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Journal of Alzheimer’s Disease 43 (2015) 465–477 DOI 10.3233/JAD-140078 IOS Press

Camptothecin and its Analogs Reduce Amyloid-␤ Production and Amyloid-␤42-Induced IL-1␤ Production Ju Wang1 , Zi-Qi Shi1 , Mu Zhang1 , Gui-Zhong Xin, Tao Pang, Ping Zhou, Jun Chen, Lian-Wen Qi, Hua Yang, Xiaojun Xu∗ and Ping Li∗ State Key Laboratory of Natural Medicines, Department of Pharmacognosy, China Pharmaceutical University, Nanjing, China Handling Associate Editor: Yi Zhun Zhu

Accepted 12 June 2014

Abstract. Compounds derived from natural products are becoming promising alternative drugs/tools in Alzheimer’s disease (AD) therapeutics. From an in-house natural products library, seventeen hits were selected for their inhibitory effect on the production of amyloid-␤ (A␤) with IC50 lower than 10 ␮M without causing obvious toxicity. Among these compounds, camptothecin (CPT) and its analogs showed inhibitory effects on amyloid-␤ 1-42 (A␤42 ) with the IC50 value in the nanomolar range in HEKsw cells and SHSY5Ysw cells. Further studies showed that CPT and its analogs inhibited A␤42 via a p53 dependent pathway. Meanwhile, CPT and its analogs could also inhibit A␤42 induced IL-1␤ production in the THP-1 cells. Taken together, our results indicate that CPT and its analogs would be a promising therapeutic candidates for AD. Keywords: Alzheimer’s disease, amyloid-␤, camptothecin, IL-1␤, p53

INTRODUCTION Alzheimer’s disease (AD) is the most common form of dementia and leads to death within 3 to 9 years after being diagnosed [1]. With increasing elderly population, it is estimated that over 100 million patients will develop AD by 2050 [2]. The most common pathologically patterns of AD patients include amyloid-␤ (A␤) plaques and neurofibrillary tangles [3]. A␤ generation and deposition are considered as the one of triggering factors for neural death of AD patients [4]. A␤ peptides 1 These

authors contributed equally to this work. to: Ping Li, PhD, Professor, Director, State Key Laboratory of Natural Medicines, Department of Pharmacognosy, China Pharmaceutical University; 24 Tongjia Lane, Nanjing 210009, China. Tel./Fax: +86 25 83271379; E-mail: [email protected] and Xiaojun Xu, PhD, Associated Professor, State Key Laboratory of Natural Medicines, Department of Pharmacognosy, China Pharmaceutical University; 24 Tongjia Lane, Nanjing 210009, China. Tel./Fax: +86 25 83271379; E-mail: [email protected]. ∗ Correspondence

originate from proteolysis of the amyloid-␤ protein precursor (A␤PP) by the sequential enzymatic actions of ␤-secretase (BACE1) and ␥-secretase [5]. An imbalance between production, clearance, and aggregation of peptides causes A␤ to accumulate, and this excess may be the initiating factor in AD. This theory was defined as “amyloid hypothesis” [6]. As reported, most of the A␤-centric approaches that reached Phase III clinical trials have failed; tramiprosate and tarenflurbil have now been discontinued [6] due to their poor preclinical outcome [7]. Semagacestat, a ␥–secretase inhibitor (GSI), reduced A␤ deposition potently both in transgenic mice and human volunteers [8, 9], but the Phase III clinical trials demonstrated that patients who were treated with semagacestat displayed an increased deterioration in cognition and activities compared to placebo-treated controls [6]. In fact, GSIs such as semagacestat have a very complex mechanism. This class of compounds is able to inhibit the production of

