Matrigel Chamber Assay Research Articles

An in vitro study of the relationship between KiSS-1 expression and hepatoma carcinoma cell proliferation, adhesion, and invasion

To investigate the impact of expression of kisspeptin-1 (KiSS-1) metastasis-suppressor gene on the proliferative, adhesive and invasive abilities of human hepatocellular carcinoma (HCC) using an in vitro cell system. The highly metastatic human hepatoma cell line MHCC97-H was transiently transfected with the pcDNA3.1/HisC vector expressing the KiSS-1 gene (experimental group) or the vector without the KisS-1 gene (blank control group). Untransfected cells served as the negative control group. Proliferative abilities of the three groups were assessed by flow cytometry and MTT assay. Adhesive abilities were assessed by MTT assays using matrigel and fibronectin. Invasive abilities and cell motility were assessed by chemoinvasion chamber assay using reconstituted matrigel and migration chamber assay using polycarbonate filters, respectively. The experimental group showed significantly lower adhesion capacity to matrigel (0.257+/-0.029) than either the blank control group (0.374+/-0.016; t=-7.90345, P less than 0.01) or the negative control group (0.394+/-0.031; t=-7.22752, P less than 0.01). Similarly, the experimental group showed significantly lower adhesion capacity to fibronectin (0.292+/-0.004) than either the blank control group (0.394+/-0.010; t=-20.93138, P less than 0.01) or the negative control group (0.412+/-0.023; t=-11.31371, P less than 0.01). The experimental group also showed significantly lower numbers of cells with invasive capacity (42.40+/-1.14) than either the blank control group (66+/-1.58; t=-27.0711, P less than 0.01) or the negative control group (67.80 +/- 1.92; t=-25.4, P less than 0.01). Similarly, the experimental group showed significantly lower numbers of cells with chemotactic movement (65.80+/-1.92) than either the blank control group (93.80+/-2.28; t=-30.11750, P less than 0.01) or the negative control group (96.40+/-2.07; t=-24.19142, P less than 0.01). The experimental group showed slightly, but not significantly, lower cell proliferation (0.644+/-0.027) than either the blank control group (0.669+/-0.022; t=-1.60371, P?>?0.05) or the negative control group (0.678+/-0.027; t=-1.97828, P?>?0.05). In addition, there were no obvious differences between the three groups in the amounts of cells arrested in either the G1 phase or the S phase. KiSS-1 overexpression suppresses the adhesion, invasion and motility, but not the proliferation, of hepatoma carcinoma cells in vitro. These findings imply that KiSS-1 might represent a promising new candidate for gene therapy against human hepatocellular carcinoma.

In Vitro and In Vivo Prostate Cancer Metastasis and Chemoresistance Can Be Modulated by Expression of either CD44 or CD147

CD44 and CD147 are associated with cancer metastasis and progression. Our purpose in the study was to investigate the effects of down-regulation of CD44 or CD147 on the metastatic ability of prostate cancer (CaP) cells, their docetaxel (DTX) responsiveness and potential mechanisms involved in vitro and in vivo. CD44 and CD147 were knocked down (KD) in PC-3M-luc CaP cells using short hairpin RNA (shRNA). Expression of CD44, CD147, MRP2 (multi-drug resistance protein-2) and MCT4 (monocarboxylate tranporter-4) was evaluated using immunofluorescence and Western blotting. The DTX dose-response and proliferation was measured by MTT and colony assays, respectively. The invasive potential was assessed using a matrigel chamber assay. Signal transduction proteins in PI3K/Akt and MAPK/Erk pathways were assessed by Western blotting. An in vivo subcutaneous (s.c.) xenograft model was established to assess CaP tumorigenecity, lymph node metastases and DTX response. Our results indicated that KD of CD44 or CD147 decreased MCT4 and MRP2 expression, reduced CaP proliferation and invasive potential and enhanced DTX sensitivity; and KD of CD44 or CD147 down-regulated p-Akt and p-Erk, the main signal modulators associated with cell growth and survival. In vivo, CD44 or CD147-KD PC-3M-luc xenografts displayed suppressed tumor growth with increased DTX responsiveness compared to control xenografts. Both CD44 and CD147 enhance metastatic capacity and chemoresistance of CaP cells, potentially mediated by activation of the PI3K and MAPK pathways. Selective targeting of CD44/CD147 alone or combined with DTX may limit CaP metastasis and increase chemosensitivity, with promise for future CaP treatment.

