Blockade of Inhibitors of Apoptosis Proteins in Combination with Conventional Chemotherapy Leads to Synergistic Antitumor Activity in Medulloblastoma and Cancer Stem-Like Cells

<div><p>Background</p><p>Medulloblastoma (MB) is the most common pediatric primary malignant brain tumor. Approximately one-third of MB patients succumb to treatment failure and some survivors suffer detrimental side effects. Hence, the purpose of this study is to explore new therapeutic regimens to overcome chemotherapeutic agent resistance or reduce chemotherapy-induced toxicity.</p><p>Methods</p><p>We detected the expression of inhibitors of apoptosis proteins (IAPs) in MB and CD133+ MB cell lines and MB tissues using immunoblotting and immunohistochemical staining. The antitumor effects of inhibitors against IAPs on MB or CD133+ MB cells were evaluated by MTT assay, Annexin V/PI analysis, and caspase-3/7 activity. Autophagy was assessed by the conversion of light chain (LC) 3-I to LC3-II and Cyto-ID autophagy detection kit.</p><p>Results</p><p>MB cells showed higher expression of IAPs compared to normal astrocytes and normal brain tissues. Conventional chemotherapeutic agents combined with small-molecule IAP inhibitors (LCL161 or LBW242) showed a synergistic effect in MB cells. Combined treatments triggered apoptosis in MB cells through activation of caspase-3/7 and autophagic flux simultaneously. In addition, we found that CD133+ MB cells with features of cancer stem cells displayed higher levels of X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis 1/2 (cIAP1/2), and were hypersensitive to treatment with IAP inhibitors.</p><p>Conclusions</p><p>These results shed light on the biological effects of combination therapy on MB cells and illustrate that IAP inhibitors are more effective for CD133+ stem-like MB cells.</p></div

Combination of IAP inhibitors and chemotherapeutic agents promotes autophagy-dependent apoptosis.

<p>(A) DAOY and D283MED cells were treated with chemotherapeutic agent (vincristine or cisplatin) only or in combination with DMSO, LCL161 (10μM) or LBW242 (10μM) for 72 hr. Autophagic flux was determined by cyto-ID and analyzed by FACS. (B) Autophagic flux was based on percentage of right shift and has been shown in bar graphs (*p < 0.05, **p < 0.001, and ***p < 0.005). (C) LC3-I/II conversion was determined by immunoblotting after these treatments for 72 hr. (D) (E) The autophagy inhibitor (chloroquine, 10 μM) reduced combination therapy-induced apoptosis, as determined by Annexin V/PI and FACS. The bar graphs show the proportions of Annexin V positive cells (*p < 0.05, **p < 0.001, and ***p < 0.005).</p

Addition of IAP inhibitors enhances chemotherapy-induced apoptosis in MB cells.

<p>DAOY and D283MED cells were treated with vincristine (1.25 nM for DAOY, 2.5 nM for D283MED) or cisplatin (0.3125 μM for DAOY, 0.625 μM for D283MED), or combination of the chemotherapeutic drug with an IAP inhibitor (LCL161 or LBW242, 10 μM) for 72 hr. (A)(B) Apoptosis was assessed by Annexin V/PI and FACS. The apoptotic proportions (Annexin V positive cells) are shown in bar graphs. (C) MB cells were treated with these chemotherapeutic agents combined with or without IAP inhibitors for 72 hr, and cell lysates were then subjected to immunoblotting analysis. (D) DAOY cells were treated with vincristine or cisplatin only, or in combination with LCL161 for 72 hr. The bar graph represents Caspase-3/7 activity, which was analyzed by FACS. The error bars represent mean ± SEM (*p < 0.05, **p < 0.001, and ***p < 0.005).</p

ED50 values of chemotherapeutic agents alone or in combination with IAP inhibitors in DAOY and D283MED cells.

<p>ED50 values of chemotherapeutic agents alone or in combination with IAP inhibitors in DAOY and D283MED cells.</p

IAP inhibitors sensitize CD133+ stem-like MB cells to chemotherapeutic agents.

