Selective Androgen Receptor Modulators (SARMs) Negatively Regulate Triple-Negative Breast Cancer Growth and Epithelial:Mesenchymal Stem Cell Signaling

Introduction

The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer with 75–95% of estrogen receptor (ER)-positive and 40–70% of ER-negative breast cancers expressing AR. Though historically breast cancers were treated with steroidal androgens, their use fell from favor because of their virilizing side effects and the emergence of tamoxifen. Nonsteroidal, tissue selective androgen receptor modulators (SARMs) may provide a novel targeted approach to exploit the therapeutic benefits of androgen therapy in breast cancer.

Materials and Methods

Since MDA-MB-453 triple-negative breast cancer cells express mutated AR, PTEN, and p53, MDA-MB-231 triple-negative breast cancer cells stably expressing wildtype AR (MDA-MB-231-AR) were used to evaluate the in vitro and in vivo anti-proliferative effects of SARMs. Microarray analysis and epithelial:mesenchymal stem cell (MSC) co-culture signaling studies were performed to understand the mechanisms of action.

Results

Dihydrotestosterone and SARMs, but not bicalutamide, inhibited the proliferation of MDA-MB-231-AR. The SARMs reduced the MDA-MB-231-AR tumor growth and tumor weight by greater than 90%, compared to vehicle-treated tumors. SARM treatment inhibited the intratumoral expression of genes and pathways that promote breast cancer development through its actions on the AR. SARM treatment also inhibited the metastasis-promoting paracrine factors, IL6 and MMP13, and subsequent migration and invasion of epithelial:MSC co-cultures.

Conclusion

1. AR stimulation inhibits paracrine factors that are important for MSC interactions and breast cancer invasion and metastasis. 2. SARMs may provide promise as novel targeted therapies to treat AR-positive triple-negative breast cancer.     

The Expression of the Nectin Complex in Human Breast Cancer and the Role of Nectin-3 in the Control of Tight Junctions during Metastasis

Introduction

Nectins are a family of integral protein molecules involved in the formation of functioning Adherens and Tight Junctions (TJ). Aberrant expression is associated with cancer progression but little is known how this effects changes in cell behaviour. This study aimed to ascertain the distribution of Nectins-1 to -4 in human breast cancer and the effect on junctional integrity of Nectin-3 modulation in human endothelial and breast cancer cells.

Methods

A human breast tissue cohort was processed for Q-PCR and immunohistochemistry for analysis of Nectin-1/-2/-3/-4. Nectin-3 over-expression was induced in the human breast cancer cell line MDA-MB-231 and the human endothelial cell line HECV. Functional testing was carried out to ascertain changes in cell behaviour.

Results

Q-PCR revealed a distinct reduction in node positive tumours and in patients with poor outcome. There was increased expression of Nectin-1/-2 in patients with metastatic disease, Nectin-3/-4 was reduced. IHC revealed that Nectin-3 expression showed clear changes in distribution between normal and cancerous cells. Nectin-3 over-expression in MDA-MB-231 cells showed reduced invasion and migration even when treated with HGF. Changes in barrier function resulted in MDAN3 cells showing less change in resistance after 2h treatment with HGF (p<0.001). Nectin-3 transformed endothelial cells were significantly more adhesive, irrespective of treatment with HGF (p<0.05) and had reduced growth. Barrier function revealed that transformed HECV cells had significantly tighter junctions that wildtype cells when treated with HGF (p<0.0001). HGF-induced changes in permeability were also reduced. Overexpression of Nectin-3 produced endothelial cells with significantly reduced ability to form tubules (p<0.0001). Immunoprecipitation studies discovered hitherto novel associations for Nectin-3. Moreover, HGF appeared to exert an effect on Nectin-3 via tyrosine and threonine phosphorylation.

Conclusions

Nectin-3 may be a key component in the formation of cell junctions and be a putative suppressor molecule to the invasion of breast cancer cells.     

