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1 Department of Microbiology and Immunology and 2 Leo Jenkins Cancer Center, Brody School of Medicine at East Carolina University, Greenville, North Carolina
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The clinical progression of CaP involves a complex array of bio-molecular events, many of which are not currently understood. The standard treatment for metastatic CaP is androgen ablation therapy. Patients generally respond well, with measurable decreases in tumor volume and prostate-specific androgen levels; however, this positive response is short-lived and relapse generally occurs within 18 months. During relapse, the tumor acquires the ability to grow in the absence of testosterone, thereby enabling advancement of the disease despite castrate levels of androgen. For androgen-independent CaP, chemotherapy is the standard treatment option for palliation of symptoms associated with disease (1) . However, the drug-resistant nature of CaP minimizes the effectiveness of such therapies, and consequently, most patients die within 12 months (2 , 3) .
Resistance to chemotoxic compounds is most often associated with the action of ATP-binding cassette drug transporters (4) . MDR-1, the gene encoding the p-glycoprotein, is perhaps the most well-characterized drug efflux pump. Alternately, the multidrug resistance protein (MRP) family is another class of drug pumps that are responsible for acquired drug resistance in solid tumors. Taken together, these families of genes have become primary targets of interest for investigations into overcoming drug resistance in a variety of cancer types (5) .
The phosphatidylinositol 3'-kinase (PI3K) signal transduction cascade has been heavily implicated in cellular proliferation and survival (6)
. The negative regulator of this pathway, PTEN, is also a known tumor suppressor protein (7)
. Furthermore, it has been reported to be mutated in 
60% of all CaP patients (8)
. Consequently, many prostatic tumors have high levels of activity within this pathway, including its primary signaling molecule Akt (9)
. Efforts to inhibit signals transduced by this pathway are currently being investigated in many laboratories and clinics for therapeutic benefits.
A definitive relationship between PI3K signaling and drug resistance has yet to be established. However, there are data that correlate a relationship between resistance to antineoplastic therapeutics and PI3K signaling. For example, a recent publication described the usage of a PI3K inhibitor to sensitize ovarian cancer cells to subsequent chemotherapy to levels that were nearly double that of either treatment alone (10) . Studies in bladder cancer have also yielded promising results; synergistic effects were observed when PI3K inhibition was combined with radiotherapy in vivo (11) . Another publication has shown that inhibition of the mammalian target of rapamycin with CCI-779 was able to sensitize drug-resistant prostate cancer cells to doxorubicin (12) . Taken together, these data support the hypothesis that aberrant PI3K signaling may lead to the development of drug resistance in advanced CaP.
The studies described hereafter are the first to illustrate a direct relationship between PI3K-mediated signaling and the modulation of ATP-binding cassette drug transporters, specifically MRP-1. Inhibition of PI3K signaling, through the use of dominant-negative inhibitors and LY294002, was able to sensitize drug-resistant CaP cells to both paclitaxel and doxorubicin. Moreover, down-regulation of MRP-1 expression was able to render drug-resistant CaP cells susceptible to the chemotoxic activities of both doxorubicin and paclitaxel. Because MRP-1 is the primary drug pump found in advanced CaP (13) , the findings presented herein should be instrumental in developing future strategies to down-regulate the expression and/or activity of this efflux pump, as well as increasing the effectiveness of antineoplastic compounds.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retroviral Infection of Cells.
Plasmid DNA containing recombinant retroviruses were transfected into the retroviral packaging cell lines PA317 and 
with lipofectin (Life Technologies, Inc.). Retroviruses were then passed sequentially from one cell line to the other to amplify their titers, as described previously (14, 15, 16)
. DU145, PC3, and LNCaP cells were then infected with viral stocks prepared from PA317 cells. The following G418-resistant (neor) retroviruses were used in this study: (a) PI3K p110 wild-type (wt), (b) PI3K p110 constitutively active (act), (c) PI3K p85
wt, and (d) PI3K 
p85 dominant negative (DN). In addition, some CaP cells were transfected with the following plasmids: (a) PTEN wt, (b) PTEN C124S DN, and (c) PTEN G129E DN. Each of these constructs have been previously described in detail (17, 18, 19)
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Cell Protein Lysate Preparation.
