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Pharmaceuticals 2014, 7(1), 46-57; doi:10.3390/ph7010046
Abstract: Recent observations suggest a lower incidence of malignancies in patients infected with HIV during treatment with Highly Active Anti-Retroviral Therapy (HAART) utilizing protease inhibitors. We investigated the effects of ritonavir, a FDA approved HIV protease inhibitor, on proliferation of pancreatic ductal adeno-carcinoma (PDAC) cell lines. Human PDAC cell lines BxPC-3, MIA PaCa-2, and PANC-1 were propagated under standard conditions and treated with serial dilutions of ritonavir. Ritonavir inhibited cell growth in a dose-dependent manner as well as activated the intrinsic apoptotic pathway in human pancreatic ductal adenocarcinoma (PDAC) cell lines. We observed down-modulation of cell-cycle promoting and up-regulation of cell-cycle inhibitory genes; enhanced interaction of retinoblastoma protein (RB) with E2F-1 transcription factor; inhibition of phosphorylation of RB, resulting in sequestration of E2F-1 and subsequent down-regulation of S phase genes; decreased interaction of E2F-1 with its consensus binding sites; inhibition of cell motility and invasiveness; and inhibition of the AKT pathway. Our results demonstrate a potential use of ritonavir as part of combination chemotherapy for PDAC. Since ritonavir is FDA approved for HIV, drug repositioning for PDAC would limit the costs and reduce risks.
Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer death, and more than 80% of patients present with distant metastases at the time of diagnosis, thereby precluding surgical resection. Systemic chemotherapy still relies on only a few drugs and has not significantly increased overall patient survival , underscoring the need for development of novel therapies .
The advent of highly-active anti-retroviral therapy (HAART) based on protease inhibitors has greatly improved the treatment of HIV so that it is now a manageable disease . Recent observations point to a decreasing incidence of some cancers in patients on protease inhibitors , with our previous work demonstrating inhibition of cell growth and induction of apoptosis in ovarian cancer . Along these lines, phase I clinical trials with nelfinavir showed partial remission with acceptable toxicity when used along with radiation and chemotherapy in patients with locally-advanced pancreatic cancer .
Non-phosphorylated retinoblastoma protein (RB) is a well-characterized tumor suppressor which inhibits E2F-1 transcription factor necessary for cell-cycle progression from G0/G1 to S phase . Since it has been shown that the protease inhibitor ritonavir also blocks cell-cycle progression at these points , we hypothesized that it may act via RB by inhibiting its phosphorylation and thus preventing its deactivation.
The AKT pathway is another important regulator of cell proliferation and survival which is activated in pancreatic cancer . Since inhibition of this pathway has been observed with various protease inhibitors [10,11], we also hypothesized that RNAi-mediated inhibition, an innate gene-silencing mechanism, along with ritonavir treatment, may be an effective combination therapy in promoting tumor regression of pancreatic cancer.
Therefore, the objective of the current study is to assess the anti-neoplastic impact of ritonavir on pancreatic cancer with regard to its specific effects on the AKT pathway and RB. We provide evidence here for the first time that ritonavir-induced signaling pathways at the level of the cell membrane result in nuclear events that block cell-cycle progression via RB protection.
Reagents and antibodies: Ritonavir was obtained from Sequoia Research Products Limited (Pang Bourne, UK) and dissolved in DMSO. Cell viability was assayed in 96-well plates utilizing the Cell Counting Kit-8 (CCK-8; Dojindo, Gaithersburg, MD, USA). RB and E2F-1 antibodies were purchased from Millipore (Danvers, MA, USA). Cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, Bcl-2, poly (ADP-ribose) polymerase (PARP) and β actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-AKT and caspases, as well as SignalSilence AKT siRNA inhibition kits, were purchased from Cell Signaling Technology (Beverly, MA, USA).
