Cathepsin B as a cancer target Gondi, Christopher S; Rao, Jasti S
Expert opinion on therapeutic targets,
03/2013, Letnik:
17, Številka:
3
Journal Article
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Introduction:
Cathepsin B is of significant importance to cancer therapy as it is involved in various pathologies and oncogenic processes in humans. Numerous studies have shown that abnormal ...regulation of cathepsin B overexpression is correlated with invasive and metastatic phenotypes in cancers. Cathepsin B is normally associated with the lysosomes involved in autophagy and immune response, but its aberrant expression has been shown to lead to cancers.
Areas covered:
This review highlights the oncogenic role of cathepsin B, discusses the regulation of cathepsin B in light of oncogenesis, discusses the role of cathepsin B as a signaling molecule, and highlights the therapeutic potential of targeting cathepsin B.
Expert opinion:
Targeting cathepsin B alone does not appear to abolish tumor growth, and this is probably because cathepsin B appears to have diverse functions and influence numerous pathways. It is not clear whether global suppression of cathepsin B activity or expression would produce unintended effects or cause the activation or suppression of unwanted pathways. A localized approach for targeting the expression of cathepsin B would be more relevant. Moreover, a combination of targeting cathepsin B with other relevant oncogenic molecules has significant therapeutic potential.
Since its initial characterization, pancreatic ductal adenocarcinoma has remained one of the most devastating and difficult cancers to treat. Pancreatic cancer is the fourth leading cause of death in ...the United States, resulting in an estimated 38460 deaths annually. With few screening tools available to detect this disease at an early stage, 94% of patients will die within five years of diagnosis. Despite decades of research that have led to a better understanding of the molecular and cellular signaling pathways in pancreatic cancer cells, few effective therapies have been developed to target these pathways. Other treatment options have included more sophisticated pancreatic cancer surgeries and combination therapies. While outcomes have improved modestly for these patients, more effective treatmentsare desperately needed. One of the greatest challenges in the future of treating this malignancy will be to develop therapies that target the tumor microenvironment and surrounding pancreatic cancer stem cells in addition to pancreatic cancer cells. Recent advances in targeting pancreatic stellate cells and the stroma have encouraged researchers to shift their focus to the role of desmoplasia in pancreatic cancer pathobiology in the hopes of developing newer-generation therapies. By combining novel agents with current cytotoxic chemotherapies and radiation therapy and personalizing them to each patient based on specific biomarkers, the goal of prolonging a patient’s life could be achieved. Here we review the most effective therapies that have been used for the treatment of pancreatic cancer and discuss the future potential of therapeutic options.
Pancreatic cancer is one of the most lethal types of cancer in the world. The incidence of pancreatic cancer increases each year with no significant decrease in mortality. Pancreatic cancer is a ...complex disease, and this complexity is partly attributed to late diagnosis, an aggressive phenotype, environmental factors and lack of effective treatment options. Surgical resection followed by adjuvant chemotherapy is the treatment of choice for early stage cancer, whereas gemcitabine is the standard first line therapy for patients with advanced stage disease. Treatment regimens comprising folinic acid, 5-fluorouracil, irinotecan, oxaliplatin and nab-paclitaxel have demonstrated modest effects in improving median survival rates. A number of other chemotherapeutics are currently undergoing clinical trials as components of combination therapies with gemcitabine. An increasing number of novel molecular targets and cellular pathways are being identified, which highlights the complexity of this disease. The development of chemoresistance to gemcitabine is multifactorial and there exists an interplay between pancreatic cancer cells, the tumor microenvironment and cancer stem cells. These components appear to be governed by a complex network of non-coding RNAs such as micro RNAs and long non-coding RNAs. In the present study, studies describing previous research on the understanding of the factors associated with the development of chemoresistance to gemcitabine in pancreatic cancer are reviewed. A comprehensive understanding of the multiple pathways of chemoresistance is key to develop next generation therapeutics to pancreatic cancer.
uPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts.
To investigate whether cholesterol could regulate uPAR ...and MMP-9 in breast carcinoma, we used MβCD (methyl beta cyclodextrin, which extracts cholesterol from lipid rafts) to disrupt lipid rafts and studied its effect on breast cancer cell migration, invasion, angiogenesis and signaling.