Cells were collected by centrifugation and washed twice with PBS. The cell pellet was then lysed in gold lysis buffer containing 20 mmol/L Tris (pH 7.5), 137 mmol/L NaCl, 5 mmol/L EDTA, 1% (v/v) Triton X-100, 15% (v/v) glycerol, 1 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, 1 µg/mL aprotinin, 1 mmol/L sodium orthovanadate, 1 mmol/L EGTA, 10 mmol/L sodium fluoride, 1 mmol/L tetrasodium PPI, and 0.1 mmol/L ß-glycerophosphate. Lysates were clarified by centrifugation at 14,000 rpm for 15 minutes, and the total protein was quantified using the bicinchoninic acid method (Pierce Biochemicals, Rockford, IL).
Western Blotting Analysis.
Twenty-five micrograms of cellular protein were resolved on 10% SDS polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. The membranes were first incubated with primary antibodies against Akt (1:1000), phospho-Akt (Ser473 and Thr308; 1:1000), and MRPr1 (1:500) and subsequently incubated with horseradish peroxidase-linked secondary antibodies. The phospho- and total Akt antibodies were purchased from Cell Signaling (New England Biolabs, Beverly, MA), and the MRPr1 antibody was obtained from Chemicon International (Temecula, CA). The Western blot procedure was performed as previously described elsewhere (20, 21, 22)
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Cell Viability Assays.
The effects of doxorubicin and paclitaxel on cell viability were assessed by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cells were plated in 96-well plates at 7500 cells per well in quadruplicate wells. After allowing 24 hours for seeding, cells were exposed to varying concentrations of drug (2-fold dilutions from 625 nmol/L doxorubicin and 9.1 µmol/L paclitaxel) diluted in RPMI plus 10% FBS. Ninety-six hours after addition of the drug(s), 5 mg/mL MTT were diluted in sterile PBS and added to each well at 10% (v/v) dilution. Cells were incubated at 37°C for 3 hours, at which point, the supernatant was removed from the wells. The reduced MTT dye was solubilized with 200 µL of DMSO and absorbance was measured on a plate reader at 540 nm. Analysis was carried out with Prizm software, version 3.0. Relative growth was determined with the following formula:
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A CytoTox-ONE homogeneous membrane integrity assay (Promega Corp., Madison, WI) was used to confirm results observed in MTT analysis. Briefly, cells were seeded at 10,000 cells per well in 96-well plates and allowed 24 hours for proper seeding. The following day, cells were treated with 10 µmol/L LY294002 for 2 hours, followed by administration of either 625 nmol/L doxorubicin or 9.1 µmol/L paclitaxel for 96 hours. On day 4, 2 µL of lysis solution (9% Triton X-100 w/v in sterile water) were added to each well to release intracellular lactate dehydrogenase (LDH) into the supernatant. After equilibration to 22°C, 100 µL of CytoTox-ONE reagent were added to each well, shaken for 30 seconds, and incubated for 10 minutes. Lastly, 50 µL of stop solution were added to each well to prevent additional release of LDH. Fluorescence was then measured at 560/590 nm. Relative growth was expressed as a value between 0 and 1 with the following formula:
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Reverse Transcriptase-PCR.
Total cytoplasmic RNA was prepared as described previously (20)
. One microgram of RNA was included in a 20-µL cDNA synthesis reaction containing reverse transcriptase buffer, 1 mmol/L of each deoxynucleoside triphosphate, 20 mg/mL oligo-dT, and 20 units of Mo-MuLV reverse transcriptase (Promega Corp.). After cDNA synthesis, the samples were subjected to PCR. TaqDNA polymerase (2.5 units; Life Technologies, Inc.), 0.15 µmol/L of sense and antisense primers, and PCR reaction buffer were combined to make a final volume of 100 µL for this step of the procedure. PCR was then carried out for 30 cycles. The primers used for these experiments were as follows: (a) MRP-1: sense, 5'-AATGCGCCAAGACTAGGAAG-3', and antisense, 5'-ACCGGAGGATGTTGAACAAG-3'; (b) MDR-1: sense, 5'-CCCATCATTGCAATAGCAGG-3', and antisense, 5'-GTTCAAACTTCTGCTCCTGA-3'; and (c) GAPDH: sense, 5'-ATGGTGAAGGTCGGTGTGAACGGATTTGGC-3', and antisense, 5'-GCATCGAAGGTGGAAGAGTGGGAGTTGCTG-3'. The PCR products were electrophoresed on 1% agarose gels and visualized after ethidium bromide staining of the gel.
