Trypan Blue Dye Exclusion Research Articles

Induction of apoptosis and modulation of homologous recombination DNA repair pathway in prostate cancer cells by the combination of AZD2461 and valproic acid.

Cancer therapies using defects in homologous recombination (HR) DNA repair pathway of tumor cells are not yet approved to be applicable in patients with malignancies other than BRCA1/2-mutated tumors. This study was designed to determine the efficacy of combination therapy of a histone deacetylase inhibitor, valproic acid (VPA) and a novel PARP inhibitor AZD2461 in both PC-3 (PTEN-mutated) and DU145 (PTEN-unmutated) prostate cancer cell lines. The Trypan blue dye exclusion assay and the tetrazolium-based colorimetric (MTT) assay were performed to measure the cytotoxicity while combination effects were assessed based on Chou-Talalay's principles. Flow-cytometric assay determined the type of cell death. The real-time PCR analysis was used to evaluate the alterations in mRNA levels of HR-related genes while their protein levels were measured using the ELISA method. γ-H2AX levels were determined as a marker of DNA damage. We observed a synergistic relationship between VPA and AZD2461 in all affected fractions of PC-3 cells (CI<0.9), but not in DU145 cells (CI>1.1). Annexin-V staining analysis revealed a significant induction of apoptosis when PC-3 cells were treated with VPA+AZD2461 (p<0.05). Both mRNA and protein levels of Rad51 and Mre11 were significantly decreased in PC-3 cells co-treated with VPA+AZD2461 while enhanced H2AX phosphorylation was found in PC-3 cells after 12 and 24 hours of co-treatment (p<0.05). Our findings established a preclinical rationale for selective targeting of HR repair pathways by a combination of VPA and AZD2461 as a mechanism for reducing the HR pathway sufficiency in PTEN-mutated prostate cancer cells.

Abstract 542: Anti-CD19 therapy for precursor B-acute lymphoblastic leukemia: Response and resistance mechanisms

Abstract Introduction: CD19 is a transmembrane protein restricted to B-lineage cells and follicular dendritic cells. In conjunction with CD21, CD81, and CD225, CD19 acts as a B cell co-receptor to decrease the threshold for antigen receptor-dependent stimulation. CD19 trafficking and expression are orchestrated by a multitude of molecules. Resistance to blinatumomab (Blina, anti-CD19 therapy) in acute lymphoblastic leukemia (ALL) is thought to be due in part to the emergence of CD19-negative escape variants. Loss of expression of CD81 and/or CD21, which are required for CD19 trafficking, results in the lack of CD19 expression. In addition, alternative CD19 splicing and other non-CD19-mediated resistance mechanisms have been described. Therefore, we aimed to study CD19 trafficking and response to Blina in ALL patients. Methods: Peripheral blood was collected at our Center. Mononuclear cells (PBMCs) were isolated and viably frozen. Cells were then thawed and washed with PBS with 10% FBS, and viable cells were counted using trypan blue dye exclusion. 1x10^6 cells/test were stained for CD19, CD81 (BD Biosciences, San Jose, CA) and CD21 (Biolegend, San Diego, CA). After incubation, cells were washed 2x with FACs buffer, resuspended and acquired on a BD Canto II flow cytometer. Results: Blood was obtained from 10 patients with relapsed or refractory pre-B-ALL with blood blasts prior to Blina treatment. Five patients subsequently achieved complete remission (CR). Cells from four of these patients had CD21 expression on &amp;lt;5% of cells, but CD81 expression on &amp;gt;40% of cells. Of the 5 patients whose disease did not respond to Blina, four also had CD21 expression on &amp;lt;5% of cells and had CD81 expression on an average of 40% of cells. CD19 was expressed on &amp;gt;40% of cells in all samples. Conclusion: Although CD21 and CD81 are required for CD19 trafficking and expression, loss of either of these antigens measured by flow cytometry did not correlate with the response to Blina. PatientAge(years)DiagnosisKaryotypeCD19*CD21*CD81*Response to Blinatumomab176Pre-B-ALL46,XX,t(9;22)(q34.1;q11.2)53241Yes (CR)255Pre-B-ALL46, X,inv(Y)(p11.2q11.23), dic(9;12)(p13;p13), del20(q11.2)42146Yes (CR)347Pre-B-ALL46, XX,t(4;11)(q21;q23)941256Yes (CR)484Pre-B-ALL46,XX,i(9)Iq10)65372Yes (CR)551Pre-B-ALL46, XX, t(9;22)(q34.1;q11.2)77578Yes (CR)633Pre-B-ALL46, XY, +X, der(17;18)(q10;q10)67185No725Pre-B-ALL46, XY, der(20;21)(q10;q10),+2146432No825Pre-B-ALL46, Y, t(X;4)(p11.2;p15), der(9)t(9;14)(p21;q12) del(9)(p21),del(14)q13), +mar[cp14]742429No979Pre-B-ALL46, XY48123No1073Pre-B-ALL46, XY59129No*% cells; CR = complete remission Note: This abstract was not presented at the meeting. Citation Format: Firas El Chaer, Brandon Carter-Cooper, Rena G. Lapidus, Maria R. Baer, Zeba N. Singh, Ashkan Emadi. Anti-CD19 therapy for precursor B-acute lymphoblastic leukemia: Response and resistance mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 542.

