Protective effects of s propargylcysteine (SPRC) on in vitro neuronal damage induced by amyloid beta (25 35 5

CHAPTER 5: CELL CULTURE PART II: OLIGOMERIC AΒ SPRC PRE-TREATMENT CONFERRED PROTECTION ON GLIOMA CELLS INCUBATED WITH Aβ 25-35 OLIGOMERS 5.1 Results 5.1.1 Effects of aggregated Aβ25-3

CHAPTER 5: CELL CULTURE PART II: OLIGOMERIC AΒ

SPRC PRE-TREATMENT CONFERRED PROTECTION ON GLIOMA CELLS

INCUBATED WITH Aβ 25-35 OLIGOMERS

5.1 Results

5.1.1 Effects of aggregated Aβ25-35 on cell viability

Figure 19: Effects of aggregated Aβ25-35 on C6 glioma cell viability, expressed as percentage of cell viability compared to the untreated control ± S.E.M Aggregated Aβ25-35 resulted in a dose-dependent decrease in cell viability 1 µM and 5 µM Aβ caused significant decrease in cell

viability to about 65% of the normal control N=6, *: p<0.05, **: p<0.01 compared with the untreated control

A dose range of 0.01 - 5 µM of Aβ25-35 aggregated at 4°C for 24 hours was treated to C6

glioma cells for 24 hours to investigate the extent of damage to cell viability using the MTT

assay (Figure 19) There was a significant dose-dependent decline in cell viability (F (4,18)=

12.327; p<0.01) after the treatment with aggregated Aβ25-35 1 µM and 5 µM Aβ treatments

resulted in 69 ± 4.8% and 66 ± 7.1% viability compared to the untreated control The 24-hour

treatment with 1 µM aggregated Aβ25-35 was established as the model for cytotoxicity in

5.1.2 Effects of pre-treatment of drugs on Aβ25-35 -induced cytotoxicity

5.1.2.1 SPRC on cell viability

Figure 20: Effects of SPRC pre-treatment on Aβ25-35-induced cytotoxicity (a) Time dependence

of SPRC pre-treatment (b) Pre-treatment of 0.1-100 µM SPRC restored Aβ-induced cytotoxicity significantly, but did not result in any toxicity in untreated cells The values are percentages of

cell viability compared to the untreated control ± S.E.M N>6, #: p<0.05 when compared to the untreated control; *: p<0.05, **: p<0.01 when compared with the Aβ-only control

Different doses of SPRC were pre-treated to C6 cells for 4 hours, 12 hours or 24 hours

before treating with 1 µM aggregated Aβ25-35 for another 24 hours (Figure 20a) Cells pre-treated

with SFM for 4 hours resulted in 90 ± 2.4% cell viability compared to control, while 12 hours

and 24 hours resulted in about 86 ± 2.3% and 86 ± 4.1% respectively This implied that while

pre-treatment with SFM did not affect the Aβ-induced cell death significantly at 4 hours,

viability decrease after pre-treatment with SFM reached a maximum after 12 hours 4 and 24

hours of SPRC pre-treatment did not restore viability significantly; cell viability was maintained

at about 90% for all doses However, pre-treatment of SPRC for 12 hours before the Aβ25-35

assault can significantly protect the cells (F (5,39)= 12.871; p<0.01), with the maximum

restoration of 98 ± 1.2% observed with 1 µM pre-treatment Hence the 12-hour pre-treatment

period is used as the model for further investigation

The toxicity of SPRC on the glioma cells was also checked with the pretreatment of 0.1

-100 µM to cells without the addition of Aβ25-35 (Figure 20b) This dose range did not cause

observable cytotoxicity even at higher doses (F (4, 29) = 0.833; N.S), though cell viability appeared

to increase as the dosages increase 100 µM SPRC resulted in about 104 ± 2.9% viability when

compared to control, but this difference was not significant

5.1.2.2 SAC and NaHS on cell viability

Figure 21: Effects of SAC or NaHS pre-treatments on Aβ25-35-induced cytotoxicity Pre-treating cells with 0.1-100 µM SAC restored viability most significantly at 1 µM and 100 µM Pre-

treatment of 0.1-100 µM NaHS did not seem to provide any protection against Aβ-induced damage The values are percentages of cell viability compared to the untreated control ± S.E.M

