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Cell and Tumor Biology |
Departments of 1 Radiation and 2 Biostatistics and Epidemiology, University of Pennsylvania; 3 Department of Pathology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
Requests for reprints: Anjali K. Gupta, Department of Radiation Oncology, University of Pennsylvania, 195 JMB, 3620 Hamilton Walk, Philadelphia, PA 19104. Phone: 215-898-0076; Fax: 215-898-0996; E-mail: gupta{at}xrt.upenn.edu.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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60% of all cancer patients in the United States (1). This resistance significantly reduces treatment options and makes disease-free recovery difficult (2, 3). Therefore, the ability to discern particular mechanisms governing multimodality tumor cell resistance and subsequently exploit them should result in more effective treatment strategies. One such factor known to increase cellular resistance to radiation is the presence of overexpressed or activated oncogenes, such as epidermal growth factor receptor (EGFR; refs. 4–6) and Ras (7, 8), or loss of the tumor suppressor gene PTEN (9). One critical observation is that these mutations share a common molecular signaling alteration that ultimately activates the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. In this regard, we and others have shown that blocking PI3K enhances the radiation response in vitro and in vivo. This effect occurs in cells in which this pathway is constitutively activated but does not affect cells quiescent in this pathway (10–12). Down-regulation of Akt using small interfering RNA also radiosensitizes these cells (13). Because this pathway is frequently activated in tumor cells but not in the normal host cells, it is an excellent candidate to target to increase radiation sensitivity. The problem has been to identify inhibitors of this pathway suitable for clinical use. For example, LY294002 and wortmannin, which are widely used to inhibit the PI3K pathway in vitro with significant radiosensitizing effects, however, are poorly tolerated in vivo and of little clinical value (14).
In this study, we explore the possibility that one class of drugs in common use clinically, the HIV protease inhibitors (HPIs), may interfere with PI3K-Akt signaling. These drugs given in combination with reverse transcriptase inhibitors are the mainstays of the current therapeutic regimens for HIV-infected patients. The HPIs are peptidomimetics that inhibit the HIV aspartyl protease, a retroviral enzyme that cleaves the viral gag-pol polyprotein and is necessary for the production of infectious viral particles (15). A prominent side effect of HPI treatment is insulin resistance and diabetes (16, 17). Because Akt, especially the Akt2 isoform (18), plays a key role in the coordinated regulation of growth and metabolism by the insulin/insulin-like growth factor signaling pathway (18, 19), we explored the possibility that HPIs might block the PI3K-Akt signaling axis in tumor cells and hence be used clinically as radiation sensitizers. In fact, Pajonk et al. have shown that one HPI, saquinavir, was a radiation sensitizer in tissue culture (20). Here, we found that three of the five HPIs that we tested were able to inhibit Akt at doses routinely achieved clinically. These compounds also sensitized tumor cells both in vitro and in vivo to radiation. HPIs have been used continuously in patients with well-characterized pharmacokinetics. They are well tolerated; thus, testing these HPIs as radiation sensitizers could easily proceed to clinical trial.
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Materials and Methods |
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MR4 cells were transfected with constitutively active PI3K consisting of the iSH2 domain of p85 fused to the NH2 terminus of p110 by means of a flexible glycine linker (21) inserted into the pGRE5/EBV dexamethasone-inducible plasmid vector. Because this is an episomal vector, a pool of cells was selected with hygromycin for 72 hours. Dexamethasone (Sigma, St. Louis, MO) at 1 µg/mL was added. Radiation survival experiments were done and protein samples were harvested 24 hours after the addition of dexamethasone.
Drugs. The HPIs were bought for research use from the hospital inpatient pharmacy of the University of Pennsylvania. Ritonavir, amprenavir, and saquinavir came as gelatin capsules. The capsule was punctured and the viscous liquid inside was dissolved in 100% ethanol to make a concentrated stock solution for subsequent experiments. Nelfinavir and indinavir came as solid caplets and were ground into a fine powder and subsequently dissolved in 100% ethanol.
