Microarray Analysis Gene Expression Profiles in Laryngeal Muscle After Recurrent Laryngeal Nerve Injury.

OBJECTIVES: The pathophysiology of recurrent laryngeal nerve (RLN) transection injury is rare in that it is characteristically followed by a high degree of spontaneous reinnervation, with reinnervation of the laryngeal adductor complex (AC) preceding that of the abducting posterior cricoarytenoid (PCA) muscle. Here, we aim to elucidate the differentially expressed myogenic factors following RLN injury that may be at least partially responsible for the spontaneous reinnervation. METHODS: F344 male rats underwent RLN injury (n = 12) or sham surgery (n = 12). One week after RLN injury, larynges were harvested following euthanasia. The mRNA was extracted from PCA and AC muscles bilaterally, and microarray analysis was performed using a full rat genome array. RESULTS: Microarray analysis of denervated AC and PCA muscles demonstrated dramatic differences in gene expression profiles, with 205 individual probes that were differentially expressed between the denervated AC and PCA muscles and only 14 genes with similar expression patterns. CONCLUSIONS: The differential expression patterns of the AC and PCA suggest different mechanisms of reinnervation. The PCA showed the gene patterns of Wallerian degeneration, while the AC expressed the gene patterns of reinnervation by adjacent axonal sprouting. This finding may reveal important therapeutic targets applicable to RLN and other peripheral nerve injuries.

Drug and apoptosis resistance in cancer stem cells: a puzzle with many pieces.

Resistance to anticancer agents and apoptosis results in cancer relapse and is associated with cancer mortality. Substantial data have provided convincing evidence establishing that human cancers emerge from cancer stem cells (CSCs), which display self-renewal and are resistant to anticancer drugs, radiation, and apoptosis, and express enhanced epithelial to mesenchymal progression. CSCs represent a heterogeneous tumor cell population and lack specific cellular targets, which makes it a great challenge to target and eradicate them. Similarly, their close relationship with the tumor microenvironment creates greater complexity in developing novel treatment strategies targeting CSCs. Several mechanisms participate in the drug and apoptosis resistance phenotype in CSCs in various cancers. These include enhanced expression of ATP-binding cassette membrane transporters, activation of various cytoprotective and survival signaling pathways, dysregulation of stemness signaling pathways, aberrant DNA repair mechanisms, increased quiescence, autophagy, increased immune evasion, deficiency of mitochondrial-mediated apoptosis, upregulation of anti-apoptotic proteins including c-FLIP [cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein], Bcl-2 family members, inhibitors of apoptosis proteins, and PI3K/AKT signaling. Studying such mechanisms not only provides mechanistic insights into these cells that are unresponsive to drugs, but may lead to the development of targeted and effective therapeutics to eradicate CSCs. Several studies have identified promising strategies to target CSCs. These emerging strategies may help target CSC-associated drug resistance and metastasis in clinical settings. This article will review the CSCs drug and apoptosis resistance mechanisms and how to target CSCs.

4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) targets mRNA of the c-FLIP variants and induces apoptosis in MCF-7 human breast cancer cells.

Cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor for the tumor necrosis factor-related apoptosis-inducing ligand TRAIL and in drug resistance in human malignancies. c-FLIP is an antagonist of caspases-8 and -10, which inhibits apoptosis and is expressed as long (c-FLIP(L)) and short (c-FLIP(S)) splice forms. c-FLIP is often overexpressed in various human cancers, including breast cancer. Several studies have shown that silencing c-FLIP by specific siRNAs sensitizes cancer cells to TRAIL and anticancer agents. However, systemic use of siRNA as a therapeutic agent is not practical at present. In order to reduce or inhibit c-FLIP expression, small molecules are needed to allow targeting c-FLIP without inhibiting caspases-8 and -10. We used a small molecule inhibitor of c-FLIP, 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), and show that CMH, but not its inactive analog, downregulated c-FLIP(L) and c-FLIP(S) mRNA and protein levels, caused poly(ADP-ribose) polymerase (PARP) degradation, reduced cell survival, and induced apoptosis in MCF-7 breast cancer cells. These results revealed that c-FLIP is a critical apoptosis regulator that can serve as a target for small molecule inhibitors that downregulate its expression and serve as effective targeted therapeutics against breast cancer cells.

Resistance to drugs and cell death in cancer stem cells (CSCs).

