Sensitization to the lysosomal cell death pathway upon immortalization and transformation

Tumorigenesis is associated with several changes that alter the cellular susceptibility to programmed cell death. Here, we show that immortalization and transformation sensitize cells in particular to the cysteine cathepsin-mediated lysosomal death pathway. Spontaneous immortalization increased the susceptibility of wild-type murine embryonic fibroblasts (MEFs) to tumor necrosis factor (TNF)-mediated cytotoxicity >1000-fold, whereas immortalized MEFs deficient for lysosomal cysteine protease cathepsin B (CathB) retained the resistant phenotype of primary cells. This effect was specific for cysteine cathepsins, because also lack of cathepsin L (a lysosomal cysteine protease), but not that of cathepsin D (a lysosomal aspartyl protease) or caspase-3 (the major executioner protease in classic apoptosis) inhibited the immortalization-associated sensitization of MEFs to TNF. Oncogene-driven transformation of immortalized MEFs was associated with a dramatic increase in cathepsin expression and additional sensitization to the cysteine cathepsin-mediated death pathway. Importantly, exogenous expression of CathB partially reversed the resistant phenotype of immortalized CathB-deficient MEFs, and the inhibition of CathB activity by pharmacological inhibitors or RNA interference attenuated TNF-induced cytotoxicity in immortalized and transformed wild-type cells. Thus, tumorigenesis-associated changes in lysosomes may counteract cancer progression and enhance therapeutic responses by sensitizing cells to programmed cell death.     

AKT pathway in neuroblastoma and its therapeutic implication

Neuroblastoma is a frequent pediatric tumor with a poor outcome in spite of aggressive treatment, even with autologous hematopoietic stem cell transplantation. The overall cure rate of 40% is unsatisfactory and new therapeutic strategies are urgently needed. AKT is a major mediator of survival signals that protect cells from apoptosis and regulate cell proliferation. The AKT signaling network is considered a key determinant of the biological aggressiveness of these tumors. In this article, the authors discuss the relation between activators of AKT in neuroblastoma, in particular, growth factors such as IGF-1, TRK, GDNF, VEGF and EGF, and their effects on tumoral proliferation, differentiation and apoptosis. Numerous other proteins interact with AKT in neuroblastoma. Several are relatively well characterized, such as PTEN and retinoic acid; others are new and potentially interesting, such as PKC and anaplastic lymphoma kinase. Specific inhibition of AKT has been studied, such as with LY249002, with significant effects on cell progression and apoptosis in tumoral cells. Moreover, a series of new drugs, such as geldanamycin and rapamycin, directly modify the expression of AKT in tumoral cells. Few specific inhibitors of AKT are available; less specific inhibitors are probably unsuitable therapeutic options in neuroblastoma. Drugs with a direct or indirect inhibitory effect on the AKT pathway, used alone or in combination with other drugs, seem to hold great promise as a new therapeutic modality in neuroblastoma.     

AKT pathway in neuroblastoma and its therapeutic implication

Neuroblastoma is a frequent pediatric tumor with a poor outcome in spite of aggressive treatment, even with autologous hematopoietic stem cell transplantation. The overall cure rate of 40% is unsatisfactory and new therapeutic strategies are urgently needed. AKT is a major mediator of survival signals that protect cells from apoptosis and regulate cell proliferation. The AKT signaling network is considered a key determinant of the biological aggressiveness of these tumors. In this article, the authors discuss the relation between activators of AKT in neuroblastoma, in particular, growth factors such as IGF-1, TRK, GDNF, VEGF and EGF, and their effects on tumoral proliferation, differentiation and apoptosis. Numerous other proteins interact with AKT in neuroblastoma. Several are relatively well characterized, such as PTEN and retinoic acid; others are new and potentially interesting, such as PKC and anaplastic lymphoma kinase. Specific inhibition of AKT has been studied, such as with LY249002, with significant effects on cell progression and apoptosis in tumoral cells. Moreover, a series of new drugs, such as geldanamycin and rapamycin, directly modify the expression of AKT in tumoral cells. Few specific inhibitors of AKT are available; less specific inhibitors are probably unsuitable therapeutic options in neuroblastoma. Drugs with a direct or indirect inhibitory effect on the AKT pathway, used alone or in combination with other drugs, seem to hold great promise as a new therapeutic modality in neuroblastoma.     

