Synergistic effect of rapamycin and cisplatin in endometrial cancer cells

Authors

  • Victoria L. Bae-Jump MD, PhD,

    Corresponding author
    1. Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of North Carolina, Chapel Hill, North Carolina
    • Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, CB #7572, the University of North Carolina, Chapel Hill, NC 27599-7572===

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    • The first 2 authors contributed equally to this article.

    • Fax: (919) 966-2646

  • Chunxiao Zhou MD, PhD,

    1. Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of North Carolina, Chapel Hill, North Carolina
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    • The first 2 authors contributed equally to this article.

  • John F. Boggess MD,

    1. Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of North Carolina, Chapel Hill, North Carolina
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  • Paola A. Gehrig MD

    1. Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of North Carolina, Chapel Hill, North Carolina
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Abstract

BACKGROUND:

Mammalian target of rapamycin (mTOR) inhibitors modulate signaling pathways involved in cell cycle progression, and phase 2 trials for endometrial cancer are currently being conducted. Because rapamycin is known to enhance the cytotoxicity of chemotherapeutic drugs, the authors' goal was to examine the effects of rapamycin and cisplatin in endometrial cancer cell lines.

METHODS:

By using Ishikawa and ECC-1 cells, cell proliferation was assessed after exposure to rapamycin, cisplatin, or both in combination. The combination index (CI) was calculated using the method of Chou and Talalay. Apoptosis was evaluated by flow cytometry. Immunoblot analysis was performed to assess expression of S6 kinase 1 and the DNA mismatch repair proteins, MSH2 and MSH6. mTOR small interfering (siRNA) was transfected into the cell lines, and proliferation and apoptosis were assessed after exposure to cisplatin.

RESULTS:

Cisplatin inhibited growth in a dose-dependent manner in both cell lines (median inhibition concentration of 8-13 μM). Simultaneous exposure of cisplatin in combination with rapamycin resulted in a significant synergistic antiproliferative effect (CI < 1). Rapamycin increased cisplatin-induced apoptosis and stimulated expression of MSH2 and MSH6 in the cisplatin-treated cell lines. Cell growth was significantly decreased in cells transfected with mTOR siRNA and treated with cisplatin compared with either alone (CI < 1). Transfection of mTOR siRNA did not induce apoptosis, but combined treatment with cisplatin increased apoptosis over that of cisplatin alone.

CONCLUSIONS:

The results of the current study provide evidence of a synergistic relation between rapamycin and cisplatin in both inhibition of cell growth and induction of apoptosis. This suggests that rapamycin and cisplatin may be a rational combination of a targeted therapy for endometrial cancer. Cancer 2009. © 2009 American Cancer Society.

Endometrial cancer is the fourth most common cancer in the United States and has been increasing in frequency secondary to an aging female population and changes in dietary and hormonal factors. Although the majority of women diagnosed with endometrial cancer have early stage disease, which is usually cured with surgery alone, to our knowledge the treatment of women with advanced or recurrent disease with either cytotoxic or hormonal agents has met with limited success. Thus, there has been a search for an additional agent, with hopefully a low toxicity profile, that could be used in combination with more traditional hormonal and cytotoxic chemotherapy to dramatically increase efficacy. Many novel agents are being investigated that target specific cellular signaling pathways that are believed to be essential in endometrial cancer progression and metastasis. One of the most promising of these for endometrial cancer is the mammalian target of rapamycin (mTOR) inhibitors.

