NanoStilbene

a nanoparticle formulation of pterostilbene

Resveratrol and Pterostilbene Exhibit Anticancer Properties Involving the Downregulation of HPV Oncoprotein E6 in Cervical Cancer Cells.

Kaushiki Chatterjee,1,2 Dina AlSharif,2 Christina Mazza,2 Palwasha Syar,2 Mohamed Al Sharif,2 and Jimmie E. Fata1,2,*,*

ABSTRACT
Cervical cancer is one of the most common cancers in women living in developing countries. Due to a lack of affordable effective therapy, research into alternative anticancer compounds with low toxicity such as dietary polyphenols has continued. Our aim is to determine whether two structurally similar plant polyphenols, resveratrol and pterostilbene, exhibit anticancer and anti-HPV (Human papillomavirus) activity against cervical cancer cells. To determine anticancer activity, extensive in vitro analyses were performed. Anti-HPV activity, through measuring E6 protein levels, subsequent downstream p53 effects, and caspase-3 activation, were studied to understand a possible mechanism of action. Both polyphenols are effective agents in targeting cervical cancer cells, having low IC50 values in the µM range. They decrease clonogenic survival, reduce cell migration, arrest cells at the S-phase, and reduce the number of mitotic cells. 

These findings were significant, with pterostilbene often being more effective than resveratrol. Resveratrol and to a greater extent pterostilbene downregulates the HPV oncoprotein E6, induces caspase-3 activation, and upregulates p53 protein levels. Results point to a mechanism that may involve the downregulation of the HPV E6 oncoprotein, activation of apoptotic pathways, and re-establishment of functional p53 protein, with pterostilbene showing greater efficacy than resveratrol.

Figure 1 Above: Pterostilbene is more potent in eliminating HeLa cervical cancer cells as compared to resveratrol: (A) Brightfield analysis of HeLa cells untreated (Ai) or treated for 24 h with 40 µM of resveratrol (Res; Aii) or 40 µM of pterostilbene (Pte; Aiii). Evidence of cell elimination was only seen robustly in cells treated with pterostilbene at 40 µM. (B) Analysis of IC50 values, generated by a Water Soluble Tetrazolium salt-1 (WST-1) assay after 24 h of exposure to resveratrol or pterostilbene indicates that pterostilbene (IC50 = 42.3 µM) is a more potent cytotoxic agent than resveratrol (IC50 = 83.5 µM; Bii). The graphs represent data from three independent experiments (mean ± S.E.M. (Standard error mean)). (C) Clonogenic assays performed to compare the relative effect of the two polyphenols on the clonogenicity of HeLa cells untreated (Ci) or treated with 50 µM of either resveratrol (Cii) or pterostilbene (Ciii). Results are from 15-days post-treatment and indicate that pterostilbene is more efficient in curbing the clonogenicity compared to resveratrol (Civ). Bar graph represents data from three independent experiments (mean ± S.E.M.; * p < 0.05; Civ).

Pterostilbene Is More Potent in Eliminating HPV+ HeLa Cells Compared to Resveratrol

In order to study the comparative cytotoxicity of pterostilbene and resveratrol on HeLa tumor cells, brightfield images (Figure 1A) and WST-1 cell viability assays (Figure 1B) were performed 24 h post-treatment. The brightfield images taken after 24 h of treatment (Figure 1A) showed that pterostilbene (40 µM) eliminates significantly more cells than resveratrol at the same concentration. Live imaging of cells treated with 60 µM of the two compounds show significantly more death and characteristic apoptotic blebbing in pterostilbene-treated cells when compared to untreated or resveratrol-treated cells (Supplementary Videos S1–S3). The WST-1 analysis revealed that although both pterostilbene and resveratrol eliminated HeLa cells significantly and in a dose-dependent manner, pterostilbene displayed a 1.97-fold lower IC50 when compared to resveratrol (42.3 µM vs. 83.5 µM; p < 0.05; Figure 1B). Additionally, both compounds, at 50 µM, significantly inhibited the clonogenicity of post-treated cells in a 15-day clonogenic assay (Figure 1C). Pterostilbene significantly reduced clonogenic survival by 87.5% compared to the control (p < 0.05), while resveratrol inhibited it by 63% (p < 0.05) (Figure 1C). Moreover, the difference between the survival percentages of the two treatment groups is significant (p < 0.05).