A polyherbal formulation, HC9 regulated cell growth and expression of cell cycle and chromatin modulatory proteins in breast cancer cell lines

Ethnopharmacological relevanceHC9, a polyherbal formulation, is based upon a traditional Ayurvedic formulation, Stanya Shodhana Kashaya (SSK, having 10 plant materials), formulated on Stanyashodhana gana, explained by Charaka in Charakasaṃhita Sutrasthana IV and mentioned in other texts as well. Stanyasodhana is the Sanskrit name for a group of medicinal plants, classified for “improving the quality of milk”. SSK is used by Ayurvedic practitioners for the cleansing and detoxification of breast milk in lactating mothers as well as for the management of various clinical conditions. HC9 is composed of equal ratios of nine different medicinal plants that include Picrorhiza kurroa Royle ex Benth., Cyperus rotundus L., Zingiber officinale Roscoe, Cedrus deodara (Roxb. ex D.Don) G.Don, Tinospora cordifolia (Willd.) Miers, Holarrhena antidysenterica (Roth) Wall. ex A.DC., Swertia chirata Buch.-Ham. ex Wall., Cissampelos pareira L. and Hemidesmus indicus (L.) R. Br. ex Schult.. It differs from the SSK formulation by having one ingredient [Marsdenia tenacissima (Roxb.)Moon (Murva)] less, due to its unavailability since it is mostly found in tropical hilly tracts of peninsular India and Vindhya ranges as well as in lower Himalayan tracts. All the medicinal plants in the formulation have reported activity against different types of cancers. Aim of the studyThe present study is aimed at evaluating the anticancer activity of the polyherbal formulation (HC9) and its mechanism of action against breast cancer cell lines. Materials and MethodsThe effect of HC9 on the viability of breast cancer (MCF-7 and MDAMB231) and non-cancerous (MCF-10A) cell lines was evaluated by MTT assay. The effect on cell growth and colony formation potential of cancer cells was determined by trypan blue dye exclusion method and soft agar assay, respectively. Cell cycle arrest was determined by propidium iodide (PI) staining and analyzed by flowcytometer. Scratch wound assay was used for studying cell migration. Cell invasion was determined by using BD BioCoat Matrigel invasion chambers. The gene expression of HIF-1α was examined by RT-PCR. The expression of p53, SMAR1, p16, MMP-2, CDP/Cux, p21, Rb, phospo-Rb (ppRb), VEGF, NFқB and COX-2 proteins was determined by western blotting. ResultsHC9 significantly altered growth of breast cancer cell lines, MCF-7 and MDA MB-231. It blocked the cell cycle progression at S phase in MCF-7 by up regulating the expression of p53, p21 and p16 proteins. In MDA MB-231, HC9 induced G1 phase arrest by up regulating the expression of p53, p21 and pRb proteins with simultaneous decrease in ppRb. It significantly reduced migration and invasion in both the cell lines, accompanied by decrease in the expression of MMP-2/9, HIF-1α and VEGF. HC9 decreased the expression of inflammatory markers (NF-қB, COX-2), and modulated the expression of chromatin modulators (SMAR1 and CDP/Cux) in both MCF-7 and MDA MB-231. ConclusionsHC9 exhibited potent anticancer activity against breast cancer cells, thereby warranting further pre-clinical and clinical studies in future.

Notopterol-induced apoptosis and differentiation in human acute myeloid leukemia HL-60 cells.

Purpose: This study aims to observe the effects of notopterol on the apoptosis and differentiation of HL-60 cells and to explore the underlying molecular mechanisms.Methods: Cell viability was assessed using sulforhodamine B assay. Cell proliferation was determined by the trypan blue dye exclusion test. Colony-forming units were assayed in methylcellulose. Apoptosis assays were carried out by annexin V-fluorescein isothiocyanate(FITC)/propidium iodide (PI) double staining, Hoechst 33342 staining, mitochondrial membrane potential, and Western blot. Wright–Giemsa staining, nitroblue tetrazolium (NBT) reduction assay, CD11b and CD14 and Western blot were detected for induction of differentiation. In addition, cell-cycle phase distribution was analyzed by flow cytometry and Western blot. The combination therapy of notopterol and all-trans retinoic acid (ATRA) on HL-60 cells was examined.Results: Notopterol obviously inhibited the growth of HL-60 cells with an IC50 value of 40.32 μM and remarkably reduced the number of colonies by 10, 20, and 40 µM. In addtion, notopterol induced the percentage of apoptotic HL-60 cells, reduced the mitochondrial membrane potential, decreased the protein expresstion of Bcl-2 and Mcl-1, and increased the expression of Bax, cleavage of caspase 9, caspase 3, and PARP. As for cell differentiation, notopterol clearly induced chromatin condensation; increased the nucleocytoplasmic ratio, nitroblue tetrazolium-positive cells, expression of CD14 and CD11b, and protein expression of c-Jun and Jun B, and decreased c-myc. Furthermore, notopterol induced the G0/G1 cell-cycle arrest as determined using flow cytometry, which may be related to the regulation of cell-cycle-related proteins p53, CDK2, CDK4, Cyclin D1, Cyclin E, and survivin. The combined use of notopterol and ATRA did not enhance the apoptotic effect as evidenced by cell viability test and Hoechst 33342. However, the combination of notopterol and ATRA enhanced the effect of inducing differentiation when compared with using either notopterol or ATRA alone, which can be evidenced by the increased nucleocytoplasmic ratio, NBT positive cells, and expression of CD14.Conclusion: This is the first time it has been demonstrated that notopterol could induce apoptosis, differentiation, and G0/G1 arrest in human AML HL-60 cells, suggesting that notopterol has potential therapeutic effects on AML. The combination application of notopterol (20 and 40 μM) and ATRA (2 μM) could augment differentiation of HL-60 cells.

