DNA Fragmentation Assay Research Articles

A new pyrrole based small molecule from Tinospora cordifolia induces apoptosis in MDA-MB-231 breast cancer cells via ROS mediated mitochondrial damage and restoration of p53 activity

Approximately 15% of globally diagnosed breast cancers are designated as triple negative breast cancer (TNBC). In this study, we investigated the effect of the natural compound, Bis(2- ethyl hexyl) 1H-pyrrole-3,4-dicarboxylate (TCCP), purified from Tinospora cordifolia on MDA-MB-231, a TNBC cell line. The pro-apoptotic nature of TCCP on MDA-MB-231 was determined by assessing various apoptotic markers. ROS generation, intracellular calcium, mitochondrial membrane potential (ΔΨm), MPTP, cardiolipin peroxidation and caspase activity were determined fluorometrically. BAX, BCL-2, cytochrome c, caspases, and p53 protein expressions were determined by immunoblotting. Further, the effect of TCCP on DNA and cell death was determined by DNA fragmentation assay, annexin-V staining, and cell cycle analysis. TCCP treatment caused endogenous ROS generation, increase in intracellular calcium and phosphorylation of p53 in a concentration-dependent manner, which was reverted upon pre-treatment with pifithrin-μ. This led to the downstream altered expression of Bcl-2 and Bax proteins, mitochondrial membrane depolarization, MPTP, and cardiolipin peroxidation. TCCP induced cytochrome c release into the cytosol, caspase activation, ultimately resulting in DNA fragmentation. Further, induction of apoptosis and morphological alterations were evident from the phosphatidylserine externalization and increase in sub G1 population. The in vivo Ehrlich ascites tumor (EAT) mouse study revealed the effectiveness of TCCP in reducing the tumor burden and resulted in a ~2 fold increase in mice survival with minimal hepato-renal toxicity. Overall, TCCP was shown to be efficient in inducing ROS and mitochondrial-mediated apoptosis by restoring p53 activity in MDA-MB-231 cells and also induced EAT cell death in vivo thereby inhibiting tumor proliferation.

Cell-bound Exopolysaccharide Extract from Indigenous Probiotic Bacteria Induce Apoptosis in HT-29 cell-line.

The aim of this study was to compare the cytotoxiceffects of local probiotic bacteria, including Lactobacillus paracasei, Lactobacillus brevis, while isolated from "Tarkhine" food and the induction of apoptosis in the HT-29 human colon adenocarcinoma cell line and normal fibroblasts. HT-29 and L-929 cell lines were treated with cell-bound exopolysaccharide extract (cb-EPS) from L. paracasei and L. brevis. The MTT assay was used to analyze cell viability. Cellular apoptosis was examined by flow cytometry and DNA fragmentation assay. The cb-EPS from both probiotic bacteria prevented the proliferation of HT-29 colon cancer cells. In addi- tion, the cytotoxic and anti-proliferative effects of the exopolysaccharide extract from both bacteria in L-929 fibro- blasts were much lower than HT-29 cells. The induction of apoptosis in HT-29 cells was observed at 48h compared with 72h. It seems that the exopolysaccharides extracted from both bacteria have a greater effect on the induction of apoptosis at 48h. The cb-EPS of L. brevis showed more potent anti-proliferative and apoptotic properties than the cb- EPS of L. paracasei. The ladder pattern of DNA fragmentation confirmed the induction of apoptosis in cancer cells. The results of the MTT assay and apoptosis indicate that the induction of apoptosis by the exopolysac- charide from bacteria depends on the dose, time, and strain of bacteria. Further studies may contribute toward the understanding of using these probiotic bacteria as biological products to treat and prevent cancers.

