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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC Oct 1, 2009.
Published in final edited form as:
PMCID: PMC2561224
NIHMSID: NIHMS64689

Autophagy: a novel mechanism of synergistic cytotoxicity between doxorubicin and roscovitine in a sarcoma model

Abstract

Doxorubicin is a genotoxic chemotherapy agent used in treatment of a wide variety of cancers. Significant clinical side-effects, including cardiac toxicity and myelosuppression, severely limit the therapeutic index of this commonly used agent and methods which improve doxorubicin efficacy could benefit many patients. Because doxorubicin cytotoxicity is cell cycle specific, the cell cycle is a rational target to enhance its efficacy.

We examined the direct, cyclin-dependent kinase inhibitor, roscovitine, as a means of enhancing doxorubicin cytotoxicity. This study demonstrated synergistic cytotoxicity between doxorubicin and roscovitine in three sarcoma cell lines: SW-982 (synovial sarcoma); U2OS-LC3-GFP (osteosarcoma) and SK-LMS-1 (uterine leiomyosarcoma), but not the fibroblast cell line, WI38. The combined treatment of doxorubicin and roscovitine was associated with a prolonged G2/M cell cycle arrest in the three sarcoma cell lines. Using three different methods for detecting apoptosis, our results revealed that apoptotic cell death did not account for the synergistic cytotoxicity between doxorubicin and roscovitine. However, morphologic changes observed by light microscopy and increased cytoplasmic LC3-GFP puncta in U20S-LC3-GFP cells after the combined treatment suggested the induction of autophagy. Induction of autophagy was also demonstrated in SW-982 and SK-LMS-1 cells treated with both doxorubicin and roscovitine by acridine orange staining. These results suggest a novel role of autophagy in the enhanced cytotoxicity by cell cycle inhibition following genotoxic injury in tumor cells. Further investigation of this enhanced cytotoxicity as a treatment strategy for sarcomas is warranted.

Keywords: roscovitine, doxorubicin, autophagy, synergy, cell cycle

Introduction

Sarcomas are a broad group of mesenchymal tumors that are notoriously chemoresistant. More than 50 histologic types of sarcoma are described (1) with an overall five-year survival for all stages of 50-60% (2-4). Only 20% of sarcomas respond to doxorubicin, which is the current standard of systemic therapy care for these tumors (5). Unfortunately the clinical utility of doxorubicin, which is also used to treat a wide range of solid and non-solid tumors, is limited by significant side-effects, particularly irreversible cardiac toxicity (6). For these reasons, efforts to improve the efficacy of doxorubicin are essential and could benefit many patients suffering from a variety of cancers.

The mechanism of doxorubicin cytotoxicity involves “poisoning” the topoisomerase II enzyme thereby interfering with the separation of daughter DNA strands and chromatin remodeling. In addition, doxorubicin intercalates into double-stranded DNA producing structural changes that interfere with DNA and RNA synthesis (6). Because it primarily damages double-stranded DNA, cells in the S phase of the cell cycle are more susceptible. Because of this cell cycle-specificity, the cell cycle is a rational target for enhancing doxorubicin efficacy.

Cyclin dependent kinases (Cdks) are essential cell cycle regulatory proteins which ensure accurate and appropriate transition between cell cycle phases and ultimately cell division (7). In addition, Cdk function is an important determinant of the cellular response to DNA damage, including that caused by genotoxic chemotherapies, such as doxorubicin (8). To date, 13 different Cdks have been identified (Cdk1-13) (9). The activity of individual Cdk types is restricted to specific phases of the cell cycle and dependent upon binding with activating cyclin proteins (10). Because Cdks are involved in regulating many important aspects of the cell cycle, they provide an appealing target for cell cycle-directed anticancer therapy.

Currently more than 100 direct and indirect Cdk inhibitors are available. One of the most studied, and apparently selective, direct Cdk inhibitors is the purine analog, roscovitine (11). Roscovitine directly inhibits Cdks by occupying the adenosine triphosphate binding site of the catalytic subunit of the kinase protein and is relatively more specific for Cdk1 and Cdk2 than other Cdk inhibitors (12). It is a well-tolerated, oral agent which has demonstrated ability to arrest tumor growth as a single agent in Phase I clinical trials (13) and is currently being tested in Phase II clinical trials against non-small cell lung cancer and nasopharyngeal cancer. Other preclinical studies have demonstrated enhanced cytotoxicity with a variety of chemotherapeutic agents when combined with roscovitine (14, 15). Because roscovitine can inhibit both Cdk1 and Cdk2, it has the potential to enhance the cytotoxicity of genotoxic chemotherapeutic agents by the combined effect of delayed cell cycle progression as well as inhibition of the DNA damage response pathway.