N>3, #: p<0.05 when compared to the untreated control; *: p<0.05, **: p<0.01 when compared

with the Aβ25-35 control

The effects of pre-treatment of SAC and NaHS on Aβ-induced cytotoxicity in C6 cells

were also elucidated using the same dose range as in SPRC (Figure 21) The 12-hour

pre-treatment of SAC protected the cells (F (5, 30) = 5.113; p<0.01) with a dose-dependent restoration

in Aβ-induced cell death SAC1 µM and 100 µM significantly restored cell viability (p<0.01) to

94 ± 1.2% and 98 ± 2.8% respectively The exogenous H2S donor NaHS did not protect against

the Aβ-induced cytotoxicity even at small doses of 0.1 µM and 1 µM (F (5, 24) = 4.452; p<0.01),

and viabilities were kept around 85% for all doses Individual comparisons of means with the

Aβ-only control group were not significant

5.1.2.3 Comparisons with equimolar concentrations of drugs

Figure 22: Comparison between equimolar concentrations of drugs on Aβ25-35-induced

cytotoxicity Pre-treating cells with 1 µM of SPRC or SAC restored viability significantly This cytoprotective effect was not seen in the NaHS-treated group The values are percentages of cell

viability compared to the untreated control ± S.E.M N>3, #: p<0.05 when compared to the untreated control; *: p<0.05, **: p<0.01 when compared with the Aβ-only control

Since 1 µM SPRC resulted in the highest cell viability after 12-hour pre-treatment, all

drugs were compared using this equimolar dose of 1 µM (Figure 22) Equimolar concentrations

of SPRC, SAC and NaHS were pre-treated to the cells and cell viabilities were found to be

significantly different using one-way ANOVA analysis (F (5,39) = 16.898; p<0.01) Among all

drugs, SPRC produced the highest restoration in cell viability to 98 ± 1.9% (p<0.01), compared

to 94 ± 1.2% (p<0.01) and 83 ± 2.7% for SAC and NaHS respectively These results called for

further studies to understand the underlying mechanisms for the cytoprotective effects of SPRC

in Aβ-induced decline in cell viability, and SAC is used as a comparison in subsequent studies

5.1.3 Effects on H 2 S pathway

5.1.3.1 Effects on H 2 S content in cell medium

Figure 23: Effects of pre-treatment of drugs on H2S concentrations in cell medium The values are expressed as fold increase in concentration compared to the untreated control ± S.E.M N=3,

#: p<0.05 compared to untreated control; **: p<0.01 compared to Aβ-only group

The cell media was collected after the entire treatment period and H2S concentrations

were investigated using the colorimetric method Treatment with aggregated Aβ25-35 significantly

decreased H2S concentrations in the medium to about 0.89-fold, or a reduction of about 10% in

H2S liberation (Figure 23) After the 12-hour pre-treatment with SPRC or SAC, both drugs

significantly increased the H2S concentrations in the medium SPRC-treated cells were found

with 1.06-fold increase (p<0.01) while SAC-treated cells were found with 1.10-fold increase

(p<0.01)

5.1.3.2 Effects on CBS expression

0.7 0.8 0.9 1 1.1 1.2 1.3

Figure 24: Effects of pre-treatment of drugs on CBS expression in cell lysates (a) Representative blots for CBS expression in treated cells N=3 (b) Percentage fold differences in CBS expression

± S.E.M N=3, #: p<0.05 compared to untreated control; *: p<0.05, **: p<0.01 compared to

Aβ-only group

The Aβ-only treatment significantly decreased the CBS expression to about 71 ± 3.4%

(p<0.05) from 100% in the untreated control cells (Figure 24) Pre-treatment of SPRC before the

subsequent Aβ25-35 treatment restored the CBS expression to 101 ± 7.8% (p<0.05) 1 µM SAC

pre-treatment increased the CBS expression even higher to 134 ± 1.3% (p<0.01)

