Cell Migration In Wound-healing Assay Research Articles

Uncovering a novel mechanism: Butyrate induces estrogen receptor alpha activation independent of estrogen stimulation in MCF-7 breast cancer cells.

Butyrate is a promising candidate for an antitumoral drug, as it promotes cancer cell apoptosis and reduces hormone receptor activity, while promoting differentiation and proliferation in normal cells. However, the effects of low-dose butyrate on breast cancer cell cultures are unclear. We explored the impact of sub-therapeutic doses of butyrate on estrogen receptor alpha (ERα) transcriptional activity in MCF-7 cells, using RT-qPCR, Western blot, wound-healing assays, and chromatin immunoprecipitation. Our results showed that sub-therapeutic doses of sodium butyrate (0.1 - 0.2 mM) increased the transcription of ESR1, TFF1, and CSTD genes, but did not affect ERα protein levels. Moreover, we observed an increase in cell migration in wound-healing assays. ChIP assays revealed that treatment with 0.1 mM of sodium butyrate resulted in estrogen-independent recruitment of ERα at the pS2 promoter and loss of NCoR. Appropriate therapeutic dosage of butyrate is essential to avoid potential adverse effects on patients' health, especially in the case of estrogen receptor-positive breast tumors. Sub-therapeutic doses of butyrate may induce undesirable cell processes, such as migration due to low-dose butyrate-mediated ERα activation. These findings shed light on the complex effects of butyrate in breast cancer and provide insights for research in the development of antitumoral drugs.

CK2 inhibitor CX4945 inhibits collagen degradation of HaCaT human keratinocyte cells via attenuation of MMP-1 secretion.

During skin aging, the extracellular matrix (ECM) concomitantly breaks down. Out of the various protein components that comprise ECM, collagen is the most abundant one. Matrix metalloproteinase-1 (MMP-1) is a major collagenase that can degrade collagen. Therefore, the inhibition of MMP-1 may be critical for skin aging prevention. CX4945 is an inhibitor of casein kinase 2 and shows anticancer effects on various types of cancer cells. In this report, we investigated the MMP-1-inhibiting effect of CX4945 in HaCaT human keratinocyte cells. We performed zymography assays, Western blot analysis and immunoprecipitation assay to investigate the anti-MMP-1 effects of CX4945. CX4945 was found to inhibit collagen degradation via attenuation of the MMP-1 secretion out of HaCaT cells. This activity of CX4945 may be mediated by the induction of MMP-1 ubiquitylation via c-Jun N-terminal kinase (JNK) signaling. In wound healing cell migration assay, CX4945 also showed suppressive effect on the migration of HaCaT cells. This finding was closely related to the attenuation of CREB transcription factor via the downregulation of ERK mitogen-activated protein kinase as observed in Western blot analysis. Our report suggests that the inhibitory effects of CX4945 on MMP-1 in epidermal cells may offer a basis for further studying its therapeutic potential as an anti-wrinkle agent.

In-vitro anticancer and anti-migration effect of Ficus racemosa leaves extract and its neoadjuvant combination with chemo-photodynamic therapy

Phytocompounds play a key role in prevention and treatment of cancers. The secondary metabolites are being studied due to their anticancer and antioxidant properties leading to the finding of new therapeutic agents. Like many medicinal plants, Ficus racemosa (FR) is rich in polyphenols and can be used in cancer treatment. The side effects of existing therapeutic modalities at high doses may be reduced in combination. In this study the anticancer and anti-migration potentials of FR were evaluated, along with its combinational study at low therapeutic dosage with doxorubicin-HCl (Dox-HCl), dacarbazine (DTIC) and photodynamic therapy (PDT). The effects of FR leaves extract on the rhabdomyosarcoma (RD) cells morphology, cell viability, and cell migration ability were investigated by light microscopy, MTT assay, and wound healing cell migration assay respectively. The effect of various FR concentrations on RD cells indicated the potential anticancer effect in a dose dependent manner. The results of low dose di-combinations (FR-chemo, FR-PDT) and tri-combinations (FR-chemo-PDT) were synergistic. The % cell viability was reduced to 29% for FR-Dox-HCl-PDT and 44% for FR-DTIC-PDT combinations. A pronounced cytotoxic effect and cell migration was observed in tri-combination therapy as compared to di- and mono-therapy. This study concluded that FR leaves extract has anticancer effects and gives synergy with chemo and PDT combinations.

