The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator (uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion, survival and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145 and PC3 are prostate cancer cell lines with low, moderate and high metastatic potential, respectively, as demonstrated by their capacity to invade extracellular matrix (ECM). In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs (siRNAs), to target human uPA and uPAR. These siRNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrate that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNAi for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as extracellular-signal regulated kinases 1/2 (ERK1/2) and the signal transducer and activator of transcription 3 (Stat 3). In addition, our results demonstrate that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation and survival of prostate cancer cells. Thus, RNAi-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.
RNAi; prostate cancer; invasion; survival; uPA and uPAR; siRNA (small interfering RNA); shRNA (small hairpin RNA); RNAi (RNA interference); uPA (urokinaseplasminogen activator); uPAR (uPA receptor); ANOVA (analysis of variance); RT (reverse transcription); TPBS (tween-20 (0.1%) phospate buffered saline); BSA (bovine serum albumin)
Objective: Although aspirin has been associated with a reduction of the risk of cancer when used as a nonsteroidal anti-inflammatory drug, its use to reduce the risk of ovarian cancer is controversial. Ovarian cancer cells usually express high levels of cyclooxygenase-1 (COX)-1. Because aspirin is a rather selective inhibitor of COX-1, the ability of aspirin to reduce the risk of ovarian cancer may be dependent on the level of COX-1 expression in those cells. Furthermore, epidermal growth factor receptor (EGFR) is frequently overexpressed in the malignant phenotype of ovarian cancer leading to increased cell proliferation and survival. Here we investigated if aspirin attenuates EGFR-activated ovarian cancer cell growth in a COX-1 dependent manner.
Methods: Cell viability assays and Western blot analyses were used to determine the effect of aspirin on EGF-stimulated cell proliferation. Gene silencing and gene expression techniques were employed to knockdown or to express COX-1, respectively.
Results: Aspirin inhibited cell viability induced by EGF in a dose dependent manner in COX-1 positive ovarian cancer cells. On the other hand, aspirin had no effect on cell viability in COX-1 negative ovarian cancer cells. In particular, aspirin decreased phosphorylated Akt and Erk activated by EGF. COX-1 silencing in COX-1 positive cells attenuated the inhibitory effect of aspirin on EGF-stimulated cell viability. Furthermore, we developed a COX-1 expressing cell line (SKCOX-1) by stably transfecting COX-1 expression vector into COX-1 negative SKOV-3 cells. SKCOX-1 cells were more responsive to aspirin when compared to cells transfected with empty vector, and decreased EGF-activated Akt and Erk as well as cell viability.
Conclusions: Taken together, aspirin inhibits viability of ovarian cancer cells by blocking phosphorylation of Akt and Erk activated by EGF. Thus it may potentiate the therapeutic efficacy of drugs used to treat COX-1 positive ovarian cancer subsets.
ovarian cancer; aspirin; COX-1; EGF; Erk; Akt; cell viability.
Kaposi's sarcoma (KS), an enigmatic endothelial cell vascular neoplasm, is characterized by the proliferation of spindle shaped endothelial cells, inflammatory cytokines (ICs), growth factors (GFs) and angiogenic factors. KSHV is etiologically linked to KS and expresses its latent genes in KS lesion endothelial cells. Primary infection of human micro vascular endothelial cells (HMVEC-d) results in the establishment of latent infection and reprogramming of host genes, and cyclooxygenase-2 (COX-2) is one of the highly up-regulated genes. Our previous study suggested a role for COX-2 in the establishment and maintenance of KSHV latency. Here, we examined the role of COX-2 in the induction of ICs, GFs, angiogenesis and invasive events occurring during KSHV de novo infection of endothelial cells. A significant amount of COX-2 was detected in KS tissue sections. Telomerase-immortalized human umbilical vein endothelial cells supporting KSHV stable latency (TIVE-LTC) expressed elevated levels of functional COX-2 and microsomal PGE2 synthase (m-PGES), and secreted the predominant eicosanoid inflammatory metabolite PGE2. Infected HMVEC-d and TIVE-LTC cells secreted a variety of ICs, GFs, angiogenic factors and matrix metalloproteinases (MMPs), which were significantly abrogated by COX-2 inhibition either by chemical inhibitors or by siRNA. The ability of these factors to induce tube formation of uninfected endothelial cells was also inhibited. PGE2, secreted early during KSHV infection, profoundly increased the adhesion of uninfected endothelial cells to fibronectin by activating the small G protein Rac1. COX-2 inhibition considerably reduced KSHV latent ORF73 gene expression and survival of TIVE-LTC cells. Collectively, these studies underscore the pivotal role of KSHV induced COX-2/PGE2 in creating KS lesion like microenvironment during de novo infection. Since COX-2 plays multiple roles in KSHV latent gene expression, which themselves are powerful mediators of cytokine induction, anti-apoptosis, cell survival and viral genome maintainence, effective inhibition of COX-2 via well-characterized clinically approved COX-2 inhibitors could potentially be used in treatment to control latent KSHV infection and ameliorate KS.
Kaposi's sarcoma associated herpes virus (KSHV), with a 160 kb DNA genome, has evolved with two distinct life cycle phases, namely latency and lytic replication. KS, a complex angioproliferative disease, is regulated by a balance between pro-angiogenic and anti-angiogenic factors. In our previous study, we showed that KSHV modulates host factors COX-2/PGE2 for its own advantage to promote its latent (persistent) infection. The premise that COX-2 is involved in growth and progression of several types of solid cancers and inflammation associated diseases has been well documented but has never been studied in KS. Here, utilizing COX-2 inhibition strategies, including chemical inhibition and a gene silencing approach, we systematically identified the potential role of KSHV induced COX-2/PGE2 in viral pathogenesis related events such as secretion of inflammatory and angiogenic cytokines, MMPs and cell adhesion in de novo infected or latently infected endothelial cells. We report that COX-2/PGE2 inhibition down-regulates viral latent gene expression and survival of latently infected endothelial cells. The data emanating from our in vitro studies is valuable, informative and requires further examination using an in vitro angiogenic model and in vivo nude mice model to further validate COX-2 as a novel therapeutic to target latent infection and the associated diseases like KS.