Cytoplasmic pro-apoptotic function of the tumor suppressor p73 is mediated through a modified mode of recognition of the anti-apoptotic regulator Bcl-XL

In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL or BCL2L1). This binding disrupted an interaction between Bcl-XL and the pro-apoptotic protein BH3-interacting domain death agonist (Bid). In particular, we found that a 16-mer p73-TAD peptide motif (p73-TAD16) mediates transcription-independent apoptosis, accompanied by cytochrome c release from the mitochondria, by interacting with Bcl-XL Interestingly, the structure of the Bcl-XL-p73-TAD16 peptide complex revealed a novel mechanism of Bcl-XL recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-XL, comprising the BH1, BH2, and BH3 domains in an orientation opposite to those of pro-apoptotic BH3 peptides. Taken together, our results indicate that the cytoplasmic apoptotic function of p73 is mediated through a noncanonical mode of Bcl-XL recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.

Cytotoxic Effects of Pinnatane A Extracted from Walsura pinnata (Meliaceae) on Human Liver Cancer Cells.

Background: Pinnatane A from the bark of Walsura pinnata was investigated for its anti-cancer properties by analyzing the cytotoxic activities and cell cycle arrest mechanism induced in two different liver cancer cell lines. Methods: A 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to analyze the pinnatane A selectivity in inducing cell death in cancer and normal cells. Various biological assays were carried out to analyze the anti-cancer properties of pinnatane A, such as a live/dead assay for cell death microscopic visualization, cell cycle analysis using propidium iodide (PI) to identify the cell cycle arrest phase, annexin V-fluorescein isothiocyanate (annexin V-FITC)/PI flow cytometry assay to measure percentage of cell populations at different stages of apoptosis and necrosis, and DNA fragmentation assay to verify the late stage of apoptosis. Results: The MTT assay identified pinnatane A prominent dose- and time-dependent cytotoxicity effects in Hep3B and HepG2 cells, with minimal effect on normal cells. The live/dead assay showed significant cell death, while cell cycle analysis showed arrest at the G0/G1 phase in both cell lines. Annexin V-FITC/PI flow cytometry and DNA fragmentation assays identified apoptotic cell death in Hep3B and necrotic cell death in HepG2 cell lines. Conclusions: Pinnatane A has the potential for further development as a chemotherapeutic agent prominently against human liver cells.

Roles of intracellular and extracellular ROS formation in apoptosis induced by cold atmospheric helium plasma and X-irradiation in the presence of sulfasalazine

Sulfasalazine (SSZ) is a well-known anti-inflammatory drug and also an inhibitor of the cystine-glutamate antiporter that is known to reduce intracellular glutathione (GSH) level and increase cellular oxidative stress, indicating its anti-tumor potential. However, the combination of SSZ with other physical modalities remains unexplored. Here, the effects of SSZ on cold atmospheric helium plasma (He-CAP), which produces approximately 24 x higher concentration of hydroxyl radicals (. OH) compared to X-irradiation (IR) in aqueous solution, and on IR-induced apoptosis in human leukemia Molt-4 cells were studied to elucidate the mechanism of apoptosis enhancement. Both the Annexin V-FITC/PI and DNA fragmentation assay revealed that pre-treatment of cells with SSZ significantly enhanced He-CAP and IR-induced apoptosis. Similar enhancement was observed during the loss of mitochondrial membrane potential, intracellular Ca2+ ions, and mitochondria- and endoplasmic reticulum-related proteins. The concentration of intracellular reactive oxygen species (ROS) was much higher in He-CAP treated cells than in X-irradiated cells. On the other hand, strong enhancement of Fas expression and caspase-8 and -3 activities were only observed in X-irradiated cells. It might be possible that the higher concentration of intracellular and extracellular ROS suppressed caspase activities and Fas expression in He-CAP-treated cells. Notably, pretreating the cells with an antioxidant N-acetyl-L-cysteine (NAC) dramatically decreased apoptosis in cells treated by He-CAP, but not by IR. These results suggest that IR-induced apoptosis is due to specific and effective ROS distribution since intracellular ROS formation is marginal and the high production of ROS inside and outside of cells plays unique roles in He-CAP induced apoptosis. We conclude that our data provides efficacy and mechanistic insights for SSZ, which might be helpful for establishing SSZ as a future sensitizer in He-CAP or IR therapy for cancer.