Human cancers emerge from cancer stem cells (CSCs), which are resistant to cancer chemotherapeutic agents, radiation, and cell death. Moreover, autophagy provides the cytoprotective effect which contributes to drug resistance in these cells. Furthermore, much evidence shows that CSCs cause tumor initiation, progression, metastasis, and cancer recurrence. Various signaling pathways including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), maternal embryonic leucine zipper kinase (MELK), NOTCH1, and Wnt/ß-catenin as well as the CSC markers maintain CSC properties. Several mechanisms including overexpression of ABC multidrug resistance transporters, a deficiency in mitochondrial-mediated apoptosis, upregulation of c-FLIP, overexpression of anti-apoptotic Bcl-2 family members and inhibitors of apoptosis proteins (IAPs), and PI3K/AKT signaling contribute to enhancing resistance to chemotherapeutic drugs and cell death induction in CSCs in various cancers. Studying such pathways may help provide detailed understanding of CSC mechanisms of resistance to chemotherapeutic agents and apoptosis and may lead to the development of effective therapeutics to eradicate CSCs.

Epithelial-mesenchymal transition: a hallmark in pancreatic cancer stem cell migration, metastasis formation, and drug resistance.

Metastasis, tumor progression, and chemoresistance are the major causes of death in patients with pancreatic ductal adenocarcinoma (PDAC). Tumor dissemination is associated with the activation of an epithelial-to-mesenchymal transition (EMT) process, a program by which epithelial cells lose their cell polarity and cell-to-cell adhesion, and acquire migratory and invasive abilities to become mesenchymal stem cells (MSC). These MSCs are multipotent stromal cells capable of differentiating into various cell types and trigger the phenotypic transition from an epithelial to a mesenchymal state. Therefore, EMT promotes migration and survival during cancer metastasis and confers stemness features to particular subsets of cells. Furthermore, a major problem limiting our ability to treat PDAC is the existence of rare populations of pancreatic cancer stem cells (PCSCs) or cancer-initiating cells in pancreatic tumors. PCSCs may represent sub-populations of tumor cells resistant to therapy which are most crucial for driving invasive tumor growth. These cells are capable of regenerating the cellular heterogeneity associated with the primary tumor when xenografted into mice. Therefore, the presence of PCSCs has prognostic relevance and influences the therapeutic response of tumors. PCSCs express markers of cancer stem cells (CSCs) including CD24, CD133, CD44, and epithelial specific antigen as well as the drug transporter ABCG2 grow as spheroids in a defined growth medium. A major difficulty in studying tumor cell dissemination and metastasis has been the identification of markers that distinguish metastatic cancer cells from cells that are normally circulating in the bloodstream or at sites where these cells metastasize. Evidence highlights a linkage between CSC and EMT. In this review, The current understanding of the PCSCs, signaling pathways regulating these cells, PDAC heterogeneity, EMT mechanism, and links between EMT and metastasis in PCSCs are summarised. This information may provide potential therapeutic strategies to prevent EMT and trigger CSC growth inhibition and cell death.

Etoposide induces protein kinase Cdelta- and caspase-3-dependent apoptosis in neuroblastoma cancer cells.

In this report, we reveal that etoposide inhibits the proliferation of SK-N-AS neuroblastoma cancer cells and promotes protein kinase Cdelta (PKCdelta)- and caspase-dependent apoptosis. Etoposide induces the caspase-3-dependent cleavage of PKCdelta to its active p40 fragment, and active PKCdelta triggers the processing of caspase-3 by a positive-feedback mechanism. Treatment of cells with the caspase-3-specific inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone or caspase-3-specific small interacting RNA (siRNA) prevented the etoposide-induced activation of caspase-8 and inhibited apoptosis. The silencing of the caspase-2 or caspase-8 genes using siRNAs did not affect the etoposide-induced processing of caspase-3, indicating that these caspases lie downstream of caspase-3 in this signaling pathway. Furthermore, the etoposide-induced processing of caspase-2 required the expression of caspase-8, and the etoposide-mediated processing of caspase-8 required the expression of caspase-2, indicating that these two caspases activate each other after etoposide treatment. We also observed that etoposide-mediated apoptosis was decreased by treating the cells with the caspase-6-specific inhibitor benzyloxycarbonyl-Val-Glu(OMe)-Ile-Asp-(OMe)-fluoromethyl ketone and that caspase-6 was activated by a caspase-8-dependent mechanism. Finally, we show that rottlerin blocks etoposide-induced apoptosis by inhibiting the PKCdelta-mediated activation of caspase-3 and by degrading caspase-2, which prevents caspase-8 activation. Our results add important insights into how etoposide mediates apoptotic signaling and how targeting these pathways may lead to the development of novel therapeutics for the treatment of neuroblastomas.