Abstract 4142: Differential expression of PDCD4 in response to Resveratrol in breast cancer cells

Abstract Background: Programmed cell death 4 (PDCD4) is a key protein involved in protein synthesis, and thus, it controls cell growth and proliferation. Evidence indicates that PDCD4 is often downregulated in breast cancer and is associated with disease progression and metastasis. Resveratrol (RSV), a natural polyphenolic compound present in grapes and red wine, is a well known anti-oxidant capable of modulating various signaling pathways, leading to suppression of cell proliferation and increased apoptosis. However, it remains unclear regarding the effect of RSV on PDCD4. Therefore, in the present study, we investigated the effect of RSV on PDCD4 in human breast cancer MCF-7 and MDA-MB-231 cells. Methods: Cell growth was measured by MTT assays, and cell invasion was determined by matrigel chamber assays. Soft agar assays were used to determine anchorage independent growth. Protein levels from treated cells were analyzed by western blot. Real time RT-PCR was used to determine relative expression of genes. Phosphorylated Akt (pAkt) was also determined by immunohistochemistry in RSV-treated MDA-MB-231 cells. To determine whether microRNAs are involved in the RSV-mediated induction of PDCD4, we cloned PDCD4 3′-UTR into the downstream of the luciferase gene as a reporter. The effect of RSV on luciferase activity was tested in MCF-7 cells. PDCD4 was stably knocked down by RNAi in MCF-7 and MDA-MB-231 cells. Results: We showed that ectopic expression of PDCD4 suppressed anchorage independent tumor cell growth as well as invasion of MDA-MB-231 cells. By contrast, downregulation of PDCD4 by RNAi significantly increased cell invasion. Of interest, cell invasion was suppressed by RSV; at the same time, PDCD4 was induced significantly by RSV in MDA-MB-231 cells, which was associated with suppression of pAkt. Furthermore, we showed that both phosphorylated mTOR and S6K were suppressed by RSV in a dose dependent manner. Since PDCD4 is a downstream effector of Akt and mTOR, this may suggest that up-regulation by RSV is through the Akt-mTOR pathway in MDA-MB-231. On the other hand, PDCD4 was reduced by RSV in estrogen receptor-positive (ERα+) MCF-7 cells. To determine the mechanism of RSV-mediated PDCD4 expression in these two cell lines, we measured the levels of miR-21 which has been previously shown to directly target PDCD4. Strikingly, expression of miR-21 was also regulated differentially. For example, miR-21 was up-regulated in MCF-7 cells while it was slightly suppressed by RSV in MDA-MB-231 cells. In particular, we found that RSV suppressed PDCD4 by transcriptional regulation of miR-21 possibly through interacting with ERα in MCF-7 cells. Conclusion: Together, these results suggest that, as a tumor suppressor gene, PDCD4 expression is dependent on cellular contents in response to RSV, and thus, a better understanding of this RSV-mediated PDCD4 expression will provide new insight into the clinical implication of RSV for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4142. doi:1538-7445.AM2012-4142

P1-02-06: Silencing of IGF-1R Has Paradoxical Effects in Triple Negative Breast Cancer Phenotypes.

