PI3K/AKT/mTOR pathway plays a key role in the tumorigenesis of many human cancers including prostate cancer. However, inhibitors of this pathway, such as Rad001, have not shown therapeutic efficacy as a single agent. Through a high-throughput screen of 5,000 widely used small molecules, we identified compounds that can synergize with Rad001 to inhibit prostate cancer cells. One of the compounds, propachlor, synergizes with Rad001 to induce apoptosis of castration-resistant prostate cancer cells via enhanced autophagy. This enhanced autophagic cell death is accompanied by increased Beclin1 expression as well as upregulation of Atg5–Atg12 conjugate and LC3-2. Rad001 and propachlor can also synergistically inhibit tumors in a xenograft animal model of prostate cancer. These findings provide a novel direction to develop combination therapies for advanced and metastatic prostate cancer that has failed the currently available therapies. Mol Cancer Ther; 11(6); 1320–31. ©2012 AACR.

Prostate cancer is the most common malignancy and the second most common cause of cancer-related death among men in Western countries (1, 2). Although prostate cancer in early stages can be cured by local therapies, there is no cure for advanced and metastatic prostate cancer. Therefore, there is an urgent need to develop novel and effective systemic therapies. As has been observed in many human cancers, growth factor signaling pathways, particularly PI3K/AKT/mTOR pathway, are critical for the development of prostate cancer. A genomic survey of prostate cancer identified mutations in this pathway, leading to its hyperactivity (3). Inactivation of PTEN, a negative regulator of this pathway, has been found in a significant portion of human prostate cancer (3), and tissue-specific deletion of PTEN is sufficient to initiate prostate cancer in a mouse model (4). Thus, inhibition of the PI3K/AKT/mTOR pathway will likely suppress prostate cancer and provide therapeutic benefits. Rapamycin or its derivative, Rad001 (Everolimus; Fig. 1B), can specifically and potently inhibit mTORC1. Indeed, rapamycin (we will refer to Rad001, which is a more stable derivative, thereafter) has been approved to treat advanced kidney cancer as well as neuroendocrine pancreatic cancer. However, efforts to expand its use in more prevalent cancers including prostate cancer have been unsuccessful. It is postulated that inhibition of mTORC1 by Rad001 leads to activation of compensatory signaling pathways thus countering the growth-inhibitory effects (5). Novel compounds that simultaneously inhibit several signaling pathways, including mTORC1, have been identified, promising to more effectively suppress the tumor growth (6). Alternatively, identification of compounds that synergize with Rad001 may enhance the potency of Rad001 to treat human cancers. We conducted a high-throughput screen of 5,000 compounds including more than 1,000 U.S. Food and Drug Administration (FDA)-approved drugs as well as purified natural products and other compounds with known safety profiles. We identified propachlor (Fig. 1B) as a compound that synergizes with Rad001 to induce cell death in prostate cancer cells.

Figure 1.

Identification of propachlor as a collaborating compound with Rad001. A, five thousand compounds were added alone or together with 20 nmol/L Rad001 in C4-2 cells, the survival ratio gauged by ATP measurement between cells that are treated with single compound or combination with Rad001 is plotted. B, the chemical structures of Rad001 and propachlor. C, propachlor can collaborate with Rad001 to inhibit PC3 and C4-2 cells. Single compounds or combination with Rad001 were used to treat PC3 or C4-2 cells for 72 hours followed by CellTiter-Glo measurement to measure cell number. The 2-way t test showed P < 0.05 between the 2 groups.

Figure 1.

Identification of propachlor as a collaborating compound with Rad001. A, five thousand compounds were added alone or together with 20 nmol/L Rad001 in C4-2 cells, the survival ratio gauged by ATP measurement between cells that are treated with single compound or combination with Rad001 is plotted. B, the chemical structures of Rad001 and propachlor. C, propachlor can collaborate with Rad001 to inhibit PC3 and C4-2 cells. Single compounds or combination with Rad001 were used to treat PC3 or C4-2 cells for 72 hours followed by CellTiter-Glo measurement to measure cell number. The 2-way t test showed P < 0.05 between the 2 groups.