Western blotting. Cells were lysed without trypsinization by rinsing culture dishes once with PBS followed by lysis with reducing Laemmli sample buffer. Samples were boiled, sheared, clarified by centrifugation, and stored at –20°C. Samples containing equal amounts of protein were separated on a 12% SDS-polyacrylamide gel and blotted onto nitrocellulose membranes. Membranes were blocked in PBS containing 0.1% Tween 20 and 5% powdered milk before primary antibody addition. Both polyclonal anti-phospho-Ser473 Akt and total Akt antibodies (New England Biolabs, Ipswich, MA) were used at a dilution of 1:2,000. Polyclonal anti–mitogen-activated protein kinase (MAPK) K-23 antibody (Santa Cruz Biotechnology) was used at a dilution of 1:500. Monoclonal anti-ß-actin antibody (Sigma) was used at a dilution of 1:4,000. Antibody binding was detected using the enhanced chemiluminescence kit (Amersham, Arlington Heights, IL). Images were digitized using an Arcus II scanner, and figures were assembled using Adobe Photoshop 3.0 and Microsoft Power Point.
Radiation survival determination. Cells in exponential growth phase were counted and plated in 60-mm dishes containing 4 mL medium. The cells were allowed to attach and drugs were added to cultures at least 1 hour before radiation. Cells were irradiated with a Mark I cesium irradiator (J.L. Shepherd, San Fernando, CA) at a dose rate of 1.6 Gy/min. Colonies were stained and counted 10 to 14 days after irradiation. A colony by definition had >50 cells. The surviving fraction was calculated by dividing the number of colonies formed by the number of cells plated times plating efficiency. Each point on the survival curve represents the mean surviving fraction from at least three replicates.
Cell growth curves. Cells (3 x 105) were plated in each T25 flask. The cells were allowed to attach and enter exponential growth and the drugs were added. At various times, total cell number was assessed in triplicate.
Tumor generation in nude mice. Pathogen-free female Ncr-nu/nu mice were obtained from Taconic (Germantown, NY) and housed aseptically in the animal facilities of University Laboratory Animal Resources and the Institute for Human Gene Therapy of the University of Pennsylvania. All experiments were carried out in accordance with University Institutional Animal Care and Use Committee guidelines. At 5 to 7 weeks of age, mice were inoculated by s.c. injection into the hind flank with 1 x 107 T24 or 1 x 106 SQ20B cells resuspended in 100 µL Matrigel (BD Collaborative Research, Franklin Lakes, NJ). SQ20B tumors usually appeared within 1 week and T24 tumors 2 to 3 weeks after injection.
Drug treatment of mice. Nelfinavir was formulated as 3-week continuous release pellets containing 12.6 mg drug with a release rate of 0.6 mg/d by Innovative Research of America (Sarasota, FL). Amprenavir was given s.c. by continuous micro-osmotic pump infusion (Alza Corp., Palo Alto, CA) of the drug at a dose of 0.8 mg/d. These doses are comparable with that used in HIV patients. Control animals received placebo pellet or pumps with carrier alone.
Drug measurements in serum. High-performance liquid chromatography was done using a binary gradient Jasco system. Serum was prepared by adding an equal part of 100% methanol followed by incubation on ice for 1 hour and centrifugation at 13,000 x g for 10 minutes. The supernatant (100 µL) was analyzed using an Alltima C18 column (4.6 x 250 mm, 5 µ) with buffer [0.1 mmol/L ammonium acetate (pH 4.7)] gradient for nelfinavir of 30% to 90% (4 minutes) followed by 90% (8 minutes) and for amprenavir of 30% to 90% (10 minutes) followed by 90% (4 minutes). Nelfinavir or amprenavir content was determined from the area under curve at the drug peak detected at 11 to 12 minutes with 
260 for amprenavir and 250 for nelfinavir. Absolute values were calculated from calibration curves.
Immunohistochemical staining. Paraffin-embedded tissue sections were stained with antibody for immunohistochemistry to phospho-Ser473 Akt as described by Zhou et al. (22). Slides were graded as 0 to 3+ staining as we have described previously (12).
