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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cyclophosphamide (CPA) has efficacy as a breast cancer therapy. However, toxicity to CPA limits its clinical applications. Hence there is a need to develop compounds that may be combined with it to improve the efficacy and overcome toxicity. We showed previously that Resveratrol (RES), a chemopreventive agent, increased the growth inhibitory effect of CPA-treated MCF-7 cells. Here we have explored the molecular basis of 5 mM CPA and 50 μM RES as a combination on cell-cycle progression, apoptosis and oxidative stress in MCF-7 breast cancer cells. Efficacy of the combination was also evaluated in a serum-free tumor explant culture model. The combination elicited enhanced anti-proliferative action coupled with differential expression of cell-cycle, apoptosis and stress factors. Furthermore, co-treatment superiority in histologically validated ER positive breast cancer explants suggests that this combination may be a worthy future clinical anti-neoplastic regimen. (Cancer Sci 2011; 102: 1059–1067)

Strategies of cancer treatment using combined therapies or agents with distinct molecular mechanisms show greater potential for higher efficacy resulting in superior survival.(1–6) In recent years, more dietary compounds (Resveratrol, Genistein, Curumin, etc.) have been recognized as cancer chemopreventive agents because of their anticancer activity.(1–6) These compounds also afford antitumor activities through regulating various signaling pathways. Therefore, cytotoxic therapies combined with these dietary compounds may exert enhanced antitumor effects through synergistic actions and may also decrease the systemic toxicities.(7) Thus, the combined treatment of Resveratrol (RES) and Cyclophosphamide (CPA) in human MCF-7 cells shows maximal cytotoxic effect, which was almost equivalent in effect to that when either drug was administered in double doses.(8)

Apoptosis assessment based on morphological changes, flow cytometric analysis and DNA fragmentation results indicate that more cells treated with CPA in combination with RES underwent apoptosis than with CPA alone.(8) These results opened up new vistas for studies of natural products like RES (a chemopreventive agent) in the treatment of neoplasms with highly cytotoxic drugs like CPA. However, the molecular mechanism or the apoptotic intermediates through which RES potentiates the cytotoxic effect of CPA against tumor cells are not known. We therefore investigated the various apoptotic pathways in MCF-7 human breast cancer cells (HBCC) and explants derived from breast cancer patients on treatment with 50 μM RES either alone or in combination with 5 mM CPA.

This study characterizes the involvement of cell-cycle factors, extrinsic (Fas L), intrinsic pathway (Bax, Bid, Bcl-2, Bcl-XL) and other apoptogenic molecules (PARP, AIF) in 5 mM CPA plus 50 μM RES-induced apoptosis in MCF-7 HBCCs. Additionally, oxidative stress potentiating the action of the combination as early at 24 h by activating MAPK and antioxidant enzymes was also investigated. Furthermore, RES (100 μM) proved its apoptotic function in breast explants in serum-free ex vivo situations through electron microscopic studies. Hence, this model was further explored for the efficacy of the combination. Synergistic tumor growth inhibition was shown by the combination through histological studies. This study further provides insight into the use of such combination (CPA plus RES) as a novel anti-neoplastic regimen of therapy for breast cancer.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Cyclophosphamide, Dulbecco’s modified Eagle’s medium (DMEM), N-(2-hydroxyethyl) piperazine-N′-2-ethanesulphonic acid (HEPES), penicillin, streptomycin, gentamicin sulfate, sulforhodamine-B (SRB), phosphate buffered saline (PBS), propidium iodide (PI), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethylether)-N,N,N′,N-tetra-acetic acid (EGTA), trichloroacetic acid (TCA), Z-VAD-FMK, protease inhibitor cocktail, phosphatase inhibitor cocktail, antibodies against β-Actin, p21Waf1/Cip1 and p27Kip1, CDK-6, Cyclin-D, Cyclin-E, Fas L and extrAvidin alkaline phosphatase staining kits were used (Sigma Chemical, St. Louis, MO, USA). Antibodies against Bcl-XL, Bcl-2, phosphorylated-p53 (Ser 15), p53, Bax, AIF, PARP, Bid, phosphorylated c-Jun, JNK1/2, phosphorylated p38, p38, superoxide dismutase (SOD) and catalase (CAT) were purchased (BioVision Research Products, Mountain View, CA, USA). Inhibitors specific to JNK (SP600015), p38 (SB203580) and Erk (U0126) were obtained (Alexis, San Diego, CA, USA), BCIP/NBT was obtained (Merck-Calbiochem, San Diego, CA) and FCS was obtained (Gibco BRL Laboratories, New York, NY, USA). All other chemicals used were of analytical grade.

