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Keywords:

  • ovarian cancer;
  • chemoresistance;
  • caspase-independent apoptosis;
  • mammalian target of rapamycin

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

BACKGROUND:

Resistance to apoptosis is 1 of the key events that confer chemoresistance and is mediated by the overexpression of antiapoptotic proteins, which inhibit caspase activation. The objective of this study was to evaluate whether the activation of an alternative, caspase-independent cell death pathway could promote death in chemoresistant ovarian cancer cells. The authors report the characterization of NV-128 as an inducer of cell death through a caspase-independent pathway.

METHODS:

Primary cultures of epithelial ovarian cancer (EOC) cells were treated with increasing concentration of NV-128, and the concentration that caused 50% growth inhibition (GI50) was determined using a proprietary assay. Apoptotic proteins were characterized by Western blot analyses, assays that measured caspase activity, immunohistochemistry, and flow cytometry. Protein-protein interactions were determined using immunoprecipitation. In vivo activity was measured in a xenograft mice model.

RESULTS:

NV-128 was able to induce significant cell death in both paclitaxel-resistant and carboplatin-resistant EOC cells with a GI50 between 1 μg/mL and 5 μg/mL. Cell death was characterized by chromatin condensation but was caspase-independent. The activated pathway involved the down-regulation of phosphorylated AKT, phosphorylated mammalian target of rapamycin (mTOR), and phosphorylated ribosomal p70 S6 kinase, and the mitochondrial translocation of beclin-1 followed by nuclear translocation of endonuclease G.

CONCLUSIONS:

The authors characterized a novel compound, NV-128, which inhibits mTOR and promotes caspase-independent cell death. The current results indicated that inhibition of mTOR may represent a relevant pathway for the induction of cell death in cells resistant to the classic caspase-dependent apoptosis. These findings demonstrate the possibility of using therapeutic drugs, such as NV-128, which may have beneficial effects in patients with chemoresistant ovarian cancer. Cancer 2009. © 2009 American Cancer Society.

Epithelial ovarian cancer (EOC) is the fourth leading cause of cancer-related deaths in women in the United States and is the most lethal of the gynecologic malignancies, with a 5-year survival rate that approaches only 15%.1 A major challenge in the management of ovarian cancer is the development of chemoresistance to most of the currently available agents. Consequently, there is an urgent need for better therapeutic options.

Although chemoresistance is multifactor, it is well accepted that resistance to programmed cell death (PCD) is a major contributing cause.2 PCD is an evolutionary conserved pathway, the activation of which leads to an energy-dependent cell suicide mechanism. In general, 2 forms of PCD have been described: 1) apoptosis or caspase-dependent cell death and 2) caspase-independent cell death.3

Apoptosis is the better characterized pathway of the 2 types of PCD, such that the terms PCD and apoptosis have been used interchangeably throughout the literature. It involves the sequential activation of a group of proteases, the caspases, and can be activated either by ligation of death receptors such as Fas and the tumor necrosis factor receptor (extrinsic pathway), or by mitochondrial depolarization (intrinsic pathway).4, 5 The Bcl-2 family of proteins, which has both proapoptotic (Bcl-2 antagonist/killer [Bak], Bcl-2–associated X protein [Bax], etc) and antiapoptotic members (Bcl-2, Bclx), controls mitochondrial integrity, and the decision to engage the intrinsic pathway depends on the ratio of the proapoptotic and antiapoptotic members in the outer mitochondrial membrane.5 Numerous studies have demonstrated that most chemotherapy agents induce cell death by activating the apoptotic pathway and that resistance to apoptosis because of high intracellular levels of antiapoptotic blockers such as X-linked inhibitor of apoptosis (XIAP) is a major cause of chemoresistance. Indeed, molecular or drug targeting of apoptotic blockers such as XIAP results in the reversal of chemoresistance.6-8

Caspase-independent cell death encompasses all events that occur when cells die in the absence of caspase activation. Autophagy is the most characterized caspase-independent PCD pathway and, hence often is called Type II PCD. It involves the controlled formation of autophagosomes, a double-membrane cytoplasmic vesicle, which can fuse with lysosomes, thus leading to the digestion of molecules within the autophagosome.9 Autophagy is controlled by the Akt-mammalian target of rapamycin (mTOR) pathway and involves key proteins such as beclin-1 and Class III phosphatidylinositol-3 kinase (PI3K).10 That pathway is activated to promote cell survival; however, because of the inherent mechanisms invoked, it also can lead to cell death.11

Cancer cells in general, and EOC cells in particular, are very resistant to apoptotic cell death mainly because of high-level expression of the apoptotic blockers XIAP and FLICE inhibitory protein.12 Thus, we hypothesized that, in addition to targeting specific apoptotic blockers, another way to overcome chemoresistance is to circumvent apoptosis and exploit nonpoptotic or caspase-independent cell death pathways.