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Materials

PC3 cells were obtained from the American Type Culture Collection (ATCC), and the C4-2 cells were kindly provided by Dr. Lily Wu at UCLA (Los Angeles, CA). Both cell lines were passaged for fewer than 6 months after resuscitation. The PC3 cells were tested and authenticated by the ATCC, and no authentication for either cell line was done by the authors. FBS, RPMI medium 1640, Dulbecco's Modified Eagle's Medium (DMEM), sodium pyruvate, l-glutamine, penicillin, and streptomycin were purchased from Hyclone; propachlor and Rad001 were from Sigma; CellTiter assay was from Promega; Caspase-3/7 Assay Kit was from Anaspec; Annexin V Apoptosis Detection Kit was from eBioscience; DharmaFECT transfection reagent was from Thermo Scientific Life Science; Lipofectamine 2000 transfection reagent was from Invitrogen; Beclin1-siRNA (5′-rGrGrA rArUrGrGrArArUrGrArrGrArUrUrArA, 5′-rArGrCrArGrCrArUrUrArArUrCrUrCrArUrU) and control-siRNA (5′-rGrArArArArArCrUrCrArUrArUrArArArUrCr, 5′-rGrUrGrGrGrGrCrGrA rUrUrUrArUrArUrGrA) were from IDT; and reverse transcriptase PCR (RT-PCR) primers for Beclin1, 5′-GGCCAATAAGATGGGTCTGA-3′ and 5′-CTGCACACAGTCCAGGAAAG-3′, for GAPDH 5′-CATGGGTGTGAACCATGAGA-3′ and 5′-CAGTGATGGCATGGACTGTG-3′, were from ValueGene. Rabbit anti-LC-3 polyclonal antibody was from GenScript; rabbit anti-Beclin1, rabbit anti-cleaved caspase-3, and rabbit anti-PARP1 polyclonal antibodies were from Cell Signaling; rabbit anti-caspase-3 polyclonal antibody was from Abgent; rabbit anti-Atg5 monoclonal antibody was from Epitomics; and mouse anti-β-actin antibody was from Sigma.

High-throughput screen

C4-2 cells were seeded in 384-well plate at 1,000 cells per well. After overnight incubation, compounds were delivered to the plates by SAGIAN Core System with a Biomek FX equipped with 500 nL pin tool, and Rad001 was added to a final concentration of 20 nmol/L using a Multidrop 384. The concentration of the compounds from the compound libraries was 10 μmol/L final, whereas the total volume was 50 μL and the dimethyl sulfoxide (DMSO) concentration was 1% or less. Ninety-six hours later, cell number was determined using CellTiter-Glo (Promega) on a VICTOR 3V (PerkinElmer), according to the manufacturers' instructions. The potential hits were identified as those that led to 50% or more reduction of cell number with the combination of Rad001 and the compound than with the compound alone. The hits were further tested in conventional tissue culture settings to verify their synergistic effects with Rad001 in decreasing cell viability.

Cell culture

The PC3 cells were maintained in the DMEM containing 10% FBS penicillin-streptomycin, l-glutamine, sodium pyruvate; and the C4-2 cells were maintained in RPMI-1640 supplemented with 10% FBS and penicillin-streptomycin. Cells were grown at 37°C with 5% CO2.

Cell viability, combination index, and growth curves

The cells were seeded into the 96-well plate at 3 × 103 cells per well. After 48 hours, PC3 cells were treated with DMSO, 0.055 to 0.88 μmol/L Rad001 for 24 hours, and 0.89 to 14.25 μmol/L propachlor or their combination for 24 hours. The C4-2 cells were treated with DMSO, 0.041 to 0.65 μmol/L Rad001 for 24 hours, and 0.25 to 4.08 μmol/L propachlor or their combination for 24 hours. Triplicates were used for each treatment group. The cell viability/relative cell number was measured with the Promega CellTiter-Glo Assay according to manufacturer's instructions. The compound interactions were analyzed with CalcuSyn software (version2.1, BIOSOFT) to determine the combination index (CI) for Rad001 and propachlor.

Equal numbers of cells were seeded into 96-well plate and maintained with normal medium. After 48 hours, the PC3 cells were treated with DMSO (control), Rad001 (0.70 μmol/L), propachlor (6.15 μmol/L), or their combination, respectively. The C4-2 cells were treated with DMSO (control), Rad001 (0.56 μmol/L), propachlor (7.05 μmol/L), or their combination, respectively. The cell viability/relative cell number was measured on days 1, 2, 3, and 4 after compound addition by CellTiter-Glo. All the experiments were carried out in triplicates. The PC3 and C4-2 cells were also treated with compounds for 1 day and analyzed with trypan blue staining.

GFP-LC3 analysis

Cells were transfected with GFP-LC3 plasmid using Lipofectamine 2000 transfection reagent. After 24 hours, the medium was changed, and the PC3 cells were treated with DMSO (control), Rad001 (0.7 μmol/L), propachlor (6.15 μmol/L), or their combination, respectively, for 1 day. The C4-2 cells were treated with DMSO (control), Rad001 (0.56 μmol/L), propachlor (7.05 μmol/L), or their combination, respectively, for 1 day. The cells were fixed in 4% paraformaldehyde for 30 minutes, washed twice with PBS and stained with 4′,6–diamidino–2–phenylindole (DAPI), and observed under a fluorescence microscope (Eclipse 90i slide scope) with ×40 lens.