Cell culture.  The MCF-7 cells were procured from the National Center for Cell Sciences (Pune, India) and cultured as reported previously.(8,9) For this study, an approximately 70–80% confluent flask of MCF-7 cells was maintained initially for the first 2 days in phenol red-free DMEM pH 7.4 containing 10% dextran coated charcoal stripped FCS (DCC/FCS). For the subsequent 24 or 48 h, the cells were exposed to 5 mM CPA with and without 50 μM RES. The cell number used has been described separately for individual experiments as below. The CPA and RES were dissolved in PBS and DMSO, respectively.

Time-dependent analysis of apoptosis.  For these studies the cells were treated with 5 mM CPA with and without 50 μM RES for varying periods (0, 6, 12, 24, 36, 48 h). Following trypsinization and washing with chilled PBS pH 7.4, the cells were processed according to Singh et al.(8) Flow cytometry was performed on a Beckton–Dickinson fluorescence activated cell sorter (FACS) using Cell Quest software (Beckton–Dickinson, Ontario, Canada). Cells with hypodiploid DNA were considered to be apoptotic (sub-G0/G1).

Modulation of mitochondrial membrane potential (Δψm) and intracellular reactive oxygen species (ROS) generation.  The Δψm was measured by the uptake of unique fluorescent cationic dye, JC-1 (excitation at 488 nm and emission at 525 nm), to signal the loss of Δψm. This fluorescent probe exists as a green fluorescent monomer (emission 527 nm) at low mitochondrial membrane potential. Mitochondrial depolarization is indicated by an increase in green fluorescence (FL-1). The MCF-7 cells (0.2 × 106 cells) were plated in a 6-well plate, exposed to CPA (5 mM) with and without RES (50 μM) for 24 and 48 h, washed and finally harvested in chilled PBS containing JC-1 (1 μM). The samples were incubated at 37°C for 30 min in the dark, washed twice with chilled PBS and finally resuspended in 200 μL PBS. Mitochondrial permeability transition was subsequently quantified on FACS (Becton–Dickinson, Franklin Lakes, NJ, USA).(10) Reactive oxygen species (ROS) generation was determined through 2′,7′-dichlorofluorescin diacetate (DCFDA, 10 μM) staining. Cells were treated as above and analyzed through a flow cytometer with excitation and emission at 490 and 530 nm, respectively.(11)

Cell morphology and viability assay.  An MTT assay(8) was used to establish the effect(s) of MAPK inhibitors (i.e. U0126 [ERK], SP600015 [JNK] and SB203580 [p38]) and on CPA with and without RES-induced apoptosis in MCF-7 cells. Briefly, 104 MCF-7 cells/well were plated in 96-well plate for 48 h in DCC/FCS media. Before the treatment of 5 mM CPA with and without 50 μM RES for 48 h, the cells were pre-incubated for 4 h with MAPK inhibitors (U0126 [10 μM], SP600015 [10 μM] and SB203580 [1 μM]). The assay was performed after completion of incubation with the drugs. Concentrations of MAPK inhibitors used were non-toxic to cells under our pre-incubation conditions (data not shown).

Protein extraction and western blotting.  The MCF-7 cells were cultured in T-75 flasks and treated with 5 mM CPA with and without 50 μM RES for different periods (24 and 48 h). Harvested cells and breast explant tissues (control and treated) were then disrupted in cell lysis buffer (0.02 M Tris–HCl pH 7.4, 1.0% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.25 M sucrose, protease and phosphatase inhibitor cocktail) and tissue lysis buffer (250 mM NaCl, 50 mM Tris at pH 7.4, 5 mM EDTA, 0.1% v/v Triton X-100, 1 mM PMSF, 50 mM NaF, 1 mM sodium orthovanadate, 10 μg/mL leupeptin, 50 μg/mL aprotinin), respectively, for 1 h at 4°C. The homogenates were sonicated for 30 s at 4°C and vigorously vortexed every 5 min for 30 min and spun at 13 000g for 15 min at 4°C. Total protein in the extracts was determined according to Lowry et al.(12) using BSA as a standard. Equal amounts of protein (50 μg) were resolved on 10–15% SDS-polyacrylamide gel electrophoresis (PAGE) and then electrotransferred onto a nitrocellulose membrane (Amersham, Aylesbury, UK). The membranes were probed with antibodies against antibodies p21Waf1/Cip1, p27Kip1, cyclin-D, cyclin-E, Fas L, Bcl-XL, Bcl-2, phosphorylated-p53 (Ser 15), p53, Bax, AIF, PARP, Bid, phosphorylated c-Jun, JNK1/2, phosphorylated p38, p38 SOD, CAT and β-Actin. Bound primary antibodies were detected with goat or sheep secondary antibody conjugated to ALP substrate. The immunoblots were detected by using colored substrate (BCIP/NBT).