The PI3K/AkT/mTOR signaling axis plays a central role in the regulation of multiple, critical cellular functions, including stress responses, cell growth and survival, and metabolism. In many human tumors, including EOC, PI3K/AKT/mTOR signaling is deregulated by a variety of oncogenic events. Therefore, the modulation of this pathway may provide alternative therapies for patients with ovarian cancer.

NV-128 is a member of the phenyl-substituted isoflavone family of compounds similar to phenoxodiol,12, 13 which, in preliminary screening, were effective in reducing viability in a series of cancer cells; suggestive of potential antitumor activity. In the current study, we investigated the molecular mechanisms by which NV-128 induces cell death. We describe in this report the activation by NV-128 of a caspase-independent cell death pathway in EOC cells. This pathway involves the inhibition of mTOR phosphorylation, mitochondrial translocation of the autophagy molecule beclin-1, and mitochondrial release and nuclear localization of the nuclease endonuclease G (EndoG).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Cell Lines and Culture Conditions

Established human EOC cell lines, A2780 and A2780/CP70 (gifts from Dr. T. C. Hamilton)14 were propagated in RPMI plus 10% fetal bovine serum (Gemini Bio-Products, Woodland, Calif). Primary EOC cell lines were isolated from malignant ovarian ascites or were explanted from ovarian tumors and cultured as described previously.6, 15

Cell Viability Assay

Cell viability was determined as reported previously6, 15 using the CellTiter 96 Aqueous 1 Solution cell proliferation assay (Promega Corporation, Madison, Wis). NV-128 (Novogen, Inc., North Ryde, New South Wales, Australia) was added to the medium from 10 mg/mL stock to yield various final concentrations. Each experiment was done in triplicate.

For experiments using the caspase inhibitor, Z-VAD-FMK (Sigma Aldrich, St. Louis, Mo), the inhibitor was added to the cultures 30 minutes before treatment to yield a final concentration of 20 μM. For experiments using the autophagy inhibitor 3 methyladenine (Sigma Aldrich), the inhibitor was added 1 hour before treatment to yield a final concentration of 10 mM.

Flow Cytometry With Hoechst and Propidium Iodide Staining

Cells were stained with 5 μg/mL Hoechst 33342 (Invitrogen-Molecular Probes, Carlsbad, Calif) and 1 μg/mL propidium iodide (Sigma Aldrich), as described previously.12 Data were acquired using a BD LSR II System (BD Biosciences, San Jose, Calif) and were analyzed using FloJo FACS analysis software (Tree Star, Inc., Ashland, Ore).

Protein Preparation and Cellular Fractionation

Protein was extracted and measured as described previously.6, 15 For separation of the cytoplasmic/mitochondrial fractions and cytoplasmic/nuclear fractions, cell pellets were processed using the ApoAlert Cell Fractionation Kit (BD Biosciences) and NE-PER Nuclear and Cytoplasmic Extraction (Pierce Biotechnology, Inc., Rockford, Ill), respectively.

Caspase-3/7, Caspase-8, and Caspase-9 Activity Assay

Caspase activity was measured using Caspase-Glo 3/7, 8, or 9 reagents (Promega) as described previously.6

Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis and Western Blot Analysis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses were performed as described previously.12

Analysis of Phoshporylated Proteins Using xMAP Technology

After treatment, cells were lysed, and lysates were used to measure the phosphorylation status of 8 proteins using the Beadlyte 8-plex Multi-Pathway Signaling Kit (Millipore, Billerica, Mass) according to the manufacturer's instructions. Data were acquired using the Bioplex System (BioRad, Hercules, Calif).

Assay of Mitochondrial Depolarization Using JC-1

Cells were trypsinized and stained with JC-1 dye using the Mitocapture mitochondrial apoptosis detection kit (BioVision Research Products, Mountain View, Calif) according to the manufacturer's instructions. Data were acquired using the BD LSR II System and were analyzed using BD FACSDiva Software (BD Biosystems).

Immunoprecipitation

Beclin-1 was immunoprecipitated from the mitochondrial fraction of cells that were treated for 1 hour with 10 μg/mL NV-128 using the Catch and Release version 2.0 Reversible Immunoprecipitation System (Millipore) and antirabbit beclin-1 (Abcam, Cambridge, Mass) according to the manufacturer's instructions.