Organoantimony(III) halide complexes with azastibocine framework as potential antitumor agents: Correlation between cytotoxic activity and N→Sb inter-coordination

The relationship between chemical structure and in vitro cytotoxic activities of a series of azastibocine-framework organoantimony(III) halide complexes against cancerous (HepG2, MDA-MB-231, MCF-7 and HeLa) and nonmalignant (HEK-293) cell lines was studied for the first time. A positive correlation between cytotoxic activity and the length of N→Sb coordinate bond on azastibocine framework of same nitrogen substituent was observed. By comparison, the organoantimony(III) complex 6-cyclohexyl-12-fluoro-5,6,7,12-tetrahydrodibenzo[c,f][1,5]azastibocine (C4) exhibited the highest selectivity index, giving a IC50(nonmalignant)/IC50(cancerous) ratio of up to 8.33. The results of cell cycle analysis indicated that the inhibitory effect of C4 on the cellular viability was caused by cell cycle arrest mainly at the S phase. The necrosis induced by C4 was confirmed by the Trypan blue dye exclusion test and the increase of lactic dehydrogenase (LDH) released in the culture medium. Furthermore, evaluation of the levels of intracellular reactive oxygen species (ROS) in MDA-MB-231 cells, by quantifying the relative fluorescence units (RFU) using spectrofluorometer, indicated that cytotoxic activity of C4 is dependent on the production of ROS. This work established the correlation between cytotoxic activity and N→Sb inter-coordination, a finding that provided theoretical and experimental basis for in-depth design of antimony-based organometallic complexes as potential anticancer agents.

Lupeol from Nyctanthes arbor-tristis Inhibits Matrix Metalloproteinase Activity, Angiogenesis and Proliferation of Glioma Cells

Angiogenesis plays a critical role in cancer progression and, hence, inhibiting angiogenesis is considered as a key strategy in cancer therapy. In this study, we screened the antiangiogenic and cytotoxic properties of Nyctanthes arbor-tristis Linn. plant of family Oleaceae, which is used in traditional medicine for the treatment of cancer, arthritis and inflammation. The antiangiogenic activity of ethanolic extract of Nyctanthes arbor-trsitis (ENA) was assessed using Chick Chorioallantoic Membrane (CAM) assay. The mechanism of antiangiogenic action was evaluated via gelatin digestion assay for matrix metalloproteinase (MMP) inhibitory activity. Cytotoxic activity of the extract on glioma cells was assessed using MTT and trypan blue dye exclusion assays. ENA impaired capillary formations in chick CAM model and inhibited MMPactivity on gelatin gels in a concentration dependent manner. The extract was found to be cytotoxic on glioma cells at higher concentrations. Bioactivity guided purification of ENA using column chromatography and thin layer chromatography afforded lupeol as the active principle. Lupeol exhibits significant MMPinhibitory activity at a concentration of 2 μg/mL and cytotoxic activity on glioma with an IC50 value of 10.75 μg/mL. The results of this study hold promise for developing this plant as a source of lupeol, a highly bioactive compound against angiogenesis.

Antiproliferative activity of longan (Dimocarpus longan Lour.) leaf extracts

Longan (Dimocarpus longan Lour.) belongs to the Sapindaceae family. We examined the antiproliferative activity of longan leaf extracts against cancer-derived cell lines in vitro. The tested samples were water extract, ethanol extract, n-hexane fraction, ethyl acetate fraction, and water fraction of longan leaf. Cytotoxicity test is against brine shrimps that were screened using Brine Shrimp Lethality Test. Antiproliferative activity assay on WEHI-164 cells (mouse fibrosarcoma cancer cell), THP-1 cells (human peripheral blood acute monocyte cell), and Vero cells (non-cancer or normal cell) that was conducted using a hemocytometer with Trypan Blue Dye exclusion. The 50% lethality concentration (LC50) value of water extract, ethanol extract, n-hexane fraction, ethyl acetate fraction, and water fraction were 854.64, 305.81, 446.55, 1313.44, and 1621.8 μg/ml. Ethanol extract exhibited significant cytotoxic due to the lowest LC50 value. The ethanol extract was then used for further examination. The highest antiproliferative activity was achieved 44.93% by 600 μg/ml ethanol extract on WEHI-164 and 57.45% by 500 μg/ml ethanol extract on THP-1. It was significantly equal to doxorubicin antiproliferative activity. Ethanol extract dose had a low effect on Vero cells. This present study confirmed that the longan leaf ethanol extract possesses marked antiproliferative activity on cancer-derived cell lines.