Small Interfering RNA (siRNA) Transfection.
siRNA was used to inhibit the expression of the MRP-1 gene. The aforementioned MRP-1 siRNA was purchased from Ambion (Austin, TX). The sequence of the MRP-1 siRNA is as follows: sense, 5'-GGCUACAUUCAGAUGACACtt-3', and antisense, 5'-GUGUCAUCUGAAUGUAGCCtc-3'. Oligofectin (Invitrogen-Life Technologies, Inc., Carlsbad, CA) was used to incorporate the siRNA (100 nmol/L) into the CaP cells. Cells were seeded at 2.5 x 106 cells per well in a 6-well plate and allowed to seed overnight. The following day, cells were washed twice with Opti-MEM media (Invitrogen-Life Technologies, Inc.) and transfected as directed by the manufacturer’s protocol for oligofectin-based transfections. After 4 hours of transfection, the growth media were subjected to RPMI plus FBS to bring the total serum concentration to 10%. Cells were then subjected to chemotoxic drugs or LY294002, as described.
Fluorescence Activated Cell Sorting (FACS) Analysis.
To assess the degree of effectiveness of siRNA transfection, FACS analysis was performed. Briefly, siRNA conjugated to FITC was administered at 10 µg/well after cells were seeded at 2.5 x 105 cells per well in a 6-well plate. Transfection was carried out as described above. Twelve hours after completion of transfection, cells were collected by trypsinization and centrifugation. Cells were then resuspended in 500 µL of FACS buffer (2.5% fetal calf serum and 0.02% NaN3 in PBS) and assayed for intracellular fluorescence using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
Determination of Statistical Significance.
Levels of statistical significance were evaluated with data derived from multiple independent experiments (
2) using a paired student t test. P < 0.05 was considered statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60% of all prostate cancers. Previous studies have demonstrated that PC3 CaP cells lack a functional PTEN protein, whereas DU145 cells are PTEN positive (12)
. To confirm these observations, western blotting was performed in these cell lines with an antibody specific for PTEN. As expected, DU145 CaP cells were positive, and PC3 were negative for the PTEN protein (Fig. 1A)
. Included in these experiments were the CWR-R1 CaP cell line and the MCF-7 breast carcinoma line, both of which were positive for PTEN expression.
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, left panel). Conversely, the PTEN-positive DU145 cells had very low basal levels of Akt phosphorylation, which were much less apparent than that observed in the PC3 cell type (Fig. 1B
, right panel). Furthermore, treatment with the PI3K inhibitor, LY294002, was able to eliminate all phosphorylation of Akt in the DU145 cells; PC3 cells, however, were not as susceptible to this inhibition, most likely attributable to the absence of regulation by PTEN in this cell line (Fig. 1B)
. Both cell types were also stably infected with either wild-type (p110 wt) or constitutively active (p110 act) mutants of the p110 catalytic subunit of PI3K. The effects of these oncogenes on Akt activation are also summarized in Fig. 1B
. Overexpression of these gene products was sufficient to induce phosphorylation of Akt in the PTEN-positive DU145 cells, whereas negligible increases in Akt activation were observed in the PC3 cell type, indicating that maximal Akt activation is achieved through the loss of PTEN in this cell line. These latter data (Fig. 1B)
provide evidence that retroviral infection of PI3K mutants is sufficient to activate Akt in both DU145 and PC3 prostate cancer cells.
PTEN-negative Cells Are More Chemoresistant to Doxorubicin and Paclitaxel Than PTEN-positive Cells.
Traditional MTT analyses were performed in PC3 and DU145 cells to determine their relative ability to grow in increasing concentrations of the chemotoxic drugs, doxorubicin and paclitaxel. In the absence of drug(s), these cell types displayed similar growth patterns over a 4-day time span (Fig. 1C)
. However, as depicted in Fig. 1D
, PC3 cells are much more resistant to death induced by both doxorubicin and paclitaxel when compared with DU145 cells. To confirm these results, LDH assays were carried out under identical conditions. At day 4, it was apparent that PC3 cells exhibited higher levels of survival in the presence of both doxorubicin and paclitaxel than DU145 cells (Fig. 2A)
. Moreover, the addition of LY294002 was able to sensitize drug-resistant PC3 cells to levels congruent with the PTEN-positive DU145 cell line, suggesting that elevated PI3K activity may be responsible for the observed patterns of chemoresistance.