ISSN 1387-2877/15/$27.50 © 2015 – IOS Press and the authors. All rights reserved

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A␤ at high concentrations but stimulates ␥–secretase at lower concentrations [10]. It should also be noted that ␥–secretase has a range of substrates, especially Notch signaling. It is suggested that the adverse effects of semagacestat on cognition are mainly related to the inhibition of Notch processing [11]. Currently symptomatic therapies for AD include the acetylcholinesterase (AChE) inhibitors (donepezil Aricept; Eisai/Pfizer), galantamine (Razadyne; Johnson & Johnson), and rivastigmine (Exelon; Novartis), and a low-affinity N-methyl-d-aspartate (NMDA) receptor antagonist (memantine) for moderate to severe AD. However, these are symptomatic treatments that do not actually slow or reverse the progression of the disease [6]. As some Chinese medicinal herbs have been used in treating dementia, many researchers are now turning to Chinese medicines to discover potential neuro-protective agents or disease modifying agents [12]. For instance, Huperzine A, an AChE inhibitor, was shown to reduce the formation of A␤ peptides by affecting A␤PP processing [13]. Clinical trials demonstrated that Huperzine A significantly improved cognitive functions [14]. In other studies, Yokukansan has been shown the therapeutic effects on the behavioral psychological symptoms of dementia in AD and Parkinson’s disease patients [15, 16]. To date, the greatest successes have come from plant-based AChE discovery programs, which have provided two of the five currently approved drugs for the treatment of AD. However, it is widely accepted that these AChE inhibitors are only effective for relieving the symptoms of AD in a short period of time, and a broader range of therapeutics is needed [17]. Given the prevalence of AD and the lack of effective long-term therapies, there is a pressing need to discover viable leads that can be developed into clinically approved drugs and powerful tools for investigation. Many natural products derived from traditional Chinese medicines have already shown their clinical therapeutic effects on anti-neurodegeneration, antiinflammation, immune-modification, and anti-cancer applications. More importantly, the multi-components and multi-targets properties of natural products make them more powerful in complex etiology and pathogenic diseases. It is promising to discover safe and effective candidates from traditional Chinese medicines. In the present study, we screened an inhouse natural products library composed of ∼500 purified chemical constituents for their inhibitory effect on the production of A␤40 /A␤42 , BACE1 activity, as well as the Notch signaling. We also confirmed such effect on A␤ production is not through cell death

or apoptosis. Promisingly, the molecular weight of the selected compounds is smaller than 500 Dalton. Most of the candidates are widely used for anti-cancer in clinical trial/investigation. Particularly, camptothecin (CPT) and three of its analogues showed very potent inhibitory effect on the production of A␤40 /A␤42 . Moreover, we did not observe any inhibitory effects on ␥–secretase. MATERIALS AND METHODS Reagents All commercial reagents are of analytical grade or the highest purity available. The ∼500 chemical constituents, composed of the in-house natural products library, were extracted from herbs and purified in-house with more than 99% purity verified by reverse-phase high pressure liquid chromatographymass spectrometry (HPLC-MS). The structures of CPT, and its analogs, 7-ethyl-10-hydroxycamptothecin (SN-38), 10-hydroxycamptothecin, 9-methoxycamptothecin are showed in Fig. 1. AlphaLISA A␤40 and A␤42 Kit (AL202C, AL203C) were purchased from Amersham/PerkinElmer (Freiburg, Germany/Waltham, MA, USA; IM249). WST-1 kit (05015944001) was purchased from Roche Applied Science (Shanghai, China). Caspase-GloR 3/7 Assay kit (G8090) was purchased from Promega (Madison, USA); Synthetic A␤42 (TFA salt), A␤42-1 (TFA salt), A␤40 (TFA salt), A␤38 (TFA salt) were purchased from American Peptide (Sunnyvale, CA). Ham’s F-12 (phenol red-free) was from BioSource (Camarillo, CA), IL-1␤ ELISA kits from R&D Systems (Minneapolis, MN). BACE1 activity kit (ab65357) was purchased from Abcam (Hong Kong, China). Cell lines and cultures All cells were cultured at 37◦ C in the presence of 5% CO2 . The HEKsw cells (Human Embryonic kidney cell line stably expressing A␤PP with the Swedish mutation) and SHSY5sw cells (human neuroblastoma cell line stably expressing A␤PP with the Swedish mutation) were purchased from Velox Pharmaceutics, Inc. (VLXSTC0001 and VLXSTC0002, Changzhou, China). Both cell lines were incubated in a Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FBS and selected by neomycin. THP-1 (Human acute monocytic leukemia cell line was purchased from ATCC (TIB-202™) cells were cultured in RPMI 1640