Mouse Xenograft Studies

Cells (1 × 106 cells) were resuspended in 200 μL total volume of 50% RPMI and 50% BD Matrigel Matrix (BD Biosciences, Bedford, Mass) and injected subcutaneously onto the right flank of NCR nude mice. Therapy commenced 8 days postinoculation as follows: paclitaxel at a dose of 10 mg/kg every 3 days intraperitoneally, carboplatin in water (Sigma Aldrich) at a dose of 40 mg/kg every 7 days, and NV-128 in 20% hydroxyproply-beta cyclodextrin (HPBCD) at a dose of 100 mg/kg every day. Control groups received 20% HPBCD in phosphate-buffered saline. Mice were treated and observed for 3 weeks. Tumor size was determined by caliper measurements, and antitumor activity was analyzed with respect to maximal tumor inhibition (treated/control) as described previously.12

Immunohistochemistry

Immunohistochemistry was performed as described previously15 using rabbit anti-EndoG (Lifespan Biosciences, Seattle, Wash) at 1:100 dilution.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

NV-128-induced Cell Death Involves Caspase-independent Chromatin Condensation

NV-128 decreased cell viability in a range of cancer cells, including the CP70 EOC cell line (Table 1). Our first objective was to determine the effect of NV-128 on a panel of primary cultures of EOC cells isolated from either ascites or tumor tissue. This panel included cultures that were resistant to paclitaxel and to carboplatin (Figs. 1A and B)15 and that expressed high levels of the antiapoptotic proteins XIAP and FLIP.12 Both chemosensitive cultures and resistant cultures revealed a significant reduction in the percentage of viable cells after treatment with NV-128, with a concentration that caused 50% growth inhibition between 5 μg/mL and 10 μg/mL (Fig. 1C).

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Figure 1. NV-128 decreases the viability of paclitaxel-resistant and carboplatin-resistant epithelial ovarian cancer (EOC) cells. EOC cells were treated with increasing concentrations of (A) paclitaxel, (B) carboplatin, and (C) NV-128 for 24 hours; and cell viability was determined as described in the text. Results shown are representative of 3 independent experiments. Note that the concentration that caused 50% growth inhibition was not reached for paclitaxel or carboplatin in cell lines that were considered resistant (dashed red line in Panels A-C).

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Table 1. Antiproliferative Activity of NV-128 Against a Range of Cancer Cell Lines
Cancer IndicationCell LineGI50: Mean/SD, μM
  1. SD indicates standard deviation; NSCLC, nonsmall cell lung cancer; GI50, the concentration that causes 50% growth inhibition.

Lung (NSCLC)NCI-H4601.15/1.41
 NCI-H8381.99/1.00
GastricMKN10.61/1.03
 NCI-N872.32/1.00
BreastMDA-MB-4681.69/3.18
 SK-BR-30.59/1.00
LiverJHH-11.86/1.00
 SK-HEP-10.98/1.00
LeukemiaCCRF-CEM1.34/1.76
OvarianCP701.28/1.56
GliomaHTB-1381.16/1.00
MelanomaIgR30.42/1.09
 MM2000.87/1.55
ProstateLNCaP21.09/1.00
 PC31.43/1.69
PancreaticMIA PaCa-20.40/1.21
 PANC-12.69/1.39

To determine whether the decrease in cell viability was because of apoptosis, we measured the activity of caspase-3/7, caspase-8, and caspase-9 and determined the status of 2 antiapoptotic molecules, XIAP and phosphorylated Akt (p-Akt). It is noteworthy that, in contrast to the increase in caspase activity observed after treatment with the known apoptotic inducer, paclitaxel, no changes in caspase-3/7, caspase-8, or caspase-9 activities were observed after treatment with NV-128 (Fig. 2A). In addition, no change in the status of XIAP was observed (Fig. 2B). However, a strong down-regulation of p-Akt was observed as early as 15 minutes after NV-128 treatment (Fig. 2C).

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Figure 2. NV-128 does not induce caspase activation. (A) Caspase activity was measured in cell lysates obtained from epithelial ovarian cancer (EOC) cells that were treated for 24 hours with increasing concentrations of NV-128 or with 2 μm paclitaxel. Results shown are for the R179 cell line, and similar results were observed in the other cell lines that were tested. (B and C) EOC cells were treated with 10 μg/mL NV-128 for the indicated times, and whole cells lysates were analyzed by Western blot analysis for the X-linked inhibitor of apoptosis protein (XIAP) and for phosphorylated Akt (p-Akt). β-Actin and total Akt (t-Akt) blots demonstrate even loading. (D) No-treatment (NT) control cells and cells that were treated with NV-128 (10 μg/mL) for 24 hours were stained with Hoechst and phosphatidylinositol (PI) and were analyzed by flow cytometry. Results shown are for the R182 cell line, and similar results were observed in the other cell lines that were tested. PE indicates phycoerythrin; PI, propidium iodide.