High levels of EGFR prevent sulforaphane-induced reactive oxygen species-mediated apoptosis in non-small-cell lung cancer cells

BackgroundSulforaphane (SFN) has been shown to induce the production of reactive oxygen species (ROS) and inhibit epidermal growth factor receptor (EGFR)–mediated signaling in non-small-cell lung cancer (NSCLC). NSCLC cells harboring constitutively active EGFR mutations are more sensitive to SFN treatment than cells with wild-type EGFR, but whether NSCLC cells with high levels of EGFR expression are more resistant or sensitive to SFN treatment is not known. Study designWe employed a pair of cell lines, CL1-0 and CL1-5, which have the same genetic background but different levels of EGFR expression, to examine the effects of high EGFR level in the sensitivity to SFN. MethodsThe effect of SFN on cell viability and tumorigenicity was examined by trypan blue dye-exclusion assay, clonogenic assays, flow cytometry, and immunoblotting in vitro as well as tumorigenicity study in vivo. ROS levels in cells were assessed by flow cytometry using the ROS-reactive fluorescent indicator CM-H2DCFDA. Knockdown of EGFR in CL1-5 cells was infected with an EGFR-targeting small hairpin (interfering) RNA (shRNA)–containing lentivirus. ResultsWe present evidence that cells with high-level EGFR expression (CL1-5) are more resistant to SFN treatment than those with low-level expression (CL1-0). SFN treatment produced a similar increase in ROS and caused arrest of a cell population at S-phase accompanied by the induction of γH2AX, a DNA damage-response marker, in both cell sublines. However, SFN induced apoptosis only in the high-EGFR-expressing CL1-0 subline. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented SFN-induced apoptosis in CL1-0 cells and production of γH2AX in both CL1-0 and CL1-5 cells. shRNA-mediated knockdown of EGFR in CL1-5 cells rendered the cells susceptible to SFN-induced apoptosis. ConclusionThe cellular effects produced by SFN in NSCLC cells are largely mediated by SFN-induced production of ROS. Cells with higher levels of EGFR were more resistant to SFN treatment and showed resistance to SFN-induced apoptosis, suggesting that high EGFR levels protect cells from SFN-induced apoptosis. Despite this, we found that SFN retained the ability to inhibit the growth of NSCLC tumors with high-level EGFR expression in vivo.

Poly(dimethylsiloxane) Induces Cytotoxicity and Genotoxicity in Human Lymphocytes

Poly-(dimethylsiloxane) (PDMS) is extensively used in edible fats and oils as an antifoaming agent. However, in vitro cyto-genotoxicity analyses of PDMS in primary human cells are limited. The present study was performed as a safety assessment encompassing the cytotoxicity and genotoxicity of PDMS on human peripheral blood lymphocytes. Isolated lymphocytes were exposed to different concentrations of PDMS from 0 to 20 mg/l at 37 °C for up to 24 h. Our results suggest a significant dose-dependent decline in cell viability at concentrations 2.5 mg/l and above as estimated by trypan blue dye exclusion test and MTT assay. The IC-50 of PDMS was found to be 20 mg/l after 24 h incubation. Moreover, a significant genotoxicity were observed at 2.5 mg/l and above as assessed by comet and DNA diffusion assays. Hence, consumption of PDMS may not be safe at concentrations above 2.5 mg/l. Further studies are needed to establish its in vivo cyto-genotoxicity mechanisms.

Mild antagonistic effect of Valproic acid in combination with AZD2461 in MCF-7 breast cancer cells.

Background: Breast cancer (BC) is a complex disease, but current treatments are not efficient enough considering increased relapse and decreased survival rate among patients. Poly (ADP-ribose) polymerase inhibitors are recently developed anticancer agents which target cells with defects in homologous recombination (HR) pathway. This study wishes to assess whether the combination of AZD2461 as a newly developed PARP1 inhibitor and valproic acid (VPA), a histone deacetylase inhibitor could effectively reduce the growth of MCF-7 cells with no fundamental DNA repair defect. Methods: Both trypan blue dye exclusion assay and MTT viability test were used to evaluate cell death. γ-H2AX levels, as a marker of DNA repair, were measured using in cell ELISA method. The Student's t-test and non-parametric analysis of variance (ANOVA) were applied for our data analyses where p-value <0.05 was considered statistically significant. Results: As calculated by CompuSyn software, IC50 values for VPA and AZD2461 were 4.89 mM and 42.83 µM respectively following 48 hours treatment. Also, the trypan blue exclusion assay results showed a concentration- and time-dependent decrease when MCF-7 cells were treated with both agents (p<0.05). Combination analysis showed a mild antagonism (CI>1.1) while γ-H2AX levels found not to be significantly increased in MCF-7 cells co-treated with VPA+AZD2461 compared to each agent alone (p=0.29). Conclusion: Our findings revealed that the combination of VPA and AZD2461 could decrease cell viability of MCF-7 cells, but it was not able to significantly increase unrepaired DNA damage sites. The mechanism responsible for drugs combination was not of synergism or addition. Determining the type of involved cell death mechanisms might be followed in further studies.