Abstract Background: Triple negative (TN) breast cancers are a heterogeneous group of breast cancers with a poor prognosis in part due to a lack of effective targeted agents. Insulin-like growth factor-1 receptor (IGF-1R) has been shown to play a role in breast cancer cell proliferation, adhesion, invasion, and migration and is overexpressed in more than a third of TN breast tumors. We hypothesized that IGF-1R could be a therapeutic target for a subset of TN breast cancers. Methods: We evaluated the effects of IGF-1R silencing on the metastatic properties of TN breast cancer cells by knock down in two morphologically distinct TN breast cancer cell lines using shRNA lentiviral techniques. Reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting were used to detect mRNA and protein expression levels, respectively, of IGF-1R signaling molecules. Anchorage-dependent growth and Matrigel chamber assays were performed to assess the effects of IGF-1R silencing on colony formation and invasion of TN breast cancer cells, respectively; wound-healing and spheroid migration were also performed to assess the effects of IGF-1R inhibition on TN breast cancer cell motility. Results: Stably transfected mesenchymal MDA-MB-231 TN cells showed effective downregulation of IGF-1R protein expression, which resulted in mesenchymal-to-epithelial transition (MET), confirmed by upregulation of the epithelial marker E-cadherin and downregulation of the mesenchymal marker vimentin. Importantly, this MET resulted in reduced colony formation (p<0.0042) and cell motility and dramatically reduced invasion (p<0.0001). Conversely, silencing of IGF-1R in epithelial MDA-MB-468 TN cells induced epithelial-to-mesenchymal transition (EMT), confirmed by downregulation of E-cadherin and upregulation of vimentin expression, with resultant increased colony formation (p<0.006), cell motility, and invasion (p<0.0001). Conclusion: Collectively, these results demonstrate a paradoxical effect of targeting IGF-1R in TN breast cancers of mesenchymal and epithelial origin. Targeting IGF-1R in TN breast cancers with a mesenchymal phenotype decreases invasion and metastatic potential. In contrast, targeting IGF-1R in TN breast cancers with an epithelial phenotype could have potentially detrimental effects. Our data suggest that IGF-1R inhibition should be explored as a therapeutic modality in TN breast cancers with a mesenchymal phenotype. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-06.

Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers – this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination.

Abstract 2350: MicroRNA-145 suppresses cell invasion and metastasis by directly targeting mucin 1

Abstract MicroRNAs (miRNAs) are important gene regulators that could play a profound role in tumorigenesis. Our previous studies indicate that miR-145 is a tumor suppressor capable of inhibiting tumor cell growth both in vitro and in vivo. In this study, we show that miR-145 exerts its function in a cell-specific manner. Although miR-145 inhibits cell growth in MCF-7 and HCT-116 cells, it has no significant effect on cell growth in metastatic breast cancer cell lines. However, miR-145 significantly suppresses cell invasion in these cells, as shown by matrigel chamber assays; in contrast, the antisense oligo against miR-145 increases cell invasion. MiR-145 is also able to suppress lung metastasis in an experimental metastasis animal model. This miR-145-mediated suppression of cell invasion is in part due to the silencing of the metastasis gene mucin 1 (MUC1). Using luciferase reporters carrying the 3′-untranslated region of MUC1 combined with western blot and immunofluorescence staining, we identify MUC1 as a direct target of miR-145. Moreover, ectopic expression of MUC1 enhances cell invasion, which can be blocked by miR-145. Of interest, suppression of MUC1 by miR-145 causes a reduction of β-catenin as well as the oncogenic cadherin 11. Finally, suppression of MUC1 by RNAi mimics the miR-145 action in suppression of invasion, which is associated with downregulation of β-catenin and cadherin 11. Taken together, these results suggest that as a tumor suppressor, miR-145 inhibits not only tumor growth, but also cell invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2350.