mTOR inhibitors such as rapamycin are known for their potent antiproliferative properties stemming from their ability to modulate signal transduction pathways involved in cell cycle progression from G1 to S‒phase and are currently under evaluation in phase 1, 2, and 3 clinical trials for a broad range of cancers, including endometrial cancer.1 Temsirolimus has been shown to increase survival of patients with metastatic renal cell carcinoma in a phase 3 trial, and is the first mTOR inhibitor to receive US Food and Drug Administration approval for cancer therapy.2 Rapamycin and its analogues exert their antiproliferative effects through the inhibition of the serine/threonine kinase, mTOR, by binding to 1 of the immunophilin family of FK 506–binding proteins, FKBP 12.3, 4 The inhibition of mTOR decreases phosphorylation and activation of S6 kinase 1 and 4E-binding protein 1 (4E-BP1), which results in the inhibition of translation of critical mRNAs involved in cell cycle progression and ultimately, cell cycle arrest in early G1 phase.3-5 mTOR is also activated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and perturbations in this cell signaling pathway have been implicated in tumorigenesis for a variety of human cancers.6, 7 It has been hypothesized that those malignancies that rely on PI3K/Akt-driven mitogen stimulation may be more sensitive to the effects of rapamycin. Given that loss of PTEN expression is 1 of the most prevalent molecular abnormalities associated with endometrial cancers, and that wild-type PTEN down-regulates the PI3K/Akt/mTOR signaling pathway, mTOR appears to be an especially promising target for endometrial cancer therapy.8-10

In addition to being used as single-agent therapy, rapamycin and its analogues have been shown to enhance the efficacy of several cytotoxic chemotherapeutic agents in a variety of different cancers that rely on the mTOR pathway.11-16 Synergistic interactions have been demonstrated in vitro between rapamycin and paclitaxel, carboplatin, and vinorelbine in breast cancer cells.13 Additive effects were also noted when rapamycin was used in combination with gemcitabine and doxorubicin.13 Rapamycin dramatically enhanced paclitaxel-induced and carboplatin-induced apoptosis, at least partially through caspase activation.13 RAD001 (everolimus), a rapamycin derivative, has also been reported to increase cisplatin-induced apoptosis in wild-type p53 gene lung cancer cells in vitro through inhibition of p21 translation.11

Cisplatin (cis-diamminedichloroplatinum[II]) is a widely used chemotherapeutic agent that exerts its cytotoxic effects by disrupting the DNA structure in cells through the formation of intrastrand adducts and interstrand cross-links.17, 18 It has proven to be 1 of the most clinically active agents for the treatment of a variety of solid tumors, including endometrial carcinoma.19, 20 However, its clinical therapeutic effect is often limited by intrinsic or acquired tumor cell resistance. In addition, cisplatin's associated nephrotoxicity and neurotoxicity, especially when administered at higher doses, have been further obstacles to the success of this treatment.17, 18 Thus, our goal was to determine whether rapamycin would potentiate the effects of cisplatin on endometrial cancer cells in regard to inhibition of cell growth and induction of apoptosis, in the hope that these 2 agents used in combination may be a more effective and less toxic treatment option for women with recurrent or advanced stage endometrial cancer.

MATERIALS AND METHODS

Cell Culture and Reagents

The endometrial cancer cell lines, Ishikawa and ECC-1, were used. The Ishikawa cells were grown in minimum essential medium supplemented with 5% fetal bovine and 5 μg/mL bovine insulin. The ECC-1 cells were maintained in RPMI‒1640 containing 5% fetal bovine serum, 200 pg/mL of estrogen, and 6 mM of sodium bicarbonate. All media were supplemented with 100 U/mL of penicillin and 100 μg/mL of streptomycin under 5% carbon dioxide. Cisplatin and rapamycin were obtained from Sigma Chemical Company (St. Louis, Mo) and were dissolved in dimethyl sulfoxide (DMSO). The M30 CytoDEATH kit was obtained from Roche (Penzberg, Germany). The polyclonal mTOR, anti–phospho-S6, non–phospho-S6 antibodies were obtained from Cell Signaling Technology (Beverly, Mass). The anti–β-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). Antibodies to the DNA mismatch repair (MMR) proteins, MSH2 and MSH6, were obtained from BD Biosciences (San Jose, Calif). The enhanced chemiluminescence Western blotting detection reagents were obtained from Amersham (Arlington Heights, Ill).

