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[Cancer Research 65, 6264-6274, July 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

LY294002 and LY303511 Sensitize Tumor Cells to Drug-Induced Apoptosis via Intracellular Hydrogen Peroxide Production Independent of the Phosphoinositide 3-Kinase-Akt Pathway

Tze Wei Poh1 and Shazib Pervaiz1,2,3

1 Department of Physiology, 2 Oncology Research Institute, National University Medical Institute, Faculty of Medicine, and 3 Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, Singapore

Requests for reprints: Shazib Pervaiz, Department of Physiology, MD902-05, Faculty of Medicine, National University of Singapore, Singapore, Singapore 117597. Fax: 65-67788161; E-mail: phssp{at}nus.edu.sg.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The phosphoinositide 3-kinase (PI3K)-Akt pathway is constitutively active in many tumors, and inhibitors of this prosurvival network, such as LY294002, have been shown to sensitize tumor cells to death stimuli. Here, we report a novel, PI3K-independent mechanism of LY-mediated sensitization of LNCaP prostate carcinoma cells to drug-induced apoptosis. Preincubation of tumor cells to LY294002 or its inactive analogue LY303511 resulted in a significant increase in intracellular hydrogen peroxide (H2O2) production and enhanced sensitivity to nonapoptotic concentrations of the chemotherapeutic agent vincristine. The critical role of intracellular H2O2 in LY-induced death sensitization is corroborated by transient transfection of cells with a vector containing human catalase gene. Indeed, overexpression of catalase significantly blocked the amplifying effect of LY pretreatment on caspase-2 and caspase-3 activation and cell death triggered by vincristine. Furthermore, the inability of wortmannin, another inhibitor of PI3K, to induce an increase in H2O2 production at doses that effectively blocked Akt phosphorylation provides strong evidence to unlink inhibition of PI3K from intracellular H2O2 production. These data strongly support death-sensitizing effect of LY compounds independent of the PI3K pathway and underscore the critical role of H2O2 in creating a permissive intracellular milieu for efficient drug-induced execution of tumor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The process of oncogenesis is a function of the balance between the rates of cellular proliferation and death. Consistent with this, deficient or defective apoptosis circuitry and/or constitutive activation of prosurvival pathways are commonly associated with most cancers. Examples of the former are overexpression of antiapoptotic members of the Bcl-2 family (Bcl-2 and Bcl-xL) or silencing of the proapoptotic counterparts, such as Bax, and overexpression of the inhibitor of apoptosis proteins (IAP; cIAP1, cIAP2, XIAP, and survivin; refs. 1–3). With respect to the prosurvival signals, a common finding reported in many cancers is the constitutive activation of Akt/protein kinase B (PKB), a downstream mediator of phosphoinositide 3-kinase (PI3K) activation signal. PI3K phosphorylates phosphatidylinositol (3,4)-diphosphate to form phosphatidylinositol (3,4,5)-triphosphate (PIP3; ref. 4). PIP3 then recruits Akt/PKB to the membrane where it becomes phosphorylated and thus activated by the phosphatidyl-dependent kinase-1. Akt/PKB has been implicated in the regulation of a variety of signal transduction pathways that mediate gene transcription (5–10), cell cycle events (11–15), cell proliferation (16–19), glycogen and protein metabolism (20–22), angiogenesis (23, 24), DNA repair (25, 26), and cell survival (27). Recent evidence strongly suggests that activated Akt/PKB not only contributes to oncogenic proliferative ability but also through phosphorylation of downstream targets, such as Bad, confers resistance to drug-induced apoptosis (28, 29).

Interestingly, aside from the conventional signals, such as activation of tyrosine receptor kinases, a causal relationship between intracellular reactive oxygen species production, in particular hydrogen peroxide (H2O2), and activation of Akt/PKB has been described recently; exogenous addition of H2O2 was shown to activate Akt/PKB phosphorylation (30–33). However, other data seem to suggest that activation of the PI3K-Akt pathway could generate other reactive species like nitric oxide and superoxide, which would further enhance the downstream effects of this pathway (33–36). These are intriguing findings, given our previous reports highlighting the role of intracellular redox status in the sensitivity of cancer cells to death stimuli (37–44). We showed that a slight pro-oxidant intracellular milieu, invariably associated with the transformed phenotype, favors cell survival either by its positive effect on cell proliferation (45) or by impeding death execution pathway(s) (41, 43, 44, 46). In contrast, a significant increase in intracellular H2O2 production and downstream acidification provides an environment conducive for apoptotic signaling (38, 39, 42).