The Actin Targeting Compound Chondramide Inhibits Breast Cancer Metastasis via Reduction of Cellular Contractility

Background

A major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent.

Methods

In vivo, the effect of Chondramide on metastasis was tested employing a 4T1-Luc BALB/c mouse model. In vitro, Chondramide was tested using the highly invasive cancer cell line MDA-MB-231 in Boyden-chamber assays, fluorescent stainings, Western blot and Pull down assays. Finally, the contractility of MDA-MB-231 cells was monitored in 3D environment and analyzed via PIV analysis.

Results

In vivo, Chondramide treatment inhibits metastasis to the lung and the migration and invasion of MDA-MB-231 cells is reduced by Chondramide in vitro. On the signaling level, RhoA activity is decreased by Chondramide accompanied by reduced MLC-2 and the stretch induced guanine nucleotide exchange factor Vav2 activation. At same conditions, EGF-receptor autophosphorylation, Akt and Erk as well as Rac1 are not affected. Finally, Chondramide treatment disrupted the actin cytoskeleton and decreased the ability of cells for contraction.

Conclusions

Chondramide inhibits cellular contractility and thus represents a potential inhibitor of tumor cell invasion.     

NAHA, a novel hydroxamic acid-derivative, inhibits growth and angiogenesis of breast cancer in vitro and in vivo

Background

We have recently synthesized novel N-alkylated amino acid-derived hydroxamate, 2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide (NAHA). Here, we evaluate the anticancer activity of NAHA against highly invasive human breast cancer cells MDA-MB-231 in vitro and in vivo.

Methodology/Principal Findings

Cell growth was evaluated by MTT and soft agar assays. Protein expression was determined by DNA microarray and Western blot analysis. Metastatic potential was evaluated by cell adhesion, migration, invasion, capillary morphogenesis, and ELISA assays. The anticancer activity in vivo was evaluated in mouse xenograft model. NAHA inhibited proliferation and colony formation of MDA-MB-231 cells together with the down-regulation of expression of Cdk2 and CDC20 proteins. NAHA inhibited cell adhesion, migration, and invasion through the suppression of secretion of uPA. NAHA suppressed secretion of VEGF from MDA-MB-231 cells and inhibited capillary morphogenesis of human aortic endothelial cells (HAECs). Finally, NAHA at 50 mg/kg was not toxic and decreased tumor volume and tumor weight in vivo. This suppression of tumor growth was associated with the inhibition of mitotic figures and induction of apoptosis, and the reduction of CD31 and VEGF positive cells in tumors.

Conclusion

NAHA could be a novel promising compound for the development of new drugs for the therapy of invasive breast cancers.     

RANKL Promotes Migration and Invasion of Hepatocellular Carcinoma Cells via NF-κB-Mediated Epithelial-Mesenchymal Transition

Background

Metastasis accounts for the most deaths in patients with hepatocellular carcinoma (HCC). Receptor activator of nuclear factor kappa B ligand (RANKL) is associated with cancer metastasis, while its role in HCC remains largely unknown.

Methods

Immunohistochemistry was performed to determine the expression of RANK in HCC tissue (n = 398). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to examine the expression of RANK, E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist and MMPs in HCC cells. Wound healing and Transwell assays were used to evaluate cell migration and invasion ability.

Results

We found that expression of RANK, the receptor of RANKL, was significantly higher in HCC tumor tissues than in peritumor liver tissues (p<0.001). Constitutive expression of RANK was detected in HCC cell lines, which can be up-regulated when HCC cells were stimulated with RANKL. Notably, in vitro experiments showed that activation of RANKL-RANK axis significantly promoted migration and invasion ability of HCC cells. In addition, RANKL stimulation increased the expression levels of N-cadherin, Snail, and Twist, while decreased the expression of E-cadherin, with concomitant activation of NF-κB signaling pathway. Moreover, administration of the NF-κB inhibitor attenuated RANKL-induced migration, invasion and epithelial-mesenchymal transition of HCC cells.