Proper Orthogonal Decomposition Analysis Reveals Cell Migration Directionality During Wound Healing.

A proper orthogonal decomposition (POD) order reduction method was implemented to reduce the full high dimensional dynamical system associated with a wound healing cell migration assay to a low-dimensional approximation that identified the prevailing cell trajectories. The POD analysis generated POD modes that were representative of the prevalent cell trajectories. The shapes of the POD modes depended on the location of the cells with respect to the wound and exposure to disturbed (DF) or undisturbed (UF) fluid flow where the net flow was in the antegrade direction with a retrograde component or fully antegrade, respectively. For DF and UF, the POD modes of the downstream cells identified trajectories that moved upstream against the flow, while upstream POD modes exhibited sideways cell migrations. In the absence of flow, no major shape differences were observed in the POD modes on either side of the wound. The POD modes also served to reconstruct the cell migration of individual cells. With as few as three modes, the predominant cell migration trajectories were reconstructed, while the level of accuracy increased with the inclusion of more POD modes. The POD order reduction method successfully identified the predominant cell migratory trajectories under static and varying pulsatile fluid flow conditions serving as a first step in the development of artificial intelligence models of cell migration in disease and development.

CircMMD_007 promotes oncogenic effects in the progression of lung adenocarcinoma through microRNA-197-3p/protein tyrosine phosphatase non-receptor type 9 axis

ABSTRACT Circular RNAs play important roles in cancer biology. In this research, we explored the underlying function and mechanism of cirMMD_007 in lung adenocarcinoma (LC). Clinical lung adenocarcinoma samples were obtained from surgery. Bioinformatic databases were used to predict miRNAs that can potentially target circRNAs and miRNA target genes. hsa_circMMD_007, miR-197-3p, and PTPN9 mRNA expressions were investigated by qRT-PCR. Protein expressions were examined using Western blot. The proliferation abilities were assessed by Cell Counting Kit-8 assays. Wound healing cell migration assay was applied to evaluate cell migration ability. Luciferase reporter assay and rescue experiments were then performed to elucidate the underlying mechanism. We found that the expression of circMMD_007 was abnormally increased in LC. The expression of circMMD_007 was higher in advanced stages. Knockout of circMMD_007 hindered the tumorigenesis of LC in vivo and in vitro. circMMD_007 could negatively regulate the expression of miR-197-3p. PTPN9 behaved to be a molecular target of miR-197-3p. In summary, this research demonstrated that circular RNA circMMD_007 could promote the oncogenic effects in the progression of LC through miR-197-3p/PTPN9 axis.

LncRNA PCAT29 suppresses cell proliferation, invasion, and migration in renal carcinoma by regulating FLOT1

Background LncRNA PCAT29 has been reported to play a role in the development of cancer; the role of FLOT1 in renal carcinoma has been also identified. This study aimed at exploring the interaction between them and their influence on the progression of renal carcinoma. M ethods The expression levels of lncRNA PCAT29 and the FLOT1 protein in tissues were determined separately using qRT-PCR and western blot, respectively. The endogenous expression of genes was modulated by recombinant plasmid and cell transfection. The cell viability, invasion, and migration were detected by MTT assay, transwell assay, and wound-healing cell migration assay, respectively. Any binding and interactions between the RNA and the proteins were determined with RNA immunoprecipitation and RNA pull-down assays. A nude mouse model for renal carcinoma was established for the i n vivo expression of PCAT29. R esults In renal carcinoma tissues, the expression of lncRNA PCAT29 was down-regulated while that of FLOT1 was up-regulated. PCAT29 negatively regulated FLOT1, and the overexpression of PCAT29 inhibited the cell proliferation viability, invasion, and migration of renal carcinoma by down-regulating FLOT1. The in vivo expression of PCAT29 inhibited tumor growth in a mouse model of renal carcinoma. C onclusion LncRNA PCAT29 inhibited the cell proliferation viability, invasion, and migration in renal carcinoma by down-regulating FLOT1, thereby suppressing tumor growth. Keywords : LncRNA PCAT29; renal carcinoma; cell proliferation; cell invasion and migration; FLOT1

