WST-1 Proliferation Assay Research Articles

Abstract P3-07-26: Anti-cancer activity of naturally formulated Trigonella foenum graecum (Fenugreek) PLGA hyaluronic acid-coated nanoparticles for treatment of breast cancer cell lines

Abstract There are several growing evidences of anticancer complementary and alternative medicines worldwide. Trigonella foenum graecum (Fenugreek) is traditionally applied to treat several disorders such as diabetes, inflammation, and gastrointestinal ailments. Previously, we reported that Fenugreek has anticancer properties against liver cancer due to its active beneficial chemical constituents. Herein, Fenugreek-loaded poly (l-lactic-co-glycolic acid) (PLGA) hyaluronic acid (HA)-coated nanoparticles have been formulated to improve and enhance the anticancer potential of Fenugreek against human breast cancer cell lines. The cytotoxic effects, physicochemical characterization in vitro and pharmacodynamic study of optimized formulation of the FCE-loaded (PLGA)-(HA)-coated nanoparticles in vivo were investigated in (ER-positive) and (triple-negative), breast cancer cell lines, ZR-75-1 and MDA-MB-231, respectively. The particle size, PDI, zeta potential, entrapment efficiency, and loading capacity of FCE-loaded nanoparticles were 188.5 ± 3.1 d.nm, 0.198 ± 0.114, −.118 mV, 38.41 ± 1.4%, and 6.37 ± 1.08%, respectively. The in vitro cytotoxicity study of FCE-loaded NPs by Wst-1 proliferation assay on both tested breast cancer cell lines showed an anti-cancer activity after 48 h of treatment in a dose-dependent manner. Furthermore, the anticancer effect of FCE-loaded nanoparticles was examined by the morphological cell changes, histone release, caspase-3 activity, and modulation of the oncogenic signal pathways of m-tor, β-catenin, cyclin D-1 and STAT3 that are constitutively active in wide variety of breast cancer cell lines which in turn could be important as therapeutic targets in breast cancer. 66 μg/mL FCE-loaded PLGA nanoparticles induced 67.3% apoptosis in both tested cell lines after 48 h that was associated with increased expression of caspase 3, Bax, Bak, p53, and cell cycle arrest at G0/G1 in a time-and dose-dependent manner compared to unloaded-nanoparticles or untreated cells. Moreover, there were significant inhibition in m-tor, β-catenin, cycline-D1, and STAT3 expression levels in both tested cell lines compared to un-loaded-nanoparticles or untreated cells. All in all, our results showed that the formulated FCE-loaded (PLGA)-(HA)-coated nanoparticles induced apoptosis in both ER-positive and triple-negative breast cancer cell lines respectively by increase the expression of pro-apoptotic genes and inhibited several oncogenic genes that might hold a promise for the complementary therapy for human breast cancer. Citation Format: Islam Yahiya, Rana Saeed, Ahmed Sultan. Anti-cancer activity of naturally formulated Trigonella foenum graecum (Fenugreek) PLGA hyaluronic acid-coated nanoparticles for treatment of breast cancer cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-26.

A16 ESOPHAGEAL ORGANOID PROLIFERATION AND DIFFERENTIATION ARE ALTERED BY LOSS OF MSH2

BackgroundThe stratified epithelium of the esophagus includes Krt15+ basal stem cells that display self-renewing and regenerative capacity, and multipotency. However, the mechanisms that specifically control their functions remain unknown. Interestingly, RNA sequencing and GSEA revealed an enrichment of a gene set associated with DNA repair in Krt15+ cells vs Krt15- cells. We also observed that Msh2 (DNA mismatch repair pathway) is the most significantly upregulated gene in Krt15+ stem cells.AimsTo determine the effect of Msh2 loss on self-renewal and differentiation of esophageal organoids.MethodsEsophageal epithelial cells were isolated from a wild-type mouse. Using flow cytometry, esophageal Krt15+ (GFP+) and Krt15- (GFP-) cells were sorted from Krt15-CrePR1 (R26mT/mG) mice. All cell populations were grown as organoids and Msh2 was depleted using a CRISPR/Cas9 approach. Impact of Msh2 loss on self-renewal and differentiation in esophageal epithelial organoids was evaluated through organoid formation assays, WST-1 proliferation assays and histological analysis.ResultsAt baseline, organoids depleted for Msh2 formed more poorly differentiated and less well-differentiated organoids than controls. Lower expression of differentiation gene Krt13 was also observed in Msh2-depleted organoids, confirming an altered differentiation pattern. Furthermore, these organoids showed a higher organoid formation rate and proliferation by WST-1 assay, suggesting that self-renewal capacity and viability are increased when Msh2 is depleted. Interestingly, following radiation, organoids depleted for Msh2 showed higher residual levels of p-H2AX (DNA damage marker), suggesting that their capacity to cope with DNA damages is altered. As mentioned above, we previously reported that Msh2 is the most upregulated gene in Krt15+ vs Krt15- cells. Therefore, to determine if Msh2 role is distinct in both populations, we depleted Msh2 in Krt15+ and Krt15- cells-derived organoids. Interestingly, our preliminary results suggest that Msh2 deletion led to increased p-H2AX and decreased Krt13 levels in Krt15+ organoids but not in Krt15- organoids.ConclusionsOur results show that Msh2 is potentially a key contributor of esophageal stemness in homeostatic and injured conditions.Funding AgenciesCIHRCanada Research Chair