Conclusions

RANKL could potentiate migration and invasion ability of RANK-positive HCC cells through NF-κB pathway-mediated epithelial-mesenchymal transition, which means that RANKL-RANK axis could be a potential target for HCC therapy.     

Expression of Nucleophosmin/NPM1 correlates with migration and invasiveness of colon cancer cells

Background

We aimed to examine the expression level of Nucleophosmin (NPM1) protein in colon cancer tissues and to investigate the potential role of NPM1 in the regulation of cell migration and invasiveness.

Methods

Immunohistochemical assay was performed to examine the expression pattern of NPM1 in 31 groups of colonic carcinoma samples, including colon tumors, adjacent normal tissues, and matched metastatic lymph nodes from the same patients. Small interfering RNA technique and exogenous expression of wild type NPM1 methods were used to further verify the function of NPM1.

Results

High-expression of NPM1 correlates with lymph node metastasis (P = 0.0003) and poor survival rate of human colon cancer patients (P = 0.017). SiRNA-mediated reduction of NPM1 was also shown to inhibit the migration and invasiveness of metastatic colon cancer HCT116 cell line. In addition, the exogenous expression of NPM1 in HT29 cells, a NPM1 low expression and low invasive colon cancer cell line, enhanced cell migration and invasiveness along with increased cell proliferation.

Conclusions

The current study uncovered the critical role of NPM1 in the regulation of colon cancer cells migration and invasion, and NPM1 may serve as a potential marker for the prognosis of colon cancer patients.     

Cathepsin K Is Present in Invasive Oral Tongue Squamous Cell Carcinoma In Vivo and In Vitro

Objectives

Cathepsin K, a lysosomal cysteine protease, is expressed in the tumor microenvironment (TME) of skin carcinoma, but nothing is known about cathepsin K in oral tongue squamous cell carcinoma (OTSCC). Our aim was to describe the expression of cathepsin K in invasive OTSCC in vitro and in a series of clinical cancer specimens.

Materials and Methods

OTSCC invasion in vitro was studied using invasive HSC-3 tongue carcinoma cells in 3D organotypic models. In total, 121 mobile tongue OTSCCs and 10 lymph node metastases were analyzed for cathepsin K expression. The association between cathepsin K expression and clinicopathological factors was evaluated.

Results

Cysteine protease inhibitor E64 and cathepsin K silencing significantly (p<0.0001) reduced HSC-3 cell invasion in the 3D models. Cathepsin K was expressed in a majority of carcinoma and metastatic cells, but the expression pattern in carcinoma cells did not correlate with clinical parameters. Instead, the weak expression of cathepsin K in the invasive TME front correlated with increased overall recurrence (p<0.05), and in early-stage tumors this pattern predicted both cancer recurrence and cancer-specific mortality (p<0.05 and p<0.005, respectively).

Conclusions

Cathepsin K is expressed in OTSCC tissue in both carcinoma and TME cells. Although the diminished activity and expression in aggressive tongue HSC-3 cells reduced 3D invasion in vitro, the amount of cathepsin K in carcinoma cells was not associated with the outcome of cancer patients. Instead, cathepsin K in the invasive TME front seems to have a protective role in the complex progression of tongue cancer.     

Variant RONΔ160 of the RON receptor tyrosine kinase promotes the growth and invasion in vitro and in vivo in gastric cancer cell lines

Background

Recepteur d’origine nantais (RON) is a receptor tyrosine kinase whose overexpression has been observed in human gastric cancers. This study aimed to determine whether overexpression of the variant RONΔ160 could induce tumorigenicity of gastric cancer cells in vitro or in vivo, and whether its specific small molecule inhibitor (Compound I) could inhibit the effect of RONΔ160.