Many epidemiological studies demonstrate that treatment with non-steroidal anti-inflammatory drugs (NSAIDs) reduce the incidence and mortality of certain malignancies, especially gastrointestinal cancer. The cyclooxygenase (COX) enzymes are well-known targets of NSAIDs. However, conventional NSAIDs non-selectively inhibit both the constitutive form COX-1, and the inducible form COX-2. Recent evidence indicates that COX-2 is an important molecular target for anticancer therapies. Its expression is undetectable in most normal tissues, and is highly induced by pro-inflammatory cytokines, mitogens, tumor promoters and growth factors. It is now well-established that COX-2 is chronically overexpressed in many premalignant, malignant, and metastastic cancers, including hepatocellular carcinoma (HCC). Overexpression of COX-2 in patients with HCC is generally higher in well-differentiated HCCs compared with less-differentiated HCCs or histologically normal liver, suggesting that COX-2 may be involved in the early stages of hepatocarcinogenesis, and increased expression of COX-2 in noncancerous liver tissue has been significantly associated with shorter disease-free survival in patients with HCC.
In tumors, overexpression of COX-2 leads to an increase in prostaglandin (PG) levels, which affect many mechanisms involved in carcinogenesis, such as angiogenesis, inhibition of apoptosis, stimulation of cell growth as well as the invasiveness and metastatic potential of tumor cells.
The availability of novel agents that selectively inhibit COX-2 (COXIB), has contributed to shedding light on the role of this molecule. Experimental studies on animal models of liver cancer have shown that NSAIDs, including both selective and non-selective COX-2 inhibitors, exert chemopreventive as well as therapeutic effects. However, the key mechanism by which COX-2 inhibitors affect HCC cell growth is as yet not fully understood.
Increasing evidence suggests the involvement of molecular targets other than COX-2 in the anti-proliferative effects of COX-2 selective inhibitors. Therefore, COX-inhibitors may use both COX-2-dependent and COX-2-independent mechanisms to mediate their antitumor properties, although their relative contributions toward the in vivo effects remain less clear.
Here we review the features of COX enzymes, the role of the expression of COX isoforms in hepatocarcinogenesis and the mechanisms by which they may contribute to HCC growth, the pharmacological properties of COX-2 selective inhibitors, the antitumor effects of COX inhibitors, and the rationale and feasibility of COX-2 inhibitors for the treatment of HCC.
Cyclooxygenase-2; Cyclooxygenase-1; Hepatocellular carcinoma; Non-steroidal anti-inflammatory drugs; Inhibit cyclooxygenase-2
Meningioma is a well-known tumor of the central nervous system, and is treated by surgical resection and/or radiation. Recently, ionizing radiation has been shown to enhance invasiveness of surviving tumor cells, and several proteolytic enzyme molecules, including urokinase plasminogen activator (uPA), seem to be upregulated after radiation. uPA and its receptor (uPAR) have been strongly implicated in tumor invasion, angiogenesis and progression. Hence, the tumor-associated uPA-uPAR system is considered a potential target for cancer therapy. In the present study, we show that radiation increases uPA levels in the IOMM-Lee meningioma cells, and subsequently, increases tumor invasion, migration and angiogenesis in vitro. Studies with signaling molecule inhibitors AG1478, U0126 and SB203580 (specific inhibitors of EGFR, MEK1/2 and p38 respectively) showed inhibition of uPA levels in both basal and irradiated-IOMM-Lee cells. The PI3K inhibitor (LY294002) and the AKT inhibitor (AKT inhibitor IV) also partially decreased uPA expression, whereas SP600125, a JNK inhibitor, did not affect uPA levels in either radiated or non-radiated cells. Further, a bicistronic plasmid construct with small interfering RNA (siRNA) against uPA and its receptor inhibited tumor invasion, migration and angiogenesis in radiation-treated IOMM-Lee cells. In addition, siRNA against uPA and its receptor inhibited subcutaneous tumor growth in athymic nude mice in combination with radiation in a synergistic manner. Thus, the specific targeting of proteases via RNA interference could augment the therapeutic effect of radiation and prevent the adverse effects resulting from tumor cells that receive sublethal doses of radiation within the tumor mass.
Cyclo-oxygenase (COX)-2 expression correlates directly with highly aggressive and metastatic breast cancer, but the mechanism underlying this correlation remains obscure. We hypothesized that invasive human breast cancer cells that over-express COX-2 have the unique ability to differentiate into extracellular-matrix-rich vascular channels, also known as vasculogenic mimicry. Vascular channels have been associated with angiogenesis without involvement of endothelial cells, and may serve as another mechanism by which tumor cells obtain nutrients to survive, especially in less vascularized regions of the tumor.
To determine whether COX-2 regulates vascular channel formation, we assessed whether treatment with celecoxib (a selective COX-2 inhibitor) or silencing COX-2 synthesis by siRNA inhibits vascular channel formation by breast cancer cell lines. Cell lines were selected based on their invasive potential and COX-2 expression. Additionally, gene expression analysis was performed to identify candidate genes involved in COX-2-induced vascular channel formation. Finally, vascular channels were analyzed in surgically resected human breast cancer specimens that expressed varying levels of COX-2.
