Matrigel Invasion Chamber Assay Research Articles

Synthesis and Characterization of Thymol Carbon Nanodot Functionalized Silver Nanoparticles (ThCND-AgNPs) and Evaluation of Their Antiproliferative, Anti-Invasive, and Apoptotic Effects on OVCAR-3 Ovarian Cancer Cells.

Ovarian cancer belongs to the category of gynecological malignancies and unfortunately holds the distinction of being the most aggressive among them. It is ranked as the fifth highest cause of cancer-related deaths in women worldwide. The utilization of metal nanoparticles (NPs) linked with natural herbal molecules in biomedical applications has been on the rise. Thymol carbon nanodot functionalized silver nanoparticles (ThCND-AgNPs) were synthesized in an original manner and subjected to thorough characterization, including analysis of their size, morphology, and elemental composition. The aim of this study is to investigate the effects of the ThCND-AgNPs on cell proliferation, invasion, and apoptotic gene expressions in OVCAR-3 ovarian cancer cells. The effect of ThCND-AgNPs on cell viability in OVCAR cells was determined in a dose- and time-dependent manner using the XTT method. The effect on the expression changes of apoptotic-related genes was assessed through the Real-time PCR method, while the anti-invasive activity was measured using the matrigel invasion chamber assay. The ThCND-AgNP molecule exhibited a dose- and time-dependent reduction in cell proliferation in OVCAR-3 cells. The IC50 values were determined to be 388.53 μg/mL at 24 h and 145.683 μg/mL at 48 h. Furthermore, the molecule was found to reduce cell invasion by 51.12% compared with the control group in OVCAR-3 cells. In terms of apoptotic-related genes, Bcl-2 expression was downregulated, while BAX, CASPASE-3, -8, and -9 expressions were unregulated. In conclusion, the obtained data reveal the potential antiproliferative, apoptotic, and anti-invasive effects of our original ThCND-AgNP molecule in ovarian cancer. While these results need further confirmation through more detailed experiments, they will provide insights for future studies.

Abstract 1718: Development of potent estrogen receptor beta agonists for treating glioblastoma

Abstract Background: Glioblastoma (GBM) is the most prevalent and aggressive type of adult brain cancers with low 5-year overall survival rates. Epidemiologic data suggest estrogen may decrease brain tumor growth. The lack of powerful, selective brain permeable estrogen receptor beta (ERβ) ligands to promote its anticancer action is limiting the therapeutic promise of ERβ. The goal of this project is to create a new class of brain permeable ERβ agonists for the treatment of GBM. Method: We have used four different assays to identify potent leads. (1) ERβ and estrogen receptor alpha (ERα) reporter assays, (2) Polar Screen Estrogen Receptor (ER) Competitive Binding Assays, (3) Cell viability assays using GBM cells, and (4) Activity in WT and ERβ-KO GBM cells. CellTiter-Glo Cell Viability and colony formation assays were used to test the efficacy of ERβ agonists. Invasion was measured by matrigel invasion chamber assays and apoptosis was measured by Caspase-Glo® 3/7 and Annexin V assays. Flow cytometry was used to evaluate the cell cycle. Western blotting, RNA-Seq, RT-qPCR, and ERβ KO cells were used in the mechanistic studies. Both orthotopic patient-derived xenografts and GBM cell line-derived (CDX) xenografts were used to test the ERβ agonist's activity in vivo. Results: Indanone and tetralone-oximes or keto-oximes have been hypothesized, produced, and evaluated as new ER agonists. Due to its high action in ERβ reporter assays, specific binding to ERβ in polar screen assays (EC50 91 nM), and potent growth inhibitory activity (IC50 7.4) in GBM cells, CIDD-97 was discovered as a possible hit by screening a library of ~60 compounds. CIDD-97 is significantly more selective for ERβ than ERα (40-fold). Treatment with CIDD-97 markedly reduced U251 cell viability while having little to no effect on U251-ER-KO cells and normal human astrocytes. Further, CIDD-97 treatment decreased the expression of stemness markers in patient derived GSC lines and promoted apoptosis. Additionally, CIDD-ERβ agonist improved TMZ's ability to reduce GBM cell survival. The activation of ERβ target genes in cells treated with CIDD-97 was verified by using RNA-Seq and RTqPCR. PK studies confirmed that CIDD-0149897 has systemic exposure, was found in the brain, and its maximal detectable levels suggested that it has good BBB permeability. Mice tolerated daily intraperitoneal treatment of CIDD-0149897 (50 mg/kg) with a 7-day repeat dosage with no evidence of toxicity. Additionally, when compared to vehicle CIDD-97 (50 mg/kg/i.p.) treatment significantly decreased tumor growth in xenograft models and extended the survival of tumor-bearing mice in orthotopic GSC models. Conclusion: Collectively, these findings pointed to CIDD-97 as a possible ERβ agonist and is easily adaptable to clinical use alongside current chemo- and radiation-therapy regimens, offering GBM patients an additional means of improving survival. Citation Format: Uday P. Pratap, Michael Tidwell, Henriette U. Balinda, Suryavathi Viswanadhapalli, Gangadhara Reddy Sareddy, Stanton McHardy, Andrew Brenner, Ratna K. Vadlamudi. Development of potent estrogen receptor beta agonists for treating glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1718.

