Early Cell Apoptosis Research Articles

Anticancer effects and the mechanism underlying 2-methoxyestradiol in human osteosarcoma in vitro and in vivo.

Osteosarcoma (OS) occurs in both children and adolescents and leads to a poor prognosis. 2-methoxyestradiol (2-ME) has a strong antitumor effect and is effective against numerous types of tumor. However, 2-ME has a low level of antitumor effects in OS. The present study investigated the effects of 2-ME on the proliferation and apoptosis of human MG63 OS cells. The potential biological mechanisms by which 2-ME exerts its biological effects were also investigated in the present study. The results of the present study demonstrated that 2-ME inhibited the proliferation of OS cells in a time- and dose-dependent manner, induced G2/M phase cell cycle arrest and early apoptosis. The expression levels of vascular endothelial growth factor (VEGF), Bcl-2 and caspase-3 were measured via western blotting and reverse transcription-quantitative PCR. As the concentration of 2-ME increased, the RNA and protein expression levels of VEGF and Bcl-2 decreased gradually, whereas the expression of caspase-3 increased gradually. In addition, tumor growth in nude mice was suppressed by 2-ME with no toxic side effects observed in the liver or kidney. Immunohistochemistry demonstrated that the expression levels of Bcl-2 and VEGF were significantly lower, and those of caspase-3 were significantly higher in test mice compared with the control group. TUNEL staining of xenograft tumors revealed that with increased 2-ME concentration, the number of apoptotic cells also gradually increased. Thus, 2-ME effectively inhibited the proliferation and induced apoptosis of MG63 OS cells in vitro and in vivo with no obvious side effects. The mechanism of the anticancer effect of 2-ME may be associated with the actions of Bcl-2, VEGF and caspase-3.

Sulfur dioxide derivatives produce antidepressant- and anxiolytic-like effects in mice

Sulfur dioxide (SO2) can be endogenously generated from sulfur-containing amino acids in animals and humans. Increasing evidence shows that endogenous SO2 may act as a gaseous molecule to participate in many physiological and pathological processes. However, the role of SO2 and its derivatives in the central nervous system remains poorly understood. The present study explored the protective effects of exogenous SO2 derivatives (Na2SO3:NaHSO3, 3:1 M/M) on cellular injury in vitro by using the cell proliferation assay (MTS), cell counting kit 8 assay (CCK-8), and cyto-flow assay in the corticosterone (CORT)-induced PC12 cell injury model. We also examined the antidepressant and anxiolytic effects of SO2 derivatives on the chronic mild stress (CMS)-induced depression mouse model by using the open field test, novelty suppressed feeding test, forced swimming test, tail suspension test, and sucrose preference test. In the MTS and CCK-8 assays, we found that preexposure of SO2 derivatives significantly blocked CORT-induced decrease of cellular survival without causing any negative effects. Results from the cyto-flow assay indicated that treatment with SO2 derivatives could reverse CORT-induced early and late apoptosis of PC12 cells. Systemic treatment with SO2 derivatives produced markedly antidepressant- and anxiolytic-like activities in mice under normal condition and rapidly reversed CMS-induced depressive- and anxiety-like behaviors. In conclusion, these findings indicate that exogenous SO2 derivatives show protective properties against the detrimental effects of stress and exert antidepressant- and anxiolytic-like actions. The present study suggests that exogenous SO2 derivatives are potential therapeutic agents for the treatment of depression, anxiety, and other stress-related diseases.

