Rosehip extracts were kind gifts from Dr. Ipek Goketpe (North Carolina A & T State University). Delbucco’s Minimal Essential Media and Minimal Essential Medium-α (MEM-α) as well as the live/dead cell viability assay were purchased from Invitrogen (USA). Fetal bovine serum (FBS) used to maintain the cells were purchased from Biowest (USA). 3-(4,5)-dimethlythiahiazo (-z-yl0-3,5-diphenytetrazoliumromide (MTT) was purchased from Calbiochem (USA). Temozolomide and tubulin primary antibody were purchased from Sigma (USA). U0126 and LY294002 were purchased from Calbiochem (USA). Antibodies against phosphorylated MAPK (Tyr 202/204), phosphorylated AKT (ser473), phosphorylated p70S6K (Thr421/Ser424), phosphorylated Rb (Ser807/811), total MAPK, Fra-1, AKT and p70S6K were purchased from Cell Signaling Technologies (Massachusetts, USA). SpectraMax M5 (Molecular Devices, USA) plate reader was used to collect MTT and live/dead data. An Accuri (Becton Dickinson, USA) Flow Cytometry was used to analyze cell cycle status.

Dried Rosehip calyces and crude Rosehip extracts were supplied by Dr. Ipeke Goktepe (North Carolina A&T State University). The freeze dried calyces were grounded to powder and then stored at −20˚C. Rosehip powder (50 g) was serially extracted with 80% methanol. The mixture of freeze dried powder and 80% aqueous methanol were sonicated for 20 minutes with continual nitrogen gas purging. The extracts were filtered through Whatman No. 2 filter paper and evaporated to dryness under reduced pressure. The extracts were and stored at –20˚C. The obtained residues of the crude extracts were tested in human glioblastomas for cellular anti-proliferative activity.

The human GBM cell lines U-1242 MG, U-251 MG, and A-172 were kindly supplied by Dr. Isa Hussaini (University of Virginia, and U-1242 MG) and Dr. Waldemar Debinski (Wake Forest University, A-172 and U-251 MG). The U-251 MG and A-172 cells were maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, USA) supplemented with 10% FBS and 1% of penicillin/streptomycin (Fisher, USA). The U-1242 cells were maintained in Minimum Essential Media-α (MEM-α) supplemented with 10% FBS and 1% penicillin/streptomycin. All cells were incubated at 37˚C in a humidified atmosphere containing 5% CO₂. Cultures were maintained in 10 cm² plastic dishes and serially passaged following trypsinization using a TrypLE Express Stable Trypsin-Like Enzyme with Phenol Red solution (Invitrogen, USA).

The effects of rosehip extracts on cell proliferation were determined by MTT assays. Briefly, 10⁴ cells were seeded into a 96-well culture plate and incubated 4 - 6 h for attachment. The cells were then treated with various concentrations of the rosehip extract (1 mg/mL, 250 µg/mL, 25 µg/mL and 25 ng/mL) for 48 h. The PI3K (Phosphoinositide3-kinase) inhibitor LY294002 (20 µM) and the MEK1/MEK2 inhibitor U0126 (10 µM) were administered for 12 - 16 h (Calbiochem). Cells treated with TMZ (100 µM) were treated for 24 h prior to MTT assays. To determine the time-course response of rosehip extracts, cells were plated into 96-well plates, and rosehip was added to the culture medium and cell viability was assessed with MTT assay at 48 h after the drug treatment. Following rosehip exposure, 10 µL of MTT reagent was added to each well of a 96-well assay plate containing the cells in 100 µL of culture medium, and the plates were incubated at 37˚C in 5% CO₂ for 4 h. A volume of 100 µL of a color development solution (0.04N HCL/ Isopropanol) was added for 90 minutes, after which the optical density at 570 nm was determined using a Spectra Max M5 microplate reader. The optical density was used to calculate proliferation rates after exposure to the different concentrations of rosehip relative to that of control cells with no rosehip exposure. All analyses were performed in triplicate, and the mean for each experiment was calculated.

