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The Human Immunodeficiency Virus Protease Inhibitor Ritonavir Inhibits Lung Cancer Cells, in Part, by Inhibition of Survivin

      Introduction:

      Ritonavir is a potential therapeutic agent in lung cancer, but its targets in lung adenocarcinoma are unknown, as are candidate biomarkers for its activity.

      Methods:

      RNAi was used to identify genes whose expression affects ritonavir sensitivity. Synergy between ritonavir, gemcitabine, and cisplatin was tested by isobologram analysis.

      Results:

      Ritonavir inhibits growth of K-ras mutant lung adenocarcinoma lines A549, H522, H23, and K-ras wild-type line H838. Ritonavir causes G0/G1 arrest and apoptosis. Associated with G0/G1 arrest, ritonavir down-regulates cyclin-dependent kinases, cyclin D1, and retinoblastoma protein phosphorylation. Associated with induction of apoptosis, ritonavir reduces survivin messenger RNA and protein levels more than twofold. Ritonavir inhibits phosphorylation of c-Src and signal transducer and activator of transcription protein 3, which are important events for survivin gene expression and cell growth, and induces cleavage of PARP1. Although knock down of survivin, c-Src, or signal transducer and activator of transcription protein 3 inhibits cell growth, only survivin knock down enhances ritonavir inhibition of growth and survivin overexpression promotes ritonavir resistance. Ritonavir was tested in combination with gemcitabine or cisplatin, exhibiting synergistic and additive effects, respectively. The combination of ritonavir/gemcitabine/cisplatin is synergistic in the A549 line and additive in the H522 line, at clinically feasible ritonavir concentrations (<10 μM).

      Conclusions:

      Ritonavir is of interest for lung adenocarcinoma therapeutics, and survivin is an important target and potential biomarker for its sensitivity. Ritonavir cooperation with gemcitabine/cisplatin might be explained by involvement of PARP1 in repair of cisplatin-mediated DNA damage and survivin in repair of gemcitabine-mediated double-stranded DNA breaks.

      Key Words

      A potentially successful approach for cancer drug development consists of repurposing existing drugs, with known tolerability, toxicity, and pharmacology.
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      MATERIALS AND METHODS

      Antisera

      Antibodies raised against human phospho-retinoblastoma protein (Rb) (no. 9308), phospho-c-Src (no. 2101S), STAT3 (clone 124H6; no. 9139), phospho-STAT3 (clone 3E2; no. 9138S), and PARP1 (no. 9542) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies raised against p53 (clone DO1; no. sc-126), cyclin-dependent kinase (CDK)2 (no. sc-163), CDK4 (no. sc-260), CDK6 (no. sc-177), cyclin D1 (sc-718), and cyclin A (clone C-19; no. sc-596) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody c-Src (clone GD11; no. 05-184) was purchased from Millipore (Billerica, MA). Antibodies p27 (no. 610242), Rb (no. 554136), and phospho-Rb were purchased from BD Biosciences (San Jose, CA). Antibody survivin (no. AF886) was purchased from R&D Systems (Minneapolis, MN). Antibody glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (clone 6C5; 10R-G109A) was purchased from Fitzgerald Industries International (Concord, MA).

      Cell Lines and Drugs

      The lung adenocarcinoma lines A549, H522, and H23 were obtained from the American Type Culture Collection (Manassas, VA). The lung adenocarcinoma line H838 was a kind gift from Dr. Manish Patel (University of Minnesota). Purified ritonavir, gemcitabine, and cisplatin were purchased from Sequoia Research Products (Pangbourne, UK).

      siRNA

      siRNA duplex SMARTpool upgrades of c-Src (no. M-003175-03), STAT3 (no. M-003544-02), survivin (no. M-003459-02), and siControl (no. D-001210-01) were purchased from Dharmacon Research, Inc. (Lafayette, CO). The sequences for siRNA duplexes used in these studies are given in Table S1 (http://links.lww.com/JTO/A62).

      Western Blotting

      Cell lysates were prepared by standard methodology. Samples containing the same amount of protein were separated on a 4 to 20% gradient SDS gel, blotted on a nitrocellulose membrane, and probed with specific antibodies. Densitometric analysis of the bands was performed using the software Alpha Imager (Alpha Innotech; San Leandro, CA), and the relative amount of protein was normalized to the corresponding signal for GAPDH.

      3, [4,5-Dimethylthiazol-2-yl]-2,5-Diphenyltetrazolium Bromide Assay

      The cells were cultured in 96-well plates for 48 hours in the presence of increasing concentration of ritonavir (ranging from 5 to 60 μM) or dimethyl sulfoxide (DMSO). Cell growth was determined by measuring the mitochondrial reduction of the tetrazolium salt, 3, [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma; St Louis, MO). Each concentration of ritonavir was tested in octuplicate, and each MTT assay was repeated three times.

