Research ArticleExperimental Studies

Phosphaplatin Anti-tumor Effect Enhanced by Liposomes Partly via an Up-regulation of PEDF in Breast Cancer

LOUIZA BELKACEMI, JASON L. ATKINS, LU YANG, PRAJAKTA GADGIL, AMY K. SATER, DIANA S. CHOW, RATHINDRA N. BOSE and SHAUN XIAOLIU ZHANG
Anticancer Research February 2018, 38 (2) 623-646;

Abstract

Background/Aim: Phosphaplatin platinum (IV) (RRD4) complex has exceptional antitumor properties. The aim of this study was to investigate the effects and the mechanism of action of free and liposome-encapsulated RRD4 in breast cancer. Materials and Methods: Liposome-encapsulated RRD4 prepared by thin-film dehydration: hydration and free RRD4 were tested in vivo and in vitro against 4T1 breast cancer cells. Cell proliferation, migration and viability were determined. Tissue and cell production and expression of pigment epithelium-derived factor (PEDF) were assessed by ELISA and western blot. 4T1 cells treated with PEDF siRNA were evaluated for viability and apoptosis. Results: RRD4 inhibited tumor growth and prevented distant metastasis. Liposome formulation enhanced this therapeutic benefit without increasing toxicity and prolonged RRD4 retention in tumor tissues. In vitro, RRD4 induced 4T1 apoptosis through up-regulation of FAS, BAX, and PUMA, and down-regulation of BCL2. RRD4 facilitates a FAS-intrinsic signaling mechanism. PEDF up-regulation represents another antitumor mechanism associated with this phosphaplatin compound. Discussion: Free RRD4 or formulated into liposomes, are excellent candidates for adjuvant therapy against breast tumor growth and metastasis.

Breast cancer is one of the most common malignancies among women worldwide (1). Breast cancer metastasis occurs when cancer cells separate from the primary tumor mass and enter blood vessels or the lymphatic system. These traveling cells subsist within the circulation by attaching to the endothelium of distant organs, crossing the endothelial barrier, and establishing new tumor colonies, which are the main cause of death (2). Indeed, according to the National Cancer Institute, while 5-year survival rate for patients diagnosed with localized breast cancer is 98.5%, it drops to a 25% for patients with distant overt metastases. Since it is not always possible to accurately forecast the risk of metastasis development in individual patients with breast cancer, the majority of patients (more than 80%) will receive adjuvant chemotherapy (1). Unfortunately, many of these patients will endure the toxic side-effects of chemotherapy unnecessarily, because local treatment alone such as surgery and radiotherapy would have been insufficient. Therefore, it is imperative to find treatment modalities with lower toxicities to treat metastatic breast cancer.

Our laboratory has developed a class of monomeric non-DNA binding platinum complexes, namely phosphaplatins that include platinum complexes ligated by diaminocyclohexane (dach) and pyrophosphate ligands. The (1R,2R diaminocyclohexane) (dihydropyrophosphato) platinum(II) (RRD2) and (1R,2R-diaminocyclohexane) (dihydropyrophosphato)(trans-dihydroxo)platinum(IV) (RRD4) (Figure 1) stereoisomers. These isomers have shown exceptional anti-tumor activities against a variety of cancers (3, 4). The remarkable physicochemical properties of phosphaplatins include high aqueous solubility, robust stability at or near neutral pH, and lack of aquation during a period of a week. Also, phosphaplatins are bound to a pyrophosphate ligand, which is abundant in the human body, and is a substrate or product of many enzymatic reactions. Among those reactions, our laboratory has found that RRD2 isomer reacts with cysteine in a consecutive 2-step process to deligate the pyrophosphate moiety from the platinum with a second order rate constant, 4.3×10−33M−1S−1 for the first step of the reaction, which is slower than that of the analogous platinum-based cisplatin response with cysteine (4, 5). Thus, the lack of aquation and phosphate hydrolysis of phosphaplatins, together with the second-order reaction of this complex with cysteine, might have significant implications in exhibiting reduced toxicities versus other platinum compounds (4). Furthermore, previous work from our laboratory showed that there were no platinum-DNA adducts, no nucleotide excision repair, homologous recombination repair, or post-replication repair in cancer cells treated with phosphaplatins (6). Instead, phosphaplatins promoted overexpression of the death receptor FAS and sphingomyelinase proteins, implying protein targeting and stimulation of an extrinsic apoptotic signaling mechanism. Collectively, these properties make phosphaplatins potentially better candidates as chemotherapeutic agents than other platinum compounds as adjuvant- and mono-therapy for breast cancer and metastatic diseases.

Figure 1.
Figure 1.

Structural formulas of Pt(II) phosphaplatin (RRD2) and Pt(IV) phosphaplatin (RRD4).

Interestingly, our laboratory has recently shown that RRD2 isomer significantly stimulates the expression of pigment epithelium-derived factor (PEDF) in various tumor cells (7). PEDF is a secreted glycoprotein (8) and a member of the serpin (serine protease inhibitors) superfamily of proteins, which is commonly expressed in normal tissues (9), and occasionally in tumorous tissues (10-11). PEDF has also been considered to possess an anti-metastatic activity (12). In vivo studies demonstrate that PEDF has the highest impact on metastases in mice bearing osteosarcoma, with a 70% reduction in the development of pulmonary metastases and a 40% decrease in primary tumor size when compared to untreated controls (13). In humans, the expression of PEDF was found to be inversely related to the metastatic potential and tumor grade of prostate adenocarcinoma (14), pancreatic adenocarcinoma (15), and glioblastoma (16). These findings led us to hypothesize that phosphaplatin-mediated PEDF up-regulation may contribute to the inhibition of breast cancer and subsequent metastasis to other organs. We tested our hypothesis using tumors established from the 4T1 murine mammary epithelial carcinoma cell line, which is a well-established and reliable metastatic breast cancer model. Orthotopically established 4T1 tumors in the syngeneic Balb/c mice are readily metastasized to different organs, such as the lungs and liver (17). More importantly, the metastatic pattern of the 4T1 tumor model closely mimics that of human breast cancer (13). We evaluated the ability of phosphaplatins to reduce regional tumor growth and metastasis. We specifically selected the RRD4 compound, since it exhibits extremely low in vitro cytotoxic activity, with IC50 values as low 0.2 μM when tested on a panel of human cancer cells (6). The compound was given either as the free form or encapsulated in liposomes (LD). The molecular mechanism underlying the effects of RRD4 on breast cancer and metastasis were also investigated by knocking down PEDF expression in 4T1 cells.

Materials and Methods

Drug preparation, and production of empty and encapsulated liposomes. The (1R,2R-diaminocyclohexane)(dihydropyrophosphato) (trans-dihydroxo)platinum(IV) (RRD4) (name abbreviations describe the R,R enantiomer of the diaminocyclohexane ligand D and the subsequent number specifies the oxidation state of the platinum center) was prepared by methods described previously (18). The purity of this compounds was 99% by HPLC and NMR spectroscopy. All platinum-based reagents were purchased from Sigma-Aldrich, Missouri, USA.

RRD4 encapsulated in liposomes (LD) or liposomes without RRD4 (liposomes; EL) were prepared by thin-film dehydration: hydration method. This method consisted of dissolving and mixing (1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):phosphatidylcoline (PC): Cholesterol:1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(4-(p-aleimidophenyl) butyramide (MPB PE)) (Avanti Lipids Inc., Alabama, USA) at 10:2:2:1 w/w ratio to yield a total weight of 20 mg/ml. Once the lipids were thoroughly mixed, the organic solvents were removed to produce a dehydrated lipid film. The lipid film was placed in an appropriate round bottom flask and dissolved into (1:1) chloroform: methanol solution. The round bottom flask containing the lipid solution was attached to a BUCHI R200 rotary evaporator and set to a speed of 40 rpm immersed into a 60°C water bath for 2 h. After the 2 h, the flask was removed from the water bath and maintained at 40 rpm under vacuum overnight to remove all residual organic solvent. The lipid film was hydrated by the addition of 3 ml phosphate buffered saline (PBS) with or without RRD4 to the flask, followed by incubation for 1 hour at 60°C at a speed of 40 rpm. Once the lipid film was hydrated, the resulting dispersion was extruded through a series of polycarbonate filter via pressurized nitrogen gas. The LD and EL were placed in PBS at 4°C for further use. LD and EL platinum contents were determined by atomic absorption spectroscopy (AAS) (Perkin Elmer, Massachusetts, USA) according to the manufacturer's instructions as described below. The data were collected from two independent experiments, and each measurement was carried out in triplicate. The efficiency of the encapsulation (EE) was calculated using the formula: Embedded Image

Cell culture. The 4T1 mammary tumor cell line was kindly provided by Dr. Fred Miller (Cancer Foundation, Michigan, USA). These cells were maintained in vitro in a medium containing RPMI 1640 medium (Invitrogen, Life Technologies Inc., New York, NY, USA) supplemented with 10% heat- inactivated fetal bovine serum (HI FBS; Invitrogen) and 100 IU/ml of penicillin and 100 μg/ml of streptomycin (PS) (Invitrogen). Non-tumorigenic human mammary epithelial cells (MCF-10 catalogue no: ATCC CRL-10317™) were cultured in DMEM/Ham's F-12 (GIBCO-Invitrogen, New York, NY, USA) supplemented with 100 ng/ml cholera toxin, 20 ng/ml epidermal growth factor (EGF), 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, 5% chelex-treated horse serum, and PS. The growth factors were purchased from Sigma Aldrich, Missouri, USA. At 80% confluency, cells were detached from the flasks using a trypsin-EDTA mixture (Invitrogen) and were either sub-cultured or used for in vivo (4T1 cells) or in vitro (4T1 and MCF-10 cells) investigations.