In an effort to study downstream effectors of the Akt/PKB signaling pathway, a variety of PI3K inhibitors are currently in use, but with varying effects. For example, wortmannin, a cell-permeant fungal metabolite, acts as a potent inhibitor of PI3K; however, it is nonspecific as it also elicits strong inhibitory effect on mitogen-activated protein kinases (MAPK; ref. 47). LY294002 (LY29) is a commonly used pharmacologic inhibitor of PI3K, where it acts on the ATP-binding site of the PI3K enzyme, thus selectively inhibiting the PI3K-Akt nexus. Consistent with this observation, LY29 has been successfully used to enhance sensitivity of cancer cells to drug-induced apoptosis, and this effect has generally been attributed to its PI3K-Akt blocking activity (29, 48–53). Although LY29 is comparatively more selective in inhibiting PI3K and would seem to be a more useful inhibitor of PI3K (54, 55), recent reports have suggested that this compound might have effects other than inhibiting PI3K (56–58). In this regard, LY29 and LY303511 (LY30), a kinase inactive analogue of LY29 that contains a single atom substitution in the morpholine ring, have each been shown to block K(V) currents via a non-PI3K-dependent mechanism in rat pancreatic ß cells (57) and inhibit monocyte chemoattractant protein-1 (MCP-1) expression in human umbilical vein endothelial cells (56). Thus, the ability of LY30 to behave like LY29 in these studies indicates that LY29 has other effects that are independent of its ability to inhibit PI3K activity. Based on these recent reports, the effects of LY29 cannot be exclusively attributed to inhibition of the PI3K-Akt pathway.

Here, we propose an alternative role for LY29 as an inducer of intracellular H2O2 generation independent of its PI3K inhibitory activity. We show that the intracellular H2O2 produced is independent of mitochondria in a variety of cell lines. At concentrations used to inhibit PI3K activity, LY29 exposure results in a significant intracellular increase in H2O2 production in tumor cell lines. Furthermore, in LNCaP prostate carcinoma cells, we also show that both LY29 and its kinase inactive analogue LY30 sensitize cancer cells to drug-induced apoptosis through intracellular H2O2 production independent of the PI3K-Akt pathway. These findings provide a hitherto undefined activity of LY29 and LY30 and highlight their potential for use for favorably tailoring the intracellular milieu of cancer cells for drug-induced death execution.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Determination of cell viability. The human lymph node carcinoma of the prostate (LNCaP) cell line was purchased from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin, and 100 µmol/L sodium pyruvate. In a typical survival assay, LNCaP cells (2.5 x 105/well) plated in 24-well plates were preincubated for 1 hour with either LY29 or LY30 (25 µmol/L) and then treated with 0.02 µmol/L vincristine overnight for 18 hours. Cytotoxicity was determined by the crystal violet assay. After drug exposure, cells were washed once with PBS and crystal violet solution (0.25 mL) was added to each well and incubated for 15 minutes. The excess crystal violet solution was washed away with distilled water and the remaining crystals were dissolved in a 1% SDS in 1x PBS solution. Viability was determined by absorbance at 595 nm wavelength using an automated ELISA reader. Cell viability experiments were thus similarly done with 50 µmol/L caspase-2 and caspase-3 inhibitors and the pan-caspase inhibitor, Z-VDVAD-fmk, Z-DEVD-fmk (R&D Systems, Minneapolis, MN), and ZVAD-fmk (Biomol, Plymouth Meeting, PA), respectively. Cells were preincubated for 1 hour with the caspase inhibitors before addition of LY29/LY30 and vincristine.

Flow cytometric analysis of intracellular hydrogen peroxide concentration. Intracellular concentration of H2O2 was determined by staining with the redox sensitive dye 5-(and-6)-chloromethyl-2',7'-dichlorofluorescin diacetate (CM-H2DCFDA; Molecular Probes, Eugene, OR), which becomes fluorescent when oxidized by H2O2 and its free radical products (40). Briefly, cells were washed once with PBS, loaded with 5 µmol/L CM-H2DCFDA at 37°C for 15 minutes, and analyzed by flow cytometry (Coulter EPICS Elite ESP) using an excitation wavelength of 488 nm. At least 10,000 events were analyzed.

Detection of total Akt/protein kinase B and Akt/protein kinase B phosphorylation levels. LNCaP cells (1.5 x 106-2 x 106) were plated in a T25 flask overnight followed by exposure to the various triggers. Cells were harvested from the flask, washed once with PBS, and then lysed with cell lysis buffer [150 mmol/L NaCl, Tris-HCl (pH 7.4), 1% NP40]. Cell lysate (200 µg) was then electrophoresed on an 8% acrylamide gel. Antibodies were used to probe for total Akt and phosphorylated Akt/PKB at the Ser473 position (Cell Signaling, Beverly, MA). Protein blots were probed with anti-ß-actin (Sigma-Aldrich, St. Louis, MO) to check for equal protein loading.