We found that invasive human breast cancer cells that over-express COX-2 develop vascular channels when plated on three-dimensional matigel cultures, whereas non-invasive cell lines that express low levels of COX-2 did not develop such channels. Similarly, we identified vascular channels in high-grade invasive ductal carcinoma of the breast over-expressing COX-2, but not in low-grade breast tumors. Vascular channel formation was significantly suppressed when cells were treated with celecoxib or COX-2 siRNA. Inhibition of channel formation was abrogated by addition of exogenous prostaglandin E2. In vitro results were corroborated in vivo in tumor-bearing mice treated with celecoxib. Using gene expression profiling, we identified several genes in the angiogenic and survival pathways that are engaged in vascular channel formation.
Antivascular therapies targeting tumor cell vasculogenic mimicry may be an effective approach to the treatment of patients with highly metastatic breast cancer.
Reactive oxygen species (ROS) production via NADPH oxidase (NOX) contributes to various types of cancer progression. In the present research, we examined the pathobiological role of NADPH oxidase (NOX)4-mediated generation of reactive oxygen species (ROS) in urothelial carcinoma (UC) of the urinary bladder, and demonstrated the utility of ROS labeling in urine cytology.
NOX4 gene was silenced in vivo and in vitro by NOX4 siRNA transfection with or without atlocollagen. Cell cycle and measurement of ROS were analyzed by flowcytometry. Orthotopic implantation animal model was used in vivo experiment. NOX4 expression in urothelial carcinoma cells was observed by immunohistochemical analysis using surgical specimens of human bladder cancer. Urine cytology was performed after treatment with ROS detection reagents in addition to Papanicolaou staining.
NOX4 was overexpressed in several UC cell lines and the NOX inhibitor, diphenylene iodonium reduced intracellular ROS and induced p16-dependent cell cycle arrest at the G1 phase. Moreover, silencing of NOX4 by siRNA significantly reduced cancer cell growth in vivo as assessed in an orthotopic mouse model. Immunohistochemistry demonstrated high expression of NOX4 in low grade/non-invasive and high grade/invasive UC including precancerous lesions such as dysplasia but not in normal urothelium. Then, we assessed the usefulness of cytological analysis of ROS producing cells in urine (ROS-C). Urine samples obtained from UC cases and normal controls were treated with fluorescent reagents labeling the hydrogen peroxide/superoxide anion and cytological atypia of ROS positive cells were analyzed. As a result, the sensitivity for detection of low grade, non-invasive UC was greatly increased (35% in conventional cytology (C-C) vs. 75% in ROS-C), and the specificity was 95%. Through ROS-C, we observed robust improvement in the accuracy of follow-up urine cytology for cases with previously diagnosed UC, especially in those with low grade/non-invasive cancer recurrence (0% in C-C vs. 64% in ROS-C).
This is the first report demonstrating that ROS generation through NOX4 contributes to an early step of urothelial carcinogenesis and cancer cell survival. In addition, cytology using ROS labeling could be a useful diagnostic tool in human bladder cancer.
The PTEN/Phosphatidylinositol 3'-kinase (PI3-kinase) growth factor signaling pathway plays a critical role in epithelial tumor development in a multitude of tissue types. Deletion of the Pten tumor suppressor gene in murine urothelial cells in vivo results in upregulation of cyclin-dependent kinase inhibitor p21. We have previously shown in mice that p21 expression blocks an increase in urothelial cell proliferation due to Pten deletion. In this study, we utilized human urothelial carcinoma cells UMUC-3 and UMUC-14 to identify the signaling pathways downstream of PI3-kinase that regulate p21.
Cells were treated with a combination of PI3-kinase stimulating growth factors and kinase inhibitors, or transfected with exogenous genes in order to identify the signaling events that are necessary for p21 induction. Mice with conditional deletion of Pten in bladder urothelium were also examined for evidence of PI3-kinase pathway signaling events that affect p21 expression.
When cells were treated with PI3-kinase activating growth factors EGF or PDGF, we found that p21 levels increased, in a manner similar to that observed in mice. We used the inhibitors LY294002, Akti-1/2, and rapamycin, to show that p21 induction is dependent upon PI3-kinase and AKT activity, and partially dependent on mTOR. We treated the cells with proteasome inhibitor MG-132 and found that p21 may be degraded in the proteasome to regulate protein levels. Importantly, our findings show that GSK-3β plays a role in diminishing p21 levels in cells. Treatment of cells with the GSK-3β inhibitor SB-216763 increased p21 levels, while exogenous expression of GSK-3β caused a decrease in p21, indicating that GSK-3β actively reduces p21 levels. We found that a combined treatment of LY294002 and SB-216763 improved the cytotoxic effect against UMUC-3 and UMUC-14 carcinoma cells over LY294002 alone, suggesting potential therapeutic uses for GSK-3β inhibitors. Immunohistochemical staining in bladders from wild-type and Pten-deleted mice indicated that GSK-3β inhibitory phosphorylation increases when Pten is deleted.
PI3-kinase and AKT cause an upregulation of p21 by suppressing GSK-3β activity and activating mTOR in both cultured human urothelial carcinoma cells and mouse urothelial cells in vivo.
AIM: To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters.
METHODS: uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a non-cancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR) mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay.
RESULTS: The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA.
CONCLUSION: uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.
Bile duct cancer; Cholangiocarcinoma; Cancer invasion; Urokinase plasminogen activator; Cancer metastasis
Increased levels of urokinase-type plasminogen activator (uPA) are associated with shortened overall survival in ovarian cancer patients. Additionally, elevated levels of the serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1), a uPA inhibitor, have also been correlated with an unfavorable prognosis in ovarian cancer. Therefore, it is critical to understand the signaling pathways that regulate PAI-1 and uPA expression in cancer cell migration-invasion.
We studied the PI3K/Akt, Rho kinase/ROCK, p38 MAPK and MEK pathways and their modulation of PAI-1 and uPA expression and wound-induced cell migration in SKOV-3 ovarian cancer cells. The PI3K/Akt pathway was further examined using pharmacological inhibitors (LY294002 and wortmannin), Akt siRNA, constitutively active Akt adenovirus and treatment with IGF-1/insulin in the SKOV-3 cells.