5.1.3.3 Effects of CBS inhibitor on cell viability

Figure 25: Effects of the CBS inhibitor AOAA

on cell viability (a) Dose dependence of 12 h pre-treatment of AOAA with and without 1 µM

(b)

Aβ25-35 treatment (b) Pre-treatment of 0.1-100 µM SPRC restored the aggravated cytoxicity by AOAA on Aβ injury significantly The values are percentages of cell viability compared to the

untreated control ± S.E.M N>6, #: p<0.05 when compared to the untreated control; &: p<0.05

when compared to the SFM + 1 µM Aβ25-35 control; *: p<0.05, **: p<0.01 when compared with

the Aβ25-35 + 100 µM AOAA control

The involvement of H2S on the Aβ injury was further investigated using the CBS

inhibitor aminoxyacetic acid (AOAA) Different doses of AOAA were pre-treated to the cells for

12 hours before the additional Aβ insult for 24 hours (Figure 25a) There was a dose-dependent

aggravation in the cell viability with increasing doses of AOAA The Aβ-only control group

resulted in cell viability of 83 ± 2.1% (p<0.05) and this was further decreased to 69 ± 3.5% in

the group with additional 100 µM AOAA (p<0.05) Increasing doses of AOAA in cells without

Aβ did not result in significant cell loss (Figure 25a) As such, 100 µM AOAA was chosen as the

investigation model for the CBS inhibitor Together with the 100 µM AOAA, increasing doses

of SPRC from 0.1µM -100 µM were pre-incubated in the cells for 12 hours (Figure 25b) After

that, 1 µM Aβ25-35 was added to the cells There was a gradual, dose-dependent increase in cell

viability following SPRC treatment 0.1 µM SPRC significantly restored the Aβ-induced

cytotoxicity from 72 ± 1.7% in the Aβ + AOAA-control to 78 ± 0.4% (p<0.05) This trend

reached a maximum at 10 µM and 100 µM SPRC, where such doses restored the cell viability to

101 ± 5.7% (p<0.01) and 101 ± 2.1% (p<0.01) respectively

5.1.4 Effects on oxidative stress

5.1.4.1 Production of DCF-DA

Figure 26: Effects of pre-treatment of drugs on DCF production, presented as percentage control

± S.E.M N=4, #: p<0.05 compared to untreated control; **: p<0.01 compared to Aβ-only group

DCF-DA fluoresces in the presence of oxidative radicals and is an indicator of oxidative

stress DCF fluorescence was significantly altered in treated cells (F (3, 15) = 9.701; p<0.01)

Aβ-treated cells increased oxidative stress, indicated by the significant increase in DCF produced to

about 124 ± 2.85% in the Aβ-only group (p<0.01) (Figure 26) However, pre-treatment of drugs

may be able to condition the cells before the Aβ-induced oxidative stress Pre-treatment of SPRC

decreased the Aβ-induced increase in DCF fluorescence to about 100 ± 3.65% (p<0.01)

Similarly, SAC-treated cells also resulted in 101 ± 3.49% (p<0.01) units of fluorescence

staining ± S.E.M N=4, #: p<0.05 compared to untreated control; **: p<0.01 compared to

Aβ-0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

The DHE stain fluoresces in the presence of free radicals, indicating cellular locations

under oxidative stress The representative photos were taken under the same brightness and

contrast between each group (Figure 27a) The Aβ-only group showed increased fluorescence

and decreased upon pre-treatment of drugs (Figure 27a, Panel II) In the presence of Aβ, cells

stained with DHE had increased fluorescence to about 1.50 ± 0.73 -fold (p<0.01) compared to

the untreated cells Such bright red fluorescence was not observed in either the SPRC- or SAC-

treated cells (Figure 27a, Panels III and IV) While SAC treatment decreased the DHE staining

slightly to 1.28 ± 0.99 -fold (p= 0.26), SPRC treatment decreased the fluorescence significantly

to 1.06 ± 0.31 -fold (p<0.01) (Figure 27b)

5.1.4.3 Expression and activities of antioxidant enzymes

Figure 28: Effects of pre-treatment of drugs on SOD-1 expression in cell lysates (a)