Cell proliferation. Since proliferation is a hallmark of cancer (20), we assessed the growth of 4T1 cells seeded in each well of 96-well plates (Sarstedt, NC, USA) without or with final free RRD4 with doses of (25, 50, or 100 μM) after 12, 24 and 48 h, or with final LD doses of (5 or 25 μM) after 12 h. The equivalent volume of EL corresponding to each LD concentration was also included. The total number of cells was calculated for control and treatments for each day using FluoReporter® Blue Fluorometric dsDNA Quantitation (Clonetech, CA, USA), and Ki-67 antibody (Cell Signaling Tech., MA, USA) immunostaining.

The FluoReporter® Blue Fluorometric dsDNA is a quantitation assay that determines the number of cells by blue-fluorescent Hoechst 33258 nucleic acid stain. In this method, wells containing 4T1 cells treated with free RRD4, LD, EL, or PBS were emptied at the desired endpoint and stored at -80°C. Once the 4T1 cells from the different groups were ready to scan, the plates were first incubated in 100 μl sterile Millipore water for 1 hour at 37°C after which the plates were placed at −80°C until frozen, then thawed at room temperature. Following the addition of 100 μl of aqueous Hoechst 33258 in TNE buffer (10 mM Tris, 2 M NaCl, 1 mM EDTA, pH 7.4, containing 2 mM sodium azide), fluorescence was measured at excitation and emission filters centered at 360 nm and 460 nm, respectively, using a microplate reader (Bio-TeK Instruments, Synergy HT, Winooski, VT, USA). A standard curve was generated using incremental numbers of untreated 4T1 cells. The level of proliferation inhibition in the platinum-treated vs. PBS-treated samples was expressed as a relative proliferation fraction of the control.

Migration assay. Scratch or wound healing assays are used as a measurement of 2-dimensional cell migration into a wound (cell- free area) that is created by a central linear scratch across the surface of a tissue culture well containing a confluent monolayer of cells (21, 22). Twelve well plates were seeded with 4T1 cells (1×106) per well and allowed to grow to 90% confluency before a wound of approximately 650 μm wide was generated in each well by scratching the plate surface using a sterile 1 ml pipette tip. The remaining cells were washed thrice with PBS to remove cell debris. To avoid cell turnover influencing the rate of wound closure, cells were serum starved overnight prior to wounding, and post-scratch media was supplemented with or without free RRD4 at a final dose of 25, 50 or 100 μM, or with LD at a final dose of 25 μM for 20 h at 37°C and under a 5% CO2 atmosphere. A 20-hour migration time was chosen because preliminary studies showed that untreated 4T1 exhibited an almost complete wound healing during this period. The wound repair process was recorded using a digital inverted microscope (Fisher Scientific, MA, USA) at 5× magnification. The level of migration was quantified using the ImageJ 1.48v program by manually defining the spaces within the wound that were not covered with cells (23-24). At least three independent experiments were performed. The percent migration inhibition was expressed as the percentage of wound closure in the platinum-treated vs. PBS plates after 20 h.

Cell viability. To measure RRD4 cytotoxicity, 4T1 or MCF-10 cells (2.5×105) per well were seeded in 6-well plates for 24 h prior to being exposed continuously to final doses of 25, 50 or 100 μM of free RRD4, or an equivalent volume of EL or PBS for an additional 12, 24 or 48 h (4T1 cells), and for 48 h (MCF-10 cells). In another set of experiments, 4T1 and MCF-10 cells (2.5×105) were treated with final doses of LD (5, 10, 25, 50, or 100 μM), or an equivalent volume of either EL or PBS for 12, 24 and 48 h. Cell viability was obtained by measuring the relative levels of intracellular ATP, a biomarker for live cells, using a CellTiter-Glo™ kit (Promega, Fitchburg, WI, USA) according to the manufacturer's instructions. Briefly, at the indicated times, control and experimental cells were mixed with the CellTiter-Glo™ reagent and incubated for 10 minutes at room temperature; luminescence intensity corresponding to the ATP levels in cells was measured using an automated plate reader (Bio-TeK Instruments) at 460 nm. The spectrophotometric values of the treated cultures were normalized to the mean spectrophotometric value of the cells that were treated with PBS, with the latter used as a control to generate relative viability fractions for each treatment group.

Activated caspase-3 Enzyme-Linked Immunosorbent (ELISA). The amount of activated caspase-3 produced by 4T1 cell lysates and supernatants was measured using an ELISA kit (Aviva Biosystems, CA, USA). After a 24-hour incubation with final doses of free RRD4 (50 μM), LD (5, 25 μM), EL (using equivalent volumes of LD), or PBS, cell lysates and supernatants were obtained for measurements of activated caspase-3 according to the manufacturer's instructions. One hundred microliters of each standard and sample lysate diluted to the appropriate concentration were incubated at 37°C in a 96-well plate. After 2 h, each well was washed at least four times using wash buffer before adding 100 μl diluted biotinylated antibody and incubated at 37°C for 1 hour. Then, 100 μl diluted streptavidin-peroxidase conjugate was added to each well and incubated at 37°C for an extra 1 h. The activation of caspase-3 was measured spectrophotometrically at a wavelength of 450 nm within 15 min. The concentration of activated caspase-3 was determined by comparing the optical density (OD) of drug-treated samples to the standard curve, and values obtained for lysates and supernatants were normalized to matched-total protein concentrations.

Immunodetection of cell proliferation and cell death. Cell proliferation was quantified using Ki-67 immunostaining. Ki-67 is absent in the resting phase of cell growth, making it a good cell proliferation marker. The 4T1 cells (2.5×105) per well cultured on cover slips in 6-well plates were treated with final doses of free RRD4 (25, 50, 100 μM), LD (5, 25 μM), EL (using equivalent volumes to LD), or PBS for 12, 24 or 48 h. In another set of experiments, MCF-10 cells (2.5×105 per well) cultured on cover slips in 6-well plates were treated with final doses of free RRD4 (50 μM), LD (5, 25 μM), EL (using equivalent volumes of either LD) or PBS for 24 h. At the indicated times, the cells were subjected to permeabilization as described previously (25). Nonspecific binding was blocked by incubating the sections with a solution containing 5% normal goat serum. The sections were incubated with a Ki-67 rabbit polyclonal antibody (1:100, Cell Signaling Tech., Danvers, MA, USA) followed by horseradish peroxidase- (HRP) conjugated anti-rabbit secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) for 1 h. Colorimetric detection was done using diaminobenzidine (DAB) as the chromogen and hydrogen peroxide as a substrate for horseradish peroxidase (Vector Laboratories). The sections were counterstained with Harris Hematoxylin (Sigma) and observed with bright field microscopy.

To complement the cell viability CellTiter-Glo™ test, we performed immunostaining of activated caspase-3, a marker for apoptotic cell death. Cells were cultured in the same conditions as for the proliferation assay. After permeabilization, nonspecific binding was blocked. The blocked sections were incubated with a non-immune serum or with a rabbit polyclonal anti-activated caspase-3 (1:100, Cell Signaling Tech.) overnight at 4°C, followed by three washes of 10 minutes in PBS and the addition of HRP conjugated anti-rabbit secondary antibody (1:200; Vector Laboratories) for 1 h. This was followed by colorimetric detection and bright field observations.

DAB immunostained Ki-67 and activated caspase-3 were observed using an Olympus BX51 microscope, and an Olympus DP73 camera (Olympus Imaging America Inc., PA, USA). The percentage of Ki-67-positive cells was calculated by counting five random fields per preparation (25) using the National Institute of Health (NIH) open-source ImageJ software (http://rsb.info.nih.gov/ij/). The ImageJ cell analysis counter can divide cells into different types according to their level of staining, and this was used to discriminate Ki-67-positive cells from unstained cells. The proliferation index was expressed as Ki-67 positive cells/total number of cells ×100. The final results were expressed as the cell proliferation fraction relative to the control.

Microscopic examination of immunofluorescently stained cells. Immunofluorescent staining of PEDF protein was performed on 4T1 or MCF-10 cells (2.5×105 per well) cultured on glass coverslips. The 4T1 cells were treated with free RRD4 (25, 50, 100 μM) for 12 h, and with LD (5, 25 μM), EL (using equivalent volume of LD), or PBS for 24 h. MCF-10 cells were incubated with free RRD4 (50 μM), LD (5, 25 μM), EL (using equivalent volume of LD) or PBS for 24 h. The cells were post-fixed in 4% (w/v) paraformaldehyde (PFA) in 0.01 M PBS (pH 7.4) for 5 min. Following extensive washes in PBS, endogenous peroxidases were quenched using 0.3% hydrogen peroxide in methanol for 10 mins. Following cell permeabilization in 0.1% Triton-X100 and 0.1% sodium citrate buffer (pH 6.0) on ice, cells were incubated in anti-rabbit polyclonal PEDF antibody (1:200, Santa Cruz Biotech., Santa Cruz, CA, USA) overnight 4°C, followed by washes with PBS and the addition of Alexa Fluor® 546-conjugated anti-rabbit (1:200; Invitrogen) for 1 hour at room temperature. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). PEDF fluorescent staining was detected using an Olympus confocal laser-scanning fluorescence microscope (Laser Scanning Confocal Imaging System Bio-Rad MRC 1024, Hercules, CA, USA).