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A dominant-negative version of the regulatory subunit of PI3K, p85, was also infected into CaP cells to examine its effects on drug resistance to paclitaxel and doxorubicin. The data show that inhibition of PI3K-mediated signaling, through the use of the dominant-negative construct, can sensitize PTEN-negative PC3 cells to chemotoxic drugs when compared with wt p85 (Fig. 2D)
. The presence of PTEN in DU145 cells, however, appears to negate the chemosensitizing effect of the dominant-negative construct, suggesting that PTEN status is an integral factor in the development of drug-resistant CaP cells (Fig. 2E)
. These data collectively indicate that inhibition of PI3K signaling, when combined with concurrent chemotherapy, may be effective in preventing drug resistance in advanced, androgen-independent CaP.
Inhibition of PI3K, in Conjunction with Chemotoxic Drugs, Can Enhance the Chemosensitivity of Advanced CaP Cells.
Figs. 1
and 2
suggested that signals transduced through the PI3K cascade might be responsible for chemoresistance patterns observed in advanced CaP cells. As such, experiments combining the use of the small molecular weight inhibitor, LY294002, and chemotherapeutics were performed. First, Fig. 2A
outlines the additive effects of this type of therapy. It is evident that endogenous regulation within the PI3K pathway, in the form of PTEN, is sufficient to minimize drug resistance to either doxorubicin or paclitaxel (DU145 cells; Fig. 2A
). On the contrary, it appears that the response to chemotherapeutics is significantly enhanced by inhibition of PI3K (via LY294002) in the PTEN-negative PC3 cell type. This observation argues that PI3K activation may help lead to the high levels of drug resistance observed in hormone-refractory prostate cancer.
Additional experiments were performed to support these data. Using trypan blue dye exclusion analyses, it was observed that inhibition of PI3K with the use of LY294002 was sufficient to chemosensitize PC3 cells to concentrations of drugs to which they would have otherwise been resistant (Fig. 3)
. In these studies, select concentrations of each drug were used to elicit an inhibitory effect on cell growth, yet allow for synergistic effects when combined with PI3K inhibition via LY294002. Parallel to data shown in Fig. 2E
, inhibition of PI3K in DU145 cells did little to enhance the efficacy of these drugs, indicating that the presence of PTEN is enough to prevent excessive resistance to chemotoxic compounds. Additionally, these data are supported by the findings shown in Fig. 2A
, where addition of the LY294002 compound was able to sensitize PC3, but not DU145 cells to both doxorubicin and paclitaxel.
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, top panel, the MDR-1 gene, which encodes for the p-glycoprotein, was not expressed in any of the three CaP cell types tested. Expression of an alternate drug efflux pump, MRP-1, was absent in the androgen-dependent LNCaP cell line. Interestingly, MRP-1 expression was minimal, at best, in the androgen-independent DU145 cell line; however, upon activation of PI3K (via stable infection), expression of MRP-1 was measurably augmented (Fig. 4A
, middle panel). The PTEN-negative PC3 cells expressed MRP-1 in the absence of transfection, indicating that their previously noted chemoresistant nature may be due to increased MRP-1 levels. Taken together, these data suggest that advanced CaP cells use the activity of PI3K signaling to increase the expression of MRP-1, initiating the development of drug resistance.
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confirmed the patterns of gene expression that were observed in the reverse transcriptase-PCR analysis. Wt DU145 cells expressed no detectable levels of MRP-1, except after infection with the constitutively active p110 mutant, indicating that PI3K activity can lead to increased MRP-1 levels (Fig. 4B
, top panel). DU145 cells were also transfected with dominant-negative varieties of PTEN to antagonize the function of PTEN in these cells. Transfection with either PTEN C124S (lipid and protein phosphatase mutant) or PTEN G129E (lipid phosphatase mutant) was able to significantly increase the levels of MRP-1 in these cells (Fig. 4B
, top panel). To ensure that these differences were not attributable to variations in protein content, the same lysates were electrophoresed, and an identical blot was probed with ß-tubulin (Fig. 4B
, bottom panel). These results support the notion that MRP-1 levels are either directly or indirectly correlated to PI3K activity in advanced CaP cells.
siRNA against MRP-1 Can Sensitize Drug-resistant CaP Cells to Chemotoxic Drugs.