<p>(A) CD133+ cells isolated from DAOY and D283MED cell lines were cultured in 96-well plates and treated with different concentrations of vincristine or cisplatin combined with or without IAP inhibitors (LCL161 10 μM) for 72 hr. The cell viability was measured by MTT assay. (B)(C) Cells were treated with chemotherapeutic drug (vincristine or cisplatin) or in combination with IAP inhibitors for 72 hours. Apoptosis was detected by Annexin V/PI and FACS. The bar graphs indicate the proportion of induced apoptosis. Similar results were obtained by three independent experiments. (*p < 0.05, **p < 0.001, and ***p < 0.005).</p

Knockdown of cIAP or XIAP enhances inhibitory effect of chemotherapies on CD133+ cell viability as well as IAP inhibitors.

<p>CD133+ MB cells were transfected with non-targeting (NT, control) siRNA and siRNA against cIAP1/2 or XIAP. The levels of XIAP and cIAP were determined using immunoblotting analysis (A), and quantified by densitometry and then shown in the bar graphs (B). (C) The transfectants were treated with different concentrations of vincristine or cisplatin for 72 hr, and cell viability was analyzed by MTT assay. The data are presented as the mean ± SEM of triplicates (*p < 0.05, **p < 0.01, and ***p < 0.005).</p

XIAP is specifically expressed in medulloblastoma (MB) tissue and both XIAP and cIAP1/2 can be enhanced by addition of chemotherapeutic agents to render MB cells hypersensitive to IAP inhibitors.

<p>XIAP levels were detected by immunohistochemical staining in normal cerebellum tissue (A), normal cerebrum tissue (B), and MB tissues (C, D). Bar scale represents 100 μm. (E) The levels of XIAP and cIAP1/2 in MB cell lines (DAOY and D283MED) and normal astrocytes (HA-h) were determined by immunoblotting. (F) DAOY, D283MED, and HA-h cells were treated with different concentrations of LCL161, LBW242, and DMSO (control) for 72 hr. Cell viability was analyzed by MTT assay. (G) The cell lysates were collected from MB cells and subjected to immunoblotting after treatment with vincristine (1.25 nM for DAOY, 2.5 nM for D283MED) or cisplatin (0.3125 μM for DAOY and 0.625 μM for D283MED) for 72hr. (H) DAOY and D283MED cells were treated with the indicated concentrations of vincristine or cisplatin in combination with LBW242 (10 μM) or LCL161 (10 μM). The cell viability was determined by MTT after these treatments for 72hr. The data are presented as the mean ± SEM of three independent experiments.</p

Combination Index (CI) of conventional chemotherapeutic agents (at ED50) combined with IAP inhibitors in medulloblastoma cells.

<p>Combination Index (CI) of conventional chemotherapeutic agents (at ED50) combined with IAP inhibitors in medulloblastoma cells.</p

High levels of XIAP and cIAP1/2 in CD133+ stem-like MB cells result in high sensitivity to IAP inhibitors.

<p>(A) Both CD133- and CD133+ cells isolated from DAOY and D283MED cells were cultured in DMEM-F12 supplemented with 10ng/ml bFGF, 10ng/ml EGF, and 1% N2. Formation of neurospheres was examined by light microscopy. Bar scale represents 100 μm. (B) On day 21, the levels of XIAP and cIAP1/2 in CD133- and CD133+ cells were detected by immunoblotting and analyzed quantitatively. (C) CD133- and CD133+ cells were respectively treated with different concentrations of LCL161 or LBW242 for 72 hr. The cell viability was analyzed by MTT assay. (D)(E) Subsequent to treatment with LCL161 or LBW242 for 72 hr, apoptotic propitiations of CD133- and CD133+ cells were analyzed by Annexin V/PI and FACS, and then shown in bar graphs. Similar results were obtained by three independent experiments. (*p < 0.05, **p < 0.001, and ***p < 0.005).</p

A Hormone Receptor-Based Transactivator Bridges Different Binary Systems to Precisely Control Spatial-Temporal Gene Expression in <em>Drosophila</em>

<div><p>The GAL4/<em>UAS</em> gene expression system is a precise means of targeted gene expression employed to study biological phenomena in <em>Drosophila</em>. A modified GAL4/<em>UAS</em> system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (<em>LexAop</em>) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/<em>UAS</em> system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in <em>Drosophila</em>.</p> </div