Pivarubicin Is More Effective Than Doxorubicin Against Triple-Negative Breast Cancer In Vivo

Figure 1. Isosteric similarity of pivarubicin and diacylglycerol (DAG). Bold lines identify the putative pharmacophores (C1b binding site) for (A) pivarubicin, (B) diacylglycerol (DAG), and (C) phorbol 12-myristate 13-acetate (PMA).

Figure 2. Cellular localization of pivarubicin. 32D.3 cells at a density of 1 × 106/ml were exposed to 5 μM doxorubicin or 1 μM pivarubicin for 1 h. Nuclear counterstaining of pivarubicin-treated cells was performed with 16 μg/ml of bisbenzimide for 1 h. Fluorescence microscopy was performed as described in the Materials and Methods section. Images’ original magnification: 1,000×.

Figure 3. Pivarubicin-mediated rapid apoptosis. (A) Cytochrome c release by pivarubicin. 32D.3 cells were exposed to 5 μM pivarubicin for 1 h and then incubated in drug-free medium for an additional 4 h. Cells were then fractionated to isolate the cytosolic fraction, subjected to immunoblotting, and treated with anti-cytochrome c monoclonal antibody (1:250) for 2 h, followed by a 1-h treatment with 1:1,000 dilution of horseradish peroxidase-conjugated goat anti-mouse second antibody as described previously12. Proteins were detected by chemiluminescence. (B) Detection of DNA fragmentation in pivarubicin-treated 32D.3 cells by TUNEL assay. Cells were treated with 5 μM pivarubicin for 1 h, washed twice in warm PBS, incubated in drug-free medium for 6 h, and then prepared for 3′-biotinylation of fragmented DNA using the TUNEL assay procedure. Individual fields of cells were detected for both DNA 3′-end labeling (green) or total cellular DNA staining by propidium iodide (red). Composite image is representative of three independent experiments.

Figure 4. Pivarubicin cytotoxicity is mediated through PKC-delta (PKCd) activation. (A) 32D.3 cells, suspended in RPMI-1640 medium/10% FCS/IL-3 at a density of 5 × 105 cells/ml, were treated with either DMSO (C) or 5 μM pivarubicin (PIV) for 4 h prior to harvesting, cell fractionation, and immunoblot analysis of PKCd as described previously14. (B) 32D.3 cells were pretreated with 10 μM rottlerin for 2 h prior to exposure to 5 μM drug for 1 h at 37°C. Cells were pelleted and washed twice in large volumes of warm PBS, then resuspended in fresh, drug-free medium containing 10 μM rottlerin at 5 × 105 cells/ml and incubated at 37°C for up to 72 h. At indicated times, aliquots of cells were withdrawn and stained with trypan blue. Viable cells were scored based on the exclusion of stain and gross morphological appearance. (C) 32D.3 cells were transfected with PKCd siRNA or scrambled siRNA as described in the Materials and Methods section and assessed for pivarubicin cytotoxicity as in (A). Each datum point represents the mean and standard error of at least three independent determinations, each consisting of 300–500 cells per count, when possible.

Figure 5. Pivarubicin circumvents multiple mechanisms of MDR. (A) LNCaP cells transfected with Bcl-2 expression vector were assessed for doxorubicin or pivarubicin cytotoxicity, compared to cells transfected with empty vectors. Cells were exposed to 5 μM drug for 1 h, washed, and further incubated for 72 h in drug-free medium. Viability was determined by the MTT assay. (B) Pivarubicin cytotoxicity in HL-60 cells transfected with Bcr-Abl or Bcl-XL expression vectors were assessed for doxorubicin or pivarubicin cytotoxicity and compared as described for (A). Each datum point represents the mean and standard error of at least three independent determinations.

Figure 6. Pivarubicin is more cytotoxic than doxorubicin against triple-negative breast cancer (TNBC) monolayer cells. (A) MDA-MB-231 and SUM-159 cells were maintained under adherent monolayer growth conditions. Upon reaching 50% confluency, cells were treated for 48 h with the dose range of doxorubicin or pivarubicin as indicated. Cell viability was corrected for death observed in vehicle-only-treated cells (<1% ethanol final well concentration). (B) MDA-MB-231 monolayer culture was plated at 6,500 cells per well into a 24-well ultralow adhesion dish in tumorsphere medium with either vehicle, doxorubicin (DOX), or pivarubicin (PIV) and cultured for up to 7 days prior to obtaining digital images. Bar graph represents the mean number of tumorspheres greater than 50 mm in diameter/well (n > 3).

Figure 7. Doxorubicin treatment of TNBC-bearing NSG mice. (A) LM2 cells were implanted into the 4R and 4L cleared mammary fat pads of female NSG mice and permitted to proliferate for 26 days until a tumor volume range of 125–190 mm3 was reached based on caliper measurements. Doxorubicin was administered IP rapid push in 70% sterile saline, 15% EtOH, 15% Cremophor EL at 14-day intervals, with tumor volumes measured at indicated days postimplantation. Dosing was suspended at 47 days due to tumor size. At endpoint, mice were euthanized and primary tumors resected for volume (B), weight (C), and luminescence (D) measurements. Endpoint results are expressed as the mean ± standard error of 10 mice per group. Statistical significance was determined by the Wilcoxon rank-sum test.

Figure 8. Pivarubicin treatment of TNBC-bearing NSG mice. LM2 cells were implanted into the 4R and 4L cleared mammary fat pads of female NSG mice and permitted to proliferate for 22–24 days until a tumor volume range of 125–190 mm3 was reached based on caliper measurements. Pivarubicin was administered IP rapid push in 70% sterile saline, 15% EtOH, 15% Cremophor EL as either a single dose at 26.6 mg/kg (A) or in two 16.5-mg/kg doses at a 14-day interval (B) with tumor volumes measured at indicated days postimplantation. Dosing was suspended at 47 days due to tumor size in vehicle-treated mice. At endpoint, mice were euthanized and primary tumors resected for volume (C) and weight (D) measurements. Results are expressed as the mean ± standard error of 10 mice per group. Statistical significance was determined by the Wilcoxon rank-sum test.

Figure 9. Representative stained thin sections: cardiotoxicity analysis of nontumor-bearing NSG mice. Images show evidence of microvacuolization (arrows) in both vehicle- and doxorubicin-treated mice.