Cell Proliferation Assay

The Ishikawa and ECC-1 cells (1 × 104) were plated in triplicate onto 96-well culture plates in their corresponding media. The next day, different concentrations of cisplatin, rapamycin, or both in combination were added in each well. Cells were incubated for 1 to 7 days. Viable cell densities were determined by measuring metabolic conversion of the colorimetric dye, MTS. MTS was added to the 96-well plates at 20 μL/well, and plates were incubated for an additional 1 to 2 hours. The MTS assay results were read by measuring absorbance at 490 nanometers (nm). The effect of cisplatin and rapamycin were calculated as a percentage of control cell growth obtained from DMSO-treated cells grown in the same culture plates. Each experiment was performed in triplicate and repeated 3 times to assess for consistency of results.

Detection of Apoptosis by Flow Cytometry

The Ishikawa and ECC-1 cells were plated in 6-well plates in their respective medium. After 24 hours, the cells were treated with cisplatin, rapamycin, or both in combination for 24 or 48 hours, and then cells were washed with phosphate-buffered saline (PBS) solution and trypsinized. Cells were fixed in 70% ice-cold methanol and stored at −20°C until flow cytometric analysis. For staining with M30, the methanol fixed cells were pelleted, had the supernatant fluid removed, and were washed twice with PBS plus 0.1% Tween 20. The cells were blocked with PBS/0.1% Tween 20 of 1% bovine serum albumin for 10 minutes, pelleted, and had the supernatant fluid removed. Subsequently, 100 μL of the block solution with a 1:100 dilution of the M30 antibody was added and incubated at room temperature in the dark for 1 hour. Incubated cells were washed twice and diluted in a 1% formaldehyde/PBS solution at 4°C overnight. Unstained and isotype controls were also run to ensure lack of cross-reactivity and specificity. Fluorescence was determined by flow cytometry on a FACSCalibur (Becton Dickinson, Bedford, Mass) at 488 nm. Dead cells and debris were gated out. Each experiment was performed in triplicate to assess for consistency of results.

Western Blot Analysis

The Ishikawa and ECC-1 cells were plated at 1 × 105 cells/well in 6-well plates in their corresponding media. After 24 hours, cells were treated with rapamycin, cisplatin, or both combined for 48 hours. Cell lysates were prepared in RIPA buffer (1% NP40, 0.5 sodium deoxycholate, and 0.1% sodium dodecyl sulfate). Equal amounts of protein were separated by gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk and then incubated with a 1:1000 dilution of primary antibody overnight at 4°C. The membrane was washed and incubated with a secondary peroxidase-conjugated antibody for 1 hour after washing. Antibody binding was detected using the enhanced chemiluminescence detection system. Western blot films were digitized, and band net intensities were quantified by a densitometer using the Genegynome Image System (Sygene, Frederick, Md). After developing, the membrane was stripped and reprobed using an antibody against β-actin to confirm equal loading. Each experiment was repeated 3 times to assess for consistency of results.

Transfection of Small Interfering RNA

mTOR small interfering RNA (siRNA) kits were purchased from Cell Signaling Technology. According to the manufacturer's instructions, ECC-1 cells were plated in 6-well plates or 12-well plates at the recommended cell concentration. After 24 hours, transfections were performed at approximately 60% confluency using transfection reagent. For each transfection reaction, 100 nM mTOR siRNA or control siRNA was used for preparation of siRNA-transfection complexes at room temperature for 15 minutes. Transfections were performed in 0.5 (12-well plate) or 1.5 mL (6-well plate) serum-free medium for 8 hours. After incubation, transfection complexes were removed and replaced with their corresponding media. In each experiment, untreated controls receiving transfection reagent were included. Transfection efficiency (80%-90%) was determined by fluorescence microscope in fluorescein-labeled nonspecific siRNA transfected cells. Cells were assayed 24 to 48 hours after transfection.