In the present study, we investigated the effect of the combination of doxorubicin with roscovitine in three different sarcoma cell lines and one immortalized fibroblast cell line. Our results demonstrate a non-apoptotic synergistic cytotoxicity in all three sarcoma cell lines, but not the fibroblasts, treated with both doxorubicin and roscovitine. This synergistic cell death was accompanied by both a prolonged G2/M arrest and significant induction of autophagy.

Materials and Methods

Cell culture

Fetal bovine serum and cell culture medium were purchased from Hyclone Laboratories (Logan, UT, USA). All other chemicals were reagent grade. SW-982, SK-LMS-1, and WI38 (immortalized fibroblasts) were purchased from ATCC and cultured in D-MEM (SW-982, SK-LMS-1) or Alpha-MEM (WI38) with 10% FBS. U20S cells stably transfected with LC3-GFP protein (U2OS-LC3-GFP) were kindly donated by Dr. Gordon B. Mills (M.D. Anderson Cancer Center, Houston, TX) and maintained in D-MEM with 10% FBS. All cells were cultured and treated at 37°C in a humidified incubator containing 6.5% CO2. A 5mM stock solution of doxorubicin (Sigma St. Louis, MO) was made in sterile water and maintained at -20°C. A 10mM stock solution of roscovitine (synthesized and kindly provided by Drs. Herve Galons, Paris, and Laurent Meijer, Roscoff, France) was made in DMSO and maintained at -20°C. Fresh drug was prepared for each experiment.

Cell cycle analysis

For DNA content analysis, harvested cells were centrifuged at 1000g for 5 minutes, washed with phosphate buffered saline (PBS) and fixed in 70% ethanol. Cells were then treated with RNase (10μg/ml) for 30 minutes at 37°C, washed with PBS, re-suspended, and stained in 1ml of 69μM propidium iodide (PI) in 38 mM sodium citrate for 30 minutes. The cell cycle phase distribution was determined by analytical DNA flow cytometry as described by Keyomarsi et al (16). The percentage of cells in each phase of the cell cycle was analyzed using Modfit software (Verity Software House, Topsham, ME, USA).

High throughput clonogenic assay and combination index

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were modified for use as high-throughput clonogenic assays (HTCA) to determine cell viability. Cells were plated in 96-well plates at a density of 1.5-3×103 cells/well. After 24 hours, cells were treated with drug-free media, doxorubicin 0.001-0.02 μM for 24 hours, roscovitine 5-25 μM for 48 hours or doxorubicin for 24 hours then roscovitine for 48 hours. Drug-free media was replaced every 48 hours after treatment. Six replicates were plated for each treatment group. Ten days after plating, 50 μl of 2.5mg/ml MTT solution was added to each well and incubated for 4 hours. The resultant blue formazan crystals were solubilized in 100 μl of buffer (0.04N HCl and 1% SDS in isopropyl alcohol) for 1 hour. Absorbance was read at 590 nm using a Wallac-1420 plate reader. Drug interactions were assessed using CalcuSyn software version 2.1 (Biosoft Inc.) to determine the combination index of the combined treatment of doxorubicin and roscovitine.

Clonogenic assays

1×103 cells were plated onto 100mm2 dishes. After 24 hours, cells were treated with fresh drug-free media, doxorubicin 0.01 μM (SK-LMS-1), 0.005 μM (U2OS-LC3-GFP and SW-982) for 24 hours, roscovitine 20 μM (SK-LMS-1) or 10 μM (U2OS-LC3-GFP and SW-982) for 48 hours or doxorubicin for 24 hours then roscovitine for 48 hours. Drug-free media was applied at the end of each treatment. After 14 days, the cells were fixed and stained with crystal violet in 100% ethanol suspension. Plates were scored for the number of visible colonies ≥ 2 mm.

Apoptosis assays

Both APC-conjugated Annexin V and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to determine the presence of apoptosis. 2×105 cells were plated on 100mm2 plates. After 24 hours, cells were treated with fresh drug-free media, doxorubicin for 24 hours, roscovitine for 48 hours or doxorubicin for 24 hours then roscovitine for 48 hours as described above. Cells were harvested and stained with Annexin V-APC and PI (Trevigen, Inc, Gaithersburg, MD) or by TUNEL using the APO-DIRECT™ Kit (BD Biosciences Pharmingen, San Diego, CA) according to the manufacturer’s instructions (Trevigen, Inc, Gaithersburg, MD). Apoptosis was detected by flow cytometry.