Protein analysis

The cultured cells were washed with cold PBS and lysed with lysis buffer (20 mmol/L KCl, 150 mmol/L NaCl, 1% NP-40, 50 mmol/L NaF, 50 mmol/L Tris-HCl, pH 7.5, 1 mmol/L dithiothreitol, 1 mmol/L EGTA, 1 × Protease Inhibitor, 10% glycerol) for 10 minutes on ice. The cells were centrifuged for 15 minutes at 4°C. The protein concentration in the supernatant was determined with the Bradford Assay (Bio-Rad). Equal amount of protein was loaded on 8% or 15% SDS-PAGE and transferred to polyvinylidene fluoride membrane. The membrane was blocked with nonfat dry milk for 1 hour, incubated with primary antibody in nonfat dry milk overnight, washed with PBS for 30 minutes, incubated with secondary antibody for 30 minutes, washed with PBS/0.1%Tween-20 for 2 hours, and detected with enhanced chemiluminescence (Pierce).

Caspase-3/7 activity analysis

Equal number of PC3 and C4-2 cells was seeded into the 96-well plates. After 48 hours, cells were treated the DMSO, Rad001, propachlor, or their combination for 15 hours. The caspase-3/7 activity was measured by the SensoLyte Homogeneous AMC Caspase-3/7 Assay Kit after reaction for 8 hours. All the experiments were carried out in triplicates.

Analysis of apoptosis

After 15-hour treatment, the PC3 and C4-2 cells were collected. The apoptosis was quantified by fluorescence-activated cell-sorting (FACS) analysis (BD FACSDiva Software v6) with Annexin-V/7-AAD staining following the manufacturer's guidelines. The percentage of Annexin-V–positive cells was analyzed by FlowJo (Version 7.6.4).

Quantitative RT-PCR

The PC3 and C4-2 were treated with compounds for 1 day and total RNA was purified from the cells with Fermentas Gene RNA Purification Kit. Equal amounts of RNA were reverse-transcribed by reverse transcriptase (Fermentas) according to the manufacturer's instructions. Quantitative real-time PCR was carried out with SA Biosciences RT2 Real-time SYBR Kit with the following parameters: 15 μL, 95°C for 8 minutes for one cycle followed by 43 cycles of 95°C for 15″/60°C for 60″.

siRNA transfection

A total of 4 × 103 of cells were seeded into the 96-well plates and transfected with siRNA (100 nmol/L) by the DharmaFECT General Transfection Reagent. The combination of Rad001 and propachlor was added to the cells for 1 day after transfection followed by cell viability/relative cell number measurement. To determine the knockdown efficiency, cells were seeded into the 12-well plate followed by transfection with Beclin1-siRNA and control-siRNA, respectively, and collected after another 24 hours.

Beclin1 mRNA half-life analysis

To determine the metabolic stability of Beclin1 mRNA, the C4-2 cells were treated with DMSO, Rad001, propachlor, and their combination. Actinomycin D was added at 10 μg/mL to inhibit transcription. After 0, 1, 2, and 4 hours, cells were collected and washed with PBS, and total RNA was extracted. Relative levels of mRNAs were determined by real-time PCR and normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the experiments were carried out in triplicates.

Cell-cycle analysis

After 24-hour treatment, PC3 and C4-2 cells were harvested, washed with PBS, and fixed in 70% ethanol. After 1-day fixation, the cells were washed with PBS twice, treated with RNase (50 μg/mL), and stained with propidium iodide (50 μg/mL) for 30 minutes at 37ºC. The cell-cycle phase distribution was determined by Flow Cytometry (BD FACSDiva Software v6). The percentage of cells in each phase was analyzed by FlowJo (Version 7.6.4).

Prostate cancer xenograft

Five- to 6-week-old SCID (severe combined immunodeficient) mice were obtained from the UCLA Division of Laboratory Animal Medicine. All the mice were inoculated with 100 μL (50% Matrigel/PBS) of PC3 cells suspension (7 × 106) to each dorsal flank with 25-gauge syringe. When the tumor size reached between 90 and 100 mm3, the mice were divided into the control, Rad001, propachlor, and combination group, with 6 mice per group. The Rad001 and propachlor were delivered intraperitoneally (i.p.) 1 and 5 mg/kg daily. The tumor size and mouse weight were measured every 4 days. These tumor measurements were converted to tumor volume using the formula, V = 0.52 × L × W2, where W and L are the smaller and larger diameters. At the 44th day, all mice were sacrificed; tumors were dissected and collected. All the animal experiments were conducted according to the protocol approved by the UCLA Animal Research Committee.

Statistical analysis

The normalized isobologram analysis and bars were conducted with CalcuSyn software (version 2.1, BIOSOFT) and Microsoft Excel 2003, respectively. With median effects model described by Chou (7), the multiple compound dose–effect calculations were conducted. CI values of <0.9, >0.9 to <1.2, >1.2 were considered as being synergistic, additive, and antagonistic, respectively. Statistical analysis was conducted by 2-sided t test. P value less than 0.05 was considered to be statistically significant.

