria play an important role in cell death induced by PDT.

CucurbitacinI induces apoptosis in ovarian cancer cells through oxidative stress and the p190B‑Rac1 signaling axis.

Ovarian cancer is a serious threat to women's life and health, with a high mortality rate. Therefore, in addition to improving surgery for ovarian cancer, it is particularly important to develop novel drug treatments. In the present study, the anticancer effects of cucurbitacinI, a natural product, were investigated. CucurbitacinI impaired the viability of SKVO3 cells in a concentration‑ and time‑dependent manner. Apoptosis was involved in the process of cucurbitacin I‑induced cell death, with an increase observed in cleaved‑caspase 3 and BAX, and a decrease in Bcl‑2. CucurbitacinI caused a notable increase in intracellular reactive oxygen species, and regulated Kelch‑like ECH‑associated protein 1 and nuclear factor erythroid‑derived 2‑like 2 to decrease the expression of antioxidant‑related genes. In addition, CucurbitacinI induced cell shrinkage by regulating the p190BRhoGAP (p190B)‑Rac1 signaling axis related to the cytoskeleton. In brief, these results suggested that cucurbitacinI induced cell death through oxidative stress and the p190B‑Rac1 signaling axis in SKVO3 cells. The results may provide novel evidence for the treatment of ovarian cancer.

Alpinoid c analog inhibits angiogenesis and induces apoptosis in COLO205 cell line

Diarylheptanoids display an array of biological and pharmacological properties. We previously reported the synthesis of a diarylheptanoid Alpinoid c and a series of its derivatives, evaluated their cytotoxicity against various human cancer cells. We found some of these derivatives were significantly more potent than Alpinoid c in preventing the proliferation of various cancer cell lines. Among these, (S, E)-1-(3, 4 dimethoxyphenyl)-6-hydroxy-7-phenylhept-4-en-3-one (DPHP) showed most potent cytotoxicity against COLO205 cells, however, the mechanism by which DPHP prevents the growth of these colon cancer cells remains unknown. In the current study, we investigated the molecular mechanism of DPHP on colon cancer cells. DPHP inhibited the proliferation of COLO205 (IC50 7.01 ± 0.62 μM) and A549 (IC50 20.03 ± 3.11 μM) cells more specifically than normal human colon epithelial cell line NCM460 (IC50 55.6 ± 4.02 μM). In COLO205 cells, DPHP induced cell shrinkage, membrane blebbing, chromatin condensation, phosphatidylserine externalization, and an accumulation of cells at sub-G1 phase. Further analysis these cells treated with DPHP revealed a decrease in mitochondrial membrane potential, an increase in Bax/Bcl2 ratio, the release of cytochrome c, activation of caspases −9, −3/7, and cleavage of the poly-ADP-ribose polymerase. DPHP treatment resulted in inhibition of hypoxia induced VEGF downstream signaling pathway in COLO205 cells is concurrent with inhibition of angiogenesis in CAM. Based on these data we suggest that DPHP significantly induced apoptosis possibly via intrinsic mitochondrial apoptosis pathway and inhibited angiogenesis. Our study suggests DPHP could be a therapeutic agent in treating colon cancer.

Characteristics of enterovirus 71-induced cell death and genome scanning to identify viral genes involved in virus-induced cell apoptosis

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease and severe neural complications in infants and young children. Viral pathogenesis is associated with virus-induced cell death and inflammatory cytokine production, which is usually correlated with the type of programmed cell death. EV71-infected cells were analyzed through microscopy, cell staining, and immunoblotting to determine the characteristics of EV71-induced cell death. Results demonstrated that EV71 infection induced cell shrinkage, nuclear condensation, decreased mitochondrial potential, and membrane phosphatidylserine translocation. Caspase-9 activation, poly (ADP-ribose) polymerase cleavage, and lactate dehydrogenase release were also observed during virus-induced cell death. The activated gasdermin D (GSDMD) and the phosphorylated mixed lineage kinase domain-like protein (p-MLKL) were not detected. These observations indicated that EV71-induced cell death was mainly executed by apoptosis through the intrinsic pathway rather than by GSDMD-mediated pyroptosis and p-MLKL-mediated necroptosis. Genome scanning analysis identified that EV71 2A, 2B, and 3C might be the determinant genes of virus-induced cell death. Further experiments showed that EV71 2A- and 3C-induced cell death exhibited dependence on their protease activities but involved different mechanisms. EV71 2A-induced cell death was correlated with the shut-off of host cap-dependent translation, whereas EV71 3C-induced cell death might not be ascribed to this mechanism. These findings would enhance our understanding of EV71 infection and viral pathogenesis, and help identify antiviral targets.