Nonradioactive Akt/protein kinase B immunoprecipitation kinase activity assay. Determination of the Akt/PKB kinase activity was done via the Akt kinase assay kit (Cell Signaling). LNCaP cells (3 x 106) were plated in a 100 mm Petri dish and treated with increasing concentrations of LY29. The culture medium was then removed and the cells were washed once with ice-cold PBS and cell lysis buffer (1 mL) was added to each plate and incubated for 10 minutes. Cells were then scraped off the plates and immobilized Akt monoclonal antibody was used to immunoprecipitate Akt from cell extracts. Then, an in vitro kinase assay was done using glycogen synthase kinase (GSK)-3 fusion protein as a substrate. Phosphorylation of GSK-3 was detected by Western blot analysis using a phospho-GSK-3{alpha}/ß (Ser21/Ser9) antibody (Cell Signaling).

Determination of the colony-forming ability of tumor cells. LNCaP cells (4,000-8,000) were pretreated with LY29 or LY30 and then treated with 0.02 µmol/L vincristine for 18 hours. As overnight treatment with LY29 or LY30 had a slight effect on cell proliferation, cell numbers were adjusted accordingly to take this into account when cells were plated. After treatment, cells were then washed and replated into 100 mm culture dishes and left to form colonies over a period of 7 to 10 days. Culture dishes were stained with crystal violet and the number of colonies in a 2 x 2 cm grid (on the culture plates) was scored to determine the colony-forming ability of cells. Only colonies containing >50 cells were counted.

Determination of caspase-2 and caspase-3 activities. Caspase-2 and caspase-3 activities were assayed by using 7-amino-4-trifluoromethyl coumarin (AFC)–conjugated and amino-4-methyl coumarin (AMC)–conjugated substrates, respectively (Biomol). LNCaP cells (2.5 x 105/mL) were preincubated with LY29 or LY30 (25 µmol/L) for 1 hour and then incubated with 0.02 µmol/L vincristine for various time points. Cells were then harvested and washed with 1x PBS, resuspended in chilled cell lysis buffer (BD PharMingen, San Diego, CA), and incubated on ice for 10 minutes. Reaction buffer (50 µL; 2x; 10 mmol/L HEPES, 2 mmol/L EDTA, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L DTT) and fluorogenic caspase-specific substrate (1 µL; DEVD-AFC for caspase-2 and VDVAD-AMC for caspase-2) were added to each sample and incubated at 37°C for 1 hour. Detection of caspase-3 activity was determined by the relative fluorescence intensity at 505 nm following excitation at 400 nm using a spectrofluorometer, whereas detection of caspase-2 activity was determined by the relative fluorescence intensity at 460 nm following excitation at 380 nm.

Propidium iodide staining for DNA fragmentation. Cells were fixed then stained with propidium iodide for DNA content analysis as described elsewhere (40). A total of 10,000 events were analyzed by flow cytometry using an excitation wavelength set at 488 nm and emission at 610 nm.

Transient transfection of LNCaP cells with human catalase. LNCaP cells were transiently transfected with the pzeoSV plasmid vector with the 1.6-kb human catalase cDNA cloned into the HindIII sites. Briefly, the pzeoSV-h-catalase (generous gift from Dr. J.D. Lambeth, Emory University School of Medicine, Atlanta, GA) was transfected into LNCaP cells using the SuperFect Transfection reagent from Qiagen GmbH (Germany) as per the manufacturer's instructions. Overexpression of human catalase occurred at 24 hours after transfection and decreased to basal levels subsequently. Cells transfected with the empty vector pzeoSV were used as controls. Expression of human catalase protein was detected using human anti-catalase antibody (Calbiochem, Darmstadt, Germany), which recognizes the 60- to 65-kDa catalase subunit.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LY294002 triggers intracellular hydrogen peroxide production in tumor cells. Incubation of human prostate carcinoma (LNCaP) cells with LY29 (25 µmol/L) for 1 hour (concentration commonly used to show PI3K inhibitory activity) resulted in a significant increase in DCF fluorescence by flow cytometry (Fig. 1), which could be reversed by 1,000 units/mL catalase, thus indicating intracellular H2O2 production. More importantly, this effect was not exclusive to only LNCaP cells as evidenced by the similar effect of LY29 on HL60 (human leukemia), CEM (human leukemia), and T24 (human bladder carcinoma) cells (Fig. 1). This increase in H2O2 also persisted even in cells stably overexpressing Bcl-2 (CEM-Bcl-2; Fig. 1). Similar experiments with LY29 were also done on isolated mitochondria; however, the increase in H2O2 was not observed in this particular system.