The statistical significance of different tumor sizes between each treatment and control group was determined by one-way ANOVA followed by the Dunnett test. Statistical analyses on body weights were conducted by one-way ANOVA followed by Tukey test. The level of significance was set at P < 0.05. Statistical calculations were carried out by the SPSS version 13.0.

Identification of compounds that synergize with Rad001 to inhibit prostate cancer cells

PI3K/AKT/mTOR signaling pathway plays a central role in many human cancers including prostate cancer. However, inhibitors of this pathway, such as Rad001, have failed to show efficacy for prostate cancer as a single agent likely due to activation of compensatory pathways (5). We reasoned that certain chemical compounds may synergize with Rad001 to inhibit multiple pathways and thus restore the full therapeutic potential of Rad001, a clinically useful drug with known safety profiles. Therefore, we chose 5,000 compounds available through our screening facility and conducted a screen in castration-resistant prostate cancer (CRPC) cells using individual compound alone or in combination with Rad001 (Fig. 1A). The primary screen yielded a number of compounds that decreased cell numbers by more than 50% in the presence of Rad001 compared with when the compounds were used alone. Several compounds collaborated with Rad001 in reducing the cell numbers in secondary and tertiary screens, and propachlor (Fig. 1B) was chosen for detailed studies.

Synergy of Rad001 and propachlor in inhibiting prostate cancer cell lines PC3 and C4-2

To confirm that Rad001 and propachlor can synergistically inhibit prostate cancer cells, we added each of them at varying concentrations and monitored the effects in PC3 and C4-2 cells, 2 prostate cancer cell lines that resist androgen withdrawal. As shown in Fig. 2A, dose-dependent decrease of cell numbers was seen in both cell lines treated with either Rad001 or propachlor. For PC3 cells, the ED50 for propachlor was 6.55 μmol/L, and we did not reach ED50 for Rad001 under the experimental conditions. For C4-2 cells, we did not reach ED50 for propachlor, whereas the ED50 for Rad001 was 0.43 μmol/L. However, when the cells were treated with the combination of the 2 compounds, there was significantly more decrease in cell numbers in both cell lines (z-axis) compared with treatment with either Rad001 or propachlor alone (Fig. 2A). CI, a measure of synergistic activity, was determined with the CalcuSyn software program, which confirmed significant synergism of the 2 compounds (Fig. 2B). For example, for PC3 cells, treatment with 0.44 μmol/L Rad001 or 3.56 μmol/L propachlor alone resulted in 10.45% and 17.15% reduction in the cell number, respectively. However, when the 2 compounds were combined, the cell number was reduced by 67.21% (CI < 0.9). Mixture-algebraic estimate analysis, conducted with CalcuSyn software, showed a strong synergism for most of the combinations of 2 compounds at various concentrations for the 2 cell lines, suggesting that the synergism is not limited to a particular threshold concentration of either compound. We also determined whether these compounds synergize at different time points after compound addition. As shown in Fig. 2C, both PC3 and C4-2 cells exhibited pronounced reduction of cell numbers after combination treatment at multiple time points.

Figure 2.

The combination of Rad001 and propachlor induces a synergistic reduction of cell number in prostate cancer cells. A, decreasing doses of Rad001 alone, propachlor alone, and their combination were used, and cell numbers measured as in Fig. 1 (x-axis, Rad001 μmol/L; y-axis, propachlor μmol/L; z-axis, 1 − (treatment luminescence unit/control luminescence). B, normalized isobologram analysis showed synergistic interactions in PC3 and C4-2 cells. The analysis was done with CalcuSyn software, which conducts the drug dose–effect calculation with the median effect method described by Chou (7). A large number of combination groups were below the line, indicating synergism. C, growth curves by measuring ATP level shows more reduction of cell number when cells were treated with the drug combination.

Figure 2.

The combination of Rad001 and propachlor induces a synergistic reduction of cell number in prostate cancer cells. A, decreasing doses of Rad001 alone, propachlor alone, and their combination were used, and cell numbers measured as in Fig. 1 (x-axis, Rad001 μmol/L; y-axis, propachlor μmol/L; z-axis, 1 − (treatment luminescence unit/control luminescence). B, normalized isobologram analysis showed synergistic interactions in PC3 and C4-2 cells. The analysis was done with CalcuSyn software, which conducts the drug dose–effect calculation with the median effect method described by Chou (7). A large number of combination groups were below the line, indicating synergism. C, growth curves by measuring ATP level shows more reduction of cell number when cells were treated with the drug combination.