Synergistic antitumor effect of suberoylanilide hydroxamic acid and cisplatin in osteosarcoma cells.

Cisplatin, as a first-line chemotherapy drug, has been widely applied for therapy of osteosarcoma. However, its application is limited by drug resistance and serious side effects, including nephrotoxicity and ototoxicity. Suberoylanilide hydroxamic acid (SAHA) is a newly developed histone deacetylase (HDAC) inhibitor, which is the first Food and Drug Administration-approved HDAC inhibitor for the treatment of cutaneous manifestations of T-cell lymphoma. However, SAHA as a monotherapy was revealed to be limited, particularly in solid tumors. In the present study, 143B osteosarcoma cells were treated with multiple concentrations of SAHA or cisplatin, either alone or combined. The morphological characteristics of the treated cells were observed using an inverted microscope. The cytotoxicity effects of the combination of SAHA and cisplatin on 143B cells were analyzed by MTT assay, colony formation assay, wound healing cell migration assay, cell apoptosis assay and cell cycle analysis. Western blot analysis was performed to detect the protein expression levels of B cell lymphoma-2 (Bcl-2)-associated X protein (Bax), Bcl-2, cleaved-caspase-3, cleaved-caspase-8 and cleaved-poly (ADP-ribose) polymerase (PARP). The experimental data indicated that the inhibition of cell proliferation in the combination group was significantly increased compared with that in single drug groups. Expression levels of pro-apoptotic protein were upregulated, whereas anti-apoptotic Bcl-2 was downregulated significantly in 143B cells following SAHA/cisplatin treatment. Taken together, the results revealed that the combination of SAHA and cisplatin inhibited the proliferation of 143B cells and induced their apoptosis synergistically, and this effectiveness may be mediated by caspase activation.

Down-regulated paxillin suppresses cell proliferation and invasion by inhibiting M2 macrophage polarization in colon cancer.

The paxillin and M2 macrophage are all involved in cell proliferation and tumor progression, and this study aims to explore the interaction between them in colon cancer and the role of paxillin in cancer progression. Expression of mRNAs and proteins was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot, separately. Endogenous expression of genes was modulated by recombinant plasmids and cell transfection. The levels of cytokines were determined by enzyme-linked immunosorbent assay (ELISA). The cell viability, invasion and migration were detected using the MTT assay, the transwell assay and the wound-healing cell migration assay, respectively. A nude mouse model for human colon cancer was constructed for tumor orthotopic expression. Paxillin was up-regulated in tumor-associated macrophages (TAMs). Paxillin was up-regulated in process of M2 macrophage polarization. M2 macrophage polarization was inhibited with paxillin suppressed. Down-regulated paxillin inhibited cell proliferation and invasion in colon cancer through suppressing M2 macrophage polarization. PI3k/Akt inhibitor repressed M2 macrophage polarization through down-regulating paxillin. PI3k/Akt inhibitor inhibited the function of the macrophage in promoting cell proliferation and invasion of colon cancer through down-regulating paxillin. Down-regulated paxillin in macrophages inhibited tumor growth of colon cancer. With the PI3K/AKT pathway inhibited, down-regulated paxillin suppressed colon cancer cell proliferation and invasion by inhibiting the M2 macrophage polarization, thereby restraining the tumor progression.