Live imaging of cells treated with 60 µM of pterostilbene (S1).

Of the two compounds, pterostilbene showed significantly more death and characteristic apoptotic blebbing with the pterostilbene-treated cells than when compared to untreated or resveratrol-treated cells.

Supplementary Videos S1-S3

Inhibition of Cell Migration of HeLa Cells Treated with Pterostilbene and Resveratrol

To determine the comparative efficacy of resveratrol and pterostilbene in inhibiting HeLa cell migration, two different sub-lethal concentrations of each compound were used in a 48-h scratch assay (Figure 2). Based on the WST-1 results and brightfield images (unpublished), we found that cells treated with a concentration below 25 µM showed no signs of cellular toxicity. To avoid any cytotoxicity, we used lower concentrations of 5 µM and 20 µM. At sub-lethal concentrations of 5 µM and 20 µM, both resveratrol and pterostilbene significantly inhibited HeLa cell migration relative to untreated cells (p < 0.05; Figure 2). Pterostilbene was more effective in inhibiting HeLa cell migration at 20 µM when compared to resveratrol; however, this result was not significant and no differences were seen between the two compounds at 5 µM (Figure 2). In an effort to analyze the effects of resveratrol and pterostilbene on cell migration, we normalized the amount of migration into the scratch (wound) by untreated cells, to 100%. Relative to this control, resveratrol-treated cells migrated only 71.2% (5 µM) and 63.7% (20 µM), while cells treated with pterostilbene migrated only 69.5% (5 µM) and 49.2% (20 µM) (Figure 2).

Figure 2 Above: Resveratrol and pterostilbene inhibit cell migration: (A) HeLa cells were monitored for cell migration into a scratched “wound”. Cells were either untreated or treated with sub-lethal concentrations (5 µM and 20 µM) of resveratrol (Res) or pterostilbene (Pte). The extent of migration into the scratched area was calculated after 48 h and revealed that both resveratrol and pterostilbene significantly inhibit cell migration, although pterostilbene had greater anti-migratory effect. (B) The graphs represents data from triplicate sample experiments normalized to the control (mean % migrated cells ± S.E.M.; * p < 0.05). Scale bar: 0.05 µm.

Cell Cycle Arrest at S-Phase in HeLa Cells Treated with Low Concentrations of Resveratrol and Pterostilbene

In order to compare the effect of sub-lethal doses of either resveratrol or pterostilbene on the cell cycle of HeLa cells, treatment was carried out with three different concentrations (5 µM, 10 µM, and 15 µM) of the two compounds for 18 h prior to flow cytometric analysis (Figure 3A). Flow cytometry analysis showed that the cells treated with either compound exhibited a significant decrease in the number of cells in the G2-M phase with respect to the control cells (p < 0.05) (Figure 3A,B, Table 1), indicating an S-phase cell cycle arrest. This effect corresponded with an increase in the number of cells arrested at the S-phase. Pterostilbene was significantly more potent than resveratrol in inhibiting cell cycle progression, showing effects at concentrations as low as 5 µM (p < 0.05) (Figure 3A,B, Table 1). At this concentration, pterostilbene had these percentages of cells in each phase: G1 = 53.4 ± 1.4, S = 34 ± 1.4, G2 =12.5 ± 0.2, while resveratrol had values of G1 = 64.8 ± 2.0, S = 16.3 ± 1.0, G2 = 18.3 ± 2.3. At a higher concentration (15 µM) both compounds significantly inhibited cells from entering into G2-M by arresting them in the S-phase, and difference between the extent of the arrest at this phase induced by the two compounds was significant (p < 0.05) (Figure 3A,B, Table 1).