3-O-(E)-p-Coumaroyl betulinic acid possess anticancer activity and inhibit Notch signaling pathway in breast cancer cells and mammosphere

Activation of Notch signaling is associated with tumor aggressiveness, poor clinical outcome and drug resistance in breast cancer patients. Targeting Notch signaling with small molecule inhibitors may be a better strategy for anticancer drug development. We identified 3-O-(E)-p-Coumaroylbetulinic acid (CB) as a lead compound and potent inhibitor of Notch signaling pathway. Treatment of human breast cancer MBA-MD-231 and T47D cells with CB resulted in a dose- and time-dependent inhibition of cell viability and G0/G1-phase cell cycle arrest. This effect was associated with a marked decrease in the expression of cyclin D1 and its activating partner, cyclin-dependent kinase 2 with concomitant increase in cyclin kinase inhibitor p21, operative in G1-phase of the cell cycle. CB treatment induced early apoptosis in breast cancer cells as evident by increase in cleaved caspase-3, decrease in Bcl2 and survivin, surge in reactive oxygen species and disruption of mitochondrial membrane potential. CB treatment altered Notch target genes viz. Hes1, Hey1 and E-cadherin at mRNA and protein level in time-dependent manner along with decrease in Notch promoter activity at IC50 concentration. Furthermore, CB treatment decreased mammosphere formation in MCF-7 cells through down-modulation of the Notch signaling pathway and suppression of self-renewal markers such as c-Myc, SOX-2 and CD44. Our findings demonstrate that CB possess anticancer activity in breast cancer cells and suppresses self-renewal ability in the mammosphere as a result of modulation in cell-cycle machinery, disruption of mitochondrial function, induction of apoptosis, and Notch inhibition.

Outer inflammatory protein of Helicobacter pylori impacts IL-8 expression, adherence, cell apoptosis and cell cycle of gastric cells independent of its copy number.

Outer inflammatory protein (OipA) is an important virulence factor of Helicobacter pylori (H. pylori), but the correlation between oipA copy number and its virulence remains unknown. The study was designed to investigate whether the duplicate oipA gene loci showed more virulent than one oipA gene in vitro. H. pylori strain CCS9803 (China Chongqing Strain 9803) that carries duplicate oipA loci was used to construct one or two oipA knockout mutant strain, which was further verified by qPCR and western blot. Gastric epithelial cells AGS and GES-1 were infected with wild-type (WT) or oipA mutants for 6 or 24h. The expression levels of IL-8, bacterial adhesion, cell apoptosis and cell cycle were performed to analyze the function of oipA. The WT and oipA mutant strains induce significantly higher mRNA and protein levels of IL-8 than the uninfected group (P < 0.05), but only oipA2 mutants induced significantly decreased expression levels than the WT-infected group (P < 0.05). Adherence to gastric cells was significantly decreased by inactivated two oipA loci (P < 0.05). The WT strain caused a significant rising proportion of early apoptosis cell, which had dropped after duplicate oipA genes were both knockout (P < 0.05). WT and oipA1 mutants failed to affect cell cycle; however, the oipA2 mutants increased M phase and reduced S phase when compared to the uninfected group. In conclusion, our study demonstrated that oipA impacts IL-8 expression, adherence, cell apoptosis and cell cycle of gastric cells independent of its gene copy number.

Exploration of certain 1,3-oxazole- and 1,3-thiazole-based hydroxamic acids as histone deacetylase inhibitors and antitumor agents

Several novel series of hydroxamic acids bearing 2-benzamidooxazole/thiazole (5a-g, 6a-g) or 2-phenylsulfonamidothiazole (8a-c) were designed and synthesized. The compounds were obtained straightforwards via a two step pathway, starting from commercially available ethyl 2-aminooxazole-4-carboxylate or ethyl 2-aminothiazole-4-carboxylate. Biological evaluation showed that these hydroxamic acids generally exhibited good cytotoxicity against three human cancer cell lines (SW620, colon; PC-3, prostate; NCI-H23, lung cancer), with IC50 values in low micromolar range and comparable to that of SAHA. These compounds also comparably inhibited HDACs with IC50 values in sub-micromolar range (0.010–0.131 µM) and some compounds (e.g 5f, IC50, 0.010 µM) were even more potent than SAHA (IC50, 0.025 µM) in HDAC inhibition. Representative compounds 6a and 8a appeared to arrest the SW620 cell cycle at G2 phase and significantly induced both early and late apoptosis of SW620 colon cancer cells. Docking experiments on HDAC2 and HDAC6 isozymes revealed favorable interactions at the tunnel of the HDAC active site which positively contributed to the inhibitory activity of synthesized compound. The binding affinity predicted by docking program showed good correlation with the experimental IC50 values. This study demonstrates that simple 1,3-oxazole- and 1,3-thiazole-based hydroxamic acids are also promising as antitumor agents and HDAC inhibitors and these results should provide valuable information for further design of more potent HDAC inhibitors and antitumor agents.

