RON receptor tyrosine kinase as a target for delivery of chemodrugs by antibody directed pathway for cancer cell cytotoxicity.

Overexpression of the RON receptor tyrosine kinase exists in various cancers and contributes to malignant progression. To validate RON as a targeting moiety for delivery of chemoagents for enhanced tumor cytotoxicity, immunoliposomes (IL) loaded with doxorubicin (Dox) were formulated followed by postinsertion of monoclonal antibodies Zt/g4, Zt/c1, or their Fab fragments specific to the RON extracellular domains. Flow cytometry analysis showed that Zt/g4 or Zt/c1-IL binds to cancer cells and causes RON internalization as evident in confocal analysis of intracellular fluorescence intensity. The antibody-directed IL uptake by cancer cells is in both dose and time-dependent manners. Studies of cytotoxicity of individual IL in vitro against colon or breast cancer cell lines revealed that Zt/g4 directed Dox-IL displayed increased cytotoxic activities with a significant reduction of IC(50) values. An average of 8-fold increases in cytotoxic efficiency was achieved among four cell lines tested. Moreover, Zt/g4 directed Dox-IL also displayed the effective killing of cancer cells that are insensitive to pegylated liposomal doxorubicin. The effect of Zt/c1-Dox-IL was not as strong as Zt/g4-Dox-IL, and only moderate activities were observed. IL coupled with the Fab fragments of Zt/g4 or Zt/c1 show moderate activities against cancer cells. The ineffectiveness seemed to be related to the weak activities of the Fab fragments in the induction of RON internalization, which resulted in reduced drug uptakes. We conclude that anti-RON antibody-directed drug delivery is effective for increased uptake of cytotoxic drugs. Antibody-based RON targeting could be developed into a potential therapeutic for treatment of malignant cancers.

Targeting acute hypoxic cancer cells by doxorubicin-immunoliposomes directed by monoclonal antibodies specific to RON receptor tyrosine kinase.

PURPOSE: Hypoxia contributes to acquired drug resistance in various cancer cells. The underlying mechanism is cellular insensitivity regulated by hypoxia-inducible factors (HIF), which impairs drug uptake, transport, and metabolism. The current study determines anti-RON antibody-directed cytotoxicity of doxorubicin (Dox)-immunoliposomes (IL) in hypoxic colon cancer cells. METHODS: Cells were cultured under hypoxia (1% O(2), 5% CO(2), and 96% N(2)) for 24 h. Dox-loaded IL were formulated followed by post-insertion of monoclonal antibody Zt/g4 specific to RON. Western blotting was used to detect HIF-1α and RON expression. Cellular uptake of Zt/g4-conjugated IL was determined by confocal and internalization assays. Cell viability was assessed by the MTT assay. RESULTS: RON and HIF-1α expression were observed in hypoxic colon HCT116 and SW620 cells. Resistance to Dox-induced cytotoxicity was acquired in hypoxic cells with increased IC(50) values. However, acquired resistance was attenuated by Zt/g4-directed Dox-IL, which displays increased cytotoxic activities. IL binding and uptake revealed that hypoxic RON expression is functional, which mediates high levels of Zt/g4-Dox-IL binding and cytoplasmic internalization. Zt/g4-Dox-IL is effective in killing hypoxic HCT116 and SW620 cells with reduced IC(50) values compared to Dox and pegylated-liposomal Dox. These effects were dependent on hypoxic RON expression. HCC1937 cells with diminished RON expression under hypoxia were insensitive to Zt/g4-Dox-IL-induced cytotoxic effect. CONCLUSIONS: RON expressed by hypoxic colon cancer cells is thus a potential targeting molecule for delivery of chemotherapeutics. The ability of anti-RON mAb to direct Dox-IL cytotoxicity could be developed for attenuating hypoxia-acquired drug resistance in various cancer cells.