Lovastatin induces apoptosis of ovarian cancer cells and synergizes with doxorubicin: potential therapeutic relevance

<p>Abstract</p> <p>Background</p> <p>Ovarian carcinoma is a rarely curable disease, for which new treatment options are required. As agents that block HMG-CoA reductase and the mevalonate pathway, the statin family of drugs are used in the treatment of hypercholesterolemia and have been shown to trigger apoptosis in a tumor-specific manner. Recent clinical trials show that the addition of statins to traditional chemotherapeutic strategies can increase efficacy of targeting statin-sensitive tumors. Our goal was to assess statin-induced apoptosis of ovarian cancer cells, either alone or in combination with chemotherapeutics, and then determine these mechanisms of action.</p> <p>Methods</p> <p>The effect of lovastatin on ovarian cancer cell lines was evaluated alone and in combination with cisplatin and doxorubicin using several assays (MTT, TUNEL, fixed PI, PARP cleavage) and synergy determined by evaluating the combination index. The mechanisms of action were evaluated using functional, molecular, and pharmacologic approaches.</p> <p>Results</p> <p>We demonstrate that lovastatin induces apoptosis of ovarian cancer cells in a p53-independent manner and synergizes with doxorubicin, a chemotherapeutic agent used to treat recurrent cases of ovarian cancer. Lovastatin drives ovarian tumor cell death by two mechanisms: first, by blocking HMG-CoA reductase activity, and second, by sensitizing multi-drug resistant cells to doxorubicin by a novel mevalonate-independent mechanism. This inhibition of drug transport, likely through inhibition of P-glycoprotein, potentiates both DNA damage and tumor cell apoptosis.</p> <p>Conclusions</p> <p>The results of this research provide pre-clinical data to warrant further evaluation of statins as potential anti-cancer agents to treat ovarian carcinoma. Many statins are inexpensive, off-patent generic drugs that are immediately available for use as anti-cancer agents. We provide evidence that lovastatin triggers apoptosis of ovarian cancer cells as a single agent by a mevalonate-dependent mechanism. Moreover, we also show lovastatin synergizes with doxorubicin, an agent administered for recurrent disease. This synergy occurs by a novel mevalonate-independent mechanism that antagonizes drug resistance, likely by inhibiting P-glycoprotein. These data raise important issues that may impact how statins can best be included in chemotherapy regimens.</p

Mutations in UBA3 Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924 in Human Leukemic Cells

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562MLN, R-U937MLN) were selected. R-K562MLN and R-U937MLN cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme’s affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562MLN cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors

Identifying molecular features that distinguish fluvastatin-sensitive breast tumor cells

Statins, routinely used to treat hypercholesterolemia, selectively induce apoptosis in some tumor cells by inhibiting the mevalonate pathway. Recent clinical studies suggest that a subset of breast tumors is particularly susceptible to lipophilic statins, such as fluvastatin. To quickly advance statins as effective anticancer agents for breast cancer treatment, it is critical to identify the molecular features defining this sensitive subset. We have therefore characterized fluvastatin sensitivity by MTT assay in a panel of 19 breast cell lines that reflect the molecular diversity of breast cancer, and have evaluated the association of sensitivity with several clinicopathological and molecular features. A wide range of fluvastatin sensitivity was observed across breast tumor cell lines, with fluvastatin triggering cell death in a subset of sensitive cell lines. Fluvastatin sensitivity was associated with an estrogen receptor alpha (ERa)-negative, basal-like tumor subtype, features that can be scored with routine and/or strong preclinical diagnostics. To ascertain additional candidate sensitivity-associated molecular features, we mined publicly available gene expression datasets, identifying genes encoding regulators of mevalonate production, nonsterol lipid homeostasis, and global cellular metabolism, including the oncogene MYC. Further exploration of this data allowed us to generate a 10-gene mRNA abundance signature predictive of fluvastatin sensitivity, which showed preliminary validation in an independent set of breast tumor cell lines. Here, we have therefore identified several candidate predictors of sensitivity to fluvastatin treatment in breast cancer, which warrant further preclinical and clinical evaluation.Fil: Goard, Carolyn A.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. University Of Toronto; CanadáFil: Chan Seng Yue, Michelle . University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. Ontario Institute of Cancer Research. Informatics and Biocomputing Platform; CanadáFil: Mullen, Peter J.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; CanadáFil: Quiroga, Ariel Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tandil. Centro de Investigaciones en Física e Ingeniería del Centro de la Provincia de Buenos Aires; Argentina. University of Alberta; CanadáFil: Wasylishen, Amanda R.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. University Of Toronto; CanadáFil: Clendening, James W.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. University Of Toronto; CanadáFil: Sendorek, Dorota H. S.. Ontario Institute of Cancer Research. Informatics and Biocomputing Platform; CanadáFil: Haider, Syed. Ontario Institute of Cancer Research. Informatics and Biocomputing Platform; CanadáFil: Lehner, Richard. University of Alberta; CanadáFil: Boutros, Paul C.. University Of Toronto; Canadá. Ontario Institute of Cancer Research. Informatics and Biocomputing Platform; CanadáFil: Penn, Linda Z.. University Health Network. Princess Margaret Cancer Centre. Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research; Canadá. University Of Toronto; Canad