Development of a novel direct dental pulp-capping material using 4-META/MMA-TBB resin with nano hydroxyapatite

In the case of dental pulp exposure, direct pulp capping is often performed to preserve vital dental pulp tissue. Numerous studies regarding the development of direct pulp-capping materials have been conducted, but materials with an appropriate sealing ability, which induce dense reparative dentin formation, have not been developed. Although nano hydroxyapatite (naHAp) is a bone-filling material with bioactivity and biocompatibility, the inductive effects of naHAp on reparative dentin formation remain unclear. In the present study, the effects of dental adhesive material 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate tri-n-butylborane [4-META/MMA-TBB or Super-bond (SB)], which included 10%, 30%, and 50% naHAp (naHAp/SB) on odontoblastic differentiation of dental pulp stem cells (DPSCs) and reparative dentin formation were investigated. Scanning electron microscopy (SEM) and energy dispersive X-ray spectrometer analysis were performed to verify the existence of naHAp particles on the surface of naHAp/SB discs. The tensile adhesive strength of naHAp/SB was measured using a universal testing machine. As a result, 10% naHAp/SB and 30% naHAp/SB showed almost the same tensile adhesive strength as SB but 50% naHAp/SB showed significantly lower than the other experimental group. WST-1 proliferation assay and SEM analysis revealed that naHAp/SB did not affect the proliferation of DPSCs. Calcium release assay, quantitative RT-PCR, and western blotting analysis demonstrated that naHAp/SB did not release calcium ion but 30% naHAp/SB increased the expression of calcium-sensing receptor (CaSR) in DPSCs. Additionally, quantitative RT-PCR, western blotting analysis, Alizarin Red S- and von Kossa staining revealed that 30% naHAp/SB induced odontoblastic differentiation of DPSCs, which was inhibited by a MEK/ERK inhibitor and CaSR antagonist. Furthermore, 30% naHAp/SB promoted dense reparative dentin formation in an experimentally-formed rat dental pulp exposure model. These findings suggest that 30% naHAp/SB can be used as an ideal direct pulp capping material.

Abstract 1944: Navitoclax enhances the efficacy of copanlisib predominantly through inhibition of BCLxL in PIK3CA mutant colorectal cancer

Abstract Background: Mutations in PIK3CA occur in 18% of colorectal cancers (CRC) and result in constitutive activation of the PI3K pathway. Previous work from our lab has determined that MTORC1/2 inhibition, with copanlisib (cop), a PI3K/MTOR inhibitor, is sufficient to induce a treatment response. We hypothesized that the BCL2 family inhibitor navitoclax (nav), a BCL2/BCLxL inhibitor, would enhance the therapeutic response of cop and increase the induction of apoptosis. Methods: Murine derived cancer organoids (MDCOs) were generated from adenocarcinomas arising in Apc and Pik3ca mutant transgenic mice ((FVB x B6) F1 Apcfl/+ Pik3caH1047R/+). MDCOs were plated and matured for 24 hours. MDCOs were treated for 48 hours. Change in diameter was used to assess response. Additionally, human 2D isogenic cell lines SW48 and SW48PIK3CA-H1047R (SW48PK) were plated and treated for 48 hours. WST-1 proliferation assay was used to determine response. In vivo response was assessed as median relative change (MRC) in tumor volume of SW48 and SW48PK xenografts in immunocompromised Rag2-Il2rg-/- (R2G2) mice. Induction of apoptosis was determined with immunoblotting (IB) for PARP cleavage. Results: MDCOs were treated with cop (200nM) or nav (250nM) alone and in combination. Single agent cop resulted in -16% MRC (p<0.001). Nav alone did not have an effect (+95% p<0.07). Enhanced therapeutic response was seen in the combination therapy compared to cop alone (cop -16% vs combo -100%; p<0.001). In SW48 and SW48PK with cop (1nM or 10nM) or nav (250nM) alone and in combination, a greater response was seen in the combination therapy in both cell lines compared to either single agent alone (p<0.005). SW48 and SW48PK xenograft studies demonstrated significant activity of this combination in vivo. SW48 tumor growth was limited with either cop (10mg/kg), nav (80mg/kg), or the combination compared to control (control +5.0 MRC, cop +3.4, nav +2.6, combo +2.6; control vs combo p<0.01; cop vs combo p=0.29). Enhanced activity of this combination was observed in SW48PK tumors compared to SW48. Growth was significantly more limited with the combination (control +5.0 MRC, cop +3.4, nav +4.5, combo +0.7; control vs combo p<0.01; cop vs combo p=0.06). IB of cleaved PARP showed induction of apoptosis was the highest in the combination therapy in all in vitro models and seen as early as 6 hours post treatment. To identify which BCL2 family member nav was primarily targeting, cop was combined with WEHI539 (BCLxL inhibitor; 250nM) or ABT199 (BCL2 inhibitor; 250nM). BCL2 inhibition did not enhance the efficacy of cop in the MDCOs or isogenic lines. However, BCLxL inhibition did enhance the efficacy of cop in both in vitro models. Conclusion: These data indicate that navitoclax, through BCLxL inhibition, enhances the efficacy of copanlisib with a greater induction of apoptosis in several models of PIK3CA mutant CRC. Citation Format: Rebecca Anna DeStefanis, Alyssa DeZeeuw, Gioia Sha, Autumn Olson, Samantha J. Anderson, Christopher P. Babiarz, Cheri A. Pasch, Linda Clipson, Dustin A. Deming. Navitoclax enhances the efficacy of copanlisib predominantly through inhibition of BCLxL in PIK3CA mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1944.