(+)-Cyanidan-3-ol inhibits epidermoid squamous cell carcinoma growth via inhibiting AKT/mTOR signaling through modulating CIP2A-PP2A axis

Background: Despite recent advances in the treatment of squamous cell skin cancer (SCSC), the disease persists, and treatment resistance develops. Thus, identifying new targets and developing new therapeutic approaches showing low vulnerability to drug resistance is highly needed.Purpose: This study aimed to reveal a novel targeted phytotherapeutic strategy for SCSC treatment alone or in combination with standard targeted anticancer molecules.Study design: A library of natural products was utilized to identify molecules that inhibit the growth of skin cancer cells. The anticancer potential of the selected compound was evaluated in human skin squamous carcinoma models, in vitro and in vivo. A comprehensive ingenuity pathway analysis (IPA) strategy and molecular biology technology was adopted to investigate the therapeutic mechanisms in human SCSC.Methods: The Matrigel invasion chamber, foci formation and soft agar colony formation assays were employed to study the cells invasion and migration potential in vitro. In vivo antitumor effects were evaluated in DMBA/TPA-induced skin papilloma and A431 human skin squamous carcinoma xenograft tumor models. An integrative IPA was employed to identify mechanisms and protein targets in human SCSC.Compounds synergies were determined by the bliss model and evaluated using human SCSC cell lines and xenograft tumors. Histological staining, immunofluorescence imaging, real-time PCR, Western blots, and flow cytometric analyses were employed to analyze apoptosis and cell signaling mechanisms.Results: We identified (+)-cyanidan-3-ol (CD-3) as a selective compound for inhibiting the growth of SCSC cell lines. CD-3 inhibited tumor growth and burden without apparent toxicity and prolonged the survival of tumor-bearing mice. CD-3 inhibitory effects on SCSC growth are mediated via cell cycle arrest and caspase-dependent apoptosis induction. Mechanistic studies showed that CD-3 activates PP2A via inhibiting CIP2A and produces tumor growth inhibitory effects via promoting dephosphorylation of oncogenic AKT/mTOR signaling proteins in SCSC cells and xenograft tumors in a PP2A dependent manner. Furthermore, the combination of CD-3 and mTOR inhibitors (mTORi) synergistically reduced oncogenic phenotypes.Conclusions: Our study suggests that PP2A activation is an effective strategy for SCSC treatment and the CD-3 and mTORi combination may serve as a promising treatment for SCSC.

In vitro anticancer activity of extracted oil from Parrotiopsis jacquemontiana (Decne) Rehder

BackgroundParrotiopsis jacquemontiana, commonly referred to as “Beranj” in the local community, is widely used traditionally and has numerous health benefits. However, no studies have been conducted to investigate its anticancer potential, particularly its extracted oil. PurposeThe present study was put forth to appraise the anticancer potential of Parrotiopsis jacquemontiana extracted oil against liver (hcclm3 and hepg2) and breast cancer (mda-mb 231 and mcf-7) cell lines relative to normal cell lines (lo2 and mcf-10a) via MTT assay. MethodsFlow cytometry indicated the apoptotic effect whereas invasion and migration capabilities of oil against cancer cells were determined by Matrigel invasion chamber and wound-scratch assays. ResultsThe results of oil revealed a time and dose-dependent increase in cell proliferation inhibition, conferring to least IC50 shown against hcclm3 (144.9 ± 0.75 μg/ml) and mda-mb 231 (145.7 ± 0.32 μg/ml) cell line at 72 h, whereas no cytotoxic effect on normal cells was observed. In addition, the oil significantly (p < 0.001) suppressed the migration and invasion of hcclm3 and mda-mb 231 cells, showing noteworthy anti-metastatic potential. Furthermore, cell death was confirmed by Annexin‒V/PI staining where the maximum apoptotic percentage was calculated for oil (200 μg/ml) alongside mda-mb 231 conferring to 15.36 ± 1.22, 26.7 ± 1.2, and 36.43 ± 1.65 at 24, 48, and 72 h whereas 12.33 ± 1.05, 19.36 ± 1.62, and 29.3 ± 0.79 was recorded alongside hcclm3 at similar time intervals, respectively. ConclusionIn conclusion, the extracted oil exhibited strong anti-proliferative, anti-metastatic, and apoptotic effects and therefore may have potential applications in cancer treatment, however, further studies of oil regarding the action mechanisms and compounds involved in anticancer therapy are necessary.

Preclinical efficacy of ribavirin in SHH and group 3 medulloblastoma.