Dynamic assessment of mitoxantrone resistance and modulation of multidrug resistance by valspodar (PSC833) in multidrug resistance human cancer cells.

P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, is one of the major causes for multidrug resistance (MDR). We report using confocal microscopy to study the roles of Pgp in mediating the efflux of the anticancer agent mitoxantrone and the reversal of MDR by the specific Pgp inhibitor valspodar (PSC833). The net uptake and efflux of mitoxantrone and the effect of PSC833 were quantified and compared in Pgp-expressing human cancer MDA-MB-435 (MDR) cells and in parental wild-type cells. The MDR cells, transduced with the human Pgp-encoding gene MDR1 construct, were approximately 8-fold more resistant to mitoxantrone than the wild-type cells. Mitoxantrone accumulation in the MDR cells was 3-fold lower than that in the wild-type cells. The net uptake of mitoxantrone in the nuclei and cytoplasm of MDR cells was only 58 and 67% of that in the same intracellular compartment of the wild-type cells. Pretreatment with PSC833 increased the accumulation of mitoxantrone in the MDR cells to 85% of that in the wild-type cells. In living animals, the accumulation of mitoxantrone in MDA-MB-435mdr xenograft tumors was 61% of that in the wild-type tumors. Administration of PSC833 to animals before mitoxantrone treatment increased the accumulation of mitoxantrone in the MDR tumors to 94% of that in the wild-type tumors. These studies have added direct in vitro and in vivo visual information on how Pgp processes anticancer compounds and how Pgp inhibitors modulate MDR in resistant cancer cells.

RNA interference in cancer: targeting the anti-apoptotic protein c-FLIP for drug discovery.

The silencing of genes by RNA interference (RNAi) has identified proteins involved in the resistance of cancers to chemotherapeutic drugs. Resistance is associated with defects in the apoptotic signaling pathways. In this article, we examine using RNAi to target the anti-apoptotic protein cellular FLICE-like inhibitory protein (c-FLIP) for drug development.

The Xpc gene markedly affects cell survival in mouse bone marrow.

The XPC protein (encoded by the xeroderma pigmentosum Xpc gene) is a key DNA damage recognition factor that is required for global genomic nucleotide excision repair (G-NER). In contrast to transcription-coupled nucleotide excision repair (TC-NER), XPC and G-NER have been reported to contribute only modestly to cell survival after DNA damage. Previous studies were conducted using fibroblasts of human or mouse origin. Since the advent of Xpc-/- mice, no study has focused on the bone marrow of these mice. We used carboplatin to induce DNA damage in Xpc-/- and strain-matched wild-type mice. Using several independent methods, Xpc-/- bone marrow was approximately 10-fold more sensitive to carboplatin than the wild type. Importantly, 12/20 Xpc-/- mice died while 0/20 wild-type mice died. We conclude that G-NER, and XPC specifically, can contribute substantially to cell survival. The data are important in the context of cancer chemotherapy, where Xpc gene status and G-NER may be determinants of response to DNA-damaging agents including carboplatin. Additionally, altered cell cycles and altered DNA damage signalling may contribute to the cell survival end point.

Human beta-galactoside alpha-2,3-sialyltransferase (ST3Gal III) attenuated Taxol-induced apoptosis in ovarian cancer cells by downregulating caspase-8 activity.

Taxol triggers apoptosis in a variety of cancer cells, but it also upregulates cytoprotective proteins and/or pathways that compromise its therapeutic efficacy. In this report, we found that Taxol treatment resulted in caspase-8-dependent apoptosis in SKOV3 human ovarian cancer cells. Moreover, Taxol-induced apoptosis was associated with caspase-3 activation. Interestingly, Taxol treatment upregulated alpha-2,3-sialyltransferase (ST3Gal III) expression and forced expression of ST3Gal III attenuated Taxol-induced apoptosis. Furthermore, ST3Gal III overexpression inhibited Taxol-triggered caspase-8 activation, indicating that ST3Gal III upregulation produces cellular resistance to Taxol and hence reduces the efficacy of Taxol therapy.

c-FLIP, a Novel Biomarker for Cancer Prognosis, Immunosuppression, Alzheimer's Disease, Chronic Obstructive Pulmonary Disease (COPD), and a Rationale Therapeutic Target.