Biomimetic Matrix Stiffness Modulates Hepatocellular Carcinoma Malignant Phenotypes and Macrophage Polarization through Multiple Modes of Mechanical Feedbacks.

The extracellular matrix (ECM) stiffening is an important sign of local microenvironment change, which is considered as a hallmark of many diseases including hepatocellular carcinoma (HCC). The fates of both cancer cells and immune cells can be regulated by mechanical feedbacks acquired from ECM, but there is a lack of a precise study of mechanical feedback modes in different cell phenotypes following with the progressively increasing ECM stiffness. Herein, we used a biopolymeric film without further modification of adhesive molecules, as a natural local niche to mimic a gradually stiffening manner from HCC onset in liver cirrhosis to its metastasis in the spinal cord. Three distinct manners of mechanical feedbacks were found: the gradual manner in HCC cell spreading, migration and early apoptosis to oxaliplatin, the stepwise manner in HCC cell adhesion, proliferation, focal adhesion (FA) formation, drug resistance, and macrophage M1 polarization; the specific manner in the stages of the progression of epithelial-mesenchymal transition at different stiffness ranges. Further investigation of molecular mechanisms confirmed those mechanical feedback manners by signaling activation of FA kinase, phosphatidylinositol 3-kinase, and expression of pro-/antiapoptotic and pro-/anti-inflammatory genes. Our results pave a novel avenue to know about mechanical feedbacks from ECM, which could be used for future cancer studies and in vitro drug screening applications.

Hellebrigenin anti-pancreatic cancer effects based on apoptosis and autophage.

Hellebrigenin is a natural product found in the toad skin secretions and plants of Urginea, including Hellebores and Kalanchoe genera. It has been shown to be active against Leishmania chagasi promastigotes and Trypanosoma cruzi trypomastigotes and also reported to play an anti-tumor effect on several cancer cell lines in vitro, including pancreatic cancer. This study is aimed to investigate the effects of Hellebrigenin on pancreatic carcinoma cells, SW1990 and BxPC-3 in vitro and its molecular mechanism involved in antitumor activities. Our results showed that Hellebrigenin effectively inhibited the proliferation of SW1990 and BxPC-3 cells in dose- and time-dependent manner. Flow cytometry results showed that Hellebrigenin induced the G0/G1 arrest in both of SW1990 and BxPC-3 cells and promoted cell early apoptosis and autophagy according to morphological observation. Immunofluorescence staining results further confirmed that cell apoptosis and autophagy also increased upon the Hellebrigenin treatment. Moreover, higher dose of Hellebrigenin further increased the cell apoptosis rate while decrease the mitochondrial membrane potential 24 h after treatment. The autophagy rate increased 48 h after treatment with significant difference (P < 0.05). Western blot analysis showed that the expression of caspase 3, 7, cleaved caspase 7, Atg 12, LC3 proteins were increased in SW1990 cell after treatment with Hellebrigenin. In addition, increasing expression of caspase 3, 7, 9, PARP, cleaved caspase 3, 7, 9, PARP, the sub basic protein of the PI3K family, Beclin-1, LC 3, Atg 3, 5, 12, 16 L were also observed after BxPC-3 cells treated with Hellebrigenin. In summary, this study reported for the first time that Hellebrigenin effectively induced autophagy and apoptosis especially the early apoptosis in SW1990 and BxPC-3 cells.