Medulloblastoma, the most common pediatric brain malignancy, has Sonic Hedgehog (SHH) and group 3 (Myc driven) subtypes that are associated with the activity of eukaryotic initiation factor 4E (eIF4E), a critical mediator of translation, and enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and master regulator of transcription. Recent drug repurposing efforts in multiple solid and hematologic malignancies have demonstrated that eIF4E and EZH2 are both pharmacologically inhibited by the FDA-approved antiviral drug ribavirin. Given the molecular overlap between medulloblastoma biology and known ribavirin activity, the authors investigated the preclinical efficacy of repurposing ribavirin as a targeted therapeutic in cell and animal models of medulloblastoma. Multiple in vitro assays were performed using human ONS-76 (a primitive SHH model) and D425 (an aggressive group 3 model) cells. The impacts of ribavirin on cellular growth, death, migration, and invasion were quantified using proliferation and Cell Counting Kit-8 (CCK-8) assays, flow cytometry with annexin V (AnnV) staining, scratch wound assays, and Matrigel invasion chambers, respectively. Survival following daily ribavirin treatment (100 mg/kg) was assessed in vivo in immunodeficient mice intracranially implanted with D425 cells. Compared to controls, ribavirin treatment led to a significant reduction in medulloblastoma cell growth (ONS-76 proliferation assay, p = 0.0001; D425 CCK-8 assay, p < 0.0001) and a significant increase in cell death (flow cytometry for AnnV, ONS-76, p = 0.0010; D425, p = 0.0284). In ONS-76 cells, compared to controls, ribavirin significantly decreased cell migration and invasion (Matrigel invasion chamber assay, p = 0.0012). In vivo, ribavirin significantly extended survival in an aggressive group 3 medulloblastoma mouse model compared to vehicle-treated controls (p = 0.0004). The authors demonstrate that ribavirin, a clinically used drug known to inhibit eIF4E and EZH2, has significant antitumor effects in multiple preclinical models of medulloblastoma, including an aggressive group 3 animal model. Ribavirin may represent a promising targeted therapeutic in medulloblastoma.

Nutritional Composition and Antioxidant, Antimicrobial, and Cytotoxic Activities of the Scaly Tooth Wild Mushroom, Sarcodon squamosus (Agaricomycetes), from Turkey.

Among natural products, mushrooms are well known for their nutritional value and health-improving features. In this study, the total phenolic content, total oxidant status, total antioxidant capacity, lipid hydroperoxides, and total free sulfhydryl levels of wild edible mushroom Sarcodon squamosus were investigated. The agar disc diffusion method was applied to test the antimicrobial effect of S. squamosus methanolic extract against Gram-positive bacteria (Micrococcus luteus NCIBM, Staphylococcus aureus ATCC 25923, and Bacillus subtilis ATCC 6633), Gram-negative bacteria (Proteus vulgaris RSKK 96026, Escherichia coli ATCC 35218, and Yersinia enterocolitica RSKK 1501), and yeast (Candida albicans ATCC 10231). According to the obtained results, S. squamosus methanolic extract had strong antioxidant and antimicrobial activity. Antiproliferative effects of the time and dose of S. squamosus extract on human hepatocellular carcinoma HepG2 cells were determined with the XTT assay. The anti-invasive and apoptotic potential of the extract was evaluated with the Matrigel invasion chamber and TUNEL assays, respectively. S. squamosus extract also showed antiproliferative and anti-invasive activity and induced apoptosis in HepG2 hepato-cellular carcinoma cells in vitro. In conclusion, S. squamosus can be considered as both a functional food and a source of nutraceuticals.

Abstract 5997: Increased connexin 43 expression and gap junction communication correlates with invasion following reduced glucose metabolism in breast cancer cells

Abstract Dysregulation of gap junction intercellular communication (GJIC) is a common feature during cancer progression. GJIC is a means of direct cell-cell communication mediated by regulated membrane channels composed of connexin proteins. This communication is frequently lost between primary tumor cells but may be upregulated at secondary metastatic sites with stromal cells. Control of this process by cancer cells has been shown to facilitate aggressive qualities both in vitro and in vivo. During the process of metastasis, cells encounter numerous metabolic challenges that must be overcome, particularly during growth of primary tumors. In this study, we set out to evaluate if changes to cancer cell metabolism affect GJIC in breast cancer cells. To address this question, we generated a metabolic variant of the MDA-MB-231 cell line conditioned to grow in glucose-limiting conditions. These cells were grown in FBS supplemented RPMI with &amp;lt;0.130mM glucose compared to 2mM in control conditions for more than 4 weeks. Substantial cell death over this time revealed a small population of cells capable of surviving in glucose reduced conditions. Growth of these cells normalized following a period of quiescence and exhibited stable viability and proliferative capacity. Following STR validation, these cells were designated MDA-MB-231LG for their ability to grow in low glucose media. MDA-MB-231LG exhibited a larger and more rounded morphologic appearance with formation of strong cell-cell contacts as demonstrated by scanning electron microscopy in contrast to parental cells which showed a higher degree of membrane overlap. Further comparison of the two cell lines through western blot and immunofluorescence analysis of connexin 43, a major connexin expressed in breast tissue, revealed higher levels in MDA-MB-231LG and increased membrane localization. Using a double label dye transfer technique, the gap junction permeable fluorescent dye calcein showed increased movement from CM-DiI labeled donor cells into neighboring cells in MDA-MB-231LG indicating functional gap junction coupling while MDA-MB-231 had little to no dye movement. To evaluate phenotypic qualities, both cell lines were grown in Matrigel and MDA-MB-231LG displayed increased stellate morphology. Use of a Matrigel invasion chamber assay confirmed the increased invasive qualities with significantly more MDA-MB-231LG invading through the lower portion of the membrane compared to MDA-MB-231 parental cells. Our data demonstrate a clear upregulation of gap junction activity following metabolic adaptation to reduced glucose availability. It also suggests a possible connection between GJIC and invasive qualities in breast cancer cells and may represent an inducible phenotype that occurs in primary tumors when tumor growth limits blood vessel penetration and nutrient availability. Citation Format: Jennifer C. Jones, Amanda M. Miceli, Mary M. Chaudhry, Mallika A. Jai, Romel N. Pancho, Alan Lazzar, Bradley S. Taylor, Vishnupriya Bodempudi, Prarthana P. Jain, Sheeri Hanjra, Alexander E. Urban, Brian Zanotti, Ellen K. Kohlmeir, Thomas M. Bodenstine. Increased connexin 43 expression and gap junction communication correlates with invasion following reduced glucose metabolism in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5997.