Immunoprecipitation.  For immunoprecipitation, cells were cultured and treated in similar conditions as mentioned for Western blot. Cell pellets were washed twice with chilled PBS and incubated in lysis buffer (50 mM Tris–HCl pH 7.4, 0.5% Nonidet P-40, protease and phosphatase inhibitor cocktail, 1 mM DTT and 1 mM phenylmethylsulfonyl fluoride) for 1 h. Lysates were clarified by centrifugation at 18,000g for 10 min. Protein content was determined as described for Western blotting. Cellular extracts (200 μg) were then incubated with primary antibody (CDK-6, PCNA) overnight at 4°C. Immune complexes were collected by incubation with Protein A sepharose (50% v/v; Pharmacia LKB, Uppsala, Sweden) for 1 h with gentle rocking. Immunoprecipitates were washed three times with lysis buffer and eluted in 2 × SDS sample buffer (50 mM Tris–HCl pH 6.8, SDS 2%, glycerol 10%, 5%β-mercaptoethanol, bromophenol blue). All the steps were performed at 4°C unless stated otherwise. Finally, samples were resolved on 15% SDS-PAGE and Western blotting was performed with p21Waf1/Cip1 as previously described.(13)

Superoxide dismutase and catalase activities.  Superoxide dismutase (SOD) and catalase (CAT) activities were determined according to McCord and Fridovich(14) and Beutler,(15) respectively. Untreated as well as treated MCF-7 cells were collected by scraping. Cells were then centrifuged and the pellets sonicated in chilled 1.5% KCl. The cell extract was spun at 1500g for 5 min at 4°C. The resulting supernatants were analyzed for enzyme activities. Each data point was performed in triplicate and the results reported as mean absorption ± SE.

Explant culture.  Human breast tissues were acquired from three female patients aged 49–64 years, undergoing breast surgery for breast tumors (n = 3) with written informed consent prior to the sampling. Immuno-histologically, these tissues were positive for estrogen and progesterone receptor (ER and PR+). The breast tissue was transported, excised and explanted, according to Sharma et al.(16) Approximately 20 explants were plated in each 35-mm Petri dish containing 3 mL serum-free M-199 in a humidified CO2 incubator (95% O2, 5% CO2) at 37°C. The explants were cultured up to day 4, and the media replenished daily. For evaluating the role of CPA/RES/CPA with RES, the explants were incubated in medium (M-199) alone for 2 days, followed by medium containing 5 and 10 mM CPA alone, 50 and 100 μM RES alone and combinations of 5 mM CPA plus 50 μM RES and 10 mM CPA plus 100 μM RES for the next 2 days. The study was approved by the Medical Ethics Committee of Central Drug Research Institute.

Electron microscopic analysis.  Electron microscopic analysis of control and treated explants was carried out according to Sharma et al.(16) The sections were double stained with uranyl acetate and lead citrate and observed under a FEI Tecnai-12 twin transmission electron microscope equipped with a SIS Mega View II CCD camera at 80 kV (FEI Company, Hillsboro, OR, USA).

Histological analysis.  Histological analysis was conducted followed by staining with H&E for evaluating the cytoarchitectural features using a light microscope (HFX-DX II; Nikon, Tokyo, Japan).(17)

Statistical analysis.  The results are expressed as mean ± SE of one of three similar experiments each performed in triplicate. A Student’s t-test was used to determine the level of significance and P < 0.05 was regarded as significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Time-dependent apoptosis.  Our initial studies have already revealed that concurrent exposure of 5 mM CPA and 50 μM RES in MCF-7 cells enhances their susceptibility to undergo apoptosis as compared to either drug alone.(8) On the basis of significant synergistic inhibition, we hypothesized that this combination might lead to early enhanced inhibition as compared to drugs alone. The possibility was investigated using flow cytometric time-dependent analysis of drug induced apoptosis (Fig. 1). Control cells displayed only basal apoptosis. A significant rise in apoptotic fraction (P < 0.01) was observed after 12 h of 5 mM CPA with and without 50 μM RES-treated MCF-7 cells, which increased with time. The significant observation here is that at 12 h, the enhanced effect of the combination was seen; that is, approximately 50% of the MCF-7 cells were found to be apoptosed. At 48 h, the combination regimen led approximately 80% of apoptosis (P < 0.001) in MCF-7 cells. Hence, co-treatment was more efficacious in inducing early cytotoxicity as compared to the drugs alone.