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Fig. 1. Structures of CPT and its analogs. A) CPT, B) SN-38, C) 10-Hydroxycamptothecin, D) 9-Methoxycamptothecin.

with 10% FBS and 2 mM L-Glutamine. Chinese hamster ovary (CHO) cell line was purchased from from Velox Pharmaceutics, Inc. (VLXC0001, Changzhou, China) and incubated in DMEM with 10% FBS.

selected from the AlphaLISA assay. The WST kit from Promega was used for cell viability assessment following the manufacturer’s instruction. The positive control molecular used in WST assay was 10 ␮M staurosporine (STS) (S6942, Sigma-Aldrich).

AlphaLISA assay for Aβ detection To detect extracellular A␤, HEKsw cells and SHSY5sw cells were cultured in 96-well plates at a density of 5 × 104 cells/well. After 24 hours treatment with various concentrations of the purified chemical constituents derived from natural sources or N-[N-(3,5-difluorophenacetyl-L-alanyl)]S-phenylglycine t-butyl ester (DAPT) (D5942, sigmaAldrich), the supernatants free of cellular debris were collected and stored at −80◦ C until use. The concentrations of A␤40 and A␤42 in the medium were measured using the AlphaLISA A␤40 and A␤42 kits from PerkinElmer following the manufacturer’s instruction. To detect intracellular A␤40 , HEKsw cells were cultured in 6-well culture plates and treated with the compounds, which decreased extracellular A␤ in the screen or DAPT for 24 hours. Cells were washed thoroughly with phosphate-buffered saline (PBS) and scraped into RIPA buffer containing protease inhibitors. Lysates were sonicated and spun at 100,000 × g for 20 minutes at 4◦ C. A␤ peptides from RIPA lysates were captured and measured with A␤40 AlphaLISA kit. Assessment of cell viability SHSY5sw cells were cultured in the 96-well plates at a density of 5 × 104 cells/well and treated with 100 ␮M selected compounds which were potential hits

Caspase 3/7 activity assay SHSY5sw cells were cultured in the 96-well plates at a density of 2 × 104 cells/well and treated with 100 nM and 10 ␮M selected compounds which were potential hits selected from the AlphaLISA assay. The caspase 3/7 activity assay kit from Promega was used for caspase activity assessment following the manufacturer’s instruction. 5 ␮M STS was served as positive control. BACE1 activity assay SHSY5Ysw cells were cultured in the 96-well plates at a density of 5 × 104 cells/well and treated with 10 ␮M compounds for 24 hours. Then the inhibition effect on BACE1 activity was tested according to the procedures provided by the manufacture. Briefly, cells were collected and lysed by adding 0.1 ml of ice-cold Extraction Buffer and incubated on ice for 10 minutes. Then 50 ␮l of cell lysates were added to each well in a 96-well plate. For positive control, 2 ␮l of reconstituted active ␤-secretase were added to 48 ␮l of Extraction Buffer. For negative control, 2 ␮l of the ␤-secretase inhibitor were added to 48 ␮l of Extraction Buffer. 2 ␮l of ␤-secretase substrate were added and then incubation in the dark at 37◦ C for 2 hours. The samples were read in the SpectroMax with Ex. = 335–355 nm and Em. = 495–510 nm.

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Preparation of Aβ peptides and controls

for multiple comparisons. p < 0.05 was considered significant.