Representative blots for SOD-1 expression in treated cells (b) Percentage fold difference in SOD-1 expression ± S.E.M N=6 (c) Effects of pre-treatment of drugs on total SOD activity The values are expressed as a percentage of the activity in the untreated cells ± S.E.M N=4, #:

p<0.05 compared to untreated control; *: p<0.05 compared to Aβ-only group

The SOD-1expression in the cells (F (3, 28) = 4.789; p<0.05) was changed significantly

after drug treatments (Figure 28a) Aβ-only treatment decreased the SOD-1 expression to 0.73 ±

0.05-fold (p<0.05) (Figure 28b) SPRC treatment restored this decrease to 0.95 ± 0.07 -fold

(p<0.094) while SAC only slightly increased the expression to 0.81 ± 0.08-fold (p= N.S) Total

SOD activity was significantly halved upon Aβ treatment to 47 ± 2.5% (p<0.01) compared to the

100% in the untreated control cells (Figure 28c) This mirrored the decrease in SOD expression

in the cell lysates On the other hand, pre-treatment with SPRC or SAC both increased the SOD

activity (F (3, 15) = 19.115; p<0.01) to 78.3 ± 9.3% (p<0.05) and 67 ± 2.7% respectively Again,

this trend was similar to the expression patterns of SOD

Figure 29: Effects of pre-treatment of drugs on catalase expression in cell lysates (a)

Representative blots for catalase expression in treated cells (b) Percentage fold difference in catalase expression ± S.E.M N=3 (c) Effects of pre-treatment of drugs on catalase activity The

values are expressed as units of activity/ µg protein ± S.E.M N=4, #: p<0.05 compared to

SFM SFM SPRC 1

µM

SAC 1 µM

Aβ treatment generally decreased the catalase expression in all groups, as depicted in the

weaker bands in Figure 29a However, all drugs treatments did not change the catalase

expression significantly (F (3,12)= 0.825; p=0.505) The catalase expressions remained lower than

the untreated control at about 0.75-fold (Figure 29b) The catalase activity was measured by the

ability to catalyse the decomposition of H2O2 in the cells The untreated control had an activity

of 0.124 ± 0.013 units/mg protein and this was decreased drastically upon treatment with

aggregated Aβ (Figure 29c) The treatment groups were statistically different (F (3,15) = 23.401;

p<0.01) as both SPRC or SAC treatment only increased the Aβ-induced decrease in activity very

slightly SPRC treatment resulted in activity of about 0.034 ± 0.007 units/mg protein (p= N.S),

whilst SAC treatment resulted in 0.048 ± 0.009 units/ mg protein (p= N.S); slight enhancements

of the enzyme activities from the low activity of 0.028 ± 0.003 (p<0.01) in the Aβ-only control

Figure 30: Effects of pre-treatment of drugs on GPx expression in cell lysates (a) Representative blots for GPx expression in treated cells (b) Percentage fold difference in GPx expression ± S.E.M N=4 (c) Effects of pre-treatment of drugs on GPx activity The values of activity are

SFM SFM SPRC 1

µM

SAC 1 µM

expressed as µmol/NADPH/min/mg protein ± S.E.M N=3, #: p<0.05 compared to untreated control; *: p<0.05 compared to Aβ-only group

Analysis with one-way ANOVA has found the differences in the GPx expressions

between the various groups to be significant (F (3, 16) = 6.214; p<0.01) The GPx expression in

cell lysates decreased to 0.59 ± 0.06-fold upon treatment with 1 µM Aβ25-35 The Aβ-only group

demonstrated a weaker band than the control group (Figure 30a) While both drug treatments did

not significantly increase the fold differences, but SPRC and SAC pre-treatments increased GPx

expression to 0.68 ± 0.05- fold and 0.89 ± 0.15- fold respectively (Figure 30b) GPx activity was

significantly altered after treatment with various drugs (F (3,11)= 22.13; p<0.01) (Figure 30c) The

Aβ25-35 insult decreased the inherent activity of 26.9 ± 1.83 NADPH/min/mg protein to 15.1 ±