SDS-PAGE and Western blot analysis. Liver, lung, and tumor tissues from the groups of mice described earlier, were sonicated in tissue protein extraction buffer T-PER (Pierce, MA, USA), whereas the 4T1 cell samples were lysed using RIPA buffer (Piece, Cambridge, MA, USA). Total protein concentration was then measured using a BCA assay kit (Pierce). For each sample, 30 μg of protein were separated on a 12% polyacrylamide gel (Invitrogen Corp.). The separated proteins were transferred electrophoretically to Immobilon-P membranes (BioRad). Membranes were blocked for 1 h in a 5% milk buffer before overnight incubation with the first antibody described in Table I overnight at 4°C followed by three 10-minute washes in PBS-Tween 20 (PBS-T). This was followed by incubation with appropriate secondary antibody (Table I) and the Enhanced Chemiluminescence Signaling system (ECL; GE Healthcare, PA, USA). The resulting bands were compared by scanning densitometry (LI-COR Inc., NE, USA). To ensure equal loading, protein blots were stripped and re-probed with β-actin (Table I).

PEDF quantification. We quantified PEDF production in liver, lung, and tumor tissues using an ELISA kit (Life Span Biosciences, Inc., Seattle, WA, USA). Experimental procedures were performed as per manufacturer's instructions. Briefly, 100 μl of each standard and tissue sample lysate diluted to the appropriate concentration were incubated at 37°C in a 96-well plate. After 2 h, each well was washed at least four times using wash buffer before adding 100 μl diluted biotinylated antibody and incubated at 37°C for 1 h. Then 100 μl diluted streptavidin-peroxidase conjugate was added to each well and incubated at 37°C for an extra 1 h. The quantity of PEDF was measured spectrophotometrically at a wavelength of 450 in a microplate reader, and values obtained for tissue samples were normalized to the total protein concentration. The concentration of PEDF was determined by comparing the OD of drug-treated samples to the standard curve, and values obtained for lysates were normalized to matched-total protein concentrations.

PEDF knockdown transient transfection. 4T1 cells were transfected with siRNA targeting mouse PEDF (PEDFi) (Santa Cruz Biotechnology, CA, USA). The mouse PEDFi is a pool of 3 target-specific 19-25 nt siRNA using Lipofectamine LTX transfection reagent (Invitrogen), as per manufacturer's instructions. Briefly, 2.5 μg of PEDFi was combined with 1.25 μl of Plus reagent (Invitrogen) in 500 μl of serum-free media for 5 minutes, after which 5 μl of Lipofectamine LTX (Invitrogen) was added, mixed, and incubated for an additional 10 minutes. Then, it was added to the well containing 4T1 cells (2.5×105 per well). The transfected cells were incubated at 37°C in humidified air with 5% CO2 for 24 h. Twenty-four hours following transfection, the extent of PEDF knockdown was assessed by western blot.

For studying the mechanism of PEDF action, the PEDFi transfected cells were treated with final concentrations of either 50 μM free RRD4, or LD (5 μM, 25 μM) for a further 24 h. EL (using equivalent volumes of LD), and PBS were used as negative and positive controls, respectively. Cell viability levels, activated caspase-3 and PEDF expression were determined using methods described above.

Mouse model of breast cancer. Four-week-old female Balb/c mice were obtained from Jackson Laboratories (IN, USA). They were housed in ventilated cages (five per cage) that were exposed to natural 12-h light and dark cycles. The animals were maintained on ad libitum diet and water. All animal procedures were subjected to review and approval by the University's Institutional Animal Care and Use Committee (IACUC), which also complies with the NIH guidelines for humane treatment of laboratory animals. Animal experiments were carried out at the Animal Care Operations (ACO) facility of the University of Houston.

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Table I.

Antibodies used in the Western blot analysis and their properties.

After a week of acclimation, 4T1 cell suspension (1×105) was injected into mammary fat pad adjacent to the left forefoot (day 0) of each mouse. Once the tumors were palpable (volume was >1.2 cm3), the mice were divided into four groups (N=5 per group); Group 1 received 100 μl PBS (vehicle), Group 2 received 100 μl EL, Group 3 received 100 μl from 1 mM stock fee RRD4 solution, and Group 4 received 100 μl of 1 mM stock LD solution. Each group was injected through the tail with one of the solutions described above every other day. Each mouse received six injections over a 13-day period, except for the mice treated with free RRD4, which were given only five doses over 11 days. Similarly, mice in the control group were injected with six doses of 100 μl of PBS. Local tumor growth was monitored every other day by measuring two perpendicular tumor diameters with a caliper. Tumor volume was calculated by the formula tumor volume (mm3)=(length (mm)×(width (mm)2×0.52 (26- 27). The mice were examined every other day for maladies including rough coat appearance, discoloration of the skin, and swollen abdomen. At the time of animal sacrifice, either 11 days (free RRD4) or 13 days (PBS, EL, or LD) after treatment, mouse body, organ, and tumor weights were documented.

Organ and tumor collection, storage, and fixed tissue processing. Liver and lungs were carefully removed using forceps to avoid shearing of the tissues. The organs were first blotted on paper then weighed, after which each sample was hemisected; one-half was flash-frozen in liquid nitrogen and stored at −80°C for platinum quantification and protein extraction, as described in subsequent sections. The other half was fixed in 10% formalin solution for 48 h, then, stored in 70% ethanol for an additional 48 h prior to tissue processing and paraffin embedding. Tissues from livers, lungs, and orthotopic tumors were sectioned into 4 μm pieces using a rotary microtome knife (Leica RM2135, Germany), dewaxed at 60°C for 45 min before being subjected to sample processing and standard Haematoxylin and Eosin (H&E) staining (28). Stained sections were subjected to histological examination and photographed using an Olympus BX51 microscope with an attached Olympus DP73 camera (Olympus Imaging America Inc., Malville, NY, USA).

Tissue processing for Atomic Absorption Spectroscopic (AAS) measurement. For quantification of platinum content in tumors, minced tissues (0.5-1 g) (N=3) from mice treated with free RRD4, LD, EL, or PBS as described above, were each added to 4 ml aqua regia solution (3:1 M ratio, HCl:HNO3) (29), and heated to 90°C for 5 min to accelerate tissue disintegration. Once the volume of the suspension had decreased to 300 μl, 20 ml of sterile Millipore water (Millipore, Waltham, MA, USA) was added. Supernatants were collected after a 10-min centrifugation at 4,000 rpm.

Platinum content in tumor tissues was measured using a graphite furnace AAS (AAS-600, Perkin Elmer, MA, USA) (4). Calibration curves were established by using a Pt 10% HCl AAS standard from Perkin Elmer in 0.1% HNO3. The instrument parameters include a drying time of 30 sec at 125°C, an ashing time of 30 sec at 1300°C, and an atomizing time of 10 sec at 2,800°C in purge Argon gas atmosphere at wavelength of 265.9 nm. The data were collected from two to three independent experiments, and each measurement was carried out in duplicate or triplicate.

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Table II.

Mean concentration and efficiency of before and after encapsulation of drug.

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Table III.

Mean fraction of 4T1 proliferative cells treated with RRD4 vs. PBS treated control using Ki-67 immunostaining.

Statistical analysis. Comparisons among multiple groups were carried out by one-way ANOVA, followed by Dunnett post-hoc and Student's t-test was used for comparison between two groups using GraphPad PRISM 7.02 software. p-Values of less than 0.05 were considered statistically significant. Bars represent±SD unless indicated otherwise.

Results

Liposome formulation of RRD4 enhances the compound stability and storability. RRD4 encapsulated in liposomes (LD) or empty liposomes (EL) were prepared as described in the methods section. The initial free RRD4 drug concentration used for encapsulation, as quantified by AAS prior to the hydration step, was 49.5 μg/l (Table II). The measured concentration of LD based on platinum concentration after encapsulation and lysis of the liposomes was 45.2 μg/l, with encapsulation efficiency of 91% (Table II). Remarkably, after a month of storage at 4°C, the initial free drug and the measured drug after encapsulation efficiencies were only decreased by 25% and 18%, respectively, implying that both free and encapsulated drugs were still stable after a month at 4°C, with the encapsulated drug having 7% higher efficiency, compared to free RRD4. EL did not reveal any platinum content during any period of time (Table II).

Liposome formulation of RRD4 increases the inhibitory effect of the compound on 4T1 cell proliferation. We determined the effect of free RRD4 and LD on 4T1 cell proliferation in vitro using Ki-67 immunostaining. At 12 h, samples containing 25, or 50 μM free RRD4 did not have any effect on 4T1 cell proliferation vs. control (Figure 2A). In contrast, samples containing 100 μM free RRD4 resulted in substantially lower cell proliferation (Figure 2A) at 12 h. At 24 h, samples including 50 or 100 μM free RRD4 showed significantly decreased cell proliferation vs. control (Table III). At 48 h, all tested free RRD4 doses greatly reduced cell proliferation vs. control (Table III). Notably, samples containing 25 μM LD markedly reduced 4T1 cell proliferation after only 12 h (Figure 2B), suggesting that liposomes may contribute to prolonging the biological half-life of the encapsulated drug vs. the free drug. Conversely, 5 μM LD (5LD) and 5EL had no effect on cell proliferation at 12 h vs. control (Figure 2B). The Ki-67 results were supported by Hoechst fluorometric assay, showing that 4T1 proliferation levels were significantly reduced with final 50 or 100 μM free RRD4 doses at 24 h, and with final doses of 25, 50 or 100 μM free RRD4 at 48 h (Table IV).

The effect of the drug was also investigated in the non-tumorigenic breast MCF-10 line. The results revealed that at 24 h, cell proliferation was not affected by LD (5, 25 μM) (Figure 3B, D, E), compared to the control (Figure 3A, E) or EL (Figure 3C, E) by Ki-67 immunostaining assay. Similarly, we observed no change in cell proliferation vs. control regardless of the drug concentration or whether it was encapsulated in liposomes or not at 24 h using Hoechst nuclei acid stain (Figure 4).

Collectively, these results indicate that both free RRD4 and LD consistently inhibit breast cancer cell proliferation in a concentration- and time-dependent manner, while sparing normal breast cells for at least 24 h. Liposome formulation seems to enhance the antitumor activity of RRD4 without increasing the toxicity to normal cells.