Dysregulation of c-FLIP (cellular FADD-like IL-1ß-converting enzyme inhibitory protein) has been shown in several diseases including cancer, Alzheimer's disease, and chronic obstructive pulmonary disease (COPD). c-FLIP is a critical anti-cell death protein often overexpressed in tumors and hematological malignancies and its increased expression is often associated with a poor prognosis. c-FLIP frequently exists as long (c-FLIPL) and short (c-FLIPS) isoforms, regulates its anti-cell death functions through binding to FADD (FAS associated death domain protein), an adaptor protein known to activate caspases-8 and -10 and links c-FLIP to several cell death regulating complexes including the death-inducing signaling complex (DISC) formed by various death receptors. c-FLIP also plays a critical role in necroptosis and autophagy. Furthermore, c-FLIP is able to activate several pathways involved in cytoprotection, proliferation, and survival of cancer cells through various critical signaling proteins. Additionally, c-FLIP can inhibit cell death induced by several chemotherapeutics, anti-cancer small molecule inhibitors, and ionizing radiation. Moreover, c-FLIP plays major roles in aiding the survival of immunosuppressive tumor-promoting immune cells and functions in inflammation, Alzheimer's disease (AD), and chronic obstructive pulmonary disease (COPD). Therefore, c-FLIP can serve as a versatile biomarker for cancer prognosis, a diagnostic marker for several diseases, and an effective therapeutic target. In this article, we review the functions of c-FLIP as an anti-apoptotic protein and negative prognostic factor in human cancers, and its roles in resistance to anticancer drugs, necroptosis and autophagy, immunosuppression, Alzheimer's disease, and COPD.

A Dose-escalation Study of Recombinant Human Interleukin-18 in Combination With Ofatumumab After Autologous Peripheral Blood Stem Cell Transplantation for Lymphoma.

Interleukin-18 (IL-18) is an immunostimulatory cytokine that augments antibody-dependent cellular cytotoxicity mediated by human natural killer cells against antibody-coated lymphoma cells in vitro and that has antitumor activity in animal models. Ofatumumab is a CD20 monoclonal antibody with activity against human B-cell lymphomas. A phase I study of recombinant human (rh) IL-18 given with ofatumumab was undertaken in patients with CD20 lymphoma who had undergone high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Cohorts of 3 patients were given intravenous infusions of ofatumumab 1000 mg weekly for 4 weeks with escalating doses of rhIL-18 as a intravenous infusion weekly for 8 consecutive weeks. Nine male patients with CD20 lymphomas were given ofatumumab in combination with rhIL-18 at doses of 3, 10, and 30 µg/kg. No unexpected or dose-limiting toxicities were observed. The mean reduction from predose levels in the number of peripheral blood natural killer cells after the first rhIL-18 infusion was 91%, 96%, and 97% for the 3, 10, and 30 µg/kg cohorts, respectively. Serum concentrations of interferon-γ and chemokines transiently increased following IL-18 dosing. rhIL-18 can be given in biologically active doses by weekly infusions in combination with ofatumumab after peripheral blood stem cell transplantation to patients with lymphoma. A maximum tolerated dose of rhIL-18 plus ofatumumab was not determined. Further studies of rhIL-18 and CD20 monoclonal antibodies in B-cell malignancies are warranted.

Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell-like spheroids, and tumor xenografts through multiple cell signaling pathways.