Anaerobic Cancer Cell Metabolism and Viability

IntroductionNormal internal human physiologic oxygen levels are hypoxic, ranging from about 0.5 to 14%. However, in solid tumors, there are intra‐tumor niches, which like stem cell niches, are anaerobic. A historical roadblock to studying anaerobic cancer cell metabolism is the inability to maintain cell viability in an oxygen‐free environment for longer than 36 hrs. We previously showed that anaerobic cancerous and non‐cancerous cells, which survive at least 14d of anaerobic culture, exhibit an altered cell phenotype, i.e. diminished attachment capacity, slowed growth rate, and inverse hypoxia inducible factor‐1α mRNA and protein expression relative to anaerobic endogenous reactive oxygen species production. Since bacteria (mitochondrial precursors) use Nitric Oxide (NO) and Hydrogen Sulfide (H2S) as terminal electron acceptors, we focused on determining the role NO and H2S generating pathways play in sustaining anaerobic cancer cell viability.MethodsHeLa 229 cells were grown to 80% confluence in 24 well plates (normoxic conditions, 5% CO2 in air; 10% FBS; DMEM medium) then transferred to degassed PS‐74656 medium (Whitley A35, anaerobic gas mixture: H2, CO2, N2; 37°C). Viability was determined by trypan blue dye exclusion. Anoxic nitric oxide production was determined by fluorescent microscopy (DAF‐2; excitation 491 nm; emission 513 nm), with NO positive cells counted (10 random fields; Mean ± SEM; p &lt; 0.05). Controls consisted of cells from the same lot processed in parallel but anaerobically incubated in control PS‐74656 medium without additional NO and H2S pathway precursors or inhibitors.ResultsUpon anaerobic incubation, NO was detected at days 3 and 10 of culture. NO is a by‐product of the Xanthine Oxidase (XO) and Nitric Oxide Synthase (NOS) pathways. Addition of pathway precursors xanthine (4 × 10−5 M) or arginine (50 mM), respectively, significantly (p &lt; 0.05) increased HeLa cell viability by 3 and 20.9 fold, respectively, at day 10 of anaerobic incubation. Allopurinol (200 μM), an XO pathway inhibitor, inhibited the xanthine induced increase in viability while L‐NAME had a similar effect on arginine‐mediated viability, which was also reduced to control level.To determine if H2S supports HeLa cell anaerobic viability, the effects of H2S pathway precursor cysteine (0.4 mM), and H2S donors JK‐2 (300 μM) and GYY4137 (400 μM) were tested. Overall, any treatment that should increase cellular H2S levels caused significantly (p &lt; 0.05) enhanced viability, as compared to medium alone (cysteine, 3.6; JK‐2, 6.3; and GYY4137, 7.6 fold increases).These results confirm our hypothesis that similar to bacteria, terminal electron donors other than oxygen can function to support tumor cell anaerobic respiration.Support or Funding InformationThis project was supported by research funds from Office of Research and Sponsored Programs (ORSP), Midwestern University, IL, College of Osteopathic Medicine (CCOM) at Midwestern University and College of Graduate Studies (CGS) at Midwestern University with assistance from the MWU Core Facility.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of SAHA and RG7388 on p21WAF1/CIP1 and p27KIP1 mediated Pathways in Cancer Cells