The peptides were prepared according to the protocols described before [18]. Briefly, the A␤ peptides were initially dissolved to 1 mM in hexafluoroisopropanol (HFIP) and aliquoted in sterile microcentrifuge tubes. The HFIP was removed in a Speed Vacuum, and the peptide film was stored dessicated at −80◦ C. For oligomeric conditions, Ham’s F-12 (phenol red-free) was added to bring the peptide to a final concentration of 100 ␮M and incubated at 4◦ C for 24 hours. Vehicle controls were made by combining the same volumes of DMSO with F12, used for the peptide preparations. Measurement of levels of IL-1β THP-1 cells seeded on 96-well plate at a density of 1 × 105 cells/well were treated with A␤42 , A␤42-1 , A␤40 , A␤38 , and/or CPT and its analogs at the dose and time respectively. Following the treatment, 100 ␮l supernatant was collected and stored at −80◦ C. IL-1␤ levels were detected by human IL-1␤ ELISA kits according to the procedures provided by the manufacturer. Statistical analysis Data were expressed as the mean ± SEM. IC50 was plotted using GraFit5 (Erithacus Software limited, UK). Statistical significance was conducted using the one-way ANOVA followed by the Tukey test

RESULTS CPT and its analogs reduce Aβ production in the range of nanomolar Initially, we screened an in-house natural products library for their inhibitory effect on A␤40 and A␤42 production using the described AlphaLISA method. About 500 single compounds were evaluated at 10 ␮M using the HEKsw cell line. Twenty five compounds were selected with more than 50% inhibition of A␤42 level at 10 ␮M in the primary screening. CPT and its analogs showed 100% inhibition of A␤40 and A␤42 levels at 10 ␮M in the primary screen. To further confirm the potency, various concentrations of the compounds and DAPT (a known inhibitor of ␥secretase) were titrated in HEKsw cells [19]. After 24 hours of treatment, CPT and its analogs inhibited the production of A␤40 and A␤42 in a dose-dependent manner with the IC50 values under the range of nanomolar (Table 1). DAPT decreased A␤40 and A␤42 levels with the IC50 values of ∼200 nM and ∼260 nM consistent with a previous report [19]. Totally there were 17 compounds showed IC50 under micormolar range (Table 1), interestingly, most of them are anticancer compounds which have been used in clinical trial/investigation. The hits rate in primary screening was 3.4% (17 candidates from 500 compounds). The screening parameters such as Z’ factor for A␤40

Table 1 Inhibition effect on A␤ production by the compounds Chemical names Camptothecin SN-38 10-Hydroxy camptothecin 9-Methoxycamptothecin Cinobufagin Digoxin Deslanoside Harmine artemisinin Lycorine Hydrochloride Roburic acid Resibufogenin Artesunate Timosaponin A3 Gossypol Cantharidin Nitidine Chloride

MWa 348 392 364 378 443 781 927 212 282 324 441 385 384 741 519 196 384

HEKsw (IC50 nM) A␤42

A␤40

4.1 ± 0.3 0.4 ± 0.1 0.8 ± 0.02 0.6 ± 0.1 99 ± 12 110 ± 30 200 ± 66 750 ± 200 1600 ± 500 >10000 ±700 3000 ± 1000 3100 ± 1200 3100 ± 800 3400 ± 2000 3800 ± 1500 5100 ± 2000

34 ± 6.7 3.3 ± 0.2 13 ± 2.6 7.7 ± 1.5 120 ± 23 120 ± 44 360 ± 100 3300 ± 1500 6500 ± 3000 3000 ± 1400 >10000 3500 ± 2000 >10000 6800 ± 1500 4200 ± 2200 4800 ± 2000 >10000

SHSY5Ysw (IC50 nM) A␤42 A␤40 8.0 ± 1.5 1.1 ± 0.2 0.9 ± 0.3 1.3 ± 0.4 120 ± 23 130 ± 55 330 ± 120 870 ± 300 1700 ± 300 1900 ± 800 2000 ± 400 3200 ± 1200 4200 ± 2000 3300 ± 1400 3800 ± 2100 4000 ± 2100 5500 ± 1600

52 ± 22 10 ± 1 23 ± 7 15 ± 7.9 200 ± 50 190 ± 24 420 ± 140 4100 ± 2200 7200 ± 3700 3200 ± 1400 >10000 5000 ± 1900 >10000 7500 ± 3100 4500 ± 2600 4400 ± 1700 >10000

All the data was representative of three independent experiments and expressed as the mean ± SEM. a MW, molecular weight (Dalton).