SAT-684 A Deadly Triad: Coexistence of Acute Pancreatitis, Hypertryglyceridemia and Diabetic Ketoacidosis

The coexistence of diabetic ketoacidosis (DKA), hypertriglyceridemia and acute pancreatitis (AP) represent a complex phenomenon described as the enigmatic triad. The exact initial culprit and pathophysiologic mechanism of this chain of events are still unclear posing a challenge in management. DKA may lead to glucose and lipid metabolism dysregulation which can result in hypertriglyceridemia leading to AP. On the other hand, hypertriglyceridemia may induce AP which may decompensate diabetes and lead to DKA. In both scenarios, this triad results in an uncommon clinical presentation with up to 80% mortality rate. Most frequently reported in children, this entity accounts for only a handful of cases reported in the literature.Case of an obese, non alcoholic 57 year old male without history of systemic illness who visits the emergency room due to mid-upper abdominal pain for the past day. Pain radiates to the back, worsens upon laying flat, and is associated with bloating and nausea. He denies previous similar episodes, vomiting, fever or bowel habit changes. Laboratory workup revealed lipidemic sample with hyperglycemia, metabolic acidosis, positive serum ketones, and normal amylase and lipase. Lipid panel revealed hypertriglyceridemia at 6,260 mg/dL (35-150). Glycated hemoglobin at 14.7%. Abdominal computed tomography showed peripancreatic inflammation consistent with pancreatitis. Given clinical and imaging criteria the diagnosis of severe hypertriglyceridemia induced AP and DKA were made. The patient was admitted to ICU and treated with intravenous insulin drip and supportive management. Resolution of DKA and successful decrease in triglycerides to less than 500 mg/dL was achieved by the third day of admission. After six days, the patient was discharged home with insulin and lipid lowering regimens.This case demonstrates an extremely rare initial presentation of diabetes mellitus. This triad is the result of a toxic chain of events that may be lethal if not promptly identified. This case makes an exemplary lesson as to always take under consideration atypical etiologies to potentially life threatening conditions and also remarks that while uncommon, pancreatitis with normal pancreatic enzymes is a possible phenomenon. Even though false negative amylase has been associated with hypertriglyceridemia induced AP, only a few cases with negative lipase have been described. While no definite explanation has been yet discovered, negative lipase may be explained by early acinar cell apoptosis in AP. More research efforts are necessary in order to improve early diagnosis, treatment, and mortality rate for this rare but potentially deadly triad and to better understand the mechanisms underlying AP and the role that digestive enzymes play.

A Digital Controlled Pulse Generator for a Possible Tumor Therapy Combining Irreversible Electroporation With Nanosecond Pulse Stimulation.

The irreversible electroporation with microsecond electric pulses is a new ablation technique adopted in the tumor therapy worldwide. On the other hand, the nsPEF (nanosecond pulsed electric field) has been proved to provide a means to induce immunogenic cell death and elicits antitumor immunity, which is under intensive in-vitro and in-vivo studies and in clinical trials. Normally, one needs two different types of electric pulse generators for producing the pulses in the ranges of nanosecond and microsecond, respectively. In order to realize these two types of tumor treatments in complementary and optimize electrical pulse parameters, we have developed a compact high-voltage pulse generator with a wide pulse width tuning range, based on a capacitor discharging configuration digitally controlled by a silicon carbide MOSFET switching array through a pair of optic-coupler drivers. The developed digital pulse generator is capable of adjusting: pulse width over 100-100μs, voltage over 0-2kV and repetition rate up to 1.2 kHz. The pulse generator is designed in simulation, implemented and verified in experiments. The pulse generator is shown to deliver a complementary treatment on Murine melanoma B16 cell lines, i.e., triggering the cell early apoptosis under the 300ns pulse stimulation while a complete killing under the 100ns pulses. The pulse generator is further demonstrated to induce antitumor immunity in a preliminary in vivo study on the mice model.