      Measurement of Clonogenic Efficiency

      Clonogenic efficiency of lung adenocarcinoma lines was measured by exposing cell monolayers (20% confluent) to varying concentrations of ritonavir (ranging from 10 to 100 μM) or DMSO for 24 hours. Each ritonavir concentration was tested in triplicate. At the end of the 24-hour treatment, cell monolayers were treated with trypsin and replated (500 cells/100-mm plate) in the absence of ritonavir in complete medium (CM) consisting of RPMI 1640 (American Type Culture Collection; Manassas, VA) supplemented with 10% fetal calf serum (Hyclone/Thermo Fisher Scientific; Logan, UT). The medium was changed every 3 days for 21 days. At the end of the 21-day incubation period, the colonies were fixed, stained with Wright-Giemsa stain (Thermo Fisher Scientific; Waltham, MA), and counted.

      Cell Cycle Analysis

      Lung adenocarcinoma cell lines 30 to 50% confluent were treated with trypsin, washed, and resuspended in CM. Cells were plated at a density of 5 × 105 cells/100-mm plate and grown for 24 and 48 hours in CM in the presence of ritonavir (half maximal inhibitory concentration [IC50]) or DMSO. Profiling of propidium iodide (PI) incorporation was performed by FACScan analysis (Becton Dickinson; San Jose, CA). Adherent and nonadherent cells were included in the profile. Cell cycle distribution was determined using ModFit software (Becton Dickinson; San Jose, CA).

      Measurement of Apoptosis

      Apoptotic cells were detected using an Annexin-V-FITC/PI apoptosis detection kit (Oncogene; Boston, MA). Cells were plated at a density of 5 × 105 cells/100-mm plate and grown for 48 hours in CM in presence of ritonavir (IC50) or DMSO. After 48 hours, cells were harvested and stained with PI and Annexin-V-FITC. Cells were then acquired by FACScan (1 × 104 cells/assay) and analyzed using CellQuest software (Becton Dickinson; San Jose, CA). Each assay was performed in duplicate.

      Real-Time Polymerase Chain Reaction

      RNA was isolated from A549 and H522 cell lines treated for 48 hours with ritonavir (40 μM) or DMSO by RNeasy mini kit (QIAGEN; Valencia, CA), following manufacturer's instructions. First-strand complementary DNA (cDNA) was synthesized with high-capacity cDNA archive kit (Applied Biosystems; Foster City, CA). The polymerase chain reaction consisted of 40 cycles (2 minutes at 50°C, 10 minutes at 95°C, 15 seconds at 95°C, and 1 minute at 60°C).

      siRNA Transfection and Cell Survival Assay

      Cells were plated in six-well poly-d-lysine-coated tissue culture plates (Becton Dickinson; Bedford, MA) at 2.5 × 104 cells/well, grown for 24 hours, and then transfected with 50 pmol/well of siRNA duplexes for c-Src, STAT3, survivin, or control siRNA using oligofectamine (Invitrogen; Carlsbad, CA) and OPTI-MEM (Invitrogen; Carlsbad, CA). The efficacy of gene silencing was evaluated by examining the protein product with Western blotting 72 hours after transfection. Each assay was performed in triplicate. Twenty-four hours after the siRNA transfection, the cells were exposed for 48 hours to ritonavir (ranging from 0 to 60 μM), and cell viability was measured by an MTT assay.

      Generation of Survivin Expression MIEG3 Vectors

      Human survivin cDNA was cloned upstream of the internal ribosomal entry site for enhanced green fluorescence protein (EGFP). The survivin sense orientation expression plasmid (S-IRES-EGFP-MIEG3) was constructed using an Eco RI adaptor as described.
      • Fukuda S
      • Foster RG
      • Porter SB
      • et al.
      The antiapoptosis protein survivin is associated with cell cycle entry of normal cord blood CD34(+) cells and modulates cell cycle and proliferation of mouse hematopoietic progenitor cells.
      Empty MIEG3 plasmid was used as control vector. Flow cytometry was used to sort A549 or H522 cells transiently transfected with the S-IRES-EGFP-MIEG3.

      Effects of Drug Combination on Cell Growth

      Cells were exposed to ritonavir and/or gemcitabine at a fixed constant ratio corresponding to the ratio of the IC50 values for each single drug. The CalcuSyn software (Biosoft; Cambridge, UK)—which uses the median-effect analysis method of Chou and Talalay—was used to determine the possible synergic effect of the drugs. According to this method, a combination index (CI) can be calculated from dose-response curves obtained using the drugs as single agent or in combination. CI value less than 1.0 indicates synergy, CI = 1.0 indicates that the drugs act in an additive fashion, and CI more than 1.0 indicates antagonism.

      Statistical Analysis

      Statistical analysis was conducted using two-tailed Student's t test (Excel, Microsoft). A difference was considered significant if p value was less than 0.05.