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Next, we evaluated whether the decrease in cell viability was associated with changes in the DNA. Thus, cells were stained with Hoecsht 33442 and propidium iodide (PI) and were analyzed by flow cytometry. Hoechst 33442 is a DNA binding dye that stains healthy DNA blue and fluoresces more intensely when it is bound to condensed chromatin. PI staining, conversely, is indicative of membrane permeabilization, which usually is observed during the process of cell death. Flow cytometry analysis of NV-128-treated cells revealed that cell death was accompanied by chromatin condensation. NV-128 induced a significant increase in double-positive cells, and 95% of cells were stained positively for both Hoechst and PI after 24 hours (Fig. 2D).

Chromatin condensation in the absence of caspase activation points to a caspase-independent pathway. To demonstrated conclusively that NV-128-induced cell death is caspase-independent, cells were treated with increasing concentrations of NV-128 in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK. Inhibition of caspases had no impact on NV-128-induced cell death (Fig. 3). Taken together, these results suggest that NV-128-induced cell death proceeds through a caspase-independent (but a possibly p-Akt–dependent) pathway, culminating in chromatin condensation.

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Figure 3. NV-128 induces caspase-independent and autophagy-independent cell death. Cells were treated with increasing concentrations of NV-128 for 24 hours in the presence or absence of either the caspase inhibitor ZVADFMK (20 μM) or the autophagy inhibitor 3 methyladenine (3MA) (10 μM), and cell viability was determined as described. Data shown are for the R182 cell line, and similar results were obtained with the other lines that were analyzed.

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NV-128 Down-regulates the mTOR Pathway

Our next objective was to identify and characterize the molecular pathways involved in NV-128-induced, caspase-independent cell death. Thus, EOC cells were treated with NV-128 (10 μg/mL), and cell lysates were used to measure the levels of a panel of phosphorylated proteins (phospho-proteins), as described earlier. The only phospho-protein the was down-regulated in the panel was phosphorylated ribosomal p70 S6 kinase (p-S6k), suggesting that NV-128 may affect the mTOR pathway. NV-128 had minimal effect on the proteins phosphorylated IκB kinase, phosphorylated signal transducer and activator of transcription 3 (pSTAT3), or phosphorylated p38 kinase and even induced the up-regulation of phosphorylated mitogen-activated protein kinase 1, phosphorylated c-Jun N-terminal kinase, pSTAT5a/b, and phosphorylated CAN-binding response regulator CreB (Fig. 4A), which most likely represented a compensatory response.

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Figure 4. NV-128 specifically down-regulates the mammalian target of rapamycin (mTOR) pathway (A) Levels of 8 phosphorylated proteins were measured in the lysates described). Data shown are for the R182 cell line, and similar results were obtained with the other lines that were analyzed. MFI indicates mean fluorescence intensity; pERK, phosphorylated mitogen-activated protein kinase 1; pIκB, phosphorylated IκB kinase; pJNK, phosphorylated c-Jun N-terminal kinase; pSTAT5a/b, phosphorylated signal transducer and activator of transcription 5a/b; p-S6k, phosphorylated 70 S6 kinase; pCREB, phosphorylated CAN-binding response regulator CreB; pSTAT3, phosphorylated signal transducer and activator of transcription 3; p38, phosphorylated p38 kinase. (B and C) EOC cells were treated with 10 μg/mL NV-128 for the indicated times, and the lysates were analyzed by Western blot analysis for phosphorylated mTOR (p-mTOR), pS6 kinase, and the autophagic marker LC3-II. NT indicates no treatment.

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To confirm the effect on the mTOR pathway, we evaluated the levels of p-S6k and phosphorylated mTOR (p-mTOR) using Western blot analysis. Figure 4B illustrates that p-mTOR and p-S6k were down-regulated 15 minutes after NV-128 treatment (Fig. 4B).