Synergistic Analysis

Statistical analysis on synergy was used to evaluate the effects of combined drug treatments. The results from the MTS assays were calculated by the CalcuSyn for Windows computer program (Biosoft, Cambridge, United Kingdom) to determine the presence of synergy between rapamycin and cisplatin. The software uses a median-effect method, which is a well-established procedure to quantify the effects of drug combinations and to determine whether they produce greater effects together than expected from simple summation of their individual effects. This methodology was originally described by Chou and Talalay.21 The combination index (CI) values are obtained from the data, and reflect the nature of the interaction between rapamycin and cisplatin, that is, < 1, synergistic activity; =1, additive; >1, antagonism.

RESULTS

Synergistic Antiproliferative Effects of Rapamycin and Cisplatin

We examined the effects of rapamycin used in combination with cisplatin in 2 endometrial cancer cell lines (Ishikawa and ECC-1), with respect to cell proliferation, cytotoxicity, and the induction of apoptosis. Rapamycin alone inhibited proliferation in a dose-dependent manner in both the Ishikawa (median inhibition concentration [IC50] = 0.5) and ECC-1 (IC50 = 0.05) cell lines after 48 to 96 hours of exposure (Fig. 1A). As expected, cisplatin alone also potently inhibited growth in a dose-dependent manner in both of these cell lines, with IC50 values of 8 μM and 13 μM, respectively (Fig. 1B). These cell lines were then treated with serial dilutions of both cisplatin and rapamycin, and their individual and combined effects on growth inhibition were evaluated. Median-effect plot analyses and calculation of the multiple drug effect/CI was performed using the well-established method of Chou and Talalay via a commercial software package obtained from Calcusyn (Biosoft).21 In these analyses, CI values are calculated for different dose-effect levels based on parameters derived from median-effect plots of rapamycin alone, cisplatin alone, or both of these agents used in combination at fixed molar ratios. A CI value significantly < 1 indicates synergy, a CI not significantly different from 1 indicates addition, and a CI significantly >1 indicates antagonism. Synergy is defined as a combination of 2 agents that has a greater therapeutic effect than would be expected by the simple addition of the individual effects of each drug.

Figure 1.

Effect of cisplatin (PDD) with and without rapamycin (Rap) on the proliferation of endometrial carcinoma cells is shown. Ishikawa and ECC-1 cells were cultured in the presence of various concentrations of (A) rapamycin and (B) cisplatin for 96 hours. Rapamycin enhanced sensitivity to cisplatin after 48 hours of exposure in both the (C) Ishikawa and (D) ECC-1 cell lines. Relative growth of cells was determined by MTS. The results are shown as the mean ± the standard error of triplicate samples and are representative of 3 independent experiments.

Simultaneous exposure of various doses of cisplatin in combination with 1 nM rapamycin on these endometrial cancer cell lines resulted in a significant synergistic antiproliferative effect, with a CI of < 1, at a range of 0.023 to 0.479 (Table 1). Synergistic effects on cell proliferation were also observed when different concentrations of cisplatin were combined with 0.1 nM and 10 nM rapamycin in both of these cell lines (data not shown).

Table 1. Combination Index Values for Ishikawa and ECC-1 Cell Lines Treated With Rapamycin and Cisplatin
Cisplatin, μMRapamycin, nMCI for Ishikawa CellsCI for ECC-1 Cells
  1. CI indicates combination index.

0.0110.0760.023
0.110.1360.061
110.1120.104
1010.2120.360
2510.2980.305
5010.3200.142
10010.4790.140