OBJECTIVE Defects in the apoptotic machinery and augmented survival signals contribute to drug resistance in glioblastoma (GBM). Moreover, another complexity related to GBM treatment is the concept that GBM development and recurrence may arise from the expression of GBM stem cells (GSCs). Therefore, the use of a multifaceted approach or multitargeted agents that affect specific tumor cell characteristics will likely be necessary to successfully eradicate GBM. The objective of this study was to investigate the usefulness of sulforaphane (SFN)-a constituent of cruciferous vegetables with a multitargeted effect-as a therapeutic agent for GBM. METHODS The inhibitory effects of SFN on established cell lines, early primary cultures, CD133-positive GSCs, GSC-derived spheroids, and GBM xenografts were evaluated using various methods, including GSC isolation and the sphere-forming assay, analysis of reactive oxygen species (ROS) and apoptosis, cell growth inhibition assay, comet assays for assessing SFN-triggered DNA damage, confocal microscopy, Western blot analysis, and the determination of in vivo efficacy as assessed in human GBM xenograft models. RESULTS SFN triggered the significant inhibition of cell survival and induced apoptotic cell death, which was associated with caspase 3 and caspase 7 activation. Moreover, SFN triggered the formation of mitochondrial ROS, and SFN-triggered cell death was ROS dependent. Comet assays revealed that SFN increased single- and double-strand DNA breaks in GBM. Compared with the vehicle control cells, a significantly higher amount of γ-H2AX foci correlated with an increase in DNA double-strand breaks in the SFN-treated samples. Furthermore, SFN robustly inhibited the growth of GBM cell-induced cell death in established cell cultures and early-passage primary cultures and, most importantly, was effective in eliminating GSCs, which play a major role in drug resistance and disease recurrence. In vivo studies revealed that SFN administration at 100 mg/kg for 5-day cycles repeated for 3 weeks significantly decreased the growth of ectopic xenografts that were established from the early passage of primary cultures of GBM10. CONCLUSIONS These results suggest that SFN is a potent anti-GBM agent that targets several apoptosis and cell survival pathways and further preclinical and clinical studies may prove that SFN alone or in combination with other therapies may be potentially useful for GBM therapy.

Cellular FLICE-like inhibitory protein (c-FLIP): a novel target for Taxol-induced apoptosis.

It is known that by binding to the FAS-associated death domain (FADD) protein and/or caspases-8 and -10 at the level of the death-inducing signaling complex (DISC), cellular FLICE-like inhibitory protein (c-FLIP) can prevent apoptosis triggered by death-inducing ligands. We investigated whether the c-FLIP splice variants, c-FLIP long [c-FLIP(L)] and c-FLIP short [c-FLIP(S)], play a role in Taxol-induced apoptosis. Our results showed that low Taxol concentrations triggered caspase-8- and caspase-10-dependent apoptosis in the CCRF-HSB-2 human lymphoblastic leukemia cell line, and induced the down-regulation of c-FLIP(S) and c-FLIP(L). Taxol decreased the expression of c-FLIP by a post-transcriptional and caspase-independent mechanism. To explore the distinct functions of the c-FLIP variants in Taxol-induced apoptosis, we transfected the cells with expression vectors carrying c-FLIP(L) and c-FLIP(S) in the sense orientation or c-FLIP(S) in the antisense orientation [c-FLIP(S)-AS]. Caspases-8 and -10 were more efficiently activated in the c-FLIP(S)-AS strain treated with 5-50nM Taxol, which revealed that c-FLIP regulates Taxol-induced apoptosis by interacting with these caspases. Furthermore, our data showed that increased expression of c-FLIP(L) or c-FLIP(S) reduced apoptosis at 5-50nM Taxol concentrations suggesting that both isoforms of c-FLIP prevent Taxol-induced apoptosis. These results revealed that Taxol induces apoptosis by down-regulating c-FLIP(S) and c-FLIP(L) expression.

P-glycoprotein enhances TRAIL-triggered apoptosis in multidrug resistant cancer cells by interacting with the death receptor DR5.

The death-inducing cytokine TRAIL is a promising agent for anticancer therapy since it preferentially kills cancer versus normal cells; however, some cancer cells are TRAIL-resistant. We initially explored whether overexpression of the MDR1 gene product P-glycoprotein (P-gp), which causes multidrug resistance (MDR) in cancer cells, also contributes to TRAIL-resistance. Surprisingly, our results revealed that P-gp-overexpression enhances TRAIL-induced apoptosis not only in neoplastic cells transfected with the MDR1 gene but also in MDR variants selected with cytotoxic anticancer agents. Mechanistic analysis of TRAIL-induced apoptosis in the MDR1-transfected MCF-7 breast cancer cell line BC-19 revealed that TRAIL-triggered significantly more apoptosis in these cells compared with parental MCF-7 cells by binding to the TRAIL receptor DR5. DR5 but not DR4 engagement by TRAIL attenuated cellular ATP levels by robustly stimulating P-gp ATPase activity, and thus triggered P-gp-dependent apoptosis by depletion of the cellular ATP pool. In addition to hyperactive P-gp-mediated ATP hydrolysis, TRAIL-induced, P-gp-potentiated apoptosis was associated with activation of caspases-6, -7, -8, and -9; Bid cleavage; and mitochondrial depolarization. P-gp interacted with the TRAIL receptors DR4, DR5, and DcR1 in plasma membranes and enhanced TRAIL binding to DR5. Interestingly, the decreased level of the decoy TRAIL receptor, DcR1, in BC-19 cells further sensitized these cells to TRAIL. Therefore, both extrinsic and intrinsic apoptosis pathways are involved in this process. These findings for the first time reveal that TRAIL treatment preferentially causes apoptosis in P-gp-overexpressing MDR cells, and suggests significant clinical implications for the use of TRAIL in treating neoplasms that have failed chemotherapy.