Molecular pathological mechanism of blue ray induces murine photoreceptor cell 661W apoptosis

Modern life is full of artificial light in day and night. Smart phones, laptops, televisions, and high definition monitors provide unnatural human light source into human eye and cause ocular defects in modern human being. Understanding the molecular mechanism of the interplay between artificial light and ocular cells may help researcher society understanding the methods that preventing artificial light‐induced visual impairment. Here, we reported a short‐term exposure at short wavelength light source from blue and green light‐emitting diode (LED) could cause the murine photoreceptor cells 661W cells death, but not in red LED. However, the molecular mechanism remaining unclear. Using the molecular probe of caspase 3 reporter, we found the blue and green LED caused caspase 3 activation even as short as 10 minutes exposure and subsequently cell death at 16 hours later. We further confirmed 661W cells apoptosis by cleavage form caspase 3 and PARP formation. Moreover, we found the decreasing of mitochondria membrane potential and increasing of cellular relative‐oxygen species in 661W cells both indicate early cell apoptosis under short‐term exposure. Up to our knowledge, there was no transcriptome analysis which revealed immediately early gene expression in light‐induced cell death. Thus, we analyzed the 661W transcriptome at 2 hours after 10 minutes’ short wavelength light exposure and validated the candidate genes expression by RT‐Q‐PCR. Using the ingenuity pathway analysis, we found valuable up‐stream regulators, pathways, and potential mechanisms in short wavelength induced photoreceptor cell death and may provide novel therapy niches. In conclusion, our preliminary results prove that short wavelength light exposure can cause the photoreceptor cells death and the potential molecular clues. Further pathohistological analysis of key protein expression in the animal models may help us to prove the mechanism inside the eye damage from short wavelength light in future.Support or Funding InformationThis study was support by Shu Zen College of Medicine and Management with grant number (SZL10800012) and Academia Sinica

Mapping the Multi‐Modal Distribution of Craniofacial Phenotypes in NOSIP Mutants

Nitric Oxide Synthase Interacting Protein (NOSIP) is a E3 mono‐ubiquitin ligase that regulates the activity of protein phosphatase 2A. Deletion of NOSIP leads to severe and variable malformations of the craniofacial complex. Our goal was to use NOSIP mutant mice to understand of mechanisms of phenotypic variation. The phenotype of an individual embryo is the product of the developmental trajectory that it previously traversed. We propose that this concept of canalization is essential for understanding the etiology of complex phenotypic outcomes. We have previously shown that the brain patterns the face, so sequential perturbations affecting the brain and face may create unique developmental trajectories that each embryo transits. We tested this by creating a 3D morphospace of NOSIP heterozygous and homozygous mutants to determine if embryos occupy unique morphospace regions based on affected craniofacial structures. Embryos between embryonic day 10.5 (E10.5) to E17.5 were collected, fixed, and imaged (mCT). Anatomical features were reconstructed using Avizo. NOISP expression appears to be restricted to the neuroepithelium, lower jaw mesenchyme, and mesenchyme and epithelium in tissue adjacent to the frontonasal ectodermal zone between stage E10.5‐11.5. Embryos with a single telencephalic lobe had more severe facial malformations, including micrognathia and obstructed airway without significant changes in early cell apoptosis (via TUNEL staining). Embryos with normal telencephalic vesicle separation had less severe facial phenotypes, including left‐right mild asymmetry and clefting. Overall, it appears that NOSIP‐KO creates a multi‐modal distribution of craniofacial phenotypes along the developmental timeline by affecting early brain development, suggesting that understanding canalization may provide insight into the complexity of genotype‐phenotype map (Fig. 1).Support or Funding InformationFunding was provided by NIH F32DE08476 and R01DE019638 grants.Schematic of the changing phenotypic landscape describing the multi‐modal distribution of craniofacial phenotypes within NOSIP+/− and NOSIP−/− embryos compared to wildtype (light blue).Figure 1

Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants

BackgroundThis study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids. MethodsA differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously. ResultsThe screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%. ConclusionThis study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.