Figure 2.
Figure 2.

4T1 cell proliferation fraction relative to the control post –treatment with free RRD4 or LD at 12 h. (A) The cell proliferation fraction relative to the control was unchanged with 25 and 50 μM free RRD4 at 12 h. (B) The cell proliferation fraction relative to the control was unchanged except in the cell containing 25LD at 12 h. The fraction of proliferation was expressed using PBS treated wells as 1. Columns in the graph represent the mean±SD (N=6-8, ANOVA followed by Dunnett posthoc test, p<0.05 is shown with one asterisk).

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Table IV.

Mean fraction of 4T1 proliferative cells treated with RRD4 vs. PBS treated control using blue-fluorescent Hoechst nucleic acid stain.

Both free and liposome formulated can efficiently induce apoptotic death of 4T1 tumor cells. Our early studies showed that one of the key antitumor mechanisms of phosphaplatins is by inducing apoptotic death of tumor cells of ovarian origin (6). We first sought to determine the viability of 4T1 cells in the presence and absence of RRD4 using the CellTiter-Glo™ luminescence assay. Incremental final doses of free RRD4 (25, 50, 100 μM) resulted in reduced 4T1 cell viability relative to controls in a concentration- and a time-dependent manner (Figure 5A). Although no significant decrease in cell viability was observed with low EL volumes (the equivalent volume to 5 and 10 μM LD), higher volumes of EL (the equivalent volume to 25, 50, 100 μM of LD) induced significant cell death at 48 h (Figure 5B). At 12 h, only samples containing 25, 50, or 100 μM LD, caused significantly higher 4T1 cell death (Figure 5C). But, at 24 and 48 h, samples including even the lowest doses of LD (5, 10 μM) decreased 4T1 cell viability. The results of the CellTiter-Glo™ assay at the 12 hour-time point were replicated with Trypan Blue with free RRD4 (Figure 6B-E), LD (Figure 7C, E-F), or EL equivalent volume of LD (Figure 7B, D, E) vs. matched controls (Figure 6A, E and 7A, F, respectively).

We also measured the impact of RRD4 on the viability of normal breast cells. We tested various final doses of free RRD4 (25, 50, 100 μM) and LD (5, 10, 25, 50, 100 μM) on MCF-10 cells for 12, 24, and 48 h. We observed a significant reduction in cell viability only at 100 μM free RRD4 at 48 h (Figure 8A). EL had no effect on MCF-10 cells throughout the incubation time that lasted for 48 h (Figure 8B). Low doses of LD (5 and 10 μM) had no effect on cell viability, whereas higher final doses of LD (25, 50, or 100 μM) resulted in substantially lower cell viability (Figure 8C).

As 4T1 cell viability was substantially diminished in the presence of RRD4, we evaluated the level of expression of a marker of apoptotic cell death (activated caspase-3) using immunohistochemistry and western blot. As expected, immunostaining of activated caspase-3 was substantially increased in the 4T1 cells in the presence of free RRD4 (Figure 9B-D) (48 h) or LD (Figure 9H-I) (24 h), compared to cells treated with either EL (data not shown), or PBS (Figure 9A, G). Negative controls where the first antibody was replaced with a non-immune serum did not reveal any significant positive immunostaining for activated caspase-3 (data not shown). These findings were confirmed by western blot analysis, showing a clear band for catalytically active caspase-3, representing endogenous levels of the large fragment (19-kDa) of activated caspase-3 (Figure 9E, J). The band was markedly up-regulated in total sample lysates treated with free RRD4 (Figure 9E-F) or LD (Figure 9J-K), compared to their respective matched controls. We further analyzed the apoptotic induction by quantitatively measuring activated caspase-3 in 4T1 cells using an ELISA assay. We found that activated caspase-3 production was greatly increased in cell lysates with 50 μM free RRD4 (2.7-fold), or 5 μM and 25 μM LD (1.8- and 2.8-fold, respectively) (Table V). Similarly, activated caspase-3 was stimulated in the supernatant of samples containing 50 μM free RRD4 (3.1-fold), or 5 μM and 25 μM LD (3.1- and 3.8-fold, respectively) (Table V). Neither lysates nor supernatants from 4T1 cultures treated with EL produced significant amounts of activated caspase-3 relative to cultures treated with PBS (Table V).

Figure 3.
Figure 3.

Effect of LD on MCF10 after 24 h. (A-D) Representative photomicrographs of MCF-10 cells stained with Ki-67 antibody showing a high number of positive nuclei at 24 h. (E) The cell proliferation fraction relative to the control was unchanged with all drug treatments using Ki-67. Columns in the graph represent the mean±SD (N=3, ANOVA followed by Dunnett posthoc test, p=0.05). Scale bar: 100 μm.

Figure 4.
Figure 4.

MCF-10 cell proliferation post free RRD4 or LD treatments by blue-fluorescent Hoechst 33258 nucleic acid stain at 24 h. The fraction of proliferation was expressed using PBS treated wells as 1. Columns in the graph represent the mean±SD (N=3). No significant change has been observed between treated and untreated control groups.

Previous data from our laboratory showed that TNF receptor superfamily member 6 (FAS) protein was overexpressed in phosphaplatin-treated ovarian tumor cells (6). Thus, we sought to determine whether FAS protein was also present in 4T1 cells treated with and without RRD4 using Western blot analysis. As expected, we detected a single 50-kDa immunoreactive band for FAS protein. We also found that FAS expression was higher in the presence of either 50 μM free RRD4 (1.7-fold) or 5 and 25 μM LD (1.4- and 1.8-fold, respectively), compared to their matched controls. EL-treated groups had little or no expression of FAS protein (Figure 9L).

View this table:
Table V.

Concentration of activated caspase-3 secreted in cell lysate and supernatant vs. Controls.

We also evaluated the expression of pro-apoptotic proteins such as PUMA and BAX, and the anti-apoptotic regulator BCL2 in total 4T1 cell lysates. For each of these proteins, we detected a single immunoreactive band of the expected molecular weight (Figure 9L). Samples containing free RRD4 (50 μM), or LD (5, 25 μM) had increased pro-apoptotic BAX (6.1, 7.6, 2.5-fold, respectively), whereas PUMA was increased only with 25 μM LD (1.8-fold), compared to matched controls. Conversely, samples treated with 50 μM free RRD4, or 5 and 25 μM LD had reduced anti-apoptotic BCL2 expression (3.2-, 2.8- and 1.4-fold, respectively), compared with matched controls.

Collectively, these results indicate that RRD4 is a selective and potent inducer of apoptotic cell death in a concentration- and time-dependent manner, preferentially targeting cancer cells, while sparing normal cells at low LD concentrations. The action mechanism of RRD4 is likely through the activation of the FAS intrinsic pathway.

Figure 5.
Figure 5.

4T1 viability assay using CellTiter-Glo™ luminescence assay. (A) 4T1 treated with increasing final doses of free RRD4 (25, 50, 100 μM) yielded reduced 4T1 cell viability relative to controls in a time-dependent manner. (B) 4T1 cells treated with EL show no significant decrease in cell viability with low EL volumes (the equivalent volume to 5 and 10 μM LD), higher volumes of EL (the equivalent volume to 25, 50, and 100 μM of LD) induced significant cell death at 48 h. (C) Doses of LD (25, 50, or 100 μM) caused significantly higher 4T1 cell death at 12 h. At 24 and 48 h even the lowest doses of LD (5, 10 μM) decreased 4T1 cell viability.

RRD4 in both free and liposome formulated form can inhibit 4T1 cell migration. We used a scratch assay to evaluate the effect of RRD4 on the ability of 4T1 to cross an artificial wound, which is a good mimic of cellular migration behavior in vivo (22). As shown in Figure 10A, the cell culture plates that did not have the compound exhibited a high degree of cell migration (i.e., 4T1 showed almost complete coverage of the wound). Conversely, cells treated with free RRD4 (Figure 10C-E, G) or LD (Figure 10F-G) showed incomplete wound closure. 4T1 migration was quantified by measuring the size of the gap that was still open after 20 h relative to the area formed from the initial scratch. Compared to the control (1±0.02, migration), samples containing 25, 50, 100 μM free RRD4, or 25 μM LD had undergone 0.95±0.02, 0.77±0.04, 0.51±0.04, and 0.43±0.08 cell migration respectively (Figure 10G). The data clearly indicate that both free RRD4 and LD hamper cell migration.

Evaluation of the RRD4 antitumor effect in vivo. 4T1 cells were implanted orthotopically into the mammary fat pad of mice. Once tumors reached an approximate size of 5 mm in diameter, mice were treated with free RRD4, LD, EL or PBS as described in the Materials and Methods. The local tumor growth was monitored by measuring the tumor every other day, and the lung and liver metastasis was determined at the end of the experiment by histology examination. The results show that LD was the most effective treatment among the groups in slowing tumor growth (Figure 11A). Mean tumor volumes from mice treated with free RRD4 or LD were significantly reduced by 61%, and 64%, respectively, compared to the PBS control (Figure 11A). Mice-treated with EL equivalent volume of LD had a trend towards a decrease in tumor size, compared to those of PBS-treated after 13 days, however, the results were not statistically different (Figure 11A).

