Search of Neuroprotective Polyphenols Using the “Overlay” Isolation Method

Previous studies of the neuroprotective activity of polyphenols have used ununiform culture systems, making it difficult to compare their neuroprotective potency. We have established a new and simple method for preparing differentiated PC12 cells by removing the toxic coating step. Cells were induced to differentiate with the nerve growth factor (NGF) in a serum-free medium, without a medium change, but with a one-time overlay supplementation of NGF. The optimal inoculation density of the cells was 6–12 × 103 cells/cm2, and the presence of serum inhibited the differentiation. Neuroprotective activity could be quantified by the specific index (SI) value, that is, the ratio of the 50% cytotoxic concentration to the 50% effective concentration. Alkaline extract from the leaves of Sasa senanensis Rehder (SE), having had hormetic growth stimulation, showed the highest SI value, followed by epigallocatechin gallate. The SI value of curcumin and resveratrol was much lower. This simple overly method, that can prepare massive differentiated neuronal cells, may be applicable for the study of the differentiation-associated changes in intracellular metabolites, and the interaction between neuronal cells and physiological factors.


Introduction
Improvement in the daily nutritional supply and the living environment resulted in the prolongation of our life span, but necessarily increased the number of elderly populations having cognitive diseases [1][2][3]. Alzheimer's disease, the most common form of dementia, is characterized by the accumulation of amyloid-β (Aβ) in the brain. Since the neurotoxicity of Aβ has been well established [4,5], preventing the accumulation [6] and early oligomerization [7] of Aβ may be a promising cognitive behavioral therapy.
Using PC12 cells prepared by the overlay method, we have re-investigated the neuroprotective activity of various polyphenols. This was compared with that of plant extracts and antioxidants, based on the specific index (SI) (defined as the ratio of 50% cytotoxic concentration (CC 50 ) to 50% protective concentration (EC 50 )).

Optimal Concentration of Ngf and Fbs for the Induction of PC12 Cell Differentiation
We first confirmed that NGF stimulated the growth of PC12 cells at the optimal concentration of 50 ng/mL in our assay system. The cell number increased up to day five, and thereafter, slightly declined, possibly due to it reaching the terminal differentiation ( Figure 1A). NGF also stimulated the formation of neurite-the marker of neuronal differentiation. When differentiated cells were defined as the cells in which the extended neurite exceeds the longest diameter of each cell ( Figure 1B), the percentage of differentiated cells reached a plateau at day five, with a maximum of 50 ng/mL NGF ( Figure 1C).

FBS Inhibited the Differentiation of PC12 Cells
When PC12 cells were cultured in a serum-free medium, neuronal differentiation reached a maximum level. When inoculation of cell density was reduced to 3.125 × 10 3 /cm 2 , the incidence of cell death slightly increased (Figure 2), which suggests the importance of continuous nutritional supply from neighboring cells. It was unexpected that the maximum differentiation can be achieved in the absence of FBS, and the addition of 1% or 10% FBS inhibited the differentiation induction. Thus, the optimal inoculation of cell density was determined to be between 6 and 12 × 10 3 /cm 2 in the absence of serum.

FBS Inhibited the Differentiation of PC12 Cells
When PC12 cells were cultured in a serum-free medium, neuronal differentiation reached a maximum level. When inoculation of cell density was reduced to 3.125 × 10 3 /cm 2 , the incidence of cell death slightly increased (Figure 2), which suggests the importance of continuous nutritional supply from neighboring cells. It was unexpected that the maximum differentiation can be achieved in the absence of FBS, and the addition of 1% or 10% FBS inhibited the differentiation induction. Thus, the optimal inoculation of cell density was determined to be between 6 and 12 × 10 3 /cm 2 in the absence of serum.

Exploration of the Overlay Method
Based on this experimental data, we explored the overlay method to isolate differentiating PC12 cells. PC12 cells were incubated for a total of six or seven days in serum-free DMEM, containing 50 ng/mL NGF, with one more time overlay of NGF ( Figure 3). The differentiated cells were well attached to the plate and were not detached by gentle pipetting.

Exploration of the Overlay Method
Based on this experimental data, we explored the overlay method to isolate differentiating PC12 cells. PC12 cells were incubated for a total of six or seven days in serum-free DMEM, containing 50 ng/mL NGF, with one more time overlay of NGF ( Figure 3). The differentiated cells were well attached to the plate and were not detached by gentle pipetting.

Apoptosis-Inducing Activity of Neurotoxic Agents
We have previously reported that cisplatin showed potent cytotoxicity against differentiated PC12 cells [34], while taxanes (paclitaxel, docetaxel) and amyloid peptides (Aβ1-42, Aβ25-35) showed cytostatic effects [34,35]. A cell sorter analysis demonstrated that cisplatin induced the apoptosis, characterized by the accumulation of the subG1 population ( Figure 4). On the other hand, paclitaxel, Aβ1-42 and Aβ25-35 reduced the population of S-phase cells-reflecting their cytostatic growth inhibition-and accumulated the G2 + M phase cells, in accordance with the reported mitotic arrest by taxanes [36,37]. It was unexpected that differentiated cells were resistant to actinomycin D-a popular apoptosis inducer for various cancer cell lines.