Cell lines and culture: Human pancreatic tumor cell lines BxPC-3, MIA PaCa-2, and PANC-1 American Type Culture Collection (Manassas, VA, USA) were grown in sub-confluent monolayer cultures in DMEM medium containing 10% FBS, supplemented with 2 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
Cytotoxicity assays: BxPC-3, MIA PaCa-2, and PANC-1 cells were treated with serial dilutions of ritonavir ranging from 5–30 µM dissolved in DMSO. In addition, PANC-1 cells were treated with gemcitabine 0.5 µM with or without ritonavir 20 µM. Normal human fibroblasts were purchased as a cell line from ATCC (CRL-2522) and propagated in DMEM medium. The same serial dilutions of ritonavir dissolved in DMSO were added as described for the pancreatic cell lines. Standard prototype growth curves and number of viable cells were determined for each cell line (treated and control groups) in triplicate experiments using CCK-8 according to manufacturer’s instructions and absorbance was read at 450 nm in a plate reader (FluoStar Optima, BMG Labtech, Cary, NC, USA). Growth curves were plotted over 72 h as a percentage of the value of DMSO-treated controls minus the value of untreated cells on day 0. IC50 values were calculated by entering the raw data into the “Sigmaplot” software program.
Analysis of apoptosis: For fluorescent microscopic image analysis of the apoptotic cell fraction, ritonavir-treated and control cells (1 × 106/mL) were mixed with annexin V-biotin and medium-binding reagent and incubated in the dark for 15 min at room temperature. Cells were then centrifuged and medium was replaced with 1× Binding Buffer containing FITC-streptavidin. Propidium iodide was added to discriminate early apoptotic from late apoptotic or necrotic cells. A portion of cell suspension (50 µL) was placed on a glass slide with cover slip and viewed immediately using a fluorescence microscope (Zeiss, AXio CamMRm Observer. A1, One Zeiss Drive, Thornwood, NY, USA) equipped with FITC and propidium iodide.
Western blot and co-immunoprecipitation assays: Cells were trypsinized, rinsed twice in PBS, and the pellet suspended in CelLytic MT cell lysis reagent (Sigma, St. Louis, MO, USA) containing a protease inhibitor cocktail tablet (Complete, Roche Applied Science, Mannheim, Germany). After a 30 min incubation, the pellet was collected by centrifugation at 4 °C for 15 min at 12,000 g. Blots and co-immunoprecipitations were conducted as previously described . Ritonavir-treated samples were immunoprecipitated with anti-E2F-1 polyclonal antibodies, and the complex was probed with RB monoclonal antibodies.
Gene expression profiling: Briefly, PANC-1 cells, either untreated or treated with ritonavir 15 µM for 48 h, were harvested and total RNA was isolated utilizing an RNeasy kit (Qiagen Inc., Valencia, CA, USA) as described by the manufacturer. Total RNA was sent to MOgene Company (MOgene, LC, St. Louis, MO, USA) for analysis.
Electromobility shift assay (EMSA): Nuclear extracts were prepared using a nuclear and cytoplasmic extraction kit from G-Biosciences (St. Louis, MO, USA). EMSA assays were performed using the non-radioactive EMSA kit according to the manufacturer’s protocol (Light-Shift Chemiluminescent EMSA kit, Thermo Scientific, Rockford, IL, USA). The samples were run on 6% Tris-borate-EDTA gels and subsequently blotted onto nylon membranes. Biotinylated E2F-1 gel shift oligonucleotides (5′-ATT TAA GTT TCG CGC CCT TTC TCA A-3′, Santa Cruz Biotechnology, Dallas, TX, USA) were used for probing, followed by streptavidin detection.
E2F-1 knockdown PANC-1 cell line generation with Lentiviral E2F-1 shRNA: Lentiviral shRNA for E2F-1 was obtained from Sigma-Aldrich and used to infect the PANC-1 cell line. Twenty-four h post-infection, 2.5 mg/mL of puromycin was added to select for infected cells. Infection efficiency was approximately 95%. Two weeks post-selection, the resulting clones were expanded and cells were harvested.
In vitro cell invasion/migration and wound-healing assays: Cell migration was determined using a modified Boyden chamber and wound healing assays were conducted using the cell-scratch method, both as previously described .
Transfection of siRNA: SignalSilence AKT siRNA inhibition kit (Cell Signaling Technology) that specifically inhibits the expression of both AKT1 and AKT2 was used for this experiment. Briefly, PANC-1 cells were transfected with 100 nM siRNA of AKT. Cells were harvested after 48 h and analyzed for expression of AKT, and Bcl-2.
