Mitochondrial Targeting of Doxorubicin Eliminates Nuclear Effects Associated with Cardiotoxicity

The highly effective anticancer agent doxorubicin (Dox) is a frontline drug used to treat a number of cancers. While Dox has a high level of activity against cancer cells, its clinical use is often complicated by dose-limiting cardiotoxicity. While this side effect has been linked to the drug’s direct activity in the mitochondria of cardiac cells, recent studies have shown that these result primarily from downstream effects of nuclear DNA damage. Our lab has developed a mitochondrially targeted derivative of Dox that enables the selective study of toxicity generated by the presence of Dox in the mitochondria of human cells. We demonstrate that mitochondria-targeted doxorubicin (mtDox) lacks any direct nuclear effects in H9c2 rat cardiomyocytes, and that these cells are able to undergo mitochondrial biogenesis. This recovery response compensates for the mitotoxic effects of Dox and prevents cell death in cardiomyocytes. Furthermore, cardiac toxicity was only observed in Dox but not mtDox treated mice. This study supports the hypothesis that mitochondrial damage is not the main source of the cardiotoxic effects of Dox

Activity of tigecycline (1 mg/mL) when freshly prepared in saline.

<p>*Tigecycline solution was preincubated at room temperature for 4 days, which was then used to determine the minimum inhibitory concentration (MIC) in <i>Escherichia coli</i> (MG1655) and mean half-maximal inhibitory concentration (IC₅₀) in TEX cells using the CellTiter viability assay following incubations with bacteria (24 hours) or cells (72 hours), respectively.</p>†<p>Tigecycline solution was preincubated at room temperature for 5 days, and TEX cells were then incubated with 5<b> µ</b>M tigecycline for 48 hours. The IC₅₀ for cell viability was then assessed by the Alamar blue assay, and respiratory complex IV activity was determined in cell lysates. Activity was normalized to citrate synthase content as a proxy for mitochondrial mass.</p><p>All solutions were adjusted to pH 7 and all incubations were performed in the dark. Numbers indicate mean ± standard deviation of at least 3 independent experiments.</p><p>Tig, tigecycline; Pyr, pyruvate; CD, 2-hydroxypropyl-β-cyclodextrin; AA, ascorbic acid.</p

Antileukemic activity of tigecycline reconstituted in saline with pyruvate and ascorbic acid.

<p>*Tigecycline solution (1 mg/mL) was preincubated at room temperature for 4 days, which was then used to determine the mean half-maximal inhibitory concentration (IC₅₀) in OCI-AML2 and HL60 cells using the CellTiter viability assay following 72 hour incubation.</p><p>Pyr, pyruvate; AA, ascorbic acid.</p

The novel ascorbic acid- and pyruvate-containing formulation displays similar tolerability in mice to saline.

<p>NOD/SCID mice (n = 3 per group) were administered saline or the novel formulation (60 mg/mL pyruvate (Pyr), 3 mg/mL ascorbic acid (AA) in saline, pH 7.0) by intraperitoneal injection 5 of 7 days over 3 weeks. At the end of the experiment, mice were sacrificed and serum and organs were collected. (<b>A</b>) Serum levels of total bilirubin, aspartate transaminase (AST) and alkaline phosphatase (ALP) were measured as indicators of liver function, while creatine kinase levels were measured as an indicator of muscle, heart or brain injury. (<b>B</b>) Heart, liver, kidney and muscle organs were sectioned and stained with hematoxylin and eosin. Representative sections from organs are shown of 1 section from 1 of 3 mice per group. Images were collected using a ScanScope XT microscope at 10× magnification. Scale bars are 100 µm.</p

The novel ascorbic acid- and pyruvate-containing formulation displays efficacy in AML cells grown <i>in vivo</i>.

<p><b>(A)</b> Mice were administered 50/kg tigecycline or 50 mg/kg novel tigecycline formulation by intraperitoneal injection and plasma was collected at increasing times after treatment. Plasma tigecycline concentration was determined using HPLC. The peak plasma concentration (<i>C</i>_max), the terminal half-life (<i>t</i>_1/2), area under the plasma concentration-time curve (AUC), clearance (CL) and volume of distribution (<i>V</i>z) were evaluated using WinNonlin 6.2.1. Data represent the mean ± standard deviation of a representative experiment with 3 mice per group. Human leukemia OCI-AML2 cells were injected subcutaneously into the flank of NOD/SCID mice. Eleven days after injection, once tumors were palpable, mice were treated with 50 mg/kg of tigecycline, novel formulation of tigecycline, or vehicle controls (saline or formulation) by intraperitoneal injection twice a day for 11 days (n = 9 per group). Tigecycline in each formulation was prepared fresh twice a day. <b>(B)</b> Tumor volume was monitored over time. Eleven days after injection, mice were sacrificed and tumors excised. <b>(C)</b> Tumor weight was measured. ** indicates p<0.01 and * indicates p<0.05 as determined by Tukey's post-test and one-way ANOVA analysis. Lines represent median. <b>(D)</b> Total proteins were extracted and analyzed by immunoblotting for Cox-1, Cox-2 and Cox-4 expression. PVDF membrane was stained with 0.1% Amido Black.</p

DHR leads to mitochondrial membrane potential collapse in human plasma cells.