Germinal Center Kinase As a Therapeutic Target in Multiple Myeloma

Introduction: Despite many new drug approvals, Multiple Myeloma remains an incurable disease due to development of drug resistance. This process is driven by clonal evolution in which kinases play a major role. Germinal center kinase (GCK) is the founding member of the GCKs, a group of Ser/Thr kinases homologous to the saccharomyces cerevisiae Ste20p, and participates in B cell differentiation. Among human tissues, GCK is highly expressed in transformed lymphocytes, spleen and whole blood. However, the expression and function of GCK in human myeloma is totally unknown.Methods and Results: First we analyzed the expression level of GCK in human MM cells and found that GCK is abundantly expressed in all the MM cell lines we tested. To determine whether GCK is required for MM cell growth, we introduced inducible shRNA to knock down GCK expression in MM cells by lentiviral mediated transduction. MM cell lines MM.1S and RPMI-8266 were stably transduced with shRNA lentivirus generated from a robust inducible knockdown vector pLKO-tet-On. Doxycycline-induced GCK-tet-on-shRNA expression resulted in GCK protein knockdown and significant inhibition of cell growth by 75% in WST-1 proliferation assay. In accordance, cell cycle analysis revealed that the knockdown of GCK decreased cells in S-phase with concomitant increase in G0/G1 growth arrest (55% vs 67%). Importantly, GCK knockdown resulted in significant increase (3.21% vs 60.8%) of apoptotic cells suggesting that inhibition of GCK is a potential therapeutic target in MM cells. This was further confirmed by treating MM cells lines (MM.1S, RPMI-8266 and H929) with GCK inhibitors TL4-12, NG25 and Bay 61-3606. We found that these drugs dose dependently and significantly inhibit multiple myeloma cell growth, with IC50 7, 1.6 and 0.5 µM, respectively in H929 cells by WST-1 proliferation assay. Mechanistic studies showed that Bay 61-3606 dose-dependently decreases the protein levels of IKZF1/IKZF3 and c-Myc, increases p53 level and induces PARP cleavage resulting in cell cycle G1 arrest (60.8 vs. 69.8 with 1 µM) and apoptosis (9.16 vs. 29.4 at 1 µM) in MM.1S cells. To confirm the critical role of GCK in in vivo growth of MM cells, we generated subcutaneous MM xenografts in SCID Beige (CB17.Cg-PrkdcscidLystbg-J/Crl) mice using doxycycline-inducible GCK shRNA MM.1S cells. Doxycycline or vehicle treatment was started after the tumor was established on day 16. In contrast to vehicle-treated MM.1S-tet-on-shGCK or doxycycline-treated MM.1S-tet-on-sh-control tumors, doxycycline treated animals bearing MM.1S-tet-on-shGCK xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 30 days. Immunohistochemistry staining of tumors confirmed the decrease of GCK and IKZF1 expression in doxycycline-treated mice bearing MM.1S-tet-on-shGCK tumors compared with tumors of vehicle- or non-doxycycline treated mice.Conclusion: Our findings show that GCK is a potential target for multiple myeloma therapy. GCK inhibition results in significant anti-MM effects by inducing cell cycle arrest and cell apoptosis accompanied by IKZF1/3 down regulation. Furthermore, the inhibition of in vivo tumor growth correlates with the doxycycline-induced GCK knockdown, further confirming the critical role of GCK in MM tumorigenesis. DisclosuresLentzsch:Amgen: Consultancy; BMS: Consultancy; Caelum Biosciences: Other: leadership position and stock.