New azaphilones, phomopsones A-C with biological activities from an endophytic fungus Phomopsis sp. CGMCC No.5416

Three undescribed azaphilones, phomopsones A-C (1–3) and two known azaphilones (4–5) were isolated from the culture of endophytic fungus Phomopsis sp. CGMCC No.5416 from the stems of Achyranthes bidentata. Their structures were determined by spectroscopic analysis (HRESIMS, 1D and 2D NMR), and the absolute configurations were determined by CD spectroscopy. Compounds 2 and 3 showed significant inhibitory activities against HIV-1 with against HIV-1 with IC50 values of 7.6 and 0.5 μmol/L, respectively. Compounds 2 and 3 also displayed moderate cytotoxicity with CC50 values of 3.2–303 μmol/L against A549, MDA-MB-231 and PANC-1 cell lines. Moreover, compound 3 can induce the early apoptosis of PANC-1 cancer cells with the apoptosis rate of 28.54%.

Discovery of potential targets of Triptolide through inverse docking in ovarian cancer cells.

Triptolide (TPL) is proposed as an effective anticancer agent known for its anti-proliferation of a variety of cancer cells including ovarian cancer cells. Although some studies have been conducted, the mechanism by which TPL acts on ovarian cancer remains to be clearly described. Herein, systematic work based on bioinformatics was carried out to discover the potential targets of TPL in SKOV-3 cells. TPL induces the early apoptosis of SKOV-3 cells in a dose- and time-dependent manner with an IC50 = 40 ± 0.89 nM when cells are incubated for 48 h. Moreover, 20 nM TPL significantly promotes early apoptosis at a rate of 40.73%. Using a self-designed inverse molecular docking protocol, we fish the top 19 probable targets of TPL from the target library, which was built on 2,250 proteins extracted from the Protein Data Bank. The 2D-DIGE assay reveals that the expression of eight genes is affected by TPL. The results of western blotting and qRT-PCR assay suggest that 40 nM of TPL up-regulates the level of Annexin A5 (6.34 ± 0.07 fold) and ATP syn thase (4.08 ± 0.08 fold) and down-regulates the level of β-Tubulin (0.11 ± 0.12 fold) and HSP90 (0.21 ± 0.09 fold). More details of TPL affecting on Annexin A5 signaling pathway will be discovered in the future. Our results define some potential targets of TPL, with the hope that this agent could be used as therapy for the preclinical treatment of ovarian cancer.

"French fries"-like luminescent metal organic frameworks for the fluorescence determination of cytochrome c released by apoptotic cells and screening of anticancer drug activity.

A luminescent metal organic framework was prepared by encapsulating Zn-Ag-In-S quantum dots into "French fries"-like MIL-68(In) metal organic frameworks (ZAISQDs@MIL-68(In)). The ZAISQDs@MIL-68(In) had a maximum excitation wavelength at 370nm and maximum emission wavelength at 620nm. It was found that the ZAISQDs@MIL-68(In) was efficiently quenched by cytochrome c (Cyt c), which is an important biomarker of early cell apoptosis. The quenching mechanism was ascribed to be aninner filter effect and dynamic quenching of Cyt c towards the ZAISQDs@MIL-68(In), and the enrichment effect of MIL-68(In). Benefiting from the multiple advantages, ZAISQDs@MIL-68(In) was developed as an assay strategy of Cyt c withlogarithmic relation between signal quenching andconcentration in the range0.02 to 3.5μM. The linear equation was (F0-F)/F0= 0.5043 + 0.2678 × logcCyt c with a detection limit of 8nM. Cyt c released by drug induced apoptotic cells was determined by ZAISQDs@MIL-68(In), and this strategy has been utilized for the screening of anticancer drug activity. Graphical abstract Schematic representation of the synthesis of ZAISQDs@MIL-68(In) and its application for Cyt c and screening anticancer drug activity.

Induction of Apoptosis by Gluconasturtiin-Isothiocyanate (GNST-ITC) in Human Hepatocarcinoma HepG2 Cells and Human Breast Adenocarcinoma MCF-7 Cells.