Methods

We constructed human gastric cancer cell line MGC-803 that was stably transfected with a recombinant plasmid expressing RONΔ160, and the effect of RONΔ160 overexpression and macrophage-stimulating protein (MSP) activation on proliferation, migration and invasion abilities of MGC-803 cells were evaluated. Tumor-bearing mice with gastric cancer cells were used to analyze the effects of RONΔ160 overexpression and Compound I on implanted tumor growth.

Results

In vitro, overexpression of RONΔ160 in MGC-803 cells resulted changes to their cell morphology, and promoted cell proliferation, migration and invasion. In addition, overexpression of RONΔ160 increased the proportion of cells in the S phase. The effect of RONΔ160 was significantly enhanced by induction of MSP inducing (p < 0.05). In vivo, RONΔ160 promoted the growth of MGC-803 cells in nude mice, including increased tumor size and weight, and lower tumor incubation period. The Compound I inhibited the tumorigenic abilities of RONΔ160 (p <0.05).

Conclusions

The results indicate that overexpression of the variant RONΔ160 altered the phenotype and tumorigenicity of MGC-803 cells. Its specific small molecule inhibitor could inhibit the effect of RONΔ160. Therefore, the variant RONΔ160 may become a potential therapeutic target for gastric cancer.     

Metadherin mediates lipopolysaccharide-induced migration and invasion of breast cancer cells

Background

Breast cancer is the most prevalent cancer in women worldwide and metastatic breast cancer has very poor prognosis. Inflammation has been implicated in migration and metastasis of breast cancer, although the exact molecular mechanism remains elusive.

Principal Findings

We show that the pro-inflammatory endotoxin Lipopolysaccharide (LPS) upregulates the expression of Metadherin (MTDH), a recently identified oncogene, in a number of breast cancer lines. Stable knockdown of MTDH by shRNA in human breast MDA-MB-231 cells abolishes LPS-induced cell migration and invasion as determined by several in vitro assays. In addition, knockdown of MTDH diminishes Nuclear Factor-kappa B (NF-κB) activation by LPS and inhibited LPS-induced IL-8 and MMP-9 production.

Conclusions

These results strongly suggest that MTDH is a pivotal molecule in inflammation-mediated tumor metastasis. Since NF-κB, IL-8 and MMP-9 play roles in LPS-induced invasion or metastasis, the mechanism of MTDH-promoted invasion and metastasis may be through the activation of NF-κB, IL-8 and MMP-9, also suggesting a role of MTDH in regulating both inflammatory responses and inflammation-associated tumor invasion. These findings indicate that MTDH is involved in inflammation-induced tumor progression, and support that MTDH targeting therapy may hold promising prospects in treating breast cancer.     

A putative novel protein,DEPDC1B,is overexpressed in oral cancer patients,and enhanced anchorage-independent growth in oral cancer cells that is mediated by Rac1 and ERK

Background

The DEP domain is a globular domain containing approximately 90 amino acids, which was first discovered in 3 proteins: Drosophila disheveled, Caenorhabditis elegans EGL-10, and mammalian Pleckstrin; hence the term, DEP. DEPDC1B is categorized as a potential Rho GTPase-activating protein. The function of the DEP domain in signal transduction pathways is not fully understood. The DEPDC1B protein exhibits the characteristic features of a signaling protein, and contains 2 conserved domains (DEP and RhoGAP) that are involved in Rho GTPase signaling. Small GTPases, such as Rac, CDC42, and Rho, regulate a multitude of cell events, including cell motility, growth, differentiation, cytoskeletal reorganization and cell cycle progression.

Results

In this study, we found that it was a guanine nucleotide exchange factor and induced both cell migration in a cultured embryonic fibroblast cell line and cell invasion in cancer cell lines; moreover, it was observed to promote anchorage-independent growth in oral cancer cells. We also demonstrated that DEPDC1B plays a role in regulating Rac1 translocated onto cell membranes, suggesting that DEPDC1B exerts a biological function by regulating Rac1. We examined oral cancer tissue; 6 out of 7 oral cancer tissue test samples overexpressed DEPDC1B proteins, compared with normal adjacent tissue.