Morphological evidence showed the association of uPAR with lipid rafts in breast carcinoma cells. MβCD treatment significantly reduced the colocalization of uPAR and MMP-9 with lipid raft markers and also significantly reduced uPAR and MMP-9 at both the protein and mRNA levels. Spheroid migration and invasion assays showed inhibition of breast carcinoma cell migration and invasion after MβCD treatment. In vitro angiogenesis studies showed a significant decrease in the angiogenic potential of cells pretreated with MβCD. MβCD treatment significantly reduced the levels of MMP-9 and uPAR in raft fractions of MDA-MB-231 and ZR 751 cells. Phosphorylated forms of Src, FAK, Cav, Akt and ERK were significantly inhibited upon MβCD treatment. Increased levels of soluble uPAR were observed upon MβCD treatment. Cholesterol supplementation restored uPAR expression to basal levels in breast carcinoma cell lines. Increased colocalization of uPAR with the lysosomal marker LAMP1 was observed in MβCD-treated cells when compared with untreated cells.
Taken together, our results suggest that cholesterol levels in lipid rafts are critical for the migration, invasion, and angiogenesis of breast carcinoma cells and could be a critical regulatory factor in these cancer cell processes mediated by uPAR and MMP-9.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
With the lack of specific signs and symptoms, pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at late metastatic stages, resulting in poor survival outcomes. Among various biomarkers, ...microRNA-21 (miR-21), a small non-coding RNA, is highly expressed in PDAC. By inhibiting regulatory proteins at the 3′ untranslated regions (UTR), miR-21 holds significant roles in PDAC cell proliferation, epithelial–mesenchymal transition, angiogenesis, as well as cancer invasion, metastasis, and resistance therapy. We conducted a systematic search across major databases for articles on miR-21 and pancreatic cancer mainly published within the last decade, focusing on their diagnostic, prognostic, therapeutic, and biological roles. This rigorous approach ensured a comprehensive review of miR-21’s multifaceted role in pancreatic cancers. In this review, we explore the current understandings and future directions regarding the regulation, diagnostic, prognostic, and therapeutic potential of targeting miR-21 in PDAC. This exhaustive review discusses the involvement of miR-21 in proliferation, epithelial–mesenchymal transition (EMT), apoptosis modulation, angiogenesis, and its role in therapy resistance. Also discussed in the review is the interplay between various molecular pathways that contribute to tumor progression, with specific reference to pancreatic ductal adenocarcinoma.
Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are ...strongly correlated with invasive cancer phenotypes.
In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results.
In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Medulloblastoma is a highly invasive cancer of central nervous system diagnosed mainly in children. Matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator receptor (uPAR) are over ...expressed in several cancers and well established for their roles in tumor progression. The present study is aimed to determine the consequences of targeting these molecules on medulloblastoma progression.
Radiation is one of the foremost methods applied for treating cancer and considerable evidence showed that radiation elevated uPAR and MMP-9 expression in medulloblastoma cell. Therefore efforts are made to target these molecules in non-irradiated and irradiated medulloblastoma cells. Our results showed that siRNA-mediated knockdown of uPAR and MMP-9, either alone or in combination with radiation modulated a series of events leading to apoptosis. Down regulation of uPAR and MMP-9 inhibited the expression of anti-apoptotic molecules like Bcl-2, Bcl-xL, survivin, XIAP and cIAPI; activated BID cleavage, enhanced the expression of Bak and translocated cyctochrome C to cytosol. Capsase-3 and -9 activities were also increased in uPAR- and MMP-9-downregulated cells. The apoptosis induced by targeting MMP-9 and uPAR was initiated by inhibiting epidermal growth factor receptor (EGFR) mediated activation of STAT3 and NF-κB related signaling molecules. Silencing uPAR and MMP-9 inhibited DNA binding activity of STAT3 and also reduced the recruitment of STAT3 protein at the promoter region of Bcl-2 and survivin genes. Our results suggest that inhibiting uPAR and MMP-9 reduced the expression of anti-apoptotic molecules by inactivating the transcriptional activity of STAT3. In addition, treating pre-established medulloblastoma with siRNAs against uPAR and MMP-9 both alone or in combination with radiation suppressed uPAR, MMP-9, EGFR, STAT3 expression and induced Bak activation leading to apoptosis.
Taken together, our results illustrated that RNAi mediated targeting of uPAR and MMP-9 might have therapeutic potential against medulloblastoma.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cathepsin B belongs to a family of cathepsins and plays an important role in normal physiological functions in the cell. However, overexpression of cathepsin B has been associated with different ...malignancies, and this has made it an attractive pharmacological target. The advent of CRISPR-Cas9 technology has allowed researchers to efficiently knock down genes with very less nonspecific activity compared to earlier methods. The protocol described below will enable investigators to develop cathepsin B knockdown stable cells and explains ways to study the knockdown.
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 and 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 the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA 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 demonstrated 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. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated 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, RNA interference-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.
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