Abstract P3-10-01: Development and characterization of a first-in-class small molecule inhibitor of PELP1

Abstract Background: Breast cancer (BCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in women. BCa is composed of distinct molecular subtypes, such as ER positive BCa (ER+ BCa) and triple negative BCa (TNBC). Development of novel effective therapies for patients with therapy resistant breast cancer (TR-BC) and TNBC remains the highest unmet need in patient treatment and survivorship. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) plays a critical role in multiple nuclear receptor functions leading to TR-BC and TNBC progression. PELP1 expression is dysregulated in BCa, is a prognostic indicator of poorer BCa survival, and its deregulation contributes to BCa therapy resistance. The objective of this study is development and characterization of a small molecule inhibitor of PELP1 (SMIP) as novel therapeutic for treating BCa. Methods: Using yeast two-hybrid screening, we identified PELP1 Inhibitory Peptide (PIP1) from a library of peptides. PIP1 binds PELP1 with high affinity and functions to inhibit PELP1 oncogenic activity. Direct binding of PIP1 to PELP1 was confirmed using biotin pull-down assays and inhibition of BCa proliferation confirmed using MTT assays. We used the Hit-Ligand interaction site with PIP1 hot spot residues based on 3D alignment and morphology to generate a library of peptidomimetics (small chemical molecules). In vitro activity was assessed using Celltiter Glo, MTT, and matrigel invasion chamber assays in multiple BCa models. Mechanistic studies were conducted using Western blot, reporter gene assays, and peptide competition assays. Xenograft and patient derived explant (PDEX) assays were used for preclinical evaluation and preliminary toxicity analysis. Results: Bioactivity screens revealed PELP1 Inhibitory Peptide (PIP1) significantly attenuates PELP1-mediated proliferation with an IC50 of 10µM across multiple BCa cell lines. We confirmed PIP1 binding to PELP1 using peptide pull-down assays with nuclear lysates from BCa cells. Using Hit-Ligand-Based interaction site with the PIP1 hot spot residues, we identified 61 potential hits using a 10,000 Diverse Set. Screening of these 61 potential hits using the MTT assays lead to the selection of SMIP34 (tetrahydropyrazolo [1,5a) pyrazole) as lead inhibitor of PELP1. SMIP34 treatment reduced proliferation at an IC50 of 3-10µM in ER+ BCa (ZR-75, MCF-7, and T- 47D); TR-BC (MCF-7-TamR, MCF-7-LTLT, ZR-75-MT-ER537S, and ZR-75-MT-ER538G); and TNBC (MDA- MB-231, and BT549) models. Additionally, SMIP34 showed no activity in human mammary epithelial cells. Specificity of SMIP34 was confirmed using PELP1 knockdown BCa cell lines. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. In matrigel invasion chamber assays, SMIP34 significantly reduced the invasiveness of TR-BC and TNBC models. In combination studies, SMIP34 displayed synergy and enhanced the efficacy of current chemotherapeutics Cisplatin and Paclitaxel. In PDEX assays, Ki67 staining revealed SMIP34 significantly decreased tumor proliferation. In xenograft models, SMIP34 (10mg/kg/s.c.) treatment resulted in significant reduction in tumors compared to vehicle treatment. Furthermore, overall mouse body weight in both control and SMIP34 treated groups were similar, suggesting no overt signs of toxicity. Conclusion: We have developed a first-in-class small molecule inhibitor of PELP1 (SMIP) displaying effectivity against TR-BC and TNBC in vitro and in vivo. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) Citation Format: Kristin A Altwegg, Suryavathi Viswanadhapalli, Monica Mann, Uday P Pratap, Mengxing Li, Junhao Liu, Yiliao Luo, Gangadhara R Sareddy, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Development and characterization of a first-in-class small molecule inhibitor of PELP1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-01.

SNP rs710886 A>G in long noncoding RNA PCAT1 is associated with the risk of endometriosis by modulating expression of multiple stemness-related genes via microRNA-145 signaling pathway.