Lipopolysaccharides induce Smad2 phosphorylation through PI3K/Akt and MAPK cascades in HSC-T6 hepatic stellate cells

AimsEndotoxemia and its pro-fibrogenic signaling play a significant role in the development of hepatic fibrosis. This study investigated whether lipopolysaccharide (LPS) directly activate cultured HSC-T6 hepatic stellate cells (HSCs) through triggering Smad-dependent pro-fibrogenic signaling pathway. Main methodsDirect cell counting and assays for cell proliferation and migration were used to measure the effects of LPS on HSC behaviors. Quantitative PCR, Western blot, and gelatin zymography were used to quantify the molecular effects of LPS on expression of HSC activation markers and signaling activity. Key findingsLong-term exposure to LPS exhibited moderately stimulatory effect on HSC cell growth. A wound-healing cell migration assay showed that LPS suppressed HSC-T6 cell migration. qPCR and Western blotting detection indicated that LPS treatment induced upregulation of type I and IV collagens, α-smooth muscle actin (α-SMA), and matrix metalloproteinase-9 (MMP-9). Gelatin zymography confirmed that LPS elevated MMP-9, but not MMP-2 gelatinolytic activity. Moreover, LPS immediately stimulated Akt, EKR1/2, JNK, p38 MAPK, and Smad2 hyperphosphorylation, supporting that LPS directly triggers pro-fibrogenic Smad signaling cascade without TGF-β1 stimulation. Kinase blockade experiments demonstrated the involvement of PI3K/Akt, JNK, p38 MAPK, but not ERK1/2 signaling activation in the LPS-elicited Smad2 phosphorylation as well as the overexpression of type I collagen and α-SMA in HSC-T6 cells. SignificanceThese findings demonstrate that LPS exerts pro-fibrogenic effect through activation and transformation of HSCs. The tissue-remodeling effect of LPS may be attributable to its ability to activate non-canonical Smad pathway through PI3K/Akt and MAPK signaling cascades.

Expression and purification of FGF21 in Pichia pastoris and its effect on fibroblast-cell migration.

Fibroblast growth factor (FGF)21 functions in the maintenance of glucose homeostasis and exerts protective effects on the liver, heat and kidneys. However, the roles of FGF21 in other tissue types are yet to be fully elucidated. The present study detected elevated expression levels of FGF21 in skin tissue. Furthermore, it was revealed that FGF21 expression in the skin was induced upon wounding. In addition, β‑klotho expression was detected in the skin tissue. To examine the role of FGF21 in the wound healing process, recombinant human (h)FGF21 was expressed in a the yeast strain Pichia (P.) pastoris, a well‑known system for recombinant protein production. Based on the sequence of hFGF21 and the optimal codon of P. pastoris, codon‑optimized FGF21 open reading frame sequences were obtained using seven pairs of 55‑59‑nt primers with seven rounds of PCR. The recombinant FGF21 was purified and its function was examined in human fibroblast cells using a wound healing cell migration assay. Treatment with FGF21 promoted cell migration, which is an important step in wound healing. Furthermore, FGF21 treatment enhanced the activity of c‑Jun N‑terminal kinase, a key regulator in fibroblast‑cell migration. In conclusion, FGF21 is induced after wounding and FGF21 expressed and purified from yeast markedly accelerates wound healing. The present study was the first to elucidate the function of FGF21 in skin tissues and provided a theoretical basis for the use of FGF21 in the treatment of skin wounds.