Gluconasturtiin, a glucosinolate present in watercress, is hydrolysed by myrosinase to form gluconasturtiin-isothiocyanate (GNST-ITC), which has potential chemopreventive effects; however, the underlying mechanisms of action have not been explored, mainly in human cell lines. The purpose of the study is to evaluate the cytotoxicity of GNST-ITC and to further assess its potential to induce apoptosis. GNST-ITC inhibited cell proliferation in both human hepatocarcinoma (HepG2) and human breast adenocarcinoma (MCF-7) cells with IC50 values of 7.83 µM and 5.02 µM, respectively. Morphological changes as a result of GNST-ITC-induced apoptosis showed chromatin condensation, nuclear fragmentation, and membrane blebbing. Additionally, Annexin V assay showed proportion of cells in early and late apoptosis upon exposure to GNST-ITC in a time-dependent manner. To delineate the mechanism of apoptosis, cell cycle arrest and expression of caspases were studied. GNST-ITC induced a time-dependent G2/M phase arrest, with reduction of 82% and 93% in HepG2 and MCF-7 cell lines, respectively. The same treatment also led to the subsequent expression of caspase-3/7 and -9 in both cells demonstrating mitochondrial-associated cell death. Collectively, these results reveal that GNST-ITC can inhibit cell proliferation and can induce cell death in HepG2 and MCF-7 cancer cells via apoptosis, highlighting its potential development as an anticancer agent.

Triclocarban exposure affects mouse oocyte in vitro maturation through inducing mitochondrial dysfunction and oxidative stress

Triclocarban (TCC), a broad-spectrum lipophilic antibacterial agent, is the main ingredient of personal and health care products. Nonetheless, its ubiquitous presence in the environment has been established to negatively affect the reproduction in humans and animals. In this work, we studied the possible toxic effects of TCC on mouse oocytes maturation in vitro. Our findings revealed that TCC-treated immature mouse oocytes had a significantly reduced rate of polar body extrusion (PBE) compared to that of control. Further study demonstrated that the cell cycle progression and cytoskeletal dynamics were disrupted after TCC exposure, which resulted in the continuous activation of spindle assembly checkpoint (SAC). Moreover, TCC-treated oocytes had mitochondrial damage, reduced ATP content, and decreased mitochondrial membrane potential (MMP). Furthermore, TCC exposure induced oxidative stress and subsequently triggered early apoptosis in mouse oocytes. Besides, the levels of histone methylation were also affected, as indicated by increased H3K27me2 and H3K27me3 levels. In summary, our results revealed that TCC exposure disrupted mouse oocytes maturation through affecting cell cycle progression, cytoskeletal dynamics, oxidative stress, early apoptosis, mitochondria function, and histone modifications in vitro.

Supercritical CO2 Processing Generates Aqueous Cisplatin Solutions with Enhanced Cancer Specificity.

Cisplatin is a highly toxic material used clinically as a potent chemotherapeutic. While effective against some cancers, toxicity limits widespread use and low solubility confounds delivery. To formulate a better tolerated and more water-soluble form of cisplatin, we designed a rapid expansion of supercritical solutions (RESS) technique with supercritical carbon dioxide (sc-CO2) to collect nanoclusters of cisplatin embedded in dry ice, in a dual-stage collection vessel cooled to liquid nitrogen temperature. These nanoclusters were solubilized in deionized water and further concentrated (up to 51.3 mM) by a Rotovap process, yielding stable cisplatin solutions with solubility up to 15 × (w/w) greater than that of normal cisplatin. Extensive material characterizations of the solutions were carried out to determine any chemical and/or structural changes of the RESS-processed cisplatin. In vitro cytotoxicity studies of these aqueous solutions showed increased cell viability and early apoptosis compared to equivalent concentrations of standard cisplatin solutions. In vivo studies using zebrafish embryos revealed that standard cisplatin solutions were acutely toxic and caused death of rapidly proliferating cells compared to RESS-processed cisplatin, which were better tolerated with reduced general cell death. Increased water solubility and matched chemical identity of RESS-processed aqueous cisplatin solutions indicate the potential to open up novel drug-delivery routes, which is beneficial for new pharmaceutical design and development.