Induction of the lysosomal apoptosis pathway by inhibitors of the ubiquitin‐proteasome system

The lysosomal apoptosis pathway is a potentially interesting therapeutic target. Since apoptosis involving the lysosomal pathway has been described to involve cathepsins, we screened a drug library for agents that induce cathepsin‐dependent apoptosis. Using pharmacological inhibitors and siRNA, we identified 2 structurally related agents (NSC687852 and NSC638646) that induced cathepsin D‐dependent caspase‐cleavage activity in human breast cancer cells. Both agents were found to induce the mitochondrial apoptosis pathway. NSC687852 and NSC638646 were found to inhibit the activity of ubiquitin isopeptidases and to induce the accumulation of high‐molecular‐mass ubiquitins in cells. We show that 3 other inhibitors of the proteasome degradation pathway induce lysosomal membrane permeabilization (LMP) and that cathepsin‐D siRNA inhibits apoptosis induced by these agents. We conclude that a screen for cathepsin‐dependent apoptosis‐inducing agents resulted in the identification of ubiquitin isopeptidase inhibitors and that proteasome inhibitors with different mechanisms of action induce LMP and cathepsin D‐dependent apoptosis. © 2008 Wiley‐Liss, Inc.     

Induction of cell death in neuroblastoma by inhibition of cathepsins B and L

A specific irreversible inhibitor of both cathepsins B and L, Fmoc-Tyr-Ala-CHN₂ (FYAD) induced apoptosis of neuroblastoma cells but not other tumor cells. Cysteine protease inhibitors that were not efficient inhibitors of both proteases did not cause death of any cell line tested. Apoptosis was preceded by accumulation of large electron dense vesicles and multivesicular bodies in the cytoplasm. Exposure of cells to the cathepsin D inhibitor, pepstatin, failed to rescue cells from FYAD-induced death. These results indicate that inhibition of cathepsins B and L may provide a unique mechanism for selectively inducing death of neuroblastoma with limited toxicity to normal cells and tissues.     

Cathepsin B in infiltrated lymph nodes is of prognostic significance for patients with nonsmall cell lung carcinoma

BACKGROUND: Tumor cells require specific proteolytic enzymes for invasion and metastasis, including lysosomal peptidases--cathepsins. Cathepsin B is a lysosomal cysteine peptidase, which appears to play a major role in invasion and metastasis of human tumors. In this study, the authors focused on the possible role of cathepsin B in lymphogenic metastasis by investigating the enzyme localization and its activity in lung tumors and corresponding tumor-infiltrated lymph nodes. METHODS: Cathepsin B activity was determined in lung tumors, lung parenchyma, and tumor cell-infiltrated and noninfiltrated regional lymph nodes of the same patient. The authors investigated 35 cancer patients suffering from nonsmall cell lung carcinoma. Cathepsin B throughout activity was measured by cleavage of the fluorogenic substrate Z-Arg-Arg-AMC at pH 6.0. RESULTS: The median specific cathepsin B activity was highest in tumors, followed by the infiltrated lymph nodes, noninfiltrated lymph nodes, and lung parenchyma. The authors showed a significant 1.8-fold increase in cathepsin B activity in tumor-infiltrated lymph nodes compared with noninfiltrated regional lymph nodes and a 4.5-fold increase in lung tumor tissue compared with lung parenchyma. High cathepsin B activity, both in tumors and tumor cell-infiltrated lymph nodes, indicated poor prognosis for overall survival. Immunohistochemical analysis showed the presence of cathepsin B in histiocytes and tumor cells but not in lymphocytes of lymph node tissue. CONCLUSIONS: The authors' findings on higher cathepsin B levels in tumor cell-infiltrated lymph nodes show that increased level of cathepsin B activity is characteristic of the invasive tumor cell phenotype. This corroborates the hypothesis, that tumor cell associated cathepsin B may play a role in lymphogenic metastasis. The authors' results support the use of lymph node associated cathepsin B as a prognostic factor for survival of patients with lung carcinoma.     