In vivo clonogenic assays. Animals were assigned randomly to treatment groups (control, radiation alone, drug alone, or radiation and drug) when tumors attained a volume of 300 to 400 mm3. Drug treatment was started 3 days (amprenavir) or 5 days (nelfinavir) before radiation. Mice were irradiated with doses of 6 to 8 Gy with a Mark I cesium irradiator at a dose rate of 1.6 Gy/min under anesthesia. Unirradiated animals were anesthetized and sham irradiated. One hour after irradiation, animals were sacrificed. Tumors were then excised, minced, and dissociated for 30 minutes at 37°C in HBSS containing 166 units/mL collagenase XI, 0.25 mg/mL protease, and 255 units/mL DNase. Cells were recovered after straining through an 80-µm mesh. The cells were pelleted by centrifugation at 500 x g and resuspended in culture medium. Cells were counted by hemocytometer using trypan blue to determine viability, and the counts were verified by Coulter counter analysis. Cells were then plated in 100-mm dishes and cultured for 14 to 21 days, after which colonies were stained and counted. Results are expressed as the plating efficiency determined from replicate dishes plated at multiple initial cell densities.
Determinations of tumor regrowth delay. Mice bearing established tumors were randomized and treated with amprenavir/nelfinavir as described above. Irradiation (6 or 8 Gy) of the flank bearing the tumor was done using a 250 kV orthovoltage irradiator (Philips RT 250) at a dose rate of 2.63 Gy/min through a 0.2-mm copper filter. The source-to-tumor target distance was 30 cm with adequate shielding of nontumor sites. Mice were examined twice weekly for evaluation of tumor growth. Tumors were measured with calipers in three mutually perpendicular diameters (a, b, and c) and the volume was calculated as V = (
/ 6) x a x b x c.
Statistical analysis. A two-sided Student's t test was used to determine significance between SF2s in Table 1. A regression model was fit to log10-transformed plating efficiency data that included terms to estimate the individual (main) effects of radiation and HPI and the interaction of these two treatments on the mean plating efficiency of tumor cells. Replicate measurements were averaged for each mouse. The linear model took the form:
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Results |
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SQ20B cells were treated with 25 µmol/L saquinavir from 5 to 60 minutes and Western blot analysis showed a time-dependent decrease in immunoreactive Akt phosphorylation levels with a complete loss of detectable phosphorylation of Akt within 20 minutes (Fig. 1A). There was no change in the total Akt levels using actin as a loading control. At lower concentrations (5-10 µmol/L range), saquinavir took 24 hours to have the same effect (data not shown). However, saquinavir was highly toxic in tissue culture with cell death within 2 hours of treatment at 25 µmol/L and cell death at 24 to 48 hours for doses between 1 and 10 µmol/L.
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Similar to saquinavir and amprenavir, nelfinavir (5 µmol/L) down-regulated Akt phosphorylation at Ser473 in both SQ20B and T24 cells (Fig. 1D) without change in total intracellular Akt protein levels. Exposure to increasing the concentration of nelfinavir resulted in an earlier onset of this response but resulted in cell toxicity at 20 µmol/L. Finally, similar to the other HPIs tested, no changes in cell growth kinetics or cell death was observed at concentrations that inhibit Akt phosphorylation (data not shown). The other HPIs tested, indinavir and ritonavir, did not appreciably affect cell growth, toxicity, or regulation of Akt signaling at concentrations up to 100 µmol/L (data not shown).
Radiation sensitization by HIV protease inhibitors in vitro. We have shown previously that inhibition of the PI3K-Akt pathway increases radiosensitivity of tumor cells (11). Insulin resistance and diabetes are recognized side effects of HPIs, suggesting that these agents may inhibit of Akt signaling. Because activation of the PI3K-Akt signaling pathway seems to confer resistance to IR-induced cell death, it seemed logical to hypothesize that HPIs can inhibit Akt activity resulting in increased tumor cell sensitivity to IR-induced cell death. To address this idea, two cell lines were tested, SQ20B and T24, which contain constitutively active Akt, as determined by increased Akt phosphorylation. Experiments were subsequently expanded to include two additional cell lines, MIAPACA2 (pancreatic tumor cells) and A549 (lung tumor cells), both of which contain an activating K-Ras mutation. Initially, clonogenic cell survival experiments were done to determine any changes in radiosensitivity in vitro. Results from these experiments showed that both T24 and SQ20B cells were sensitized to the cytotoxicity of IR following exposure to either amprenavir (10 µmol/L) or nelfinavir (5 µmol/L; Fig. 2A). The SF2 results from these survival experiments are shown (Table 1). Cell survival experiments with saquinavir were limited due to its toxicity, however; exposure of T24 cells inhibited Akt and resulted in radiosensitization. REF cells, which do not have increased Akt phosphorylation and showed no change in radiation-induced cell death, suggests that normal tissues are not affected by exposure to HPIs. In every cell line with increased signaling through Akt, there was at least 15% reduction in surviving fraction with the drug, and in many cases, the reduction was as large as 40%. These results are significant as there is no overlap in the SEs.