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Combination of Rad001 and propachlor synergistically induces apoptosis in PC3 and C4-2 cells

The synergistic reduction of cell number can be the result of a cell-cycle block or an induction of cell death or their combination. We conducted a cell-cycle analysis of the cells treated with the compounds but did not detect a significant change in the distribution of cell-cycle phases. We conducted trypan blue staining of the cells after the drug treatment. As shown in Fig. 3A, there was a significant decrease in the percentage of cells negative for intracellular blue staining in both PC3 and C4-2 cells compared with single compound treatment (P < 0.005), suggesting that the combination treatment resulted in decreased cell viability. This was further confirmed with the semiquantitative analysis of the abundance of proteins involved in cell apoptosis. As shown in Fig. 3B, when PC3 and C4-2 cells were treated with vehicle (DMSO), Rad001, propachlor, or their combination for 15 hours, there was a significant increase of the cleaved form of apoptosis marker PARP1 (Fig. 3B, top; ref. 8). This was accompanied by the induction of cleaved caspase-3 (Fig. 3B, middle), reduced expression of Bcl-2 (Fig. 3B, bottom), and induced activity of caspase-3/7 (Fig. 3C), suggesting that apoptotic pathway was activated in response to the combination treatment. To provide another level of confirmation, we conducted flow cytometric analysis of cells stained with Annexin-V and 7-AAD to examine the population level of apoptotic response to the compounds. As shown in Fig. 3D, combination of Rad001 and propachlor increased the fraction of apoptotic cells significantly more than single treatment, as quantified by the percentage of both early and late apoptotic cells in the 2 cell lines. These data strongly suggest that combination of Rad001 and propachlor results in synergistic cytotoxicity through the activation of the apoptotic pathway.

Figure 3.

Combination of Rad001 and propachlor synergistically induces cell death in prostate cancer cells. PC3 cells were treated with DMSO (control), Rad001 (0.70 μmol/L), propachlor (6.15 μmol/L), or their combination, and C4-2 cells were treated with DMSO (control), Rad001 (0.56 μmol/L), propachlor (7.05 μmol/L), or their combination. A, trypan blue (TB) assay. Combination treatment resulted in lower cell viability than either compound alone (2-way t test: P < 0.005). B, increased levels of cleaved PARP1 and caspase-3 and decreased Bcl-2 in PC3 and C4-2 cells treated for 15 hours with the combination of Rad001 and propachlor. Lysates from cells were separated by SDS-PAGE followed by immunoblotting with respective antibodies. C, higher activities of caspase-3/7 in PC3 and C4-2 cells treated for 15 hours with the drug combination. D, flow cytometric analysis of apoptosis with Annexin-V and 7-AAD staining. There was a significant increase in the percentage of apoptotic cell after combination treatment versus single compound (P < 0.05).

Figure 3.

Combination of Rad001 and propachlor synergistically induces cell death in prostate cancer cells. PC3 cells were treated with DMSO (control), Rad001 (0.70 μmol/L), propachlor (6.15 μmol/L), or their combination, and C4-2 cells were treated with DMSO (control), Rad001 (0.56 μmol/L), propachlor (7.05 μmol/L), or their combination. A, trypan blue (TB) assay. Combination treatment resulted in lower cell viability than either compound alone (2-way t test: P < 0.005). B, increased levels of cleaved PARP1 and caspase-3 and decreased Bcl-2 in PC3 and C4-2 cells treated for 15 hours with the combination of Rad001 and propachlor. Lysates from cells were separated by SDS-PAGE followed by immunoblotting with respective antibodies. C, higher activities of caspase-3/7 in PC3 and C4-2 cells treated for 15 hours with the drug combination. D, flow cytometric analysis of apoptosis with Annexin-V and 7-AAD staining. There was a significant increase in the percentage of apoptotic cell after combination treatment versus single compound (P < 0.05).

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Combination of Rad001 and propachlor synergistically induces autophagy in PC3 and C4-2 cells

It is important to understand the mechanism by which the combination of the 2 compounds synergistically activates the apoptotic pathway. Because programmed cell death can be induced by autophagy, we determined whether the 2 compounds synergistically induce autophagy. Enhanced conversion of microtubule-associated protein 1 light chain 3 (LC3-1) to its faster-migrating form LC3-2 is a hallmark of autophagy induction (9). As shown in Fig. 4A, there was substantially more LC3-2 conversion after combination treatment compared with Rad001 or propachlor treatment alone in both PC3 and C4-2 cell lines, suggesting that they can synergistically induce autophagy. We also examined the abundance of autophagosomes induced by the compounds. Autophagosomes are characterized by membranous structures with double or multiple membrane layers. Transmission electron microscopic analysis of the cells treated with various compounds showed that the cells treated with the 2 compounds in combination had increased number of autophagosomes in comparison with single-agent treatment (Fig. 4B, top), again confirming that there was a synergistic induction of autophagy. The quantification of this increase is shown in Fig. 4B (bottom).