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  		Figure 1. LY29 triggers intracellular H2O2 production in tumor cells. Tumor cells (1 x 106) from a variety of cell lines were treated with 25 µmol/L LY29 for 1 hour in the presence or absence of 1,000 units/mL catalase. Cells were loaded with the H2O2-sensitive probe DCFH-DA (5 µmol/L) for 15 minutes and intracellular H2O2 was determined by the shift in DCFH-DA fluorescence detected by flow cytometry as detected by flow cytometry.

 
LY294002-induced hydrogen peroxide production is independent of its phosphoinositide 3-kinase-Akt inhibitory activity in LNCaP cells. LNCaP prostate cancer cells that express constitutively active form of Akt/PKB were incubated with increasing concentrations of LY29 (1-25 µmol/L) and the effect on Akt/PKB activation and intracellular H2O2 production was assessed. As expected, concentrations of LY in excess of 5 µmol/L effectively blocked Akt phosphorylation, assayed by Western blot analysis with the phosphospecific antibody and by the ability of active Akt/PKB to phosphorylate its substrate GSK-3{alpha}/ß (Fig. 2A). Intriguingly, LY29 was still able to trigger intracellular H2O2 production at a concentration (1 µmol/L; Fig. 2B) that did not affect Akt/PKB phosphorylation status. Furthermore, the fact that LY-induced H2O2 production was independent of PI3K activity was corroborated by the inability of wortmannin, another commonly used PI3K inhibitor, to trigger increase in intracellular H2O2 (Fig. 3B) at doses that effectively inhibited Akt phosphorylation (Fig. 3A).



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  		Figure 2. LY29-induced H2O2 production is independent of its PI3K-Akt inhibitory activity in LNCaP cells. A, ability of the varying doses of LY29 to dephosphorylate and deactivate Akt was shown by Western blot. LNCaP cells (1.5 x 106-2 x 106) were incubated with varying doses of LY29 (1-25 µmol/L) for 1 hour and cell lysates were obtained for Western blot analysis of the Akt phosphorylation status in these cell extracts. In a similar experiment, LNCaP cells (3 x 106) were incubated with varying doses of LY29 (1-25 µmol/L) for 1 hour, after which the cell lysates were obtained and the Akt immunoprecipitation kinase activity assay was done on these cell extracts as described in Materials and Methods. The total Akt and total GSK bands indicated equal loading of protein for analysis of the Akt phosphorylation status and activity, respectively. B, LNCaP cells (0.8 x 106) were treated with 1 µmol/L LY29 for 1 hour in the presence or absence of 1,000 units/mL catalase. Cells were then loaded with DCFH-DA (5 µmol/L) for 15 minutes and the amount of intracellular H2O2 generated was indicated by the shift in fluorescence as detected by flow cytometry.

 


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  		Figure 3. Wortmannin inhibits PI3K but has no effect on intracellular H2O2 production in LNCaP cells. A, LNCaP cells (1.5 x 106-2 x 106) were incubated with varying concentrations of wortmannin (10-200 nmol/L) for 1 hour and cell lysates were obtained for Western blot analysis of the Akt phosphorylation status. B, LNCaP cells (0.8 x 106) were treated with varying doses of wortmannin (10-200 nmol/L) for 1 hour. Cells were then loaded with DCFH-DA (5 µmol/L) for 15 minutes and intracellular H2O2 was assayed by flow cytometry.

 
LY303511, a LY294002 analogue, also produces hydrogen peroxide, inhibits proliferation, and sensitizes LNCaP cells to drug-induced apoptosis. To provide further evidence that the ability of LY29 to produce H2O2 was not linked to its PI3K inhibitory effect, we investigated an analogue of LY29 (i.e., LY30), which has a high degree of structural similarity with LY29 (Fig. 4A) but completely lacks the ability to inhibit the PI3K enzyme (refs. 56, 57, 59, 60; Fig. 4C). Indeed, similar to the results obtained with LY29, incubation of LNCaP cells for 1 hour with varying concentrations of LY30 (1-25 µmol/L) resulted in a significant increase in intracellular H2O2 (Fig. 4B).



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  		Figure 4. LY30, a LY29 analogue, also produces H2O2 in LNCaP cells. A, structures of both LY29 and LY30. B, LNCaP cells (0.8 x 106) were treated with varying doses (1-25 µmol/L) of LY30 for 1 hour in the presence or absence of 1,000 units/mL catalase. Cells were then loaded with DCFH-DA (5 µmol/L) for 15 minutes and intracellular H2O2 was detected by flow cytometry. C, LNCaP cells (1.5 x 106-2 x 106) were incubated with 25 µmol/L LY29 or LY30 for 1 hour and cell lysates were obtained for Western blot analysis of the Akt phosphorylation status.