      RESULTS

      Ritonavir Inhibits Growth and Colony Formation of Lung Adenocarcinoma Lines

      The effect of ritonavir on the growth of the A549 and H522 lines was assayed by MTT assay and compared with vehicle (DMSO). Ritonavir inhibits the growth of the lung adenocarcinoma lines A549 and H522, exhibiting IC50 values of 35 and 42 μM, respectively (Figure 1A). Ritonavir also inhibits the growth of the lung adenocarcinoma lines H23 and H838, exhibiting IC50 values of 44 and 35 μM, respectively, confirming that ritonavir is active across a range of lung adenocarcinoma lines (results for H23 not shown; results for H838 shown in Figure S1; http://links.lww.com/JTO/A61). Ritonavir was also effective at inhibiting adhesion-dependent colony formation of the A549 and H522 lines, exhibiting IC50 values of 30 and 40 μM, respectively (Figure 1B).
      Figure thumbnail gr1
      FIGURE 1Panel A, Ritonavir inhibits growth of the lung adenocarcinoma lines A549 and H522. Ritonavir inhibited the growth of both lines, exhibiting an half maximal inhibitory concentration (IC50) of 35 μM for A549 (open circles) and 42 μM for H522 (open squares). Results are representative of three consistent experiments. Panel B, Ritonavir reduces the clonogenic efficiency of the lung adenocarcinoma lines A549 and H522. Ritonavir was efficient in reducing the clonogenic efficiency in both the A549 (open circles) and H522 (open squares) lines, exhibiting an IC50 of 30 and 40 μM, respectively. Panel C, Ritonavir induces apoptosis of the lung adenocarcinoma lines A549 and H522. Ritonavir significantly increased early- and late-apoptotic events in both lines. The results are representative of two consistent experiments.

      Ritonavir Induces Apoptosis of Lung Adenocarcinoma Lines

      To determine whether ritonavir induces apoptosis, the adenocarcinoma lines A549 and H522 were exposed to ritonavir at the IC50 for 48 hours, stained with PI/Annexin-V-FITC and analyzed by flow cytometry. Ritonavir treatment increased the percentage of early-apoptotic cells 10-fold from 1.6 to 16%, for the A549 line, and threefold, from 3.2 to 10%, for the H522 line (Figure 1C). The percentage of late-apoptotic cells increased 10-fold, from 0.6 to 5.9% for the A549 line, and twofold from 0.6 to 1.1% for the H522 line.

      Ritonavir Induces a G0/G1 Cell Cycle Arrest

      To determine whether ritonavir inhibits cell cycle progression of lung adenocarcinoma cells, the A549 and H522 lines were grown in the presence of ritonavir at either its IC50 (40 μM) or half the IC50 (20 μM) or in the presence of vehicle (DMSO) for 24 and 48 hours. The cell cycle distribution was determined by flow cytometry. For both lines, incubation with ritonavir for 48 hours resulted in a dose-dependent G0/G1 cell cycle block, as demonstrated by increase of the G0/G1 population and by a corresponding reduction of the S and G2/M populations (Figure 2A, Table S2 [http://links.lww.com/JTO/A62]). Similar results were obtained after 24 hours incubation with ritonavir (results not shown).
      Figure thumbnail gr2
      FIGURE 2Panel A, Ritonavir causes G0/G1 cell cycle arrest in the A549 and H522 lung adenocarcinoma lines. Ritonavir was able to induce a G0/G1 block as demonstrated by increase of the G0/G1 population and by a corresponding reduction of the S and G2/M populations. Cells grown with ritonavir or vehicle are indicated with (+) and (−), respectively. For quantitation of the flow cytometry analysis, see Table 2S [http://links.lww.com/JTO/A62]. Panel B, Ritonavir down-regulates the expression of numerous cell cycle-associated proteins in the lung adenocarcinoma lines A549 and H522. CDK2, CDK4, CDK6, cyclin D1, cyclin A, phospho-retinoblastoma protein (Rb), and Rb levels were reduced in both lines treated with ritonavir. Levels of p53 were reduced in the H522 line but increased in the A549 line. The levels of p27Kip1 were increased in both lines. For quantitation of the Western blots, see Table S3 [http://links.lww.com/JTO/A62].