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Figure 1.  For time-dependent apoptosis analysis, cells were treated with 5 mM CPA with and without 50 μM RES for varying periods. Following incubation the cells were harvested, permeabilized, stained with propidium iodide (40 μg/mL) and analyzed by flow cytometry. All charts are typical of three independent experiments each performed under identical conditions. *< 0.05, **< 0.01, ***< 0.001 (as compared to control cells); $(< 0.001) co-treatment versus CPA; #(< 0.001) co-treatment versus RES.

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Mitochondrial events induced by 5 mM CPA with and without 50 μM RES.  Changes in Δψm and ROS levels were again used as assays for functional efficacy. Control MCF-7 cells elicited basal fluorescence reflecting intact, functional mitochondria (Fig. 2A). Drugs alone at 24 h showed no change in Δψm while the combination treatment resulted in rapid dissipation of Δψm as detected by consequent decrease in mean fluorescence (FL-1). Significant decrease in Δψm was noticeable upon treatment with CPA and RES (< 0.001) alone at 48 h, which further deteriorated with co-treatment (< 0.001).

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Figure 2.  Analysis of 5 mM CPA without and with 50 μM RES-induced alterations in mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS). Briefly, 2 × 106 MCF-7 cells were pre-cultured for 48 h in phenol red-free DMEM (DCC treated FCS treated) and then exposed to different doses of CPA/RES/CPA plus RES. (A) For the assessment of loss in Δψm, trypsinized cells were incubated with the fluorescent cationic dye, JC-1 (excitation at 488 nm and emission at 525 nm) (1 μM) for 30 min at 37°C in the dark, washed twice with chilled PBS and finally resuspended in 200 μL PBS. The mitochondrial permeability transition was subsequently quantified on FACS (Becton–Dickinson). (B) For measuring ROS production, the cells were exposed to the ligands for 24 and 48 h, washed twice with chilled PBS. Following this, the cells were incubated with 10 μM DCFDA fluorophore for 30 min at 37°C in the dark, washed twice with chilled PBS and trypsinized. Finally, the stained cells were analyzed through flow cytometry. (C) 5 mM CPA without and with 50 μM RES-treated MCF-7 cells at 24 h showed increase in expression of SOD and CAT as compared to basal expression of these enzymes in control untreated cells. The level of expression of antioxidative enzymes was optimal at 48 h in comparison to 24 h. (D,E) Assays of SOD and CAT activities were performed as described in “Materials and Methods”. At 24 h, 5 mM CPA showed activity similar to control while 50 μM RES-induced the activity of SOD and CAT. The increase in activity of SOD was maximal upon co-treatment accompanied with unaltered CAT activity. By 48 h, the SOD and CAT activity was unaltered as compared to that at 24 h CPA exposure. However, in RES-exposed cells, both the enzymes activity decreased. Finally, the co-treated cells displayed decrement of activity of SOD while CAT expression and activity was similar to that observed at 24 h. Data shown are mean ± SE of one of three similar experiments each performed in triplicate. *< 0.05, **< 0.01, ***< 0.001 (as compared to control cells); $(< 0.001) co-treatment versus CPA; #(< 0.001) co-treatment versus RES.

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At 24 h, intracellular ROS induction following CPA treatment revealed minimal effects whereas RES alone showed an anti-oxidative effect. Co-treatment increased ROS greater than the drugs alone (< 0.001). However, CPA and RES alone generated significant amount of ROS at < 0.001 and 0.05, respectively, at 48 h. Nevertheless, ROS production unexpectedly declined in the combination group in MCF-7 cells (< 0.001) (Fig. 2B) at 48 h.

