Ritonavir blocks AKT signaling, activates apoptosis and inhibits migration and invasion in ovarian cancer cells
- Sanjeev Kumar†2, 3,
- Christopher S Bryant†3,
- Sreedhar Chamala2, 1,
- Aamer Qazi1, 3,
- Shelly Seward2, 3,
- Jagannath Pal1, 3,
- Christopher P Steffes3,
- Donald W Weaver1, 3,
- Robert Morris2, 3,
- John M Malone2, 3,
- Masood A Shammas1, 3,
- Madhu Prasad1, 3 and
- Ramesh B Batchu1, 3Email author
© Kumar et al; licensee BioMed Central Ltd. 2009
Received: 18 December 2008
Accepted: 22 April 2009
Published: 22 April 2009
Ovarian cancer is the leading cause of mortality from gynecological malignancies, often undetectable in early stages. The difficulty of detecting the disease in its early stages and the propensity of ovarian cancer cells to develop resistance to known chemotherapeutic treatments dramatically decreases the 5-year survival rate. Chemotherapy with paclitaxel after surgery increases median survival only by 2 to 3 years in stage IV disease highlights the need for more effective drugs. The human immunodeficiency virus (HIV) infection is characterized by increased risk of several solid tumors due to its inherent nature of weakening of immune system. Recent observations point to a lower incidence of some cancers in patients treated with protease inhibitor (PI) cocktail treatment known as HAART (Highly Active Anti-Retroviral Therapy).
Here we show that ritonavir, a HIV protease inhibitor effectively induced cell cycle arrest and apoptosis in ovarian cell lines MDH-2774 and SKOV-3 in a dose dependent manner. Over a 3 day period with 20 μM ritonavir resulted in the cell death of over 60% for MDAH-2774 compared with 55% in case of SKOV-3 cell line. Ritonavir caused G1 cell cycle arrest of the ovarian cancer cells, mediated by down modulating levels of RB phosphorylation and depleting the G1 cyclins, cyclin-dependent kinase and increasing their inhibitors as determined by gene profile analysis. Interestingly, the treatment of ritonavir decreased the amount of phosphorylated AKT in a dose-dependent manner. Furthermore, inhibition of AKT by specific siRNA synergistically increased the efficacy of the ritonavir-induced apoptosis. These results indicate that the addition of the AKT inhibitor may increase the therapeutic efficacy of ritonavir.
Our results demonstrate a potential use of ritonavir for ovarian cancer with additive effects in conjunction with conventional chemotherapeutic regimens. Since ritonavir is clinically approved for human use for HIV, drug repositioning for ovarian cancer could accelerate the process of traditional drug development. This would reduce risks, limit the costs and decrease the time needed to bring the drug from bench to bedside.
Ovarian cancer is the second most common gynecologic malignancy, but the most common cause of death among women who develop gynecologic cancers . It is the fifth leading cause of cancer death in females in the United States. It is estimated that 22,430 new cases along with 15,280 deaths were attributed to ovarian cancer in 2007 in the United States . Although current management strategies have resulted in a several fold increase in the median survival for ovarian cancer over past few decades, mortality from the disease still remains high . Up to one third of the patients who receive the first line platinum based chemotherapy for ovarian cancer fail to achieve clinical remission and approximately 50% patients who achieve clinical remission in first course of chemotherapy, eventually have relapse of their disease. Both of the above mentioned categories of patients have exceedingly poor 5 year survival rates indicating the need to develop novel chemotherapeutic drugs which could find their use either as sole therapy or in combination with already existing drugs.
