We have created ERroGFP-S4, a novel ER redox probe suitable for monitoring redox dynamics in the oxidative environment of the ER. ERroGFP-S4 can be used for detection of aberrant ER redox states related to various physiological and pathological conditions.
Follicular lymphoma (FL) commonly recurs and is difficult to cure. Obinutuzumab is a humanized glycoengineered type II anti-CD20 antibody with a mode of action that includes induction of antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and direct cell death. There is no evidence on the effectiveness of re-treatment with obinutuzumab in patients with prior obinutuzumab treatment. Using obinutuzumab-induced-direct-cell-death-resistant cells, we investigated the efficacy of obinutuzumab re-treatment in combination with chemotherapeutic agents used in FL treatment. Human non-Hodgkin lymphoma (NHL) SU-DHL-4 cells were sustainably exposed to obinutuzumab in vitro, and seventeen resistant clones expressing CD20 and showing 100-fold higher IC 50 of obinutuzumab than parental cells were established. The growth inhibition effect of obinutuzumab in combination with bendamustine, 4hydroperoxy-cyclophosphamide, doxorubicin, vincristine, or prednisolone was estimated using an interaction index based on the Bliss independence model. For each clone, there were various combinations of obinutuzumab and chemotherapeutic agents that showed supra-additive effects. Obinutuzumab combined with doxorubicin enhanced caspasedependent apoptosis and growth inhibition effect. Obinutuzumab combined with prednisolone enhanced DNA fragmentation and G0/G1 arrest. These combinations also had an antitumor effect in mouse xenograft models. Our results indicate that re-treatment with obinutuzumab, when it is combined with chemotherapeutic agents, is effective in the CD20-positive obinutuzumab-induced-direct-cell-death-resistant cells.
Purpose
Trastuzumab emtansine (T-DM1) is the standard treatment in the current second-line therapy of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. However, a useful therapy after T-DM1 resistance has not been established. In this study, we established two different HER2-positive T-DM1-resistant cancer cells and evaluated the antitumor effect of trastuzumab in combination with pertuzumab (TRAS + PER).
Methods
Single-cell-cloned OE19 and BT-474 cells were cultured with increasing concentrations of T-DM1 to generate T-DM1-resistant OE19bTDR and BT-474bTDR cells, respectively. HER2 expression was assessed by immunohistochemistry. Multidrug resistance proteins (MDR1 and MRP1) were evaluated by real-time polymerase chain reaction and western blotting. Intracellular trafficking of T-DM1 was examined by flow cytometry and immunofluorescence staining. Efficacy of TRAS + PER was evaluated by cell proliferation assay, HER3 and AKT phosphorylation, caspase 3/7 activity, and antitumor activity.
Results
HER2 expression of both resistant cells was equivalent to that of the parent cells. Overexpression of MDR1 and MRP1 was observed and affected the T-DM1 sensitivity in the OE19bTDR cells. Abnormal localization of T-DM1 into the lysosomes was observed in the BT-474bTDR cells. In BT-474bTDR cells, TRAS + PER inhibited the phosphorylation of AKT involved in HER2–HER3 signaling, and apoptosis induction and cell proliferation inhibition were significantly higher with TRAS + PER than with the individual drugs. TRAS + PER significantly suppressed tumor growth in the OE19bTDR xenograft model compared with each single agent.
Conclusions
The results suggest that the TRAS + PER combination may be effective in T-DM1-resistant cancer cells where HER2 overexpression is maintained.
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