The PI3K/Akt pathway negatively regulates PAI-1 expression and positively correlates with migratory abilities and uPA expression in SKOV-3 cells. A reduction in active Akt results in an increase in PAI-1 expression coupled with a decrease in uPA expression to ultimately result in reduced cell migration and invasion. By contrast, an increase in Akt activity reduces PAI-1 expression and results in an increase in SKOV-3 wound-induced cell migration. Furthermore, IGF-1 and insulin stimulated SKOV-3 migration by altering the balance between uPA and PAI-1 to favor uPA, and the enhanced migration was attenuated by treatment with LY294002 indicating PI3K/Akt in this pathway.
These results suggest an overall ovarian tumor-protective role for PAI-1, and that the PI3K/Akt signaling pathway regulates the ratio of PAI-1:uPA to either increase or decrease cell migration.
Plasminogen activator system; PAI-1; Urokinase; Migration and invasion; SKOV-3 cells; PI3K/Akt signaling system
Cyclooxygenase (COX)-2 has been implicated in tumour progression, angiogenesis and metastasis in non-small cell lung cancer (NSCLC). We speculated that inhibition of COX-2 activity might reduce expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in lung cancer cells.
The levels of IL-8, VEGF and prostaglandin E2 (PGE2) were measured by ELISA. Expression of COX-1 and COX-2 was determined by Western blotting. Inhibition or knockdown of COX-2 was achieved by treating NSCLC cells with specific COX-2 inhibitor NS-398 or COX-2 siRNA, respectively.
We found that NSCLC cell lines produced more IL-8 than VEGF (p < 0.001). In contrast, small cell lung cancer (SCLC) cell lines produced more VEGF than IL-8 (p < 0.001). COX-1 was expressed in all cell lines, but COX-2 was expressed only in NSCLC cell lines. Consistent with this, PGE2 was significantly higher in NSCLC cell lines than SCLC cell lines (p < 0.001). We tested these cell lines with a potent specific COX-2 inhibitor NS-398 at concentrations of 0.02, 0.2, 2, 20 μM for 24 or 48 h. The COX-2 activity was reduced in a dose-dependent fashion as shown by reduced PGE2 production. VEGF was significantly reduced following the treatment of NS-398 in A549 (by 31%) and MOR/P (by 47%) cells lines which expressing strong COX-2, but not in H460 cell line which expressing very low COX-2. However, IL-8 was not reduced in these cell lines. To confirm these results, we knocked down COX-2 expression with COX-2 siRNA in these cell lines. VEGF was significantly decreased in A549 (by 24%) and in MOR/P (by 53%), but not in H460 whereas IL-8 was not affected in any cell line.
We conclude that NSCLC cells produce much higher levels of IL-8 than SCLC cells whereas both NSCLC and SCLC cells produce similar levels of VEGF. COX-2 is only expressed in NSCLC cells, but not in SCLC cells. VEGF is produced in both NSCLC and SCLC cells regardless of COX-2 expression. However, VEGF production is, at least partly, COX-2 dependent in NSCLC cells expressing COX-2. In contrast, IL-8 production is COX-2 independent in both NSCLC and SCLC cells. We speculate that combined targeting of COX-2 and IL-8 may be useful in the treatment of patients with NSCLC and targeting VEGF may be useful in the treatment of patients with SCLC.
Previous studies have shown that class-I histone deacetylase (HDAC) 8 mRNA is upregulated in urothelial cancer tissues and urothelial cancer cell lines compared to benign controls. Using urothelial cancer cell lines we evaluated whether specific targeting of HDAC8 might be a therapeutic option in bladder cancer treatment.
We conducted siRNA-mediated knockdown and specific pharmacological inhibition of HDAC8 with the three different inhibitors compound 2, compound 5, and compound 6 in several urothelial carcinoma cell lines with distinct HDAC8 expression profiles. Levels of HDAC and marker proteins were determined by western blot analysis and mRNA levels were measured by quantitative real-time PCR. Cellular effects of HDAC8 suppression were analyzed by ATP assay, flow cytometry, colony forming assay and migration assay.
Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%. The HDAC8 specific inhibitors compound 5 and compound 6 significantly reduced viability of all urothelial cancer cell lines (IC50 9 – 21 μM). Flow cytometry revealed only a slight increase in the sub-G1 fraction indicating a limited induction of apoptosis. Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected. The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.
Deregulation of HDAC8 is frequent in urothelial cancer, but neither specific pharmacological inhibition nor siRNA-mediated knockdown of HDAC8 impaired viability of urothelial cancer cell lines in a therapeutic useful manner. Accordingly, HDAC8 on its own is not a promising drug target in bladder cancer.
Histone deacetylase 8; Histone deacetylase inhibitor; Urothelial bladder cancer; Cell cycle arrest
uPA/uPAR is a multifunctional system that is over expressed in many cancers and plays a critical role in glioblastoma (GBM) invasion. Previous studies from our lab have also shown that uPA/uPAR down regulation inhibits cancer cell invasion in SNB 19 GBM cells.
As Notch 1 is known to be over expressed and promotes invasion in glioblastoma, we therefore tested our hypothesis of whether down regulation of uPA/uPAR, singly or in tandem, attenuates GBM invasion via Notch 1 receptor. Targeted down regulation of uPA/uPAR, either singly or simultaneously, inhibited the anchorage independent growth of U251MG and GBM xenograft cell lines 4910 and 5310 as assessed by soft agar colony formation assay. Expression of all four Notch receptors was confirmed in GBM tissue array analysis by immunohistochemistry.