The HIV (human immunodeficiency virus) infection is characterized by inherently increased risk of multiple blood and solid organ malignancies. Highly Active Anti-Retroviral Therapy (HAART) is the term used for intensive combination therapy used to treat patients with HIV infection. The combination typically consists of reverse transcriptase inhibitors (e.g. Zidovudine) and protease inhibitors (e.g. ritonavir, nelfinavir). Use of HAART has resulted in substantial reductions in progression of HIV to AIDS, reduction in opportunistic infections, hospitalizations, and deaths . Interestingly, recent observations point to a decreasing incidence of neoplastic lesions in patients using HAART. [4–6] In the Swiss HIV Cohort Study Clifford et. al.,  reported that in HAART users, the standardized incidence ratio for Kaposi Sarcoma decreased to 25.3 (95% CI = 10.8 to 50.1) as compared to 239 (95% CI = 211 to 270) in non HAART users. Many other investigators have subsequently reported similar associations of potential anti-neoplastic impact of HAART. [8–10] Even before the above mentioned studies were published, the anti-neoplastic properties of ritonavir (which is a protease inhibitor and forms an integral part of HAART), had already been demonstrated in some cancers. Specifically, Ritonavir induced apoptosis in tumor cell lines of lymphoblastoid origin, including lymphoma cells and myeloid leukemia cells, fibrosarcoma and mastocytoma cells as well as immortalized Kaposi's-sarcoma cell lines [11, 12]. No effect on proliferation or survival was observed with non-tumor cells, including non-transformed immortalized fibroblasts or primary macrophages [13, 14].
PI3K/AKT pathway is an important regulator of cellular proliferation and survival, and plays a central role in the progression and metastasis of various human cancers [15, 16]. This pathway is activated in wide range of tumors but not in normal tissues. We hypothesize that the inhibition of this pathway with RNAi together will ritonavir treatment may offer better tumor regression. RNAi is an innate gene-silencing mechanism evolved to protect against viruses initiated by 19 bpl double-stranded RNA molecules (short interfering RNA) homologous to the sequence of the target gene that mediate post-transcriptional gene-silencing [17, 18]. Introduction of chemically synthesized siRNAs can mimic gene silencing target genes. Loss of function studies can be done using siRNA technology for any given gene to assess the function of a gene.
The objective of the current study is to assess the anti-neoplastic impact of ritonavir, and to delineate the underlying mechanisms. Development of clinically approved HIV drug, ritonavir as an effective adjuvant therapy in ovarian cancer by drug repositioning could accelerate the process of traditional drug development in oncology.
Ritonavir acts as antiproliferating agent for ovarian cancer cell lines
Ritonavir induces apoptosis MDAH-2774 cells
Ritonavir enhances pro-apototic signals while inhibiting anti-apotitic bcl-2 expression
Ritonavir causes cell cycle arrest and blocks S phase entry of MDAH-2774 cells in cultures
Ritonavir mediated perturbations in cell cycle regulatory proteins
Ritonavir inhibits AKT pathway leading to apoptosis in MDAH-2774 cells
Ritonavir inhibits cell motility and invasiveness
Finding new indications for the already existing compounds, called drug repositioning that takes advantage of the existing data on pharmacokinetics, toxicity and dosage escalation studies in humans. Drug repositioning can potentially have tremendous cost savings and can expedite movement of a drug from bench to bedside in a relatively short amount of time . For example lenalidomide, an analogue of thalidomide was originally marketed for morning sickness that is now 'repositioned' and approved for therapy of multiple myeloma . This is a prime example of the immense potential of drug repositioning. Likewise oral hypoglycemic rosiglitazone , immunosuppressant drug rapamycin , and the birth control hormone medroxy-progesterone acetate  are also being tested for 'repositioning' to be used as anti-cancer agents. Ritonavir is an FDA approved drug for HIV treatment, being used well over a decade with tolerable side effects .
Ovarian cancer is the deadly form of gynecologic malignancy with exceedingly poor 5 year survival rates  and is the subject of intense research for development of newer antineoplastic compounds which could be used either as a sole or adjuvant therapy. Further, newer compounds may hold even greater promise in drug resistant and relapsing ovarian cancer where the efficacy of the existing chemotherapeutic agents is marginal, at best.
Here for the first time, we show that ritonavir acts as an effective anti-proliferating agent for the ovarian cancers cells in vitro by inducing growth arrest and apoptosis providing insights into molecular mechanisms. Further, it also exhibits the potential to inhibit invasion and migration of these cell lines. Although paclitaxel and carboplatin have good response rates, there are limited treatment options in case of disease relapse where majority of patients become refractory to conventional chemotherapy due to the generation of drug resistant phenotype . In addition we document an additive effect of cell killing when ritonavir combined with paclitaxel.