Further, to analyze the role of oxidative stress induced apoptosis, we scrutinized the expression and activities of anti-oxidative enzymes SOD and catalase (CAT). The SOD leads to dismutation of the superoxide anion to molecular oxygen and H2O2 and is further detoxified by CAT, whose ratio is critical for the cell for ROS generation.(18) At 24 h, CPA-treated cells showed increased expression in the absence of activity for both the enzymes (Fig. 2C–E). On the other hand, RES increased both the expression and activity of SOD and CAT (Fig. 2C–E). The increase in expression and activity of SOD was optimal upon co-treatment (Fig. 2C,D). This was accompanied by unaltered CAT expression and activity (Fig. 2C,E). Moreover, at 48 h, the expression of SOD further declined accompanied with unaltered activity upon CPA exposure (Fig. 2C,D). On the contrary, the expression and activity of CAT remained unaltered (Fig. 2C,E). In RES-exposed cells, both the enzyme expression and activity declined as compared to 24 h (Fig. 2C–E). Finally, the co-treated cells displayed decrement of both expression as well as activity of SOD as compared to 24 h (Fig. 2C,D). The CAT expression and activity were similar to that observed at 24 h (Fig. 2C,D). Hence, the SOD/CAT ratio sustained by RES at 24 h was lost at 48 h while co-treatment altered the ratio early at 24 h, accounting for enhanced ROS generation.

Mitogen activated protein kinases (MAPK) and oxidative stress.  Next, we analyzed the role of oxidative stress in CPA/RES/CPA plus RES-induced apoptosis by examining the activation levels of stress activated MAPK. Administration of MEK/ERK inhibitor (U0126) potentiated, whereas JNK (SP600015) and p38 (SB203580) inhibitors suppressed CPA/RES/CPA + RES-induced apoptosis (P < 0.05) (Fig. 3A,B) in MCF-7 cells. These results were corroborated by Western blot analysis, which showed that drugs alone as well as with co-treatment induced phospho-p38, which peaked at 48 h (Fig. 3C). Moreover, the drugs also induced phosphorylation of phospho-c-Jun at 24 h, which further decreased at 48 h. This lends credence to the existence of early synergism between CPA and RES in the context of activation of stress activated kinases, which may further induce apoptotic events.

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Figure 3.  The ERK, JNK and p38MAPK are differentially responsive to 5 mM CPA without and with 50 μM RES-induced apoptosis of MCF-7 cells. Briefly, 104 cells/well were plated in 96-well plates for 48 h in DCC/FCS media. Prior to their exposure to the drugs for 48 h the cells were pre-incubated for 2–4 h with MAPK inhibitors. (A) U0126 (10 μM), (B) SB203580 (1 μM) and SP600015 (10 μM). After termination of the incubation, MTT assay was conducted to assess the cytostatic/toxic/proliferative effect(s) of inhibitors. Data shown are the mean ± SE of one of three similar experiments each performed in triplicate. *< 0.05, **< 0.01, ***< 0.001 (as compared to control cells); $(< 0.001) CPA/RES/CPA + RES without and with MAPK inhibitors. (C) Western blot analysis for analyzing the time-dependent alterations in expression level of phospho-p38/p38, phospho-c-Jun/JNK1/2.

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Activation of p21Waf1/Cip1 and p27Kip1 and phosphorylation of p53 at Ser 15.  As single agents, RES and CPA have been reported to induce cell-cycle arrest.(8) To assess the mechanism responsible for RES mediated cell-cycle arrest, CPA plus RES-induced alterations in the expression of p21Waf1/Cip1 and p27Kip1 were analyzed. Figure 4A shows increases in expression of p21Waf1/Cip1 and p27Kip1 in CPA with and without RES-treated MCF-7 cells, in comparison to basal expression in control cells. Both these factors increased significantly at 24 h to decline subsequently at 48 h. However, the drugs alone and in combination decreased the levels of cyclin-D and -E in a time-dependent manner (Fig. 4A). Interestingly, RES alone increased the level of G1 cyclins in cells at 24 h to decline significantly at 48 h (Fig. 4A). Furthermore, the results indicate that CPA and RES alone induced maximal expression of phospho-p53 (Ser 15) in the cells which peaked at 48 h (Fig. 4B). Finally, the combination evidenced enhanced cytotoxicity through maximal phosphorylation of p53 as compared to the individual drugs alone, and hence proves its efficacy.

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Figure 4.  Cell growth inhibition with 5 mM CPA is potentiated by 50 μM RES. Western blot was performed with 50 μg protein for (A) cell-cycle regulators (cyclin-D and -E as well as cyclin dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1), (B) phosphorylated-p53 (Ser 15) and total p53 isolated from treated (5 mM CPA without and with 50 μM RES for 24 and 48 h) and untreated control MCF-7 cells. β-Actin was used as control. The data are representative of three identical experiments.