Western blot analysis for apoptosis

For Western blot analysis, cells were homogenized by sonication and high-speed centrifugation. Cell lysate supernatant was assayed for total protein content and subjected to western blot analysis as described by Rao et al (17). Briefly, 25 μg of protein from each condition was electropheresed on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to Immunobilon P for 2 hours. Membranes were washed at 4°C in Blotto (5% nonfat dry milk in 20mM Tris, 137mM NaCl, 0.25% Tween, pH 7.6) overnight. After six washes in TBST (20mM Tris, 137mM NaCl, 0.05% Tween, pH 7.6), membranes were incubated in primary antibody (Parp, Cell Signaling, Danvers, MA) for 2 hours (1μg/ml in Blotto). Membranes were washed and incubated with goat anti-mouse horseradish peroxidase conjugate at a dilution of 1:5000 for 1 hour, washed and developed with Renaissance chemiluminescence system as directed by the manufacturers (NEN Life Sciences Products, Boston, MA, USA).

Autophagy assays

1×104 U2OS-LC3-GFP cells were plated on coverslips in 6-well plates. After 24 hours, cells were treated with fresh drug-free media, doxorubicin for 24 hours, roscovitine for 48 hours or doxorubicin for 24 hours then roscovitine for 48 hours.. The cells were fixed with 4% paraformaldehyde, stained with 4’,6-diamidino-2-phenylindole, dilactate (DAPI, Invitrogen, Eugene, OR) and observed with fluorescence microscopy (Leica DM 4000 B).

To detect autophagy in SK-LMS-1 and SW-982 cells, 1×104 cells were plated on coverslips in 6-well plates. After 24 hours, cells were treated with fresh drug-free media, doxorubicin for 24 hours, roscovitine for 48 hours or doxorubicin for 24 hours then roscovitine for 48 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature then stained with acridine orange (AO) (Polysciences Inc., Warrington, PA), 1μg/ml in PBS at 37°C, in the dark, and observed immediately with fluorescence microscopy. To quantify the number of cell with acidic vesicles, cells were seeded into 6-well plates at a density of 4×104 cells/well and cultured overnight. The cells were stained with 1μg/ml AO in DMEM at 37°C for 15 minutes. After incubation, the cells were washed with PBS and removed with trypsin-EDTA, re-suspended and analyzed by flow cytometry.

Statistical analysis

Each experiment was repeated at least three times. The isobologram analysis and graphs were carried out using Calcusyn software (version 2.1, Biosoft, Inc), which performs multiple drug dose-effect calculations using the median effects method described by T-C Chou and P. Talalay (18). Combination index (CI) values <0.9 indicates synergy; CI >0.9 and <1.2 indicate additivity; and CI>1.2 indicate antagonism. Comparisons among groups were analyzed by two-sided t test. A difference of P ≤ 0.05 was considered to be statistically significant. All analyses were done with SPSS software, Version 12.0. The data represent the means of 3 or more samples with S.E.M.

Results

Synergistic cytotoxicity between doxorubicin and roscovitine

HTCA was used to compare the cytotoxic effects of doxorubicin alone, roscovitine alone and doxorubicin followed by roscovitine in SW-982, U2OS-LC3-GFP, SK-LMS-1 sarcoma and WI38 immortalized fibroblast cell lines. A sequential drug treatment strategy (see methods) was chosen based on previous reports in the literature demonstrating sequence-specific synergistic effects with administration of combination chemotherapy, and Cdk inhibitors (19, 20). As shown in a 3-D graph in Figure 1A, dose-dependent increases in cell death were seen in all four cell lines when treated with doxorubicin alone (x axis). For SW-982, the percent of cell death increased from 5% to 60% as cells were treated with increasing doxorubicin doses from 0.001 to 0.015 μM (IC50 ~ 0.0125μM). For U2OS-LC3-GFP the percent of cell death increased from 5% to 85% as cell were treated with increasing doxorubicin doses from 0.001 to 0.01 μM (IC50 between 0.005 and 0.0075 μM). For SK-LMS-1, the percent of cell death increased from 7% to 25% as cell were treated with increasing doxorubicin doses from 0.001 to 0.02 μM (IC50 not reached). For WI38 the percent of cell death increased from 13% to 50% as cell were treated with increasing doxorubicin doses from 0.001 to 0.01 μM (IC50 0.01μM). The percent of cell death for all three sarcoma cell lines treated with roscovitine alone ranged between 5% and 30% as cells were treated with increasing roscovitine doses from 5 to 20 μM (y axis). For WI38 the percent of cell death increased from 25% to 95% as cell were treated with increasing roscovitine doses from 5 to 25 μM (IC50 10 μM). When the two drugs were combined, there was a significant increase in cell death (z axis) in all three sarcoma cell lines as compared with either doxorubicin or roscovitine alone in isobologram determinations. However, in the WI38 cells, the combination of doxorubicin and roscovitine did not result in such a synergistic cell death. Combination indices (CI), as determined using the CalcuSyn software, demonstrated synergistic cytotoxicity between doxorubicin and roscovitine in the three sarcoma cell lines, but not the WI38 cells (Figure 1B). For example, in SW-982 cells, treatment with either 0.005 μM doxorubicin alone or 10 μM roscovitine alone resulted in a cell death rate of only 15%. However, when treated first with 0.005 μM doxorubicin then 10 μM roscovitine, the percent of cell death increased to 55% (CI<0.9), suggesting synergism. Similarly, in U2OS-LC3-GFP, treatment with either 0.005 μM doxorubicin alone or 10 μM roscovitine alone resulted in a cell death of 30% and 7%, respectively. However, when treated first with 0.005 μM doxorubicin then 10 μM roscovitine, the percent of cell death increased to 75% (CI<0.9). For SK-LMS-1, treatment with either 0.02 μM doxorubicin alone or 20 μM roscovitine alone resulted in a cell death of 25%. However, when treated first with 0.02 μM doxorubicin then 20 μM roscovitine, the percent of cell death increased to 64% (CI<0.9). Hence, in all 3 sarcoma cell lines, the combination of doxorubicin and roscovitine resulted in synergistic cell killings. On the other hand, for the WI38 immortalized fibroblast cells, the CIs were predominantly antagonistic (CI>1.2) and not synergistic. This finding suggests that the increased cytotoxicity of the combined treatment is tumor cell-specific.