Figure 3 Above: S-phase arrest in HeLa cells treated with low concentrations of resveratrol and pterostilbene: (A) Flow-cytometric evaluation of HeLa cells untreated or treated with sub-lethal doses of resveratrol (Res) and pterostilbene (Pte) for 18 h. Treated cells exhibited S-phase arrest and a subsequent decrease in the number of cells in G2/M. Pterostilbene was a more potent compound than resveratrol, showing a capacity to arrest cells at the S-phase at concentrations as low as 5 µM. (B) Graphical representation of the dose-dependent cell cycle effects induced by resveratrol and pterostilbene at three different concentrations (5 µM, 10 µM, and 15 µM). (B) The graph represents data from triplicate sample experiments normalized to the control (mean % cells in each phase ± S.E.M.) (C) Immunofluorescent images of HeLa cells probed for the M-phase marker phiso-histone-H3 (serine10). HeLa cells were untreated or treated with 5 µM and 10 µM of resveratrol or pterostilbene. Immunofluorescent images display a decrease of histone-H3 in cells treated with both the compounds, the effects at 5 µM of pterostilbene is much greater than those of resveratrol (at 5 µM). (D) Graphical representation of the percent of mitotic cells calculated from immunofluorescent images reveal that resveratrol and to a greater extent pterostilbene are effective in decreasing the number of mitotic HeLa cells. The graph represents data from experiments obtained from triplicate samples normalized to the control (mean % mitotic cells ± S.E.M.;* p < 0.05).

Downregulation of Viral Oncoprotein E6 and Upregulation of Active-Caspase-3 in HeLa Cells Treated with Pterostilbene and Resveratrol

In order to investigate how resveratrol and pterostilbene were affecting HeLa cell survival and cell cycle progression, we treated cells with either of the two compounds at sub-lethal (10 µM) and higher (50 µM) concentrations prior to analysis by immunostaining for E6, active caspase-3, and p53 (Figure 4A–C). At 10 µM, both resveratrol and pterostilbene failed to significantly affect levels of E6 and active caspase-3 levels relative to the control (Figure 4A,B). However, at 50 µM both compounds significantly suppressed E6 levels and elevated cleaved caspase-3 levels in treated cells relative to the untreated cells (Figure 3A–C). At this concentration (50 µM), pterostilbene was significantly more potent than resveratrol at suppressing E6 levels (resveratrol = 0.77 ± 0.11: 23% decrease vs. pterostilbene = 0.57 ± 0.06: 43% decrease; p < 0.05) and simultaneously elevating active caspase-3. It should be noted that we were unable to detect any noticeable differences in the sub-cellular localization of E6 in treated cells (Figure 4A).

Figure 4 Above: Downregulation of viral oncoprotein E6 and upregulation of active-caspase-3 in HeLa cells treated with resveratrol or pterostilbene: (A) HeLa cells immunostained for E6 levels (green) and counterstained with the nuclear dye 4’,6-diamidino-2-phenylindole (DAPI) (blue) after treatment with resveratrol (Res) and pterostilbene (Pte; 10 µM and 50 µM). Loss of E6 proteins are visually evident in cells treated with 50 µM of either resveratrol or pterostilbene. (B) Cell image analysis of the E6 fluorescent data revealed a significant 43% decrease of E6 protein levels in HeLa cells treated with pterostilbene at 50 µM and a 23% decrease of E6 levels in cells treated with resveratrol, both relative to the control. The graph represents data from experiments obtained from three independent experiments normalized to the control (mean % normalized to DAPI ± S.E.M.; * p < 0.05). (C) Immunofluorescent images probing for active-caspase-3 (green) shows a corresponding enhanced activation of this mediator of apoptosis by both resveratrol and pterostilbene.

Upregulation of Tumor Suppressor Protein p53 in HeLa Cells Treated with Pterostilbene and Resveratrol

Concomitant with E6 suppression, 50 µM pterostilbene treatment for 22 h caused an upregulation of p53 in HeLa cells (Figure 5A,B). When compared to the control, pterostilbene treatment elicited a 2-fold increase in p53 levels (staining normalized to DAPI; Figure 5B; p < 0.05). In comparison to the control, HeLa cells treated with 50 µM of resveratrol also caused an upregulation of p53 (1.75-fold increase; Figure 5A,B; p < 0.05) at 22 h.