Neuroprotective Effects of Polyphenols
Alkaline extract from the leaves of Sasa senanensis Rehder (SE) protected the cytotoxicity induced by paclitaxel and Aβ 25-35 , but not the cytotoxicity induced by cisplatin. Epigallocatechin gallate (EGCG) and curcumin showed some protective activity when induced by Aβ 25-35 ( Figure 5A).

Neuroprotective Effects of Polyphenols
Alkaline extract from the leaves of Sasa senanensis Rehder (SE) protected the cytotoxicity induced by paclitaxel and Aβ25-35, but not the cytotoxicity induced by cisplatin. Epigallocatechin gallate (EGCG) and curcumin showed some protective activity when induced by Aβ25-35 ( Figure 5A). Table 2 shows the SI (=CC50/EC50) value of polyphenols, plant extracts and antioxidants, calculated using the data from our original papers [33,35,38,39] and the present study ( Table 2). The higher the SI value, the stronger the neuroprotective activity would be expected.  Angelica shikokiana Makino extract [38] and the hot water extract of Miso-a traditional Japanese fermented food that has supported our diet for many years [39]-showed neuroprotective effects against paclitaxel and amyloid peptides ( Figure 5B). Especially, a significant (p < 0.05) growth stimulation effect of Miso on Aβ1-42 and Aβ25-35-treated cells was observed above 0.04%.

Neuroprotective Activity of Plant Extracts (Exp. 3)
Angelica shikokiana Makino extract [38] and the hot water extract of Miso-a traditional Japanese fermented food that has supported our diet for many years [39]-showed neuroprotective effects against paclitaxel and amyloid peptides ( Figure 5B). Especially, a significant (p < 0.05) growth stimulation effect of Miso on Aβ 1-42 and Aβ 25-35 -treated cells was observed above 0.04%.

Growth Stimulation by SE
There was a possibility that neuroprotective effects may be related to the growth stimulation against PC12 cells. To test this possibility, PC12 cells were cultured for 24 h in a serum-free medium containing various sample concentrations. The removal of serum from the cultured medium reduced the growth potential of PC12 cells to 10% of the control level ( Figure 6). By the addition of SE, the viability returned back to 40% of the control level, whereas the growth stimulation effects of EGCG, resveratrol, and curcumin was much less (Figure 6).

Discussion
We have explored a new and simple method that enabled us to prepare differentiated neuronal cells by the repeated overlay of the NGF-containing medium without a medium change, omitting the use of expensive collagen-coated plates, or the toxic coating step. Using the overlay method, we reinvestigated the neuroprotective activity of polyphenols.
SE, a group of three over-the-counter drugs, showed the highest neuroprotective activity against both undifferentiated and differentiated PC12 cells, as well as undifferentiated SH-SY5Y cells. This seems to be linked to growth stimulation at lower doses, known as hormesis [40], since we have experienced similar growth stimulation of SE towards human gingival epithelial fibroblast (HGEP) [41] and PC12 cells [33]. SE showed prominent anti-HIV [42] and anti-UV activity [43], like the lignincarbohydrate complexes extracted with alkaline solution. The present study further adds that SE showed potent neuroprotective activity. SE contains the lignin-carbohydrate complex and the various degradation products, and thus not a purified material. The lignin-carbohydrate complex showed poor bioavailability [44], and its biological activity might be mediated through one of the patternrecognition receptors (dectin-2) in the mucosa of oral cavities [45] and intestinal duct. We found that p-coumaric acid, one of the lignin precursors present in SE (unpublished data), has no neuroprotective activity. Further fractionation of SE is necessary to identify the neuroprotective substances present in SE.
EGCG, a main component of green tea, showed some neuroprotective activity. We found that higher concentrations of EGCG led to false-positive coloring with MTT reagent-indicated by the red circle in Figures 5 and 6-and were possibly due to the non-specific binding of tannin to protein [46,47]. Neuroprotective effects of EGCG may be mediated through cell-surface receptors [48] or by the direct binding to neurotoxic agents.
Curcumin showed some neuroprotective activity against differentiated PC12 cells, but not against undifferentiated cells. The fluctuation in the SI value of this compound may be due to its potent cytotoxicity, narrowing the chemotherapeutic range.
Food polyphenols, at low and nontoxic concentrations, slow down the progression of neurodegenerative diseases and therefore may be a promising approach [49]. It was unexpected that Miso extract also showed neuroprotective ( Figure 5B) and growth promotion activity [38], further substantiating the possible link between neuroprotective activity and hormesis. Since Miso is a daily food, it is very much meaningful to identify the active principle for the future research aiming at alleviating the neuronal diseases.