Molecular Genetic Dissection of Inflammatory Linear Verrucous Epidermal Naevus Leads to Successful Targeted Therapy

Molecular Genetic Dissection of Inflammatory Linear Verrucous Epidermal Naevus Leads to Successful Targeted Therapy

N-acetylcysteine attenuates PGE2 and ROS production stimulated by 4-META/MMA-based resin in murine osteoblastic cells.

This study examined the effects of N-acetylcysteine (NAC) on the inflammatory reactions of murine osteoblastic cells cultured on the 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate (4-META/MMA)-based resin. Superbond C&B (SB) was used as the 4-META/MMA-based resin and placed in a 48-well cell culture plate. The cells were cultured in αMEM (control) as well as on SB and SB in αMEM with NAC (SB+NAC). They were examined using the WST-1 proliferation assay, real-time PCR, enzyme-linked immunosorbent assay (ELISA), intracellular reactive oxygen species (ROS) measurements, and cellular glutathione (GSH) detection. COX-2 and IL-6 gene expressions were upregulated in SB; however, they were suppressed by NAC. Furthermore, PGE2 production in the culture medium was increased in SB, whereas NAC decreased the PGE2 production. NAC lowered the ROS level in the culture medium and significantly increased the intracellular GSH level. The present in vitro study demonstrated that NAC might be effective for dental material detoxification.

Human laryngeal squamous cell carcinoma cell line release of endogenous anandamide and 2-arachidonoylglycerol, and their antiproliferative effect via exogenous supplementation: an in vitro study.

The level of the major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are altered in several types of carcinomas, and are known to regulate tumor growth. Thusly, this study hypothesized that the HEp-2 human laryngeal squamous cell carcinoma (LSCC) cell line releases AEA and 2-AG, and aimed to determine if their exogenous supplementation has an anti-proliferative effect in vitro. In this in vitro observational study a commercial human LSCC cell line (HEp-2) was used to test for endogenous AEA and 2-AG release via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The anti-proliferative effect of AEA and 2-AG supplementation was evaluated via WST-1 proliferation assay. It was observed that the HEp-2 LSCC cell line released AEA and 2-AG; the median quantity of AEA released was 15.69ngmL-1 (range: 14.55-15.95ngmL-1) and the median quantity of 2-AG released was 2.72ng -1 (range: 2.67-2.74ngmL-1). Additionally, both AEA and 2-AG exhibited an anti-proliferative effect. The anti-proliferative effect of 2-AG was stronger than that of AEA. These findings suggest that AEA might function via a CB1 receptor-independent pathway and that 2-AG might function via a CB2-dependent pathway. The present findings show that the HEp-2 LSCC cell line releases the major endocannabinoids AEA and 2-AG, and that their supplementation inhibits tumor cell proliferation in vitro. Thus, cannabinoid ligands might represent novel drug candidates for laryngeal cancers, although functional in vivo studies are required in order to validate their potency.

Innovative One-Shot Paradigm to Tune Filler–Polymer Matrix Interface Properties by Plasma Polymer Coating in Osteosynthesis Applications

The present study aims to improve the interfacial bonding between hydroxyapatite particles (HAs) and polylactide (PLA) to enhance the mechanical performance and biocompatibility of bone implants based on HA/PLA. For this, one-shot surface functionalization of HA via plasma polymerization is developed. Taking advantage of acetylene plasma chemistry, the hydrophobicity of HA particles was finely tuned prior to their introduction into a PLA matrix via an extrusion process. The effect of the plasma power (20 or 100 W) on the composition of the plasma polymer film (PPF) formed on the HA surface was studied via Fourier transform infrared (FTIR) spectroscopy, time-of-flight secondary-ion mass spectrometry (ToF-SIMS), and X-ray photoelectron spectroscopy (XPS). The amount of PPF formed was evaluated via thermogravimetric analyses (TGA). Cytotoxicity of the modified HA particles was monitored by the WST-1 proliferation assay and lactate dehydrogenase (LDH) release and showed that independent on the studied conditions, cell viability remained above the 70% threshold and LDH accumulation changes were insignificant, suggesting good biocompatibility. Contact angle measurements and morphological and rheological analyses showed that the low working power promoted more hydrophobic surfaces and a better HA/PLA interface. Dynamic mechanical analyses revealed that the storage modulus at 37 °C increased for the composite containing functionalized particles by 1.5 times compared to the neat particle's composites. This work opens a route toward further one-shot development of improved scaffolds for bone tissue engineering.