Figure 1
Synergistic effect of doxorubicin and roscovitine in sarcoma cells. (A) HTCA was used to compare the cytotoxic effects of doxorubicin alone, roscovitine alone and doxorubicin followed by roscovitine in SW-982, U2OS-LC3-GFP, SK-LMS-1 and WI38 cell lines ...

The HTCA was complemented with conventional clonogenic assays in the sarcoma cell lines, and both demonstrated a synergistic effect between doxorubicin and roscovitine in all three sarcoma cell lines (Figures 1C and D). For SW-982 cells, there was a 10% decrease in the number of colonies formed after treatment with 10 μM roscovitine alone; a 30% decrease after treatment with 0.005 μM doxorubicin alone; and a 64% decrease in the number of colonies formed after treatment with doxorubicin (0.005 μM) then roscovitine (10 μM) (p≤ 0.006). For U2OS-LC3-GFP cells, there was a 6% decrease in the number of colonies formed after treatment with 10 μM roscovitine alone; a 64% decrease after treatment with 0.005μM doxorubicin alone; and a 82% decrease in the number of colonies formed after treatment with doxorubicin (0.005 μM) then roscovitine (10 μM) (p≤ 0.001). For SK-LMS-1 cells, there was no significant change in the number of colonies formed after treatment with 20 μM roscovitine alone. There was a 47% decrease after treatment with 0.01 μM doxorubicin alone and a 85% decrease in the number of colonies formed after treatment with doxorubicin (0.01 μM) then roscovitine (20 μM) (p≤ 0.008). Panel C shows representative clonogenic plates from each experiment and panel D shows quantization of the clonogenic assays under each condition.

Treatment with doxorubicin followed by roscovitine induces prolonged G2-arrest

To determine the cell cycle effects of doxorubicin and roscovitine, both alone and in combination, DNA content was evaluated using PI staining followed by flow cytometry. Cell cycle distributions for the three sarcoma cell lines, both untreated and treated, are shown in Figures 2A. After 24 hour treatment with doxorubicin alone, all three sarcoma cell lines showed an increase in the percentage of cells in G2/M phase: 5% in SW-982, 6% in U20S-LC3-GFP, and 9% in SK-LMS-1 (Figure 2B) as compared with untreated cells. After 48 hour treatment with roscovitine alone, there was an increase in the percentage of cells in G1 by 13% for SW-982. U2OS-LC3-GFP and SK-LMS-1demonstrated a 7% and 25% increase in the percentage of cells in G2/M phase compared with untreated cells, respectively. Combined treatment with doxorubicin for 24 hours followed by roscovitine for 48 hours resulted in an increase in the percentage of cells in G2/M phase compared with untreated cells by 5% for SW-982, 24% for U2OS-LC3-GFP and 45% for SK-LMS-1. Collectively, comparison of the changes in cell cycle distribution in response to each drug alone versus in combination suggests significant modulation of the cell cycle pathway by the combination of the two drugs. To assess the involvement of the cell cycle as the modulator of the observed synergistic response, we next performed cell cycle analysis at various time points: at the completion of drug treatment (T1) and 72-96 hours after the completion of drug treatment (T2).

Figure 2
Effect of doxorubicin and roscovitine on cell cycle. (A) SW-982 cells, U2OS-LC3-GFP cells and SK-LMS-1 cells were treated with either doxorubicin (Dox) 0.005μM (SW-982 and U20S-LC3-GFP) or 0.01 μM (SK-LMS-1) for 24 hours, roscovitine (Rosc) ...