2-Methylpyridine-1-ium-1-sulfonate as an Inducer of Apoptosis and Cell Cycle Arrest: A comparative in vitro and Computational Study

“Let food be thy medicine and thy medicine be thy food” was expressed by Hippocrates and the health benefits of medicinal plants and natural products have been considered by humans since historic times. The current study aims to investigate the anti-cancer activity of 2-Methylpyridine-1-ium-1-sulfonate (MPS) isolated from bulbs of Allium hirtifolium. The MPS compound (in a dose-dependent manner) induced arrest the AGS cells in G1 and G2/M phases, and Caco-2 cells in G1 and S phases. These findings were associated with the down-regulation of cyclin D1, CDK4, and up-regulation of p21, p27 and p53. According to the morphological observations and DNA fragmentation assay, the MPS compound induced apoptosis in both cell lines, and also cause a significant increase in the expression of Bax/Bcl-2. In this context, our molecular docking results unveiled that the MPS compound has considerable affinity to interact with the minor groove of ctDNA and also with cell cycle kinases. To approve and find the accurate MPS mode of action against cancer cell lines (especially in gastrointestinal cancer) further studies is highly recommended.

Exposure to non‐ionizing electromagnetic radiation of public risk prevention instruments threatens the quality of spermatozoids

The use of artificial insemination in cattle breeding has evolved to global extent, and insemination doses are often shipped via air transport which requires strict radiation-based examinations. For the determination of effect of non-ionizing radiation (NIR), to which are beings frequently exposed due to protection of airport or cultural event security, freshly ejaculated and cryopreserved bovine spermatozoa were used as experimental model. Following radiation with hand-held metal detector in various exposition times (0, 10s, 15, 30 and 60min-groups FR, FR10, FR15, FR30 and FR60) the spermatozoa underwent motility and DNA fragmentation analyses. Study on cryoconserved semen treated with NIR was performed in time intervals 0, 10s, 1 and 5min (insemination doses radiated before cryoconservation-CB, CB10, CB1, CB5; samples radiated after freezing-CA, CA10, CA1 and CA5). Fresh semen and insemination doses radiated after cryoconservation showed significantly lower total and progressive motility. No effect on motility parameters was detected in semen extended with cryopreservative medium and radiated prior to freezing. Surprisingly, NIR showed a potential to stimulate spermatozoa velocity; however, the effect was modulated throughout the post-thawing incubation. Based on the DNA fragmentation assay, sperm DNA stayed intact. Present study underlines the potential harm of NIR, which is frequently used in everyday life, with overall adverse impact on human and animal reproduction. Current study also points out on interesting short-term spermatozoa stimulation induced by NIR.

Redox balance and DNA fragmentation in arsenic-exposed occupational workers from different industries of Pakistan.

AbstractOccupational exposure accounts for a contact between workers and different toxicants. Present study was designed to measure the arsenic-induced DNA fragmentation and oxidative stress in exposed workers. Blood, hair, and nail samples were collected from welding, brick kiln, furniture, pesticide, and paint industries (n = 50/industry) of Pakistan along with 200 controls. DNA damage was calculated using DNA fragmentation assay. Antioxidant enzymes (CAT, SOD, GPx) were measured using ELISA. Results revealed that arsenic exposure induced DNA fragmentation in brick kiln, furniture, and welding industries. Enzyme activity was reduced in five industries compared to control. In exposed group, significant depletion of enzymes was observed in furniture, welding, and brick kiln workers. Based on age and time of exposure, significant difference was observed in welding and brick kiln group. Smokers of exposed group showed significantly reduced levels of enzymes compared to controls. Arsenic deposition was observed higher in the hair, nail, and blood samples of exposed group (P < 0.001) compared to control. Likewise, lead and cadmium contents were higher in the blood samples of industrial workers compared to control. This study suggests increased trend of cellular damage and oxidative stress in occupational workers profoundly in welding, furniture, and brick kiln industries. Moreover, this study recognizes the contribution of age, exposure time, and smoking status toward arsenic-induced oxidative stress and DNA fragmentation.