1.37 NADPH/min/mg protein (p<0.01) This significant decline mirrored the decrease in GPx

protein expression as reported In the treated groups, SPRC restored the decline to 22.9 ± 0.63

NADPH/min/mg protein (p<0.05), comparable to the untreated control This restoration was not

seen in the SAC-treated group, where activity remained at about 11.4 ± 1.49 NADPH/min/mg

protein (p= N.S)

1β expressed as concentration (pg/ml) ± S.E.M N≥3, #: p<0.05 compared to untreated control;

**: p<0.01 compared to Aβ-only group

Treatment with 1 µM Aβ25-35 increased the mRNA (Figure 31a) and protein expressions

(Figure 31b) of pro-inflammatory IL-1β The mRNA expression of IL-1β was increased to 1.54

± 0.07-fold compared to untreated control (p<0.01) while the protein expression was increased

to 2047 ± 27 pg/ml compared to 1632 ± 98 pg/ml in the untreated control 12-hour pre-treatment

of 1 µM SPRC to the cells significantly decreased both the mRNA and protein expressions of

IL-1β The mRNA expression was decreased to 0.25 ± 0.01-fold (p<0.01) while the protein

expression was reduced to 1607 ± 53 pg/ml (p<0.01) Likewise, pre-treatment with SAC also

significantly decreased the mRNA expression to 0.55 ± 0.04- fold (p<0.01) and the protein

5.1.5.2 Expression of pro-inflammatory IL-6

Figure 32: Effects of pre-treatment of drugs on IL-6 expression (a) mRNA expression of IL-6 expressed as fold difference of the untreated control group ± S.E.M (b) Protein expression of IL-

6 expressed as concentration (pg/ml) ± S.E.M N≥3, #: p<0.05 compared to untreated control; **: p<0.01 compared to Aβ-only group

The pro-inflammatory IL-6 mRNA expression was increased to 1.64 ± 0.17-fold (p<0.05)

following Aβ treatment (Figure 32a) Similarly, the IL-6 protein expression increased slightly to

59 ± 5.5 pg/ml compared to 52 ± 0.7 pg/ml in the untreated control (Figure 32b), although it was

not significant Pre-treatment of SPRC decreased both the mRNA and protein expressions

significantly to 0.70 ± 0.08- fold (p<0.01) and 23 ± 0.85 pg/ml (p<0.01) respectively Although

the mRNA expression of IL-6 decreased to 0.34 ± 0.11- fold (p<0.01) following SAC

pre-treatment, there was no observable change to the protein expression of IL-6, remaining at a high

The TNF-α mRNA expression significantly changed after Aβ treatment; the mRNA

expression of the Aβ-only control was significantly increased to 1.42 ± 0.09-fold (p<0.05)

(Figure 33a) Pre-treatment of SPRC and SAC both significantly attenuated the Aβ-induced

increase in TNF-α mRNA to 0.19 ± 0.006-fold (p<0.01) and 0.48 ± 0.03- fold expression for

TNF-α (p<0.01) This was not reflected in the protein expressions, where the concentrations of

TNF-α increased following the Aβ treatment (Figure 33b) Aβ treatment increased the TNF-α

concentration significantly to 94 ± 6.6 pg/ml (p<0.05) compared to 56 ± 5.0 pg/ml in the

untreated control Pre-treatment with SPRC or SAC did not significantly decrease the TNF-α

protein concentration, although 1 µM SPRC resulted in a slight decline in the TNF-α

SFM SFM SPRC 1

µM

SAC 1 µM

5.1.5.4 Expression of anti-inflammatory IL-10

Figure 34: Effects of pre-treatment of drugs on IL-10 expression of inflammatory factors (a) mRNA expression of IL-10 expressed as fold difference of the untreated control group ± S.E.M

(b) Protein expression of IL-10 expressed as concentration (pg/ml) ± S.E.M N≥3, #: p<0.05 compared to untreated control; **: p<0.01 compared to Aβ-only group

