Fei Chu

Control of Multidrug Resistance Gene mdr1 and Cancer Resistance to Chemotherapy by the Longevity Gene sirt1

Irreversible growth arrest (also called senescence) has emerged recently as a tumor suppressor mechanism and a key determinant of cancer chemotherapy outcome. Previous work from our laboratory suggested that the cellular ability to undergo or to escape senescence dictates its fate to become drug-sensitive or drug-resistant, respectively. In the present study, we made the hypothesis that longevity genes, by virtue of their ability to inhibit senescence, may contribute to the onset of drug resistance. We report that expression of the longevity gene sirt1 increased both at the RNA and protein levels in all the five drug-resistant cell lines tested when compared with their drug-sensitive counterparts. In addition, biopsies from cancer patients treated with chemotherapeutic agents also expressed high levels of this molecule. These changes were specific for sirt1 because the expression of other members of its family was not affected. More importantly, small interfering RNA-mediated down-regulation of sirt1 significantly reversed the resistance phenotype and reduced expression of the multidrug resistance molecule P-glycoprotein. This was further confirmed by ectopic overexpression of sirt1, which induced expression of P-glycoprotein and rendered cells resistant to doxorubicin. Collectively, these findings uncovered a novel function for the longevity gene sirt1 as a potential target for diagnosis and/or treatment of cancer resistance to chemotherapy. They also describe a proof of principle that signaling pathways implicated in longevity may share similarities with those leading to development of drug resistance in cancer.

Identification of midkine as a mediator for intercellular transfer of drug resistance.

Resistance to cytotoxic agents is a major limitation for their clinical use to treat human cancers. Tumors become resistant to chemotherapy when a subset of cells undergoes molecular changes leading to overexpression of drug transport proteins, alterations in drug–target interactions or reduced ability to commit apoptosis. However, such changes may not be sufficient to explain why both resistant and nonresistant cells survive drugs action in tumors that ultimately become drug resistant. We hypothesized that, in such tumors, a cytoprotective relationship may exist between drug-resistant and neighboring drug-sensitive cells. The present study addresses the possibility that drug-resistant cells secrete in their culture medium factors able to protect sensitive cells from drug toxicity. A survival molecule, midkine, was identified by cDNA array to be expressed only in drug-resistant cells. Midkine-enriched fractions obtained by affinity chromatography exert a significant cytoprotective effect against doxorubicin in the wild-type drug-sensitive cells. Moreover, transfection of these cells with the midkine gene caused a decreased response to doxorubicin. The underlying mechanism of this cytoprotection appeared to imply activation of the Akt pathway and inhibition of drug-induced proliferation arrest as well as apoptotic cell death. These findings provide evidence for the existence of intercellular cytoprotective signals such as the one mediated by midkine, originating from cells with acquired drug resistance to protect neighboring drug-sensitive cells and thus contribute to development of resistance to chemotherapy.

Role of the proteolytic hierarchy between cathepsin L, cathepsin D and caspase-3 in regulation of cellular susceptibility to apoptosis and autophagy

The present investigation was undertaken to measure the relative abilities of pro-death versus pro-survival proteases in degrading each other and to determine how this might influence cellular susceptibility to death. For this, we first carried out in vitro experiments in which recombinant pro-death proteases (caspase-3 or cathepsin D) were incubated with the pro-survival protease (cathepsin L) in their respective optimal conditions and determined the effects of these reactions on enzyme integrity and activity. The results indicated that cathepsin L was able to degrade cathepsin D, which in turn cleaves caspase-3, however the later enzyme was unable to degrade any of the cathepsins. The consequences of this proteolytic sequence on cellular ability to undergo apoptosis or other types of cell death were studied in cells subjected to treatment with a specific inhibitor of cathepsin L or the corresponding siRNA. Both treatments resulted in suppression of cellular proliferation and the induction of a cell death with no detectable caspase-3 activation or DNA fragmentation, however, it was associated with increased accumulation of cathepsin D, cellular vaculolization, expression of the mannose-6-phosphate receptor, and the autophagy marker LC3-II, all of which are believed to be associated with autophagy. Genetic manipulations leading either to the gain or loss of cathepsin D expression implicated this enzyme as a key player in the switch from apoptosis to autophagy. Overall, these findings suggest that a hierarchy between pro-survival and pro-death proteases may have important consequences on cell fate.