NV-128 Induces Autophagy

Morphologically, NV-128-treated EOC cells contained large intracellular vacuoles (Fig. 5B), which stained positively with acridine orange (data not shown), suggesting that NV-128 induces autophagic-like cell death. Autophagy is 1 of the best characterized caspase-independent forms of cell death, and it has been demonstrated that autophagy is controlled in part by the Akt-mTOR pathway.16 To determine whether the process of autophagy is involved, we evaluated the autophagic marker LC3-II. Western blot analyses demonstrated a significant increase in the level of LC3-II 8 hours after NV-128 treatment (Fig. 4C), confirming activation of the autophagic pathway.

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Figure 5. NV-128 induces intracellular vacuole formation and mitochondrial depolarization. (A and B) These are confocal microscope images of CP70 cells that either were unstimulated (A) or were treated with 10 μg/mL NV-128 for 2 hours (B). Note the presence of intracellular vacuoles in B (red arrows) but not in A. (C and D) Theses are fluorescent microscope images of cells that were stained with JC-1 (a cationic fluorescent dye that stains intact mitochondria red and stains depolarized mitochondria green.) and either were unstimulated (C) or were treated with 10 μg/mL NV-128 for 2 hours (D). The rounded arrow indicates punctuate, red staining in unstimulated cells; the diamond arrow indicates a cell with some mitochondrial depolarization; and the pointed arrow indicates a cell with bright green fluorescence, suggesting that most mitochondria have depolarized.

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Autophagy Is Not Required for NV-128-induced Cell Death

To determine whether the process of autophagy is the primary mechanism of NV-128-induced cell death, cells were treated with NV-128 in the presence or absence of the well characterized autophagy inhibitor, 3 methyladenine (3-MA), which inhibits the earliest step in the autophagosome formation.17 Cell viability studies using 3-MA (10 μM) indicated that the compound is not able to inhibit NV-128-induced cell death (Fig. 3). These data suggest that, although some characteristics of autophagy were observed, it is not the primary mechanism of cell death induced by NV-128.

NV-128 Induces Mitochondrial Depolarization

Next, we determined whether NV-128-induced cell death involved the mitochondria, because it contains nucleases that may be responsible for the observed chromatin condensation. Thus, control and treated cells were stained with JC-1 dye, a cationic fluorescent dye that stains intact mitochondria red and stains depolarized mitochondria green. Immunofluorescent images of control cells revealed mostly red fluorescence representative of intact mitochondria (Fig. 5C), whereas images of NV-128-treated cells mostly were green (Fig. 5D), suggesting that NV-128 is able to induce mitochondrial depolarization. This spectral shift was confirmed by flow cytometry, which demonstrated that NV-128-treated cultures had 59% and 84% depolarized cells (more than control) at 1 hour and 4 hours, respectively (Fig. 6).

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Figure 6. Quantitative analysis of mitochondrial depolarization using flow cytometry. Epithelial ovarian cancer cells were treated with NV-128, stained with JC-1 dye and were analyzed by flow cytometry to quantify mitochondrial depolarization.

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Beclin-1 Mitochondrial Translocation Is Associated With Mitochondrial Depolarization

Our next goal was to determine the upstream pathway responsible for NV-128-induced mitochondrial depolarization. The stability of the mitochondrial membrane is controlled in part by the Bcl-2 family of proteins.5 The proteins BH3 interacting domain death agonist (Bid) and Bax are cytoplasmic proteins that translocate to the mitochondria to initiate depolarization. In contrast, Bcl-2 is a resident mitochondrial protein that stabilizes the membrane. Treatment with NV-128 induces mitochondrial translocation of Bax 2 hours after treatment but does not induce the activation of Bid (Fig. 7A).

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Figure 7. NV-128 induces beclin-1 mitochondrial translocation and endonuclease G (EndoG) nuclear translocation. (A) Western blot analysis of cell lysates and mitochondrial fractions that were prepared from epithelial ovarian cancer (EOC) cells treated with 10 μg/mL NV-128. Total cell lysates were analyzed for full-length BH3 interacting domain death agonist (Bid), and mitochondrial fractions were analyzed for beclin-1 and Bcl-2 antagonist/killer (Bax). β-Actin and the voltage-dependent, anion-selective channel protein (VDAC) are shown as loading controls. NT indicates no treatment. (B) Western blot analysis of anti-beclin immunoprecipitates (IP) derived from mitochondrial fractions of EOC cells that were treated with NV-128 (10 μg/mL) for 1 hour and then probed with anti-Bcl-2 and anti-Bax. (C) Western blot analysis of nuclear fractions for apoptosis-inducing factor (AIF) and EndoG. Topoisomerase I (Topo-I) is shown as loading control.