Figure 4.

Rad001 and propachlor combination synergistically induces autophagy in PC3 and C4-2 cells. Autophagy was analyzed after 1 day of treatment. PC3 cells were treated with DMSO (control), Rad001 (0.70 μmol/L), propachlor (6.15 μmol/L), or their combination, and C4-2 cells were treated with DMSO (control), Rad001 (0.56 μmol/L), propachlor (7.05 μmol/L), or their combination. A, upregulation of LC3-2 in PC3 and C4-2 cells treated with combination of the 2 compounds. Immunoblotting was conducted as in Fig. 3. B, increased autophagosomes (arrows) were observed by electron microscopy in cells treated with Rad001/propachlor compared with each compound alone. Quantified data from 30 cells are shown in the bottom. C, autophagosome analysis through GFP-LC3 expression. C4-2 and PC3 cells expressing GFP-LC3 were treated with DMSO, Rad001, propachlor, or their combination for 24 hours. The cells were fixed with paraformaldehyde and visualized with epifluorescence. Yellow arrows indicate the punctate pattern of GFP-LC3, representative of autophagosome, and nuclei were visualized through DAPI staining. The number of GFP-LC3 punctuate dots/cells was quantitated in 50 GFP+ cells from each group (bottom).

Figure 4.

Rad001 and propachlor combination synergistically induces autophagy in PC3 and C4-2 cells. Autophagy was analyzed after 1 day of treatment. PC3 cells were treated with DMSO (control), Rad001 (0.70 μmol/L), propachlor (6.15 μmol/L), or their combination, and C4-2 cells were treated with DMSO (control), Rad001 (0.56 μmol/L), propachlor (7.05 μmol/L), or their combination. A, upregulation of LC3-2 in PC3 and C4-2 cells treated with combination of the 2 compounds. Immunoblotting was conducted as in Fig. 3. B, increased autophagosomes (arrows) were observed by electron microscopy in cells treated with Rad001/propachlor compared with each compound alone. Quantified data from 30 cells are shown in the bottom. C, autophagosome analysis through GFP-LC3 expression. C4-2 and PC3 cells expressing GFP-LC3 were treated with DMSO, Rad001, propachlor, or their combination for 24 hours. The cells were fixed with paraformaldehyde and visualized with epifluorescence. Yellow arrows indicate the punctate pattern of GFP-LC3, representative of autophagosome, and nuclei were visualized through DAPI staining. The number of GFP-LC3 punctuate dots/cells was quantitated in 50 GFP+ cells from each group (bottom).

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Autophagosomes can also be visualized through the expression of GFP-LC3, which upon autophagosome formation becomes clustered on the membrane vesicles and visible as a ring structure under a fluorescent microscope. Figure 4C (top) shows that there were more punctuate GFP-positive vesicles in response to the combination treatment in both PC3 and C4-2 cells, and the quantification of the results is shown in Fig. 4C (bottom). Consistent with the increase of autophagy in response to the combination treatment, we also found that Atg5–Atg12 conjugate, which is involved in the first of the two ubiquitination-like reactions that control autophagy (10), was increased in the 2 cell lines treated with the 2 compounds in combination (Fig. 5A). Taken together, we conclude that there is a synergistic induction of autophagy in response to the combinatorial treatment of Rad001 and propachlor, resulting in autophagic cell death.

Figure 5.

Regulation of autophagy proteins in response to combination treatment of Rad001 and propachlor. A, Atg5–Atg12 conjugates and Beclin1 were induced in response to combination treatment for 24 hours. Immunoblotting was conducted as in Fig. 3. B, Beclin 1 mRNA was more significantly upregulated by the combination treatment as shown by qRT-PCR analyses (left). Right, shows that Beclin1 mRNA stability does not increase in response to the combination treatment. C, Beclin1 is critical for the autophagic death induced by the combination treatment. PC3 and C4-2 cells were transfected with control siRNA or siRNA for Beclin1 (right). Cells were treated with combination of Rad001 and propachlor 24 hours after transfection. The cell number was determined with ATP measurement 24 hours after the combination treatment.

Figure 5.

Regulation of autophagy proteins in response to combination treatment of Rad001 and propachlor. A, Atg5–Atg12 conjugates and Beclin1 were induced in response to combination treatment for 24 hours. Immunoblotting was conducted as in Fig. 3. B, Beclin 1 mRNA was more significantly upregulated by the combination treatment as shown by qRT-PCR analyses (left). Right, shows that Beclin1 mRNA stability does not increase in response to the combination treatment. C, Beclin1 is critical for the autophagic death induced by the combination treatment. PC3 and C4-2 cells were transfected with control siRNA or siRNA for Beclin1 (right). Cells were treated with combination of Rad001 and propachlor 24 hours after transfection. The cell number was determined with ATP measurement 24 hours after the combination treatment.