MiR-145 has been shown to suppress cell invasiveness and proliferation in endometriosis, whereas prostate cancer-associated transcript 1 (PCAT1) was reported to act as a sponge of miR-145 with one single-nucleotide polymorphism (SNP), rs710886, located in the chromosomal segment of PCAT1. Therefore, this study aimed to explore the association between rs710886 SNP and the risk of endometriosis, as well as the effect of this SNP on the activation of the signaling pathway downstream of PCAT1. Real-time polymerase chain reaction (PCR) was performed to observe the expression of miR-145 in transfected cells, while Matrigel invasion chamber assays and MTT assay were conducted to examine the invasiveness/proliferation among different cell groups. Moreover, bioinformatics tools, luciferase assays, real-time PCR, and Western blot analysis were used to measure the expression of these target genes in the presence of miR-145. Finally, a statistical analysis was conducted to compare the genotypes of rs710886 SNP between fertile healthy women and infertile women with endometriosis. PCAT1 small interfering RNA (siRNA) evidently increased the expression of miR-145 but reduced the invasiveness/proliferation of cells. P-PCAT1 exhibited an opposite effect as that of PCAT1 siRNA, indicating PCAT1 could promote the proliferation and invasiveness of endometriosis stem cells via inhibiting the expression of miR-145. Meanwhile, FASCIN1, SOX2, MSI2, SERPINE1, and JAM-A were identified as target genes of miR-145 via computational analysis and luciferase assays. Finally, a significant genetic effect was observed in both the dominant (AG+GG vs AA) and recessive models (GG vs AG+AA), indicating the presence of an association between the genotype of SNP rs710886 and the risk of endometriosis. SNP rs710886 A>G could lower the expression of PCAT1, thus leading to the overexpression of miR-145. Highly expressed miR-145 would inhibit the invasiveness and proliferation of endometriosis stem cells via targeting specific genes, thus decreasing the risk of endometriosis.

Centipede Scolopendra suppresses cell growth in human epidermoid carcinoma cell A431

The aim of this study was to examine the anti-proliferation and anti-migration effect of Centipede Scolopendra extracts (CSE) on human epidermoid carcinoma cells (high-EGFR expression) A431 and elucidate the underlying signaling mechanisms. MTT and colony formation assays were used. Migration and invasion potential of A431 cells were examined by wound-healing assays and matrigel invasion chamber assays. To investigate the underlying molecular mechanisms, we used ELISA to analyze the expression of EGF, Western blotting to analyze the expression of MMP2 (matrix metalloproteinase 2), MMP9 and EGFR, PCR to analyze the mRNA expression of EGFR pretreated with CSE. The results showed that CSE effectively inhibited the proliferation of A431. Furthermore, CSE-mediated cell cycle arrest in S phase. We also observed that CSE treatment led to down-regulation of MMP2 and MMP9 and suppress the migration and invasion in A431. CSE exerted its anti-proliferation and anti-migration by targeting EGFR and related metastasis factors, thus could be a useful therapeutic candidate for high-EGFR expression cancer intervention.&#x0D; Video Clip of Methodology:&#x0D; Cell migration and invasion assay: 2 min 41 sec Full Screen Alternate

Upregulation of miR-101 enhances the cytotoxic effect of anticancer drugs through inhibition of colon cancer cell proliferation.

This study investigated the effect of miR-101 on proliferation, migration, invasion, and chemotherapy sensitivity in colon cancer cell lines HT-29 and RKO. MicroRNAs are a class of small noncoding RNA molecules, which play important roles in diverse biological processes of human cancers, such as carcinogenesis, development, differentiation, and apoptosis. The expression of miR-101 in colon cancer and adjacent non-tumor tissues were examined by quantitative real-time polymerase chain reaction. The expression of miR-101 was upregulated by recombinant adenovirus Ad-miR-101. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cloning methods. Cell migration and invasion potential were examined using Transwell migration and Matrigel invasion chamber assays. Drug sensitivity to 5-fluorouracil (5-FU) and cisplatin (DDP) was explored using MTT assays and l acridine orange/ethidium bromide double staining. The expression of miR-101 decreased in colon cancer tissues compared with adjacent non-tumor tissues. The upregulated expression of miR-101 suppressed cell proliferation and inhibited cell migration and invasion in HT-29 and RKO colon cancer cell lines. The overexpression of miR-101 promoted the inhibitory effect of 5-FU and DDP on HT-29 cells. The expression of miR-101 was downregulated in colon cancer. The upregulated expression of miR-101 inhibited proliferation and migration, and increased the sensitivity of colon cancer cells to chemotherapy.

Investigation of the effects of a sulfite molecule on human neuroblastoma cells via a novel oncogene URG4/URGCP.

The aim of this study is to determine the anticancer effect of sulfite on SH-SY5Y neuroblastoma cells in vitro conditions and elucidate underlying molecular mechanism of sulfite and explore its therapeutic activity. In this study, cytotoxic effects of sulfite in SH-SY5Y cels were detected over time in a dose dependent manner with the IC50 doses ranging from 0.5 to 10 mM. Genotoxic effect of sulfite was shown by comet assay. IC50 doses in the SH-SY5Y cells were detected as 5 mM. Expression profiles of the target genes related to apoptosis and cell cycle control were determined by quantitative RT-PCR. Protein changes were determined by western blot analysis. URG4/URGCP, CCND1, CCND2, CDK4, CDK6, E2F4 and BCL-2 gene expression levels were significantly reduced and RB1, TP53, BAX, BID, CASP2, CASP3, CASP9 and DIABLO gene expressions were significantly increased in dose group cells. The mechanism of this result may be related to sulfite dependent inhibition of cell cycle at the G1 phase by down-regulating URG4/URGCP or CCND1, CDK4, CDK6 gene expression and stimulating apoptosis via the intrinsic pathway. Sulfite suppressed invasion and colony formation in SH-SY5Y cell line using matrigel invasion chamber and colony formation assay, respectively. It is thought that sulfite demonstrates anticarcinogenesis activity by affecting cell cycle arrest, apoptosis s, invasion, and colony formation on SH-SY5Y cells. Sulfite may be an effective agent for treatment of neuroblastoma as a single agent or in combination with other agents.