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

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

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

MLN4924-resistant K562 cells show decreased inhibition of cullin neddylation by MLN4924 but remain sensitive to knockdown of NEDD8.

<p><b>A)</b> Cells were treated with increasing concentrations of MLN4924 as indicated for 24 hours. After treatment, total cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting using anti-NEDD8 and anti-α-tubulin antibodies to detect NEDD8-cullin complexes and equal protein loading, respectively. <b>B)</b> Cells were infected with lentiviral vectors expressing three independent shRNA sequences targeting NEDD8 (shNEDD8) or a control sequence (shControl), and successfully transduced puromycin-resistant populations were selected. Total cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting with antibodies against NEDD8 and α-tubulin. <b>C)</b> Cells infected with vectors containing shNEDD8 or control sequences and selected for puromycin resistance were seeded in 6-well plates (1×10⁵ cells/ml). After incubation for 2, 3, and 6 days, cell growth and viability were assessed by trypan blue exclusion. Values represent the mean percentage ± SD of viable cells relative to cells infected with control sequences.</p

MLN4924-resistant U937 leukemia cells show reduced sensitivity to MLN4924 and are heterozygous for a UBA3 mutation Y352H.

<p><b>A)</b> Cells (1×10⁴ cells/well) were seeded in 96-well plates and incubated with increasing concentrations of MLN4924 for 72 hours. After incubation, cell viability was measured by the CellTiter-Glo assay. Values shown are the mean percentage ± SD of viable cells relative to controls. <b>B)</b> Cells were treated with increasing concentrations of MLN4924 as indicated for 24 hours followed by isolation of total cellular proteins. Equal amounts of proteins were fractionated by SDS-PAGE and analyzed by immunoblotting with anti-NEDD8 and anti-α-tubulin antibodies. <b>C)</b> UBA3 cDNA in parental and MLN4924-resistant R-U937_MLN cells was sequenced as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093530#pone-0093530-g004" target="_blank">Figure 4</a>. Shown are the nucleotide sequence from codons 347 to 354 for U937 (left panel) and R-U937_MLN cells (right panel), three letter amino acid codes, and codon numbers. Arrows depict the position of the first nucleotide of the codon 352 in which the single nucleotide shift (T → C) in R-U937_MLN occurred. <b>D)</b> Sequence alignment of UBA3s from different organisms was performed, and residues corresponding to the Y352 are shaded. <b>E)</b> The Y352H mutation. To the left the NAE heterodimer surface is shown in grey and its subdomains labelled. Phe352 is shown in magenta. Bound NEDD8 is shown in green ribbon format. On the right, a close-up of the interface between NAE (cyan) and NEDD8 (green) is shown in ribbon format. Shown in line format are residues within proximity.</p