Retinoblastoma protein (RB) is an important tumor suppressor protein that control progression through the late G1 phase of the cell cycle and, thereby, the commitment to enter the S phase[32, 33] Also, E2F-1 transcription factor that is necessary t o drive the cell into S phase.[32, 34, 35] Cyclins and cyclin-dependent kinases (CDKs) regulate the activity of RB by phosphorylation that controls the progression through G1 [34–36]. Since we observed elevated levels of under-phosphorylated RB, we speculated the lower levels of CDK-2, 4 and 6, one of the important proteins responsible driving cell cycle progressions through G0/G1 phase of cell cycle by phosphorylation of RB. As expected, gene profile analysis of ritonavir treated cells showed down modulation of CDKs and cyclins that are gate keepers of G0/G1 phase of cell cycle. The p21WAF-1/Cip1 inhibits CDK-4, thus preventing both phosphorylation of RB and the release of transcriptionally active E2F-1. Our observations of lower levels of CDK inhibitors and inhibition of RB phosphorylation along with increased levels of expression of E2F-1 transcription factor in response to ritonavir corroborate the results of cell cycle analysis where cells entering S-phase were reduced by over 25%.
Numerous studies show that the PI3K/AKT pathway is constitutively over-expressed in ovarian cancers, apart from several other common human cancers . Activation of AKT is a key event in producing chemo-resistant phenotype through a variety of pathways , whereas inhibition of AKT sensitizes chemo-resistant cells to cisplatin induced apoptosis. Our data corroborates with the observations that increased levels of AKT contributes to chemo-resistance by attenuating p53-mediated PUMA up-regulation and phosphorylation of p53, which are essential for sensitivity to cisplatin-induced apoptosis . Our data indicated that ritonavir inhibits phosphorylation of AKT followed by effective apoptosis. Taken together, these findings suggest that ritonavir may have useful role as an anti-AKT agent in treatment of ovarian cancer in general, but more specifically in relapsed patients due to drug resistance phenotype generation which is attributed to AKT. Further, the Bcl-2 inhibition appeared to be mediated through an AKT dependent pathway, as treatment of anti-AKT siRNA had similar results as ritonavir in Bcl-2 down regulation.
One of the important reasons for high mortality in ovarian cancer patients is the late stage at which the disease is diagnosed, namely FIGO III , where tumor cells would have already traversed the peritoneal cavity by migration and invasion. Effect of ritonavir in inhibiting the invasion and migration of the ovarian cancer cell lines adds another dimension in its anticancer properties and may be especially useful in ovarian cancer as trans-peritoneal spread is a key event in this cancer. We are currently studying the precise molecular pathways involved in migration inhibition seen with ritonavir in in-vivo models.
Ritonavir has been in use for over a decade in the treatment of HIV patients with acceptable toxicity profiles, the primary impetus for this work has been to evaluate the re-positioning of the drug to ovarian cancer chemotherapy. An important aspect of repositioning of ritonavir from HIV therapy to cancer therapy will be the achievable dose in cancer patients. Ritonavir blood plasma levels in HIV patients normally observed at 15 μM  and much higher concentrations of over 45 μM were also observed in individual patients [41, 42]. We observed the growth inhibitory effects of ritonavir in ovarian cancer patients in the range of 5 to 20 μM which is lower than the plasma concentrations observed in HIV patients.
Studies to further elucidate mechanism, specifically cell signaling target modulation, are ongoing in our lab. The determination of synergistic or additive effects in conjunction with conventional chemotherapeutic regimens represents a putative application for ritonavir at its so far known non-toxic concentrations. This would accelerate the process of drug development for a disease that has highest mortality rate among gynecological cancers.
Ovarian cancer poses many treatment difficulties as it is often undetectable in its early stages, and therefore diagnosis usually occurs when surgical treatment is no longer an effective option emphasizing the need for novel, non-toxic and effective treatments. Here we present the evidence that ritonavir; an FDA approved drug for human use for HIV effectively induces cell cycle arrest and apoptosis by inhibiting AKT pathway and retinoblastoma phosphorylation. Further we observed an additive effect of ritonavir in killing ovarian cancer cells when used in conjunction with paclitaxel showing its potentials to be 'repositioned' for ovarian cancer as an adjuvant therapy.