Evaluation of Paeonia emodi and its gold nanoparticles for cardioprotective and antihyperlipidemic potentials

Paeonia emodi Wall. ex Royle is an important member of family Paeoniaceae and folklorically used for constipation, hysteria, respiratory diseases, epilepsy and cardiac diseases like hypertension, palpitations, congestive heart failure and atherosclerosis. In the present study, ethyl acetate fraction of P. emodi (Pe.EA) was subjected to column chromatography to obtain sub- fractions. These sub-fractions were screened for their cardioprotective activity in isoproterenol hydrochloride (ISO) induced myocardial infarction (MI) in mice. The most active fraction Pe. EA 40 was used for its gold nanoparticles synthesis (Pe.EA 40-AuNPs). Pe.EA 40 and Pe.EA 40-AuNPs were investigated for their cardioprotective, antihyperlipidemic, DNA fragmentation assay and histopathological study. Pe.EA 40 (80 mg/kg body weight) significantly reduced the serum levels of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH), Creatine Phosphokinase (CPK) to 66.07 ± 1.54, 77.08 ± 1.79, 84.86 ± 1.34 and 265.34 ± 4.34 IU/L respectively as compared to ISO treated group. Pe.EA 40-AuNPs (40 mg/kg) reduced the levels of ALT, AST, CPK and LDH to 60.74 ± 2.79, 75.47 ± 1.67, 80.48 ± 2.64 and 247.54. ± 5.57 IU/L respectively. A significant reduction was observed in lipid profile, protection in DNA damage and restoration of histopathological changes as compared to ISO treated group. Based on the results, it can be suggested that preparation of Pe.EA 40-AuNPs enhances the therapeutic potential of plant extract for the treatment of atherosclerosis and MI.

Metabolic alterations induced by attenuated Zika virus in glioblastoma cells

BackgroundZika virus (ZIKV) has recently become a major matter of concern since its association with microcephaly cases in newborns was determined. From then on, ZIKV has been untiringly studied, and several scientific data have shown the virus’ tropism to neural cells. Previous studies have proposed that ZIKV induced glioblastoma cells death, suggesting that ZIKV and ZIKV-associated molecules might induce intracellular biochemical alterations, and thereby be alternatives for neural cancer management. In this sense, the present contribution presents a prospective approach for glioblastomas management: producing a ZIKV-based therapy that stimulates glioblastoma control. We developed an attenuated ZIKV prototype based on bacterial outer membrane vesicles (OMV). The attenuated ZIKV was applied on glioblastoma cells, where cytopathic and cytostatic effects were evaluated. Glioblastoma cells, with and without treatment, were submitted to mass spectrometry analysis.ResultsMicroscopic analysis showed cytopathic effects induced by ZIKV prototype on glioblastoma cells after 24 and 48 h post-treatment. DNA fragmentation assay and TNF-alpha expression were indicative that ZVp induced cell damage and death. The metabolomics investigation elected 5 different biomarkers that might be associated with cell cytopathic effects, highlighting intracellular biochemical modifications induced by the attenuated ZIKV. The remarkable evidence of cell death in glioblastoma stimulated further studies that rendered a preliminary screening with other tumor cell lineages, though anti-proliferative activity test. Among the 11 tumors evaluated, prostate and ovarian tumors were the most affected by ZIKV prototype, and other 6 cell lines also presented cytostatic effects.ConclusionsResults ultimately demonstrated that this prototype not only emerges as a potential alternative for glioblastoma management, but may also be an important tool against other important tumors.

Cytotoxic and apoptosis inducing effect of some pyrano [3, 2-c] pyridine derivatives against MCF-7 breast cancer cells.