Targeting cathepsin S induces tumor cell autophagy via the EGFR-ERK signaling pathway

Cathepsin S is a cellular cysteine protease, which is frequently over-expressed in human cancer cells and plays important role in tumor metastasis. However, the role of cathepsin S in regulating cancer cell survival and death remains undefined. The aim of this study was to determine whether targeting cathepsin S could induce autophagy/apoptosis in cancer cells. In this study, we demonstrated that targeting cathepsin S by either specific small molecular inhibitors or cathepsin S siRNA induced autophagy and subsequent apoptosis in human cancer cells, and the induction of autophagy was dependent on the phosphorylation of EGFR and activation of the EGFR-related ERK/MAPK-signaling pathway. In conclusion, the current study reveals that cathepsin S plays an important role in the regulation of cell autophagy through interference with the EGFR-ERK/MAPK-signaling pathway.     

Bobel-24 and derivatives induce caspase-independent death in pancreatic cancer regardless of apoptotic resistance

The poor prognosis of pancreatic cancer and poor sensitivity to current therapeutics, associated with resistance to apoptosis, urge the search for new drugs. We previously described the induction of caspase-independent mithochondrial death in leukemia cells by Bobel-24 (AM-24) and derivatives. Here, we explored whether these compounds induce a similar cytotoxicity in human pancreatic carcinoma cell lines (NP18, NP9, NP31, and NP29). Bobel-24 or Bobel-16 induced cytotoxicity and DNA synthesis inhibition in all cell lines and apoptosis in all lines, except for NP9. Caspase and/or poly(ADP-ribose) polymerase-1 (PARP-1) activity inhibition experiments showed that cytotoxicity was mainly induced through apoptosis in NP18 and through a caspase-independent process in NP9. Moreover, in NP29 or NP31 cell lines, both caspase-dependent and caspase-independent cell death mechanisms coexisted. Cell death was associated with reactive oxygen species (ROS) production, mitochondrial depolarization, cytochrome c and apoptosis-inducing factor (AIF) release, AIF nuclear translocation, and lysosomal cathepsin release. Inhibition of ROS production, mitochondrial pore permeability, PARP-1, or phospholipase A2 partially prevented cell death. Moreover, cathepsin B inhibition or down-regulation by small interfering RNA partially blocked cell death. In conclusion, Bobel-24 and derivatives trigger caspase-independent lysosomal and mitochondrial death in all tested human pancreatic cancer lines, irrespective of their degree of apoptotic sensitivity, becoming the only active cytotoxic mechanism in the apoptosis-resistant NP9 line. This mechanism may overcome the resistance to apoptosis observed in pancreatic carcinoma when treated with current genotoxic drugs.     

Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis

Invasion and metastasis of certain tumors are accompanied by increased mRNA protein levels and enzymatic activity of cathepsin L. Cathepsin L has also been suggested to play a role in the proteolytic cascades associated with apoptosis. To investigate the role of cathepsin L in brain tumor invasion and apoptosis, the human glioma cell line, IPTP, was stably transfected with full-length antisense and sense cDNA of cathepsin L. Down-regulation of cathepsin L by antisense cDNA significantly impaired (up to 70%) glioma cell invasion in vitro and markedly increased glioma cell apoptosis induced by staurosporine. Compared to control and parental cell lines, antisense down-regulation of cathepsin L was associated with an earlier induction of caspase-3 activity. Up-regulation of cathepsin L activity by sense cDNA was associated with reduced apoptosis and later induction of caspase-3 activity. Moreover, down-regulation of cathepsin L lowered the expression of antiapoptotic protein Bcl-2, whereas up-regulation increased the expression of Bcl-2, indicating that cathepsin L acts upstream of caspase-3. These data show that cathepsin L is an important protein mediating the malignancy of gliomas and its inhibition may diminish their invasion and lead to increased tumor cell apoptosis by reducing apoptotic threshold.     