Rapamycin Enhanced Cisplatin-induced Apoptosis

The induction of apoptosis was analyzed by flow cytometry with the M30 antibody after treatment with cisplatin in the Ishikawa and ECC-1 cells. The M30 antibody detects a cytokeratin neoepitope generated after cleavage at position 387 to 396, and is considered to be a specific marker for epithelial apoptosis.22-24 After exposure to various concentrations of cisplatin (1 μM, 10 μM, and 50 μM) for 48 hours, a significant dose-dependent increase in the expression of M30 was observed in both cell lines. We further investigated whether rapamycin in combination with cisplatin would enhance cisplatin-induced apoptosis. Previously, we have demonstrated that rapamycin alone had no effect on apoptosis in an endometrial cancer cell lines.25 Treatment with rapamycin (1 nM) with different concentrations of cisplatin induced increases in the percentage of expression of M30 in both cell lines (Fig. 2). Similar results were found when rapamycin at 0.1 nM and 10 nM were used in combination with cisplatin. These results indicate that rapamycin enhances the efficacy of cisplatin by increasing cisplatin-induced apoptosis.

Figure 2.

Rapamycin (Rap) was found to enhance cisplatin-induced apoptosis. (A) Ishikawa and (B) ECC-1 cells were cultured for 24 hours and then treated with different concentrations of cisplatin (PDD) with or without rapamycin (1 nM) for 48 hours. Apoptosis was determined by flow cytometric analysis of M30 expression. Data shown are representative of at least 2 independent experiments.

Effect of Rapamycin and Cisplatin on mTOR Pathway

To investigate the mechanisms underlying the synergistic antiproliferative effect between rapamycin and cisplatin, we characterized the effect of this combination treatment on relevant cell signaling targets. Previous studies suggest that p70S6K is a downstream target of the mTOR pathway.26 p70S6K kinase directly phosphorylates the 40S ribosomal protein S6, which results in enhanced translation of proteins that contain a polypyrimidine tract in the 5′-untranslated region.26 Therefore, we studied the effect of rapamycin and cisplatin on the phosphorylation of the S6 ribosomal protein in both cell lines. As would be expected, rapamycin alone reduced the phosphorylation of S6 and resulted in decreased levels of total S6 after 48 hours of exposure. We have previously demonstrated that rapamycin rapidly inhibits S6 phosphorylation as early as 15 minutes after exposure, and that this effect persists for 24 to 48 hours after initial treatment.25, 27 In contrast, cisplatin did not affect phosphorylation of S6 or expression of total S6 protein. Rapamycin in combination with cisplatin decreased phosphorylation of S6 and total protein expression of S6, similar to treatment with rapamycin alone (Fig. 3). This suggests that rapamycin may exert its synergistic effect through the mTOR pathway by regulating phosphorylation of the S6 protein.

Figure 3.

Rapamycin (Rap) inhibits S6 phosphorylation in Ishikawa and ECC-1 cells. The cells were treated with cisplatin (PDD) (10 μM) alone, rapamycin (1 nM) alone, or in combination for 48 hours. Total protein was analyzed by Western blot analysis with antibodies against phospho-S6 (ser235 of 236), total S6, and β-actin as indicated. Note that cisplatin was not found to affect phosphorylated S6 (P-S6) expression.

Selective Gene Silencing of mTOR-enhanced Cisplatin-induced Apoptosis

To specifically determine whether mTOR is required for increased cisplatin sensitivity and enhanced cisplatin-induced apoptosis, mTOR siRNA was transfected into each of these endometrial cancer cell lines. Control siRNA was transfected into these same lines as the control for these experiments. A significant decrease in cell growth after 48 to 96 hours was found in those cells transfected with mTOR siRNA and treated with cisplatin (10 μM), as compared with either of these treatments used alone (Fig. 4A). Synergy was also found between cisplatin and mTOR siRNA in these cell lines (CI < 1; range, 0.565-0.850) (Table 2). Transfection of mTOR siRNA alone did not induce apoptosis, but combined treatment with cisplatin increased apoptosis at higher levels than noted in cells treated with cisplatin only (Fig. 4B). Western immunoblot analysis showed that cells transfected with mTOR siRNA specifically reduced mTOR protein expression and decreased phosphorylation of S6 protein, a downstream target of this pathway (Fig. 4C). These results indicate that mTOR is a key target in regulation of rapamycin sensitization of endometrial cancer cells to cisplatin-induced apoptosis.