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Induction of interactions of p21Waf1/Cip1 with CDK6 and PCNA.  To investigate the implication of p21Waf1/Cip1 in CPA with and without RES-induced cell-cycle arrest, we analyzed the interaction of p21Waf1/Cip1 with CDK6 and proliferating cell nuclear antigen (PCNA). Immunoprecipitations with anti-CDK6 and anti-PCNA was followed by immunoblotting for p21Waf1/Cip1. Results demonstrate that in CPA with and without RES-treated cells, p21Waf1/Cip1 interacted with CDK6 and PCNA in a time-dependent manner with significant increase at 48 h as compared to controls (Fig. 5). Furthermore, significant increase of p21Waf1/Cip1/CDK6 and PCNA complex in combination treated cells is suggestive of enhanced cell-cycle arrest as compared to drugs alone.

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Figure 5.  CPA (5 mM) without and with 50 μM RES-treated MCF-7 cells for 24 and 48 h induces time-dependent interaction of p21Waf1/Cip1 with CDK6 and PCNA as compared to control cells. The cell-lysates were immunoprecipitated (IP) with anti-CDK6 or anti-PCNA antibody and subjected to Western blot analysis with p21Waf1/Cip1 antibody.

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Expression of Fas, Bcl-2 family members and other down-stream apoptogenic factors.  Blotting studies indicated that the drug treatment up-regulated CD95L or Fas L (Fas Ligand), Bax, a 18-kDa fragment of Bcl-XL and a 15-kDa cleaved form of t-Bid, although they down-regulated Bcl-2 in MCF-7 cells as compared to control in a time-dependent manner (Fig. 6A,B). However, synergism in combination was evidenced by up-regulation of all the proapoptotic factors like Fas, Bax, cleaved Bcl-XL and cleaved Bid accompanied with down-regulation of Bcl-2 suggests its efficacy over the drugs alone.

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Figure 6.  Role of time-dependent differential expression of (A) Fas, Bcl-2 family members, (B) proapoptotic Bid, and (C) other downstream apoptogenic factors (PARP and AIF) in 5 mM CPA without and with 50 μM RES-induced apoptosis. Phenol red-free DMEM (DCC treated FCS) MCF-7 cells were treated with drugs for 24 and 48 h and the whole cell lysate (50 μg) was separated on SDS-polyacrylamide gel. β-Actin was used as control.

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We further evaluated the downstream factors of apoptosis by studying the cytosolic release of AIF from the mitochondria and the cleavage of PARP as a substrate of caspases. As shown in Figure 6C, the absence of the 57-kDa AIF in control cells was conspicuous but the band was clearly discernible in drug-treated samples both at 24 and 48 h. In addition, cleaved PARP, a 89-kDa fragment in drug-treated cells as early at 24 h as compared to control (Fig. 6C), showed uncleaved protein. Hence, in MCF-7 cells, the combination is comparatively more efficient than drugs alone in significantly inducing other apoptogenic molecules.

Transmission electron microscopic (TEM) studies.  As shown in representative electron micrographs (Fig. 7), the 4-day control human breast explant revealed organelle-rich stromal fibroblasts with prominent nucleoli and intra-cytoplasmic lipid droplets. The cytoplasm copiously contained structurally intact Golgi vesicles, rough ER and healthy mitochondria with well defined cristae and an electron-dense mitochondrial matrix. Bundles of intermediate filaments were also observed in the cytoplasm. However, cellular damage was evident in a few areas (Fig. 7A). In contrast, the RES (100 μM) treated explants (Fig. 7B,C) revealed mostly rounded apoptotic cells having lost cell–cell contact, large inter-cellular spaces, chromatin condensation, pkynotic nuclei and loss of nucleoli. Disruption in mitochondrial structural integrity was observed with loss of cristae and mitochondrial swelling. Apoptotic bodies and membrane blebbing were also visible. Bundles of intermediate filaments, vacuolation and myelin whorls were also present.

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Figure 7.  Photomicrographs of human breast explant tissue cultured in serum-free medium. (A) Ultrastructural analysis of the 4 day control showing stromal fibroblasts with distinct nucleoli and intra-cytoplasmic lipid droplets. Abundant cytoplasm consisting of intact Golgi vesicles, rough ER, mitochondria with well defined cristae and electron-dense matrix can also be observed. Stacks of intermediate filaments were also visible in the cytoplasm. However, cellular damage was evident in a few areas. (Bi–iii,C) RES (100 μM) treated explants revealed mostly rounded apoptotic cells, lost intercellular contacts, large inter-cellular spaces, chromatin condensation, pkynotic nuclei and loss of nucleoli. Mitochondrial swelling, loss of cristae and disruption in structural integrity, apoptotic bodies and membrane blebbing was also evidenced. Vacuolation of the cytoplasm, myelin whorls and intermediate filament bundles were the other noticeable features. At least 10 grids prepared from four different blocks were analyzed for each sample.