Abstract 33: In vitro functional study of Ang 2 in colorectal cancer cell line and the significance of Ang 2 expression in colorectal cancer patients

Abstract Objectives: Angiopoietin 2 (Ang-2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang-2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang-2 an ideal drug target. The function and mechanism of Angiopoietin 2 in colorectal cancer proliferation, invasion, metastasis and angiogenesis have not been fully elucidated to date. The aim of present study was to evaluate the biological roles of Ang-2 in the colorectal cancer cell line and to correlate the Ang-2 overexpression with the clinicopathological factors of colorectal cancers. Methods: The biological roles of Ang-2 were investigated in LoVo colorectal cancer cell line that expresses high level of Ang-2 by knockdown Ang-2 with shRNA transfection. The functional roles of Ang-2 were evaluated by invasion assay, wound healing cell migration assay, MTT assay and tube formation assay. Moreover, Ang-2 expression was investigated by immunohistochemistry on tissue microarrays of 415 cases of colorectal cancer and the association of Ang-2 expression was examined with clinicopathologic features. Results: The LoVo cell line knockdowned Ang-2 by shRNA revealed significant decreases of viability, invasion, migration and tube formation compared to that of control (p<0.005) respectively. The Ang-2 was overexpressed in 45.8% of colorectal cancers. The overexpression of Ang-2 was significantly associated with pT (p=0.048), pN (p<0.001), vascular invasions (p-=0.023), lymphatic invasions (p<0.001) and TNM stages (p=0.022). Multivariate Cox regression analysis showed that the overexpression of Ang-2 in pN stage 1 and 2 were an independent prognostic factor of decreased survival [pN stage 1, HR=5.17 (95% CI: 6.36-19.59, p=0.016); pN stage 2, HR=7.01 (95% CI: 1.76-27.96, p=0.006)]. The overall survival was significantly decreased in patients of Ang-2 high expression [HR=6.78 (95% CI: 4.16-11.05, p<0.001)]. The cumulative survival was significantly decreased in patients of Ang-2 high expression compared to those of Ang-2 low expression by Kaplan-Meier analysis (log rank test, p<0.001). Conclusion: The overexpression of Ang-2 is significantly associated with tumor progression rather than carcinogenesis in colorectal cancer. It can be a valuable poor prognostic marker of colorectal cancer and the Ang-2 can be used in therapeutic strategies. Citation Format: Moo Jun Baek, Dongjun Jeong, Chang-Jin Kim, Tae Hyun Kim, Han Jo Kim, Tae Sung Ahn, Sang Byung Bae, Sang Han Lee. In vitro functional study of Ang 2 in colorectal cancer cell line and the significance of Ang 2 expression in colorectal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 33. doi:10.1158/1538-7445.AM2014-33

Piecewise linear approximation to Fisher's equation

A simple method is presented which allows the replacement of a nonlinear differential equation with a piecewise linear differential equation. The method is based on the idea that a curve of the nonlinear terms of the dependent variable in a differential equation can be replaced by an approximate curve consisting of a set of line segments tangent to the original curve. We apply this method to the ubiquitous Fisher's equation and demonstrate that accurate solutions are obtained with a relatively small number of line segments. References R. A. Fisher. The wave of advance of advantageous genes. Ann. of Eugenics. , 7 , 355--369, 1937. V. M. Kenkre and M. N. Kuperman. Applicability of the Fisher equation to bacterial population dynamics. Phys. Rev. E. , 67 , 2003, 051921. doi:10.1103/PhysRevE.67.051921 D. A. Kessler, Z. Ner and L. M. Sander. Front propagation: precursors, cutoffs, and structural stability. Phys. Rev. E. , 58 , 1998, 107--114. P. K. Maini, D. L. S. McElwain and D. I. Leavesley. Traveling wave model to interpret a wound-healing cell migration assay for human peritoneal mesothelial cells. Tissue Eng. , 10 , 2004, 475--482. M. J. Ablowitz and A. Zeppetella. Explicit solutions of Fisher's equation for a special wave speed. Bull. Math. Biol. , 41 , 1979, 835--840. X. Y. Wang. Exact and explicit solitary wave solutions for the generalised Fisher equation. Phys. Lett. A , 131 , 1988, 277--279. J. Rinzel and J. B. Keller. Traveling wave solutions of a nerve conduction equation. Biophys. J. , 13 , 1973, 1313--1337. Z. Jovanoski and S. Soo. Piecewise linear approximation of nonlinear ordinary differential equations. ANZIAM J. (E) , 51 , 2010, C570--C585. http://journal.austms.org.au/ojs/index.php/ANZIAMJ/article/view/2622 T. Hagstrom and H. B. Keller. The numerical calculation of traveling wave solutions of nonlinear parabolic equations. SIAM J. Sci. Stat. Comput. , 7 , 1986, 978--988.

Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates class I ADP ribosylation factors (ARFs) by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus-end-directed motor protein, moves cargo away from the microtubule-organizing center (MTOC) on microtubules. Because KANK1, a protein containing N-terminal KN, C-terminal ankyrin-repeat, and intervening coiled-coil domains, has multiple actions in cells and also interacts with KIF21A, we explored a possible interaction between it and BIG1. We obtained evidence for a functional and physical association between these proteins, and found that the effects of BIG1 and KANK1 depletion on cell migration in wound-healing assays were remarkably similar. Treatment of cells with BIG1- or KANK1-specific siRNA interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, which was not mimicked by KIF21A depletion. Although colocalization of overexpressed KANK1 and endogenous BIG1 in HeLa cells was not clear microscopically, their reciprocal immunoprecipitation (IP) is compatible with the presence of small percentages of each protein in the same complexes. Depletion or overexpression of BIG1 protein appeared not to affect KANK1 distribution. Our data identify actions of both BIG1 and KANK1 in regulating cell polarity during directed migration; these actions are consistent with the presence of both BIG1 and KANK1 in dynamic multimolecular complexes that maintain Golgi/MTOC orientation, differ from those that might contain all three proteins (BIG1, KIF21A, and KANK1), and function in directed transport along microtubules.

Aberrant Overexpression of the Cell Polarity Module Scribble in Human Cancer

Human Scribble (Scrib) is an evolutionary-conserved cell polarity protein, but its potential role in human cancer is controversial. Herein, we show that Scrib is nearly universally overexpressed in cultured tumor cell lines and genetically disparate cancer patient series compared with matched normal tissues in vivo. Instead of a membrane association seen in normal epithelia, tumor-associated Scrib is mislocalized and found predominantly in the cytosol. Small-interfering RNA silencing of Scrib in model lung adenocarcinoma A549 cells inhibited cell migration in wound-healing assays, suppressed tumor cell invasion across Matrigel-coated inserts, and down-regulated the expression of cell motility markers and mediators of epithelial-mesenchymal transition. These data uncover a previously unrecognized exploitation of Scrib for aberrant tumor cell motility and invasion, thus potentially contributing to disease progression in humans.

Abstract 2363: Dual effects of unique oversulfated dermatan sulfates from ascidians (Chordata-Tunicata) on tumor invasion: inhibition of ephitelial-mesenchymal transition and hematogeneous metastasis