Materials and methods
Reagents and antibodies
Ritonavir was obtained from Sequoia Research Products Limited (Pangbourne, UK) and dissolved in dimethyl sulfoxide (DMSO). Stock solutions were freshly prepared in DMSO (0.001%) and added to the cell cultures to obtain the indicated final concentrations. DMSO alone (0.001%) was found to have no significant effect on cellular function. Following antibodies were used: Retinoblastoma & E2F-1 antibodies from Millipore (Danvers, MA); Cyclins, CDKs, poly (ADP-ribose) polymerase (PARP) and actin antibodies from Santa Cruz Biotechnology, (Santa Cruz, CA). Antibodies against phospho-AKT, caspases, Insulin like growth factor 1 (IGF-1) were obtained from Cell Signaling Technology (Beverly, MA). SignalSilence AKT siRNA inhibition kits were purchased from Cell Signaling Technology (Beverly, MA).
Cell lines and culture
Ovarian cancer cell lines, MDAH-2774 and SKOV-3 (American Type Culture Collection, Manassas, VA) were propagated in McCoy's 5A medium and normal human fibroblasts were propagated in DMEM medium, both were supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (ThermoFisher Scientific, Pittsburgh, PA). Cells were cultured in a humidified atmosphere with 5% CO2 at 37°C. Trypsin (0.25%)/EDTA solution was used to detach the cells from the culture flask for passing the cells.
Cell proliferation assays
Standard prototype growth curves and number of viable cells were determined for each cell line (treated and control groups) in triplicate experiments by the Cell Counting Kit-8 (CCK-8) (Dojindo, Gaithersburg, MD) according to manufactures' instructions. Briefly, cells were plated at a density of 3,000 per well in 96-well plates in a total of 100 μl medium and allowed to grow for 24 h. Ritonavir dissolved in DMSO was added, and the cells were allowed to grow for the indicated time. Growth of the cells in each set of a group was terminated by adding 10 ml of CCK-8 reagent, incubated for an hour and absorbance was read at 450 nm in a plate reader (FluoStar Optima, BMG Labtech). Growth curves were plotted as a percentage of the value of DMSO-treated controls minus the value of untreated cells on day 0. Day 2 & 3 values were considered for the determination of the 50% cell proliferation inhibition (IC50) for a given treatment. In some cases parallel manual count was also performed with trypan blue and counting by exclusion method using a Hemocytometer. The findings confirmed CCK-8 assay results. Human fibroblasts were similarly treated as cancer cells to display differential cytotoxicity at any given dose.
Analysis of apoptotic cells
Apoptotic cells were analyzed by using Annexin V FITC apoptosis detection kit (Calbiochem, Gibbstown, NJ). Ritonavir treated MDAH-2774 cells (1 × 105 cells) were trypsinized, washed with cold PBS, fixed with 70% ethanol, and stored at -20°C until use. The fixed cells were stained with propidium iodide (20 μg/ml) with RNaseA (10 μg/ml) and incubated at room temperature for 30 min in the dark. The DNA content of the cells was analyzed by flow cytometer using the fluorescence-activated cell sorter system (FACS) and sub G1 population was considered to represent apoptotic cells. Fluorescence microscope (Zeiss, AXio CamMRm Observer. A1) was used for visual analysis of apoptotic cells. Propidium iodide (PI) was added to discriminate early apoptotic cells from late apoptotic or necrotic cells. For the fluorescent microscopy, after incubating the cells with Ritonavir at the indicated dose concentrations for 48 hours, the cells were trypsinized and washed twice with cold PBS (0.15 mol/L, pH 7.2). Centrifugation was performed at 5000 c/min for 5 min, and the pellet was resuspended in 1 × binding buffer at a density of 1.0 × 105 –1.0 × l06 cells per mL. Further incubation was performed with 5 μL of FITC-conjugated annexin V and 5 μL of PI for 15 min in the dark. 400 μL of 1 × binding buffer was added to each sample tube, and the samples were analyzed by FACS.