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Fig. 2. Effects of CPT and its analogs on A␤ production. SHY5Ysw cells and HEKsw cells were treated with variety concentration of DAPT, CPT and its analogs for 24 hours, the supernatants free of cellular debris were collected to measure the A␤42 concentration, the RIPA lysates of HEKsw cells were collected to measure intracellular A␤40 concentration. A) IC50 determination of CPT and its analogs for A␤40 production in HEKsw cells. The IC50 of CPT, SN-38, 10-Hydroxycamptothecin, 9-Methoxycamptothecin and DAPT were 34 ± 6.7 nM, 3.3 ± 0.2 nM, 13 ± 2.6 nM, 7.7 ± 1.5 nM, and 260 ± 120 nM, respectively. B) IC50 determination of CPT and its analogs for A␤42 production in HEKsw cells. The IC50 of CPT, SN-38, 10-Hydroxycamptothecin, 9-Methoxycamptothecin, and DAPT were 4.1 ± 0.3 nM, 0.43 ± 0.1 nM, 0.84 ± 0.02 nM, 0.62 ± 0.08 nM, and 200 ± 81 nM, respectively. C) IC50 determination of CPT and its analogs for A␤40 production in SHSY5Ysw cells. The IC50 of CPT, SN-38, 10-Hydroxycamptothecin, 9-Methoxycamptothecin, and DAPT were 52 ± 22 nM, 10 ± 1 nM, 23 ± 7 nM, 15 ± 3.8 nM, and 410 ± 150 nM, respectively. D) IC50 determination of CPT and its analogs for A␤42 production in SHSY5Ysw cells. The IC50 of CPT, SN-38, 10Hydroxycamptothecin, 9-Methoxycamptothecin, and DAPT were 8.0 ± 1.5 nM, 1.1 ± 0.2 nM, 0.9 ± 0.3 nM, 1.3 ± 0.4 nM, and 400 ± 300 nM, respectively. E) IC50 determination of CPT and its analogs for intracellular A␤40 production in HEKsw cells. The IC50 of CPT, SN-38, 10-Hydroxycamptothecin, 9-Methoxycamptothecin and DAPT were 38 ± 14 nM, 4.3 ± 0.7 nM, 15 ± 7.9 nM, 4.9 ± 1.1 nM, and 190 ± 66 nM, respectively. Data are means ± SD from 3 separate experiments performed in triplicate.

and A␤42 were 0.74 and 0.76, respectively. The CV% values were 7.5% and 11% for A␤40 and A␤42, respectively. And the Signal to Background (S/B) ratio was more than 20 in both assays. All the parameters mean the results of both assays are reliable. To eliminate the possibility that the observed reduction of A␤ was cell-line-specific, we treated different doses of the selected compounds as well as DAPT in

the SHSY5Ysw cell line. As shown in Table 1, CPT and its analogs inhibited A␤40 (Table 1) and A␤42 (Fig. 2) dose-dependently with IC50 values in the range of nanomolar. DAPT showed approximately 2-fold elevation in IC50 values for A␤40 (400 nM) and A␤42 (400 nM). The observed elevation in the IC50 values could due to the intrinsic cell line differences in metabolism of A␤PP or the different expression

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Chemical names Camptothecin SN-38 10-Hydroxycamptothecin 9-Methoxycamptothecin Cinobufagin Digoxin Deslanoside Harmine artemisinin Lycorine Hydrochloride Roburic acid Resibufogenin Artesunate Timosaponin A3 Gossypol Cantharidin Nitidine Chloride