Down regulation of uPA/uPAR, either singly or simultaneously, in U251 MG and tumor xenografts inhibited the cleavage of the Notch receptor between the Gly 1743 and Val 1744 positions, thereby suggesting inhibition of activated cytosolic fragment-related Notch gene transcription. Morphological analysis confirmed inhibition of NICD when U251 MG cells were treated with puPA, puPAR or pU2. uPA/uPAR down regulation inhibited Notch 1 mRNA in all three examined cell lines. uPA/uPAR shRNA down regulated nuclear activation of NF-κB subunits and phosphorylation of AKT/mTOR pathway in U251 MG and GBM xenografts. puPA down regulated NICD and HES induced phosphorylation of AKT/ERK and NF-κB. Down regulation of Notch 1 using siRNA inhibited uPA activity as shown by fibrinogen zymography. It also decreased uPA expression levels as shown by western blotting. Exogenous addition of uPA activated Notch 1 in uPAR antisense U251 MG cells and also in uPAR antisense cells transfected with siRNA against Delta and Jagged. The Notch 1 receptor co-localized with LAMP-1, a marker for lysosomes in uPA, uPAR and U2, down regulated U251 MG cells which probably indicates inhibition of Notch 1 receptor trafficking in GBM cells. Notch 1 expression was significantly inhibited in puPA- and pU2-treated pre-established intracranial tumors in mice.
Overall our results show that down regulation of uPA/uPAR, either singly or simultaneously, could be an effective approach to attenuate Notch 1 receptor cleavage, signaling and endosomal trafficking in U251MG cells and xenografts, and ultimately inhibiting GBM invasion.
uPA/uPAR; invasion; glioblastoma; Notch 1 receptor
Bladder cancer is the most common malignant tumor of the urinary tract and remains one of the major causes of cancer death worldwide. In this study, we investigated the effect and mechanism of Artesunate (ART), a traditional Chinese medicine, on inducing apoptosis of human bladder cancer cells. In vivo antitumor activity was investigated in bladder cancer in rat by subcutaneous injection of different concentration of ART. The effect of ART on growth inhibition and apoptosis of bladder cancer cells was evaluated using dimethylthiazoly-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. Cyclooxygenase-2 (COX-2) and miR-16 expression levels were determined with real-time PCR. The concentrations of prostaglandin E2 (PGE2) in the supernatants of bladder cancer cells were measured with an ELISA kit. The miR-16 inhibitor or mimic were transfected into cells to up- or down-regulate miR-16 expression. ART efficiently inhibited orthotopic tumor growth in the bladder cancer rat, which is accompanied with an increase of miR-16 expression and a decrease of COX-2 expression. In vitro, ART could induce cytotoxicity and apoptosis in bladder cancer cells, but presented a much lighter toxicity effect against normal human urothelial cells. ART significantly increased miR-16 expression and decreased the expression of COX-2 and the production of PGE2. More importantly, down-regulation of miR-16 expression could reverse the effect of ART on apoptosis and COX-2 expression in bladder cells. Moreover, exogenous PGE2 could inhibit apoptosis of bladder cancer cells treated with ART. In conclusion, ART can elicit an anti-tumor effect against bladder cancer by up-regulation of miR-16 expression, which resulted in the decrease of COX-2 expression and PGE2 production. Hence, ART might be an effective drug for the treatment of bladder cancer.
Artesunate; bladder cancer; apoptosis; miR-16; COX-2
Cyclooxygenase (COX)-2 is overexpressed in many types of cancers including hepatocellular carcinoma (HCC). Meloxicam, a selective COX-2 inhibitor, has shown potential therapeutic effects against HCC, but the mechanisms accounting for its anti-cancer activities remain unclear.
Methods and Findings
Meloxicam inhibited the ability of human HCC cells expressing higher levels of COX-2 to migrate, invade, adhere and form colonies through upregulating the expression of E-cadherin and downregulating the expression of matrix metalloproteinase (MMP) -2. Meloxicam induced cell apoptosis by upregulating pro-apoptotic proteins including Bax and Fas-L, and downregulating anti-apoptotic proteins including survivin and myeloid cell leukemia-1 (Mcl-1), through inhibiting phosphorylation of AKT. Addition of prostaglandin E2 (PGE2), the major product of COX-2, could abrogate the effects of meloxicam on the expression of survivin and myeloid cell leukemia-1 (Mcl-1), but not Bax and Fas-L, indicating that meloxicam induces cell apoptosis via both COX-2-dependent and -independent pathways. Meloxicam also induced cell autophagy by upregulating Beclin 1 and light chain 3-II. Specific inhibition of autophagy by 3-methyladenine and chloroquine had little effect on cell apoptosis but could enhance the pro-apoptotic effects of meloxicam by further upregulating the expression of Bax.
Meloxicam executes its antitumor effects by targeting the COX-2/MMP-2/E-cadherin, AKT, apoptotic and autophagic pathways in COX-2-dependent and -independent pathways, and inhibition of cell autophagy could help to overcome the resistance to meloxicam-induced apoptosis in HCC.
Members of the urokinase-type plasminogen activator (uPA) system including uPA, its receptor uPAR and the plasminogen activator inhibitor 1 (PAI-1) play an important role in tumour invasion and progression in a variety of tumour types. Since the majority of clear cell renal cell carcinoma (ccRCC) shows distant metastasis at time of diagnosis or later, the interplay of uPA, uPAR and PAI-1 might be of importance in this process determining the patients’ outcome.
Corresponding pairs of malignant and non-malignant renal tissue specimens were obtained from 112 ccRCC patients without distant metastasis who underwent tumour nephrectomy. Tissue extracts prepared from fresh-frozen tissue samples by detergent extraction were used for the determination of antigen levels of uPA, uPAR and PAI-1 by ELISA. Antigen levels were normalised to protein concentrations and expressed as ng per mg of total protein.