The therapeutic effect on tumor metastasis was assessed by histological examination on sections prepared from the collected organ tissues by the end of this in vivo study. Microscopic analysis of tissues revealed the presence of micrometastases in the liver (whit arrows Figure 11B-E) and the lungs (white arrows in Figure 11F-I), where the cancer cells (indicated by white arrows) invaded the normal tissues. We found fewer tumor metastases in the free RRD4-treated samples, and fewest in the LD-treated groups in both liver and lungs, compared to their matched control groups (Figure 11B, F). We also examined the primary tumors histologically. The primary tumors from mice treated with either PBS or EL consisted of sheets of cells with nuclei (Figure 11I-J), whereas tumors from animals treated with free RRD4 or LD (grey arrows, Figure 11J-M) revealed fewer nuclei in areas with a high degree of apoptosis or necrosis. Using Western blots, we confirmed that the liver, lung, and tumor tissue protein lysates from free RRD4- or LD-treated mice had significantly increased levels of apoptosis, as reflected by the up-regulation of activated caspase-3 expression, a marker of apoptosis (Figure 12A-C).

Figure 6.
Figure 6.

Trypan Blue staining of 4T1 at 12 h. (A-D) Photomicrographs of 4T1 stained with Trypan Blue post-incubation in different concentrations of free RRD4 showing prominent staining with 100 μM free RRD4. (E) Graphical representation of the fraction of cell death after treatments with free RRD4 using Trypan blue. Scale bar: 50 μm.

We also examined the total protein binding ability of free RRD4 and LD extracted from cell tumor lysates by AAS. Platinum taken up by tumors treated with LD was more than twice as much as that of tumors treated with free RRD4 (Figure 12 Q; p<0.05), indicating that liposome formulation can improve RRD4 delivery as well as retention in tumor tissues. Platinum content from tumor tissues treated with EL was comparable to that of tumor tissues from PBS at the background level (Figure 12D).

As a part of efforts to determine the in vivo toxicity of RRD4, we measured the animal body weights during the experiment. Prior to the initiation of the treatments, there was no difference in body weights among the mice (data not shown). The weights of animals treated with 100 μl of final dose 1 mM LD or the EL equivalent volume of LD, were unchanged, compared to those of PBS-treated mice (Figure 13) after 13 days of treatment. Also, liver and lung weights of these animals were not affected by either LD or EL (Table VI). Although, the body weight of animals treated with free RRD4 was reduced by 5%, compared to the PBS control group (Figure 13; p<0.05) after 11 day-treatments, their liver and lung weights were comparable to those of their matched controls at the time of recording (Table VI). Of note, the group of mice treated with free RRD4 were sacrificed after the fifth injection because they showed signs of illness.

The antitumor mechanism of RRD4 includes the up-regulation of PEDF expression and secretion. PEDF plays an important part in the inhibition of tumor growth and metastasis (30). To assess the role of PEDF induction by RRD4 in contributing to the observed therapeutic benefit on both primary and metastatic tumors, we determined the in vivo production of PEDF in liver, lung, and tumor tissues from mice exposed to free RRD4, LD, EL and PBS using an ELISA assay. Our results revealed a marked increase in PEDF in response to both free RRD4 and LD in the liver (3.9-fold and 4.1-fold respectively) (Figure 14A), lungs (2.2-fold and 2.4-fold respectively) (Figure 14B), and tumors (3.7-fold and 4.5-fold respectively) (Figure 14C), compared to matched PBS-treated controls.

Figure 7.
Figure 7.

Trypan Blue staining of 4T1 at 12 h. (A-E) Photomicrographs of 4T1 stained with Trypan Blue post incubation with different doses of LD or the equivalent volume of EL revealing prominent staining with (E) 25 μM LD. (F) Graphical representation of the fraction of cell death using Trypan Blue after treatments with LD. Scale bar: 50 μm.

Figure 8.
Figure 8.

MCF-10 viability assay using CellTiter-Glo™ luminescence assay. (A) MCF-10 cells treated with free RRD4 showing significant cell death at 100 μM at 48 h. (B) MCF-10 treated with EL demonstrated insignificant cell death at all the time periods tested. (C) MCF-10 treated with LD (25, 50 or 100 μM) yielded reduced 4T1 cell viability relative to controls in a time-dependent manner, whereas lower LD concentrations (5, 10 μM) had no impact on cell death at 48 h. For all the cell death experiments, the fraction of cell death was expressed using PBS treated wells as 1. The graphs represent the mean±SD (N=3-8, ANOVA followed by Dunnett posthoc test, p<0.05).

Figure 9.
Figure 9.

Caspase-3 activation and apoptosis pathway. 4T1 cells were probed for the presence of activated caspase-3. Cell treated with final doses of (B-D) free RRD4 (25, 50 or 100 μM) for 48 h increased activated caspase-3 staining vs. (A) PBS control, as revealed by immunocytochemistry. Cells treated with (C-D, K) LD (5 or 25 μM) for 24 h resulted in increased activated caspase-3 staining vs. (A) PBS control for using immunocytochemistry. EL and PBS controls had no significant effect on caspase-3 immunostaining. Western blot and densitometric analysis of activated caspase-3 in cells treated with (E-F) free RRD4 for 48 h, or (J-K) LD for 24 h. (L) Western blot analysis of pro-apoptotic FAS, PUMA, BAX, and anti-apoptotic BCL2 after treatment with either 50 μM free RRD4 or LD (5, 25 μM) for 24 h resulted in stimulation of the FAS apoptotic intrinsic pathway. Columns in the graphs represent the mean±SEM (N=4-8, ANOVA followed by Dunnett posthoc test, p<0.05 is shown with one asterisk. Scale bars: 50 or 100 μm.

Figure 10.
Figure 10.

Free RRD4 and LD inhibit wound closure. (A-F) Effects of free RRD4 and LD on wound healing in scratch assays as shown in the bright-field microscope images of 4T1 migration. 4T1 migration was inhibited by the addition of (D-E) 50 and 100 μM free RRD4, or (F) 25 μM LD, but not by (C) 25 μM free RRD4 or (B) PBS after 20 h. (G) Graphical representation of the fraction of cell migration after treatments with free RRD4 or LD. The fraction of cell inhibition was expressed using vehicle treated wells as 1. Columns in the graph represent the mean±SD (N=3; ANOVA followed by Dunnett posthoc test, p<0.05 is shown with one asterisk). Scale bar: 200 μm.

Figure 11.
Figure 11.

In vivo effect of free RRD4 and LD. (A) 4T1 cells were implanted orthotopically into the mammary fat pad. Mouse treatments were administrated via tail vein every other day for a total of either 11 days (free RRD4) or 13 days (LD, EL, or PBS). Tumor size were recorded every other day. Grey arrows indicate days of injection of the free RRD4 drug. Grey arrows show days of injection of PBS, EL, and LD. The graph represents the mean±SD (N=5, ANOVA followed by Dunnett posthoc test, p<0.05 and p<0.01 are shown with one and two asterisks, respectively). (B-M) H& E staining showing lower micrometastases (white arrows) in (B-E) Liver, (F-I) and lung treated with free RRD4 or LD. (J-M) Primary tumors from mice treated with either EL or PBS consisted of sheets of cells. White arrows indicate metastatic deposits in liver and lungs, while grey arrows indicate zones of fewer nuclei in tumors. Black arrows represent clumps of liver tumor cells in inserts. Scale bars: panels (100 μm) and inserts (50 μm).

Figure 12.
Figure 12.

Tissue activated caspase-3 expression in (A) liver, (B) lung, and (C) tumor tissue protein lysates from free RRD4- or LD-treated mice had significantly increased levels of apoptosis, as reflected by the up-regulation activated caspase-3 using western blot. (D) Accumulation of platinum in the tumor after injection of free RRD4, EL or LD. Columns in the graphs represent the mean±SD (N=3-5, ANOVA followed by Dunnett posthoc test, p<0.05 and p<0.01 are shown with one and two asterisks, respectively.

The in vivo data were supported by in vitro measurement of PEDF expression from cultures of 4T1 cells treated with increasing concentrations of free RRD4 or LD. Adding either drug resulted in a significant increase in nuclear PEDF expression in a concentration- and time-dependent manner. For example, at 12 h, densitometric analysis of the bands revealed a final dose of 50 or 100 μM free RRD4 resulted in significant induction of PEDF expression (3.6- and 3.5-fold, respectively) (Figure 15A-B). Moreover, cell samples containing 5 or 25 μM LD yielded markedly increased levels of PEDF protein, but not with EL vs. control at 24 h as shown by western blotting (Figure 16A-B). Western results for 4T1 were confirmed by immunofluorescence confocal microscopy for free RRD4 (Figure 17B-L) vs. controls (Figure 17 A-C) at 12 h, and for LD (Figure 18B, D) vs. control (Figure 18A) at 24 h. Notably, PEDF is generally considered a secreted protein. However, several immunochemical studies (31-32), including our present work showed strong nuclear staining for this protein by immunofluorescence confocal microscopy.

The expression of PEDF was also up-regulated in MCF-10 cell samples containing free RRD4 (50 μM) or LD (5 μM) by Western blot (Figure 19F). Using confocal microscopy, we also found that free RRD4 (50 μM) (Figure 19B) or LD (5, 25 μM) (Figure 19C, E) increased mainly nuclear immunostaining vs. controls (Figure 19A). In contrast, EL had no significant impact on PEDF expression (Figure 19D) vs. controls (Figure 19A). Collectively, these results indicate that RRD4 mediates increased levels of PEDF in liver, lung, and tumor tissues in vivo, and in 4T1 and MCF-10 cells in vitro.

To investigate the functional significance of PEDF induction by RRD4 treatment, we applied siRNA-mediated gene silencing of PEDF on protein expression from 4T1 cultures. We initially examined PEDF expression using western blot analysis. Twenty-four hours post PEDFi transfection, PEDF expression levels were reduced by 98% in 4T1 cells, compared to the control (Figure 20A-B). The effect of RRD4 on the expression of PEDF protein in the presence of PEDFi was then assessed by western blot analysis. In the presence of PEDFi, 4T1 cells treated with 50 μM free RRD4 or LD (5, 25 μM) resulted in no significant increase in PEDF expression, compared to matched controls (Figure 21).