Senescence-initiated reversal of drug resistance: specific role of cathepsin L

The present study was undertaken to verify whether induction of senescence could be sufficient to reverse drug resistance and, if so, to determine the underlying mechanism(s). Our findings indicated that cotreatment of drug-resistant neuroblastoma cells with doxorubicin, at sublethal concentrations, in combination with the pan-caspase inhibitor, Q-VD-OPH, elicited a strong reduction of cell viability that occurred in a caspase-independent manner. This was accompanied by the appearance of a senescence phenotype, as evidenced by increased p21/WAF1 expression and senescence-associated beta-galactosidase activity. Experiments using specific inhibitors of major cellular proteases other than caspases have shown that inhibition of cathepsin L, but not proteasome or cathepsin B, was responsible for the senescence-initiated reversal of drug resistance. This phenomenon appeared to be general because it was valid for other drugs and drug-resistant cell lines. A nonchemical approach, through cell transfection with cathepsin L small interfering RNA, also strongly reversed drug resistance. Further investigation of the underlying mechanism revealed that cathepsin L inhibition resulted in the alteration of intracellular drug distribution. In addition, in vitro experiments have demonstrated that p21/WAF1 is a substrate for cathepsin L, suggesting that inhibition of this enzyme may result in p21/WAF1 stabilization and its increased accumulation. All together, these findings suggest that cathepsin L inhibition in drug-resistant cells facilitates induction of senescence and reversal of drug resistance. This may represent the basis for a novel function of cathepsin L as a cell survival molecule responsible for initiation of resistance to chemotherapy.     

Loss of responsiveness to IGF-I in cells with reduced cathepsin L expression levels

The lysosomal cysteine proteinase cathepsin L is involved in proteolytic processing of internalized proteins. In transformed cells, where it is frequently overexpressed, its intracellular localization and functions can be altered. Previously, we reported that treatment of highly metastatic, murine carcinoma H-59 cells with small molecule cysteine proteinase inhibitors altered the responsiveness of the type I insulin-like growth factor (IGF-I) receptor and consequently reduced cell invasion and metastasis. To assess more specifically the role of cathepsin L in IGF-I-induced signaling and tumorigenicity, we generated H-59 subclones with reduced cathepsin L expression levels. These clonal lines showed an altered responsiveness to IGF-I in vitro, as evidenced by (i) loss of IGF-I-induced receptor phosphorylation and Shc recruitment, (ii) reduced IGF-I (but not IGF-II)-induced cellular proliferation and migration, (iii) decreased anchorage-independent growth and (iv) reduced plasma membrane levels of IGF-IR. These changes resulted in increased apoptosis in vivo and an impaired ability of the cells to form liver metastases. The results demonstrate that cathepsin L expression levels regulate cell responsiveness to IGF-I and thereby identify a novel function for cathepsin L in the control of the tumorigenic/metastatic phenotype.     