As shown in Figure 2A, untreated cells in all three sarcoma cell lines showed persistent cell division over the time course with increased percentage of cells residing in G1 phase at the time of final analysis (T2), consistent with a contact-induced G1 cell cycle arrest (compare control cells in T1 with T2). In addition, the percentage of cells in G1 phase also increased in all three sarcoma cell lines treated with either doxorubicin or roscovitine alone, suggesting the persistence of cell division after drug treatment (compare Dox alone cells in T1 with T2). In comparison, after combined treatment with doxorubicin and roscovitine, SW982 cells showed a smaller percentage of cells in G1 compared to the initial time point (-19%) and a persistence of cells in G2/M phase compared to untreated cells at the final time point (T2, 13% versus 3% of untreated cells). For U2OS-LC3-GFP cells, there was no significant change in the percentage of cells in G1 compared to the initial time point (41% versus 46%, respectively) but a persistence of cells in G2/M phase compared to untreated cells at the final time point (14% versus 2%). SK-LMS-1 cells demonstrated an increase in the percentage of cells in G1 as compared with the combined treatment at the initial time point (19%) as well as a persistence of cells in the G2/M phase (15% versus 3%) as compared with untreated cells at the final time point (Figure 2B). These findings translated into a 4-fold, 7-fold and 5-fold increase in the percentage of SW982, U2OS-LC3-GFP and SK-LMS-1 cells remaining in G2/M phase, respectively, as compared with untreated cells at the final time point with the combination treatment. These results suggest that treatment by roscovitine after doxorubicin not only inhibits the cell cycle but also the ability of the cell to recover from cytotoxic injury. All of these experiments were performed at least three times in triplicate with similar results. Figure 2B is representative of a single experiment in the SK-LMS-1 cell line.

Synergistic cytotoxicity by doxorubicin and roscovitine is not due to increased apoptosis

We detected unique morphologic changes by light microscopy of untreated and treated cells particularly in U2OS-LC3-GFP and SK-LMS-1 cells (Figure 3 A-B). As compared with untreated cells, cells treated with both doxorubicin and roscovitine became larger, displayed prominent fibrils, and were often multinucleated (see arrows). Based upon the synergistic cytotoxicity observed with the combined drug treatment, it was clear that cell death was present, but not through apoptosis. Three different methods were used to determine the degree of cell death due to apoptosis after treatment with either the single agents or the combination: Annexin V-APC staining, TUNEL staining, and expression of Parp (Figure 4).

Figure 3
Morphological changes observed by light microscopy. (A) U2OS-LC3-GFP cells and (B) SK-LMS-1 cells were treated with either doxorubicin 0.005μM (U20S-LC3-GFP) or 0.01μM (SK-LMS-1) for 24 hours, roscovitine 10μM (U20S-LC3-GFP) or ...
Figure 4
(A) Fluorescence-activated cell sorting-based apoptosis analyses. SW-982 (upper panel), U20S-LC3-GFP (middle panel) and SK-LMS-1 cells (lower two panels) were treated with either doxorubicin (Dox) 0.005μM (SW-982 and U20S-LC3-GFP) or 0.01 μM ...

As shown in Figure 4A, there was a significant increase in Annexin V-APC positive SW-982 cells after treatment with either doxorubicin or roscovitine alone or in combination as compared with untreated cells (p≤ 0.004). Roscovitine treatment alone lead to the greatest fold increase in Annexin V-APC positive SW-982 cells (4-fold) and this was significant when compared with either doxorubicin treatment alone or in combination with roscovitine (p≤ 0.021). However, as shown previously, the combined treatment resulted in greater cell death than either doxorubicin or roscovitine alone, and because apoptosis was not significantly increased after the combination treatment, this finding supports the hypothesis of a non-apoptotic mechanism of synergistic cell death.

In comparison, there was no significant difference in Annexin V-APC positive U2OS-LC3-GFP cells after treatment with either doxorubicin or roscovitine alone or in combination compared with untreated cells. For SK-LMS-1 cells, there was a significant increase in the Annexin V-APC positive cells treated with the combination of doxorubicin and roscovitine as compared with untreated cells and cells treated with doxorubicin alone (p≤0.05). This increase was not significantly different when compared with roscovitine alone. However, because the percentage of Annexin V-APC positive cells was only 12% in cells treated with the combination of doxorubicin and roscovitine, these findings also support an alternative mechanism of synergistic cell death due to the combination treatment. Similar results were found using the TUNEL assay. SK-LMS-1 cells showed a slight increase in the TUNEL positive cells treated with the combination of doxorubicin and roscovitine. This difference was statistically significant when compared with doxorubicin alone (p=0.05) but not roscovitine alone. However, similar to the Annexin-APC analysis, the percentage of TUNEL positive cells was low (4%) in cells treated with the combination of doxorubicin and roscovitine again supporting an alternative mechanism of synergistic cell death due to the combination treatment.

