Abstract
BackgroundXIAP (X-linked inhibitor of apoptosis protein) is one of the most important members of the apoptosis inhibitor family. XIAP is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth and survival of malignant cells. We hypothesized that downregulation of XIAP by human umbilical cord blood mesenchymal stem cells (hUCBSC) in glioma cells would cause them to undergo apoptotic death.Methodology/Principal FindingsWe observed the effect of hUCBSC on two malignant glioma cell lines (SNB19 and U251) and two glioma xenograft cell lines (4910 and 5310). In co-cultures of glioma cells with hUCBSC, proliferation of glioma cells was significantly inhibited. This is associated with increased cytotoxicity of glioma cells, which led to glioma cell death. Stem cells induced apoptosis in glioma cells, which was evaluated by TUNEL assay, FACS analyses and immunoblotting. The induction of apoptosis is associated with inhibition of XIAP in co-cultures of hUCBSC. Similar results were obtained by the treatment of glioma cells with shRNA to downregulate XIAP (siXIAP). Downregulation of XIAP resulted in activation of caspase-3 and caspase-9 to trigger apoptosis in glioma cells. Apoptosis is characterized by the loss of mitochondrial membrane potential and upregulation of mitochondrial apoptotic proteins Bax and Bad. Cell death of glioma cells was marked by downregulation of Akt and phospho-Akt molecules. We observed similar results under in vivo conditions in U251- and 5310-injected nude mice brains, which were treated with hUCBSC. Under in vivo conditions, Smac/DIABLO was found to be colocalized in the nucleus, showing that hUCBSC induced apoptosis is mediated by inhibition of XIAP and activation of Smac/DIABLO.Conclusions/SignificanceOur results indicate that downregulation of XIAP by hUCBSC treatment induces apoptosis, which led to the death of the glioma cells and xenograft cells. This study demonstrates the therapeutic potential of XIAP and hUCBSC to treat malignant gliomas.
Highlights
Apoptosis is the cell’s intrinsic death program that controls normal tissue homeostasis
In order to make sure that inhibition of cell proliferation, induced cytotoxicity, observed apoptotic phenotype and decreased protein expressions of X-Linked Inhibitor of Apoptosis Protein (XIAP) and Akt in glioma cells, the co-cultures were grown in the same media which was used for growing glioma cells alone, as described in Materials and Methods
We observed that human umbilical cord blood mesenchymal stem cells (hUCBSC) inhibited proliferation of the glioma cell lines and the inhibition was more than 50% in all the cell lines tested (Fig. 1A)
Summary
Apoptosis is the cell’s intrinsic death program that controls normal tissue homeostasis. One mechanism through which tumor cells are believed to acquire resistance to apoptosis is by overexpression of XIAP (X-linked inhibitor of apoptosis protein). XIAP interacts with and inhibits active caspases-3, -7 and -9 [6,7,8]. Recent studies confirmed that overexpression of XIAP confers resistance to multi-agent chemotherapy, including stimuli of the mitochondrial and death receptor pathways of caspase activation [9,10,11]. Through its ability to inhibit caspases, overexpression of XIAP renders cells resistant to multi-agent chemotherapy. There have been very few reports of the downregulation of XIAP in human glioblastoma cells. XIAP is upregulated in various malignancies, including human glioblastoma. It promotes invasion, metastasis, growth and survival of malignant cells. We hypothesized that downregulation of XIAP by human umbilical cord blood mesenchymal stem cells (hUCBSC) in glioma cells would cause them to undergo apoptotic death
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