FIGURE 6 from Metronomic Administration of Topotecan Alone and in Combination with Docetaxel Inhibits Epithelial–mesenchymal Transition in Aggressive Variant Prostate Cancers

Metronomic topotecan treatment as a single agent and in combination with DTX reduces EMT/stemness in AR^Low/mCRPC/NEPC clonally derived taxane-resistant prostate cancer subtypes. A, Assessment of posttreated “stem-like” cells (CD44^high/CD133^high) population in taxane-resistant AR^Low/mCRPC cell lines by Flow cytometry: DUTXR cells line stained with stemness markers (CD44, CD33, and both CD44/133) after CONV-TOPO, METRO-TOPO, and combination (CONV-DTX+METRO-TOPO) treatments. CONV-TOPO, METRO-TOPO, and combination (CONV-DTX+METRO-TOPO) treatments reduced CD44^high cells 81.0%, 71.5%, and 63.4%, respectively, in taxane-resistant AR^Low mCRPC DUTXR cells. Therefore, METRO-TOPO as single agent and in combination treatment reduced the highest percentage of “stem like” cell population load (P < 0.05; Table 2). B, Colony-forming assay: Number and size of colonies were reduced after all treatments compared with control (no-drug treatment). Next, combination treatment with CONV-DTX+METRO-TOPO reduced the number and size of colonies the greatest compared with CONV-TOPO, CONV-DTX, and METRO-TOPO treatments in the DUTXR cell line. Posttreatment colony number and size reduction in the following order: CONV-TOPO>CONV-DTX>METRO-TOPO>Combination (METRO-TOPO+CONV-DTX). Therefore, METRO-TOPO as a single agent or in combination reduced highest number of the colony and size compared with other treatments. Colonies were developed for 21 days (*, P ≤ 0.05). C, Immunoblot analysis: Proteins representing top EMT markers were downregulated significantly after METRO-TOPO treatment compared with CONV-TOPO treatment in AR^Low/mCSPC/NEPC^{taxane-resistant} (DUTXR) prostate cancer cell lines. Combination treatment (CONV-DTX+METRO-TOPO) exhibited the highest downregulation of EMT marker proteins compared with other treatments. Posttreatment protein expression downregulation was following orders: CONV-TOPO>CONV-DTX>METRO-TOPO. Beta-actin was used as a control housekeeping gene (*, P ≤ 0.05). D, Assessment of treatment effect on “stem-like” cells (CD44^high) population in taxane-resistant AR^Low/mCRPC cell lines by FACS: DUTXR cell line stained with stemness markers (CD44) and sorted CD44⁺ versus CD44⁻ cells, followed by CONV-TOPO, METRO-TOPO, CONV-DTX, and combination (CONV-DTX+METRO-TOPO) treatments. Next, cell cytotoxicity (MTT) and caspase 3/7 activity (apoptosis) were assessed. (I) Cytotoxicity profiling by MTT showed that combination (CONV-DTX+METRO-TOPO) treatment reduced highest cell survival or cell growth compared with other treatments. Posttreatment cell survival or cell growth reduction was following orders: CONV-TOPO>CONV-DTX>METRO-TOPO. (II) Combination (CONV-DTX+METRO-TOPO) treatment induced greater apoptosis compared with other treatments in taxane-resistant AR^Low/mCRPC-DUTXR cell lines. (III and IV) CD44⁻ cells showed no significant differences for all treatments (*, P ≤ 0.05). E, Pretreatment and posttreatment microscope imaging: Results showed significantly higher cell death in METRO-TOPO and combination (CONV-DTX+METRO-TOPO) treatments compared with CONV treatment for both the drugs (TOPO and DTX). Images were captured by Cytation5 Cell Imaging Multimode Reader at a 1,000 μm scale. ImageJ analysis showed a significant difference in cell density for CONV versus METRO versus combination TOPO treatments (*, P ≤ 0.05).