Supplementary Figure Legends from Novel Targeting of Phospho-cMET Overcomes Drug Resistance and Induces Antitumor Activity in Multiple Myeloma (original) (raw)
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Purpose: The aim of the study was to verify the hypothesis that the cMet oncogene is implicated in chemio- and novel drug resistance in multiple myeloma.Experimental Design: We have evaluated the expression levels of cMET/phospho-cMET (p-cMET) and the activity of the novel selective p-cMET inhibitor (SU11274) in multiple myeloma cells, either sensitive (RPMI-8226 and MM.1S) or resistant (R5 and MM.1R) to anti–multiple myeloma drugs, in primary plasma cells and in multiple myeloma xenograft models.Results: We found that resistant R5 and MM.1R cells presented with higher cMET phosphorylation, thus leading to constitutive activation of cMET-dependent signaling pathways. R5 cells exhibited a higher susceptibility to the SU11274 inhibitory effects on viability, proliferation, chemotaxis, adhesion, and to its apoptogenic effects. SU11274 was able to revert drug resistance in R5 cells. R5 but not RPMI-8226 cells displayed cMET-dependent activation of mitogen-activated protein kinase pathwa...
This was performed by SYBR Green and the StepOne system (Applied Biosystems, Foster City, CA, USA) (1). The PCR was conducted using HGF (5'-TCCACGGAAGAGGAGATGAGA-3'/5'-GGCCATATACCAGCTGGGAAA-3'), cMET (5'-GCTAAAATGCTGGCACCCTAA-3'/5'-ATATCCGGGACACCAGTTCAGA-3'), and control glyceraldehyde 3 phosphate dehydrogenase (GAPDH; 5′-GAAGGTGAAGGTCGGAGT-3′/5′-CATGGGTGGAATCATATTGGAA-3′) primers (Invitrogen, Paisley, UK). The mRNA was quantified using GAPDH as the reference gene and the 2-∆∆CT formula (2). Immunoprecipitation and Western blot Total proteins from the MM patients' plasma cells (PCs, 5×10 5 /patient) and the MM cell lines (2×10 6 /sample) were incubated with anti-cMET antibody, and antigen-antibody complexes immunoprecipitated by Protein G/agarose. Protein aliquots (50 μg) were immunoblotted with anti-cMET and anti-phospho(p)-cMET (p-cMET, Tyr1349) antibodies (both from Cell Signaling Technology, Danvers, MA, USA) (3). Immunoreactive bands were detected with enhanced chemiluminescence (LiteAblot ® , Euroclone, Milan, Italy) and the Gel-Logic1500 system (Eastman Kodak Co., Rochester, NY, USA), and quantified as optical density (OD) units by the Kodak imaging software. Florescence-activated cell sorting (FACS) Patients' bone marrow mononuclear cells (BMMCs, 1×10 6 cells/tube) and the MM cell lines (5×10 5 cells/tube) were incubated with fluorescein isothiocyanate (FITC)-conjugated mouse anti-cMET, rabbit anti-p-cMET (Y1234/1235), and isotype matched control antibodies (R&D Systems, Inc.,
Development of target-specific treatments in multiple myeloma
British Journal of Haematology, 2010
In the last decade, the novel agents lenalidomide, bortezomib, and thalidomide have dramatically improved outcomes for patients with multiple myeloma (MM). A number of new therapies with precise targets involved in MM cell growth and replication are now in development and have the potential for further improvements. Second-generation proteasome inhibitors and thalidomide derivatives may offer increased efficacy and safety. Investigational therapies with rationally selected targets in MM include inhibitors of histone deacetylase, heat shock protein 90, mammalian target of rapamycin, BCL2, Akt, mitogen-activated protein kinase, and telomerase. In addition, monoclonal antibodies directed against several targets have been developed and many are showing promise in initial clinical trials in MM. Interest in the ancient remedy of arsenic trioxide has been revived because of its proapoptotic effects on mitochondria, despite its established toxicities. In general, combination regimens are proving the most efficacious, which is to be expected given the multiple overlapping pathways responsible for MM growth and progression.