Abstract Metastatic disease is responsible for most cancer-associated deaths and epithelial-mesenchymal transition (EMT) along with hematogeneous metastasis are critical steps in cancer progression. Therefore, inhibition of these events could be an effective approach to reduce the metastatic disease. Heparin has been show to modulate EMT-associated growth factors and also to inhibit P-selectin, leading to attenuation of metastasis. Previously, we showed that unique dermatan sulfates (DS) from Styela plicata and Pallusia nigra, composed by 2,4-O-sulfated and 2,6-O-sulfated disaccharide units, respectively, bind with high affinity (KD of ∼ 33nM) to the EMT-related growth factor, hepatocyte growth factor (HGF), modulating its MET-dependent intracellular signaling. In the present work, the effect of the ascidian DSs on EMT-mediated cell migration and P-selectin-mediated metastasis in experiments in vitro and in vivo were evaluated. To investigate migration, confluent human mammary cells were scraped with a pipette tip and cultured for 3 days in the presence of the ascidian DSs. The migration of human mammary cells in the wound-healing cell migration assay was drastically inhibited by the glycans. The anti-selectin activity was evaluated by the binding of LS180 cells to immobilized P-selectin in the presence of the ascidian DSs. The ascidian DSs inhibited the binding of tumor cells to P-selectin with IC50 values of 13.51 μg/mL (2,4-DS) and 12.19 μg/mL (2,6-DS) and were better inhibitors than unfractionated heparin (IC50 of 24.51 μg/mL). DS from porcine skin had no effect. To investigate the effect of ascidian DSs in vivo, mice were injected with LS180 cells 10 minutes after treatment with PBS, UFH (1 mg/mouse) or ascidian DSs (100 μg/mouse) and the presence of platelets-tumor cells aggregates in the lung microvasculature was evaluated. The ascidian DSs inhibited the adhesion of platelets to tumor cells in vivo in a concentration 10-fold lower than heparin. The inhibitory effect of glycans could be observed as early as 30 minutes upon cells injection or 3 hours later. Metastasis mice in vivo was investigated by injecting 100 μg of each ascidian DSs or PBS 10 minutes prior injection of MC-38 cells. Twenty-eight days later, lungs were harvested for macroscopic evaluation of metastasis. Both ascidian DSs drastically reduced metastasis of MC-38 cells. PBS had no effect. Metastasis was drastically reduced in P-sel-/- mice and neither 2,4- nor 2,6-DS had any additional effect. In conclusion, ascidian DSs inhibit cell migration and reduce hematogeneous metastasis by a EMT- and P-selectin mediated events and could be used therapeutically to prevent tumor invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2363. doi:10.1158/1538-7445.AM2011-2363

Effects of BIG1 and KANK1 on cell polarity and directed migration during wound healing

Brefeldin A‐inhibited guanine nucleotide‐exchange protein (BIG)1 activates class I ADP‐ribosylation factors by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus‐end‐directed motor protein, moves cargo away from the microtubule‐organizing center (MTOC) on microtubules. Here, we describe a newly recognized relationship between BIG1 and KANK1, a protein that has multiple actions in cells and interacts with KIF21A. Microscopically, overexpressed KANK1 appeared partially colocalized with endogenous BIG1, consistent with potentially functional interactions as was their reciprocal immunoprecipitation (IP), but direct interaction of KANK1 and BIG1 was not established. Depletion or overexpression BIG1 protein appeared not to affect KANK1 distribution. Effects of BIG1‐ and KANK1‐depletion on cell migration in wound‐healing assays were, however, remarkably similar. Depletion of BIG1 or KANK1 interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, but KIF21A depletion did not have such effects. These studies identify novel functions of both BIG1 and KANK1 in regulating cell polarity during directed migration.

The effect of versican G3 domain on local breast cancer invasiveness and bony metastasis

IntroductionIncreased versican expression has been associated with local breast cancer invasiveness and a more aggressive tumor phenotype. The cellular mechanisms are not fully understood and this study evaluated versican G3 domain with its EGF-like motifs in influencing tumor invasion and metastasis.MethodsOne recombinant construct was synthesized (a signal peptide for product secretion and the versican G3 domain). The construct was stably transfected into human breast carcinoma MT-1 cells. Cell viability in vitro was evaluated in low serum and serum starvation conditions. In vivo study of tumor growth was evaluated in a nude mouse model. G3 effects on rodent vascular endothelial cells were evaluated in vitro on cell survival, apoptosis, migration, and vascular formation. The effects of VEGF, fibronectin, and G3 on vascular formation were examined. An intracardiac injection model of metastatic human breast carcinoma tested the effect of G3 on distant bony and soft tissue metastasis. Analysis of metastatic burden included histology, radiographs, and micro-CT quantification of osteolysis.ResultsA greater viability of cancer cells was observed in low serum and serum-free conditions in the presence of versican G3. Larger subcutaneous tumors were obtained in the G3 group following tumor cell injection into CD1 mice. G3 induced a greater degree of rodent vascular endothelial cell proliferation and migration in vitro. Simultaneous presence of fibronectin, VEGF, and G3 promoted endothelial cell migration in wound-healing assays as compared to the treatments containing none, one or two of these molecules. Systemic tumor burden to distant bony and soft tissue metastatic sites was greater in the G3 group using the intracardiac injection metastatic modelConclusionVersican G3 domain appears to be important in local and systemic tumor invasiveness of human breast cancer. Effects include enhancing cell viability, proliferation, migration and enhancing local tumor growth. Potential effects on angiogenesis include enhancing vascular endothelial proliferation, migration, and vessel formation. The interactions between tumor cells, surrounding stromal components and neo-vascularization in breast cancer may include interactions with VEGF and fibronectin. The propensity of versican G3 to influence tumor invasion to bone and the mechanisms of G3 mediated osteolysis warrants ongoing studies.