Effect of exposure of osteoblast-like cells to low-dose silver nanoparticles: uptake, retention and osteogenic activity

Objective Silver nanoparticles (AgNPs) are widely used in orthopaedic implants because of their excellent antimicrobial properties. However, the effects of AgNPs on bone cells and osteogenic activity are still poorly understood. Method Here, we investigated the effect of AgNPs on the cell viability, uptake, and osteogenic activity of osteoblast-like cells (MG-63 cells) at low concentrations. Results Our results showed that uptake and retention of AgNPs reduced the cell viability and increased cell membrane penetrability even after termination of exposure of MG-63 cells to AgNPs. In addition, AgNPs induced cell shrinkage, reduced the expressions of ALP, COL-I, OCN and OPG, and enhanced the expressions of Runx2 and RANKL. Conclusion Collectively, our work demonstrated that the cytotoxic effects of low-dose AgNPs on MG-63 cells persisted even after termination of exposure. AgNPs may interfere with bone formation. More attention should be paid to the toxicity of AgNPs during the design of future orthopaedic implants.

Enhancement of antitumor activity by using 5-ALA-mediated sonodynamic therapy to induce apoptosis in malignant gliomas: significance of high-intensity focused ultrasound on 5-ALA-SDT in a mouse glioma model.

OBJECTIVEHigh invasiveness of malignant gliomas frequently causes early local recurrence of the tumor, resulting in extremely poor outcome. To control such recurrence, novel therapies targeted toward infiltrating glioma cells around the tumor border are required. Here, the authors investigated the antitumor activity of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas to explore the possibility for clinical use of 5-ALA-mediated SDT (5-ALA-SDT).METHODSIn vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251Oct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry and TUNEL staining. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined by analysis of the effect of pretreatment with the radical scavenger edaravone. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated.RESULTSThe 5-ALA-SDT inhibited cell growth and changed cell morphology, inducing cell shrinkage, vacuolization, and swelling. Flow cytometric analysis and TUNEL staining indicated that 5-ALA-SDT induced apoptotic cell death in all gliomas. The 5-ALA-SDT generated significantly higher ROS than in the control group, and inhibition of ROS generation by edaravone completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. The proliferative activity of the entire tumor was markedly decreased. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact.CONCLUSIONSThe 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field, whereas the surrounding brain tissue remained normal, resulting in longer survival of the HIFU-treated mice compared with that of untreated mice. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-specific therapy, not only for a tumor mass but also for infiltrating tumor cells in malignant gliomas.

Chemical Constituents of Thai Citrus hystrix and Their Antiausterity Activity against the PANC-1 Human Pancreatic Cancer Cell Line.

Human pancreatic cancer cells have an extreme tolerance to nutrition starvation, enabling them to survive in a hypovascular tumor microenvironment. Searching for agents that preferentially inhibit cancer cell viability under nutrition starvation conditions is a novel antiausterity strategy in anticancer drug discovery. In the present study, a hexane extract of the peels of Citrus hystrix fruits showed preferential cytotoxicity against PANC-1 human pancreatic cancer cells using a nutrient-deprived medium. Phytochemical investigation of this bioactive extract led to the isolation of 10 coumarins (1-10) including a new furanocoumarin (1). The isolated compounds were tested for their preferential cytotoxic activity against three different human pancreatic cancer cell lines [PANC-1, MIA PaCa-2, and PSN-1]. Among these, bergamottin (7) was identified as the most active constituent. In real-time live imaging, 7 was found to induce cell shrinkage, membrane blebbing, and disintegration of organelles in PANC-1 cells. Bergamottin (7) was also found to inhibit PANC-1 cell migration and colony formation. Mechanistically, 7 inhibited key survival proteins in the Akt/mTOR signaling pathway. Bergamottin (7) and related compounds are potential antiausterity candidates for drug development against pancreatic cancer.