Following exposure to rosehip extracts at the various concentrations, the cells were washed with cold phosphate-buffered saline containing 0.2 mM sodium orthovanadate. The cells were then harvested and lysed on ice with 125 µL of pre-cooled Triton lysis buffer containing 1% Triton X-100, 0.2% Nonidet P-40, in the presence of 2 mm EDTA, 2 mg/mL sodium orthovanadate, and 1x protease cocktail inhibitor. Cells were centrifuged at 15,000 rpm for 1 min at 4˚C. The protein concentration of the supernatant was determined by the BCA assay (BioRad, USA) and equal amounts of protein from control and drug-treated cells were boiled for 5 min in SDS-PAGE buffer. Proteins were separated by SDS-PAGE on 8% polyacrylamide gels and subsequently transferred onto PVDF membranes. For immunoblotting, PVDF membranes were incubated with specific antibodies recognizing target proteins overnight at 4˚C. The membranes were then incubated with HRPconjugated secondary antibody (1:2000) for 1 h at room temperature and subsequently analyzed by enhanced chemiluminescence (ECL) detection system (Thermo Scientific, USA) and visualized by autoradiography. Tubulin (1:5000) was used as loading controls.

The viability of the cell lines following extract treatment was assessed using the LIVE-DEAD assay (Molecular Probes, USA). The assay was performed according to the manufacturer’s instructions to examine rosehip extract cytotoxicity after 48 h. The assay utilizes two fluorescent dyes to label live and dead cells. The cytoplasm of live cells was stained with 2 μm Calcein AM (green) the nucleus of dead cells stained with 4 μm EthD-1 (red). Stained cells were viewed using the Olympus IX71 inverted fluorescent microscope. Images were captured within 45 min of labeling using the Olympus DP 71 digital camera. The amount of dye in each cell was quantitatively measured using the SpectraMax M5 microplate reader.

Cells were plated in 6 cm plates and incubated under the conditions described above. After 4 - 6 hr incubation, to allow cell attachment, the cells were rinsed and incubated in serum free media for 24 h. After 24 h, the media was discarded and the cells were treated with various concentrations of the rosehip extract (250 µg/mL and 25 µg/mL) for 48 hr. Staurosporine (1 µM) was administered for 18 h (Sigma, USA). After treatment, the cells were trypsinized and pooled, then pelleted by centrifugation at 300 × g for 6 min. Cell pellets were washed with PBS, then fixed in ice cold 70% ethanol, and stored at −20˚C for 2 hr. Fixed cells were washed with PBS followed by a 30 minute incubation at room temperature in the dark in a staining solution consisting of PBS with 20 µg/mL propidium iodide (Sigma) (Missouri, USA), 0.1% Triton X-100 and 200 µg/mL RNase A (Sigma, USA). Stained cells were analyzed on the Accuri C6 Flow Cytometer. Using DNA histograms, FCS Express 4 software (Becton Dickson, USA) was used to quantify cell cycle compartments to estimate the percentage of cells distributed in the different cell cycle phases. Each experiment was repeated 4 times and the results are indicative of 4 independent studies.

Experiments examining cell proliferation using MTT, Live/Dead apoptotic analysis and cell cycle determination using flow cytometry were averaged and means and s.e.m. from three individual experiments were analyzed for statistical significance using a one-tailed ANOVA and the Newman-Kuels multiple comparison test to determine statistical significance.

We determined the rate of GBM cell proliferation in vitro by measuring the rate of MTT incorporated into three human GBM cell lines, A-172, U-251 MG and U-1242 MG cells following varying concentrations of rosehip extracts (1 mg/mL, 250 µg/mL, 25 µg/mL and 25 ng/mL). Each one of the human brain tumor cell lines assayed demonstrated a significant decrease in cell proliferation following treatment with various concentrations of rosehip extracts. Specifically, rosehip extract concentrations ranging from 25 µg/mL to 1 mg/mL significantly decrease the rate of cell proliferation in A-172 cells (Figure 1(a)). U-251 MG and U-1242 MG cells demonstrated higher sensitivity to rosehip extracts. In these cells the rosehip concentrations ranging from 25 ng/mL to 1 mg/mL show a decrease of cell proliferation (Figures 1(b) and (c)). These data suggest that crude rosehip extracts decrease the rate of cell proliferation in human brain tumor cell lines. The ability of rosehip extracts to prevent cell proliferation was compared to the ability of known PI3K/AKT (LY294002, 20 µM) and MAPK (U0126, 10 µM) inhibitors. In each of the cell lines tested the higher concentrations of rosehip extracts demonstrated an equal or greater decrease of cell proliferation than LY294002 and U0126 (Figures 1(a)-(c)). These data suggest that rosehip extracts have a greater capacity to prevent GBM cell proliferation than known inhibitors of cell proliferation.