Figure 4.

Treatment of Ishikawa and ECC-1 cells with mammalian target of rapamycin (mTOR) small interfering RNA (siRNA) was found to increase sensitivity to cisplatin (PDD). (A) Cells transfected with 100 nM of mTOR siRNA ± 10 μM of cisplatin were compared with control siRNA-transfected cells treated with rapamycin (Rap) (1 nM), cisplatin (10 μM), or both combined. Cell proliferation was determined by MTS at 72 hours. (B) The effects of mTOR siRNA and cisplatin on apoptosis were examined using the M30 antibody by flow cytometry. Cells were transfected with 100 nM of mTOR siRNA or control siRNA for 8 hours. Cisplatin at 10 μM was added to both mTOR siRNA–transfected and control siRNA–transfected cells for an additional 48 hours. (C) ECC-1 cells transfected with mTOR siRNA were compared with control siRNA-transfected cells and with cells having received a 48-hour exposure to 1 nM of rapamycin by Western blot analysis. Protein expression was analyzed for mTOR, phosphorylated-S6 (p-S6), 4E-binding protein 1 (4E-BP1), and β-actin.

Table 2. Combination Index Values for Ishikawa and ECC-1 Cell Lines Treated With mTOR siRNA and Cisplatin
Cisplatin, μMmTOR siRNA, nMCI for Ishikawa CellsCI for ECC-1 Cells
  1. mTOR indicates mammalian target of rapamycin; siRNA, small interfering RNA; CI, combination index.

11000.8500.795
101000.5650.680

Effect of Rapamycin on DNA MMR Protein Expression

Although treatment with rapamycin alone does not affect apoptosis in the endometrial cancer cell lines, rapamycin did appear to enhance cisplatin-induced apoptosis (Fig. 2). To determine a possible underlying mechanism for this finding, we assessed expression of the DNA MMR proteins, MSH2 and MSH6, after exposure to rapamycin (1 nM), cisplatin (10 μM), or both in combination. We initially performed a time course experiment and studied their individual and combined effects at 12 hours, 24 hours, 48 hours, and 72 hours of exposure. Although we noted a small effect at 12 hours, greater effects were observed between 24 and 72 hours after treatment with either cisplatin alone or combination treatment with cisplatin and rapamycin (data not shown). Different doses of each of these drugs were also evaluated, with greater effects noted with increasing doses, particularly of cisplatin. On the basis of these initial experiments, we decided to use the IC50 doses for each drug and the 48-hour time point for the majority of the work. Cisplatin increased expression of both MSH2 and MSH6 in the ECC-1 and Ishikawa cell lines (Fig. 5). As quantified by a densitometer and normalized to control, cisplatin treatment resulted in a 1.9–fold to 2.3–fold increase in MSH2 expression and a 1.3–fold to 2.1–fold increase in MSH6 expression. The combination of cisplatin and rapamycin resulted in greater stimulation of MSH2 and MSH6 expression than cisplatin alone. Treatment with cisplatin and rapamycin increased MSH2 expression by 2.5–fold to 2.8–fold and increased MSH6 expression by 1.5–fold to 2.4–fold. These effects were more pronounced in the Ishikawa cell line. Rapamycin alone consistently appeared to have no effect on MSH2 and MSH6 expression, even at different time points or doses. Thus, the increased expression of MSH2 and MSH6 by rapamycin in cisplatin-treated endometrial carcinoma cell lines may contribute to rapamycin's observed enhancement of cisplatin-induced apoptosis. These results, although subtle, were found to be consistent among 3 independent Western blot analyses.

Figure 5.

Rapamycin (Rapa) increased expression of DNA mismatch repair proteins in cisplatin (PDD)-treated endometrial carcinoma cell lines. Both Ishikawa and ECC-1 cells were cultured in the presence of cisplatin (10 μM), rapamycin (1 nM), or both in combination for 48 hours. Total proteins were analyzed by Western blot analysis with antibodies against MSH2 and MSH6. Note rapamycin alone did not affect MSH2 and MSH6 expression. The corresponding densitometer values are included for the MSH2 and MSH6 proteins.