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MR4 cells transfected with active PI3K treated with dexamethasone showed a significant increase in resistance to IR-induced cell death, whereas no change in clonogenic cell survival was seen in control cells. Expression of the vector alone and exposure to dexamethasone did not alter the plating efficiency of the cells. In addition, treatment of transfected cells with amprenavir and nelfinavir in the presence of dexamethasone sensitized them to radiation (Fig. 2C).
HIV protease inhibitors inhibit Akt signaling in vivo. Although in vitro data provide an important preliminary background to advance the potential use of new anticancer agents, these results must be confirmed and validated in an in vivo tumor model system. Before regrowth experiments, we initially determined if nelfinavir and amprenavir can down-regulate Akt in xenografts. As such, nelfinavir was given at 0.6 mg/d via a continuous releasing pellet and it was observed that a minimum of 5 days of exposure was necessary for serum levels of nelfinavir to equilibrate in the 2 to 6 µmol/L range. The tumors were stained with anti-phospho-Ser473 Akt antibody and the phosphorylation was down-regulated from 3+ to 0-1+ staining. Figure 3A shows representative sections from SQ20B xenografts, one from a mouse treated with nelfinavir (right) and one without (left). The serum level of nelfinavir in the mouse treated with drug was 4.5 µmol/L (Fig. 3B) and Western blotting with anti-phospho-Ser473 Akt confirms the decrease in phospho-Akt seen in tumor slides.
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Radiation sensitization in vivo measured by clonogenic assay. Tumor cell colony formation assays were used to measure radiation response after amprenavir and nelfinavir treatment of SQ20B or T24 tumor-bearing animals. The clonogenicity of tumor cells was compared after isolation from tumors treated with radiation plus amprenavir/nelfinavir, radiation alone, amprenavir/nelfinavir alone, or mock treatment. The radiation dose was 8 Gy for SQ20B tumors and 6 Gy for T24 tumors. The animals were pretreated with nelfinavir pellets for 5 days or with amprenavir pumps for 2 days. Both amprenavir and nelfinavir treatment of SQ20B (Fig. 4A) or T24 (Fig. 4B) xenografts resulted in reduction in clonogenicity after irradiation. As seen in Table 2, both amprenavir and radiation and nelfinavir and radiation in SQ20B xenografts exhibited highly statistically significant synergy (P < 0.001 each). In T24 xenografts (Table 3), nelfinavir and radiation exhibited highly statistically significant synergy (P = 0.003) and the synergistic effect of amprenavir and radiation nearly reached statistical significance (P = 0.053).
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SQ20B tumor xenografts treated with nelfinavir and the results for mean tumor volumes (Fig. 5A) and each individual tumors (Fig. 5B) are shown. In the radiation plus nelfinavir group, two slowly growing tumors reached a volume of 1,000 mm3 at 70 and 78 days (data not shown in Fig. 5B). The mean time to tumor volume of 1,000 mm3 was 11 days in the control group and 12 days in the nelfinavir alone group. As expected, mean values increased in both radiation alone (15 days) and radiation and nelfinavir (41 days) groups. As seen in Table 4, a statistically significant synergistic effect between radiation and nelfinavir was detected by linear regression analysis (P = 0.03).