Cathepsin L inhibition suppresses drug resistance in vitro and in vivo: a putative mechanism

Cathepsin L is a lysosomal enzyme thought to play a key role in malignant transformation. Recent work from our laboratory has demonstrated that this enzyme may also regulate cancer cell resistance to chemotherapy. The present study was undertaken to define the relevance of targeting cathepsin L in the suppression of drug resistance in vitro and in vivo and also to understand the mechanism(s) of its action. In vitro experiments indicated that cancer cell adaptation to increased amounts of doxorubicin over time was prevented in the presence of a cathepsin L inhibitor, suggesting that inhibition of this enzyme not only reverses but also prevents the development of drug resistance. The combination of the cathepsin L inhibitor with doxorubicin also strongly suppressed the proliferation of drug-resistant tumors in nude mice. An investigation of the underlying mechanism(s) led to the finding that the active form of this enzyme shuttles between the cytoplasm and nucleus. As a result, its inhibition stabilizes and enhances the availability of cytoplasmic and nuclear protein drug targets including estrogen receptor-alpha, Bcr-Abl, topoisomerase-IIalpha, histone deacetylase 1, and the androgen receptor. In support of this, the cellular response to doxorubicin, tamoxifen, imatinib, trichostatin A, and flutamide increased in the presence of the cathepsin L inhibitor. Together, these findings provided evidence for the potential role of cathepsin L as a target to suppress cancer resistance to chemotherapy and uncovered a novel mechanism by which protease inhibition-mediated drug target stabilization may enhance cellular visibility and, thus, susceptibility to anticancer agents.

Cellular conditioning with trichostatin A enhances the anti-stress response through up-regulation of HDAC4 and down-regulation of the IGF/Akt pathway

Evidence is accumulating that chromatin plays a major role in the control of cellular response to stress. This is best illustrated by the recent findings that chromatin‐modifying factors of class III histone deacetylases (sirtuins) are capable of protecting cells from oxidative and genotoxic stress. In particular, Sirt1 has been shown to mimic the action of caloric restriction for the prevention of aging‐associated diseases. In the present study, we have investigated the potential role of class I and II histone deacetylases (HDACs) in cellular protection against various stresses, including those caused by nutrient deprivation. For this, we utilized a cellular model in which expression of class I and II HDACs was altered as a result of cellular adaptation to trichostatin A (TSA), a selective inhibitor of these deacetylases. Our results indicated that TSA‐resistant cells also developed resistance to H2O2, DNA‐damaging agents, and to nutrient deprivation. Interestingly, the insulin signaling pathway mediated by Akt was inhibited in the TSA‐resistant cells, mirroring the effect of glucose deprivation on this pathway. Since expression of HDAC4 was consistently enhanced in the TSA‐resistant cell lines, we suggest that this enzyme may contribute to their anti‐stress response. In agreement with this, siRNA‐mediated knockdown of HDAC4 in stress‐resistant cells enhanced their sensitivity to the DNA‐damaging drug doxorubicin and also to glucose deprivation. Akt phosphorylation was also up‐regulated in response to HDC4 knockdown. Together, these findings suggest that cellular conditioning with TSA may represent a useful approach to mimic the effects of caloric restriction.