DISCUSSION

We have demonstrated a synergistic relation between rapamycin and cisplatin in regard to both the inhibition of cell growth and induction of apoptosis in human endometrial cancer cell lines. This was further substantiated by blocking mTOR expression through mTOR siRNA and demonstrating that decreased mTOR signaling is an essential mechanism for rapamycin chemosensitization and cisplatin-induced apoptosis. Furthermore, rapamycin increased the expression of DNA MMR proteins in cisplatin-treated endometrial carcinoma cell lines, and this may be a possible underlying mechanism for rapamycin's enhancement of cisplatin-induced apoptosis. This suggests that the combination of rapamycin and cisplatin may be an effective targeted therapy for endometrial cancer.

Synergy in the current study was quantified using the combination index equation of Chou and Talalay, which allows for the evaluation of 2 or more chemotherapeutic agents at different concentrations and effect levels.21 Through this methodology, combinations of drugs can be analyzed for synergy versus antagonism as well as their maximal antitumor efficacy. Such analyses have been the scientific basis for clinical protocol designs, such as the use of trastuzumab with docetaxol and cisplatin in patients with human epidermal growth factor receptor-2(HER-2)–positive advanced breast cancer.28, 29 Traditionally, new combination therapeutic treatments for cancer have evolved through the addition of a new drug to a pre-existing established regimen. Given the number of new and old drugs available, this strategy is useful and efficient in identifying which combinations have statistically significant synergistic interactions in vitro, thus narrowing the potential choices to those combinations that may have the greatest clinical benefit and deserve testing in future human trials.

DNA-damaging agents, such as cisplatin, have had a major impact on the treatment of a wide range of cancers, including gynecologic malignancies, but the use of cisplatin is often limited by its intolerable side effects and inevitable chemoresistance. Rapamycin and its derivatives have been proposed as potentially potent chemotherapeutic chemosensitizers. Cisplatin and rapamycin have exhibited synergy through the inhibition of cell proliferation and induction of apoptosis in many types of cancer cells, including lung cancer, breast cancer, ovarian cancer, and primitive neuroectodermal tumor cells.11, 12, 14, 16 We have demonstrated that even low doses of rapamycin (1 nM) in combination with low doses of cisplatin (0.01 μM) resulted in a strong synergistic effect and a substantial 2–fold to 3–fold decrease in the IC50 of cisplatin (Fig. 1) (Table 1). Although rapamycin does not significantly induce apoptosis in these endometrial cancer cell lines,25 we found that treatment with rapamycin and cisplatin together increased the induction of apoptosis and caspase activity, well above the effects of cisplatin alone (Fig. 2). Thus, mTOR inhibition by rapamycin may have the potential to inhibit tumor growth as well as enhance the effects of cisplatin at lower doses, culminating in overall decreased multiorgan toxicities for endometrial cancer patients.

Although rapamycin and its derivatives clearly act as cytostatic agents by arresting cells in the G1 phase, these agents are generally not believed to induce apoptosis, except potentially in tumor cells lacking functional p53.30, 31 Both of the endometrial cancer cell lines in this study express functional p53 (data not shown), which allows us to believe that the results shown in Figure 2 are not a result of an independent increase in rapamycin-induced apoptosis. Thus, the mechanism by which rapamycin enhances cisplatin-induced apoptosis is poorly understood. It has been demonstrated that mTOR inhibitors may sensitize tumor cells to DNA-damaging agents by blocking up-regulation of p21, thus shifting the equilibrium of the cell toward apoptosis.11 This was shown to be true for wild-type p53 human lung cancer cell lines, but not mutant p53 tumor cells.11 In contrast, in p53-deficient human ovarian cancer cell lines, cisplatin in combination with the mTOR inhibitor RAD001 resulted in induction of p21.32, 33 In a subsequent study, RAD001 enhanced the sensitivity of these ovarian cancer cell lines to cisplatin, independent of p53 status.14 This discrepancy between studies may reflect inherent molecular differences between tumor types, resulting in interruption of specific signaling cascades, and thus their potentially varied response to mTOR inhibition in combination with DNA-damaging agents.