Conclusions

DEPDC1B was a guanine nucleotide exchange factor and induced both cell migration in a cultured embryonic fibroblast cell line and cell invasion in cancer cell lines; moreover, it was observed to promote anchorage-independent growth in oral cancer cells. We also demonstrated that DEPDC1B exerts a biological function by regulating Rac1. We found that oral cancer samples overexpressed DEPDC1B proteins, compared with normal adjacent tissue. Suggest that DEPDC1B plays a role in the development of oral cancer. We revealed that proliferation was linked to a novel DEPDC1B-Rac1-ERK1/2 signaling axis in oral cancer cell lines.     

MiR-20a Is Upregulated in Anaplastic Thyroid Cancer and Targets LIMK1

Background

There have been conflicting reports regarding the function of miR-20a in a variety of cancer types and we previously found it to be dysregulated in sporadic versus familial papillary thyroid cancer. In this study, we studied the expression of miR-20a in normal, benign and malignant thyroid samples, and its effect on thyroid cancer cells in vitro and in vivo.

Methodology/Principal Findings

The expression of miR-20a in normal, benign and malignant thyroid tissue was determined by quantitative RT-PCR. Thyroid cancer cells were transfected with miR-20a and the effect on cellular proliferation, tumor spheroid formation, and invasion was evaluated. Target genes of miR-20 were determined by genome-wide mRNA expression analysis with miR-20a overexpression in thyroid cancer cells and target prediction database. Target genes were validated by quantitative PCR and immunoblotting, and luciferase assays. MiR-20a expression was significantly higher in anaplastic thyroid cancer than in differentiated thyroid cancer, and benign and normal thyroid tissues. MiR-20a significantly inhibited thyroid cancer cell proliferation in vitro (p<0.01) and in vivo (p<0.01), tumor spheroid formation (p<0.05) and invasion (p<0.05) in multiple thyroid cancer cell lines. We found that LIMK1 was a target of miR-20a in thyroid cancer cell lines and direct knockdown of LIMK1 recapitulated the effect of miR-20a in thyroid cancer cells.

Conclusions/Significance

To our knowledge, this is the first study to demonstrate that miR-20a plays a role as a tumor suppressor in thyroid cancer cells and targets LIMK1. Our findings suggest the upregulated expression of miR-20a in anaplastic thyroid cancer counteracts thyroid cancer progression and may have therapeutic potential.     

CD166/ALCAM Expression Is Characteristic of Tumorigenicity and Invasive and Migratory Activities of Pancreatic Cancer Cells

Background

CD166, also known as activated leukocyte cell adhesion molecule (ALCAM), is expressed by various cells in several tissues including cancer. However, the role of CD166 in malignant tumors is controversial, especially in pancreatic cancer. This study aimed to clarify the role and significance of CD166 expression in pancreatic cancer.

Methods

We performed immunohistochemistry and flow cytometry to analyze the expression of CD166 in surgical pancreatic tissues and pancreatic cancer cell lines. The differences between isolated CD166+ and CD166- pancreatic cancer cells were analyzed by invasion and migration assays, and in mouse xenograft models. We also performed quantitative RT-PCR and microarray analyses to evaluate the expression levels of CD166 and related genes in cultured cells.

Results

Immunohistochemistry revealed high expression of CD166 in pancreatic cancer tissues (12.2%; 12/98) compared with that in normal pancreas controls (0%; 0/17) (p = 0.0435). Flow cytometry indicated that CD166 was expressed in 33.8–70.2% of cells in surgical pancreatic tissues and 0–99.5% of pancreatic cancer cell lines. Invasion and migration assays demonstrated that CD166- pancreatic cancer cells showed stronger invasive and migratory activities than those of CD166+ cancer cells (p<0.05). On the other hand, CD166+ Panc-1 cells showed a significantly stronger colony formation activity than that of CD166- Panc-1 cells (p<0.05). In vivo analysis revealed that CD166+ cells elicited significantly greater tumor growth than that of CD166- cells (p<0.05) in both subcutaneous and orthotopic mouse tumor models. mRNA expression of the epithelial-mesenchymal transition activator Zeb1 was over-expressed in CD166- cells (p<0.001). Microarray analysis showed that TSPAN8 and BST2 were over-expressed in CD166+ cells, while BMP7 and Col6A1 were over-expressed in CD166- cells.