We then examined the impact of RRD4 in the presence of PEDFi on accumulation of apoptotic cell markers. Activated caspase-3 production was not increased by the addition of 50 μM free RRD4, or LD (5, 25 μM) in either PEDFi cell lysates or supernatants (Table V). When using CellTiter-Glo™ viability assay (Figure 22) or Western blot (Figure 23), we found that despite the presence of PEDFi, caspase-3 was significantly activated in cells treated with 25 μM LD. All samples treated with EL produced no significant increase in activated caspase-3 in the presence of PEDFi, compared to matched controls with PEDFi (Figure 23, Table V).

Figure 13.
Figure 13.

Mean weights from animals treated with and without RRD4. Black arrows indicate days of injection of the free RRD4 drug. Grey arrows show days of injection of the PBS, EL or LD. The graph represents the mean±SD (N=5, ANOVA followed by Dunnett posthoc test, p<0.05 is shown with one asterisk).

Figure 14.
Figure 14.

PEDF production in vivo. (A-C) PEDF production in liver, lung and tumor tissues as determined by an ELISA assay. Columns in the graphs represent the mean±SEM (N=3, ANOVA followed by Dunnett posthoc test, p<0.05 and p<0.01 are shown with one and two asterisks, respectively).

Collectively, these findings indicate that PEDF protein may contribute to the increase in apoptosis elicited by RRD4. Consistent with this, PEDF protein may mediate, at least partly, the reduced tumor size and tumor metastasis to liver and lungs.

Discussion

The present study was initiated to evaluate the therapeutic effects of phosphaplatins on breast tumor growth and metastasis. We formulated RDD4 encapsulated liposomes with high encapsulation efficiency and which were stable even after one-month of storage at 4°C. Moreover, unlike free RRD4, RRD4-encapsulated liposomes (LD) have no impact on animal body weights. On the other hand, both free RRD4 and LD treatments resulted in primary tumor regression and a lower level of metastases to the liver and the lungs. At the cellular level, RRD4-mediated cell death was associated with up-regulation of pro-apoptotic proteins such as FAS, BAX, PUMA, and caspase-3, and down-regulation of anti-apoptotic protein BCL2, suggesting a FAS intrinsic signaling pathway as a mechanism to apoptosis. The RRD4-mediated induction of PEDF protein may contribute to the increase in apoptosis, which in turn may, at least partly, promote the reduced tumor size and tumor metastasis to liver and lungs. Therefore, free RRD4, and more importantly LD, could be excellent candidates as an adjuvant therapy for breast cancer and for preventing its metastasis to other organs.

Figure 15.
Figure 15.

(A-B) Representative western blots and densitometric analysis of PEDF expression in 4 T1 cells with and without free RRD4 at 12 h. Columns in the graphs represent the mean±SEM (N=3, ANOVA followed by Dunnett posthoc test, p<0.05 is shown with one asterisk).

The benefit of carrier systems such as liposomes to improve the therapeutic activity of anticancer agents is usually achieved by reducing drug-related toxicities to normal tissues, thereby permitting elevated drug doses to be employed and/or boosting the antitumor potency of the drug (33). We found that the intermittent injection of LD to a 4T1 mouse model of breast cancer resulted in weight comparable to that of animals treated with vehicle (PBS), implying that the drug was not toxic, unlike in animals treated with free RRD4. The fact that lower doses of LD were needed to impact breast tumor cells with a minimal effect on normal breast cells in vitro supports the use of liposomes as a carrier.

Additionally, our studies suggest that enhanced antitumor activity observed with LD preparations is related to the ability of the liposome carrier system to increase tumor drug accumulation and retention at solid tumor sites over an extended period of time, leading to increased therapeutic activity. The lack of effect of the empty liposomes was not surprising due to their similarity to biological membranes, which makes them safe and biocompatible (34).

Figure 16.
Figure 16.

PEDF expression in 4T1 cells. (A) Representative western blots and (B) densitometric analysis of PEDF expression in 4T1 with and without LD or EL (5 and 25 μM) at 24 h. Columns in the graphs represent the mean±SEM (N=3, ANOVA followed by Dunnett posthoc test, p<0.05 is shown with one asterisk).

At the cellular level, several studies have described a correlation between apoptosis and proliferation (35-37) in various cancers. However, there are also incidences where an apoptotic index was not strictly associated with proliferation index (38-40). In our in vitro cell culture systems, we observed both decreased breast cancer cell proliferation and viability, together with increased activation of caspase-3 in the presence of RRD4, particularly in the group treated with LD. We speculate that a decreased proliferation coupled with increased cell death contribute to the inhibition of primary tumor growth, with subsequent negative effects on tumor cell migration from primary tumors to distant organs. In vitro studies showing lower levels of cell migration in the scratch assay corroborated this conclusion.

Figure 17.
Figure 17.

Confocal microscopy of mainly nuclear PEDF expression in 4T1 cells following treatment with (D-L) free RRD4 and (A-C) PBS control after 12 h. Scale bar: 50 μm.

Previous work from our laboratory revealed that decreased cell viability was accompanied by FAS overexpression in cells treated with RRD2 (6). Similarly, we found that decreased cell viability accompanied by increased cell apoptosis in 4T1 cells treated with either free RRD4 or LD was paralleled by an overexpression of FAS protein. FAS protein can activate apoptosis through the extrinsic and the intrinsic pathways, both of which lead to caspase-3 activation and apoptosis. This is mediated by the cytoplasmic domain of FAS that has a protein-protein interaction motif, the FAS-activated death domain protein (FADD), allowing the induction of downstream signal transduction machinery. In the intrinsic pathway, apoptosis is initiated by the cleavage of BID (BH3) interacting-domain death agonist, which in turn activates the mitochondrial pathway by inhibiting anti-apoptotic BCL2, activating pro-apoptotic BAX or PUMA, and releasing cytochrome c to the cytosol, leading to the stimulation of initiator caspases such as caspase-9 with downstream cleavage of effector caspase-3 to yield apoptosis (41). Our current study showed that BAX and PUMA were up-regulated, while BCL2 was down-regulated in the RRD4-treated samples, supporting a role for FAS in the intrinsic pathway to apoptosis in the 4T1 breast cancer cells.

Figure 18.
Figure 18.

Confocal microscopy of nuclear PEDF expression following incubation with (B, D) LD, (C) 25EL (equivalent volume of 25 μM LD), and (A) PBS control in 4T1 cells at 24 h. Scale bar: 20 μm.

Figure 19.
Figure 19.

Confocal microscopy showing mainly nuclear PEDF expression following MCF-10 cells incubation with (B) 50 μM free RRD4, (C, E) 5 and 25LD (5 and 25 μM) or (D) 25EL (equivalent volume of 25 μM LD) at 24 h. Scale bar: 20 μm. (F) Representative western blots of PEDF expression in MCF-10 cells treated with RRD4 or LD at 24 h.

Figure 20.
Figure 20.

PEDF inhibition produces robust and specific target knockdown. (A) Representative western blot and (B) corresponding densitometric analysis demonstrating significant knockdown of the target protein by PEDFi with no significant effect elicited in the control by the transfection reagent alone in the 4T1 cells. Columns in the graph represent the mean±SD (N=2, Student't-test, p<0.05 is shown with one asterisk).

There is strong evidence to support a role for PEDF as a tumor suppressor (42). PEDF inhibits the development of various cancers, including lung (43) liver (44), prostate (45), ovarian (45), and pancreatic carcinoma (45), melanoma (46), osteosarcoma (47), and neural tumors (16). In the current study, decreased breast tumor sizes and lower incidence of secondary tumors in liver and lung tissues in the 4T1 mouse model of breast cancer were accompanied by PEDF up-regulation in the RRD4-treated hosts, implicating PEDF as a potentially important molecule that could contribute to the reduction in tumor growth and metastasis. This hypothesis that elevated production of PEDF may represent a mechanism driving tumor regression is corroborated by two reports. The first one shows that intratumoral injection of a PEDF-expressing plasmid in athymic mouse models caused significant inhibition on the growth of the pre-established tumor (48). The other report reveals that lung-derived endothelial cells from PEDF-deficient mice exhibit enhanced migratory capacity and adherent ability relative to cells from wild-type mice (49), consistent with an inhibitory effect of PEDF in cancer progression.

Figure 21.
Figure 21.

Representative western blot demonstrating significant knockdown of PEDF target protein by PEDFi in the presence of free RRD4 or LD.

View this table:
Table VI.

Balb/c organ and tumor weights with and without drug treatment.

Although the exact mechanism of PEDF action on metastatic cancers has yet to be fully elucidated, some previous studies may shed some light on how PEDF may hamper metastasis (50). Besides reducing angiogenesis (51), and decreasing VEGF expression (52), some investigations have demonstrated that PEDF is a potent inducer of cell apoptosis (2, 53).

Specifically, it has been suggested that PEDF can target malignant tumor cells directly by attenuating tumor proliferation and promoting apoptosis (2), reducing the likelihood of migration In the current work, increased amounts of PEDF protein and unchanged or reduced levels of proliferation and apoptosis in breast tumor cells transfected with PEDFi and treated with RRD4 indicated that PEDF may play a pivotal role on inhibiting primary breast tumor growth, as well as preventing metastasis. This proof-of-principle test through RNA interference confirms the notion that PEDF production and expression can specifically and robustly attenuate breast tumor growth and metastasis, and that such inhibition is due to potentiation of the effects of PEDF by chemotherapeutic agents such as RRD4.

Figure 22.
Figure 22.