      Ritonavir Down-Regulates CDK2, CDK4, CDK6, and Cyclin D1 Levels and Inhibits pRb, While Up-Regulating p27Kip1and Wild-Type p53

      Because ritonavir induces a G0/G1 block in lung adenocarcinoma lines (Figure 2A), it was investigated whether ritonavir affects the levels of proteins known to regulate the G1/S checkpoint. Both the A549 and H522 lines exhibit reduction of G1/S checkpoint regulators CDK2, CDK4, CDK6, and cyclin D1, after 48 hours of treatment at the ritonavir IC50 (Figure 2B; Table S3 [http://links.lww.com/JTO/A62]). Correlating with reduction of CDKs and cyclin D1, phospho-Rb levels were reduced by more than 80% (Figure 2B; Table S3 [http://links.lww.com/JTO/A62]). Cyclin A, involved in S phase progression, was also decreased by ritonavir (Figure 2B; Table S3 [http://links.lww.com/JTO/A62]). Furthermore, p27Kip1, an important inhibitor of cyclin E/CDK2 complexes, was increased in both lines. The A549 line is p53 wild type, whereas the H522 line is p53 mutant (codon 191), and the effects of ritonavir differed, with p53 increasing in the wild-type line and decreasing in the mutant line (Figure 2B and Table S3 [http://links.lww.com/JTO/A62]).

      Effects of Ritonavir on Survivin, c-Src, STAT3, and Cleaved PARP1 Levels in Lung Adenocarcinoma Lines

      The potential roles of survivin, its up-stream regulatory proteins c-Src and STAT3 and their phosphorylated forms, and its down-stream signaling molecule PARP were studied in the A549 and H522 lines. Ritonavir treatment significantly reduced survivin expression in both cell lines (Figure 3A; Table S4a [http://links.lww.com/JTO/A62]). In both lines, ritonavir reduced fractional phosphorylation of c-Src by more than half (Figure 3A; Table S4a [http://links.lww.com/JTO/A62]). Total c-Src was also reduced in the H522 line but not A549 (Figure 3A; Table S4a [http://links.lww.com/JTO/A62]). Ritonavir treatment reduced the fractional phosphorylation of STAT3 by 40% in both cell lines but did not affect STAT3 levels (Figure 3A; Table S4a [http://links.lww.com/JTO/A62]). In both lines, ritonavir significantly increased the levels of cleaved PARP1 (Figure 3A; Table S4a [http://links.lww.com/JTO/A62]).
      Figure thumbnail gr3
      FIGURE 3Panel A, Ritonavir down-regulates the expression of phospho-c-Src, Src, phospho signal transducer and activator of transcription protein (STAT)3, and survivin and increases cleaved PARP in the lung adenocarcinoma lines A549 and H522. Both in the A549 and H522 line, ritonavir reduced phospho-c-Src, phospho-STAT3, and survivin levels, whereas it increased the cleaved PARP levels; it did not affect total STAT3 levels, and it down-regulated total c-Src only in the H522 line. Cells growth with ritonavir or vehicle are indicated with (+) and (−), respectively. For quantitation of the Western blots, see Table S4a [http://links.lww.com/JTO/A62]. Panel B, Ritonavir down-regulates survivin mRNA in the lung adenocarcinoma lines A549 and H522. After 48 hours of treatment, ritonavir drastically reduced the levels of survivin messenger RNA (mRNA) in both lines.

      Effects of Ritonavir on Survivin mRNA Levels in Lung Adenocarcinoma Lines

      To determine whether reduction in survivin protein levels by ritonavir corresponds to a down-regulation of its message, survivin mRNA levels were measured by real-time polymerase chain reaction. Survivin mRNA was reduced by 70 to 80% in ritonavir-treated lung adenocarcinoma lines (Figure 3B). These results indicate that ritonavir-mediated reduction of survivin is mediated largely through reduction of survivin mRNA.

      RNAi Profiling of Survivin or Its Regulators c-Src and STAT3 Reveals that Survivin is the Most Important Ritonavir Target