Anti-cancer activities of some pyrano-pyridines have been previously reported. Herein, we investigated anti-proliferative and apoptotic effects of the novel pyrano [3, 2-c] pyridine (P.P, TPM.P, 4-CP.P and 3-NP.P) compounds against MCF-7 breast cancer cells. The MCF-7 cells were cultured in the presence of various concentrations (20-200 μM) of the tested compounds for 3 days and the cell viability was determined by MTT assay. Induction of apoptosis was qualitatively assayed by acridine orange/ethidium bromide (AO/EtBr) staining, DNA fragmentation assay, as well as quantitatively by Annexin V/PI double staining and cell cycle analysis. These compounds inhibited growth and proliferation of the MCF-7 cells in a dose- and time-dependent manner. The IC50 values of P.P, TPM.P, 4-CP.P and 3-NP.P after 24 h of exposure were calculated to be 100±5.0, 180±6.0, 60±4.0 and 140±5.0 μM, respectively. 4-CP.P was determined as the most potent compound and was chosen for further studies. The result of flow cytometric cell cycle analysis indicated an increase in sub-G1 population after 72 h treatment of the cells. Furthermore, this was accompanied by exposure of phosphatidylserine (PS) in the outer cell membrane after time course of treatment with the 4-CP.P. Based on these observations, the pyrano [3, 2-c] pyridines can be regarded as a valuable candidate for further pharmaceutical evaluations.

Feline panleukopenia viral infection in cats: Application of some molecular methods used for its diagnosis.

Forty diseased cats and seven healthy control cats from different sex, ages and breeds had examined clinically to confirm presence or absence of clinical symptoms of Feline panleukopenia disease (FP). Several tools including ELISA, gene expression analysis (qRT-PCR), DNA fragmentation test and apoptosis assay were conducted to determine the Feline panleukopenia disease in cat tissues. Clinical symptoms in the form of depression, fever, anorexia, vomiting, diarrhea, dehydration, anaemia and leucopenia were recorded in the diseased cats while no clinical sings were observed in control healthy cats. ELISA results showed that all of diseased (n = 40) cats were positive while control cats (n = 7) were negative for FP viral antigen. After carrying out of ELISA assay, supportive treatment trials including fluid therapy, immunostimulant, antibiotics to overcome dehydration, restoring electrolytes imbalances, combating secondary bacterial infection were conducted but all diseased cats were died and control cats exposed to soft death. Gene expression analysis detected high levels of FP viral gene in several cat tissues in which ilium exhibited high viral expression levels compared with jejunum. Also, viral expression levels in jejunum were higher than in mesenteric lymph nodes. In addition, viral expression levels were not detected in tissues of control cats. The results of the DNA fragmentation assay observed that DNA extracted from different tissues of infected cats exhibited damaged DNA bands as compared with DNA of control cats. DNA fragmentation rates in infected tissues increased significantly (P < 0.01), the highest rates were showed in ilium and jejunum tissue than in mesenteric lymph nodes. Determination of apoptosis in cat tissues showed that rate of apoptosis/necrosis increased significantly (P < 0.05) in infected cats tissues in comparison to control cats. Moreover the highest apoptotic ratios of infected cats were observed in ilium and jejunum tissues compared with mesenteric lymph nodes.

Vicenin-2: a potential radiosensitizer of non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is a major form of cancer and is resistant to chemo- and radio-therapy. Vicenin-2 (VCN-2) is a flavonoid obtained from Ocimum sanctum L. and it has been reported to have radioprotective and anti-cancer properties. This study was conducted to check for the radiosensitizing potential of VCN-2 in the NSCLC cell line, NCI-H23. NCI-H23 cells were exposed to VCN-2 singularly, and to X-rays with and without prior VCN-2 treatment. Cytotoxicity assay, cell proliferation assay, caspase-3 activity assay, DNA fragmentation assay and Western blotting for Rad50, MMP-2 and p21 were performed to investigate the radiosensitizing properties of VCN-2. Fibroblast survival assay was performed using HEK293T cells to check for any adverse effects of VCN-2 on normal fibroblast cell line. VCN-2 singularly and in combination with radiation reduced the surviving cancer cells, increased caspase-3 activity, increased DNA fragmentation, increased the levels of Rad50 and lowered levels of MMP-2 and p21 proteins while being non-toxic and radioprotective to the fibroblast cells. VCN-2 showed a potent radiosensitizing property while also showing a chemotherapeutic property against NSCLC cell line NCI-H23.