Resistance to Cell Death and Its Modulation in Cancer Stem Cells.

Accumulating evidence has demonstrated that human cancers arise from various tissues of origin that initiate from cancer stem cells (CSCs) or cancer-initiating cells. The extrinsic and intrinsic apoptotic pathways are dysregulated in CSCs, and these cells play crucial roles in tumor initiation, progression, cell death resistance, chemo- and radiotherapy resistance, and tumor recurrence. Understanding CSC-specific signaling proteins and pathways is necessary to identify specific therapeutic targets that may lead to the development of more efficient therapies selectively targeting CSCs. Several signaling pathways-including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), maternal embryonic leucine zipper kinase (MELK), NOTCH1, and Wnt/Β-catenin&and expression of the CSC markers CD133, CD24, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain CSC properties. Studying such pathways may help to understand CSC biology and lead to the development of potential therapeutic interventions to render CSCs more sensitive to cell death triggered by chemotherapy and radiation therapy. Moreover, recent demonstrations of dedifferentiation of differentiated cancer cells into CSC-like cells have created significant complexity in the CSCs hypothesis. Therefore, any successful therapeutic agent or combination of drugs for cancer therapy must eliminate not only CSCs but differentiated cancer cells and the entire bulk of tumor cells. This review article expands on the CSC hypothesis and paradigm with respect to major signaling pathways and effectors that regulate CSC apoptosis resistance. Moreover, selective CSC apoptotic modulators and their therapeutic potential for making tumors more responsive to therapy are discussed. The use of novel therapies, including small-molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of CSCs, immunotherapy, and noncoding microRNAs may provide better means of treating CSCs.

Emerging targets for glioblastoma stem cell therapy.

Glioblastoma multiforme (GBM), designated as World Health Organization (WHO) grade IV astrocytoma, is a lethal and therapy-resistant brain cancer comprised of several tumor cell subpopulations, including GBM stem cells (GSCs) which are believed to contribute to tumor recurrence following initial response to therapies. Emerging evidence demonstrates that GBM tumors are initiated from GSCs. The development and use of novel therapies including small molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of GSCs, immunotherapy, and non-coding microRNAs may provide better means of treating GBM. Identification and characterization of GSC-specific signaling pathways would be necessary to identify specific therapeutic targets which may lead to the development of more efficient therapies selectively targeting GSCs. Several signaling pathways including mTOR, AKT, maternal embryonic leucine zipper kinase (MELK), NOTCH1 and Wnt/ß-catenin as well as expression of cancer stem cell markers CD133, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain GSC properties. Moreover, the data published in the Cancer Genome Atlas (TCGA) specifically demonstrated the activated PI3K/AKT/mTOR pathway in GBM tumorigenesis. Studying such pathways may help to understand GSC biology and lead to the development of potential therapeutic interventions to render them more sensitive to chemotherapy and radiation therapy. Furthemore, recent demonstration of dedifferentiation of GBM cell lines into CSC-like cells prove that any successful therapeutic agent or combination of drugs for GBM therapy must eliminate not only GSCs, but the differentiated GBM cells and the entire bulk of tumor cells.

Proteinase-3, a serine protease which mediates doxorubicin-induced apoptosis in the HL-60 leukemia cell line, is downregulated in its doxorubicin-resistant variant.