      Recently, RNAi silencing has been used to identify genes whose expression affects drug responses in cancer.
      • Zhang YW
      • Jones TL
      • Martin SE
      • et al.
      Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response.
      In this study, the regulatory pathway for survivin expression, which is mediated by c-Src and STAT3, was profiled by RNAi with the purpose of testing the relative importance of the members of this pathway as ritonavir targets. To verify the effectiveness of the RNAi approaches, the protein levels of survivin, c-Src, and STAT3 were determined. Knock down of c-Src, STAT3, and survivin reduced their corresponding proteins by 50, 70, and 50%, respectively (Figure 4A, Table S4b [http://links.lww.com/JTO/A62]). Knock down of c-Src significantly reduced survivin and STAT3 levels in both lines, indicating that there is a direct relationship between c-Src levels and its downstream signaling proteins (Figure 4A, Table S4b [http://links.lww.com/JTO/A62]). Knock down of STAT3 reduced survivin in the H522 line only (Figure 4A, Table S4b [http://links.lww.com/JTO/A62]). These results indicate that survivin levels are determined, in part, by c-Src and STAT3 levels, confirming a model of a signaling hierarchy between these regulatory proteins in NSCLC. Inhibitory effects of survivin, c-Src, and STAT3 knock down on the ritonavir-sensitive lung adenocarcinoma lines were verified by MTT assay. siRNA targeting c-Src, STAT3, or survivin significantly inhibits the growth of both the A549 and H522 lines at 48 hours (Figure 4B). After establishing the effectiveness of the RNAi silencing of c-Src, STAT3, and survivin, this approach was used to determine the relative importance of the survivin pathway members as ritonavir targets. Only survivin knock down shifted the MTT dose-response curve significantly to the left for the A549 and H522 lines, indicating the importance of survivin levels for ritonavir sensitivity (Figure 4C). In contrast, despite the hierarchy placing c-Src and STAT3 up-stream of survivin, c-Src, STAT3, and nontarget RNAi failed to affect the ritonavir IC50 for either line. After survivin RNAi treatment, the ritonavir IC50 decreased to 25 μM for both lines (Figure 4C). Testing ritonavir at its IC50 in combination with survivin siRNA confirmed that reduction of survivin sensitizes lung cancer lines to ritonavir (Figure 4D).
      Figure thumbnail gr4
      FIGURE 4Panel A, c-Src, STAT3, and survivin siRNA reduce the levels of their target proteins. The (−) column indicates nontarget siRNA, whereas the (+) column indicates specific siRNA as labeled. The siRNA to c-Src, STAT3, and survivin reduced the level of the corresponding protein in all three lines. For quantitation of the Western blots, see Table S4b [http://links.lww.com/JTO/A62]. Panel B, siRNA targeting c-Src, STAT3, and survivin reduces growth of the lung adenocarcinoma lines A549 and H522. Cells treated with siRNA targeting c-Src, STAT3, and survivin exhibited reduced growth, when compared with cells treated with nontargeting siRNA. p value of <0.05 is indicated with an asterisk. Panel C, siRNA targeting c-Src or STAT3 does not reduce ritonavir half maximal inhibitory concentration (IC50) for the lung adenocarcinoma lines A549 and H522. A549 and H522 cells transfected with siRNA targeting c-Src, STAT3, survivin, or nontargeting siRNA were grown in the presence of increasing concentrations of ritonavir. Silencing of survivin mRNA expression increased the cell sensitivity to ritonavir, reducing its IC50 to 25 μM for the A459 line and 27 μM for the H522 line. Panel D, siRNA targeting survivin increases the sensitivity to ritonavir of the lung adenocarcinoma lines A549 and H522. A549 and H522 cells transfected with siRNA targeting survivin were treated with ritonavir at the appropriate IC50. Survivin siRNA and ritonavir acted together to reduce cell growth in both lines. p value of <0.05 is indicated with an asterisk.

      Overexpression of Survivin Induces Resistance to Ritonavir in Lung Adenocarcinoma Lines

      Because survivin knock down sensitizes lung adenocarcinoma lines to ritonavir, it was determined whether survivin overexpression leads to ritonavir resistance. Survivin expression was increased threefold in both lines by the S-IRES-EGFP-MIEG3 transfection (Figure 5A). Transfection with the survivin expression plasmid resulted in significant increases of the ritonavir IC50 for both lines (by 10–20 μM) (Figure 5B). Therefore, survivin overexpression causes resistance to ritonavir, whereas survivin knock down causes sensitivity, identifying survivin as a critical target for this drug.
      Figure thumbnail gr5
      FIGURE 5Panel A, Overexpression of survivin in the lung adenocarcinoma lines A549 and H522. Cells transiently transfected with human survivin complementary DNA (cDNA) had significantly higher levels of survivin, when compared with cells transfected with the empty vector. Panel B, Overexpression of survivin increases resistance of the lung adenocarcinoma lines A549 and H522 to ritonavir. Overexpression of survivin reduced the cell sensitivity to ritonavir, increasing its half maximal inhibitory concentration (IC50) to 45 μM for the A549 line and 55 μM for the H522 line.

      Ritonavir Exhibits Enhanced Anticancer Activity When Combined with Gemcitabine and/or Cisplatin

      After identifying ritonavir inhibitory activity in lung adenocarcinoma lines and survivin as its major target, it was important to learn effective ways of combining ritonavir with other chemotherapy agents. This goal can be accomplished by identifying chemotherapy drugs that do not interact pharmacokinetically with ritonavir, thereby avoiding drug interactions that could cause unpredictable toxicity. Ritonavir is known to interact with drugs metabolized by CYP3A4, by either decreasing or increasing their metabolism.
      • Barry M
      • Gibbons S
      • Back D
      • et al.
      Protease inhibitors in patients with HIV disease. Clinically important pharmacokinetic considerations.
      For instance, as a CYP3A4 inhibitor, ritonavir is known to boost the levels of chemotherapy drugs, such as taxanes, vinca alkaloids, and etoposide, making toxicity difficult to predict.
      • Bardelmeijer HA
      • Ouwehand M
      • Buckle T
      • et al.
      Low systemic exposure of oral docetaxel in mice resulting from extensive first-pass metabolism is boosted by ritonavir.