 
LNCaP cells were treated for 18 hours with a low dose (0.02 µmol/L) of vincristine that does not, by itself, significantly affect tumor cell viability (Fig. 5A). However, preincubation of cells with LY29 and LY30 significantly increased cell sensitivity to 0.02 µmol/L vincristine as indicated by the reduction in the viable cell fraction (Fig. 5B). At the same time, incubation of cells with LY29 or LY30 overnight for 18 hours alone reduced cell viability by ~40% and 15%, respectively, suggesting perhaps an antiproliferative effect of these compounds on the doubling rate of the cells as well (Fig. 5B). In addition, whereas treatment with 0.02 µmol/L vincristine alone did not induce intracellular caspase activation, pretreatment of cells with LY29 or LY30 triggered robust increases in caspase-2 and caspase-3 activities on subsequent exposure to the same concentration of vincristine (Fig. 5C). The involvement of caspases in this system was further evidenced by the use of the tetrapeptide inhibitors of caspase-2 (VDVAD-fmk), caspase-3 (DEVD-fmk), and the pan-caspase inhibitor (ZVAD-fmk). Interestingly, addition of ZVAD-fmk was able to block LY-induced sensitization of LNCaP cells to drug-induced apoptosis more effectively than the addition of caspase-2 or caspase-3 inhibitor alone, as seen most significantly in an increase in cell viability over a longer period of study at 48 hours (Fig. 5D). The activation of caspase-8 or caspase-9 was minimal and a similar sensitizing effect of LY30 on LNCaP cells was observed with other commonly used chemotherapeutic compounds, etoposide and daunorubicin (data not shown). As an additional marker of apoptosis, analysis of fragmented DNA was also carried out. LNCaP cells incubated with 0.02 µmol/L vincristine were again preincubated in the presence or absence of LY29 or LY30. Incubation with 0.02 µmol/L vincristine alone did not alter the cell cycle profile; however, addition of LY29 or LY30 to vincristine-treated cells increased the sub-G1 and G2-M fractions of these cells, albeit to different extents at 18 and 48 hours. Extent of DNA fragmentation is shown as a histogram where LY30 increased the amount of DNA fragmentation observed in vincristine-treated cells to levels similar to that observed with LY29 at 48-hour incubation (Fig. 5E). Addition of caspase-2 inhibitor (VDVAD-fmk), caspase-3 inhibitor (DEVD-fmk), and the pan-caspase inhibitor (ZVAD-fmk) reduced the amount of DNA fragmentation as shown in Fig. 5E and cell cycle profiles (Fig. 5F and G). The pan-caspase inhibitor ZVAD-fmk reduced the extent of DNA fragmentation effectively as depicted in the cell cycle profiles (Fig. 5F and G). Additionally, whereas a low nonlethal dose of vincristine (0.02 µmol/L) did not produce any detectable H2O2, preincubation of tumor cells with 25 µmol/L LY30 produced a significant burst of intracellular H2O2 as seen with a rightward shift in DCF fluorescence (Fig. 5H).




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  		Figure 5. LY30 can reduce cell proliferation and sensitize cells treated with low doses of vincristine to apoptosis via an increase in caspase activity. A, LNCaP cells (2.5 x 105) were exposed to doses of 0.02 to 1 µmol/L vincristine for 18 hours, after which the crystal violet assay was done to establish a cell viability dose-response curve. B, LNCaP cells (2.5 x 105) were exposed to 0.02 µmol/L vincristine (vin) for 18 hours in the presence or absence of a 1-hour pretreatment either with 25 µmol/L LY29 or LY30. The crystal violet assay was then done on these cells as described in Materials and Methods to determine cell viability after 18 hours. C, LNCaP cells (2.5 x 105) were treated similarly for a time period of 6 to 18 hours and lysed to obtain whole-cell lysates. Caspase-2 and caspase-3 activities were assayed by using AFC- and AMC-conjugated substrates, respectively, on the whole-cell lysates. D, for experiments with the caspase inhibitors, cells were preincubated for 1 hour with 50 µmol/L of the respective caspase inhibitors before treatment with LY29/LY30 and vincristine for 18 and 48 hours, after which cell viability was assessed using the crystal violet assay. Columns, mean of at least three independent experiments; bars, SD. Viability of cells treated with vincristine and LY29/LY30 was calculated as a percentage against cells treated only with vincristine. E, LNCaP cells were incubated with 0.02 µmol/L vincristine in the presence or absence of 25 µmol/L LY30 together with various caspase inhibitors as described previously and stained with propidium iodide for cell cycle analysis by flow cytometry as described in Materials and Methods. The percentage of cells in the sub-G1 population was shown as an indication of the extent of DNA fragmentation occurring in the cells. Columns, mean of at least three independent experiments; bars, SD. F, actual cell cycle profiles as analyzed by flow cytometry of LNCaP cells treated for 18 hours to show the effect of ZVAD on cells treated with 0.02 µmol/L vincristine and LY29/LY30. Representative of at least three independent experiments. G, actual cell cycle profiles as analyzed by flow cytometry of LNCaP cells treated for 48 hours to show the effect of ZVAD on cells treated with 0.02 µmol/L vincristine and LY29/LY30. Representative of at least three independent experiments. H, LNCaP cells (0.8 x 106) were treated with 0.02 µmol/L vincristine for 1 hour in the presence or absence of a 1-hour preincubation with 25 µmol/L LY30. Cells were then loaded with DCFH-DA (5 µmol/L) for 15 minutes and intracellular H2O2 was detected by flow cytometry.