Antigen levels of uPA, uPAR, and PAI-1 correlated with each other in the malignant tissue specimens (rs=0.51-0.65; all P<0.001). Antigen levels of uPA system components were significantly higher in tissue extracts of non-organ confined tumours (pT3+4) compared to organ-confined tumours (pT1+2; all P<0.05). Significantly elevated levels of uPAR and PAI-1 were also observed in high grade ccRCC. When using median antigen levels as cut-off points, all three uPA system factors were significant predictors for disease-specific survival (DSS) in univariate Cox’s regression analyses. High levels of uPA and uPAR remained independent predictors for DSS with HR=2.86 (95% CI 1.07-7.67, P=0.037) and HR=4.70 (95% CI 1.51-14.6, P=0.008), respectively, in multivariate Cox’s regression analyses. A combination of high antigen levels of uPA and/or uPAR further improved the prediction of DSS in multivariate analysis (HR=14.5, 95% CI 1.88-111.1, P=0.010). Moreover, high uPA and/or uPAR levels defined a patient subgroup of high risk for tumour-related death in ccRCC patients with organ-confined disease (pT1+2) (HR=9.83, 95% CI 1.21-79.6, P=0.032).
High levels of uPA and uPAR in tumour tissue extracts are associated with a significantly shorter DSS of ccRCC patients without distant metastases.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-974) contains supplementary material, which is available to authorized users.
PAI-1; Prognostic biomarker; Renal cell carcinoma; uPA; uPAR; uPA system
Background & Aims
Resistance to apoptosis is essential for cancer growth. We previously reported that hepatic co-expression of c-Myc and E2F1, two key regulators of proliferation and apoptosis, enhanced HCC development in transgenic mice. Here, we investigated the molecular mechanisms underlying oncogenic cooperation between c-Myc and E2F1 in relationship to human liver cancer.
Activation of pro- and anti-apoptotic cascades was assessed by immunoblotting in in vivo and in vitro HCC models, and in primary human HCC. Effect of antisense oligodeoxy nucleotides against c-Myc and E2F1 was studied in human HCC cell lines. Suppression of PIK3CA/AKT, mTOR, and COX-2 pathways was achieved by pharmacological inhibitors and specific siRNAs in human and mouse HCC cell lines.
Co-expression with E2F1 did not increase proliferation triggered by c-Myc overexpression but conferred a strong resistance to c-Myc-initiated apoptosis via concomitant induction of PIK3CA/Akt/mTOR and c-Myb/COX-2 survival pathways. COX-2 was not induced in c-Myc and rarely in E2F1 tumors. In human HCC, PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways were similarly activated, with levels of PIK3CA/Akt, mTOR, and c-Myb being inversely associated with patients’ survival length. Knocking down c-Myc and E2F1 oncoproteins reduced PIK3CA/Akt and mTOR and completely abolished c-Myb and COX-2 expression in human HCC cell lines. Finally, simultaneous inhibition of PIK3CA/Akt/mTOR and COX-2 activity in in vitro models caused massive apoptosis of neoplastic hepatocytes.
E2F1 may function as a critical anti-apoptotic factor both in human and rodent liver cancer through its ability to counteract c-Myc-driven apoptosis via activation of PIK3CA/Akt/mTOR and c-Myb/COX-2 pathways.
Urokinase-type plasminogen activator (uPA) is associated with cancer recurrence where the most evidence comes from studies in breast cancer. According to the European Organization for Research and Treatment of Cancer, uPA is considered one of the most prominent biomarkers for cancer recurrence and therefore new agents are needed to inhibit it. Whether uPA is also expressed in pediatric cancers is yet unknown. If it is then uPA inhibitors might also help children with recurrent cancers. In this study, we addressed whether the integrin-linked kinase inhibitor (ILK), QLT0267, could suppress uPA. We previously showed that uPA expression is maximally inhibited when both the Akt and MAP kinase pathways were blocked which we anticipated can be achieved via QLT0267. In MDA-MB-231 breast cancer cells, QLT0267 blocked signaling through Akt and MAP kinase with a correlative decrease in uPA protein and mRNA, which corresponded to an inhibition of c-Jun phosphorylation. Consistent with these findings, cellular invasion was inhibited with either QLT0267 or with small interfering RNA against ILK. We then questioned whether uPA was commonly expressed in childhood sarcomas and if QLT0267 might be effective in this setting. We determined for the first time that uPA was highly expressed in rhabdomyosarcomas (RMS), but not Ewings sarcomas by screening cell lines (n = 31) and patient samples (n = 200) using Affymetrix microarrays. In alveolar RMS (ARMS) cell lines, QLT0267 blocked cell signaling, uPA production, invasion and ultimately survival. We concluded that QLT0267 blocks the production of uPA providing a new target for the management of recurrent cancers.
urokinase-type plasminogen activator; integrin-linked kinase; breast cancer; rhabdomyosarcoma; recurrence
Osteopontin (OPN) is one of important molecular targets in cancer progression, metastasis as a calcium-binding, extracellular-matrix-associated protein of the small integrin-binding ligand and, N-linked glycoprotein. In the present study, anti-metastatic mechanism of ethanol extracts of Ocimum sanctum (EEOS) was elucidated on OPN enhanced metastasis in NCI-H460 non- small cell lung cancer cells.
Cell viability was measured by MTT assay. Adhesion and invasion assays were carried out to see that EEOS inhibited cell adhesion and invasion in OPN treated and non-treated NCI-H 460 cells. RT-PCR was used to determine the mRNA levels of uPA, uPAR, and EGFR.