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Histological analysis.  The results derived from in vitro studies on MCF-7 cells were also extrapolated in breast cancer tissue explants of day 4 displaying infiltrating ductal carcinoma upon H&E staining (Fig. 8A). Slides from 5 mM CPA alone cultured tissues harvested from day 4 clearly indicated necrosis of malignant cells with reduction of tumor load (Fig. 8B). The 10-mM CPA group indicated dissolution of ductal cells with fibrosis and cellular degeneration around tumor (Fig. 8C). The 50-μM RES-exposed tissue did not show any cytotoxic effect (Fig. 8D) while 100 μM RES exposure led to complete degeneration in the tumor area (Fig. 8E). Drug combination (5 mM CPA plus 50 μM RES) showed residual tumor with coagulative necrosis (Fig. 8F). Data of 10-mM CPA plus 100-μM RES treatment has not been shown because there was a reduction of the tissue to a small, incipient necrosed mass, disallowing its sectioning.

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Figure 8.  Breast cancer tissue was excised, explanted and cultured in serum-free M-199 in a humidified CO2 incubator. For evaluating the role of drugs (CPA/RES/CPA with RES), the explants were incubated in M-199 alone for 2 days, followed by in medium containing 5 mM CPA, 50 μM RES and combination of both 5 mM CPA plus 50 μM RES for the next two days. Histological analysis was conducted following staining with H&E using a Nikon light microscope (Model HFX-DX II). (A) Breast explants of day 4 control showing infiltrating ductal carcinoma with malignant cells in the lining of the milk duct. (B) Tissues cultured with 5 mM CPA clearly indicated necrosis of malignant cells with reduction of tumor load. (C) 10 mM CPA indicated dissolution of ductal cells confined with fibrosis and cellular degeneration around tumor. (D) 50 μM RES showed no cytotoxic effect. (E) 100 μM RES lead to complete degeneration in the tumor area. (F) The combination shows residual tumor with coagulative necrosis. Magnification ×100. (G) The homogenates prepared from control and drug-treated explants showed dose-dependent increase in p53 phosphorylation at residue serine 15. Western blot was performed with specific phosphorylated-p53 (Ser 15) and p53 antibody using 50 μg protein. β-Actin was used as control. Independent experiments were repeated at least three times.

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Homogenates from 5 and 10 mM CPA, 50 and 100 μM RES alone and their combination (5 mM CPA plus 50 μM RES, 10 mM CPA plus 100 μM RES) treated as well as control explant tissues harvested after day 4 indicated that CPA and RES alone evoke transient dose-dependent increases in p53 phosphorylation at residue serine-15 (Fig. 8G). Here, the combination enhanced p53 phosphorylation as compared to that in explants from drugs alone and control groups (Fig. 8G).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Deriving combination therapies to improve anti-tumor effect is an area of interest in breast cancer treatment. We have previously observed that co-treatment with the phytoestrogen Resveratrol (RES, 50 μM) enables the use of a lower dose of cyclophosphamide (CPA, 5 mM) to maximally inhibit ER+ MCF-7 breast cancer cell growth, promoting apoptosis in part via caspases.(8)

Earlier reports have indicated that the balance between the pro-apoptotic and anti-apoptotic proteins, dysfunction of mitochondria, up-regulation of death receptors, activation of caspases and increase in reactive oxygen species (ROS) are the important factors deciding apoptotic cell death in tumor cells.(19–21) Besides, phosphorylation of specific serine residues of p53 regulates the transcription of more than 150 genes involved in various cellular processes (apoptosis and cell-cycle regulation).(22) The cyclin dependent kinase (CDK) inhibitors p21 and p27 are also involved in p53-mediated growth arrest. Induction of p21 protein expression abrogates cell-cycle progression by inhibiting the activity of CDK and by interacting with proliferating cell nuclear antigen (PCNA), thereby directly preventing DNA synthesis.(23–25) Furthermore, some studies have reported that certain upstream elements of the different mitogen activated protein kinase (MAPK) cascades are targeted and cleaved by the caspases.(26) The three kinases, such as the p42/44 extracellular signal-related kinases (ERK1/2), c-Jun N-terminal protein kinase (JNK) and p38MAPK directly modulate the phospho-active levels of pro-apoptotic factors.(27,28) Therefore, a secondary apoptotic pathway may exist in certain cells dependent upon the activation of MAPK.(28,29) Moreover, ROS and MAPK together play an important role in determining responsiveness of the cells to apoptotic signals. Exposure of cancer cells to ROS generating anticancer agents exhausts the cellular antioxidant capacity, leading to apoptosis.