IC50 of intracellular WST assay Caspase 3/7 BACE1 A␤40 (nM) (% Inhibition at 100 ␮M) (% activity at 10 ␮M (% Inh at 10 ␮M) 38 ± 14 4.3 ± 0.7 15 ± 7.9 4.9 ± 1.1 88 ± 33 96 ± 27 190 ± 89 2500 ± 700 5700 ± 2100 3300 ± 1200 >10000 2900 ± 900 >10000 5900 ± 1500 4300 ± 2000 4700 ± 1600 >10000

23 ± 7.3 35 ± 9.3 0.7 ± 2.8 16 ± 2.7 0 ± 1.2 11 ± 3.3 0 ± 0.9 33 ± 10 0 ± 2.5 0 ± 3.2 42 ± 7.5 0 ± 2.2 0 ± 3.0 86 ± 4.8 75 ± 6.9 35 ± 4.7 0 ± 1.6

0 ± 3.8a 44 ± 4.2a 16 ± 3.0a 20 ± 2.4a 0±2 0 ± 5.4 0 ± 2.1 0 ± 4.5 6.9 ± 5.6 0 ± 0.9 45 ± 3.6 0 ± 4.5 3.3 ± 4.0 5.0 ± 3.6 34 ± 10 29 ± 11 27 ± 4.0

3.5 ± 3.1 0 ± 8.5 0 ± 4.5 0 ± 0.8 15 ± 9.2 0 ± 4.6 0 ± 5.7 0 ± 13 12 ± 3.6 22 ± 11 0 ± 0.9 0 ± 3.8 0 ± 4.1 13 ± 2.8 11 ± 9.0 5 ± 3.2 0 ± 5.1

Notch signaling (% Inh at 10 ␮M) IC50 = 3.7 ± 1.3 ␮M 50 ± 8.2 IC50 = 6.3 ± 1.6 ␮M IC50 = 4.2 ± 0.9 ␮M 50 ± 11 33 ± 3.4 25 ± 6.5 28 ± 5.3 12 ± 3.8 0 ± 2.3 28 ± 11 23 ± 2.8 0 ± 3.4 IC50 = 9.0 ± 2 ␮M 23 ± 13 0 ± 4.5 12 ± 5

All the data was representative of three independent experiments and expressed as the mean ± SEM. a Detect the % activity of caspase3/7 at 100 nM.

levels of exogenous A␤PP. The IC50 values of CPT and its analogs are still in the range of nanomolar, which indicates the effect of CPT and its analogs are specific to A␤. Furthermore, to confirm whether the observed change in A␤ levels was due to reduction on its production and/or secretion, the intracellular A␤40 in HEKsw cells was quantified at various concentrations of the compounds and DAPT. All of the compounds reduced the intracellular A␤40 in a dose-dependent manner (Table 2). The equivalent inhibition of both extracellular and intracellular A␤40 argues strongly that these compounds interfere with the production of A␤. As the selected compounds induced the reduction of A␤ could also be led by A␤ degradation, we made 10 ␮M of the selected compounds and 1% DMSO in conditioned media from HEKsw cells which provided an exogenous source of A␤ and incubated with CHO cells. There was no significant alteration of A␤40 in the selected compounds versus control cells (Supplementary Table 1, 1% DMSO in conditioned media from HEKsw cells was served as control), thus exclude the possibility of regulating A␤ degrading enzymes. CPT and its analogs inhibit Aβ production not through cell death and apoptosis CPT and its analogs are known inhibitors of Nuclear DNA topoisomerase I used as anti-cancer drugs in clinical/trial [20]. However, the clinical application of CPT is limited by several factors, especially toxicity [21]. Moreover, as reported, CPT induced neuronal cell