Curcumin as a Novel Nanocarrier System for Doxorubicin Delivery to MDR Cancer Cells: In Vitro and In Vivo Evaluation.

Curcumin (CRC) has been widely used as a therapeutic agent for various drug delivery applications. In this work, we focused on the applicability of CRC as a nanodrug delivery agent for doxorubicin hydrochloride (DOX) (commercially known as Adriamycin) coated with poly(ethylene glycol) (PEG) as an effective therapeutic strategy against multidrug-resistant cancer cells. The developed PEG-coated CRC/DOX nanoparticles (NPs) (PEG-CRC/DOX NPs) were well localized within the resistant cancer cells inducing apoptosis confirmed by flow cytometry and DNA fragmentation assays. The PEG-CRC/DOX NPs suppressed the major efflux proteins in DOX-resistant cancer cells. The in vivo biodistribution studies on HCT-8/DOX-resistant tumor xenograft showed improved bioavailability of the PEG-CRC/DOX NPs, and thereby suppressed tumor growth significantly compared to the other samples. This study clearly shows that curcumin nanoparticles could deliver DOX efficiently into the multidrug-resistant cancer cells to have potential therapeutic benefits.

Pinostrobin Exerts Neuroprotective Actions in Neurotoxin-Induced Parkinson's Disease Models through Nrf2 Induction.

The aim of the present study was to assess the neuroprotective effects of pinostrobin (PSB), a dietary bioflavonoid, and its underlying mechanisms in neurotoxin-induced Parkinson's disease (PD) models. First, PSB could attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons and improve behavior deficiency in zebrafish, supporting its potential neuroprotective actions in vivo. Next, PSB could decreased apoptosis and death in the 1-methyl-4-phenylpyridinium (MPP+)-intoxicated SH-SY5Y cells, evidenced by MTT, LDH, Annexin V-FITC/PI, and DNA fragmentation assay. PSB also blocked MPP+-induced apoptotic cascades, including loss of mitochondrial membrane potential, activation of caspase 3, and reduced ratio of Bcl-2/Bax. In addition, PSB suppressed MPP+-induced oxidative stress but increased antioxidant enzymes, evidenced by decrease of reactive oxygen species generation and lipid peroxidation and up-regulation of GSH-Px, SOD, CAT, GSH/GSSG, and NAD/NADH. Further investigations showed that PSB significantly enhanced Nrf2 expression and nuclear accumulation, improved ARE promoter activity and up-regulated expression of HO-1 and GCLC. Furthermore, Nrf2 knockdown via specific Nrf2 siRNA abolished PSB-induced antioxidative and antiapoptotic effects against MPP+ insults. Interestingly, we then found that PSB promoted phosphorylation of PI3K/AKT and ERK, and pharmacological inhibition of PI3K/AKT or ERK signaling diminished PSB-induced Nrf2/ARE activation and protective actions. In summary, PSB confers neuroprotection against MPTP/MPP+-induced neurotoxicity in PD models. Promoting activation of Nrf2/ARE signaling contributes to PSB-mediated antioxidative and neuroprotective actions, which, in part, is mediated by PI3K/AKT and ERK.

Abstract 4924: Evaluation of the efficacy of curcumin and Y15 in platinum resistant ovarian cancer cells