Figure 5 Above: Upregulation of the tumor suppressor protein p53 in HeLa cells treated with resveratrol and pterostilbene: (A) Immunoflourescent images of p53 protein (green) untreated or after treatment with 50 µM of either resveratrol (Res) or pterostilbene (Pte) for 22 h. Levels of p53 are elevated in cells treated with either polyphenol. (B) Image analysis of p53 immunofluorescence indicates that pterostilbene treatment at 50 µM elicited a significant 2-fold increase in p53, while resveratrol exposure at similar concentrations induced a significant 1.75 increase in p53. The graph represents data from experiments obtained from three independent experiments normalized to the control (mean % normalized to DAPI ± S.E.M. * p < 0.05). (C) Western blot analysis also revealed that the elevation of p53 protein levels is evident in HeLa cells treated with 50 µM of resveratrol and pterostilbene; however, significant differences relative to the control were only reached with HeLa cells treated pterostilbene at 50 µM. (D) The graph represents data from experiments obtained from three independent experiments normalized to the control (mean % normalized to beta-actin ± S.E.M.; * p < 0.05).

Conclusion

Cervical cancer is a major concern in developing countries due to lack of affordable prophylaxis and treatment. As present modes of treatment like surgery, chemotherapy, or radiation involve high systemic toxicity, there is an urgent need to find affordable alternative therapies. Diet-based polyphenols like resveratrol and pterostilbene are therefore potential candidates for the effective therapy of cervical cancer with significantly low toxicity. We found pterostilbene to be a more potent anticancer agent than resveratrol in HeLa cells. This difference may be a function of pterostilbene being capable of upregulating p53 and downregulating E6 significantly more than resveratrol. As pterostilbene is non-toxic to normal cells [20], it has the potential to be a robust, cost-effective anti-E6+ tumor drug. Others have found that that pterostilbene possess greater bioavailability and stability [45] than resveratrol in vivo (80% vs. 20%). Resveratrol has been shown to be non-toxic to several cells lines like glial cells and neurons, even after a treatment dose of 100 µM for 48 h [46]. Other studies on normal fibroblasts also state the non-toxicity of resveratrol at our observed potent anticancer concentrations [47]. Additionally, pterostilbene shows no toxicity at these concentrations in normal skin fibroblasts and myoblasts [48]. According to clinical studies, the safe dosage for resveratrol and pterostilbene is 5 g/day [49] and 250 mg/day [20], respectively. Our initial in vivo studies in the laboratory using a non-toxic dosage of both resveratrol and pterostilbene has shown promising results in inhibiting tumor growth in a model of cervical cancer [32]. Taken together, our findings support the further evaluation of pterostilbene as a possible therapy against cervical cancer.

Here, we show that pterostilbene potently suppresses HPV E6 expression (Figure 4) and efficiently eliminates HPV+ cells in culture by p53-mediated apoptosis (Figure 1 and Figure 5) while suppressing cell proliferation (Figure 3) and migration (Figure 2). We find that pterostilbene is a more promising agent against cervical cancer when compared to resveratrol. Based on such properties, the use of pterostilbene presents a relatively economical but highly hopeful therapeutic approach to treat HPV infections and cervical cancers. Our future studies will include signaling studies using HPV+ murine tumor models to confirm these observations in vivo.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852819/ 

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What is Pterostilbene?

Pterostilbene is a natural dietary compound found in blueberries. We use the syntheic version due to limited amounts occuring naturally in the berries.

how much nanostilbene should i take daily?

We recommend up to 300mg daily which is approx 1.5 milliliters. However many find that 200mg daily is also suffice, therefore a range of 200-300mg daily is recommended.

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Yes small amounts may be applied locally but remember at 200mg per ml it is highly concentrated with a single drop containing 10mg.

What is nanostilbene?

NanoStilbene  is an easily absorbed nanoemulsion of nanoparticle pterostilbene in the range of 75-100nm at a concentration of 200 milligrams per milliliter

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NanoStilbene contains 200mg per ml or approximately 10mg per drop.

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