Aspirin and P2Y12 inhibition attenuate platelet-induced ovarian cancer cell invasion

BackgroundPlatelet-cancer cell interactions play a key role in successful haematogenous metastasis. Disseminated malignancy is the leading cause of death among ovarian cancer patients. It is unknown why different ovarian cancers have different metastatic phenotypes. To investigate if platelet-cancer cell interactions play a role, we characterized the response of ovarian cancer cell lines to platelets both functionally and at a molecular level.MethodsCell lines 59 M and SK-OV-3 were used as in vitro model systems of metastatic ovarian cancer. Platelet cloaking of each cell line was quantified by flow cytometry. Matrigel invasion chamber assays were used to assess the invasive capacity of the cell lines. The induction of an EMT was assessed by morphology analysis and by gene expression analysis of a panel of 11 EMT markers using TaqMan RT-PCR.ResultsSK-OV-3 cells adhered to and activated more platelets than 59 M cells (p = 0.0333). Platelets significantly promoted the ability of only SK-OV-3 cells to invade (p ≤ 0.0001). Morphology and transcritpome analysis indicated that platelets induce an epithelial-to-mesenchymal transition phenotype in both cells lines, with a more exaggerated response in SK-OV-3 cells. Next, we investigated if antiplatelet agents could abrogate the platelet-induced aggressive phenotype in SK-OV-3 cells. Both aspirin (p ≤ 0.05) and 2-methylthioadenosine 5′-monophosphate triethylammonium salt hydrate (P2Y12 inhibitor; p ≤ 0.01) significantly decreased their invasion capacity, and effectively reverted invasion to levels comparable to SK-OV-3 cells alone.ConclusionWhile there is increasing evidence for the cancer-protective effect of aspirin, this study suggests P2Y12 inhibition may also play a role. Understanding these complex interactions between platelets and cancer cells could ultimately allow the establishment of therapies tailored to inhibiting metastasis, thus significantly reducing cancer morbidity.

Anti-proliferative and anti-invasive effects of ferulic acid in TT medullary thyroid cancer cells interacting with URG4/URGCP.

Ferulic acid (4-hydroxy-3-methoxycinnamic acid; FA), a common dietary plant phenolic compound, is abundant in fruits and vegetables. The aim of present study is to investigate the effects of FA on cell cycle, apoptosis, invasion, migration, and colony formation in the TT medullary thyroid cancer cell line. The effect of FA on cell viability was determined by using CellTiter-Glo assay. IC50 dose in the TT cells was detected as 150μM. URG4/URGCP (upregulated gene-4/upregulator of cell proliferation) is a novel gene in full-length mRNA of 3.607kb located on 7p13. It was determined that FA caused a decrease in the expression of novel gene URG4/URGCP, CCND1, CDK4, CDK6, BCL2, MMP2, and MMP9, a significant increase in the expression of p53, PARP, PUMA, NOXA, BAX, BID, CASP3, CASP9, and TIMP1 genes in TT human thyroid cancer cell line by using real-time PCR. It was found that FA in TT cells suppressed invasion, migration, and colony formation by using matrigel invasion chamber, wound healing, and colony formation assay, respectively. In conclusion, it is thought that FA indicates anticarcinogenesis activity by affecting cell cycle arrest, apoptosis, invasion, migration, and colony formation on TT cells.

Eupolyphaga sinensis Walker Ethanol Extract Suppresses Cell Growth and Invasion in Human Breast Cancer Cells.

To examine the antiproliferation and anti-invasion of Eupolyphaga sinensis Walker 70% ethanol extract (ESWE) on breast cancer and elucidate the underlying signaling mechanisms. MTT and colony formation assays were used to investigate the effect of ESWE on proliferation of breast cancer cells in vitro. The xenograft mouse tumor model was used to determine the effect of ESWE on breast cancer in vivo. To investigate the underlying molecular mechanisms, we used western blotting to analyze the expression of ERK1/2, CXCR4, matrix metalloproteinase 2 (MMP2), and MMP9 pretreated with ESWE. The stromal cell-derived factor (SDF)-1α-induced migration and invasion potential of breast cancer cells were examined by wound-healing assays and Matrigel invasion chamber assays. ESWE effectively inhibited the proliferation of MDA-MB-435s and MDA-MB-231 cells and exhibited antitumor effects in an MDA-MB-231 xenograft mice model. Furthermore, ESWE suppressed the activity of ERK1/2, a key molecule of MAPK signaling. We also observed that ESWE treatment led to downregulation of CXCR4 expression as well as greatly reduced MMP2 and MMP9. ESWE affected CXCR4 expression partially through the modulation of autocrine vascular endothelial growth factor. However, suppression of CXCR4 expression was the result of downregulation of mRNA expression. Inhibition of CXCR4 expression by ESWE further correlated with the suppression of SDF-1α-induced migration and invasion in breast cancer cells. ESWE exerted its antiproliferation and antiinvasion by regulating MAPK signaling and related metastasis factorsand thus could be a useful therapeutic candidate for breast cancer intervention.