Polyhexamethylene biguanide and chloroquine induce programmed cell death in Acanthamoeba castellanii

Several chemotherapeutic drugs have been described as amoebicidal agents acting against Acanthamoeba trophozoites and cysts. However, the underlying mechanism of action is poorly characterized. Here, we describe programmed cell death (PCD) in A. castellanii induced by polyhexamethylene biguanide (PHMB) and chloroquine. We used four types of amoebicidal agents including 0.02% PHMB, 0.02% chlorhexidine digluconate, 100 μM chloroquine, and 100 μM 2,6-dichlorobenzonitrile to kill Acanthamoeba trophozoites and cysts. Exposure to PHMB and chloroquine induced cell shrinkage and membrane blebbing in Acanthamoeba, observed microscopically. Externalization of phosphatidyl serine on the membranes of Acanthamoeba was detected by annexin V staining. Apoptotic cell death of Acanthamoeba by PHMB and chloroquine was confirmed by FACS analysis. Nuclear fragmentation of Acanthamoeba was demonstrated by DAPI staining. PHMB induced PCD in trophozoites and cysts, and chloroquine induced PCD in cysts. These findings are discussed to establish the most effective treatment for Acanthamoeba-induced keratitis.

Resveratrol suppresses human cervical carcinoma cell proliferation and elevates apoptosis via the mitochondrial and p53 signaling pathways.

Numerous studies have demonstrated the apoptotic and anti-proliferative effects of resveratrol, a natural polyphenolic phytoalexin, on various cancer cell lines. However, the effects of resveratrol on the regulation of human cervical carcinoma, and the mechanisms underlying these effects, remain to be elucidated. In the present study, the potential mechanisms underlying the effects of resveratrol in HeLa cervical carcinoma cells were investigated. The results revealed that resveratrol inhibited proliferation and induced apoptosis in HeLa human cervical cancer cells in a dose-dependent and time-dependent manner. Resveratrol induced cell shrinkage in HeLa cells and apoptosis accompanied by the activation of caspase-3 and −9. Furthermore, resveratrol upregulated the expression of the pro-apoptotic B-cell lymphoma (Bcl)-2-associated X protein and downregulated the expression of the anti-apoptotic proteins Bcl-2 and Bcl-extra large in HeLa cells. In addition, p53, a protein that is essential for cell survival and cell cycle progression, exhibited elevated expression levels in resveratrol-treated HeLa cells. Therefore, resveratrol may be a promising novel inhibitor of human cervical cancer.

Apigenin induces cell shrinkage in Candida albicans by membrane perturbation.

Apigenin, a natural flavone, has been well characterized for its their anticarcinogenic property; however, its bioactivity against pathogenic fungi has not been investigated in detail. In this study, we examined the antifungal activity and mode of action of apigenin. Apigenin inhibited the growth of fungal pathogens, which induced superficial infection and reduced biofilm mass. Three-dimensional flow cytometric analysis demonstrated that apigenin induced morphological changes, especially cell shrinkage, in Candida albicans. We investigated the cause of cell shrinkage using the cyanine dye 3,3΄-dipropylthiacarbocyanine iodide. Results revealed that apigenin altered the cell membrane potential. Apigenin also induced membrane dysfunction, and increased cell permeability to 1,6-diphenyl-1,3,5-hexatriene and propidium iodide. We observed the influx and efflux of fluorescent molecules of varying molecular weights and radii across large unilamellar vesicles and live cells that had been treated with apigenin. Membrane disruption facilitates the release of small intracellular constituents such as ions and sugars, but not proteins. These findings suggested that apigenin exerted an antifungal activity by inducing membrane disturbances, which led to cell shrinkage and leakage of intracellular components.

Effects of naringin on the expression of miR-19b and cell apoptosis in human hepatocellular carcinoma.

The effects of naringin on the expression of miR-19b and cell apoptosis were investigated in the human hepatocellular carcinoma cell line HepG2. HepG2 cells were treated with varied concentrations of naringin. The effects of naringin on the proliferation of HepG2 cells were observed by an MTT assay, morphological changes of cells were observed by an inverted microscope, cell apoptosis was detected by DAPI staining, miR-19b mRNA levels were determined with RT-PCR, and the expression of Bax and Bcl-2 proteins was examined by western blot assay. MTT results showed that naringin significantly inhibited the proliferation of HepG2 cells. Apoptotic HepG2 cells showed obvious changes in morphology under inverted microscope. DAPI staining suggested that naringin could induce cell shrinkage and nuclear chromatin condensation. RT-PCR results showed that naringin could upregulate the expression of miR-19b mRNA. Finally, western blot suggested that naringin upregulated the expression of Bax protein, but downregulated the expression of Bcl-2 protein. In conclusion, naringin can upregulate the expression of miR-19b mRNA and induce HepG2 cell apoptosis. In addition, it can also upregulate the expression of Bax protein and downregulate the expression of Bcl-2 protein during the process of apoptosis.