DNA MMR is a highly conserved pathway that follows a stepwise sequence of events, involving the recognition and correction of DNA errors occurring spontaneously during replication. Several proteins are involved in the initiation of MMR, including the 3 MutS homologs (MSH2, MSH3, and MSH6). The MutS homologs form a heterodimer that recognizes DNA damage. This process eventually leads to excision of the damaged DNA strand and resynthesis. A functional MMR system is required for the detection of DNA damage created by cisplatin-induced adducts.34, 35 Platinum complexes interfere with normal MMR activity, resulting in failure of DNA repair and ultimately apoptosis. Loss of MMR function, possibly by altering adduct detection, has been linked to cisplatin-resistance in a variety of different tumor types.36

As would be predicted, cisplatin increased expression of MSH2 and MSH6 in the endometrial cancer cell lines. However, surprisingly, treatment with rapamycin appeared to stimulate MSH2 and MSH6 expression in cisplatin-treated endometrial carcinoma cell lines (Fig. 5). This finding, although subtle as demonstrated by Western immunoblot analysis, was found to be consistent between 3 independent experiments. To our knowledge, this is the first reported link between rapamycin and regulation of MMR proteins. These results, in part, may be an alternative explanation for rapamycin's enhancement of cisplatin-induced apoptosis that warrants further investigation. Given that the abortive efforts to repair DNA damage are paramount to cisplatin's cytotoxicity, we theorize that rapamycin facilitates this process by augmenting production of the MMR proteins.

Rapamycin is known to inhibit mTOR kinase activity, resulting in dephosphorylation of S6 and 4E-BP1, which are 2 critical downstream translational regulators. Inhibition of S6 and 4E-BP1 activity leads to decreased global translation and ribosome biogenesis of proteins that are essential for cell cycle progression from G1 to S‒phase.3, 4 Cisplatin treatment alone did not affect phosphorylation of S6 (Fig. 3), whereas combining treatment with rapamycin reduced phosphorylation of S6 with a concomitant reduction in total S6 protein. Knockout of mTOR by targeted siRNA increased cellular sensitivity to cisplatin, as manifested through the inhibition of proliferation and induction of apoptosis (Fig. 4) (Table 2). This suggests that decreased mTOR signaling is an essential step for rapamycin-enhanced chemosensitivity to cisplatin in endometrial cancer cells, but the underlying molecular mechanism still remains to be determined. However, given that these 2 agents appear to exert their antitumorigenic effects through distinct but somehow convergent pathways in relation to cell growth and death, this is the probable basis for their synergistic relationship and lends credence for their potential clinical application.

Rapamycin and its analogues have been shown to increase the efficacy of several cytotoxic and hormonal agents in other tumor types, but to our knowledge have not been systematically studied in endometrial cancer cells. mTOR inhibitors have demonstrated great promise in endometrial cancer as single agents, but our hypothesis is that combination therapy may afford better alternatives for a cancer in which cytotoxic and hormonal agents have had limited success. Thus, our future goals are to evaluate mTOR inhibitors in combination with other chemotherapeutic agents commonly used in the treatment of endometrial cancer to assess potential synergy in vitro and in vivo. This work should potentially provide the scientific foundation for rational combinations to test in subsequent clinical trials of women with recurrent or metastatic endometrial cancer.

Acknowledgements

We thank Gleta Carswell for her help with the flow cytometry experiments.

Conflict of Interest Disclosures

Supported by the University of North Carolina Clinical Translational Science Award-K12 Scholars Program (KL2 RR025746), the V Foundation, and the Steelman Fund.

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