Lysosomotropic compounds and spermine enzymatic oxidation products in cancer therapy (review)

Apoptotic pathways represent the mechanisms of programmed cell death that counteract initiation and progression of cancer. New therapeutic targets are currently being explored on the basis of our detailed knowledge of the mechanisms and factors involved in apoptosis. In recent years, numerous proteins have been identified, which act as tumour suppressors or as oncoproteins in caspase-independent programmed cell death mechanisms, in which lysosomes are implicated for their lysosomal functions in cancer, mainly attributed to lysosomal proteinases, particularly the cathepsins. If cathepsins are released from the lysosomal lumen into the cytoplasm they initiate a number of processes that may cause either apoptotic or non-apoptotic (necrotic) cell death. The release of cathepsin D into the cytoplasm by vacuolar-type ATPase (V-ATPase) inhibitors produces the characteristic signs of apoptotic cell death, including caspase-3 activation and DNA laddering. For the destabilisation of the lysosomal membrane, two methods are available having therapeutic potential: the formation of reactive oxygen species (ROS) by irradiation or by enzymatic reactions and the lysosomal membrane permeabilisation by lysosomotropic compounds. Findings also suggest that the deregulation of polyamine metabolism or cytotoxic metabolites generated from the oxidative deamination of spermine by amine oxidases in association with lysosomotropic compounds may induce apoptosis. Cross-resistance of cells to cytotoxic actions of a wide variety of natural and synthetic anticancer drugs is the well-known phenomenon called multidrug resistance (MDR), due to glycoprotein P that functions as an ATP-dependent pump. The sensitisation of tumour cells to anticancer drugs by lysosomotropic compounds, and particularly the sensitisation of MDR-resistant cells recommend scrutinizing the potential of lysosomotropic drugs in cancer therapy.     

Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39

In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.     

Targeting GD2 ganglioside and aurora A kinase as a dual strategy leading to cell death in cultures of human neuroblastoma cells

The mechanism of the inhibitory effect of anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) on human neuroblastoma cells survival was studied in vitro. It was recently shown in IMR-32 cells that death induced by this antibody exhibited several characteristics typical of apoptosis. In this study we used cytotoxixity assays, qRT-PCR and immunoblotting to evaluate the response of several human neuroblastoma cell lines to the anti-GD2 14G2a mAb. We showed that the mAb decreases all three aurora kinases expression and phosphorylation in IMR-32 and LA-N-1 cells. Most importantly, we show, that MK-5108 specific aurora A kinase inhibitor decreases neuroblastoma cell survival, and when used in combination with the mAb, significantly potentiates cytotoxicity against IMR-32, CHP-134, and LA-N-5 neuroblastoma cells in vitro. It was shown that downregulation of aurora A kinase by the therapeutic antibody is associated with decreased levels of MYCN protein in cytoplasm, and induced expression of PHLDA1 and P53 proteins.     

Cyclooxygenase-2 is expressed in neuroblastoma, and nonsteroidal anti-inflammatory drugs induce apoptosis and inhibit tumor growth in vivo

Neuroblastoma is the single most common and deadly tumor of childhood and is often associated with therapy resistance. Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins. COX-2 is up-regulated in several adult epithelial cancers and is linked to proliferation and resistance to apoptosis. We detected COX-2 expression in neuroblastoma primary tumors and cell lines but not in normal adrenal medullas from children. Treatment of neuroblastoma cells with nonsteroidal anti-inflammatory drugs, inhibitors of COX, induced caspase-dependent apoptosis via the intrinsic mitochondrial pathway. Treatment of established neuroblastoma xenografts in nude rats with the dual COX-1/COX-2 inhibitor diclofenac or the COX-2-specific inhibitor celecoxib significantly inhibited tumor growth in vivo (P < 0.001). In vitro, arachidonic acid and diclofenac synergistically induced neuroblastoma cell death. This effect was further pronounced when lipooxygenases were simultaneously inhibited. Proton magnetic resonance spectroscopy ((1)H MRS) of neuroblastoma cells treated with COX inhibitors demonstrated accumulation of polyunsaturated fatty acids and depletion of choline compounds. Thus, (1)H MRS, which can be performed with clinical magnetic resonance scanners, is likely to provide pharmacodynamic markers of neuroblastoma response to COX inhibition. Taken together, these data suggest the use of nonsteroidal anti-inflammatory drugs as a novel adjuvant therapy for children with neuroblastoma.     