NF-κB–Dependent Plasticity of the Epithelial to Mesenchymal Transition Induced by Von Hippel-Lindau Inactivation in Renal Cell Carcinomas

The critical downstream signaling consequences contributing to renal cancer as a result of loss of the tumor suppressor gene von Hippel-Lindau (VHL) have yet to be fully elucidated. Here, we report that VHL loss results in an epithelial to mesenchymal transition (EMT). In studies of paired isogenic cell lines, VHL silencing increased the levels of N-cadherin and vimentin and reduced the levels of E-cadherin relative to the parental VHL(+) cell line, which displayed the opposite profile. VHL(+) cells grew as clusters of cuboidal and rhomboid cells, whereas VHL-silenced cells took on an elongated, fibroblastoid morphology associated with a more highly invasive character in Matrigel chamber assays. Based on earlier evidence that VHL loss can activate NF-kappaB, a known mediator of EMT, we tested whether NF-kappaB contributed to VHL-mediated effects on EMT. On pharmacologic or molecular inhibition of NF-kappaB, VHL-silenced cells regained expression of E-cadherin, lost expression of N-cadherin, and reversed their highly invasive phenotype. Introducing a pVHL-resistant hypoxia-inducible factor 1alpha (HIF1alpha) mutant (HIFalpha(M)) into VHL(+) cells heightened NF-kappaB activity, phenocopying EMT effects produced by VHL silencing. Conversely, inhibiting the heightened NF-kappaB activity in this setting reversed the EMT phenotype. Taken together, these results suggest that VHL loss induces an EMT that is largely dependent on HIFalpha-induced NF-kappaB. Our findings rationalize targeting the NF-kappaB pathway as a therapeutic strategy to treat renal tumors characterized by biallelic VHL inactivation.

Targeting prostate cancer progenitor cells with telomerase interference: A novel therapeutic strategy

e22029 Background: We investigated whether telomerase, which is critical for benign stem cell activation, also plays a role in prostate cancer progenitor cells (PCPCs), which are thought to mediate therapy resistance and cancer progression, and we tested whether telomerase interference can effectively inhibit PCPC proliferation. Methods: A putative PCPC population was isolated from human prostatectomy specimens via collagen attachment and FACS selection for integrin α2β1 and CD44. PCPCs were characterized for gene expression (RT-PCR), clonogenicity (colony formation), invasiveness (matrigel chamber), and telomerase activity (qPCR-TRAP). PCPC telomerase interference was accomplished by lentiviral expression of 2 constructs: telomerase RNA with an altered template region (MT-Ter) and siRNA targeting wild-type telomerase RNA (anti-Ter siRNA). The effects of these constructs were assessed by measuring PCPC viability (MTS) and apoptosis (TUNEL assay). Results: An integrin α2β1+CD44+ putative PCPC population was isolated from 6 human prostate tumors. This population expressed high levels of “progenitor phenotype” genes (ABCG2, β-catenin, NANOG, Oct3/4) and low levels of “differentiated phenotype” genes (AR and PSA). PCPCs yielded >50 colonies per 1000 cells seeded on collagen after 3 weeks vs. none from FACS- cells, and matrigel chamber assay showed 10% of the PCPC population invading over 24 hours vs. none of the FACS- population. Most importantly, PCPCs possessed at least 20- fold greater telomerase activity than FACS- cells, and induction of telomerase interference in PCPCs via MT-hTer and anti- hTer siRNA expression elicited a brisk apoptotic response (TUNEL) by day 3 in >90% of cells, with concomitant near-complete growth inhibition (MTS). Conclusions: We have shown that human prostate tumors contain a subpopulation of prostate cancer progenitor cells (PCPCs) marked by an undifferentiated gene expression profile, vigorous clonogenicity and invasiveness, and high levels of telomerase activity that can be successfully exploited to neutralize these cells. Ongoing studies are investigating the in vivo effects of telomerase interference on PCPC tumorigenicity in mouse models. No significant financial relationships to disclose.