To investigate the intracellular mechanisms controlling rosehip extract sensitivity observed in the brain tumor cell lines, we examined the phosphorylation state of several proteins. We first determined the inhibitory effect of rosehip extracts on the phosphorylation levels of MAPK (Erk1/2; Tyr 202/04) and the total protein levels of Fra-1, a downstream target of MAPK and a regulator of GBM formation and progression [29,30]. All cell lines were serum starved for 18 h to reduce the basal phosphorylation levels, and then incubated in the presence of various concentrations of rosehip extracts (1 mg/mL, 250 µg/mL, 25 µg/mL and 25 ng/mL), and 10% FBS. Additional samples were serum starved as described above and incubated in the presence of 10% FBS and either U0126 (10 µM) or LY294002 (20 µm), known chemical inhibittors of the MAPK and AKT signaling pathways, respecttively. MAPK (Erk1/2; Tyr 202/04) phosphorylation in A-172 (Figure 2(a)), U-251 MG (Figure 3(a)), and U-1242 MG (Figure 4(a)) cells was decreased following exposure to rosehip extracts and U0126, but not LY294002. In addition to a decrease in MAPK (Erk1/2) phosphorylation, Fra-1 (total) protein levels were also decreased in response to rosehip treatment and U0126 treatment, but not LY294002. Previous studies have shown that MAPK (Erk1/2) phosphorylation results in

increased Fra-1 protein levels and increased transcripttional activity [31-33]. Taken together these data suggest that rosehip extracts prevent MAPK (Erk1/2) phosphorylation and this inhibition of MAPK (Erk1/2) phosphorylation correlates with the reduction of cell proliferation observed in A-172 cells (Figures 1(a)-(c)).

Additional studies investigated whether rosehip extracts could decrease the activity of the AKT cell proliferation regulatory pathway. Each cell line was treated in a similar manner as described above. Rosehip extracts (all concentrations tested) prevent AKT phosphorylation (ser 473) and p70S6K (Thr 421/Ser 424) phosphorylation in A-172 cells, in a similar fashion as LY294002 (Figure 2(b)). AKT and p70S6K phosphorylation is only slightly decreased by higher concentrations of rosehip extracts (1 mg/mL and 250 µg/mL) in U-251MG cells (Figure 2(b)).

In U-1242 MG cells AKT and p70S6K phosphorylation is decreased by rosehip extracts (Figure 4(b)). Specifically, rosehip extracts are able to decrease the phosphorylation of p70S6K at all concentrations tested in U-1242 MG cells (Figure 4(b)), but only at the highest tested concentrations in U-251 MG cells (Figure 3(b)). These data indicate that rosehip can also prevent the activation on the AKT cell signaling regulatory pathway in A-172 and U-251MG cells (Figures 2(b) and 3(b)). More importantly, these data indicate that rosehip extracts are able to inhibit multiple proliferation regulatory mechanisms in the cell lines assayed.

In order to examine whether rosehip extracts were promoting cell death in A-172, U-251 MG and U-1242 MG cell lines, cells were cultured in the presence of 250 µg/mL rosehip extract and 10% FBS and the amount of cell death was measured using a fluorescence colorimetric analysis (as described in the materials and methods section). The mean percentage of dead cells was determined following rosehip extract treatment and these values were compared to the values generated by A-172, U-251 MG and U-1242 MG cells that were cultured in 10% FBS and staurosporine (1 µM) for either 3 h (U-1242MG cells) or 24 h (A-172 and U-251MG cells) in order to induce cell death. Since in cell proliferation assays there was no significant difference observed between the 1 mg/mL and 250 µg/mL concentration of rosehip extracts we used 250 µg/mL of rosehip extracts to determine the apoptosis promoting capacity of the extracts. Each cell line tested with rosehip extracts (250 µg/mL) did not promote cell death, but staurosporine treatment did result in cell death (Figure 5). Figure 6 (these data correlate to the A-172 panel displayed in Figure 5) shows A-172 cells treated with staurosporine have a much higher amount of cell death than cells treated with rosehip extracts or cells cultured in serum containing medium without inhibitors (Figure 6, panels C, F, I). These data suggests the prevention of cell proliferation observed in cells treated with rosehip extracts is due to inhibition/decrease of cell cycle progression and not cell death.