Graphical representation of the fraction of viable cells post-transfection with PEDFi and treated with a final dose of 50 μM free RRD4, LD (5, 25 μM), EL, or PBS for 24 h. No significant cell death was observed in the presence of PEDFi except for LD (25 μM). Conversely, with no PEDFi, there was a significant decrease in cell viability in cell treated with 50 μM free RRD4 or LD (5 or 25 μM) vs. control treated with PBS. The fraction of cell death was expressed using PBS treated wells as 1. Columns in the graphs represent the mean±SD (N=3; ANOVA followed by Dunnett posthoc test, *p=0.05, **p=0.0005, ***p=0.0001 shown with one, two or three asterisks).

PEDF exerts its anti-angiogenic activity in part by inducing endothelial cell death. Volpert et al. (2) showed that the mechanism by which PEDF does this is via the activation of the FAS/FASL death pathway. Takenaka et al. (54) and Abe et al. (46) demonstrated that PEDF administration in vitro caused a significant degree of cell apoptosis in both melanoma (G361) and osteosarcoma (MG63) cell lines, and this effect could be reversed by administration of anti-FASL antibody. Therefore, we postulate that in breast tumors, increased PEDF may also stimulate tumor cell death through the FAS pathway. Elevated FAS expression in the 4T1 in the presence of RRD4 corroborates this premise. However, this requires further investigation.

Figure 23.
Figure 23.

Representative western blot revealing that in contrast with 25 μM LD treatment, cell incubation with 50 μM free RRD4 or 5 μM LD resulted in no activation of caspase-3, compared to control cells treated with transfection reagent. EL has no effect on cell death.

In summary, we established that liposome formulation enhances the potency of RRD4 against breast tumor growth and metastasis with reduced toxicity over the free form of the drug. Our findings provide evidence that RRD4, especially when encapsulated in liposomes, may represent an excellent candidate for adjuvant- and mono-therapy to alleviate breast tumor burden.

Acknowledgements

The Authors would like to thank Mr. Jeff Spencer for molecular biology technical advice, Dr. Fu Xinping and Ms. Jin Aiwu for help with the 4T1 animal model. The Authors would also like to acknowledge the contribution of the Division of Research (DOR) at the University of Houston. This study was sponsored by a grant from The Cancer Prevention and Research Institute of Texas (RP130553 to XZ and RNB) and a grant from William and Ella Owen Medical Research Foundation.

Footnotes

  • This article is freely accessible online.

  • Received October 26, 2017.
  • Revision received November 19, 2017.
  • Accepted November 29, 2017.