Ambivalent Effects of Tumor Necrosis Factor Alpha on Apoptosis of Malignant and Normal Human Keratinocytes

Introduction: Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that may paradoxically induce either apoptosis or cell survival. It mediates its activity through binding of TNF-receptor (TNFR) 1 or 2. TNFR1 is mainly responsible for transmitting apoptotic signals. The activation of apoptotic mechanisms can either be intrinsic (mitochondrial) or extrinsic (death receptors). Death ligands such as TNF-related apoptosis-inducing ligand (TRAIL) specifically induce extrinsic apoptosis, while cytostatic drugs such as 5-fluorouracil (5FU) induce intrinsic apoptosis. Objectives: To investigate the effects of TNFα on apoptosis in malignant and normal human keratinocytes. Methods: Human cutaneous squamous cell carcinoma (SCC) cell line SCC-13 and immortalized human keratinocytes HaCaT as well as primary normal human keratinocytes (PNHK) were stimulated with TNFα and then treated either with TRAIL or 5FU. Cell viability and cell proliferation, DNA fragmentation, apoptosis, and cytotoxicity were determined by WST-1 proliferation assay, ELISA, flow cytometry, and colorimetric analysis of lactate dehydrogenase, respectively. In addition, Western blotting was performed for analysis of caspase-3. Results: TNFα affected viability of SCC-13 and HaCaT cells in combination with 5FU or TRAIL. In contrast, TNFα did not influence cell viability of PNHK. It enhanced the apoptotic effects of both extrinsic and intrinsic stimuli in SCC-13 and HaCaT. In clear contrast, TNFα protected PNHK against TRAIL- and 5FU-induced apoptosis. The effects were dose-dependent and TNFα-specific; furthermore, the apoptosis pathway was caspase-dependent. Conclusions: In summary, opposing effects of TNFα in malignant versus normal human keratinocytes were observed with possibly relevant clinical implications, when patients are treated with TNFα inhibitors.

The antiproliferative potential of isolated emodin and aloe-emodin from Rheum ribes on different cancer cell lines

Herbal compounds are important resources that used in the treatment of many diseases, and are inspiring in drug design and development. Rheum ribes, a perennial native plant, has potentially diverse medicinal effects, including antioxidant, antibacterial and anti-tumor activity. The key molecules associated with anticancer activity of R. ribes in tumor formation still remains unclear. The present study was aimed to elucidate the antiproliferative effects of two purified compounds, emodin and aloe-emodin, from R. ribes. In our study, chromatographic separation was used to purification of compounds. Experimental 1D and 2D 1H and 13C-NMR spectra helped us to confirm the chemical structures of emodin and aloe-emodin. The viability of human glioblastoma cell line (U373), human breast carcinoma cells (MCF-7), and human colorectal cancer cell line (HT-29) were detected by WST-1 proliferation assay as dose- and time-dependent. The isolated emodin and aloe-emodin from R. ribes, reduced viability of human glioblastoma, breast and colorectal cancer cell lines in dose- and time-dependent manner significantly. These antiproliferative effects may contribute as a possible strategy to the development of new therapeutic approaches in the treatment of cancer.

A novel class of small molecule inhibitors with radioprotective properties

The goal of this study was to develop novel radioprotective agents targeting the intrinsic apoptotic pathway and thus decreasing the radiation-induced damage. For that purpose, we designed, synthesized and analyzed ten new compounds based on the 1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-phenoxypropan-2-ol leading structure. The cytotoxicity of the newly synthesized substances was tested in vitro on cell lines derived from different progenitor cells by WST-1 proliferation assay. MTT test was utilized to assess half-maximal inhibitory concentrations and maximum tolerated concentrations of novel compounds in A-549 cells. Screening for radioprotective properties was performed using flow-cytometry in MOLT-4 cells exposed to 60Co ionizing gamma radiation. Selected candidates underwent in vivo testing in C57Bl/6 J mice having a positive impact on their immunological status. In summary, we report here promising compounds with radioprotective effect in vivo.

General toxicity assessment of the novel aldose reductase inhibitor cemtirestat.