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Because Bax translocation (normally 1 of the first mediators of mitochondrial membrane destabilization) occurs after the onset of NV-128-induced mitochondrial depolarization (observed 1 hour after treatment), an alternative mechanism must be responsible for the initiation of mitochondrial instability. Beclin-1 is a tumor suppressor gene, and it has been demonstrated that beclin-1 interacts with Bcl-2 through its BH domain.18 Analysis of beclin-1 messenger RNA by reverse transcriptase-polymerase chain reaction analysis and of beclin-1 protein levels from whole cell lysates indicated that there were no changes in beclin-1 message or protein expression after NV-128 treatment (data not shown). However, analysis of mitochondrial fractions revealed that beclin-1 translocates to the mitochondria as early as 1 hour after NV-128 treatment (Fig. 7A), which correlates with the onset of mitochondrial depolarization.

Mitochondrial Beclin-1 Associates With Bcl-2

Apart from its role in the initiation of autophagy, in which it associates with Class III PI3K, it has been demonstrated that the BH3 domain of beclin-1 is able to interact with both Bcl-2 and Bclxl.18-20 In light of this finding, we hypothesized that beclin-1 mitochondrial translocation can lead to its interaction with Bcl-2 and, thus, can interfere with the ability of Bcl-2 to stabilize the mitochondria. To demonstrate this association, we immunoprecipitated beclin-1 from the mitochondrial fraction of untreated cells or from cells that were treated for 1 hour with NV-128. Then, the presence of Bcl-2 and Bak in the immunecomplex was determined by Western blot analysis. We observed higher levels of Bcl-2 associated with beclin-1 after NV-128 treatment compared with a no-treatment control (Fig. 7B), demonstrating that NV-128-induced beclin-1 mitochondrial translocation can lead to a beclin-1–Bcl-2 interaction. Conversely, Bak was not observed in the complex, suggesting that this is a specific interaction between beclin-1 and Bcl-2. These findings suggest that beclin-1 may have the ability to initiate mitochondrial depolarization by translocating to the mitochondria, where it binds to and inactivates Bcl-2, potentially in a manner similar to that described for the Bid-Bak or Bid-Bax interaction.

NV-128 Induces EndoG Nuclear Translocation

In the absence of caspase activation, our next goal was to determine how NV-128-induced mitochondrial depolarization and chromatin condensation are linked. Mitochondrial nucleases represent a family of proteins that, once released from the mitochondria, can translocate to the nucleus and cleave DNA. The observation that NV-128 is able to induce mitochondrial depolarization suggests that a mitochondrial nuclease may be responsible for the observed chromatin condensation. Thus, we used Western blot analysis to quantify the levels of the mitochondrial nucleases, apoptosis-inducing factor (AIF) and EndoG, in nuclear fractions of NV-128-treated cells. An increase in the level of nuclear EndoG, but not AIF, was observed after NV-128 treatment (Fig. 7C), suggesting that EndoG is released from the mitochondria, translocates to the nucleus, and cleaves DNA, resulting in chromatin condensation.

Antitumor Activity of NV-128 on an EOC Xenograft Model

Next, we assessed the in vivo antitumor activity of NV-128. We established a nude mouse EOC xenograft model using EOC cells that had been isolated from malignant ovarian cancer ascites. The antitumor activity of NV-128 was compared with that of carboplatin and paclitaxel as described earlier. NV-128 induced a significant decrease in tumor proliferation kinetics and final tumor volume in a dose-dependent manner compared with carboplatin and paclitaxel (Fig. 8A,B). The treated/control values were 30%, 58%, and 58% for NV-128, carboplatin, and paclitaxel, respectively.

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Figure 8. In vivo activity of NV-128: Epithelial ovarian cancer (EOC) tumors were established in subcutaneously in NCR nude mice, and treatments were given as described). Tumor size was determined by caliper measurements. (A) EOC tumor proliferation kinetics. (B) Excised tumors from representative mice dosed with either vehicle control or NV-128. (C) Representative tumors from each group were lysed and analyzed by Western blot. PBS indicates phosphate-buffered saline; p-S6K, phosphorylated ribosomal p70 S6 kinase; t-S6K, total p-S6K. (D) Paraffin-embedded sections of representative mouse tumors were analyzed for the localization of endonuclease G by immunohistochemistry.

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Finally, to demonstrate that the in vivo antitumor activity of NV-128 parallels the observed in vitro mechanisms, tumors were lysed, the level of p-S6k was determined by Western blot analysis, and paraffin-embedded sections of mouse tumors were immunostained for EndoG. The blots in Figure 8C reveal that tumors taken from animals treated with NV-128 had a significant decrease on p-S6k compared with the vehicle control. In addition, we observed the nuclear localization of EndoG in tumors from animals treated with NV-128, whereas tumors from control animals only had cytoplasmic staining (Fig. 8D).