The mRNA expression of the anti-inflammatory IL-10 was significantly down-regulated

to 0.36 ± 0.04- fold (p<0.01) following Aβ treatment (Figure 34a) SPRC pre-treatment

enhanced the IL-10 mRNA expression drastically to 1.73 ± 0.07- fold (p<0.01) Moreover, the

expression of the anti-inflammatory IL-10 was increased drastically to 1.73 ± 0.07-fold (p<0.01)

However, the SAC pre-treatment did not have an effect on IL-10 expression, with the mRNA

expression maintaining at 0.25 ± 0.03- fold The protein expression of IL-10 however, did not

show a similar trend The IL-10 protein expression generally declined slightly following Aβ

treatment (Figure 34b), but pre-treatment of SPRC or SAC both did not result in observable

changes to the protein expressions The IL-10 expression dropped to 174 ± 0.54 pg/ml in the

Aβ-only control from 178 ± 0.21 pg/ml in the untreated control While SPRC pre-treatment did not

change the IL-10 protein expression (174 ± 0.93 pg/ml) noticeably, SAC pre-treatment restored

the IL-10 protein expression to 178 ± 1.23 pg/ml

SFM SFM SPRC 1

µM

SAC 1 µM

5.1.6 Effects on cell death mechanisms

5.1.6.1 Cell cycle analysis

Figure 35: Effects of pre-treatment of drugs on cell cycle analysis N=3, #: p<0.05 compared to untreated control; *: p<0.05 compared to Aβ-only group

Cells were stained with propidium iodide and counted using flow cytometry to determine

the cell cycle status upon treatment of drugs (Figure 35) Upon pre-treatment of drugs,

significant difference was only observed in the sub-G1 phase (F(3,12)= 8.178; p<0.01), but not

amongst the other phases Cells treated with Aβ25-35 only significantly increased the percentage

of cells in the sub-G1 phase from 1.09 ± 0.2% to 4.34 ± 0.3% (p<0.01) Pre-treatment with

SPRC or SAC both decreased the sub-G1 cell population to 1.64 ± 0.5% and 1.39 ± 0.7%

respectively Though treatment with Aβ25-35 resulted in a decline in the G1 and G2/M phases

while arresting at the S phase, pre-treating with SPRC and SAC restored the percentage of cells

in the G1 phase and eliminated the increase in the S phase There was no observable increase in

the G2/M phase following drug treatments These results suggested that while the treatment of

Aβ25-35 resulted in some changes in the cell cycle status, pre-treatment of the drugs may only

minimally prevent arresting the cells at sub-G1 phase but do not enhance other cell cycle phases

#

5.1.6.2 Apoptosis

(a)

Figure 36: Effects of pre-treatment of drugs on DNA fragmentation using TUNEL staining (a) Representative photos of drug-treated groups at 20X magnification where fragmented DNA is stained green and indicated with white arrows The scale bar of 100 µm is indicated at the lower left of each panel The nucleus is counter-stained with DAPI and presented as blue fluorescence I: Untreated control; II: Aβ-only control; III: SPRC-treated group; IV: SAC-treated group (b) Proportion of green/blue fluorescence ± S.E.M, normalized to the untreated control N=3, #:

p<0.05 compared to untreated control; *: p<0.05 compared to Aβ-only group

The effects of drug treatment on apoptosis are elucidated using TUNEL staining, where

fragmented DNA is an indication of apoptosis and stained green (Figure 36a) The various

treatments generally altered the ratio of apoptotic cells when calculated with one-way ANOVA

(F (3,11)= 8.884; p<0.01) (Figure 36b) Cells treated with only serum-free medium (untreated

control) showed some degree of apoptosis, where a small percentage of cells (normalized to 1)

fluoresced in green (Figure 36a, Panel I) Upon treatment with Aβ25-35, the percentage of green

fluorescence increased to 3.74 ± 0.3-folds (p<0.05), implying a large increase in the proportion

of apoptotic cells (Figure 36b) However, pre-treatment with SPRC significantly reversed the

Aβ-induced increase in apoptosis by decreasing the number of cells with green fluorescence