Abstract 2647: Anti-tumor activity of sHA8k, a HYAL1 hyaluronidase inhibitor, in bladder cancer cells

Abstract INTRODUCTION AND OBJECTIVE: Tumor cell-derived hyaluronidase HYAL1, which degrades hyaluronic acid (HA) into angiogenic fragments, promotes tumor growth and metastasis. HYAL-1 is an independent prognostic marker for predicting metastasis. Small molecular mass sulfated hyaluronic acid (sHA) derivatives (sHA8k) inhibit HYAL1 activity through a mixed inhibition mechanism. Antitumor activity of sHA8k and mechanism of action was evaluated in BCa models.. METHODS: Effect of sHA8k (0-40 ug/ml) on cell proliferation and apoptosis was examined in BCa cells (253J-Lung, HT1376, UMUC-3, T24, RT4) by cell counting and Cell Death ELISA kit. Matrigel invasion and Boyden chamber assays were used to test the effect of sHA8k on invasive activity. Effect of sHA on signaling, apoptosis cascade, HA receptor (CD44, RHAMM), EMT markers (β-catenin, E-cadherin, Snail, Twist) levels, was evaluated by Q-pCR and immunoblotting assays. Angiogenic fragment addition and mAkt transfection were performed to elucidate mechanism of action. Athymic mice bearing 253J-Lung xenografts were treated with sHA (25 and 50 mg/kg) by i.p. injection. RESULTS: sHA8k inhibited proliferation, motility and invasion in BCa cells that expressed HYAL-1. At IC50 for HAase activity inhibition (∼ 20-μg/ml), sHA induced &amp;gt; 3-fold apoptosis and inhibited invasive activity of BCa cells. sHA8k induced caspase-3, -8, -9 activation, up-regulation of Fas, Fas-L, FADD, DR4, DR5 and E-cadherin. sHA8k downregulated CD44, RHAMM, bcl-2, phospho(p)-Akt, pGSK3β, pβcatenin(ser552), snail and Twist expression. Effect of sHA8k were attenuated by angiogenic HA fragments or overexpression of m-Akt and downregulation of CD44 and RHAMM mimicked sHA8k effects. sHA significantly inhibited 253J-Lung xenograft growth. The majority of the animals did not form palpable tumors at 50-mg/kg dose. No weight loss or serum and organ toxicity was observed in sHA treated animals. Biochemical analysis of tumors also showed the same alterations in Akt and EMT pathways as observed cell culture. CONCLUSION: This is the first study that shows sHA8k, a small molecular mass HYAL-1 inhibitor has potent antitumor activity. Support: R01 CA 72821-14 (VBL) Citation Format: Andre R. Jordan, Juan Chipollini, Luis Lopez, Travis Yates, Vinata Lokeshwar. Anti-tumor activity of sHA8k, a HYAL1 hyaluronidase inhibitor, in bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2647. doi:10.1158/1538-7445.AM2015-2647

Abstract P5-07-07: Combination of IGF1R/FAK inhibition and PI3K/mTOR inhibition in triple-negative breast cancers

Abstract Introduction: Triple-negative breast cancers (TNBCs) account for 15%-20% of all breast cancers with limited treatment options and poor prognosis. The poor outcomes seen with TNBCs are in part due to a lack of viable therapeutic targets. Overexpression of insulin-like growth factor 1 receptors (IGF1R) and focal adhesion kinase (FAK) are closely associated with invasive breast carcinomas. However, in our previous study, we found that the combined use of IGF1R inhibitor and FAK inhibitor had produced limited effects on TNBC cells inhibition. NVP-BEZ235 is a potent PI3K/mTOR dual inhibitor and has been shown to be effective in TNBC cell lines, especially for the mesenchymal-like and luminal-androgen receptor subtypes. Unlike rapamycin, which produces a feedback activation of Akt, NVP-BEZ235 alone successfully blocks the Akt activation and effectively inhibits the cells proliferation. Our hypothesis is that the combined inhibition of IGF1R/FAK and PI3K/mTOR produces greater suppression on TNBC cell growth. Methods: We examined the effects of NVP-TAE226, the dual inhibitor of IGF1R and FAK, in combination with NVP-BEZ235 on human MDA-MB-231 and BT549 TNBC cell lines. SRB cell survival assays were performed following NVP-TAE226 or NVP-BEZ235 treatments alone and NVP-TAE226 in combination with NVP-BEZ235. Western blotting was used to detect expression and phosphorylation of down-stream signaling proteins and epithelial to mesenchymal transition (EMT)-related markers. Matrigel invasion chamber assay was performed to evaluate the TNBC cells invasion patterns under treatments of either NVP-TAE226 or NVP-BEZ235 alone or the two drugs in combination. Spheroid migration assay will also be used to assess combination effect on the metastatic nature of TNBC cells. Results: The combined IGF1R/FAK inhibition with NVP-TAE226 and PI3K/mTOR inhibition with NVP-BEZ235 resulted in significantly greater cytotoxicity than either single agent alone in MDA-MB231 and BT549 cell lines (P&amp;lt;0.05). The combination of IGF1R/FAK and PI3K/mTOR inhibition suppressed the PI3K/Akt and MEK/ERK signaling cascades, reduced FAK and ZEB1 activity and significantly decreased the cell invasion for TNBC cell lines (p&amp;lt;0.05). Our data indicated that the combination treatment targeting both PI3K/Akt pathway and EMT related protein molecules (IGF1R/FAK) lead to greater cytotoxic effect and suppression of EMT and invasion. Conclusion: These results suggest that the combined inhibition of IGF1R/FAK and PI3K/mTOR may be an effective strategy for TNBC and warrant further investigation in in vivo animal studies. Citation Format: LaTonia D Taliaferro-Smith, Tongrui Liu, Tiffanie Y Alcaide, Tanisha Z McGlothen, Ruth M O'Regan. Combination of IGF1R/FAK inhibition and PI3K/mTOR inhibition in triple-negative breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-07.