Cell cycle phase determination
MDAH-2774 cells were seeded at 1 × 106 cells in 10 cm dishes and the culture medium changed to serum-free medium for 24 h to facilitate cell cycle synchronization. Cell cycle analysis was conducted using Cell cycle phase determination kit (Cayman chemical company, Ann Arbor, MI). Cells were treated with 5 or 20 μM ritonavir and further incubated in medium containing 10% serum. Later cells were trypsinized, washed with PBS and re-suspended in 1 ml of assay buffer and 1 ml of fixative both provided with the kit. After 2 h incubation, cells were centrifuged at 500c for 5 min and cell pellet was retained. Pellet was suspended in 0.5 ml of staining solution that contained PI and incubated for 30 min at room temperature in the dark. Samples were analyzed in FL2 channel of flow cytometer with a 488 nm excitation laser.
With 4–15% SDS Tris-Glycine gels, western transfer of proteins to nitrocellulose papers was conducted with iBlot dry blotting device (InVitrogen Corp, Carlsbad, CA). Blocking agent was 3% non fat milk powder and the secondary antibodies conjugated to HRPO (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Enhanced chemiluminescence detection kit from Amersham Pharmacia, Uppsala, Sweden was used.
Transfection of Akt small interfering RNA (siRNA)
SignalSilence AKT siRNA inhibition kit (Cell Signaling Technology Beverly, MA) that specifically inhibits the expression of both AKT 1 and AKT 2, was utilised to downregulate AKT protein in MDAH-2774. Briefly, MDAH-2774 cells were transfected with 100 nM siRNA with the transfection reagent provided in the kit. Cells were harvested after 48 hrs and analyzed for the expression of AKT, Bcl-2 and actin antibodies. Controls were transfected with non-specific SiRNA and grown under similar conditions. Similar to SiRNA, inhibition of AKT signaling was achieved by LY294002 whereas forced induction of the Akt pathway was achieved by IGF-1 treatment. The treatment with AKT SiRNA, IGF-1 and LY294002 was conducted to investigate the AKT/PIK pathway involvement in Ritonavir treatment of cell cultures.
In vitro migration and wound-healing Assays
Cell migration was determined with a modified Boyden chamber assay with filters of 8-μm pore size were used. Briefly, MDAH-2774 cells (105/500 μL) were added into the upper compartment of a migration chamber with various concentrations of ritonavir. The chamber was then incubated at 37°C in a 5% CO2. After 18 h, the non-migrated cells on the upper surface of the membrane were removed using cotton buds. The underside of the chamber was washed twice with in PBS, fixed by 4% formaldehyde for 20 min and stained by DIPA or crystal violet (0.1% w/v) for 15 min. The number of migrated cells at other side of the compartment was counted under a microscope for nine random fields. The assays were performed in triplicate. For wound healing assays, MDAH-2774 cells were plated at equal density in and grown to 80% confluency. Using a sterile pipette tip, wounds were generated. Cells were then rinsed with medium and replaced with the fresh medium. Areas of wound were marked and photographed at various time points with a phase-contrast microscope.
Gene Expression Profiling
MDAH-2774 cells treated with 15 μM ritonavir for 24 h, were harvested and total RNA was isolated utilizing an RNeasy kit (Qiagen Inc., Valencia, CA), reverse-transcribed to get cDNA using the "Superscript II RT kit" (Invitrogen™ Life Technologies, Carlsbad, CA). cDNA was used in an in vitro transcription reaction to synthesize cRNA utilizing "ENZO RNA labeling kit (Enzo Diagnostics, Inc., Farmingdale, NY). Labeled cRNA was purified with the RNeasy Mini-kit (Qiagen Inc., Valencia, CA) and quantitated. Purified cRNA (15 μg) was hybridized to Whole Human Genome (G4112A) arrays (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocol. The G4112A set consists of arrays representing approximately 44,000 human genes. Total RNA was amplified using Agilent Low Input Linear Amplification Kit according to the process outlined by the manufacturer (Agilent Technologies, Santa Clara, CA). Amplified target cRNA (1–5 μg) was labeled with either cyanine-5 or cyanine-3 using ULS RNA Flurorescent Labeling Kit according to the manufacturer's protocol (Kreatech Biotechnology, Amsterdam, The Netherlands). Concentration of labeled cRNA and the label incorporation was determined by Nanodrop-1000 spectrophotometer. Labeled cRNA was fragmented and hybridized overnight to Agilent whole Human Genome arrays (4 × 44 K format) according to Agilent protocol. The arrays were scanned using Agilent scanner (G2505B) and data was extracted using Agilent's Feature Extraction Software.
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