Epigenetic modifications can lead to significant influence on cancer development, growth and progression. The main epigenetic modifications observed in human are methylation and acetylation. Histone acetylations are largely regulated by histone deacetylases (HDACs) and, several HDAC inhibitors (HDACi) such as SAHA (Vorinostat), which are already approved for human use or currently in different stages of clinical trials, belong to the new class of drugs with promising effects on the cancer growth and metastatic process. The small molecule RG7388 is a newly developed inhibitor that is specific for an oncogene‐derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. One of the unique characteristics of these two drugs is their ability to induce p21 expression through distinct mechanisms. A clear understanding related to the molecular mechanism whereby SAHA can induce cell cycle arrest and trigger necrosis, apoptosis or necroptosis is still evolving. Similarly, the ability of RG7388 for producing anticancer effect is undergoing thorough investigation, since it can produce p53 dependent and p53 independent effects. So far, our experiments with MCF‐7 breast cancer cells and LNCaP prostate cancer cells have confirmed the abilities of these two drugs to induce p21 expression through distinct mechanisms. In addition, these two drugs were expected trigger cell cycle arrest and cell death through mechanisms that are similar. Therefore, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 by using the MCF‐7 and LNCaP cells. The cytotoxicity, cell cycle arrest and apoptosis/necroptosis effects of the treatments were assessed by using Trypan Blue Dye Exclusion (TBDE) method, and fluorescence assay with caspase 3/7 specific DEVD‐amc fluorogenic substrate. In addition, cell cycle and apoptosis related proteins were analyzed by immunoblotting methods. To this point, our results from MCF‐7 and LNCaP cells suggest that the cell death caused by SAHA treatment in both cell lines are through induction of p21WAF1/CIP1 and p27Kip1 levels, that could be independent of p53. On the other hand, treatment of LNCaP cells with RG7388 was able to induce p21WAF1/CIP1 expression through inhibition of MDM2 that was also causing significant cell death. However, the RG7388 treatment was not able to elevate p21WAF1/CIP1 in MCF‐7 cells, even though there was clear evidence of p53 elevation in these cells. Hence, we are suspecting that, there is some level of uncoupling of p53 mediated transcriptional induction of p21WAF1/CIP1 in MCF‐7 cells. As of now, our studies confirm that two different drugs, which have the ability to induce p21 expression, exhibit distinct mechanisms of cell death through p53 dependent and independent pathways. Since it points to an interesting interplay between multiple pathways, confirmation of the actual cell death mechanism induced by RG7388 in MCF‐7 and LNCaP cancer cells requires additional exploration.Support or Funding InformationThis research was supported by the Royal Dames of Cancer Research Inc., Fort Lauderdale, FloridaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anti‐biofilm and cytoprotective activities of quercetin againstPseudomonas aeruginosaisolates

Increase in infection with multidrug resistant Pseudomonas aeruginosa is a serious global challenge in healthcare. Pseudomonas aeruginosa is capable of causing human infection in various sites and complicates the infection due to its virulence factors. This study was aimed to investigate the effect of quercetin, a dietary flavonoid against the virulence factors of P. aeruginosa and its cell protective effects on epithelial cells. Bactericidal activity, anti-biofilm activity and effect on different virulence factors were carried out using standard methods by using five P. aeruginosa isolates. Cytotoxicity and cell protective effect of quercetin was evaluated by trypan blue dye exclusion assay. All the tested isolates were completely inhibited (100%) by quercetin at a concentration of 500μgml-1 . It showed significant (P<0·05) inhibitory effect on virulence factors including biofilm formation and showed significant protective effect on HEK 293T cells infected with P. aeruginosa strains. This study supports the role of quercetin against P. aeruginosa, by inhibiting virulence factors as well as its cytoprotective activity during bacterial infection either by attenuating the virulence or providing direct protective effect to the host cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The increase in infections caused by opportunistic pathogen Pseudomonas aeruginosa is a serious concern in the health care system. This study describes the beneficial effects of a dietary flavonoid, quercetin against pathogenic P. aeruginosa strains and its protective effect against the P. aeruginosa infection in HEK 293T cells invitro.

Lentiviral interferon: A novel method for gene therapy in bladder cancer.

456 Background: Gene therapy for bladder cancer (BLCA) is rapidly evolving. We reported that intravesical adenoviral interferon-alpha (Ad-IFNα) produced a complete response in 35% of patients with BCG-unresponsive BLCA enrolled in a Phase II trial. Lentivirus (LV) is another potential vector for intravesical delivery of IFNα. Unlike the adenovirus, LV can infect non-dividing cells and integrate into the host’s genome, making it one of the most efficient gene delivery vectors. The objective of this study was to investigate lentiviral interferon-alpha (LV-IFNα) BLCA gene therapy in preclinical models. Methods: Murine BLCA cell lines were transduced in-vitro with LV-IFNα using a multiplicity of infection (MOI) of 2:1. IFNα levels were measured by ELISA. Cell viability was assessed using Trypan blue dye exclusion. qPCR was used to identify expression of IFNα target genes. A LV-βGalactosidase reporter construct was delivered intravesically, and urinary IFNα levels were measured in mice treated with LV-IFNα or control virus to assess gene transfer. To assess survival benefit, p53+/- C57/B6 mice were exposed to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) to induce CIS and then treated with LV-IFNα or control virus, and sacrificed when moribund. Results: Efficient LV-IFNα transduction of BLCA cells was observed at an MOI of 2:1, resulting in increased expression of IFNα and its target genes PDL-1, TRAIL, and IRF7 (p&lt;0.001), and reduced cell viability vs. controls (p&lt;0.001). Mechanistically, TRAIL dependent cytotoxicity in the LV-IFNα cells was rescued by Caspase 8 inhibition. βGal expression confirmed efficient transduction of murine urothelium. Urinary IFNα levels were elevated in mice receiving LV-IFNα compared with control virus. BBN mice treated with LV-IFNα had longer overall survival than mice treated with control virus (p=0.04). LV-IFNα induced intratumoral CD8+ T cell infiltration, high expression of PD-L1, and inhibited angiogenesis. Conclusions: LV-IFNα effectively upregulated IFNα target genes, was cytotoxic to murine BLCA cells, and improved the survival of BBN tumor-bearing mice. LV appears to be a promising vector for intravesical gene delivery.