Discussion
We have explored a new and simple method that enabled us to prepare differentiated neuronal cells by the repeated overlay of the NGF-containing medium without a medium change, omitting the use of expensive collagen-coated plates, or the toxic coating step. Using the overlay method, we re-investigated the neuroprotective activity of polyphenols.
SE, a group of three over-the-counter drugs, showed the highest neuroprotective activity against both undifferentiated and differentiated PC12 cells, as well as undifferentiated SH-SY5Y cells. This seems to be linked to growth stimulation at lower doses, known as hormesis [40], since we have experienced similar growth stimulation of SE towards human gingival epithelial fibroblast (HGEP) [41] and PC12 cells [33]. SE showed prominent anti-HIV [42] and anti-UV activity [43], like the lignin-carbohydrate complexes extracted with alkaline solution. The present study further adds that SE showed potent neuroprotective activity. SE contains the lignin-carbohydrate complex and the various degradation products, and thus not a purified material. The lignin-carbohydrate complex showed poor bioavailability [44], and its biological activity might be mediated through one of the pattern-recognition receptors (dectin-2) in the mucosa of oral cavities [45] and intestinal duct. We found that p-coumaric acid, one of the lignin precursors present in SE (unpublished data), has no neuroprotective activity. Further fractionation of SE is necessary to identify the neuroprotective substances present in SE.
EGCG, a main component of green tea, showed some neuroprotective activity. We found that higher concentrations of EGCG led to false-positive coloring with MTT reagent-indicated by the red circle in Figures 5 and 6-and were possibly due to the non-specific binding of tannin to protein [46,47]. Neuroprotective effects of EGCG may be mediated through cell-surface receptors [48] or by the direct binding to neurotoxic agents.
Curcumin showed some neuroprotective activity against differentiated PC12 cells, but not against undifferentiated cells. The fluctuation in the SI value of this compound may be due to its potent cytotoxicity, narrowing the chemotherapeutic range.
Food polyphenols, at low and nontoxic concentrations, slow down the progression of neurodegenerative diseases and therefore may be a promising approach [49]. It was unexpected that Miso extract also showed neuroprotective ( Figure 5B) and growth promotion activity [38], further substantiating the possible link between neuroprotective activity and hormesis. Since Miso is a daily food, it is very much meaningful to identify the active principle for the future research aiming at alleviating the neuronal diseases.
We have recently manufactured highly tumor-specific derivatives from the chromone core structure found in flavones, isoflavones and 2-styrylchromones, and discovered that their tumor-specificity was correlated with molecular shape and hydrophobicity [53,54]. By analogy, for searching new neuroprotective substances, it may be one way to introduce various functional groups to the core substance present in the natural kingdom, repeat the synthesis, and verify the process using QSAR analysis.

Cell Culture
PC12 cells were purchased from Riken Cell Bank (Tsukuba, Japan). These cells were cultured in DMEM and were supplemented with 10% FBS, 100 units/mL, penicillin G and 100 µg/mL streptomycin under a humidified 5% CO 2 atmosphere.

Determination of Viable Cell Numbers
To the culture medium, a fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny ltetrazolium bromide (MTT) (final concentration: 0.1 mg/mL) with (for differentiated cells) or without (for non-differentiated cells) of 50 ng/mL NGF. The cells were incubated for 1 h, and the formazan precipitate was dissolved in DMSO to measure their absorbance at 560 nm with a plate reader (Infinite F 50 R, TECAN, Kawasaki, Japan).

Induction of Differentiation Toward Neurons
For the induction of differentiation, PC12 cells were incubated for the indicated times with a differentiating inducing medium (DMEM, supplemented with 0% or 1% FBS, 50 ng/mL NGF and antibiotics). Differentiated cells were defined as the cells in which the extended neuritis exceeded the longest diameter of each cell, assessed under the light microscope (EVOS FL, ThermoFisher Scientific, Waltham, MA, USA) ( Figure 1B).

Cell Cycle Analysis
Cells (approximately 10 6 ) were harvested, fixed with a 1% paraformaldehyde in phosphate-buffered saline without calcium or magnesium ions [PBS(−)]. Fixed cells were washed twice with PBS(−), and then treated for 30 min with 400 µL of 0.2 mg/mL RNase A (preheated for 10 min at 100 • C to deactivate DNase) to degrade RNA. Cells were then washed twice with PBS(−) and stained for 15 min with 0.005% propidium iodide (PI) in the presence of 0.01% NP-40 in PBS(−), which prevents cell aggregation. After filtering through Falcon ® cell strainers (40 µM) (Corning Inc., Corning, NY, USA) to remove aggregated cells, PI-stained cells were subjected to a cell sorter (SH800 Series, SONY Imaging Products and Solutions Inc., Kanagawa, Japan). Cell cycle analysis was performed with the Cell Sorter Software version 2.1.2 (SONY Imaging Products and Solution Inc., Kanagawa, Japan).

Statistical Treatment
Experimental values are expressed as the mean ± standard deviation (SD) of triplicate or quadruplicate samples. Statistical analysis was performed using Student's t-test. A p-value of <0.05 was considered to be significant.

Conclusions
The present study demonstrated that plant extracts, having hormetic growth stimulation, showed higher neuroprotective activity than lower molecular weight polyphenols. The overlay method, that can prepare massive differentiated neuronal cells, may be applicable for the study of differentiation-associated changes in intracellular metabolites by metabolomics, and the interaction between neuronal cells and physiological factors.