References

  1. ↵
    1. Weigelt B,
    2. Peterse JL,
    3. van't Veer LJ
    : Breast cancer metastasis: markers and models. Nat Rev Cancer 5(8): 591-602, 2005.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Volpert OV,
    2. Zaichuk T,
    3. Zhou W,
    4. Reiher F,
    5. Ferguson TA,
    6. Stuart PM,
    7. Amin M,
    8. Bouck NP
    : Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor. Nat Med 8(4): 349-357, 2002.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Paronetto F
    : Immunocytochemical observations on the vascular necrosis and renal glomerular lesions of malignant nephrosclerosis. Am J Pathol 46: 901-915, 1965.
    OpenUrlPubMed
  4. ↵
    1. Bose RN,
    2. Maurmann L,
    3. Mishur RJ,
    4. Yasui L,
    5. Gupta S,
    6. Grayburn WS,
    7. Hofstetter H,
    8. Salley T
    : Non-DNA-binding platinum anticancer agents: Cytotoxic activities of platinum-phosphato complexes towards human ovarian cancer cells. Proc Natl Acad Sci USA 105(47): 18314-18319, 2008.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Bose RN,
    2. Ghosh SK,
    3. Moghaddas S
    : Kinetic analysis of the cis-diamminedichloroplatinum(II)--cysteine reaction: implications to the extent of platinum--DNA binding. J Inorg Biochem 65(3): 199-205, 1997.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Bose RN,
    2. Moghaddas S,
    3. Belkacemi L,
    4. Tripathi S,
    5. Adams NR,
    6. Majmudar P,
    7. McCall K,
    8. Dezvareh H,
    9. Nislow C
    : aAbsence of activation of DNA repair genes and excellent efficacy of phosphaplatins against human ovarian cancers: implications to treat resistant cancers. J Med Chem 58(21): 8387-8401, 2015.
    OpenUrl
  7. ↵
    1. Yang L,
    2. Moghaddas S,
    3. Dezvareh H,
    4. Belkacemi L,
    5. Bark SJ,
    6. Bose RN,
    7. Do LH
    : Insights into the anti-angiogenic properties of phosphaplatins. J Inorg Biochem 164: 5-16, 2016.
    OpenUrl
  8. ↵
    1. Becerra SP
    : Focus on Molecules: Pigment epithelium-derived factor (PEDF). Exp Eye Res 82(5): 739-740, 2006.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Kuncl RW,
    2. Bilak MM,
    3. Bilak SR,
    4. Corse AM,
    5. Royal W,
    6. Becerra SP
    : Pigment epithelium-derived factor is elevated in CSF of patients with amyotrophic lateral sclerosis. J Neurochem 81(1): 178-184, 2002.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Zhang L,
    2. Chen J,
    3. Ke Y,
    4. Mansel RE,
    5. Jiang WG
    : Expression of pigment epithelial derived factor is reduced in non-small cell lung cancer and is linked to clinical outcome. Int J Mol Med 17(5): 937-944, 2006.
    OpenUrlPubMed
  11. ↵
    1. Feng CC,
    2. Ding Q,
    3. Zhang YF,
    4. Jiang HW,
    5. Wen H,
    6. Wang PH,
    7. Wu Z
    : Pigment epithelium-derived factor expression is down-regulated in bladder tumors and correlates with vascular endothelial growth factor and matrix metalloproteinase-9. Int Urol Nephrol 43(2): 383-390, 2011.
    OpenUrlPubMed
  12. ↵
    1. Belkacemi L,
    2. Zhang SX
    : Anti-tumor effects of pigment epithelium-derived factor (PEDF): implication for cancer therapy. A mini-review. J Exp Clin Cancer Res 35: 4, 2016.
    OpenUrl
  13. ↵
    1. Dass CR,
    2. Ek ET,
    3. Contreras KG,
    4. Choong PF
    : A novel orthotopic murine model provides insights into cellular and molecular characteristics contributing to human osteosarcoma. Clin Exp Metastasis 23(7-8): 367-380, 2006.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Halin S,
    2. Wikstrom P,
    3. Rudolfsson SH,
    4. Stattin P,
    5. Doll JA,
    6. Crawford SE,
    7. Bergh A
    : Decreased pigment epithelium-derived factor is associated with metastatic phenotype in human and rat prostate tumors. Cancer Res 64(16): 5664-5671, 2004.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Uehara H,
    2. Miyamoto M,
    3. Kato K,
    4. Ebihara Y,
    5. Kaneko H,
    6. Hashimoto H,
    7. Murakami Y,
    8. Hase R,
    9. Takahashi R,
    10. Mega S,
    11. et al
    : Expression of pigment epithelium-derived factor decreases liver metastasis and correlates with favorable prognosis for patients with ductal pancreatic adenocarcinoma. Cancer Res 64(10): 3533-3537, 2004.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Guan M,
    2. Yam HF,
    3. Su B,
    4. Chan KP,
    5. Pang CP,
    6. Liu WW,
    7. Zhang WZ,
    8. Lu Y
    : Loss of pigment epithelium derived factor expression in glioma progression. J Clin Pathol 56(4): 277-282, 2003.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Yoneda T,
    2. Michigami T,
    3. Yi B,
    4. Williams PJ,
    5. Niewolna M,
    6. Hiraga T
    : Actions of bisphosphonate on bone metastasis in animal models of breast carcinoma. Cancer 88(12 Suppl): 2979-2988, 2000.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Mishur RJ,
    2. Zheng C,
    3. Gilbert TM,
    4. Bose RN
    : Synthesis, X-ray crystallographic and NMR characterizations of platinum(II) and platinum(IV) pyrophosphato complexes. Inorg Chem 47(18): 7972-7982, 2008.
    OpenUrlPubMed
    1. Eloy JO,
    2. Petrilli R,
    3. Topan JF,
    4. Antonio HM,
    5. Barcellos JP,
    6. Chesca DL,
    7. Serafini LN,
    8. Tiezzi DG,
    9. Lee RJ,
    10. Marchetti JM
    : Co-loaded paclitaxel/rapamycin liposomes: Development, characterization and in vitro and in vivo evaluation for breast cancer therapy. Colloids Surf B Biointerfaces 141: 74-82, 2016.
    OpenUrl
  20. ↵
    1. Ahn CS,
    2. Metallo CM
    : Mitochondria as biosynthetic factories for cancer proliferation. Cancer Metab 3(1): 1, 2015.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Cory G
    : Scratch-wound assay. Methods Mol Biol 769: 25-30, 2011.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Liang CC,
    2. Park AY,
    3. Guan JL
    : In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2(2): 329-333, 2007.
    OpenUrlCrossRefPubMed
  23. ↵
    1. van der Meer AD,
    2. Vermeul K,
    3. Poot AA,
    4. Feijen J,
    5. Vermes I
    : A microfluidic wound-healing assay for quantifying endothelial cell migration. Am J Physiol Heart Circ Physiol 298(2): H719-725, 2010.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Rodriguez LG,
    2. Wu X,
    3. Guan JL
    : Wound-healing assay. Methods Mol Biol 294: 23-29, 2005.
    OpenUrlPubMed
  25. ↵
    1. Belkacemi L,
    2. Hebb MO
    : HSP27 knockdown produces synergistic induction of apoptosis by HSP90 and kinase inhibitors in glioblastoma multiforme. Anticancer Res 34(9): 4915-4927, 2014.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Kotoh T,
    2. Dhar DK,
    3. Masunaga R,
    4. Tabara H,
    5. Tachibana M,
    6. Kubota H,
    7. Kohno H,
    8. Nagasue N
    : Antiangiogenic therapy of human esophageal cancers with thalidomide in nude mice. Surgery 125(5): 536-544, 1999.
    OpenUrlPubMed
  27. ↵
    1. Willson JK,
    2. Pretlow TG,
    3. Zaremba JL,
    4. Brattain MG
    : Heterogeneity among preparations of crude trypsin used to disaggregate the human tonsil. Immunology 30(1): 157-160, 1976.
    OpenUrlPubMed
  28. ↵
    1. Cardiff RD,
    2. Miller CH,
    3. Munn RJ
    : Manual hematoxylin and eosin staining of mouse tissue sections. Cold Spring Harb Protoc 2014(6): 655-658, 2014.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Meeravali NN,
    2. Madhavi K,
    3. Manjusha R,
    4. Kumar SJ
    : Sequential extraction of platinum, cisplatin and carboplatin from environmental samples and pre-concentration/separation using vesicular coacervative extraction and determination by continuum source ETAAS. Talanta 118: 37-44, 2014.
    OpenUrl
  30. ↵
    1. Hong H,
    2. Zhou T,
    3. Fang S,
    4. Jia M,
    5. Xu Z,
    6. Dai Z,
    7. Li C,
    8. Li S,
    9. Li L,
    10. Zhang T,
    11. Qi W,
    12. Bardeesi AS,
    13. Yang Z,
    14. Cai W,
    15. Yang X,
    16. Gao G
    : Pigment epithelium-derived factor (PEDF) inhibits breast cancer metastasis by down-regulating fibronectin. Breast Cancer Res Treat 148(1): 61-72, 2014.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Tombran-Tink J,
    2. Aparicio S,
    3. Xu X,
    4. Tink AR,
    5. Lara N,
    6. Sawant S,
    7. Barnstable CJ,
    8. Zhang SS
    : PEDF and the serpins: phylogeny, sequence conservation, and functional domains. J Struct Biol 151(2): 130-150, 2005.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Gupta RK,
    2. Barton RP
    : Letter: pneumothorax after general anaesthesia for panendoscopy. Br J Anaesth 47(5): 643, 1975.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Mayer LD,
    2. Bally MB,
    3. Cullis PR,
    4. Wilson SL,
    5. Emerman JT
    : Comparison of free and liposome encapsulated doxorubicin tumor drug uptake and antitumor efficacy in the SC115 murine mammary tumor. Cancer Lett 53(2-3): 183-190, 1990.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Treat J,
    2. Damjanov N,
    3. Huang C,
    4. Zrada S,
    5. Rahman A
    : Liposomal-encapsulated chemotherapy: preliminary results of a phase I study of a novel liposomal paclitaxel. Oncology (Williston Park) 15(5 Suppl 7): 44-48, 2001.
    OpenUrl
  35. ↵
    1. Mattern J,
    2. Volm M
    : Imbalance of cell proliferation and apoptosis during progression of lung carcinomas. Anticancer Res 24(6): 4243-4246, 2004.
    OpenUrlAbstract/FREE Full Text
    1. Ito Y,
    2. Matsuura N,
    3. Sakon M,
    4. Takeda T,
    5. Umeshita K,
    6. Nagano H,
    7. Nakamori S,
    8. Dono K,
    9. Tsujimoto M,
    10. Nakahara M,
    11. Nakao K,
    12. Monden M
    : Both cell proliferation and apoptosis significantly predict shortened disease-free survival in hepatocellular carcinoma. Br J Cancer 81(4): 747-751, 1999.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Hao X,
    2. Du M,
    3. Bishop AE,
    4. Talbot IC
    : Imbalance between proliferation and apoptosis in the development of colorectal carcinoma. Virchows Arch 433(6): 523-527, 1998.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Tormanen U,
    2. Eerola AK,
    3. Rainio P,
    4. Vahakangas K,
    5. Soini Y,
    6. Sormunen R,
    7. Bloigu R,
    8. Lehto VP,
    9. Paakko P
    : Enhanced apoptosis predicts shortened survival in non-small cell lung carcinoma. Cancer Res 55(23): 5595-5602, 1995.
    OpenUrlAbstract/FREE Full Text
    1. Hindermann W,
    2. Berndt A,
    3. Wunderlich H,
    4. Katenkamp D,
    5. Kosmehl H
    : Quantitative evaluation of apoptosis and proliferation in renal cell carcinoma. Correlation to tumor subtype, cytological grade according to thoenes-classification and the occurrence of metastasis. Pathol Res Pract 193(1): 1-7, 1997.
    OpenUrlPubMed
  40. ↵
    1. Tannapfel A,
    2. Geissler F,
    3. Kockerling F,
    4. Katalinic A,
    5. Hauss J,
    6. Wittekind C
    : Apoptosis and proliferation in relation to histopathological variables and prognosis in hepatocellular carcinoma. J Pathol 187(4): 439-445, 1999.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Cryns V,
    2. Yuan J
    : Proteases to die for. Genes Dev 12(11): 1551-1570, 1998.
    OpenUrlFREE Full Text
  42. ↵
    1. Becerra SP,
    2. Notario V
    : The effects of PEDF on cancer biology: mechanisms of action and therapeutic potential. Nat Rev Cancer 13(4): 258-271, 2013.
    OpenUrlCrossRefPubMed
  43. ↵
    1. He SS,
    2. Shi HS,
    3. Yin T,
    4. Li YX,
    5. Luo ST,
    6. Wu QJ,
    7. Lu L,
    8. Wei YQ,
    9. Yang L
    : AAV-mediated gene transfer of human pigment epithelium-derived factor inhibits Lewis lung carcinoma growth in mice. Oncol Rep 27(4): 1142-1148, 2012.
    OpenUrlPubMed
  44. ↵
    1. Lattier JM,
    2. Yang H,
    3. Crawford S,
    4. Grossniklaus HE
    : Host pigment epithelium-derived factor (PEDF) prevents progression of liver metastasis in a mouse model of uveal melanoma. Clin Exp Metastasis 30(8): 969-976, 2013.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Doll JA,
    2. Stellmach VM,
    3. Bouck NP,
    4. Bergh AR,
    5. Lee C,
    6. Abramson LP,
    7. Cornwell ML,
    8. Pins MR,
    9. Borensztajn J,
    10. Crawford SE
    : Pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas. Nat Med 9(6): 774-780, 2003.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Abe R,
    2. Shimizu T,
    3. Yamagishi S,
    4. Shibaki A,
    5. Amano S,
    6. Inagaki Y,
    7. Watanabe H,
    8. Sugawara H,
    9. Nakamura H,
    10. Takeuchi M,
    11. Imaizumi T,
    12. Shimizu H
    : Overexpression of pigment epithelium-derived factor decreases angiogenesis and inhibits the growth of human malignant melanoma cells in vivo. Am J Pathol 164(4): 1225-1232, 2004.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Ek ET,
    2. Dass CR,
    3. Contreras KG,
    4. Choong PF
    : PEDF-derived synthetic peptides exhibit antitumor activity in an orthotopic model of human osteosarcoma. J Orthop Res 25(12): 1671-1680, 2007.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Matsumoto K,
    2. Ishikawa H,
    3. Nishimura D,
    4. Hamasaki K,
    5. Nakao K,
    6. Eguchi K
    : Antiangiogenic property of pigment epithelium-derived factor in hepatocellular carcinoma. Hepatology 40(1): 252-259, 2004.
    OpenUrlCrossRefPubMed
  49. ↵
    1. He X,
    2. Cheng R,
    3. Benyajati S,
    4. Ma JX
    : PEDF and its roles in physiological and pathological conditions: implication in diabetic and hypoxia-induced angiogenic diseases. Clin Sci (Lond) 128(11): 805-823, 2015.
    OpenUrl
  50. ↵
    1. Orgaz JL,
    2. Ladhani O,
    3. Hoek KS,
    4. Fernandez-Barral A,
    5. Mihic D,
    6. Aguilera O,
    7. Seftor EA,
    8. Bernad A,
    9. Rodriguez-Peralto JL,
    10. Hendrix MJ,
    11. Volpert OV,
    12. Jiménez B
    : Loss of pigment epithelium-derived factor enables migration, invasion and metastatic spread of human melanoma. Oncogene 28(47): 4147-4161, 2009.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Dawson DW,
    2. Volpert OV,
    3. Gillis P,
    4. Crawford SE,
    5. Xu H,
    6. Benedict W,
    7. Bouck NP
    : Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science 285(5425): 245-248, 1999.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Zhang T,
    2. Guan M,
    3. Xu C,
    4. Chen Y,
    5. Lu Y
    : Pigment epithelium-derived factor inhibits glioma cell growth in vitro and in vivo. Life Sci 81(16): 1256-1263, 2007.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Guan M,
    2. Jiang H,
    3. Xu C,
    4. Xu R,
    5. Chen Z,
    6. Lu Y
    : Adenovirus-mediated PEDF expression inhibits prostate cancer cell growth and results in augmented expression of PAI-2. Cancer Biol Ther 6(3): 419-425, 2007.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Takenaka K,
    2. Yamagishi S,
    3. Jinnouchi Y,
    4. Nakamura K,
    5. Matsui T,
    6. Imaizumi T
    : Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells. Life Sci 77(25): 3231-3241, 2005.
    OpenUrlCrossRefPubMed
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Phosphaplatin Anti-tumor Effect Enhanced by Liposomes Partly via an Up-regulation of PEDF in Breast Cancer
LOUIZA BELKACEMI, JASON L. ATKINS, LU YANG, PRAJAKTA GADGIL, AMY K. SATER, DIANA S. CHOW, RATHINDRA N. BOSE, SHAUN XIAOLIU ZHANG
Anticancer Research Feb 2018, 38 (2) 623-646;
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