Cemtirestat, 3-mercapto-5H-[1,2,4]-triazino[5,6-b] indole-5-acetic acid was recently designed and patented as a highly selective and efficient aldose reductase inhibitor endowed with antioxidant activity. The aim of the present study was to assess the general toxicity of cemtirestat using in silico predictions, in vitro and in vivo assays. ProTox-II toxicity prediction software gave 17 “Inactive” outputs, a mild hepatotoxicity score (0.52 probability) along with a predicted LD50 of 1000 mg/kg. Five different cell lines were used including the immortalized mouse microglia BV-2, the primary human fibroblasts VH10, the insulinoma pancreatic β-cells INS-1E, the human colon cancer cells HCT116 and the human immortalized epithelial endometrial cell lines HIEEC. In contrast to the clinically used epalrestat, cemtirestat showed remarkably low cytotoxicity in several different cell culture viability tests such as MTT proliferation assay, neutral red uptake, BrdU incorporation, WST-1 proliferation assay and propidium iodide staining followed by flow cytometry. In a yeast spotting assay, the presence of cemtirestat in incubation of Saccaromyces cerevisiae at concentrations as high as 1000 μM did not affect cell growth rate significantly. In the 120-day repeated oral toxicity study in male Wistar rats with daily cemtirestat dose of 6.4 mg/kg, no significant behavioral alterations or toxicological manifestations were observed in clinical and pathological examinations or in hematological parameters. In summary, these results suggest that cemtirestat is a safe drug that can proceed beyond preclinical studies.

207 IL-17E (IL-25) alters epidermal homeostasis in a matter distinct from IL-17A

Our group has recently shown that keratinocyte-derived IL-17E (IL-25), one of six IL-17 isoforms, is overexpressed in lesional psoriatic skin. The presence of a complete receptor for IL-17E at the keratinocyte surface indicates the potential autocrine mode of action of the cytokine. We therefore aimed at addressing the influence of IL-17E on the functions of human primary keratinocytes. First, IL-17E, but not IL-17A, promoted the proliferation of keratinocytes as assessed by WST-1 proliferation assay and crystal violet staining. Surprisingly, IL-17E addition to cultured keratinocytes resulted in the concomitant up-regulation of differentiation-associated gene transcripts (i.e. keratin 10, loricrin and filaggrin), while their expression was either inhibited or not changed by IL-17A. Immunohistochemical analysis of in vitro reconstituted 3D epidermal equivalents confirmed these results. IL-17E, contrary to IL-17A, was instead not involved in the induction of an antimicrobial response. Moreover, time-lapse analysis of cell movement showed that IL-17E particularly influences cell motility increasing both cell speed and displacement. This was associated with specific changes in actin cytoskeleton organization and regulation of proteins involved in the focal contacts assembly such as vinculin. Altogether, our data point towards an important role for IL-17E in the epidermal phenotype of psoriasis; this role is clearly distinct from the role of IL-17A. Our results also reveal a possible effect of IL-17E beyond psoriasis, in disorders characterized by epidermal alterations as well as in the wound healing process.

LncRNA MNX1-AS1 promotes progression of esophageal squamous cell carcinoma by regulating miR-34a/SIRT1 axis

BackgroundLong non-coding RNAs (lncRNAs) are powerful factors influencing the tumorigenesis and metastasis of multiple carcinomas. LncRNA MNX1-AS1 plays critical roles in the progression of tumor formation according to recent research, while its roles in esophageal squamous cell carcinoma (ESCC) remains unknown. MethodsThe expression levels of lncRNA MNX1-AS1 were examined in ESCC tissues by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The role of lncRNA MNX1-AS1 was performed by WST-1 proliferation assays, migration and invasion assays. Besides, the molecular mechanism of lncRNA MNX1-AS1 was verified by online bioinformatics, qRT-PCR and rescue assays. ResultsMNX1-AS1 was signifcantly upregulated in ESCC tissues. It was conformed that high MNX1-AS1 expression was associated with ESCC lymph node metastasis. Moreover, we found that knockdown of MNX1-AS1 apparently suppressed the cell proliferation, migration, and invasion capacity. Flow cytometry analysis showed MNX1-AS1 regulated ESCC cell cycle and apoptosis progression. Mechanism analysis revealed that miR-34a inhibitor could rescue the influence of inhibiting MNX1-AS1 on ESCC cells migration by serving as competing endogenous RNA (ceRNAs). Furthermore, we found that miR-34a specifically targeted SIRTI. ConclusionsTaken together, we demonstrated that lncRNA MNX1-AS1/miR-34a/SIRT1 regulatory axis could play an important role in ESCC progression, and MNX1-AS1 may act as a novel potential biomarker for esophageal squamous cell carcinoma.