Combinatorial Use of DNA Ligase Inhibitor L189 and Temozolomide Potentiates Cell Growth Arrest in HeLa

Introduction:The issues of carcinoma drug resistance to alkylating agents such as Temozolomide (TMZ) are considered as a major concern in therapeutics. The potential ways to achieve better cancer cell growth arrest and cytotoxicity have been suggested including the combinatorial use of DNA repair protein inhibitors and genotoxic drug TMZ. Here, authors assess the ability of DNA ligase inhibitor (L189) to modulate TMZ mediated HeLa cell growth arrest and cytotoxicity.Materials and Methods:Here, authors have employed Trypan blue dye exclusion and propidium iodide (PI) using FACS to determine HeLa cell viability after exposure to TMZ with or without L189 inhibitor. Additionally, authors show the DNA ligase III protein level using ELISA and fluorescent microscopy to support the observed effects of combinatorial use of TMZ and L189.Results:In this paper, data indicate that the addition of L189 produced appreciable decrease in the growth of HeLa cells. However, combined treatment of L189 and TMZ showed enhanced TMZinduced HeLa growth arrest possibly in G2/M cell cycle phase without employing cell death mechanisms.Conclusions:These results underscore the combinatorial treatment using TMZ and L189 to bring desirable cancer cell growth arrest and future molecular study to dissect out the participating pathways.

Bufalin induces apoptosis via mitochondrial ROS-mediated caspase-3 activation in HCT-116 and SW620 human colon cancer cells

Objective: Bufalin has been reported to kill various types of cancer including human colorectal cancer. Our previous study demonstrated that bufalin induced cell death via autophagy in HT-29 and Caco-2 colon cancer cells, but the action of bufalin remains unclear. This study was conducted to investigate the role of bufalin in other colon cancer HCT-116 and SW620 cells as well as its potential mechanism.Methods: The effect of bufalin in HCT-116 and SW620 colon cancer cells was detected by assessing cell viability and cell death. Apoptotic cells were analyzed by Western blot and trypan blue dye exclusion assay. Mitochondrial ROS production was analyzed by flow cytometry after DCFDA and DHR-123 staining. The potential mechanism was investigated via pharmacological inhibitors.Results: Bufalin had high potency against HCT-116 and SW620 cells with IC50 values of 12.823 ± 1.792 nM and 26.303 ± 2.498 nM in HCT-116 and SW620 cells, respectively. Bufalin decreased cell viability, increased cell death as well as caspase-3 downstream target (cleaved PARP) accumulation, and these actions were significantly blocked by pan-caspase inhibitor zVAD-FMK. Mechanistically, ROS production, but neither the NAD(P)H oxidase, AMPK, ERK nor p38, is responsible for bufalin-induced apoptotic cell death. Moreover, bufalin-induced ROS generation is derived from mitochondria.Conclusion: Bufalin significantly induces apoptosis in HCT-116 and SW620 colon cancer cells via mitochondrial ROS-mediated caspase-3 activation. We believe that our novel findings will greatly alter our current understanding on the anti-cancer mechanism of bufalin in colon cancer cells and will pave the way for further exploiting the clinical application.

Induction of Apoptosis in HeLa by Corn Small RNAs

Insights in RNA biology have opened up a plethora of opportunities to explore the small regulatory RNAs from various natural and artificial sources. These small RNAs have been suggested to play a role too in tumor progression by either as oncogenic or tumor suppressor small RNAs. In this study, authors have attempted to evaluate the therapeutic potential of small RNAs fractionated from corn (Zea mays) upon growth and survival of HeLa. Here, authors have employed standard cellular-based approaches including microscopy, spectroscopy, and flow cytometry-based staining assays. Our data indicate that corn small RNAs fraction can appreciably decrease HeLa cell proliferation and survival, which is supported by a number of complementary assays such as Trypan blue dye exclusion, MTT, propidium iodide, and Annexin V/PI apoptotic cell death. Taken together, present finding suggests that corn small RNAs fraction may display up to 70% reduction in HeLa cell viability. Furthermore, these data indicate that around 40–50% of HeLa cells become apoptotic due to exogenous use of corn small. Overall, this finding proposes that possibility of cross-kingdom anticancer use of small RNAs from corn and present data need to be explored in depth.

Synthesis, antiproliferative and apoptosis induction potential activities of novel bis(indolyl)hydrazide-hydrazone derivatives.