 
LY303511 inhibits colony-forming ability of LNCaP cells treated with vincristine. Not only could both LY29 and LY30 sensitize cells to vincristine-induced cell death but they were also effective in reducing the colony-forming ability of LNCaP cells when treated with vincristine (Fig. 6A). LNCaP cells were pretreated with LY29 or LY30 before treatment with 0.02 µmol/L vincristine for 18 hours. As both LY29 and LY30 had an effect on cell proliferation overnight, the cell numbers were adjusted accordingly to take that into consideration. The cells were then replated onto 100 mm culture plates and incubated for 8 to 14 days to allow colonies to form. Cells pretreated with LY29 or LY30 (before vincristine exposure) showed a significant reduction in the number of colonies formed compared with cells treated with vincristine alone (Fig. 6B). It should be pointed out that treatment of tumor cells with LY30 alone for 18 hours did not seem to significantly reduce the colony-forming ability of cells; however, incubation for the same period with LY29 alone affected the colony-forming ability of LNCaP cells, suggesting an even stronger sensitizing effect of LY30, which unlike LY29 has no effect on the PI3K-Akt pathway.



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  		Figure 6. LY30 inhibits the colony-forming ability of cells treated with vincristine. A, LNCaP cells (4,000-8,000) were exposed to 0.02 µmol/L vincristine for 18 hours in the presence or absence of a 1-hour pretreatment with either 25 µmol/L LY29 or LY30. The cells were then seeded in 100 mm Petri dishes and allowed to form colonies over 8 to 14 days, after which they were stained with crystal violet as described in Materials and Methods to show colony formation. Representative of nine independent experiments, each done in triplicate. B, extent of colony formation for three independent experiments, each done in triplicate.

 
Transfection with human catalase inhibits caspase activity and increase in apoptosis sensitivity induced by LY294002 and LY303511. Having shown that the LY compounds not only increased sensitivity to drug-induced apoptosis but also stimulated a significant increase in intracellular H2O2 production, we next asked if LY-induced increases in caspase-2 and caspase-3 activities and cell sensitivity to drug-induced apoptosis were a result of intracellular H2O2 production. To do so, LNCaP cells were transiently transfected with a pzeoSV plasmid vector containing a 1.6-kb human catalase cDNA. Overexpression of catalase was confirmed by Western blot analysis and by the decrease in basal intracellular level of H2O2 by DCF fluorescence analysis (Fig. 7A). Indeed, overexpression of catalase was able to significantly revert LY-induced increases in the sensitivity (Fig. 7B) and caspase activity (Fig. 7C) of tumor cells on exposure to 0.02 µmol/L vincristine.