Conclusions

CD166+ pancreatic cancer cells are strongly tumorigenic, while CD166- pancreatic cancer cells exhibit comparatively stronger invasive and migratory activities. These findings suggest that CD166 expression is related to different functions in pancreatic cancer cells.     

Sperm Associated Antigen 9 Plays an Important Role in Bladder Transitional Cell Carcinoma

Background

Majority of bladder cancer deaths are caused due to transitional cell carcinoma (TCC) which is the most prevalent and chemoresistant malignancy of urinary bladder. Therefore, we analyzed the role of Sperm associated antigen 9 (SPAG9) in bladder TCC.

Methodology and Findings

We examined SPAG9 expression and humoral response in 125 bladder TCC patients. Four bladder cancer cell lines were assessed for SPAG9 expression. In addition, we investigated the effect of SPAG9 ablation on cellular proliferation, cell cycle, migration and invasion in UM-UC-3 bladder cancer cells by employing gene silencing approach. Our SPAG9 gene and protein expression analysis revealed SPAG9 expression in 81% of bladder TCC tissue specimens. High SPAG9 expression (>60% SPAG9 positive cells) was found to be significantly associated with superficial non-muscle invasive stage (P = 0.042) and low grade tumors (P = 0.002) suggesting SPAG9 putative role in early spread and tumorigenesis. Humoral response against SPAG9 was observed in 95% of patients found positive for SPAG9 expression. All four bladder cancer cell lines revealed SPAG9 expression. In addition, SPAG9 gene silencing in UM-UC-3 cells resulted in induction of G₀–G₁ arrest characterized by up-regulation of p16 and p21 and consequent down-regulation of cyclin E, cyclin D and cyclin B, CDK4 and CDK1. Further, SPAG9 gene silencing also resulted in reduction in cellular growth, and migration and invasion ability of cancer cells in vitro.

Conclusions

Collectively, our data in clinical specimens indicated that SPAG9 is potential biomarker and therapeutic target for bladder TCC.     

CXCR4/CXCL12 participate in extravasation of metastasizing breast cancer cells within the liver in a rat model

Introduction

Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer.

Methods

Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4.

Results

In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p<0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p<0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. Conclusion: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.     

Heat Shock Protein 60 Overexpression Is Associated with the Progression and Prognosis in Gastric Cancer

Background

Heat shock protein 60 (HSP60) is a chaperonin with essential functions for cell physiology and survival, and its expression correlates with prognosis in a number of malignancies. The aim of this study is to determine the relationship of HSP60 status with clinicopathological parameters and prognosis in gastric cancer.

Methods

The levels of HSP60 and matrix metallopeptidase 9 (MMP-9) antigen was evaluated by immunohistochemistry in 223 gastric carcinoma samples. The association between HSP60 and MMP-9, clinicopathological parameters, and prognosis of gastric cancer was examined.

Results

The level of HSP60 protein was significantly associated with depth invasion, lymph node metastasis and stage of disease (all P<0.05). Both univariate and multivariate analyses revealed that HSP60 was an independent prognostic factor for both overall survival (OS) and recurrence-free survival (RFS) (both P<0.05). Furthermore, HSP60 overexpression was associated with a poor prognosis in patients with advanced gastric cancer in different risk groups. Moreover, HSP60 was significantly correlated with MMP-9 among 223 gastric cancer tissues (P<0.001). Patients who had HSP60 overexpression, in which tumor cells displayed high invasiveness, had poor OS and shorter RFS.