<p>LP1 cells were treated with DMSO, 10 or 20 µM of DHR for 24 h, stained by TMRM alone (A) or in combination with Annexin V-FITC (B) followed by flow cytometric analysis. (C) LP1 cells were treated with 10 µM of DHR for indicated time periods, followed by TMRM and Annexin V-FITC staining and flow cytometric analysis.</p

Stability and activity of tigecycline reconstituted in saline with or without additives.

<p>*Tigecycline solution was preincubated at room temperature for 4 days, which was then used to determine the minimum inhibitory concentration (MIC) in <i>Escherichia coli</i> (MG1655) and mean half-maximal inhibitory concentration (IC₅₀) in TEX cells using the CellTiter viability assay following incubations with bacteria (24 hours) or cells (72 hours), respectively.</p>†<p>Tigecycline solution was preincubated at room temperature for 5 days, and TEX cells were then incubated with 5<b> µ</b>M tigecycline for 48 hours. The IC₅₀ for cell viability was then assessed by the Alamar blue assay, and respiratory complex IV activity was determined in cell lysates. Activity was normalized to citrate synthase content as a proxy for mitochondrial mass.</p><p>All solutions were adjusted to pH 7 and all incubations were performed in the dark. Numbers indicate mean ± standard deviation of at least 3 independent experiments, where applicable.</p><p>Tig, tigecycline; Pyr, pyruvate; CD, 2-hydroxypropyl-β-cyclodextrin; AA, ascorbic acid.</p

Glycogen Synthase Kinase-3 Inhibition Sensitizes Pancreatic Cancer Cells to TRAIL-Induced Apoptosis

<div><p>Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines with little or no effect on normal cells. However, its effect is limited as some cancers including pancreatic cancer show de novo resistance to TRAIL induced apoptosis. In this study we report that GSK-3 inhibition using the pharmacologic agent AR-18, enhanced TRAIL sensitivity in a range of pancreatic and prostate cancer cell lines. This sensitization was found to be caspase-dependent, and both pharmacological and genetic knock-down of GSK-3 isoforms resulted in apoptotic features as shown by cleavage of PARP and caspase-3. Elevated levels of reactive oxygen intermediates and disturbance of mitochondrial membrane potential point to a mitochondrial amplification loop for TRAIL-induced apoptosis after GSK-3 inhibition. Consistent with this, overexpression of anti-apoptotic mitochondrial targets such as Bcl-XL, Mcl-1, and Bcl-2 rescued PANC-1 and PPC-1 cells from TRAIL sensitization. However, overexpression of the caspase-8 inhibitor CrmA also inhibited the sensitizing effects of GSK-3 inhibitor, suggesting an additional role for GSK-3 that inhibits death receptor signaling. Acute treatment of mice bearing PANC-1 xenografts with a combination of AR-18 and TRAIL also resulted in a significant increase in apoptosis, as measured by caspase-3 cleavage. Sensitization to TRAIL occurred despite an increase in β-catenin due to GSK-3 inhibition, suggesting that the approach might be effective even in cancers with dysregulated β-catenin. These results suggest that GSK-3 inhibitors might be effectively combined with TRAIL for the treatment of pancreatic cancer.</p> </div

DHR induces human plasma cell death by activating apoptotic pathway.

<p><b>A</b>, Human plasma cells LP1 and KMS11 were treated with DHR at the indicated concentrations for 24 h. Cell lysates were then prepared and subject to immunoblotting assay against apoptosis-associated proteins caspase-3, -8 and -9. GAPDH were used as a loading control. B, LP1 and KMS11 cells were treated for 24 h with DMSO, DHR, z-VAD-fmk or DHR+Z-VAD-fmk, followed by caspase-3 analysis by Western blotting. C, LP1 and KMS11 cells were treated for 24 h with DMSO, DHR, z-VAD-fmk or DHR+Z-VAD-fmk, followed by Annexin-V-FITC/PI staining and flow cytometric analyses. Pro-casp: pro-caspase; Cle-casp: cleaved caspase.</p

Affected fractions and combination indices with RITA and DXM combination on H929 cell lines.

<p>Notes; A CI of less than, equal to, and more than 1 indicates synergy, additivity, and antagonism, respectively.</p