257 The Medical Coat: Bacterial Vector Versus Professional Traditional

The aim of the present study is to establish a bacterial clone capable of secreting an integrinαvβ3 targeting probe with bioluminescent and fluorescent activities, and to verify its specific targeting and optical activities using molecular imaging. A bacterial vector expressing a fusion of secretory Gaussia luciferase (sGluc), mCherry, and RGD (sGluc-mCherry-RGDX3; GCR), and a control vector expressing a fusion of secretoryGaussia luciferaseand mCherry (sGluc-mCherry; GC) were constructed. The GCR and GC proteins were expressed inE. coliand secreted into the growth medium, which showed an approximately 10-fold higher luciferase activity than the bacterial lysate. Successful purification of GCR and GC was achieved using the 6X His-tag method. The GCR protein bound with higher affinity to U87MG cells than CHO cells in confocal microscopy and IVIS imaging, and also showed a high affinity for integrinαvβ3 expressing tumor xenografts in anin vivoanimal model. AnE. coliclone was established to secrete an integrinαvβ3 targeting imaging probe with bioluminescent and fluorescent activities. The probe was produced feasibly and at low cost, and has shown to be useful for the assessment of angiogenesisin vitroandin vivo.

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Requirement of Caspase-8 Versus Caspase-9 during Apoptosis in Multiple Myeloma Cells Induced by Bortezomib- or a Novel Proteasome Inhibitor NPI-0052.

Blood ◽

10.1182/blood.v106.11.3378.3378 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3378-3378 ◽

Cited By ~ 1

Author(s):

Dharminder Chauhan ◽

Laurence Catley ◽

Mugdha Velankar ◽

Anthony Letai ◽

Teru Hideshima ◽

...

Keyword(s):

Multiple Myeloma ◽

Proteasome Inhibitor ◽

Proteasome Inhibition ◽

Proteolytic Processing ◽

Caspase 8 ◽

Caspase 9 ◽

Transfected Cells ◽

Viable Cells ◽

Vector Versus ◽

Induced Apoptosis

Abstract Proteasome inhibition is an effective therapy for the treatment of relapsed/refractory multiple myeloma (MM); however, the sequence of events leading to apoptosis following proteasome inhibition is unclear. Here we defined the requirement for caspase-8 versus caspase-9 during Bortezomib (VelcadeTM)- or novel proteasome inhibitor NPI-0052-induced apoptosis in MM cells. Incubation of MM.1S cells with pan-caspase inhibitor (Z-VAD-FMK) markedly abrogates both NPI-0052- and Bortezomib-induced apoptosis. Inhibition of caspase-8 (IETD-FMK) led to a significant decrease in NPI-0052-triggered cell death, whereas inhibition of caspase-9 (LEHD-FMK) only moderately blocked NPI-0052-triggered decreased viability in MM.1S cells (P < 0.005). In contrast, Bortezomib-induced decrease in viability is equally blocked by either caspase-8 or caspase-9 inhibitors (P < 0.005). These biochemical data were further examined by genetic studies using dominant-negative (DN) strategies. Treatment of DN-caspase-8-transfected MM cells with NPI-0052 (IC50: 7 nM) markedly increases survival compared to cells transfected with DN-caspase-9. In contrast, treatment of either DN-caspase-8 or DN caspase-9-tranfected MM.1S cells with Bortezomib (IC50: 5 nM) increases survival to a similar extent. The proteolytic processing of pro-caspase-8 is mediated by Fas Associated Death-Domain (FADD) protein, and we next examined blockade of FADD with DN-FADD to further confirm the role of caspase-8. DN-FADD significantly attenuated NPI-0052-induced cytotoxicity compared to empty vector-transfected MM.1S cells (42 ± 2.0% viable cells in vector- versus 76 ± 5.1% viable cells in DN-FADD-transfected cells; P < 0.05). Importantly, treatment of DN-FADD-transfected MM.1S cells with Bortezomib results in only 16% increase in survival compared to vector-transfected cells (39 ± 2.4% viable cells in vector- versus 55 ± 4.1% viable cells in DN-FADD-transfected cells; P < 0.05). These data, coupled with caspase-8 or caspase-9 inhibition studies, suggest that NPI-0052 relies more on FADD-caspase-8 signaling than does Bortezomib, confirming a differential mechanism of action of NPI-0052 versus Bortezomib in MM cells. To further address this issue, we examined alterations in Bax, a proapoptotic protein which translocates from cytosol to mitochondria during apoptosis, inhibits Bcl-2, and facilitates release of cyto-c and activation of caspase-9. NPI-0052 induces little, if any, increase in Bax levels in mitochondria, whereas Bortezomib triggers a significant accumulation of Bax in mitochondria. Experiments using Bax wild type (WT) or knockout mouse embryonic fibroblast (MEFs) show that NPI-0052 decreases viability in both Bax (WT) and Bax (knock-out) MEFs, whereas deletion of Bax confers significant resistance to Bortezomib. These findings suggest a differential requirement for Bax during NPI-0052- versus Bortezomib-induced apoptosis. Collectively, our data suggest that 1) NPI-0052-induced MM cell apoptosis is predominantly mediated by caspase-8; and 2) Bortezomib-induced apoptosis requires both caspase-8 and caspase-9 activation. These data provide rationale for combining agents based on differential signaling cascades to amplify apoptosis and enhance anti-tumor activity.

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