Involvement of syntaxin 4 in the transport of membrane-type 1 matrix metalloproteinase to the plasma membrane in human gastric epithelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) localized on the plasma membrane plays a central role in various normal biological responses including tissue remodeling, wound heeling, and angiogenesis and in cancer cell invasion and metastasis, by functioning as a collagenase and activating other matrix metalloproteinases. In order to elucidate the molecular mechanism of the MT1-MMP targeted localization on the plasma membrane, we examined the participation of syntaxin proteins in MT1-MMP intracellular transport to the plasma membrane in human gastric epithelial AGS cells. Western blotting showed that syntaxin 3 and 4 proteins, which are known to function in intracellular transport towards the plasma membrane, were expressed in AGS cells. Immunocytochemistry revealed that transient transfection of AGS cells with dominant-negative mutant syntaxin 4 decreased plasma membrane MT1-MMP expression. In contrast, transient transfection with either dominant-negative mutant syntaxin 3 or 7 did not affect MT1-MMP localization on the plasma membrane. Cell surface biotinylation assay and Matrigel chamber assay demonstrated that stable transfection with dominant-negative mutant syntaxin 4 decreased the amount of MT1-MMP on the plasma membranes and inhibited the cell invasiveness. We suggest that syntaxin 4 is involved in the intracellular transport of MT1-MMP toward the plasma membrane.

Angiogenic synergy of bFGF and VEGF is antagonized by Angiopoietin-2 in a modified in vivo Matrigel assay

A murine modification of the Matrigel chamber assay originally developed for use on rats is presented. This modified assay permits improved quantification due to subcutaneous Matrigel implants of constant shape and volume. We have quantitatively assessed the angiogenic potential of the growth factors basic fibroblast growth factor (bFGF), VEGF, and Angiopoietin-2 (Ang-2) with special emphasis on their mutual interactions. A reproducible dose–response relationship for bFGF was established for doses between 150 and 1000 ng per chamber, whereas VEGF did not display angiogenic activity on its own in the tested dose of up to 200 ng per chamber. Conversely, we found a strong synergistic action of bFGF and VEGF when combined in a 3:1 ratio. Two other combinations (ratios) with greater VEGF doses were also tested, but the synergistic effect was only observed when 50 ng of VEGF was added to 150 ng per chamber of bFGF. This synergistic effect of bFGF and VEGF was significantly reduced by further addition of 100 ng Angiopoietin-2. Inhibition of the response to bFGF and VEGF was confirmed by in vitro EC migration experiments, which, together with our in vivo results, indicates that Ang-2 may target chemotactic responses to bFGF and VEGF in vivo.

Gene Profiling of Cottontail Rabbit Papillomavirus-Induced Carcinomas Identifies Upregulated Genes Directly Involved in Stroma Invasion as Shown by Small Interfering RNA-Mediated Gene Silencing

To investigate changes in cellular gene expression associated with malignant progression, we identified differentially expressed genes in a cottontail rabbit papillomavirus (CRPV) squamous carcinoma model employing New Zealand White rabbits. The technique of suppression subtractive cDNA hybridization was applied to pairs of mRNA isolates from CRPV-induced benign papillomas and carcinomas, with each pair derived from the same individual rabbit. The differential expression of 23 subtracted cDNAs was further confirmed by quantitative reverse transcription-PCR (RT-PCR) with additional biopsies. Eight papilloma-carcinoma pairs examined showed a constant upregulation of the transcripts for the multifunctional adaptor protein 14-3-3 zeta and the Y-box binding transcription factor YB-1, whereas transcripts for m-type calpain 2 and NB thymosin beta, which are involved in cell motility and tissue invasion, as well as casein kinase 1 alpha, chaperonin, and annexin I, were found to be upregulated in the majority of the cases. RNA-RNA in situ hybridization and laser capture microdissection in combination with quantitative RT-PCR analysis verified the deregulated expression of the transcripts in the tumor cells. In contrast, CRPV E7 transcript levels remained rather constant indicating no requirement for a further upregulation of E7 expression following tumor induction. Small interfering RNA-mediated interference with expression of genes encoding YB-1, m-type calpain 2, or NB thymosin beta in a CRPV-positive cell line established from New Zealand White rabbit keratinocytes resulted in decreased cell invasion in matrigel chamber assays.