Western blot analysis was also performed to determine whether the enhanced cell death was occurring through apoptosis. As shown in Figure 4B, in all three sarcoma cell lines, the expression of the cleaved form of the apoptosis marker, Parp, decreased after treatment with the combination of doxorubicin and roscovitine as compared with either doxorubicin or roscovitine alone. This finding further suggests that apoptosis is not the only mechanism of cell death due to the combined treatment of doxorubicin and roscovitine. Since the morphological changes we observed with the drug combination were reminiscent of the homeostatic condition of autophagy, we explored whether an autophagy-mediated mechanism of cell death was associated with the synergistic cytotoxicity.

Synergistic cytotoxicity by doxorubicin and roscovitine is associated with autophagy

Microtubule-associated protein-1 light chain-3 (LC3), the homologue of the yeast Apg8/Aut7p gene, localizes on the autophagosomal membrane during autophagy (21). The LC3-GFP fused protein is used frequently to detect autophagy through the increased presence of GFP puncta within the cytoplasm (21). Figure 5A shows the difference in the presence of LC3-GFP punctuate structures in U2OS-LC3-GFP cells treated with doxorubicin alone, roscovitine alone or with doxorubicin followed by roscovitine as compared with untreated cells. The pictures clearly show that treatment with either drug alone leads to increased autophagocytic LC3-GFP puncta, as compared with untreated U2OS-LC3-GFP cells. Furthermore, cells treated with doxorubicin alone had more LC3-GFP puncta than cells treated with roscovitine alone. However, U2OS-LC3-GFP cells treated with both doxorubicin and roscovitine demonstrated the most LC3-GFP puncta as compared with cells treated with either drug alone. These findings suggested that autophagy may be associated with the mechanism of synergistic cytotoxicity of doxorubicin and roscovitine.

Figure 5
Induction of autophagy by combined treatment with doxorubicin and roscovitine. (A) U20S-LC3-GFP cells were treated with either doxorubicin 0.005 μM for 24 hours, roscovitine for 48 hours, the combination of doxorubicin and roscovitine (Dox+Rosc) ...

To determine whether autophagy was also present in SW-982 and SK-LMS-1, the cells were stained with acridine orange (AO). AO is concentrated in acidic vesicles such as the autpohagolysosome and has been used as a measure of autophagy (22). SK-LMS-1 and SW-982 cells treated with either doxorubicin or roscovitine developed significantly increased cytoplasmic AO puncta as compared with untreated cells (Figure 5B). Furthermore, SK-LMS-1 and SW-982 cells treated with both doxorubicin and roscovitine demonstrated more AO puncta as compared to cells treated with either doxorubicin or roscovitine alone. Quantization of AO staining by flow cytometry demonstrated a significant increase in both the SW-982 and SK-LMS-1 cells treated with both doxorubicin and roscovitine as compared to untreated cells and cell treated with either doxorubicin or roscovitine alone (p≤0.003 for all conditions) (Figure 5C). Taken together, these results suggested an autophagic mechanism of synergistic cytotoxicity due to the combined treatment of doxorubicin and roscovitine.

Discussion

Roscovitine synergistically increases doxorubicin cytotoxicity in sarcoma cells but not fibroblasts

Doxorubicin is a commonly used cytotoxic chemotherapy agent with a significant and use-limiting side-effect profile. The ability to increase doxorubicin efficacy would positively impact many patients suffering from a variety of cancers. In this report, we demonstrate synergistic cytotoxicity between the Cdk inhibitor roscovitine and doxorubicin in three different sarcoma cell lines. However, immortalized human fibroblast cells were not responsive to such synergistic action, suggesting that the synergism observed is tumor specific. Previous studies have shown that Cdk inhibition mediates tumor cell-specific cell cycle arrest and cell death (23, 24). These effects occur through Cdk-inhibition in both the early and late phases of the cell cycle. For example, within the G1 phase, Cdk-mediated phosphorylation of Rb leads to E2F-1 transcription factor activation and upregulation of the genes required for the transition into S phase (25). Cdk inhibition during G1 can lead to G1 arrest and in S phase can lead to inappropriately persistent E2F-1 resulting in both S-phase delay and apoptosis (26).

The G2/M transition is also susceptible to Cdk inhibition. Cdk1 activation is essential for progression from the G2 to M cell cycle phases, the progression of mitosis through metaphase and cell survival during the mitotic checkpoint (27, 28). Conditional knockdown of Cdk1 has been shown to result in extensive DNA rereplication and apoptosis (29). Inhibition of Cdk1 has also been shown to increase apoptosis following genotoxic stress, while prolonged Cdk1 inhibition alone can cause significant tumor cell-specific apoptosis (23, 24). Furthermore, Cdk1 inhibition during the spindle assembly checkpoint has been shown to cause tumor cell-specific cell cycle progression without cell division resulting in mitotic catastrophe (30). Because most conventional chemotherapy agents cause DNA damage and activate cell cycle checkpoints, it is logical that disruption of these checkpoints through a Cdk inhibitor, like roscovitine, would increase their cytotoxic effects.

