Induction of targeted necrosis with HER2-targeted platinum(iv) anticancer prodrugs

Abstract

It is well-recognized that the failure of many chemotherapeutics arises due to an inability to induce apoptosis. Most cancers acquire a myriad of pro-survival adaptations, and the vast heterogeneity and accumulation of multiple often unrelated anti-apoptotic signaling pathways have been a major stumbling block towards the development of conventional chemotherapeutics, which can overcome drug resistance. We have developed highly potent and selective HER2-targeted Pt(iv) prodrugs bearing anti-HER2/neu peptides that induce targeted necrosis as a novel strategy to circumvent apoptosis-resistance. These Pt(iv)-peptide conjugates exhibit a unique biphasic mode of cytotoxicity comprising rapid killing of cancer cells via necrosis in the first phase followed by an extended and gradual phase of delayed cell death. We demonstrate that these Pt(iv)-peptide prodrugs are more potent than their Pt(ii) congeners in direct cell-killing and exhibit comparable long-term inhibition of proliferative capacity and with greater selectivity against HER2-positive cancer cells.

Fig. 1. Synthesis of HER2-targeted Pt(iv)–AHNP conjugate consisting of an AHNP motif tethered to a cytotoxic Pt pharmacophore. The AHNP motif was separated with a small tripeptide spacer (GGK) and functionalized with an aminooxy(acetate) linker at the terminal lysine residue. AHNP sequence: H₂N-YC*DGFYAC*YMDVGGKK (aminooxy)-CONH₂ (* – linked disulfide bridge).

Fig. 2. (a) Pt(iv)–AHNP conjugates are much more efficiently taken up into cells. Whole cell (left) and nuclear (right) uptake after drug treatment (20 μM) for 4 h in NCI-N87 as measured by ICP-MS. Statistical analysis by unpaired Student's t test. (b) Pt(iv)–AHNP conjugates selectively kill HER2 over-expressing NCI-N87 over the normal HER2 expressing A2780. A co-culture of NCI-N87 and A2780 (pre-stained with CellTracker™ Green) was drug-treated for 24 h before viability staining with PI. Statistical analysis by two-way ANOVA and Bonferroni post-tests. Means ± s.e.m. (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3. Apoptosis was evaluated by Annexin V/PI staining of drug-treated cells after 24 h. (a) Scatter plot of treated NCI-N87 cells (15 μM drug) (b) histograms of apoptosis-sensitive NCI-N87 (left) and apoptosis-resistant BT-474 (right) after 24 h exposure.

Fig. 4. (a) Representative overlaid microscopy images of the control and the treated NCI-N87 (15 μM drug for 24 h and allowed to recover in fresh media for a week). Differences in nuclear morphology were visualized by Hoechst 3342 staining. Untreated cells were shown at day 1 while drug-treated cells were shown at day 6. (b) 4a-treated NCI-N87 undergoes short-term proliferation 24 h after drug treatment but displays increasingly severe nuclear fragmentation over 7 d (left to right). (c) Clonogenic assay of NCI-N87 (left) and BT-474 (right) to assess the long-term proliferation ability of single cells drug-treated for 24 h and allowed to recover in fresh complete media. Representative image of AHNP-treated NCI-N87 colonies shown in the inset. Statistical analysis by unpaired Student's t test against non-treated control. Means ± s.e.m. (***p < 0.001).