EEOS significantly inhibited cell adhesion and invasion in OPN treated and non treated NCI-H460 cells, though EEOS did not show any toxicity up to 200 μg/ml. EEOS effectively attenuated the expression of OPN and CD44 and also OPN activated the expression of CD44 in NCI-H460 cells. In addition, EEOS effectively suppressed the expression of phosphatidylinositide 3-kinases (PI3K) and cyclooxygenase 2 (COX-2) and the phosphorylation of Akt at protein level in OPN treated NCI-H460 cells. Also, EEOS significantly attenuated the expression of urokinase plasminogen activator (uPA), its receptor (uPAR) and epidermal growth factor receptor (EGFR) at mRNA level and reduced vascular endothelial growth factor (VEGF) production and MMP-9 activity in OPN treated NCI-H460 cells. Furthermore, PI3K/Akt inhibitor LY294002 enhanced anti-metastatic potential of EEOS to attenuate the expression of uPA and MMP-9 in OPN treated NCI-H 460 cells.
Overall, our findings suggest that anti-metastatic mechanism of EEOS is mediated by inhibition of PI3K/Akt in OPN treated NCI-H460 non-small cell lung cancer cells.
EEOS; Metastasis; Osteopontin; uPA; uPAR; PI3K
Non-melanoma skin cancer (NMSC) is the most common type of skin cancer in Caucasian populations. Its increasing incidence has been a major public health concern. Elevated expressions of ODC and COX-2 are associated with both murine and human NMSCs. Inhibition of these molecular targets singly employing their respective small molecule inhibitors showed limited success. Here, we show that combined blockade of ODC and COX-2 using their potent inhibitors, DFMO and diclofenac respectively abrogates growth of A431 epidermal xenograft tumors in nu/nu mice by more than 90%. The tumor growth inhibition was associated with a diminution in the proliferation and enhancement in apoptosis. The proliferation markers such as PCNA and cyclin D1 were reduced. TUNEL-positive apoptotic cells and cleaved caspase-3 were increased in the residual tumors. These agents also manifested direct target-unrelated effects. Reduced expression of phosphorylated MAPKAP-2, ERK, and Akt (ser473 & thr308) were noticed. The mechanism by which combined inhibition of ODC/COX attenuated tumor growth and invasion involved reduction in EMT. Akt activation by ODC+COX-2 over-expression was the key player in this regard as Akt inhibition manifested effects similar to those observed by the combined inhibition of ODC+COX-2 whereas forced over-expression of Akt resisted against DFMO+diclofenac treatment. These data suggest that ODC+COX-2 over-expression together leads to pathogenesis of aggressive and invasive cutaneous carcinomas by activating Akt signaling pathway, which through augmenting EMT contributes to tumor invasion.
The prognostic value of cyclooxygenase-2 (COX-2) in human renal cell carcinoma (RCC) remains unclear. The purposes of this study are to elucidate the clinical significance of COX-2 in clear cell RCC (CCRCC) and to assess the treatment effect of COX-2 inhibition on CCRCC cell lines.
Using tumor samples obtained from 137 patients who had undergone nephrectomy at Seoul National University Hospital, we evaluated COX-2 expression on immunohistochemistry. Moreover, we performed the cell proliferation assay using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H tetrazolium bromide (MTT) and cell invasion assay. Thus, we evaluated the effect of meloxicam, an inhibitor of COX-2, in two human CCRCC cell lines.
Cancer-specific survival (p=0.038) and progression-free survival (p=0.031) were shorter in the COX-2 high expression group. A multivariate logistic regression model showed that COX-2 expression was an independent risk factor for pTNM stage and Fuhrman nuclear grade. The MTT assay revealed that COX-2 inhibition led to the suppression of the proliferation of CCRCC cell lines. Moreover, it also reduced their invasion capacity.
This study postulates that COX-2 is a poor prognostic indicator in human CCRCC, suggesting that COX-2 inhibition can be a potential therapy in CCRCC.
Carcinoma, renal cell; Cyclooxygenase 2; Prognosis
An in vitro model was developed to understand if celecoxib could synergize with Mitomycin C (MMC), commonly used for the prevention of non-muscle invasive bladder cancer recurrence, and eventually elucidate if the mechanism of interaction involves multi drug resistance (MDR) transporters.
UMUC-3, a non COX-2 expressing bladder cancer cell line, and UMUC-3-CX, a COX-2 overexpressing transfectant, as well as 5637, a COX-2 overexpressing cell line, and 5637si-CX, a non COX-2 expressing silenced 5637 cell line, were used in the present study. The expression of COX-2 and MDR pumps (P-gp, MDR-1 and BCRP) was explored through western blot. The anti-proliferative effect of celecoxib and MMC was studied with MTT test. Three biological permeability assays (Drug Transport Experiment, Substrate Transporter Inhibition, and ATP cell depletion) were combined to study the interaction between MDR transporters and celecoxib. Finally, the ability of celecoxib to restore MMC cell accumulation was investigated.
The anti-proliferative effect of celecoxib and MMC were investigated alone and in co-administration, in UMUC-3, UMUC-3-CX, 5637 and 5637si-CX cells. When administered alone, the effect of MMC was 8-fold greater in UMUC-3. However, co-administration of 1 μM, 5 μM, and 10 μM celecoxib and MMC caused a 2,3-fold cytotoxicity increase in UMUC-3-CX cell only. MMC cytotoxicity was not affected by celecoxib co-administration either in 5637, or in 5637si-CX cells. As a result of all finding from the permeability experiments, celecoxib was classified as P-gp unambiguous substrate: celecoxib is transported by MDR pumps and interferes with the efflux of MMC. Importantly, among all transporters, BCRP was only overexpressed in UMUC-3-CX cells, but not in 5637 and 5637si-CX.
The UMUC-3-CX cell line resembles a more aggressive phenotype with a lower response to MMC compared to the wt counterpart. However, the administration of celecoxib in combination to MMC causes a significant and dose dependent gain of the anti-proliferative activity. This finding may be the result of a direct interaction between celecoxib and MDR transporters. Indeed, BCRP is overexpressed in UMUC-3-CX, but not in UMUC-3, 5637, and 5637si-CX, in which celecoxib is ineffective.