Caspase- and mitochondrial dysfunction-dependent mechanisms of lysosomal leakage and cathepsin B activation in DNA damage-induced apoptosis.

A lysosomal pathway, characterized by partial rupture of lysosomal membranes and cathepsin B activation, is activated during camptothecin (CPT)-induced apoptosis in U937 and Namalwa cancer cells. These lysosomal events occur simultaneously with mitochondrial permeabilization and caspase activation. In U937 cells, blocking mitochondrial permeability transition pore with cyclosporin A and bongkrekic acid reduces mitochondrial and lysosomal rupture, suggesting that lysosomal rupture may be dependent, in part, on mitochondrial disruption. Overexpressing bcl-xL, an antiapoptotic protein known to preserve mitochondrial functions, also impedes lysosomal and mitochondrial disruption in both cell lines, indicating signaling between the two organelles. In addition, no evidence was obtained of bcl-2-like proteins targeting lysosomes. Caspase activities, including caspase-2L, are required for lysosomal and mitochondrial disruption, and lysosomal cathepsin B slightly participates in apoptosis propagation after CPT, although not essential for apoptosis activation. Our study provides evidence for the participation of a lysosomal pathway during DNA damage-induced cell death. Our data suggest that caspase activation and mitochondrial disruption represent cell-context-specific mechanisms by which DNA damage leads to lysosomal rupture, and that lysosomal cathepsins could slightly participate in apoptosis propagation after CPT.     

Cathepsin-B and cathepsin-L expression levels do not correlate with sensitivity of tumour cells to TNF-alpha-mediated apoptosis

Recently, evidence has been accumulated that besides the caspase proteases, lysosomal cathepsins may play a role in apoptosis induction. This is especially significant as many human tumour cells express high levels of cathepsins, which might sensitise these cells to specific proapoptotic stimuli mediated by cathepsins. We found that TNF-alpha-mediated DNA fragmentation in tumour cells was significantly reduced in the presence of E64d and CA074Me, two inhibitors of lysosomal cysteine proteases. Transient transfection of cathepsin-B (Cath-B) and -L (Cath-L) resulting in expression levels comparable to those found in many tumours did not sensitise tumour cells to TNF-alpha-mediated apoptosis. As lysosomal proteases are thought to be activated by their release from this organelle into the cytosol, we used the lysosomotropic detergent N-dodecyl-imidazole-HCl (NDI-HCl) to disturb lysosomal integrity efficiently and trigger the release of its proteolytic content into the cytosol. Treatment of HeLa cells with NDI-HCl resulted in cell death, which, however, could also not be influenced by augmented Cath-B or -L expression levels. Therefore, our data do not support the hypothesis that the high Cath-B or -L expression levels frequently detected in tumour cells might be exploited to target selectively those tumours for an enhanced cell death effect induced by lysosomotropic agents.     

Inhibition of focal adhesion kinase and src increases detachment and apoptosis in human neuroblastoma cell lines

Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that is overexpressed in a number of human tumors including neuroblastoma, and regulates both cellular adhesion and survival. We have studied the effects of FAK inhibition upon neuroblastoma using adenovirus‐containing FAK‐CD (AdFAK‐CD). Utilizing an isogenic MYCN+/MYCN? neuroblastoma cell line, we found that the MYCN+ cells are more sensitive to FAK inhibition with AdFAK‐CD than their MYCN negative counterparts. In addition, we have shown that phosphorylation of Src is increased in the untreated isogenic MYCN? neuroblastoma cells, and that the decreased sensitivity of the MYCN? neuroblastoma cells to FAK inhibition with AdFAK‐CD is abrogated by the addition of the Src family kinase inhibitor, PP2. The results of the current study suggest that both FAK and Src play a role in protecting neuroblastoma cells from apoptosis, and that dual inhibition of these kinases may be important when designing therapeutic interventions for this tumor. © 2009 Wiley‐Liss, Inc.