Toxicology Studies

Because NV-128 induced significant cell death in all cultures that we tested in vitro, a concern was its possible toxic side effects. Thus, we determined whether NV-128 induced myelosuppression or affected liver and kidney functions in the animals. A comparison of white blood cell and erythrocyte counts, hematocrit, and hemoglobin levels in NV-128-treated animals and control animals revealed no significant differences (Fig. 9A). Comparison of alkaline phosphatase and alanine aminotransferase activities and of creatinine and urea content in sera also revealed no differences between the 2 groups (Fig. 9B). Moreover, histopathologic examination of hematoxylin and eosin-stained sections from liver, kidney, spleen, and stomach revealed no histopathologic changes that would suggest toxicity (data not shown). Taken together, these results indicate that NV-128 does not induce myelosuppression and is not hepatotoxic or nephrotoxic.

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Figure 9. In vivo toxicology studies: (A) Comparison of white blood cells (WBC) (×109/L), erythrocytes (RBC) (×1012/L), hematocrit (HCT), and hemoglobin (Hb) (g/L) levels in control and NV-128–treated animals. (B) Comparison of alkaline phosphatase (ALP) (U/L), alanine aminotransferase (ALT) (U/L), urea (mmol/L), and creatinine (Cre) (μmol/L) levels in control and NV-128–treated animals.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

In this report, we describe the antitumor activity of NV-128, which involved the inhibition of mTOR signaling both in vitro and in vivo. Furthermore, we describe the potential role of beclin-1 as a regulator of mitochondrial membrane integrity through its interaction with Bcl-2.

Chemoresistance, because of the blockade of the apoptotic pathway, is 1 of the major limitations in the successful treatment of EOC. Indeed, cancer cells in general are characterized by high levels of expression of antiapoptotic proteins such as XIAP, Bcl-2, and p-Akt. Although numerous in vitro studies have reported that knockdown of these proteins can sensitize cancer cells to apoptotic signals, the current state of gene therapy technology limits this approach.

The description of additional forms of cell death other than apoptosis suggests that an alternative approach to chemoresistance may be to bypass apoptosis and specifically target alternative pathways that are independent of antiapoptotic proteins. In this study, we used a panel of EOC cells, including paclitaxel-resistant and carboplatin-resistant cell lines, and demonstrated that treatment of these cells with NV-128 induces a caspase-independent cell death characterized by mitochondrial depolarization and chromatin condensation. We also demonstrated that this pathway involves the early down-regulation of p-mTOR and p-AKT. An important function of mTOR is the control of translation through the regulation of S6k and eukaryotic translocation initiation factor 4E-binding protein 1. This results in enhanced translation, enhanced cell mass, and cell cycle progression.21, 22 mTOR is an essential part of tumor progression and is capable of integrating proliferative, antiapoptotic, and angiogenic signaling by connecting VEGF, hypoxia-inducible factor 1 (HIF-1), and human epidermal growth factor receptor family receptors.23 In addition, signaling through mTOR is stimulated by survival pathways, including the PI3K-Akt and Ras/Raf/mitogen-activated protein kinase pathways.24 Therefore, inhibition of mTOR activity may have critical effects on many of these survival pathways.

EOC cells are resistant to rapamycin (our unpublished data), the classic mTOR complex I inhibitor, which functions as a structural competitive inhibitor. Therefore, the down-regulation of p-mTOR may represent a more suitable target to block the prosurvival pathways activated by mTOR more than competitive inhibition.

To understand the downstream consequences of mTOR inhibition, we evaluated proteins associated with autophagy and mitochondrial integrity. Although we observed the activation of the autophagic pathway, the induction of cell death by NV-128 was not inhibited by the autophagy inhibitor 3MA. We also observed changes in mitochondrial integrity, and it is noteworthy that we observed that the levels of beclin-1, a main player in the autophagic pathway, significantly increased in the mitochondria after NV-128 treatment. Beclin-1 is a Bcl-2–binding protein that was identified first by using yeast 2-hybrid systems.19 Its function as a proautophagic molecule has been characterized extensively. Beclin-1, as part of a complex with Class III PI3K, is involved in autophagosome formation, which initiates the process of autophagy. Beclin-1 is deleted monoallelically in most cancers, which is suggestive of an antitumor function.25 Indeed, reports have indicted that the overexpression of beclin-1 has proapoptotic effects. Furuya et al demonstrated that ectopic expression of beclin-1 augments cisplatin-induced caspase-9 activation,26 whereas Levine's group demonstrated that beclin-1 gene transfer in breast cancer cells results in decreased tumorigenicity.25 Because of the role played by beclin-1 in autophagy, a process known initially as a cell-survival mechanism, it is unclear how a molecule involved in a survival pathway would have antitumor activity.