Differential effect of long-term drug selection with doxorubicin and vorinostat on neuroblastoma cells with cancer stem cell characteristics

Numerous studies have confirmed that cancer stem cells (CSCs) are more resistant to chemotherapy; however, there is a paucity of data exploring the effect of long-term drug treatment on the CSC sub-population. The purpose of this study was to investigate whether long-term doxorubicin treatment could expand the neuroblastoma cells with CSC characteristics and histone acetylation could affect stemness gene expression during the development of drug resistance. Using n-myc amplified SK-N-Be(2)C and non-n-myc amplified SK-N-SH human neuroblastoma cells, our laboratory generated doxorubicin-resistant cell lines in parallel over 1 year; one cell line intermittently treated with the histone deacetylase inhibitor (HDACi) vorinostat and the other without exposure to HDACi. Cells’ sensitivity to chemotherapeutic drugs, the ability to form tumorspheres, and capacity for in vitro invasion were examined. Cell-surface markers and side populations (SPs) were analyzed using flow cytometry. Differentially expressed stemness genes were identified through whole genome analysis and confirmed with real-time PCR. Our results indicated that vorinostat increased the sensitivity of only SK-N-Be(2)C-resistant cells to chemotherapy, made cells lose the ability to form tumorspheres, and reduced in vitro invasion and the SP percentage. CD133 was not enriched in doxorubicin-resistant or vorinostat-treated doxorubicin-resistant cells. Nine stemness-linked genes (ABCB1, ABCC4, LMO2, SOX2, ERCC5, S100A10, IGFBP3, TCF3, and VIM) were downregulated in vorinostat-treated doxorubicin-resistant SK-N-Be(2)C cells relative to doxorubicin-resistant cells. A sub-population of cells with CSC characteristics is enriched during prolonged drug selection of n-myc amplified SK-N-Be(2)C neuroblastoma cells. Vorinostat treatment affects the reversal of drug resistance in SK-N-Be(2)C cells and may be associated with downregulation of stemness gene expression. This work may be valuable for clinicians to design treatment protocols specific for different neuroblastoma patients.

The role of nerve growth factor in caspase-dependent apoptosis in human BE(2)C neuroblastoma

PURPOSE The purpose of the study was to determine if nerve growth factor (NGF) stimulation induces apoptosis in the BE(2)C neuroblastoma cell line in vitro. METHODS The LPCX retroviral vector was used to achieve stable transduction of NGF complementary DNA into BE(2)C neuroblastoma cells. Wild-type and NGF-transduced cells were then incubated with varying concentrations of NGF for varying periods. A laddering assay was performed to determine the presence of DNA fragments characteristic of apoptosis. The expression of various cleaved and total caspases was determined by Western immunoblotting. RESULTS p75 receptor expression in the NGF-transduced cell line was equivalent to that in the wild-type cell line, but Trk A receptor expression was significantly decreased in BE(2)C-NGF cells. DNA laddering assay demonstrated that only BE(2)C-NGF cells underwent apoptosis after stimulation with exogenous NGF. BE(2)C-NGF cells have increased expression of cleaved caspase-3 when compared with wild-type cells. Cleaved caspase-3 expression is further increased with exogenous NGF stimulation in the transduced cells. CONCLUSION This study confirms that NGF stimulation of BE(2)C neuroblastoma cells can induce apoptosis through activation of the caspase cascade in vitro. The differential expression of the receptors Trk A and p75 between the wild-type and NGF-transduced cell lines may explain the differing effects observed.

Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

Resistance to cytotoxic agents has long been known to be a major limitation in the treatment of human cancers. Although many mechanisms of drug resistance have been identified, chemotherapies targeting known mechanisms have failed to lead to effective reversal of drug resistance, suggesting that alternative mechanisms remain undiscovered. Previous work identified midkine (MK) as a novel putative survival molecule responsible for cytoprotective signaling between drug-resistant and drug-sensitive neuroblastoma, osteosarcoma and breast carcinoma cells in vitro. In the present study, we provide further in vitro and in vivo studies supporting the role of MK in neuroblastoma cytoprotection. MK overexpressing wild type neuroblastoma cells exhibit a cytoprotective effect on wild type cells when grown in a co-culture system, similar to that seen with doxorubicin resistant cells. siRNA knockdown of MK expression in doxorubicin resistant neuroblastoma and osteosarcoma cells ameliorates this protective effect. Overexpression of MK in wild type neuroblastoma cells leads to acquired drug resistance to doxorubicin and to the related drug etoposide. Mouse studies injecting various ratios of doxorubicin resistant or MK transfected cells with GFP transfected wild type cells confirm this cytoprotective effect in vivo. These findings provide additional evidence for the existence of intercellular cytoprotective signals mediated by MK which contribute to chemotherapy resistance in neuroblastoma.