Roscovitine-enhanced doxorubicin cell death is associated with autophagy

We show here that apoptosis is not the only mechanism of synergistic cell death caused by the combination of doxorubicin and roscovitine. Instead, autophagy is significantly increased in the cells treated with the combination of doxorubicin and roscovitine. Autophagy is a membrane-trafficking process which, under normal conditions, degrades cytosolic proteins and organelles through engulfment into double-membraned vesicles (autophagosomes). Autophagosomes fuse with lysosomes to form autolysosomes and the contents are degraded (31). Autophagy is induced above basal levels by a wide variety of stimuli including nutrient deprivation and genotoxic stress and has a direct impact on cell viability. In this study, we have demonstrated that autophagy can also be induced by the direct Cdk inhibitor, roscovitine, in the setting of previous cytotoxic treatment.

Cell cycle and autophagy

One of the more intriguing findings of this study is the induction of autophagy by the combination of doxorubicin and roscovitine in all three sarcoma cell lines despite different initial cell cycle effects of the individual drug treatments. Interestingly the combined treatment with doxorubicin and roscovitine lead to a prolonged G2/M arrest in all three sarcoma cell lines. Therefore, we postulate that this prolonged arrest, caused by the combined activation of the DNA damage checkpoint by doxorubicin followed by the inhibition of the Cdk1-cyclinB complex by roscovitine, may be a trigger for the induction of autophagy and ultimately cell death (Figure 6).

Figure 6
Proposed mechanism of autophagy-mediated synergistic cell death by genotoxic chemotherapy and cyclin dependent kinase inhibition.

The relationship between cell cycle regulation and autophagy is not yet clearly defined. A variety of agents have demonstrated induction of autophagy and autophagic-cell death in association with a G2 cell cycle arrest (32, 33). Based upon our findings of the combination treatment of doxorubicin and roscovitine leading to synergistic cell death, induction of autophagy and prolonged G2 cell cycle arrest, we postulate the following model. DNA damage by doxorubicin (and potentially other genotoxic chemotherapy agents or treatments) activates the DNA-PK/ATM/ATR kinases, initiating two potential Cdk1-cyclin B inactivating cascades: i) phosphorylation of Cdc25 by CHK1 and CHK2 and ii) (when present) activation of p53 and upregulation of direct Cdk1-cyclin B inhibitors such as 14-3-3σ, GADD45, and p21. Injured cells undergo cell cycle arrest and autophagy to facilitate the repair of the damaged DNA. Once repaired, passage into mitosis occurs. However, pharmacologic inhibition of the Cdk1-cyclin B complex by roscovitine also induces cell cycle arrest and autophagy, thereby preventing the cells from entering mitosis. Furthermore, due to the pivotal role of Cdk1-cyclin B in the G2/M transition, the G2/M DNA damage checkpoint may also be inhibited by roscovitine. In this manner, autophagy induced by a G2 cell cycle arrest in the setting of genotoxic injury and Cdk inhibition, may be exploitable as a mechanism of cell death. Further investigation into the mechanism of synergy between cytotoxic agents and Cdk inhibition as well the role of the cell cycle in the initiation and outcome of autophagy (e.g. cell survival or cell death) will potentially provide new approaches to tumor-specific anticancer therapies.

Acknowledgments

This work was supported in part by grant number CA87458 from the National Institutes of Health (to KK), National Cancer Institute P50CA116199 (to K. K); and National Institutes of Health T32 CA009599 (to LAL). We gratefully acknowledge Dr. Robert Bast for helpful discussions.