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  		Figure 7. Transfection of human catalase into cells reduces the increase in caspase-2 and caspase-3 activity in cells treated with vincristine and LY29 or LY30. A, LNCaP cells were transiently transfected with human catalase and the empty zeocin vector. Overexpression of the catalase was detected by Western blot analysis as described in Materials and Methods. Catalase overexpression resulted in decreased basal levels of H2O2 in LNCaP cells assayed by DCFH-DA loading and flow cytometry. B and C, at 24 hours post-transfection, LNCaP cells (0.8 x 106) were treated with LY30/29 and 0.02 µmol/L vincristine as described previously. Activation of caspases and proliferation rate of these cells were then assayed 18 hours later as described previously to compare the effects of catalase-transfected and empty vector–transfected cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LY294002 and LY303511 sensitize LNCaP cells to drug-induced apoptosis independently of the phosphoinositide 3-kinase pathway. Defects or deficiencies in apoptotic signaling pathway(s) are a common feature of many tumors; therefore, ways to combat and overwhelm these blocks constitute a major effort of biologists and clinicians alike. One of the signaling pathways shown to be constitutively active in many tumor cells, thereby providing them with a survival advantage over their normal counterparts, is the PI3K-Akt survival circuitry. An essential component of this pathway is the active kinase Akt/PKB, which once phosphorylated can signal recruitment of many downstream targets involved in either cell survival signaling or inhibition of apoptosis. Therefore, identification and development of compounds with the ability to inhibit upstream PI3K activation and subsequent Akt phosphorylation are an attractive strategy for enhancing tumor cell sensitivity to apoptosis. To that end, LY29 has long been used as a somewhat selective inhibitor of PI3K-mediated phosphorylation of Akt/PKB (55), and most of its biological activity relevant to apoptosis sensitization has been attributed to its effect on the PI3K-Akt survival network. Indeed, the realization that the PI3K-Akt axis is abnormally active in a variety of tumor cells has been the impetus behind preclinical trials involving inhibitory agents, such as LY29. Here, we show a novel mechanism by which LY compounds (LY29 and LY30) sensitize LNCaP prostate carcinoma cells expressing constitutively active Akt/PKB to drug-induced apoptosis. Exposure of tumor cells to LY29 and more importantly to its inactive analogue LY30 (no effect on PI3K activation and Akt phosphorylation) resulted in a significant increase in intracellular H2O2 production and sensitization of prostate carcinoma cells to nonlethal concentration of the chemotherapeutic agent vincristine via caspase-2 and caspase-3 activation, increased DNA fragmentation, and a strong inhibitory effect on the colony-forming ability of tumor cells. The reduced viability of cells treated overnight for 18 hours with only LY29 or LY30 (60% and 85% viability for LY29 and LY30, respectively) could be an indication of the inhibitory effect of the LY compounds on cell proliferation as the doubling time of LNCaP prostate carcinoma cells is ~24 hours. This could be extrapolated to mean that proliferation of all of the LY29-treated cells was affected and proliferation of ~15% of LY30 treated cells was affected. LY30 also significantly enhanced the extent of DNA fragmentation in vincristine-treated cells, especially over a longer period of 48 hours. The ability of the tetrapeptide caspase inhibitors to block the observed DNA fragmentation showed again that this was a caspase-dependent pathway independent of the PI3K-Akt axis. The pan-caspase inhibitor ZVAD-fmk almost completely attenuated the process of DNA fragmentation observed in these cells, much more effectively than the presence of either caspase-2 or caspase-3 inhibitor alone, suggesting that it is probably the synergistic inhibition of caspase-2 and caspase-3 by ZVAD-fmk that protected the cells from DNA fragmentation. Interestingly, there was no significant increase in either caspase-8 or caspase-9 activation in this system, suggesting a direct effect of H2O2 on caspase-2 and/or caspase-3 activation resulting in death execution. Furthermore, the critical involvement of H2O2 in LY-induced augmentation of drug-induced apoptosis sensitivity is corroborated by transient transfection with a vector containing human catalase, scavenger of intracellular H2O2; transient transfection with catalase virtually completely reverted the death-promoting effect of LY29 and LY30. It should be pointed out that apoptosis-sensitizing effect of LY compounds was not exclusive to vincristine but applied similarly to daunorubicin and etoposide (data not shown).

Considering the strong inhibitory effect of LY29 on PI3K activation, one could argue that it is not surprising that preincubation of tumor cells resulted in an increase in sensitivity to apoptosis. However, the data reported here strongly support a novel, PI3K-independent mechanism for sensitizing tumor cells to drug-induced apoptosis through intracellular production of H2O2. Corroborating this is the ability of low concentrations of LY29 (1-5 µmol/L) that do not affect PI3K activity but still induce intracellular H2O2 production and sensitize tumor cells to drug-induced apoptosis (data not shown), thereby excluding the involvement of the PI3K pathway in this system. This is further supported by a strong sensitizing effect of LY30, which lacks PI3K inhibitory activity but, similar to LY29, triggers a significant increase in intracellular H2O2 production. Finally, the fact that wortmannin, another commonly used inhibitor of PI3K, had no effect on intracellular H2O2 production provides evidence that LY-induced H2O2 production is not a function of PI3K inhibition. In fact, incubation of LNCaP cells with wortmannin actually reduced the levels of H2O2 in the cell. Although the data at this point in time are not sufficient to explain this observation, it could be attributed to the ability of wortmannin to nonspecifically inhibit MAPK (47) and phospholipase D. Regarding the relationship between inhibition of PI3K and intracellular H2O2 production, a plausible scenario, considering the previously shown ability of LY29 to bind to the ATP-binding site of other protein kinases like the DNA-dependent protein kinase in the nucleus (61), could be that LY compounds affect transcription of intracellular redox control genes, like catalase and superoxide dismutase. However, the burst of intracellular H2O2 generated by the LY compounds in LNCaP cells was detected as early as 15 minutes after incubation (data not shown), thereby rendering this possibility highly unlikely.