death by DNA damage and apoptosis through caspase3 pathway [22]. To rule out the reported cytotoxicity and apoptosis effects, 100 ␮M, 10 ␮M, 1 ␮M, and 100 nM CPT, its analogs, and other selected compounds were incubated with SHSY5Ysw cells for 24 hours and tested in WST and caspase-3/7 apoptosis assay, respectively. Most of the compounds showed less than 50% inhibition of cell viability at 100 ␮M and less than 50% activation of apoptosis at 100 nM/10 ␮M (Table 2). CPT and its analogs showed no significant inhibition of cell viability in the WST (Fig. 3A). In the WST assay, 10 ␮M of STS was positive control served as 100% inhibition of cell viability and 1% DMSO was negative control designed as 0% inhibition. Meanwhile, CPT and its analogs did not induce apoptosis through caspase 3/7 pathway at both 1 ␮M and 100 nM (Fig. 3B). STS (100% activity of caspase 3/7 at 5 ␮M) and 1% DMSO (0% activity of caspase 3/7) were designed as positive and negative control in the apoptosis assay, respectively. However, Timosaponin A3 and Gossypol showed inhibition of cell viability in WST assay with IC50 of 7.4 ␮M and 22 ␮M, respectively. Roburic acid and Nitidine Chloride induced significant caspase 3/7 activity in apoptosis assay with the activation of (45 ± 3.6)% and (27 ± 4.0)% at 10 ␮M, respectively. The selected compounds inhibit Aβ production not through inhibition of BACE1 activity BACE1 is an attractive target for the development of inhibitor drugs to treat AD. It is the first protease that processes A␤PP in the pathway leading to the

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It has been reported that expression of A␤PP can be regulated by the tumor suppressor p53 [24]. Since CPT can strongly active p53, we evaluated if CPT and its analogs inhibit A␤PP expression in a p53-dependent manner. SHSY5Ysw cells were treated with different concentration of CPT or its analogs for 6 hours. Protein level of A␤PP was decreased dose-dependently (Fig. 5A, B). This effect can be abolished by knock down p53 through transfect cells with shRNA-p53 for 18 hours (Fig. 4C, D), which indicate that CPT and its analogs can decrease A␤PP expression level through active p53 protein. CPT and its analog did not inhibit Notch signaling at low concentrations

Fig. 3. Effects of CPT and its analogs on cell death and apoptosis. A, B) SHSY5Ysw cells were treated with CPT or its analogs for 24 hours then detected cell viability through WST assay (A) or caspase 3/7 activity (B). C) Primary neuron cells were treated with CPT or its analogs for 72 hours. CPT and its analogs did not induce primary neuron cell death at 0.03 ␮M or 0.3 ␮M significantly. **p10 ␮M). DAPT inhibited Notch Signaling with IC50 of 2.7 ± 0.4 ␮M. In the Notch reporter assay, 100 ␮M DAPT was positive control served as 100% inhibition of Notch signaling and 1% DMSO served as background control. As showed in Fig. 4D, there was almost no inhibition of Notch signaling at 140 nM CPT and its analogs relative to 100 ␮M DAPT. In conclusion, at low concentration, CPT

and its analogues already inhibit A␤ formation, but did not affect ␥-secretase activity. The inhibition effects on Notch signaling by other compounds were demonstrated in Table 2. CPT and its analogs inhibit Aβ42 induced IL-1β increase in microglia cells Next, we evaluated whether CPT and its analog could also reduce the pro-inflammatory cytokine

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Fig. 5. CPT and its analogs down-regulate A␤PP level in a p53-dependent manner. A, B) SHSY5Ysw cells were treated with different concentration of CPT or its analogs for 6 hours. A) Expression of A␤PP was examined by western blot. B) Band intensity was normalized to GAPDH and quantitated by densitometry. C, D) SHSY5Ysw cells were transfected with shRNA (p53) for 18 hours, then treated with 30 nM CPT or its analogs for 6 hours. C) Expression of A␤PP, p53, and p-p53 were examined by western blot. D) Band intensity was normalized to ␤-actin and quantitated by densitometry. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 compared to the control group; # p < 0.05, ## p < 0.01, ### p < 0.001 compared to the shRNA group. Data are means ± SD from 3 separate experiments.