      Makinson A, Pujol JL, Le Moing V, et al. Interactions between cytotoxic chemotherapy and antiretroviral treatment in human immunodeficiency virus-infected patients with lung cancer. J Thorac Oncol;5:562–571.

      In contrast, the widely used lung cancer drug gemcitabine is not known to interact with ritonavir, and as such, it represents a promising candidate for combined therapy. When gemcitabine was combined with ritonavir at the respective IC50 for each drug, reduction of survivin was still observed, indicating that gemcitabine does not interfere with ritonavir-mediated reduction of survivin (Figure 6A, Table S5 [http://links.lww.com/JTO/A62]). Furthermore, in the A549, gemcitabine alone significantly reduced survivin levels, and such reduction was enhanced by the combination ritonavir/gemcitabine. When tested for synergy by Chou-Talalay
      • Chou TC
      • Talalay P
      Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors.
      isobologram analysis, the combination ritonavir/gemcitabine exhibited strong synergy (Figure 6B) with combination index (CI) values for the A549 and H522 lines of 0.57 ± 0.33 and 0.16 ± 0.09, respectively. Ritonavir exhibits an IC50 of 15 to 20 μM in combination with gemcitabine concentrations of 15 to 60 nM, which are well below Cmax concentrations attained clinically with gemcitabine monotherapy.
      • Kroep JR
      • Giaccone G
      • Voorn DA
      • et al.
      Gemcitabine and paclitaxel: pharmacokinetic and pharmacodynamic interactions in patients with non-small-cell lung cancer.
      • Fogli S
      • Danesi R
      • De Braud F
      • et al.
      Drug distribution and pharmacokinetic/pharmacodynamic relationship of paclitaxel and gemcitabine in patients with non-small-cell lung cancer.
      Also cisplatin, which is commonly used in lung cancer chemotherapy, is not known to interact with ritonavir. The ritonavir/cisplatin combination was less active in inhibiting the growth of lung adenocarcinoma lines than the ritonavir/gemcitabine combination, exhibiting CI values of 1 ± 0.55 and 1.1 ± 1.16 for the A549 and H522 lines, respectively (Figure 6B). These CI values indicate that cisplatin and ritonavir are additive rather than synergistic for the A549 and H522 lines.
      • Chou TC
      • Talalay P
      Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors.
      Similarly to what we observed for the combination ritonavir/gemcitabine, ritonavir IC50 was reduced to ∼20 μM in combination with cisplatin at 4.5 μM.
      Figure thumbnail gr6
      FIGURE 6Panel A, Gemcitabine and ritonavir down-regulate the survivin levels of the lung adenocarcinoma lines A549 and H522. Ritonavir was able to reduce the levels of survivin either when used alone or together with gemcitabine. For quantitation of the Western blots, see Table S5 [http://links.lww.com/JTO/A62]. Panel B, Ritonavir and gemcitabine synergize in inhibiting the growth of the lung adenocarcinoma lines A549 and H522. The combination gemcitabine and ritonavir had a synergic effect and was significantly more effective in reducing growth compared with each drug alone. On the other hand, the combined effect of ritonavir and cisplatin was additive for the A549 and H522 lines. Panel C, The combination ritonavir/gemcitabine/cisplatin is synergistic in the A549 line and additive in the H522 line. The concentrations of ritonavir (R), gemcitabine (G), and cisplatin (C) are indicated on the x axis. The combination ritonavir/gemcitabine/cisplatin acted synergistically in the A549 line and additively in the H522 line. The combination gemcitabine/cisplatin was superior to ritonavir/gemcitabine/cisplatin in the H522 line but not in the A549 line.
      As the combination gemcitabine/cisplatin is a standard regimen for the treatment of lung adenocarcinoma,
      • Schiller JH
      • Harrington D
      • Belani CP
      • et al.
      Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer.
      we also tested the effect of ritonavir in combination with gemcitabine/cisplatin in the A459 and H522 lines. The ritonavir/gemcitabine/cisplatin combination acted synergistically in the A549 line (CI = 0.6 ± 0.2) and was slightly superior to the combination gemcitabine/cisplatin (Figure 6C). On the other hand, in the H522 line, the combination ritonavir/gemcitabine/cisplatin acted additively (CI = 0.9 ± 0.2), but it was inferior to the gemcitabine/cisplatin combination (Figure 6C). Importantly, in both cell lines, ritonavir IC50 was less than 10 μM in combination with concentrations of gemcitabine and cisplatin well below their IC50.