Intracellular generation of hydrogen peroxide: a novel mechanism of action of LY compounds. H2O2 has long been shown to play vastly different roles in the cell, where subtle changes in the delicate intracellular redox status could greatly affect cell physiology. For example, depending on its concentration, H2O2 can induce cell proliferation, mediate drug-induced apoptosis in many models (particularly in tumor cells), or at overwhelming levels bring about necrotic cell death. Indeed, there is a fair amount of controversy with respect to the relationship between PI3K signaling and H2O2 production, with data supporting both an upstream and a downstream role of H2O2 in PI3K signaling (62). However, in tumor cells, intracellular generation of H2O2, specifically in response to drug exposure, is independent of PI3K. Data presented here not only corroborate those findings but also underscore the inherent ability of the LY compounds to trigger a significant increase in intracellular H2O2 and provide a novel mechanism for their sensitizing effect on tumor cells. More significantly, although previous studies have shown that H2O2 is a mediator of vincristine-induced apoptotic cell death, we show here that incubation of cells with a nonlethal dose of vincristine did not generate H2O2 in the cell, an observation commensurate with the fact that there was no cell death with such low doses of vincristine. It was the preincubation with LY compounds that significantly enhanced the levels of H2O2 in the cell, which was then responsible for tipping the cell toward eventual apoptosis.

The ability of the LY compounds to generate intracellular H2O2 could perhaps be further understood by a closer look at their structures. The structures of the LY compounds were based on quercetin, a naturally occurring bioflavonoid that was shown previously to inhibit PI3K. However, quercetin was also shown to be able to inhibit other kinases like PI4K and several tyrosine and serine/threonine kinases. LY29 was synthesized using quercetin as a lead compound where the dihydroxyphenyl group at the 2-position of the chromone ring in quercetin was replaced with a morpholine moiety (55) as seen in LY29, thus conferring on LY29, its specificity in the inhibition of PI3K as it was then subsequently unable to inhibit PI4K, epidermal growth factor receptor tyrosine kinase, or other ATP-requiring kinases and ATPases. A simple replacement of oxygen with nitrogen in the 2-position of the morpholine ring gave the compound LY30, which displayed a dramatic reduction in the ability of this compound to inhibit PI3K. LY29 inhibits PI3K to <1% of its original activity at a dose of 100 µmol/L; however, LY30 at the same dose allows PI3K to retain almost all of its original activity (55). Although the H2O2 generated by both LY compounds is functionally relevant, it is probably the synergistic effect of the ability of LY29 to inhibit PI3K effectively that results in its greater potency compared with LY30 when it is used.

Although the current data seem to exclude the mitochondria as a possible source of H2O2 accumulation by these LY compounds, it does not exclude the possibility of the activation of other oxidase-like complexes in the cell, such as those found in the endoplasmic reticulum or activation of the copper/zinc superoxide dismutases in the cytosol itself. As these compounds possess structural similarity to quercetin, a bioflavonoid, it is perhaps not surprising that they generate H2O2 in the cell, given the controversial nature of flavonoids to act as both an antioxidant with free radical scavenging properties and a pro-oxidant.

Interestingly, recent reports have shown biological effects of LY compounds that are independent of its PI3K-inhibiting activity in a variety of biological systems (56, 57). For instance, both LY29 and LY30 have been shown to block K(V) currents via a direct non-PI3K-dependent mechanism in rat pancreatic ß cells (57) and to inhibit MCP-1 expression in human umbilical vein endothelial cells (56). Considering our findings, one is tempted to speculate that these PI3K-independent activities of LY compounds might be linked to the intracellular generation of H2O2.

Intracellular hydrogen peroxide production: a permissive environment for sensitization of cells to drug-induced apoptosis. We have highlighted previously the critical role of intracellular H2O2 in sensitization of tumor cells to apoptotic stimuli by creating an intracellular milieu permissive for caspase activation and death execution. In addition, we showed that priming of tumor cells with subapoptotic concentrations of H2O2 significantly enhanced the sensitivity of tumor cells to drug-induced apoptosis (63), and alternatively, blocking intracellular H2O2 production inhibited death execution (38, 44, 46). Along the same lines, intracellular production of H2O2 seems to be a common denominator in drug-induced apoptosis of tumor cells. Judging from these in vitro data, it is logical that chemotherapeutic efficacy could be significantly improved in an environment made conducive for death execution by the presence of H2O2. Therefore, identification of compounds that trigger a significant increase in intracellular H2O2 and their use in conjunction with chemotherapy agents could be an attractive strategy to enhance the sensitivity of tumor cells to drug therapy. In the light of these findings, the structures of the LY compounds could therefore be used as a model to develop chemotherapeutic compounds that could trigger intracellular H2O2, which in turn would either sensitize tumor cells to drug-induced apoptosis or directly induce apoptosis itself.


   Acknowledgments
 
Grant support: National Medical Research Council Singapore grants NMRC/0539/2001 and NMRC/0738/2003.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Marie-Veronique Clement, Andrea Holme, and Alan Kumar for useful discussions.

Received 1/17/05. Revised 4/13/05. Accepted 5/ 2/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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