Betulinic Acid-Nitrogen Heterocyclic Derivatives: Design, Synthesis, and Antitumor Evaluation in Vitro.

Betulinic acid (BA) is a star member of the pentacyclic triterpenoid family, which exhibits great prospects for antitumor drug development. In an attempt to develop novel antitumor candidates, 21 BA-nitrogen heterocyclic derivatives were synthetized, in addition to four intermediates, 23 of which were first reported. Moreover, they were screened for in-vitro cytotoxicity against four tumor cell lines (Hela, HepG-2, BGC-823 and SK-SY5Y) by a standard methylthiazol tetrazolium (MTT) assay. The majority of these derivatives showed much stronger cytotoxic activity than BA. Remarkably, the most potent compound 7e (the half maximal inhibitory concentration (IC50) of which was 2.05 ± 0.66 μM) was 12-fold more toxic in vitro than BA-treated Hela. Furthermore, multiple fluorescent staining techniques and flow cytometry collectively revealed that compound 7e could induce the early apoptosis of Hela cells. Structure–activity relationships were also briefly discussed. The present study highlighted the importance of introducing nitrogen heterocyclic rings into betulinic acid in the discovery and development of novel antitumor agents.

Abstract P1-07-01: Cholinergic receptor muscarinic 3 (CHRM3) contributes to breast cancer tumorigenesis through angiogenesis regulation

Abstract Objectives: Cholinergic muscarinic receptors are widely distributed in different human organs with various biological functionals such as synaptic transmission. Accumulating evidence revealed the overexpression of these receptors especially cholinergic receptor muscarinic 3 (CHRM3) with multiple roles in cancer behaviors such as proliferation, apoptosis, angiogenic phenotypes and even epithelial-mesenchymal transition (EMT) through various signaling pathways. However, its roles in breast cancer and angiogenesis still remain elusive. Methods: Gene expression and patient survival data were from the TCGA and the GTEx projects database. Cells were transfected with scrambled shRNA or CHRM3 shRNA to knockdown gene expression. pcDNA1.3 empty expression vector or CHRM3 expressing vector was used for ectopic expression in the cells. Cell proliferation was examined in different treatment groups by MTT assay and further subjected to cell cycle and apoptosis assay. Expression levels of CHRM3 in paired breast cancer tissues and different cell lines as well as angiogenesis-related markers in cells with different treatments were investigated by qRT-PCR. Results: CHRM3 expressions were significantly upregulated in triple negative breast cancer (TNBC) subtype, but not other subtypes of breast cancer, and was consistent with the data in TCGA database. Further investigation in different breast cancer cells revealed that CHRM3 expressions were higher in MDA-MB-231 and MDA-MB-468, which are both TNBC cell lines. After treatment with CHRM3-specific inhibitor, 4-DAMP, cell viability was significantly decreased in both MDA-MB-231 and MDA-MB-468 cells. G1-phase arrest and decreased S-phase cell populations and early apoptosis were increased upon 4-DAMP. Moreover, angiogenic markers such as ANG1 and VEGFD were significantly decreased upon CHRM3 knockdown in breast cancer cells or human umbilical vein endothelial cells (HUVEC). Gene correlation analysis from TCGA database indicated a significant positive correlation between CHRM3 and ANG1. Furthermore, CHRM3 shRNA or 4-DAMP treatment inhibited cell proliferation in both MDA-MB-231 and HUVEC. Conclusions: We found that CHRM3 is upregulated in TNBC tissues and cell lines. CHRM3 confers tumor oncogenic roles, and for the first time, we identified a potential relationship between CHRM3 and angiogenesis in breast cancer. Further experiments are warranted to explore the underlying mechanism of CHRM3 in regulating angiogenesis. Citation Format: Jiawei Chen, Vivian Y Shin, Isabella Cheuk, Jennifer Siu, Ava Kwong. Cholinergic receptor muscarinic 3 (CHRM3) contributes to breast cancer tumorigenesis through angiogenesis regulation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-07-01.