In recent years, indole-indazolyl hydrazide-hydrazone derivatives with strong cell growth inhibition and apoptosis induction characteristics are being strongly screened for their cancer chemo-preventive potential. In the present study, N-methyl and N,N-dimethyl bis(indolyl)hydrazide-hydrazone analog derivatives were designed, synthesized and allowed to evaluate for their anti-proliferative and apoptosis induction potential against cervical (HeLa), breast (MCF-7 and MDA-MB-231) and lung (A549) cancer cell lines relative to normal HEK293 cells. The MTT assay in conjunction with mitochondrial potential assays and the trypan blue dye exclusion were employed to ascertain the effects of the derivatives on the cancer cells. Further, mechanistic studies were conducted on compound 14a to understand the biochemical mechanisms and functional interactions with various signaling pathways triggered in HeLa and MCF-7 cells. Compound 14a induced apoptosis via caspase independent pathway through the participation of mitogen-activated protein kinases (MAPK) such as extracellular signal related kinase (ERK) and p38 as well as p53 pathways. It originates the activation of pro-apoptotic proteins such as Bak and Mcl-1s and also strongly induced the generation of reactive oxygen species. In downstream signaling pathway, activated p53 protein interacted with MAPK pathways, including SAPK/c-Jun N-terminal protein kinase (JNK), p38 and ERK kinases resulting in apoptotic cell death. The involvement of MAPK cascades such as p38, ERK and p38 on compound 14a induced apoptotic cell death was evidenced by the fact that the inclusion of specific inhibitors of p38, ERK1/2 and JNK MAPK (SB2035809, PD98059 and SP600125) prevented the compound 14a towards induced apoptosis. The results clearly showed that MAP kinase cascades were crucial for apoptotic response in compound 14a induced cellular killing and were dependent on p53 activity. Based on the results, compound 14a was identified as a promising candidate for cancer therapeutics and these findings furnish a basis for further in vivo experiments on anti-proliferative activity.

Cell Cycle Arrest and Cytotoxic Effects of SAHA and RG7388 Mediated through p21WAF1/CIP1 and p27KIP1 in Cancer Cells.

Background and Objective: Alterations in gene expressions are often due to epigenetic modifications that can have a significant influence on cancer development, growth, and progression. Lately, histone deacetylase inhibitors (HDACi) such as suberoylanilide hydroxamic acid (SAHA, or vorinostat, MK0683) have been emerging as a new class of drugs with promising therapeutic benefits in controlling cancer growth and metastasis. The small molecule RG7388 (idasanutlin, R05503781) is a newly developed inhibitor that is specific for an oncogene-derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. These two drugs have shown the ability to induce p21 expression through distinct mechanisms in MCF-7 and LNCaP cells, which are reported to have wild-type TP53. Our understanding of the molecular mechanism whereby SAHA and RG7388 can induce cell cycle arrest and trigger cell death is still evolving. In this study, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 using MCF-7 and LNCaP cells. Materials and Methods: The cytotoxicity, cell cycle arrest, and apoptosis/necroptosis effects of the SAHA and RG7388 treatments were assessed using the Trypan Blue dye exclusion (TBDE) method, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence assay with DEVD-amc substrate, and immunoblotting methods. Results: The RG7388 treatment was able to induce cell death by elevating p21WAF1/CIP1 through inhibition of MDM2 in LNCaP, but not in MCF-7 cells, even though there was evidence of p53 elevation. Hence, we suspect that there is some level of uncoupling of p53-mediated transcriptional induction of p21WAF1/CIP1 in MCF-7 cells. Conclusion: Our results from MCF-7 and LNCaP cells confirmed that SAHA and RG7388 treatments were able to induce cell death via a combination of cell cycle arrest and cytotoxic mechanisms. We speculate that our findings could lead to the development of newer treatments for breast and prostate cancers with drug combinations including HDACi.

Cytotoxicity and genotoxicity of size-fractionated particulate matter collected in underground workplaces

Road tunnel construction involves the use of explosives and diesel-powered machines in a very limited indoor space. Tunnel construction workers are thus exposed to a variety of toxic substances, including dust, asbestos, silica, concrete, diesel fumes, and oil mist. In this study, the low noise miniaturized Sioutas Cascade Impactor (SKC) has been used to collect PM samples as a function of particle size (i.e., aerodynamic diameter). Airborne particulates were sampled into a road tunnel (final length 2391 m) under construction near the village of Pale, Municipality of Foligno, Umbrian Apennines (State Highway 77 “Val di Chienti”). Three sampling sessions were performed: (i) in the West yard, during dislodging of rocks; (ii) in the East yard during drilling of rocks, and (iii) during scaling and grouting with quick-drying shotcrete. The aim of this study was to characterize size-fractionated PM samples for their cytotoxic/genotoxic potentials. Toxicological evaluation was carried out in vitro on A549 lung carcinoma cells: cytotoxicity was assessed by Trypan blue dye exclusion assay, whereas genotoxicity was assessed by comet assay (primary DNA damage) and cytokinesis-block micronucleus (CBMN) Cytome assay (cytogenetic effects). Primary DNA damage (i.e., strand breakage) was mainly caused by coarse fractions A (O > 2.5 μm) sampled in the West yard (sample i) and in the East yard (sample iii). Cytogenetic effects were mainly caused by the fine fractions AF (aerodynamic O < 0.25 μm) sampled in the East yard (sample ii). Results reported in this article show that particulate matter (PM) samples collected underground (i.e., during road tunnel construction) may trigger various cytotoxic/genotoxic effects. The recent classification of air pollution and PM itself as carcinogenic to humans (group 1) by the IARC makes indoor/outdoor air pollution a growing matter of concern. Our results highlight the need in this occupational setting for a reduction, if applicable, of PM emission (prevention), or an implementation of work practices by stressing the importance of proper use of protective equipment (protection).