We report here that expression of proteinase 3 (PR3), a serine protease, is down-regulated in the HL60/ADR multidrug resistant variant of the human myelogenous leukemia cell line HL-60, and that down-regulation of PR3 is associated with doxorubicin (DOX) resistance in these cells. To determine whether PR3 is involved in DOX-induced apoptosis in HL-60 cells, and whether its loss causes resistance to DOX, we inhibited PR3 expression by an anti-sense PR3 oligodeoxynucleotide and showed that inhibition of PR3 expression results in a significant reduction in DOX-induced DNA fragmentation and increased resistance to DOX-induced apoptosis. Our results revealed that PR3-mediated DOX-induced apoptosis in HL-60 cells is independent of the loss of mitochondrial membrane potential (deltapsi(m)) and activation of the caspase-8 and -9 pathways. Moreover, while PR3 is involved in the cleavage of caspase-3, PR3-mediated DOX-induced DNA fragmentation and apoptosis were not prevented by a specific inhibitor of caspase-3. These data suggest that activation of caspase-3 alone is not sufficient to trigger PR3-mediated DOX-induced apoptosis. Treatment with an anti-PR3 oligomer significantly decreased reactive oxygen species (ROS) generation in cells treated with low concentrations of DOX, revealing a role for PR3 in enhancing production of DOX-induced ROS. Moreover, DOX-induced apoptosis at 0.001-0.01 microM was only inhibited in HL-60 cells pre-treated with the antioxidant N-acetyl-cysteine in the absence of anti-PR3, revealing that DOX-induced apoptosis in these cells is PR3- and ROS-dependent. Our results show that PR3 is involved in DOX-induced ROS-dependent apoptosis and that its loss is associated with resistance to DOX in HL-60 cells.

Photoaffinity analogs for multidrug resistance-related transporters and their use in identifying chemosensitizers.

A major obstacle in cancer treatment is the development of resistance to multiple chemotherapeutic agents in tumor cells. The hallmark of this multidrug resistance (MDR) is overexpression of the MDR 1 P-glycoprotein or the multidrug resistance protein MRP1. It is well documented that these proteins confer MDR in cancer cells. Much evidence indicates that control of intracellular drug levels in MDR cells is determined by P-glycoprotein or MRP, and therefore these proteins are suitable targets for identifying MDR-reversing agents (MDR modulators). We originally explored the drug-binding ability of P-glycoprotein by synthesizing and using radioactive photoaffinity analogs of vinblastine. Since our initial discovery that P-glycoprotein binds to vinblastine photoaffinity analogs, many P-glycoprotein- and MRP-specific photoaffinity analogs have been developed. In this review, photoaffinity analogs which specifically bind to P-glycoprotein or MRP are discussed. Moreover, utilizing these photoprobes to identify, characterize and localize the drug binding sites of P-glycoprotein and MRP is described. Using P-glycoprotein-specific photoaffinity analogs in combination with site-directed antibodies to several domains of this protein has allowed the localization of the general binding domains of some of the cytotoxic agents an MDR modulators on P-glycoprotein. However, the molecular architecture of the drug binding sites, their exact location on the P-glycoprotein molecule, and the total number of the drug binding sites remain to be determined. This review discusses recent advances in delineating the structure of the drug-binding sites of P-glycoprotein. Moreover, novel MRP1 photoaffinity analogs are reviewed. Copyright 1999 Harcourt Publishers Ltd.

Glioblastoma stem cells (GSCs) epigenetic plasticity and interconversion between differentiated non-GSCs and GSCs.

Cancer stem cells (CSCs) or cancer initiating cells (CICs) maintain self-renewal and multilineage differentiation properties of various tumors, as well as the cellular heterogeneity consisting of several subpopulations within tumors. CSCs display the malignant phenotype, self-renewal ability, altered genomic stability, specific epigenetic signature, and most of the time can be phenotyped by cell surface markers (e.g., CD133, CD24, and CD44). Numerous studies support the concept that non-stem cancer cells (non-CSCs) are sensitive to cancer therapy while CSCs are relatively resistant to treatment. In glioblastoma stem cells (GSCs), there is clonal heterogeneity at the genetic level with distinct tumorigenic potential, and defined GSC marker expression resulting from clonal evolution which is likely to influence disease progression and response to treatment. Another level of complexity in glioblastoma multiforme (GBM) tumors is the dynamic equilibrium between GSCs and differentiated non-GSCs, and the potential for non-GSCs to revert (dedifferentiate) to GSCs due to epigenetic alteration which confers phenotypic plasticity to the tumor cell population. Moreover, exposure of the differentiated GBM cells to therapeutic doses of temozolomide (TMZ) or ionizing radiation (IR) increases the GSC pool both in vitro and in vivo. This review describes various subtypes of GBM, discusses the evolution of CSC models and epigenetic plasticity, as well as interconversion between GSCs and differentiated non-GSCs, and offers strategies to potentially eliminate GSCs.