Guilan Li

JNK-dependent Release of Mitochondrial Protein, Smac, during Apoptosis in Multiple Myeloma (MM) Cells

Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Previous studies have shown that c-Jun NH2-terminal kinase (JNK) is involved in regulating another mitochondrial protein, cytochrome c during apoptosis; however, the role of JNK in the release of mitochondrial Smac is unknown. Here we show that induction of apoptosis in multiple myeloma (MM) cells is associated with activation of JNK, translocation of JNK from cytosol to mitochondria, and release of Smac from mitochondria to cytosol. Blocking JNK either by dominant-negative mutant (DN-JNK) or cotreatment with a specific JNK inhibitor, SP600125, abrogates both stress-induced release of Smac and induction of apoptosis. These findings demonstrate that activation of JNK is an obligatory event for the release of Smac during stress-induced apoptosis in MM cells.

Identification of genes regulated by dexamethasone in multiple myeloma cells using oligonucleotide arrays.

Our previous studies have characterized Dexamethasone (Dex)-induced apoptotic signaling pathways in multiple myeloma (MM) cells; however, related transcriptional events are not fully defined. In the present study, gene expression profiles of Dex-treated MM cells were determined using oligonucleotide arrays. Dex triggers early transient induction of many genes involved in cell defense/repair-machinery. This is followed by induction of genes known to mediate cell death and repression of growth/survival-related genes. The molecular and genetic alterations associated with Dex resistance in MM cells are also unknown. We compared the gene expression profiles of Dex-sensitive and Dex-resistant MM cells and identified a number of genes which may confer Dex-resistance. Finally, gene profiling of freshly isolated MM patient cells validates our in vitro MM cell line data, confirming an in vivo relevance of these studies. Collectively, these findings provide insights into the basic mechanisms of Dex activity against MM, as well as mechanisms of Dex-resistance in MM cells. These studies may therefore allow improved therapeutic uses of Dex, based upon targeting genes that regulate MM cell growth and survival.

A Novel Carbohydrate-Based Therapeutic GCS-100 Overcomes Bortezomib Resistance and Enhances Dexamethasone-Induced Apoptosis in Multiple Myeloma Cells

Human multiple myeloma is a presently incurable hematologic malignancy, and novel biologically based therapies are urgently needed. GCS-100 is a polysaccharide derived from citrus pectin in clinical development for the treatment of cancer. Here we show that GCS-100 induces apoptosis in various multiple myeloma cell lines, including those resistant to dexamethasone, melphalan, or doxorubicin. Examination of purified patient multiple myeloma cells showed similar results. Specifically, GCS-100 decreases viability of bortezomib/PS-341-resistant multiple myeloma patient cells. Importantly, GCS-100 inhibits multiple myeloma cell growth induced by adhesion to bone marrow stromal cells; overcome the growth advantage conferred by antiapoptotic protein Bcl-2, heat shock protein-27, and nuclear factor-kappaB; and blocks vascular endothelial growth factor-induced migration of multiple myeloma cells. GCS-100-induced apoptosis is associated with activation of caspase-8 and caspase-3 followed by proteolytic cleavage of poly(ADP-ribose) polymerase enzyme. Combined with dexamethasone, GCS-100 induces additive anti-multiple myeloma cytotoxicity associated with mitochondrial apoptotic signaling via release of cytochrome c and Smac followed by activation of caspase-3. Moreover, GCS-100 + dexamethasone-induced apoptosis in multiple myeloma cells is accompanied by a marked inhibition of an antiapoptotic protein Galectin-3, without significant alteration in Bcl-2 expression. Collectively, these findings provide the framework for clinical evaluation of GCS-100, either alone or in combination with dexamethasone, to inhibit tumor growth, overcome drug resistance, and improve outcome for patients with this universally fatal hematologic malignancy.

Superoxide-dependent and -independent mitochondrial signaling during apoptosis in multiple myeloma cells.

Superoxide (O2−) radicals have been linked to apoptosis. Here, we show that 2-methoxyestradiol (2ME2)-induced apoptosis in multiple myeloma (MM) cells is associated with O2− generation, whereas dexamethasone (Dex)-induced apoptosis occurs without concurrent increase in O2−. In contrast, both these agents decrease mitochondrial transmembrane potential (Δψm). Treatment of MM cells with an antioxidant N-acetyl-L-cysteine blocks 2ME2, but not Dex-induced apoptosis as well as release of mitochondrial proteins cytochrome c (cyto c) and Smac. Taken together, these results demonstrate that there are at least two distinct apoptotic pathways: one dependent on O2−, which is induced by 2ME2 and is associated with release of cyto c and Smac; and the other an independent of O2−, which is triggered by Dex and associated with Smac release.

Blockade of ubiquitin-conjugating enzyme CDC34 enhances anti-myeloma activity of Bortezomib/Proteasome inhibitor PS-341.

The ubiquitin-conjugating enzyme CDC34 (UBC3) is linked to cell cycle progression in diverse cell types; however, its role in multiple myeloma (MM) pathogenesis is unclear. Here, we show that CDC34 is highly expressed in patient MM cells and MM cell lines versus normal cells. Blocking CDC34 using a dominant-negative strategy enhances the anti-MM activity of Bortezomib/Proteasome inhibitor PS-341, dexamethasone (Dex) and 2-Methoxyestradiol (2ME2). The expression of wild-type CDC34 reduces Dex-induced cytotoxicity in MM cells. Moreover, inhibition of CDC34 enzymatic activity abrogates interleukin-6-induced protection against Dex-induced apoptosis. Together, these findings provide evidence that (1) CDC34 expression is associated with growth and survival of MM cells and (2) blocking CDC34 activity not only enhances anti-MM activity of Bortezomib and 2ME2 but also overcomes IL-6-triggered Dex-resistance.

2-Methoxyestardiol and bortezomib/proteasome-inhibitor overcome dexamethasone-resistance in multiple myeloma cells by modulating Heat Shock Protein-27

Multiple myeloma (MM) remains fatal despite all available therapies. Initial treatment with conventional drugs such as, Dexamethasone (Dex) effectively induces MM cell death; however, prolonged drug exposures results in the development of chemoresistance. Our prior study demonstrated that 2-Methoxyestradiol (2ME2) induces apoptosis in multiple myeloma (MM) cells resistant to Dexamethasone (Dex). Here, we show the mechanism whereby 2ME2 overcomes Dex-resistance. The oligonucleotide array analysis demonstrates that Heat Shock Protein–27 (Hsp27) is upregulated in Dex-resistant, but not in Dex-sensitive MM cells. Proteomics analysis of Hsp27-immunocomplexes revealed the presence of actin in Dex-resistant, but not in Dex-sensitive cells. Biochemical interaction between Hsp27 and actin was examined by co-immunoprecipitation with anti-Hsp27 or actin Abs. Far western blot analysis using GST-Hsp27 fusion protein showed a direct association with actin both in vitro and in vivo. Importantly, 2ME2- or Bortezomib/Proteasome inhibitor (PS-341)-induced apoptosis in Dex-resistant MM cell lines and MM patient cells is associated with disruption of the Hsp27-actin complexes. Finally, blockade of Hsp27 by anti-sense strategy enhanced anti-MM activity of both 2ME2 and PS-341. These findings provide the clinical application of novel therapeutics targeting Hsp27 to improve patient outcome in MM.