Travelling Waves of Attached and Detached Cells in a Wound-Healing Cell Migration Assay

During a wound-healing cell migration assay experiment, cells are observed to detach and undergo mitosis before reattaching as a pair of cells on the substrate. During experiments with mice 3T3 fibroblasts, cell detachment can be confined to the wavefront region or it can occur over the whole wave profile. A multi-species continuum approach to modelling a wound-healing assay is taken to account for this phenomenon. The first cell population is composed of attached motile cells, while the second population is composed of detached immotile cells undergoing mitosis and returning to the migrating population after successful division. The first model describes cell division occurring only in the wavefront region, while a second model describes cell division over the whole of the scrape wound. The first model reverts to the Fisher equation when the reattachment rate relative to the detachment rate is large, while the second case does not revert to the Fisher equation in any limit. The models yield travelling wave solutions. The minimum wave speed is slower than the minimum Fisher wave speed and its dependence on the cell detachment and reattachment rate parameters is analysed. Approximate travelling wave profiles of the two cell populations are determined asymptotically under certain parameter regimes.

Multi-scale modeling of a wound-healing cell migration assay

A continuum model and a discrete model are developed to capture the population-scale and cell-scale behavior in a wound-healing cell migration assay created from a scrape wound in a confluent cell monolayer. During wound closure, the cell population forms a sustained traveling wave, with close contact between cells behind the wavefront. Cells exhibit contact inhibition of migration and contact-limited proliferation. The continuum model includes the two dominant mechanisms and characteristics of cell migration and proliferation, using a cell diffusivity function that decreases with cell density and a logistic proliferative growth term. The discrete model arises naturally from the continuum model. Individual cells are simulated as continuous-time random walkers with nearest-neighbor transitions, together with a birth/death process. The migration and proliferation parameters are determined by analysing individual mice 3T3 fibroblast cell trajectories obtained during the development of a confluent cell monolayer and in a wound healing assay. The population-scale model successfully predicts the shape and speed of the traveling wave, while the discrete model is also successful in capturing the contact inhibition of migration effects.

Traveling wave model to interpret a wound-healing cell migration assay for human peritoneal mesothelial cells.

The critical determinants of the speed of an invading cell front are not well known. We performed a "wound-healing" experiment that quantifies the migration of human peritoneal mesothelial cells over components of the extracellular matrix. Results were interpreted in terms of Fisher's equation, which includes terms for the modeling of random cell motility (diffusion) and proliferation. The model predicts that, after a short transient, the invading cell front will move as a traveling wave at constant speed. This is consistent with the experimental findings. Using the model, a relationship between the rate of cell proliferation and the diffusion coefficient was obtained. We used the model to deduce the cell diffusion coefficients under control conditions and in the presence of collagen IV and compared these with other published data. The model may be useful in analyzing the invasive capacity of cancer cells as well in predicting the efficacy of growth factors in tissue reconstruction, including the development of monolayer sheets of cells in skin engineering or the repair of injured corneas using grafts of cultured cells.