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Discussion |
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Here, we have shown that several HPIs have an unexpected ability to inhibit Akt phosphorylation. Both nelfinavir and amprenavir inhibited phosphorylation of Akt Ser473 in both tissue culture and in vivo experiments. Concomitantly, these drugs sensitized all four tumor cell lines tested in vitro. Because radiation is typically given in 2 Gy doses for at least 30 fractions, even a 15% reduction in surviving fraction (0.57-0.487 in A549 cells with amprenavir) translates into an increase of 2 logs of cell kill (0.5730 versus 0.48730) with the addition of the drug. The administration of amprenavir or nelfinavir to mice at levels comparable with those used in HIV patients resulted in sensitization of both T24 and SQ20B xenografts after a single dose of radiation. The effect of these HPIs was synergistic with radiation without increase in normal tissue toxicity as determined using REF clonogenicity or epidermal skin thickness in mice with drug treatment and radiation.
Although the translation research presented here clearly shows the clinical potential for amprenavir, nelfinavir, or saquinavir, the exact mechanism by which these agents interfere with the PI3K-Akt signaling pathway remains unclear. The observation that MR4 cells with transfection of active PI3K were also sensitized by the protease inhibitors leads us to think it might be a point between PI3K and Akt. PI3K phosphorylates PtdIns-4,5-P2 to yield PtdIns-3,4,5-P3. PtdIns-3,4,5-P3 in turn causes membrane localization of protein kinase B/Akt and the phosphoinositide-dependent kinase-1, which phosphorylates the Thr308 site (25). For maximal activation, however, the second site on Akt (Ser473) must also be phosphorylated. The kinase regulating phosphorylation of Ser473 is not known, but several kinases, such as ILK-1 (26) and ATM (27), have been suggested.
The use of HPIs has been associated with direct effects on tumors, including those through modulation of the cell proteasome (reviewed in ref. 28). Ritonavir and saquinavir have both been shown to affect the 26S proteasome resulting in accumulation of p21 and I
B (20, 29). This effect of saquinavir has been reported previously to correlate with radiation sensitization (20). However, in our hands, ritonavir neither inhibited Akt phosphorylation nor resulted in radiation sensitization.
HPIs have been in clinical use since 1995 and are associated with side effects, including insulin resistance and diabetes (17). Because insulin signals through Akt (18, 19), we explored a possible link of HPIs to Akt inhibition. Three of the five HPIs we tested inhibited Akt phosphorylation and radiosensitized cells at pharmacologically achievable concentrations. Our data do not directly support the hypothesis that Akt inhibition is also the mechanism of insulin resistance. Of the two HPIs that did not reduce Akt phosphorylation and did not radiosensitize cells, indinavir is associated with a higher risk of developing diabetes than the other HPI (16, 30). It is likely that the insulin resistance associated with HPIs may be multifactorial (31, 32), although effects on Akt may play a role in some cases (33). It nevertheless remains that three of the five HPIs tested resulted in down-regulation of Akt phosphorylation at Ser473 at clinically achievable concentrations. We tested two of the HPIs (amprenavir and nelfinavir) as adjuvant antitumor agents in vivo and saw synergistic sensitization with HPI and radiation. Because there is safety data on both amprenavir and nelfinavir for the last 5 years, they should be tested as radiation sensitizers in clinical trial.
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Acknowledgments |
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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.
We would like to thank Dr. A.V. Kachur for his assistance with high-performance liquid chromatography.
Received 4/ 7/05. Revised 7/ 1/05. Accepted 7/ 7/05.
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B pathway. J Biol Chem 2000;275:8027–31.
. Diabetologia 2004;47:1107–17.[Medline]This article has been cited by other articles:
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), transfected and dexamethasone (Trans + Dex; 
), transfected and dexamethasone with nelfinavir (Tran + Dex + Nel; 
), and transfected and dexamethasone with amprenavir (Tran + Dex + Amp; 
). Points for other curves (dexamethasone alone, transfected alone, nelfinavir alone, amprenavir alone, dexamethasone and amprenavir, dexamethasone and nelfinavir, transfected and nelfinavir, and transfected and amprenavir) are also shown, but the curves are not visualized as they overlapped the control curve.
), tumors treated with 8 Gy radiation (x), or 8 Gy and nelfinavir-treated tumors (
). B, data from each individual tumor, which were used to derive the data in A.