Cyclooxygenase-2; Multidrug resistance; Bladder cancer
Understanding the complex, multistep process of metastasis remains a major challenge in cancer research. Metastasis models can reveal insights in tumor development and progression and provide tools to test new intervention strategies.
To develop a new cancer metastasis model, we used DU145 human prostate cancer cells and performed repeated rounds of orthotopic prostate injection and selection of subsequent lymph node metastases. Tumor growth, metastasis, cell migration and invasion were analyzed. Microarray analysis was used to identify cell migration- and cancer-related genes correlating with metastasis. Selected genes were silenced using siRNA, and their roles in cell migration and invasion were determined in transwell migration and Matrigel invasion assays.
Our in vivo cycling strategy created cell lines with dramatically increased tumorigenesis and increased ability to colonize lymph nodes (DU145LN1-LN4). Prostate tumor xenografts displayed increased vascularization, enlarged podoplanin-positive lymphatic vessels and invasive margins. Microarray analysis revealed gene expression profiles that correlated with metastatic potential. Using gene network analysis we selected 3 significantly upregulated cell movement and cancer related genes for further analysis: EPCAM (epithelial cell adhesion molecule), ITGB4 (integrin β4) and PLAU (urokinase-type plasminogen activator (uPA)). These genes all showed increased protein expression in the more metastatic DU145-LN4 cells compared to the parental DU145. SiRNA knockdown of EpCAM, integrin-β4 or uPA all significantly reduced cell migration in DU145-LN4 cells. In contrast, only uPA siRNA inhibited cell invasion into Matrigel. This role of uPA in cell invasion was confirmed using the uPA inhibitors, amiloride and UK122.
Our approach has identified genes required for the migration and invasion of metastatic tumor cells, and we propose that our new in vivo model system will be a powerful tool to interrogate the metastatic cascade in prostate cancer.
Prostate cancer; Invasion; Migration; Metastasis; Angiogenesis; Lymphangiogenesis; Lymph node; EpCAM; Integrin; Beta4; uPA; New model
The role of human papillomavirus (HPV) in bladder carcinogenesis remains controversial. Overexpression of p16INK4a, a surrogate marker for infection with oncogenic HPV in other tumours, has been described for urothelial carcinoma in situ (UCIS). Our goal was therefore to evaluate whether overexpression of p16INK4a is associated with HPV infection and to identify mechanisms of p16INK4a upregulation in UCIS.
Materials and Methods
In 60 tissue specimens from a total of 45 patients (UCIS and controls), we performed p16INK4a immunohistochemistry followed by detection and subclassification of HPV DNA. In a subset of samples, we tested for gene amplification of p16INK4a applying fluorescence in situ hybridization (FISH). RAS/MAPK signalling and epithelial-mesenchymal transition (EMT) was assessed using immunohistochemistry. Finally, we transfected urothelial carcinoma cells with KRAS and examined the expression of p16INK4a as well as markers of EMT.
We found overexpression of p16INK4a in 92.6% of UCIS and in all cervical intraepithelial neoplasia (CIN) controls. In contrast, we detected high-risk HPV DNA in 80% of CIN, but none in UCIS. There was no gene amplification of p16INK4a. High levels of phosphorylated kinases and urokinase plasminogen activator (uPA) and loss of membraneous E-cadherin were detected in UCIS. KRAS transfection of urothelial carcinoma cells led to upregulation of p16INK4a and uPA accompanied by loss of E-cadherin that could be inhibited by application of the kinase-inhibitor Sorafenib.
Our results show that overexpression of p16INK4a in UCIS is neither associated with HPV infection nor p16INK4a gene amplification but is a consequence of enhanced RAS/MAPK signalling that promotes EMT, possibly due to Sorafenib-sensitive paracrine secretion of the EMT activator uPA. These findings might open a novel therapeutic option for localized but aggressive urothelial cancer.
Urothelial bladder cancer is the ninth most common cancer. Despite surgical and chemotherapeutic treatment the prognosis is still poor once bladder cancer progresses to a muscle-invasive state. Discovery of new diagnostic markers and pathophysiologic effectors might help to contribute to novel diagnostic and therapeutic options. The extracellular matrix microenvironment shaped by the extracellular matrix critically affects tumor cell and stroma cell functions. Therefore, aim of the present study was to assess the possible implication of the small leucine-rich proteoglycan biglycan in progression of human urothelial bladder cancer.
Methods and Results
For this purpose tumor biopsies of 76 bladder cancer patients with different tumor stages (pTa, pT1-T4) were investigated with respect to biglycan expression and correlated with a long-term (10 years) clinical follow-up. Interestingly, higher biglycan mRNA expression was associated with higher tumor stages and muscle invasiveness. In
vitro knock-down of endogenous biglycan in human urothelial carcinoma cells (J82 cells) increased proliferation, whereas addition of recombinant biglycan and overexpression of biglycan inhibited tumor cell proliferation. In line with this growth-inhibitory effect of biglycan, transplantation of J82 cells after knock-down of biglycan resulted in significantly increased growth of subcutaneous xenograft tumors in nude mice in
vivo. Furthermore, treatment with two anti-proliferative, multi-receptor tyrosine kinase inhibitors—sunitinib and sorafenib—strongly upregulated biglycan expression. Collectively, the experimental data suggest that high biglycan expression is associated with reduced tumor cell proliferation. In accordance, Kaplan-Meier analysis revealed higher 10-year survival in patients with high biglycan mRNA expression in tumor biopsies.
In conclusion, the present data suggest that biglycan is an endogenous inhibitor of bladder cancer cell proliferation that is upregulated in response to anti-proliferative tyrosine kinase inhibitors. In addition, high biglycan expression is associated with favorable prognosis.