In this paper, we further explore the mechanisms underlying improved inhibition of growth by CPA and RES co-treatment than by drugs alone, and confirm enhanced apoptosis versus the control. The magnitude of cell death increased significantly with treatment time (measured up to 2 days using sub-G0/G1-FACS [Fig. 1], mitochondrial membrane potential assays [Fig. 2A] and altered redox status [Fig. 2B–E]). Modulation of MAPK ERK (Fig. 3A), p38 and JNK (Fig. 3B,C) as well as other accompanying events (e.g. changes in cell-cycle-related factors [decrease in cyclins D/E, Fig. 4A], activation of p21, p27 and phospho-Ser 15–p53 [Fig. 4A,B]; increased p21 interaction with CDK6 and PCNA [Fig. 5] and involvement of extrinsic [Fas L] and intrinsic pathways [increased Bax/Bid, decreased Bcl-2 and cleavage of bcl-xL; Fig. 6A,B]; PARP and AIF cleavage [Fig. 6C]) were revealed by blotting and immunoprecipitation studies. Thus, such events may favorably contribute to the enhanced pro-apoptotic action of CPA plus RES co-treatment. The activated MAPK were shown to be directly involved in apoptosis promoted by RES and CPA separately and together since pharmacological inhibitors of JNK and p38 suppressed the anti-tumor effect (Fig. 3B). On the contrary, ERK inhibitor potentiated apoptosis (Fig. 3A), indicating MAPK involvement. This suggests the superiority of combination over the drugs alone since the former suppressed ERK and activated JNK and p38 pathways more significantly. Furthermore, the combination of drugs did not proportionally cause further increase in ROS generation in spite of a decrease in Δψm at 24 h and maximum decrement at 48 h (Fig. 2A). This may be due to the initiation of late stages of apoptosis as shown by annexin/PI studies,(8,30) which suggests that membrane rupture may lead to leaking of ROS that could not be detected by DCFDA dye. Western blotting revealed that increased oxidative stress (a further potential pro-apoptotic input) may also contribute to their anti-tumor effect with AIF release (Fig. 6C). Additionally, the concomitant disruption of the SOD/CAT ratio manipulates the cellular redox state in co-treatment further supports the concept of functional synergy (Fig. 2B–E).

To further explore the efficacy of these two molecules, a serum-free primary explant culture system derived from human breast tissue was used. The system retains the ability for hormonally induced cellular differentiation, similar to the in vivo situation, since the autocrine and paracrine interactions among the heterogeneous cell types are conserved.(13) The cultured explants were subjected to ultrastructural analysis. Comparison of transmission electron micrographs of control versus 100 μM of RES (IC50 dose) clearly indicated massive apoptosis of the cells, thereby justifying its use adjunct with CPA for combination therapy (Fig. 7).

The histological studies conducted on cultured explants provided evidence that ligands evoke definitive changes in the cyto-architecture leading to tumor regression (Fig. 8A–F). Drugs alone also affected the tumor load in different doses (5 mM CPA, 10 mM CPA and 100 μM RES) but combining the drugs (5 mM CPA + 50 μM RES) led to coagulative necrosis with negligible tumor residue. Additionally, enhanced up-regulation of pp53 in explants treated with a combination of CPA and RES correlated well with tumor regression (Fig. 8G). This combination may therefore provide a better option as a probable therapeutic regimen. These observations thus raise new possibilities for developing combination strategies for coordinated regulation of specific gene products associated with apoptosis for cancer therapy.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

The authors are grateful to the Director and Head, Toxicology Division, Central Drug Research Institute, for their interest in this study. We also thank Mr A L Vishwakarma for technical support and Dr. Vinita Dwivedi for help with flow cytometry and histological studies. NS is a recipient of a Women Scientist Fellowship of the Department of Science and Technology, Government of India (SR/WOS-A/LS-400/2004, SSP0183). MN and VR are recipients of Senior Research Fellowships from the Council of Scientific and Industrial Research and University Grants Commission, New Delhi. The study was also supported by the CSIR network projects CMM0018 and CMM034. This paper bears the CDRI Communication Number: 7602

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References