Measuring p-glycoprotein activity through the use of the MDR-1–specific inhibitor, verapamil, is well documented. However, MRP-1 inhibitors (MK571, benzbromarone, and indomethacin) are currently under investigation (23)
, and their specificity is questionable. In turn, results derived from assays designed to ascertain MRP-1 function are uncertain. Consequently, the use of siRNA to inhibit MRP-1 gene expression was exploited to examine the effects of MRP-1 on drug resistance in advanced CaP. Fig. 5A
depicts the siRNA transfection efficiency with FITC-labeled siRNA as measured by FACS analysis. At 12 hours posttransfection, >50% of the cells still possessed measurable levels of the siRNA, indicating that sufficient levels of siRNA gained entry into the cytosol (Fig. 5A)
. The effects of MRP-1 siRNA on MRP-1 gene expression were then assayed in DU145 and PC3 cells. In the PTEN-positive DU145 cell line, there were negligible effects of MRP-1 siRNA on MRP-1 gene expression; in PC3 cells, however, there was a significant inhibition of MRP-1 expression at 8 hours and near-complete inhibition from 12 to 72 hours (Fig. 5B)
. Subsequent Western blotting confirmed these expression patterns and MRP-1 protein levels were diminished over 72 hours in PC3 cells with patterns reflective of mRNA levels observed in Fig. 5C
.
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detail the effectiveness of the MRP-1 siRNA on advanced CaP cells. To determine whether inhibition of MRP-1 expression was sufficient to chemosensitize drug-resistant CaP cells, an additional experiment was performed. DU145 and PC3 cells were transfected with MPR-1 siRNA, then administered either doxorubicin or paclitaxel, and cells were counted at 24, 48, and 72 hours posttransfection. The addition of MRP-1 siRNA, by itself, had no effect on cellular growth proving that MRP-1 has no role in cell survival in the absence of chemotoxic compounds (Fig. 6)
. In the DU145 cell type, addition of MRP-1 siRNA had no effect on chemosensitization because these cells do not express MRP-1. Conversely, MRP-1 siRNA had measurable effects on drug resistance in PC3 cells; an additive inhibitory effect was measured when MRP-1 siRNA and either doxorubicin or paclitaxel were coadministered to these cells, particularly at the 72-hour time point (Fig. 6)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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60% of CaP patients harbor a mutant form of PTEN. Moreover, a recent report suggests that the dosage of PTEN in prostatic lesions is directly correlated to CaP progression, incidence, and overall biology (24)
. Strengthening this argument are other reports stating that prolonged androgen-ablation therapy can lead to heightened levels of PI3K-signaling activity in CaP cells (25
, 26)
. Consequently, efforts to regulate signals transduced through the PI3K pathway may provide therapeutic insight regarding progression and control of the disease.
Combining inhibition of signaling pathways with other types of therapy (i.e., radiation, cytotoxic drugs) is quickly gaining popularity in an array of cancer types (27, 28, 29, 30, 31)
. Likewise, our data indicate that inhibition of PI3K signaling is able to sensitize drug-resistant CaP cells to doxorubicin and paclitaxel (Figs. 2
and 3
). Chemotoxic drugs are the primary therapeutic option for hormone-refractory prostate cancer patients; however, patients rapidly develop resistance to these compounds, thereby negating their palliative effects. Thus, our studies have the potential to lead to the development of a clinical scenario whereby signaling inhibitors are combined with chemotherapy to augment the effectiveness of cytotoxic drugs and extend patient survival.