Abstract Curcumin, a compound derived from turmeric (Curcuma longa), inhibits the proliferation of multiple cancer cell types, including ovarian, pulmonary, brain, and breast through various mechanisms. In advanced ovarian cancer, focal adhesion kinase (FAK), a tyrosine kinase, is overexpressed and contributes to platinum resistance. The compound Y15 (1, 2, 4, 5-benzene tetra amine tetrahydrochloride) inhibits FAK phosphorylation and subsequent activation. Therefore, in the present study we evaluated the combined cytotoxic efficacy of Curcumin and Y15 in platinum resistant OVCAR3 ovarian cancer cells. Cells were initially treated with concentrations of Y15 (10-100 μM), and curcumin (0-20µM) to determine IC10 values. Cells were subsequently treated with Y15 (20 μM) and curcumin (5 µM) alone and in combination. The cytotoxic profile of each treatment was assessed using the MTT assay. DNA fragmentation and poly ADP ribose polymerase (PARP) cleavage assays were performed to evaluate the mechanism of cell death. We further evaluated the expression of total FAK, phosphorylated FAK, p53, p21, and BAX in response to treatments using western blot. Y15 significantly increased the cytotoxic efficacy of curcumin when compared to the curcumin only treatment. Cell death by apoptosis was confirmed by PARP cleavage and the presence of DNA fragments in Y15, curcumin, and curcumin plus Y15 treatment groups. The curcumin plus Y15 combination significantly downregulated the expression of phosphorylated FAK when compared to the individual treatments and control. Reduced FAK phosphorylation also correlated with increased expression of P53, P21 and BAX in curcumin and Y15 treatment groups. These results indicate that Y15 significantly enhances the cytotoxic profile of curcumin in platinum resistant OVCAR-3 cells. This study is the first to report a FAK dependent cytotoxic mechanism of curcumin in ovarian cancer and in further work we will evaluate the signaling pathways involved. Citation Format: Arkene S. Levy, Appu Rathinavelu, Nicole Coelho, Amanda Ramnot, Thanigaivelan Kanagasabai, Olena Bracho, Robert Smith. Evaluation of the efficacy of curcumin and Y15 in platinum resistant ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4924.

Knowledge gaps in male infertility: a reproductive endocrinology and infertility perspective.

Reproductive research has moved forward at a remarkable pace. Some of these advances are the result of a separation between male and female specialties, allowing focused study in specific areas of the field. However, the different training programs between male and female fertility specialists has created an environment in which some discoveries are not put in the greater context of clinical care. At times, interventions have been measured against surrogate markers of outcome that may not impact the most meaningful outcome for patients—the delivery of a healthy neonate. For example, medical and surgical interventions that use changes in semen parameters may have a limited impact on the likelihood of achieving a live birth due to the limitations inherent in the semen analysis for predicting outcomes. Other commonly used tests, such as sperm DNA fragmentation assays provide promising biological plausibility to account for subfertility of some male partners. However, until well defined thresholds for predicting outcomes in different treatment scenarios are available, changes in sperm DNA fragmentation testing is not an adequate outcome for measuring the utility of interventions. The biggest limitation for these tests remains their analysis of bulk semen. Tests allowing interrogation of the reproductive competence of a given sperm, while allowing that sperm to be used in assisted reproductive technology procedures remain elusive. Progress toward reaching this end (whether by hyaluronic acid binding, IMSI, or Ramen spectroscopy) is underway, but much remains to be learned. Achieving testing and capture of individual sperm would better facilitate studies that measure the most meaningful outcome for patients and providers—the delivery of a healthy baby.

Cleavage of caspase-12 at Asp94, mediated by endoplasmic reticulum stress (ERS), contributes to stretch-induced apoptosis of myoblasts.

Mechanical overloading can lead to skeletal muscle damage instead of remodeling. This is attributed to the excessive apoptosis of myoblasts, mechanism of which remains to be elucidated. The present study aimed to investigate the involvement of endoplasmic reticulum stress (ERS) and caspase-12 in mediating the stretch-induced apoptosis of myoblasts. Myoblast apoptosis was evaluated by Hoechst staining, DNA fragmentation assay, Annexin V binding, and propidium iodide staining, as well as caspase-3 and poly-ADP-ribose polymerase 1 cleavage. First, our results showed that apoptosis was elevated in a time-dependent manner when myoblasts were subjected to cyclic mechanical stretch (CMS) for 12, 24, and 36 hr. Concomitantly, CMS triggered theERS and caspase-12 cleavage; ERS inhibitor GSK 2606414 suppressed the CMS-induced cleavage of caspase-12 and myoblast apoptosis. Silencing caspase-12 attenuated the apoptosis of myoblasts under CMS. Furthermore, CMS-induced myoblast apoptosis was partially recovered by overexpressing wild-type caspase-12 in caspase-12-silenced myoblasts. In contrast, overexpressing mutant caspase-12 (D94N), which cannot be cleaved into the active caspase-12 fragments, failed to accomplish the same effect. Finally, C2C12 overexpressing truncated caspase-12 segment (TC-casp12-D94), which starts from Asp94 and ends at Asn419, underwent apoptosis under both static and stretched conditions. Interestingly, C2C12 myoblasts seemed to be resistant to stretch-induced apoptosis upon low-serum-induced differentiation. In conclusion, our study provided evidence that caspase-12 cleavage at Asp94, induced by ERS under mechanical stimuli, is the key molecule in initiating the stretch-triggered apoptosis of myoblasts.

