Bortezomib is a proteasome inhibitor that has direct antitumor effects. We and others have previously demonstrated that bortezomib could also sensitize tumor cells to killing via the death ligand, TRAIL. NK cells represent a potent antitumor effector cell. Therefore, we investigated whether bortezomib could sensitize tumor cells to NK cell-mediated killing. Preincubation of tumor cells with bortezomib had no effect on short-term NK cell killing or purified granule killing assays. Using a 24-h lysis assay, increases in tumor killing was only observed using perforin-deficient NK cells, and this increased killing was found to be dependent on both TRAIL and FasL, correlating with an increase in tumor Fas and DR5 expression. Long-term tumor outgrowth assays allowed for the detection of this increased tumor killing by activated NK cells following bortezomib treatment of the tumor. In a tumor purging assay, in which tumor:bone marrow cell mixtures were placed into lethally irradiated mice, only treatment of these mixtures with a combination of NK cells with bortezomib resulted in significant tumor-free survival of the recipients. These results demonstrate that bortezomib treatment can sensitize tumor cells to cellular effector pathways. These results suggest that the combination of proteasome inhibition with immune therapy may result in increased antitumor efficacy.

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Thrombospondin-4 (TSP4) is a matricellular protein found in the heart and upregulated in heart failure; however, its role in cardiac regulation is unknown. Our previous work showed that mice lacking the TSP4 gene (tsp4-/-) respond normally to acute (seconds) pressure overload, but rapidly deteriorate minutes after. Thus, we hypothesize that TSP4 is involved in the slow force response (SFR), the second phase of stretch-mediated adaptation to loading, discovered a century ago, yet not fully understood. One proposed mechanism for the SFR is an increase in ERK phosphorylation that leads to increased Na þ /H þ exchanger-1 activity. The latter increases intracellular Na þ , causing the Na þ /Ca 2þ exchanger (reverse mode) to rise intracellular Ca 2þ , therefore increasing force. To investigate this, cardiac papillary muscles were isolated from tsp4-/and wild-type (WT) littermates. Muscles were stretched from 92% of the length that generated maximum force (L max ) to 98% L max . This length was maintained for 15 min while force and Ca 2þ transients (fura-2AM) were simultaneously recorded. The immediate rise in force without change in Ca 2þ (Frank-Starling) was similar in both groups. However, whereas a positive SFR occurred in WT (33 5 7%, n=5), tsp4-/muscle displayed a negative SFR (À14 5 2%, n=5). The difference in force was accompanied by a rise in Ca 2þ in WT but not in tsp4-/-(7 5 2% vs. 15 2%, P<0.05). Next, hearts were excised from mice 15 minutes following transaortic constriction (pressure overload) and phosphorylation of ERK1/2 and Akt was found to be decreased in the tsp4-/mice (P<0.05). We conclude that TSP4 is necessary for cardiac adaptation to stretch. Its absence blunts the SFR (force and Ca 2þ rise). Moreover, TSP4 seems to be involved in this mechanism upstream of ERK1/2 and Akt.

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in width, and cultivated cardiomyocytes from mouse embryonic hearts on the electrode with same closed circuit shape assisted by agarose microstructures. After the confirmation of regular propagation of beatings, we added a 1mM Astemizole which is well known to raise ventricular arrhythmias, and found that the profile of FP changed to the abnormal beating shape and then reached to the fibrillation shape [1]. To investigate the propagation pathway of excitation on abnormal beating, we constructed the circuit on 16 electrodes with area of 50x50 mm in a similar geometry to ring electrode assay. When abnormal beating happened on convoluted FPs obtained from all the 16 channels, one directional block on propagation were occurred and induced ''re-entry'' [2]. These results suggest that abnormal propagation, such as re-entry, causes abnormal fibrillation-like signal on the ring electrode. In conclusion, a simple quasi-in vivo ECG measurement assay has been proposed and developed and the results showed the typical arrhythmia profiles, in which both the temporal depolarization information and the spatial beating propagation information were appeared. [1] Nomura F, et al.