The present study demonstrates cell cycle arrest, induction of apoptosis, and inhibition of cell migration in pancreatic cancer cell lines by ritonavir, a protease inhibitor that has been in use for over a decade in the treatment of HIV patients. Although several mechanisms are well characterized for signal transduction from AKT on the plasma membrane  to transcriptional activation of cell cycle control genes in the nucleus , the specific pathways modulated by ritonavir are not yet understood. In a recent study, ritonavir induced caspase-dependent apoptosis and suppressed NF-κB activity by inhibiting IκB phosphorylation of primary effusion lymphoma cells . We provide evidence herein that in the presence of ritonavir, phosphorylation of both AKT and RB is inhibited. As a result, the ability of E2F-1, sequestered by its binding to RB, to promote transcription of necessary S phase genes is impaired (Figure 7). Since ritonavir has been shown to augment the effect of other chemotherapeutic drugs such as docetaxel , we conducted combination studies of ritonavir and gemcitabine, the latter considered first-line therapy for PDAC. We demonstrated an additive effect on cell death in PANC-1 cells, suggesting the benefits of ritonavir as part of combination therapy. Of note, there is a beneficial effect of combining 7-allylamino-17-demethoxygeldanamycin and ritonavir as a novel therapeutic target by inhibiting the expression of heat shock factor-1 in the treatment of renal cancer .
We next assessed the effect of ritonavir on the activity of enzymes involved in the apoptotic pathway. We observed activation of caspase 9, an upstream event in the intrinsic apoptotic pathway, followed by activation of caspase 7, which in turn leads to breakdown of PARP. Although untreated PANC-1 cells showed basal levels of PARP cleavage, we demonstrated a significant, dose-dependent increase in the cleavage of PARP with ritonavir exposure, along with activation of caspases and inhibition of anti-apoptotic Bcl-2.
Gene expression profiling of PANC-1 cells indicated that ritonavir could beneficially affect critical steps in cell cycle progression. Indeed, we demonstrated down-regulation of tumor-promoting E2F transcription factors and CDKs, along with up-regulation of tumor-suppressing RB proteins. More specifically, in Western blot and immunoprecipitation analyses, we found a dose-dependent (1) increase in non-phosphorylated, active RB; (2) decrease in CDK4; and (3) increase in p21waf1/cip1 with ritonavir exposure. These all contribute to the formation of RB-E2F-1 complexes, thereby sequestrating E2F-1, preventing subsequent activation of S phase genes, and halting cell cycle progression.
Tumor metastasis is a multistep process involving basal membrane matrix degradation, detachment of proliferating tumor cells, and transport of these cells in the blood or lymph, resulting in invasion into other tissues . Our findings of decreased cell migration by the wound healing assay and inhibition of cell invasion by the Boyden chamber assay are consistent with the earlier observation that ritonavir inhibited the expression of endothelial cell adhesion molecules .
Our results showing ritonavir-mediated inhibition of AKT activity and decreased cell proliferation have relevance in light of earlier observations correlating gemcitabine-induced drug resistance with activation of the AKT pathway in pancreatic cancer cells . We found that reduction in the expression of p-AKT by Western blot analysis correlated with decreased cell proliferation and migration. Along these lines, Yamamoto et al.  showed that low p-AKT levels were a good prognostic indicator for overall survival in 65 patients who underwent surgery for pancreatic cancer. Thus, ritonavir-induced phosphorylation (inactivation) of AKT may represent a potential therapeutic approach to management of PDAC.
Ritonavir plasma levels in HIV patients normally range from 15–45 µM, providing therapeutic efficacy with negligible side effects . Of note, we observed growth inhibitory effects of ritonavir in the range of 5–20 µM, suggesting that these beneficial effects could be achieved in patients with PDAC at clinically relevant, nontoxic doses. The relatively low toxicity of these drugs, taken together with the large body of data available regarding their pharmacokinetics, tissue distribution, and safety, would allow for the rapid clinical evaluation of ritonavir alone or in combination with other chemotherapeutic agents. Indeed, recent phase I clinical trials with nelfinavir in pancreatic cancer have shown promising results .
In summary, we provide evidence that ritonavir-induced activation of RB is linked to inhibition of the AKT pathway by a common signaling mechanism, resulting in down-regulation of S phase genes. This may prove useful in the chemotherapeutic management of PDAC in conjunction with other agents.
Conflicts of Interest
The authors declare no conflict of interest.
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