References

1. Zahm SH, Fraumeni JF., Jr The epidemiology of soft tissue sarcoma. Semin Oncol. 1997;24:504–14. [PubMed]
2. Gaynor JJ, Tan CC, Casper ES, et al. Refinement of clinicopathologic staging for localized soft tissue sarcoma of the extremity: a study of 423 adults. J Clin Oncol. 1992;10:1317–29. [PubMed]
3. Jaques DP, Coit DG, Hajdu SI, Brennan MF. Management of primary and recurrent soft-tissue sarcoma of the retroperitoneum. Ann Surg. 1990;212:51–9. [PMC free article] [PubMed]
4. Singer S, Demetri GD, Baldini EH, Fletcher CD. Management of soft-tissue sarcomas: an overview and update. Lancet Oncol. 2000;1:75–85. [PubMed]
5. Verweij J, Lee SM, Ruka W, et al. Randomized phase II study of docetaxel versus doxorubicin in first- and second-line chemotherapy for locally advanced or metastatic soft tissue sarcomas in adults: a study of the european organization for research and treatment of cancer soft tissue and bone sarcoma group. J Clin Oncol. 2000;18:2081–6. [PubMed]
6. Rubin E, Hait W. Drugs That Target DNA Topoisomerases. In: Kufe D, Bast R, Hait W, editors. Cancer Medicine 7. Hamilton, Ontario: BC Decker, Inc; 2006. pp. 690–3.
7. Sherr CJ. G1 phase progression: cycling on cue. Cell. 1994;79:551–5. [PubMed]
8. Melo J, Toczyski D. A unified view of the DNA-damage checkpoint. Curr Opin Cell Biol. 2002;14:237–45. [PubMed]
9. Shi J, Feng Y, Goulet AC, et al. The p34cdc2-related cyclin-dependent kinase 11 interacts with the p47 subunit of eukaryotic initiation factor 3 during apoptosis. J Biol Chem. 2003;278:5062–71. [PubMed]
10. Meyerson M, Enders GH, Wu CL, et al. A family of human cdc2-related protein kinases. Embo J. 1992;11:2909–17. [PMC free article] [PubMed]
11. Meijer L, Borgne A, Mulner O, et al. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem. 1997;243:527–36. [PubMed]
12. Meijer L, Raymond E. Roscovitine and other purines as kinase inhibitors. From starfish oocytes to clinical trials. Acc Chem Res. 2003;36:417–25. [PubMed]
13. Benson C, White J, De Bono J, et al. A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days. Br J Cancer. 2007;96:29–37. [PMC free article] [PubMed]
14. Abal M, Bras-Goncalves R, Judde JG, et al. Enhanced sensitivity to irinotecan by Cdk1 inhibition in the p53-deficient HT29 human colon cancer cell line. Oncogene. 2004;23:1737–44. [PubMed]
15. Crescenzi E, Palumbo G, Brady HJ. Roscovitine modulates DNA repair and senescence: implications for combination chemotherapy. Clin Cancer Res. 2005;11:8158–71. [PubMed]
16. Keyomarsi K, Conte D, Jr, Toyofuku W, Fox MP. Deregulation of cyclin E in breast cancer. Oncogene. 1995;11:941–50. [PubMed]
17. Rao S, Lowe M, Herliczek TW, Keyomarsi K. Lovastatin mediated G1 arrest in normal and tumor breast cells is through inhibition of CDK2 activity and redistribution of p21 and p27, independent of p53. Oncogene. 1998;17:2393–402. [PubMed]
18. Chou T-C, Talalay P. Analysis of combined drug effects: a new look at a very old problem. Trends in Pharm Sci. 1983;4:450–4.
19. Motwani M, Delohery TM, Schwartz GK. Sequential dependent enhancement of caspase activation and apoptosis by flavopiridol on paclitaxel-treated human gastric and breast cancer cells. Clin Cancer Res. 1999;5:1876–83. [PubMed]
20. Shah MA, Kortmansky J, Motwani M, et al. A phase I clinical trial of the sequential combination of irinotecan followed by flavopiridol. Clin Cancer Res. 2005;11:3836–45. [PubMed]
21. Klionsky DJ, Abeliovich H, Agostinis P, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2008;4:151–75. [PMC free article] [PubMed]
22. Paglin S, Hollister T, Delohery T, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001;61:439–44. [PubMed]
23. Ongkeko W, Ferguson DJ, Harris AL, Norbury C. Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage. J Cell Sci. 1995;108(Pt 8):2897–904. [PubMed]
24. Vassilev LT, Tovar C, Chen S, et al. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci U S A. 2006;103:10660–5. [PMC free article] [PubMed]
25. Lundberg AS, Weinberg RA. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol Cell Biol. 1998;18:753–61. [PMC free article] [PubMed]
26. Phillips AC, Vousden KH. E2F-1 induced apoptosis. Apoptosis. 2001;6:173–82. [PubMed]
27. Nigg EA. Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol. 2001;2:21–32. [PubMed]
28. O’Connor DS, Wall NR, Porter AC, Altieri DC. A p34(cdc2) survival checkpoint in cancer. Cancer Cell. 2002;2:43–54. [PubMed]
29. Itzhaki JE, Gilbert CS, Porter AC. Construction by gene targeting in human cells of a “conditional’ CDC2 mutant that rereplicates its DNA. Nat Genet. 1997;15:258–65. [PubMed]
30. Weaver BA, Cleveland DW. Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death. Cancer Cell. 2005;8:7–12. [PubMed]
31. Baehrecke EH. Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol. 2005;6:505–10. [PubMed]
32. Aoki H, Takada Y, Kondo S, Sawaya R, Aggarwal BB, Kondo Y. Evidence that curcumin suppresses the growth of malignant gliomas in vitro and in vivo through induction of autophagy: role of Akt and extracellular signal-regulated kinase signaling pathways. Mol Pharmacol. 2007;72:29–39. [PubMed]
33. Kuo PL, Hsu YL, Cho CY. Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther. 2006;5:3209–21. [PubMed]

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