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Combination of Rad001 and propachlor increases the expression of Beclin1, which is important for the induced autophagy and apoptosis

Beclin1 (Atg6) is critical for the initiation of autophagy pathway (11). Accordingly, we found that Beclin1 protein levels increased in response to the combination treatment (Fig. 5A, right). The increase of Beclin1 protein is likely the result of increased mRNA levels of Beclin1 as shown by qRT-PCR analysis (Fig. 5B, left). To determine whether the increase of mRNA level is the result of enhanced transcription of Beclin1 gene or increased stability of Beclin1 mRNA, we examined the metabolic stability of Beclin1 mRNA by measuring its half-life. As shown in Fig. 5B (right), Rad001 decreased Beclin1 mRNA stability, whereas propachlor slightly increased its stability and the combination of the 2 compounds did not increase Beclin1 mRNA stability as compared with the control group, suggesting that the increase of Beclin1 mRNA is likely the result of increased transcription of the gene.

To determine whether the induced autophagy is necessary for the increased cell death induced by the combination treatment, we used siRNA to reduce the expression of Beclin1 and examined whether its loss of function impacts on the cell death induced by the combination treatment. As shown in Fig. 5C, the Beclin1 level was markedly reduced by siRNA treatment (right) and reduction of Beclin1 protein led to a significant rescue of cell death in response to the combination treatment (left and middle). These results suggest that synergistic induction of autophagy is likely the underlying mechanism for the increased cell death induced by the 2 compounds used in combination. Taken together, these results suggest that Beclin1 is induced in response to the combination treatment, and the enhanced autophagy plays an essential role in the synergistic induction of cell death.

Rad001 and propachlor combination inhibits prostate cancer xenograft tumor

We next determined whether the synergism also exists in a preclinical prostate cancer xenograft mouse model. The toxicity of propachlor is relatively low with LD50 at 550 mg/kg (12), so we used it at 5 mg/kg D−1 while using Rad001 at 1 mg/kg D−1, which has been commonly reported (13). We initiated tumor with s.c. injection of PC3 cells on both flanks of SCID mice. When tumor size reached approximately 100 mm3, we started daily intraperitoneal injection of the 2 compounds and measured tumor size and body weight on every fourth day. As shown in Fig. 6A, the combination of Rad001 and propachlor inhibited the growth of PC3 xenograft tumor significantly more than either compound alone (**, P < 0.05 compared with one compound administration; *, P < 0.05 compared with control). In addition, combination treatment showed no adverse effect on body weights (Fig. 6A, right; P > 0.05) and daily activities. No complications such as anaphylaxis and skin necrosis were observed throughout the course of the study. We also examined autophagosome formation in the xenograft tumors by transmission electron microscopy. Consistent with the in vitro results, combination of the 2 compounds increased autophagosome formation in the xenograft tumors (Fig. 6B and C). In addition, there was a significant increase of LC3-1, LC3-2, and Beclin1 levels in the combination treatment group, compared with groups that received either compound alone (Fig. 6D). Therefore, we have shown that propachlor can synergize with Rad001 to induce apoptosis of prostate cancer cells through synergistic induction of autophagy in vitro and in vivo, establishing a foundation for potential clinical trials of similar combinations in patients with prostate cancer.

Figure 6.

Combination of Rad001 and propachlor significantly inhibits the PC3 xenograft tumors in mice. A, antitumor activity on PC3 xenografts by various compounds. Mice were administered daily i.p. with Rad001 (1 mg/kg), propachlor (5 mg/kg), or both (6 mice/group). The tumor sizes (left) and body weights (right) were measured every 4 days. Tumors treated with 2 drugs were much smaller than single compound treatment or control. B, electron microscopic analysis of the tumors collected from various treatment groups. Arrows indicate autophagosomes. C, quantitation of the number of autophagosomes in 30 cells from the xenograft tumors. D, immunoblot analysis for LC3 and Beclin1 from the xenograft tumors.

Figure 6.

Combination of Rad001 and propachlor significantly inhibits the PC3 xenograft tumors in mice. A, antitumor activity on PC3 xenografts by various compounds. Mice were administered daily i.p. with Rad001 (1 mg/kg), propachlor (5 mg/kg), or both (6 mice/group). The tumor sizes (left) and body weights (right) were measured every 4 days. Tumors treated with 2 drugs were much smaller than single compound treatment or control. B, electron microscopic analysis of the tumors collected from various treatment groups. Arrows indicate autophagosomes. C, quantitation of the number of autophagosomes in 30 cells from the xenograft tumors. D, immunoblot analysis for LC3 and Beclin1 from the xenograft tumors.