Together with the description of the crystal structure of beclin-1 and the demonstration that its BH3 domain associates with antiapoptotic Bcl-2 family members,20 our data, which demonstrate the association between beclin-1 mitochondrial translocation and mitochondrial depolarization, provide evidence of the possible prodeath function of beclin-1, which is independent of its role in autophagy. Exactly how beclin-1 is shifted preferentially to the mitochondria and how beclin-1-Bcl-2 binding initiates mitochondrial depolarization remain to be elucidated. We can postulate that beclin-1 binding to Bcl-2 releases the inhibitory effect of Bcl-2 on Bak, leading to Bak-induced mitochondrial depolarization.

Another interesting finding is the early down-regulation of p-Akt without any associated down-regulation of XIAP. XIAP is a substrate of Akt, and it has been demonstrated that XIAP is ubiquitinated and degraded by the proteasome once it is dephosphorylated.27 Thus, it was surprising to observe stable levels of XIAP coupled with declining p-Akt. It is possible that several steps are required for proteosomal degradation of XIAP and that the ubiquitination of a dephosphorylated XIAP requires other mechanisms. Another possibility is that phosphorylated XIAP may be quite stable, allowing it to maintain its phosphorylation status for an extended time even with declining levels of p-Akt. This stability in the level of XIAP also may be the reason for the absence of caspase-9 and caspase-3 activation.

We also demonstrated that NV-128–induced mitochondrial depolarization leads to the nuclear translocation of EndoG, but not AIF. This suggests that the mitochondrial insult induced by NV-128 is specific, allowing the release of only some mitochondrial proteins and not a complete mitochondrial rupture, which would have released all resident mitochondrial proteins. Moreover, the demonstration that AIF is not present in the nucleus upon the induction of a caspase-independent pathway supports previous reports that caspase is required to cleave the attachment of AIF to the inner mitochondrial membrane before its release.28

The antitumoral activity of NV-128 was demonstrated further in our in vivo studies. NV-128 was superior to paclitaxel and carboplatin and was well tolerated at its efficacious dose. It induced a significant decrease in tumor kinetics compared with paclitaxel and carboplatin without any evident decrease in animal weight or activity. In contrast, mice that received carboplatin lost more weight than control mice and exhibited severe cachexia. Moreover, we confirmed that the pathway described in vitro also is the main pathway that is activated in vivo.

Although we have not identified the receptor/target of NV-128 in EOC cells, we believe that the capacity of NV-128 to inhibit p-mTOR triggers a cascade of events leading to caspase-independent cell death in otherwise chemoresistant EOC cells (Figs. 10A and B). This opens new possibilities for the use of NV-128 as a potential addition to conventional chemotherapy that targets EOC cells.

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Figure 10. Proposed mechanism of NV-128–induced cell death: (A) In unstimulated/healthy cells, the process of autophagy is inhibited by mammalian target of rapamycin (mTOR) and antiapoptotic proteins, such as the X-linked inhibitor of apoptosis (XIAP), inhibit apoptosis. EndoG indicates endonuclease G; Bak, Bcl-2 antagonist/killer; pS6K, phosphorylated ribosomal p70 S6 kinase; pAKT, phosphorylated AKT; Bax, Bcl-2–associated X protein. (B) NV-128 treatment induces down-regulation of phosphorylated mTOR (p-mTOR) and beclin-1 mitochondrial translocation. Mitochondrial beclin-1 inhibits Bcl-2, and the resulting mitochondrial depolarization leads to EndoG nuclear translocation and chromatin condensation. The connection between p-mTOR and beclin-1 mitochondrial translocation remains to be determined. LC3-II is an autophagic marker.

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The demonstration of a functional, caspase-independent cell death pathway in apoptotic-resistant EOC cells is an important key step toward the development of alternative targeted therapy for refractory patients. Further studies currently are ongoing to determine the main target of this compound.

Conflict of Interest Disclosures

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References

Funded by National Cancer Institute (RO1CA118678, RO1CA127913) and in part by Novogen, Inc.

David Brown is an employee of Novogen, Inc.

References

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Conflict of Interest Disclosures
  7. References