      DISCUSSION

      Survival of recurrent/metastatic NSCLC with palliative chemotherapy fails to exceed 1 year, and there is an unmet need for new drugs and drug combinations that work through novel mechanisms.
      • Janku F
      • Stewart DJ
      • Kurzrock R
      Targeted therapy in non-small-cell lung cancer–is it becoming a reality?.
      In this study, we propose ritonavir as a candidate drug for metastatic lung adenocarcinoma clinical trials, based on its inhibition of adenocarcinoma lines at concentrations in the 35 to 45 μM range. Such concentrations are clinically attainable, albeit with significant gastrointestinal toxicity,
      • Gatti G
      • Di Biagio A
      • Casazza R
      • et al.
      The relationship between ritonavir plasma levels and side-effects: implications for therapeutic drug monitoring.
      with ritonavir monotherapy at 600 mg twice daily.
      • Hsu A
      • Granneman GR
      • Witt G
      • et al.
      Multiple-dose pharmacokinetics of ritonavir in human immunodeficiency virus-infected subjects.
      Study of signaling pathways affected by ritonavir by siRNA profiling revealed that survivin is an important target, whereas c-Src and STAT3 seem to be of lesser importance. Furthermore, forced overexpression of survivin confers relative resistance to ritonavir, confirming importance of survivin as a ritonavir target. Ritonavir reduces survivin, in part, by reducing its mRNA levels. Because survivin is regulated in cancer primarily through its mRNA expression,
      • Bao R
      • Connolly DC
      • Murphy M
      • et al.
      Activation of cancer-specific gene expression by the survivin promoter.
      these results suggest that ritonavir is likely attacking a basic mechanism of survivin transcriptional regulation.
      Ritonavir inhibits lung adenocarcinoma growth and anchorage-dependent clonogenicity, in part, by inducing G0/G1 cell cycle arrest and, in part, by inducing apoptosis. Survivin is implicated in regulation of the G1/S
      • Ai Z
      • Yin L
      • Zhou X
      • et al.
      Inhibition of survivin reduces cell proliferation and induces apoptosis in human endometrial cancer.
      • Song J
      • So T
      • Cheng M
      • et al.
      Sustained survivin expression from OX40 costimulatory signals drives T cell clonal expansion.
      • Suzuki A
      • Hayashida M
      • Ito T
      • et al.
      Survivin initiates cell cycle entry by the competitive interaction with Cdk4/p16(INK4a) and Cdk2/cyclin E complex activation.
      and G2/M checkpoint, and therefore, its reduction by ritonavir is expected to cause inhibition of the cell cycle. Ritonavir may also inhibit the G1/S checkpoint through down-regulation of CDKs, cyclin D1, and associated Rb phosphorylation, as well as by induction of p27Kip and wild-type p53. Although reduction of survivin by ritonavir is expected to promote apoptosis in lung cancer presumably related to loss of the antiapoptotic effect of survivin,
      • Ulukus EC
      • Kargi HA
      • Sis B
      • et al.
      Survivin expression in non-small-cell lung carcinomas: correlation with apoptosis and other apoptosis-related proteins, clinicopathologic prognostic factors and prognosis.
      the remaining mechanisms by which ritonavir induces apoptosis remain to be determined. These mechanisms could include induction of DNA damage, in part, through increased cleavage of PARP1.
      Importantly, drug combination studies revealed that ritonavir is active at lower concentrations when combined with gemcitabine, cisplatin, and the gemcitabine/cisplatin combination. In combination with gemcitabine or cisplatin, ritonavir exhibits IC50 values in the range of 15 to 20 μM. With the gemcitabine/cisplatin combination, the ritonavir IC50 is in the 8 μM range, which should be attainable with 100 mg twice daily dosing.
      • Hsu A
      • Granneman GR
      • Witt G
      • et al.
      Multiple-dose pharmacokinetics of ritonavir in human immunodeficiency virus-infected subjects.
      Although the gemcitabine/cisplatin combination in advanced NSCLC resulted in the longest median time to progression compared with three other chemotherapy doublets, this was only 4.2 months.
      • Schiller JH
      • Harrington D
      • Belani CP
      • et al.
      Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer.
      We hypothesize, based on in vitro synergy, that addition of low-dose ritonavir to the gemcitabine/cisplatin combination may improve time to progression, with acceptable toxicity. Furthermore, because ritonavir is not myelosuppressive and potentially could be continued through the period of gemcitabine/cisplatin treatment, ritonavir could potentially inhibit regrowth of lung adenocarcinoma between cycles of chemotherapy. Therefore, a phase I study of daily ritonavir in combination with the established gemcitabine and cisplatin schedule is an important next step. Although K-ras mutation status did not affect sensitivity to ritonavir, for the H838 K-ras wild-type line, there was lack of synergy with gemcitabine and antagonism with cisplatin. These results suggest that K-ras mutant lung adenocarcinoma is the best candidate histology for future clinical trials.