Since rosehip extracts are able to prevent cell proliferation in the cell lines tested, but do not promote cell death we wanted to examine whether exposure to these extracts resulted in PARP cleavage, a marker of apoptosis. To test further whether rosehip is promoting apoptosis, we used a western blot to assay for PARP cleavage. PARP, a nuclear polymerase protein important for DNA repair in response to stress, is cleaved from its 116 kDA full size protein to a 89 kDA fragment, which in human cells has been associated with cell apoptosis [34]. In Figure 7 we show that rosehip extracts (all concentrations) do not induce PARP cleavage in neither the A-172, U-251MG nor U-1242MG cells (Figures 7(a)-(c)) when compared to control cells either cultured under serum starvation conditions or cultured in the presence of 10% FBS. Additionally, cells cultured with FBS (10%) and in the presence of U0126 and LY294002 do not display an increased amount of cleaved PARP (Figures 7(a)-(c)). However, A-172, U-251MG and U-1242MG cells treated with staurosporine (1 µM) demonstrate a drastic increase

in the amount of cleaved PARP, demonstrating the affect of apoptosis on the cleavage state of PARP in these cell lines (Figures 7(a)-(c)). These data further emphasize that rosehip extracts do not promote apoptosis in the GBM cell lines assayed.

Since GBM cells treated with rosehip extracts had decreased rates of proliferation and were not undergoing

cell death we investigated the mechanism by which these extracts were decreasing cell proliferation. Flow cytometry was used to analyze the cell cycle following exposure to rosehip extracts. A-172 cells treated with rosehip extracts at a high concentration (250 µg/mL) and a low concentration (25 µg/mL) show that cells exposed to rosehip extracts are unable to maintain a normal cell cycle (Figure 8). Specifically, rosehip extracts (both concentrations assayed) significantly prevented these cells from exiting the G₀/G₁ phase of the cell cycle (Figure 8). Cells treated with rosehip extracts have the same percentage of cells remaining in the G₀/G₁ phase of the cell cycle as serum-starved cells (Figure 8).

Since rosehip extracts were able to slow cells from exiting the G₀/G₁ phase of the cell cycle we determined the level of retinoblastoma (Rb) tumor suppressor protein phosphorylation (Ser 807/811) following exposure to rosehip extracts (1 mg/mL, 250 µg/mL, 25 µg/mL and 25 ng/mL). Rb phosphorylation by a CDK inactivates tumor suppressor (cell cycle inhibitory) function of Rb and promotes continuation of the cell cycle [35]. A-172 cells show a decrease of Rb phosphorylation at each concentration of rosehip extracts, whereas control serum-cultured (10% FBS) cells not exposed to rosehip extracts cells do not (Figure 9). U-251MG cells exhibit a lowered amount of Rb phosphorylation following exposure to higher concentrations of rosehip extracts (1mg/mL and

250 µg/mL); whereas U-1242MG cells only exhibited substantial decrease in Rb phosphorylation when exposed to the highest concentration of rosehip extracts (1 mg/mL) (Figure 9). These data demonstrate that rosehip extracts are able to slow the rate of cell proliferation in

the GBM cell lines assayed by halting the cells in the G₀/G₁ phase of the cell cycle via an Rb-mediated mechanism.

To better understand the chemotherapeutic potential of rosehip extracts against human GBM cell lines, we tested whether the antiproliferative capacity of rosehip extracts compared to that of Temozolomide (TMZ). TMZ (also known by the following brand names; Temodar, Temodal and Temcad) is one of the current first line chemotherapeutic options used in the treatment of GBMs. TMZ is an orally administered alkylating agent that can either halt the cell cycle and/or initiate cell death [36]. Using MTT assays we compared the rate of cell proliferation following exposure to either rosehip extract alone (250 µg/mL), TMZ (100 µM) alone or a combination of the rosehip extracts and TMZ [37]. We used rosehip extracts at a concentration of 250 µg/mL since our previous cell proliferation studies demonstrated that 250 µg/mL concentration of rosehip extracts significantly decreased cell proliferation (Figures 1(a)-(c)). Surprisingly, in all of the cell lines assayed, cells exposed to the rosehip extracts (250 µg/mL) demonstrated a greater decrease in cell proliferation when compared to cells exposed to TMZ (100 µM) (Figures 10(a)-(c)). These observed differences were statistically significant (P < 0.05) in A-172 and U-251 MG cell lines (Figures 10(a) and (b)). Additionally, in U-251MG cells, TMZ (100 µM) demonstrated no

inhibitory effect, whereas rosehip extracts (250 µg/mL) significantly decrease the rate of cell proliferation (Figure 10(b)). These assays also show that the combination of TMZ (100 µM) and rosehip extracts (250 µg/mL) do not synergize to decrease cell proliferation more than rosehip as a single agent (Figures 10(a)-(c)). These data demonstrate that rosehip extracts have a greater capacity to prevent in vitro GBM cell proliferation than a widely used chemotherapeutic treatment of GBMs.