Nerigoside suppresses colorectal cancer cell growth and metastatic potential through inhibition of ERK/GSK3β/β-catenin signaling pathway

BackgroundNerigoside (NG), a cardenolide isolated from a commonfolk medicine, Nerium oleander Linn. (Apocynaceae), has not been explored for its biological effects. To date, cardenolides have received considerable attention in pharmacology studies due to their direct effects of apoptosis-induction or growth-inhibitory against tumor in vitro and in vivo. Whether and how NG exerts anticancer effects against colorectal cancer remains to be elucidated. PurposeThe aim of this study was to investigate the anticancer effect of NG in human colorectal cancer cells. MethodsTo test anticancer effect, we compared potency of NG in two colorectal cancer cell lines, HT29 and SW620 by WST-1 and colony proliferation assays. And we investigated mechanism of anticancer activities by analyzing players in apoptotic and ERK/GSK3β/β-catenin signaling pathways in HT29 and SW620 cells treated with NG. ResultsIn this study, we showed that NG markedly suppressed the cell viability and colony formation of colorectal cancer cells HT29 and SW620, with no significant toxic effect on non-cancer cells NCM460. Annexin V-FITC/PI and CFSE labeling results revealed that NG suppressed cell proliferation in low concentration, along with reducing expression of PCNA, while NG induced apoptosis in high concentration,. Meanwhile, NG significantly arrested cell migration by reversal of EMT and cell cycle on G2/M. Then, we found that the ERK and GSK3β/β-catenin signaling pathway were noticeably blocked in CRC cells after treatment with NG. According to western blot, NG upregulated the expression of p-GSK3β/GSK3β and decreased especially the expression of β-catenin in nuclear. In addition, Wnt signaling and its target genes were suppressed in response to NG. Then, the Ser9 phosphorylation of GSK3β can be reduced / raised by GÖ 6983 / LiCl, respectively. Thus, we further confirmed that the GSK3β/β-catenin axis is involved in NG-prevented cell proliferation. ConclusionNG inhibited the growth of colorectal cancer cells by suppressing ERK/GSK3β/β-catenin signaling pathway. And the GSK3β/β-catenin axis is involved in preventing cell proliferation and migration by NG-treatment. These results suggest that NG may be used to treat colorectal cancer, with better outcome by combining with GSK3β inhibitor to block Wnt pathway.

Abstract B59: Race-related differential splicing of the insulin receptor: A novel target underlying prostate cancer disparities

Abstract Background: Age-adjusted incidence and mortality rates for prostate cancer (PCa) among African American (AA) men are significantly greater than among white men. Much of this disparity remains after controlling for behavioral, social, and neighborhood determinants of health, which is consistent with a biologic contribution. Evidence suggests that the genetic etiology and molecular progression of PCa are different in AAs versus whites. Our laboratory has identified novel alternatively spliced genes in AA versus white PCa biopsy tissue that track with population-based prostate tumor aggressiveness. This work addresses the urgent need to interrogate novel molecular mechanisms driving more aggressive PCa in AAs to aid in development of new biomarkers and therapeutic targets for improved prognosis and treatment of aggressive disease. Methods: We have analyzed RNA from human PCa biopsy tissue from 20 AA and 15 white patients using exon arrays. From these data, we prioritized targets for further study by identifying those associated with androgen signaling and PCa and those that have also been shown to be alternatively spliced in breast, lung, and liver cancer. To validate race-related alternative splicing of the insulin receptor (INSR) gene, we have used qPCR and targeted sequencing of RNA from population-specific PCa cell lines and patient-derived explants. In addition, we have knocked down the INSR splice variants in population-specific PCa cell lines using isoform-specific siRNAs and assessed resulting alterations in proliferation using a WST-1 proliferation assay. Current studies include assessing additional resulting alterations in expression of selected key targets in INSR signaling using Western blot analysis. In addition, studies are under way to treat population-specific PCa cell lines with INSR signaling inhibitors and assess resulting alterations in PCa cell biology. Results: Exon array analysis of RNA from AA and white PCa biopsy tissue revealed 934 differentially expressed exons in 861 corresponding genes. Thirty-nine targets have been prioritized for further study. We have validated race-related alternative splicing of INSR, involving differential exon inclusion/skipping of exon 11, in population-specific PCa cell lines and patient-derived explants, with the exclusion of exon 11 occurring more often in preclinical models of white PCa. Knockdown of INSR splice variants in population-specific PCa cell lines has shown that decreased expression of the isoform excluding exon 11 inhibits proliferation while decreased expression of the isoform including exon 11 has no effect on proliferation. Further studies to assess additional resulting alterations in expression of selected key targets in INSR signaling are under way. In addition, studies are under way to treat population-specific PCa cell lines with INSR signaling inhibitors and assess resulting alterations in PCa cell biology. Conclusions: These studies identify differential splicing of INSR in AA and white PCa and the INSR isoform excluding exon 11 as contributors to prostate tumor aggressiveness. Further interrogation of this mechanism underlying PCa disparities and its effect on targeted therapeutics has the potential to reduce PCa disparities for AA men and improve outcomes for men of all races with aggressive disease driven by this mechanism. Citation Format: April Deveaux, Bi-Dar Wang, Bonnie Lacroix, Brendon Patierno, Dadong Zhang, Kouros Owzar, Norman Lee, Daniel George, Jennifer Freedman, Steven Patierno. Race-related differential splicing of the insulin receptor: A novel target underlying prostate cancer disparities [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B59.