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Hormonal therapy has been the main treatment modality for advanced or metastatic prostate cancer for decades. However, hormonal therapy, including the newest drugs such as abiraterone and MDV3100, is palliative and nearly all patients will eventually experience tumor recurrence, for which there is no effective therapy. Thus, there is an urgent need to develop novel therapies targeting additional pathways for CRPC. It has been found that androgen enhances mTOR activity in an androgen receptor–dependent manner (14), and active mTOR signaling suppresses autophagy. Autophagosome formation is increased in the rat prostate epithelial cells upon castration (15, 16). More recently, it was reported that prostate cancer cells can enhance autophagy to survive androgen deprivation treatment (17). Therefore, it is likely that autophagy plays an important role in the development of prostate cancer, and targeting this pathway may be a novel therapeutic strategy.

Propachlor is a herbicide first marketed by Monsanto in 1965 (18) with well-established safety profile. Although this particular compound may or may not be used in patients with prostate cancer, its synergy with Rad001 and the underlying mechanism provide us novel therapeutic targets and an opportunity to increase the therapeutic efficacy and reduce the side effects of existing drugs. In this regard, the recent identification of combination treatment of rapamycin and thapsigargin for ras-driven cancer represents another excellent example (19). Identification of novel compounds that synergize with existing molecules will also expand our understanding of the molecular mechanisms of induced cancer cell death.

The main finding of our study is that the combination of Rad001 and propachlor induces apoptosis of CRPC cells in a synergistic manner, and enhanced autophagy is likely the underlying mechanism. Autophagy or macroautophagy is a major intracellular pathway for degrading and recycling cellular macromolecules, including proteins, ribosomes, and cytoplasmic organelles (20). In normal cells, autophagy functions to maintain cellular homeostasis by disposing of damaged or aged organelles. Autophagy is induced in response to stresses such as nutrient starvation, hormone treatment, chemotherapeutic agents, as well as in pathologic conditions, including neurodegenerative diseases such as Alzheimer disease, Parkinson disease, Huntington disease, hereditary myopathies, infectious diseases, and cancer (20–23). More recently, it was found that excessive autophagy can lead to cell death, especially in the absence of functional apoptotic molecules such as Bax/Bak (24). This type of cell death is termed the second type of programmed cell death (20). Thus, it is conceivable that the induction of autophagy is cell context–dependent, and the extent of autophagy dictates the cellular outcome. At an appropriate level, autophagy protects cells by recycling and disposing of toxic and nonfunctional cellular proteins and organelles, whereas excessive autophagy or a particular kind of autophagy may result in damage of the cells, ultimately leading to cell death. As such, agents have been identified that can cause tumor cell death and reduce tumor burden by causing autophagic cell death, suggesting that modulation of autophagy is a new avenue for anticancer therapeutics (25, 26). In gastric cancer cells, inhibition of caspase-3 enhances the autophagic cell death, suggesting that inhibition of apoptosis results in induction of autophagy (27). For prostate cancer, induction of autophagy and caspase-independent apoptosis by arginine deiminase appears to be a novel therapeutic modality (28). It has also been reported that inhibition of autophagy can lead to induction of apoptosis (29). Simultaneous induction of autophagy and apoptosis seems to be responsible for cell death in response to the combination treatment of histone deacetylase inhibitor and the Bcl-2 homology domain-3 mimetic GX15-070 (30). Rad001 induces autophagy but is not potent (31). Because propachlor can synergize with Rad001 to enhance autophagy and induce cell death in 2 CRPC cell lines, this may represent a new direction to develop therapies for advanced prostate cancer that has failed both traditional (e.g., LH-RH analog, casodex) and newer (e.g., abirateron, MDV3100) forms of hormonal therapy for which no effective treatment exists.

C4-2 was derived from LNCaP cells and has features of castration-resistant adenocarcinoma, such as expressing androgen receptor and prostate-specific antigen. PC3 cells are negative for androgen receptor and prostate-specific antigen but express neuroendocrine markers, similar to human prostate small cell carcinoma (32), which represents the most aggressive form of late-stage prostate cancer. Although the 2 cell lines are similarly resistant to androgen deprivation, they appear to have different sensitivities to the agents used in this study, which may have clinical implications.

In summary, we have shown that Rad001 and propachlor can synergize with each other to cause death of CRPC cells, thus providing a novel therapeutic possibility for this incurable disease. Our findings also establish autophagy induction as a novel direction of prostate cancer therapy.

No potential conflicts of interests were disclosed.

The authors thank Dr. Hong Zhang for helpful discussions.

J. Huang is supported by UCLA SPORE in Prostate Cancer (PI: Robert Reiter), Department of Defense Prostate Cancer Research Program (PC101008), a challenge award from the Prostate Cancer Foundation (PI: O. Witte), a creativity award from the Prostate Cancer Foundation (PI: Matthew Rettig), Cal-Tech-UCLA Joint Center for Translational Medicine Program, and National Cancer Institute (1R01CA158627-01; PI: Leonard Marks).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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