Temozolomide may induce cell cycle arrest by interacting with URG4/URGCP in SH-SY5Y neuroblastoma cells.

Temozolomide (TMZ) is an alkylating drug used usually in glioma treatment by inducing the apoptosis in glioma cell. The aim of the study is to investigate the anticancer mechanism of TMZ in SH-SY5Y human neuroblastoma cell line. Cytotoxic effects of TMZ were determined by using XTT assay. IC50 doses in the SH-SY5Y were detected as 5 mM. Expression profiles of novel genes URG4/URGCP, CCND1, CCND2, CDK4, and BCL2 were determined by real-time PCR. The apoptotic effects of TMZ were evaluated with TUNEL method. Furthermore, effects of TMZ on colony formation and invasion were investigated in this study. It was observed that TMZ in SH-SY5Y cell line caused a significant decrease in the gene expressions of URG4/URGCP, CCND1, CCND2, CDK4, and BCL2. According to TUNEL assay results, TMZ markedly induced apoptosis in SH-SY5Y cell line. It was found that TMZ in SH-SY5Y cell line suppressed invasion and colony formation using matrigel invasion chamber and colony formation assay, respectively. To conclude, it is thought that TMZ demonstrates anticarcinogenesis activity by affecting cell cycle arrest, apoptosis, invasion, and colony formation on SH-SY5Y cells. TMZ may be an effective agent for treatment of neuroblastoma as a single or in combination with other drugs.

Inhibition of NADPH oxidase protects against metastasis of human lung cancer by decreasing microRNA-21.

The objective of this study was to detect the effect of NADPH oxidase (NOX) inhibition on metastasis of lung cancer. Primary human lung cancer cells were isolated from surgical tissues using the Cancer Cell Isolation Kit. Invasion was detected using the BD Biocoat Matrigel Invasion Chamber assay. Expressions of microRNA-21 (miR-21), PTEN, MMP9, and p47 were detected by qPCR. Groups of nude mice were challenged with A549 cells with or without DPI and detected for tumor metastasis and survival. NOX inhibition in human lung cancer cells significantly reduced their invasive potential in vitro. NOX inhibition in vivo led to decreased metastasis of human lung cancer and prolonged the survival time of tumor-bearing nude mice. Further, NOX inhibition resulted in decreased expression of miR-21 in human lung cancer cells. Increased expression of miR-21 abrogated the effect of NOX inhibitor on metastasis of human lung cancer in vitro and in vivo. Decreased expression of miR-21 facilitated the effect of NOX inhibitor on metastasis of human lung cancer in vitro and in vivo. Furthermore, increased expression of PTEN and decreased expression of MMP9 were observed in human lung cancer cells in response to NOX inhibition. Finally, close correlations of miR-21 expression levels with NADPH oxidase expression level and differentiation state of tumor cells were observed in lung cancer patients. Inhibition of NADPH oxidase protected against metastasis of human lung cancer cells by decreasing miR-21 expression, which could facilitate the understanding of lung cancer pathogenesis and provided clues for the development of novel therapeutics for lung cancer patients.

Steroidal saponins from Paris polyphylla suppress adhesion, migration and invasion of human lung cancer A549 cells via down-regulating MMP-2 and MMP-9.

Tumor metastases are the main reasons for oncotherapy failure. Paris polyphylla (Chinese name: Chonglou) has traditionally been used for its anti-cancer actions. In this article, we focus on the regulation of human lung cancer A549 cell metastases and invasion by Paris polyphylla steroidal saponins (PPSS). Cell viability was evaluated in A549 cells by MTT assay. Effects of PPSS on invasion and migration were investigated by wound-healing and matrigel invasion chamber assays. Adhesion to type IV collagen and laminin was evaluated by MTT assay. Expression and protease activity of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were analyzed by Western blotting and gelatin zymography, respectively. PPSS exerted growth inhibitory effects on A549 cells, and effectively inhibited A549 cell adhesion, migration and invasion in a concentration-dependent manner. Western blotting and gelatin zymography analysis revealed that PPSS inhibited the expression and secretion of MMP-2 and MMP-9 in A549 cells. PPSS has the potential to suppress the migration, adhesion and invasion of A549 cells. PPSS could be a potential candidate for interventions against lung cancer metastases.