Template-assisted fabrication of tunable aspect ratio, biocompatible iron oxide pillar arrays

The extensive use of iron oxide nanomaterials in biomedical applications has prompted the development of a novel substrate for evaluating cell behaviour. This study examines the fabrication of tuneable length iron oxide pillar arrays using the porous nanochannels of anodic aluminium oxide membranes, and evaluates the biocompatibility of the substrate. The electroformed iron pillars were found to conform to the template channels with slightly larger iron oxide pillar diameters, due to the presence of an oxide shell. The biocompatibility was then confirmed with WST-1 proliferation and viability assay of cultured KT98 murine neural/progenitor stem cells on the surface of the pillar array; with no significant difference observed between viable cells after seven days of culture on iron oxide pillars, flat iron oxide, and tissue culture polystyrene. The physical properties of the pillar arrays were linked to the adhesion and spreading of the cells, and found that cells cultured on the pillar arrays had reduced spreading in comparison to tissue culture polystyrene control. In addition, it was found that protein expression was unaffected by culture on iron oxide substrates. The results of this study indicate that iron oxide pillar arrays are suitable to extended cell studies.

Control of cell proliferation by a porous chitosan scaffold with multiple releasing capabilities

The aim of this study was to develop a porous chitosan scaffold with long-acting drug release as an artificial dressing to promote skin wound healing. The dressing was fabricated by pre-freezing at different temperatures (−20 and −80 °C) for different periods of time, followed by freeze-drying to form porous chitosan scaffolds with different pore sizes. The chitosan scaffolds were then used to investigate the effect of the controlled release of fibroblast growth factor-basic (bFGF) and transforming growth factor-β1 (TGFβ1) on mouse fibroblast cells (L929) and bovine carotid endothelial cells (BEC). The biocompatibility of the prepared chitosan scaffold was confirmed with WST-1 proliferation and viability assay, which demonstrated that the material is suitable for cell growth. The results of this study show that the pore sizes of the porous scaffolds prepared by freeze-drying can change depending on the pre-freezing temperature and time via the formation of ice crystals. In this study, the scaffolds with the largest pore size were found to be 153 ± 32 μm and scaffolds with the smallest pores to be 34 ± 9 μm. Through cell culture analysis, it was found that the concentration that increased proliferation of L929 cells for bFGF was 0.005 to 0.1 ng/mL, and the concentration for TGFβ1 was 0.005 to 1 ng/mL. The cell culture of the chitosan scaffold and growth factors shows that 3.75 ng of bFGF in scaffolds with pore sizes of 153 ± 32 μm can promote L929 cell proliferation, while 400 pg of TGFβ1 in scaffolds with pore size of 34 ± 9 μm can enhance the proliferation of L929 cells, but also inhibit BEC proliferation. It is proposed that the prepared chitosan scaffolds can form a multi-drug (bFGF and TGFβ1) release dressing that has the ability to control wound healing via regulating the proliferation of different cell types.

In vitro observations and in silico predictions of xenoestrogen mixture effects in T47D-based receptor transactivation and proliferation assays

Within endocrine disruptor research, evaluation and interpretation of mixture effects and the predictive value for downstream responses still warrant more in-depth investigations.We used an estrogen receptor (ER)-mediated reporter gene assay (ER-CALUX®) and a cell proliferation assay (WST-1 assay), both based on the T47D breast cancer cell line, to test mixtures of heterogeneous xenoestrogens. Observed concentration-response curves were compared to those predicted by the concepts of concentration addition (CA), generalized concentration addition (GCA), and a novel full logistic model (FLM).CA performed better regarding mixture potency (EC50 values), whereas GCA was superior in predicting mixture efficacy (maximal response). In comparison, FLM proved to be highly suitable for in silico mixture effect prediction, combining advantages of both CA and GCA. The inter-assay comparison revealed that ER activation is not necessarily predictive for induction of cell proliferation.The results support the use of models like CA, GCA, or FLM in mixture effect evaluation. However, we conclude that reliable estimations regarding the disruptive potential of mixtures of endocrine active substances require an integrative approach considering more than one assay/endpoint to avoid misinterpretations. The formazan-based WST-1 proliferation assay might be a possible alternative to commonly used proliferation assays in endocrine disrupter research.