      Although the mechanisms behind cooperation between ritonavir and gemcitabine and/or cisplatin are not known, it is likely these mechanisms involve survivin effects on DNA repair pathways. Gemcitabine is a DNA-strand terminator that stalls replication forks, causes S-phase arrest,
      • Ewald B
      • Sampath D
      • Plunkett W
      ATM and the Mre11-Rad50-Nbs1 complex respond to nucleoside analogue-induced stalled replication forks and contribute to drug resistance.
      and double-strand breaks (DSBs) while inhibiting homologous recombination repair, which is required for repairing DSB.
      • Wachters FM
      • van Putten JW
      • Maring JG
      • et al.
      Selective targeting of homologous DNA recombination repair by gemcitabine.
      • Crul M
      • van Waardenburg RC
      • Bocxe S
      • et al.
      DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin.
      Survivin has been reported to enhance DSB repair, and we hypothesize that reduction of survivin by ritonavir may increase sensitivity to gemcitabine through this mechanism.
      • Jiang G
      • Ren B
      • Xu L
      • et al.
      Survivin may enhance DNA double-strand break repair capability by up-regulating Ku70 in human KB cells.
      Survivin reduction may also explain sensitivity of lung adenocarcinoma to ritonavir in combination with cisplatin due to increased PARP1 cleavage.
      PARP1 may be involved in repair of cisplatin-induced DNA damage. PARP1 is known to recruit XRCC1 to sites of DNA damage.
      • Dantzer F
      • Ame JC
      • Schreiber V
      • et al.
      Poly(ADP-ribose) polymerase-1 activation during DNA damage and repair.
      XRCC1 is a scaffolding factor required for base excision repair
      • Dianova II
      • Sleeth KM
      • Allinson SL
      • et al.
      XRCC1-DNA polymerase beta interaction is required for efficient base excision repair.
      and recently, nucleotide excision repair (NER).
      • Moser J
      • Kool H
      • Giakzidis I
      • et al.
      Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase III alpha in a cell-cycle-specific manner.
      Of interest, interference with NER interferes with repair of cisplatin-induced DNA damage.
      • Crul M
      • van Waardenburg RC
      • Bocxe S
      • et al.
      DNA repair mechanisms involved in gemcitabine cytotoxicity and in the interaction between gemcitabine and cisplatin.
      Although PARP1 has not been implicated as a key regulator of NER, it has been recently been located at sites of cisplatin-induced DNA damage, by two photoaffinity labeling studies.
      • Zhu G
      • Lippard SJ
      Photoaffinity labeling reveals nuclear proteins that uniquely recognize cisplatin-DNA interstrand cross-links.
      • Guggenheim ER
      • Xu D
      • Zhang CX
      • et al.
      Photoaffinity isolation and identification of proteins in cancer cell extracts that bind to platinum-modified DNA.
      This finding potentially implicates PARP1 in repair of cisplatin-mediated DNA interstrand crosslinks by NER. In addition, PARP1 reduction has also recently been demonstrated to play a critical role in chemosensitivity to the gemcitabine/cisplatin combination in triple-negative breast cancer.
      • Hastak K
      • Alli E
      • Ford JM
      Synergistic chemosensitivity of triple-negative breast cancer cell lines to poly(ADP-Ribose) polymerase inhibition, gemcitabine, and cisplatin.
      Future studies will determine the mechanisms by which ritonavir may enhance DNA damage by cisplatin and gemcitabine.
      On the basis of the importance of survivin as a ritonavir target in lung adenocarcinoma, we propose that survivin may be a useful biomarker for ritonavir sensitivity. We hypothesize that among tumors expressing survivin, those exhibiting lower survivin levels will be more likely to respond to ritonavir. Our results from forced survivin overexpression are artificial and may not reflect survivin levels in tumors occurring in patients, and therefore, we would not recommend excluding patients with high-survivin levels from clinical trials of ritonavir. Only the analysis of data from such trials would reveal whether there is a relationship between survivin levels and ritonavir sensitivity.

      ACKNOWLEDGMENTS

      Supported by the National Institute of Health (grants R01 CA113570 [to D.A.P.] and HL-079654 to [L.M.P.]). D.A.P. acknowledges a Walther Cancer Research Prize, the Flight Attendant's Medical Research Institute Clinical Innovator Award 042257, and support from the Walther Oncology Center at Indiana University, the Thoracic Oncology Program at Indiana University, a Clarian Values Foundation grant, and equipment grant from the Indiana Elks. The authors also acknowledge the Masonic Cancer Center Experimental Therapeutics Initiative.
      The authors thank Drs. Lawrence Einhorn, Hal Broxmeyer, and Patrick Loehrer for support and encouragement of this work. The authors thank Drs. Anja Bielinsky, Robert Kratzke, Manish Patel, David Donner, Janice Blum, Ann Roman, Maureen Harrington, Christine Clouser, and Reuben Harris for helpful discussions. The authors thank Dr. Manish Patel for the H838 line. The authors thank Susan Rice for help with flow cytometry experiments.

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