Conclusion

HSP60 plays an important role on tumor aggressiveness and prognosis, and may act as a promising target for prognostic prediction.     

Anti-Metastatic and Anti-Tumor Growth Effects of Origanum majorana on Highly Metastatic Human Breast Cancer Cells: Inhibition of NFκB Signaling and Reduction of Nitric Oxide Production

Background

We have recently reported that Origanum majorana exhibits anticancer activity by promoting cell cycle arrest and apoptosis of the metastatic MDA-MB-231 breast cancer cell line. Here, we extended our study by investigating the effect of O . majorana on the migration, invasion and tumor growth of these cells.

Results

We demonstrate that non-cytotoxic concentrations of O . majorana significantly inhibited the migration and invasion of the MDA-MB-231 cells as shown by wound-healing and matrigel invasion assays. We also show that O . majorana induce homotypic aggregation of MDA-MB-231 associated with an upregulation of E-cadherin protein and promoter activity. Furthermore, we show that O . majorana decrease the adhesion of MDA-MB-231 to HUVECs and inhibits transendothelial migration of MDA-MB-231 through TNF-α-activated HUVECs. Gelatin zymography assay shows that O . majorana suppresses the activities of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9). ELISA, RT-PCR and Western blot results revealed that O . majorana decreases the expression of MMP-2, MMP-9, urokinase plasminogen activator receptor (uPAR), ICAM-1 and VEGF. Further investigation revealed that O . majorana suppresses the phosphorylation of IκB, downregulates the nuclear level of NFκB and reduces Nitric Oxide (NO) production in MDA-MB-231 cells. Most importantly, by using chick embryo tumor growth assay, we also show that O . majorana promotes inhibition of tumor growth and metastasis in vivo.

Conclusion

Our findings identify Origanum majorana as a promising chemopreventive and therapeutic candidate that modulate breast cancer growth and metastasis.     

Reduced Expression of Uroplakin 1A Is Associated with the Poor Prognosis of Gastric Adenocarcinoma Patients

Background

The aim of this study was to investigate the expression and prognostic significance of Uroplakin1A (UPK1A) in gastric adenocarcinoma patients. Functional studies were also analyzed in vitro.

Methodology/Principal Findings

Real-time quantitative PCR (RT-qPCR), western blotting, and immunohistochemical (IHC) staining methods were used to analyze the expression of UPK1A in primary gastric adenocarcinoma tissue samples. Compared with matched adjacent non-tumor, the expression of UPK1A in fresh surgical specimens was reduced, which was confirmed by RT-qPCR (P<0.01) and western blotting analysis (P<0.01). The paraffin specimens from a consecutive series of 445 gastric adenocarcinoma patients who underwent surgery between 2003 and 2006 were analyzed by IHC staining. The relationship between UPK1A expression, clinicopathological factors, and survival were evaluated. IHC staining analysis revealed that the reduced expression of UPK1A was observed in 224 cases (50.3%). Additionally, the correlation analysis of clinicopathological factors demonstrated that reduced expression of UPK1A was significantly associated with histological grade (P = 0.022), node metastasis (P<0.001) and tumor node metastasis (TNM) stage (P = 0.008) (7^th edition of the International Union Against Cancer (UICC)). Furthermore, Kaplan-Meier survival analysis revealed that the reduced expression of UPK1A was significantly associated with poor prognosis (P = 0.043). Cox hazards model analysis indicated that UPK1A expression was an independent risk factor at the 0.1 level (P = 0.094). The function of UPK1A in cell cycle, migration, and invasion was investigated by overexpressing UPK1A in the MKN45 gastric cancer cell line. The elevated expression of UPK1A cells induced G1 phase arrest and significantly inhibited migration and invasion.

Conclusions/Significance

The reduced expression of UPK1A might play a role in the progression of gastric cancer. Thus, UPK1A could be a potential favorable biomarker associated with gastric cancer prognosis.