(Figure 36a, Panel III) to 1.24 ± 0.43-folds (p<0.05) (Figure 36b) Pre-treatment with SAC also

reduced the percentage of cells with green fluorescence (Figure 36a, Panel IV); however, this

decline was not as large or significant as that in the SPRC-treated group, to about 2.79 ±

0.47-folds (Figure 36b)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5

Figure 37: Effects of pre-treatment of drugs on PARP and pro-caspase 3 expressions in cell lysates (a) Representative blots for PARP and pro-caspase 3 expressions in treated cells N=6 (b)

Fold difference in pro-caspase 3 expression ± S.E.M #: p<0.05 compared to untreated control;

**: p<0.01 compared to Aβ-only group

The expressions of PARP and caspase 3 were used to elucidate the extent of apoptosis in

Aβ-treated cells Immunoblotting of PARP resulted in multiple bands corresponding to the

differently-cleaved products of the protein (Figure 37a) While the 116 kDa uncleaved protein

cannot be clearly seen in some groups, the 87 kDa main cleaved product was visible from the

blots There was an obvious increase in the expression of the 87 kDa cleaved product in the

Aβ-only group, an indication of increased apoptosis following the application of Aβ Although there

was a corresponding increase in the expression of the 116 kDa protein seen from the darker band

at a higher molecular weight, in general the expression of PARP was enhanced following Aβ

treatment In contrast, drug treatments reduced the Aβ-induced increase where both the 116 kDa

0.5 0.6 0.7 0.8 0.9 1 1.1

and 87 kDa proteins were visibly lighter This decrease was much more obvious in the

SAC-treated group than the SPRC-SAC-treated group

The antibody used was specific for pro-caspase 3 expressions and any changes were used

loosely to quantify the amounts in the activated apoptotic protein caspase 3 (Figure 37a) There

was a significant decrease in the expression in the Aβ-only group to 0.85 ± 0.02-fold (p<0.01)

(Figure 37b), suggesting a loss of the uncleaved 32 kDa protein that may possibly be cleaved

during apoptosis This loss was significantly restored in the SPRC-treated group to 0.96 ±

0.02-fold (p<0.01) There was also a slight increase in the SAC-treated group to 0.89 ± 0.05-0.02-fold (p=

N.S) although no significance was found

Figure 38: Effects of pre-treatment of drugs on autophagy using acridine orange staining (a) Representative photos of drug-treated groups at 60X magnification with a scale bar of 20 µm indicated at the lower left of each panel Acridine orange enters the cell membrane to stain the cytoplasm and nucleus green, but stains the acidic autophagic vacuoles orange I: Untreated control; II: Aβ-only control; III: SPRC-treated group; IV: SAC-treated group (b) Proportion of

orange/green fluorescence ± S.E.M, n=3, #: p<0.05 compared to untreated control; **: p<0.01

compared to Aβ-only group

To better understand the mechanisms of cell death in drug-treated cells, the involvement

of autophagy was investigated The proportion of orange/green fluorescence indicates the extent

of staining of acidic autophagic vacuoles, and hence taken as a representation for autophagy

0 0.2 0.4 0.6 0.8 1 1.2 1.4

(b)

processes The degree of staining was altered in cells treated with Aβ25-35 and also after

pre-treatments of drugs (F (3,13)= 12.443, p<0.01) The proportion in untreated cells was 0.76 ± 0.008,

and this proportion was drastically increased following the treatment with Aβ25-35 to 1.15 ± 0.008

(p<0.05) (Figure 38b) This could be seen obviously when comparing Figure 38a (Panel I) and

Figure 38a (Panel II), where bright orange dots were observed in the cytoplasm, suggesting the

presence of autophagic vacuoles Such bright orange dots were significantly fewer in number

after pre-treatment with SPRC or SAC (Figure 38a, Panels III and IV) Pre-treatment with SPRC

resulted in a proportion of 0.74 ± 0.07, comparable to that in the untreated control Similarly,

pre-treatment of SAC resulted in a proportion of 0.77 ± 0.05 Both drugs significantly decreased

the proportion when compared to the Aβ-only control (p<0.01), implying an interruption to the

autophagic processes in the disease state