Abstract 3365: Histone deacetylase inhibitor differentially regulates stemness gene expression in neuroblastoma drug resistant cells

Histone deacetylase inhibitors (HDACi) have been developed as anti-tumor agents and they may play a role in reversing drug resistance by combination treatment with other chemotherapeutic drugs. However, it has also been reported that HDACi can upregulate the cancer stem surface marker expression and promote stem cell self-renewal, which may cause inherit resistance to chemotherapeutic drugs. We investigated the effect of HDACi treatment on the stemness gene expression in drug resistant cells. Using SK-N-Be2C, high aggressive I type human of neuroblastoma cells, and SKN-SH, low aggressive mixed N and S type of human neuroblastoma cells, our laboratory generated doxorubicin drug resistant cell lines with and without intermittent histone deacetylase inhibitor Vorinostat treatment over one year. For each cell line, four groups were generated: wild type (WT), doxorubicin resistant (DoxR), vorinostat treated WT (WT-v), and vorinostat - treated doxorubicin resistant (DoxR-v). The cells’ sensitivity to chemotherapeutic drugs, the ability of forming tumorspheres and in vitro invasion were examined. Cell surface markers and side populations were analyzed by flow cytometry. The differential expressed stemness genes were identified by whole genome analysis and confirmed by real-time PCR. Vorinostat increased the sensitivity of SK-N-Be2C resistant cells to chemotherapy, made the cells lose the ability to form tumorspheres, and reduced in vitro invasion ability and the percentage of side population. In contrary, vorinostat decreased the sensitivity of SK-N-SH resistant cells to drugs, enhanced the cell9s ability to form tumorshperes and had little effect on the cells’ invasion and side population. The putative neuroblastoma marker, CD133, was not enriched in either long term or short term vorinostat/doxorubicin treatment. Nine stemness linked genes (ABCB1, ABCC4, LMO2, SOX2, ERCC5, S100A10, IGFBP3, TCF3 and VIM) were upregulated in doxorubicin resistant SK-N-Be2C cells, compared to vorinostat - treated resistant cells. Vorinostat did not have significant effect on stemness gene expression in SK-N-SH cells, except for ABCB1 and ABCC4 genes, which were deregulated in vorinostat - treated resistant cells. In conclusion, Histone deacetylase inhibitor differentially regulates stemness gene expression in neuroblastoma drug resistant cells. It has better effect on reversing drug resistance in high aggressive neuroblastoma and this may be associated with downregulated stemness gene expression. This work may be valuable for clinicians to design treatment protocols specific for different neuroblastoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3365. doi:1538-7445.AM2012-3365

Abstract 781: The effect of vorinostat on the development of resistance to doxorubicin in neuroblastoma

Histone deacetylase (HDAC) inhibitors, especially vorinostat, are currently under investigation as potential adjuncts in the treatment of neuroblastoma. The effect of vorinostat co-treatment on the development of resistance to other chemotherapeutic agents is unknown. In the present study, we treated two human neuroblastoma cell lines [SK-N-SH and SK-N-Be(2)C] with progressively increasing doses of doxorubicin under two conditions: with and without vorinsotat co-therapy. The resultant doxorubicin-resistant (DoxR) and vorinostat-treated doxorubicin resistant (DoxR-v) cells were equally resistant to doxorubicin despite significantly lower P-glycoprotein expression in the DoxR-v cells. Whole genome analysis was performed using the Ilumina Human HT-12 v4 Expression Beadchip to identify genes with differential expression unique to the DoxR-v cells. We uncovered a number of genes whose differential expression in the DoxR-v cells might contribute to their resistant phenotype, including hypoxia inducible factor-2. Finally, we used Gene Ontology to categorize the biological functions of the differentially expressed genes unique to the DoxR-v cells and found that genes involved in cellular metabolism were especially affected. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 781. doi:1538-7445.AM2012-781