Drug resistance is primarily mediated through the cell’s ability to actively efflux potentially hazardous substances from the intracellular space. Most often, this is achieved via the action of ATP-binding cassette drug transporters, namely the p-glycoprotein or MRP-1 pumps (4)
. These studies show that none of the CaP cell lines tested were positive for MDR-1 gene expression (Fig. 4A)
. Interestingly, CaP cells with constitutive PI3K activity were positive for MRP-1 expression, whereas PTEN-positive (DU145 wt) cells expressed minimal levels of this gene. However, activation of PI3K potentiated up-regulation of MRP-1, indicating that the PI3K pathway may modulate expression of the MRP-1 gene. These trends were also observed on the protein level when various dominant-negative PTEN and constitutively active PI3K mutants were observed to increase levels of the MRP-1 gene product (Fig. 4B)
.
Wang and Beck (32)
were the first to hypothesize that p53 is a negative regulator of MRP-1 expression. However, to date, there is no report of any p53-binding motifs located within the MRP-1 promoter region (33)
. Therefore, one might hypothesize that repression of MRP-1 by p53 occurs by an indirect means rather than an immediate interaction with the MRP-1 promoter. Beck also reported that expression of the specificity protein 1 (Sp1) transcription factor is a strong activator of MRP-1 expression (32)
. Given that there are three Sp1-binding sites on the MRP-1 promoter and that Sp1 activity has been reported to be PI3K dependent (34)
, it is plausible to reason that Sp1 is critical for MRP-1 expression in advanced CaP cells. Data from Fig. 4A
suggests that p53 (LNCaP: p53 positive; DU145 and PC3: p53 negative) may be the dominant regulatory molecule governing MRP-1 expression, but PI3K activity and subsequent Sp1 activation may be necessary for maximal expression of MRP-1, as shown in the DU145 + p110 (act) cell type. This conclusion is merely speculative, however, and additional research is needed to confirm or refute its veracity.
Extensive characterization of MRP-1 has revealed that it possesses the ability to confer resistance to a wide array of chemotoxic compounds, including anthracyclines, Vinca alkaloids, epipodophyllotoxins, camptothecins, and others (5)
. Taxanes, however, are not widely believed to be substrates of the MRP-1 pump despite reports that MRP-1–/– mice are moderately more sensitive to taxanes than MRP-1+/+ mice (35)
. In this study, we show that inhibition of MRP-1 expression can cause the chemosensitization of CaP cells to a taxane, paclitaxel (Fig. 6)
. An identical effect was observed when MRP-1 siRNA was combined with doxorubicin, indicating that MRP-1 is responsible for efflux of both of these drugs in CaP cells.
Proliferation of CaP cells in prostatic tumors is not an enormous problem because only between 1 and 3% of all CaP cells undergo active proliferation (36) . This may explain why prostatic disease is difficult to detect until the latter stages of life. Survival of CaP cells, however, is a problematic issue because CaP cells die at lower rates than at which they divide. Consequently, pathways that mediate survival, rather than proliferation (i.e., mitogen-activated protein kinase signaling), may be attractive targets for reducing tumor growth in CaP patients. Our data support this premise because mitogen-activated protein kinase activation had no appreciable effects on drug resistance in these CaP cell lines (data not shown).
After hormone relapse, a CaP patient typically has <18 months to live. Thus, extending those precious months into years or even decades is a meaningful objective. Single agent chemotherapy has been marginally effective; consequently, a large number of studies are currently underway to assess the effectiveness of combinatorial chemotherapy (37, 38, 39) . The drawback to the administration of multiple chemotoxic compounds has been the toxicity profiles associated with such treatment regimens. As such, other approaches that curtail toxicity while maximizing efficacy are warranted. The results presented herein imply that chemotherapy combined with PI3K inhibition may represent an alternative strategy that could be effectively used in patients with hormone-refractory prostate cancer.
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FOOTNOTES |
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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.
Requests for reprints: James A. McCubrey. Phone: (252) 744-2704; Fax: (252) 744-3104; E-mail: mccubreyj{at}mail.ecu.edu
Received 5/ 7/04. Revised 8/ 6/04. Accepted 8/20/04.
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) versus DU145 (gray circles) cells. D, MTT assay depicting comparison of chemoresistance profiles of both PC3 and DU145 wt cell lines over increasing concentrations of doxorubicin and paclitaxel (96 hours of treatment). The data are derived from three individual experiments where each assay was performed in quadruplicate.
) in PC3 cells. E, comparison of p85 wt subunit of PI3K (gray circle) versus a dominant-negative mutant of p85 (
, 24' post-treatment;
, 48' post-treatment; 
, 72' post-treatment.