Novel mechanism of cannabidiol-induced apoptosis in breast cancer cell lines

Studies have emphasized an antineoplastic effect of the non-psychoactive, phyto-cannabinoid, Cannabidiol (CBD). However, the molecular mechanism underlying its antitumor activity is not fully elucidated. Herein, we have examined the effect of CBD on two different human breast cancer cell lines: the ER-positive, well differentiated, T-47D and the triple negative, poor differentiated, MDA-MB-231 cells. In both cell lines, CBD inhibited cell survival and induced apoptosis in a dose dependent manner as observed by MTT assay, morphological changes, DNA fragmentation and ELISA apoptosis assay. CBD-induced apoptosis was accompanied by down-regulation of mTOR, cyclin D1 and up-regulation and localization of PPARγ protein expression in the nuclei and cytoplasmic of the tested cells. The results suggest that CBD treatment induces an interplay among PPARγ, mTOR and cyclin D1 in favor of apoptosis induction in both ER-positive and triple negative breast cancer cells, proposing CBD as a useful treatment for different breast cancer subtypes.

Novel pregnenolone derivatives modulate apoptosis via Bcl-2 family genes in hepatocellular carcinoma in vitro

A series of pregnenolone derivatives were synthesized and assessed for anti-cancer activity against hepatocellular carcinoma cell line (HepG2). The synthesized hetero-steroids (compounds 3, 4, 5, 6, 7, 8a and 8b) were evaluated for their cytotoxic activities using MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. Apoptotic activity was assessed using dual acridine orange/ethidium bromide staining method and DNA fragmentation assay. Pro-apoptotic genes (Bax and Bak) and anti-apoptotic genes (Bcl-2 and Bcl-xL) were analyzed using quantitative real time PCR. The results revealed that compounds 4 and 6 displayed cytotoxic activity (IC50s, 36.97 ± 2.18 and 18.46 ± 0.64 μM, respectively), while compounds 5 and 7 exhibited weak cytotoxic activity (IC50s, 93.87 ± 8.30 μM and 93.48 ± 4.14 μM, respectively). All synthesized heterocyclic pregnenolone derivatives induced apoptosis through DNA fragmentation. Compounds 4 and 6 increased early and late apoptotic cell percentages while compounds 3, 5, 7 and 8b increased either early or late apoptotic cell percentage. Moreover, compounds 3, 6 and 8b up-regulated the expression level of Bak gene. On the other hand, compounds 4, 5, 7 and 8a down-regulated the Bcl-2 expression level, besides, compounds 5, 7 and 8a down-regulated the Bcl-xL expression level. Compounds 5, 7, 8a and 8b increased the Bak/Bcl-xL ratio, besides, compound 8a raised the Bax/Bcl-xL ratio whereas compound 5 elevated Bax/Bcl-2 and Bak/Bcl-2 ratios. The present work introduced novel pro-apoptotic pregnenolone derivatives that acted against HepG2 cells through DNA fragmentation, apoptotic morphological changes and were able to increase the pro-apoptotic/anti-apoptotic ratios of Bcl-2 family genes. This study particularly revealed that the cytotoxic compound 4 is the most promising pro-apoptotic compound among other synthesized derivatives where it induced apoptosis (late and early) through the down-regulation of Bcl-2 gene expression level.