induced by oligomeric A␤42 [18]. Here we used a human monocytic THP-1 cell line as a model for microglia to delineate the inhibitory effects of CPT and its analogues to oligomeric A␤42 induced IL1␤ secretion [27]. Oligomeric A␤40 and A␤42 could induce IL-1␤ secretion but not A␤38 and the negative control A␤42-1 at 10 ␮M concentrations. The assay was validated by control compound, triptolide, which completely inhibited A␤42 induced IL-1␤ secretion at 10 nM consistent with former report [18] (Supplementary Figure 2C). THP-1 cells were pretreated with different concentrations of CPT and its analogs for 6 hours were then incubated with 10 ␮M oligomeric A␤42 for another 3 hours. The supernatants were taken for the measurement of levels of IL-1␤. As shown in Fig. 6, pretreatment with varies concentrations of CPT and its analogs dose-dependently inhibited A␤42 induced IL-1␤ production from THP-1 cells.

DISCUSSION AD is a devastating neurological disorder that affects more than 37 million people worldwide. Current FDA-

approved drugs for AD do not prevent or reverse the disease, and provide only modest symptomatic improvements [28]. Due to the complex etiology of AD, the treatment needs to consider multifactorial nature of the disease, for this reason, use of multi-functional drugs gaining increasing attention against complex diseases [29]. Several therapeutic strategies have been developed to treat AD, including anti-inflammatory, anti-oxidant, and anti-amyloid approaches. Recently, herbal treatments have been tested in cells and animal models of AD and in clinical trials with AD subjects. In AD animal models and cell models, herbal extracts appear to have fewer adverse effects than beneficial effects on cognitive functions. These extracts have multi-functional properties (pro-cholinergic, anti-oxidant, anti-amyloid, and anti-inflammatory), and their use in the treatment of AD patients seemed to be promising. The chemical compositions of herbs and their potency for alleviating or reducing symptoms of AD or for affecting the disease mechanism need to be further studied [30]. In the present study, we screened an in-house natural products library composed of ∼500 purified chemical constituents which derived from natural products.

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Fig. 6. Effects of CPT and its analogs on A␤42 induced IL-1␤ secretion. THP-1 cells were pretreated with different concentrations of CPT and its analogs for 6 hours and then incubated with 10 ␮M oligomeric A␤42 for another 3 hours, the supernatants were taken for the measurement of levels of IL-1␤. 10 ␮M A␤42 significantly induced IL-1␤ secretion compare to control (1% DMSO treated group), CPT (A), SN-38 (B), 10-Hydroxycamptothecin (C), and 9-Methoxycamptothecin (D) inhibited A␤42 induced IL-1␤ secretion dose dependently. # p < 0.05 compared to the control group (cells treated with 1% DMSO alone served as negative control), ∗ p < 0.05 compared to 10 ␮M A␤42 treated group. Data are means ± SD from 3 separate experiments.

Among the 17 potential hits, Timosaponin A3 and Gossypol showed inhibition of cell viability in WST assay with IC50 of 7.4 ␮M and 22 ␮M, respectively. Roburic acid and Nitidine Chloride induced significant caspase 3/7 activity in apoptosis assay. Interestingly, most of the hits were anti-cancer compounds. These compounds reduced A␤42 and A␤40 and with the IC50 in a dosage from nanomolar to micromolar. Moreover these compounds showed no cytotoxicity in the cell viability assay. In addition, these compounds do not regulate the Notch signaling pathway. In the meanwhile, all the compounds did not show BACE1 activity inhibition in the kinase assay. Most importantly, the molecular weight of most the selected compounds was less than 500, indicating that these compounds might be able to cross blood-brain barrier. Among these candidates, CPT and its analogs present potent A␤ inhibition effect with IC50 value of 1∼10 nanomolar. Furthermore, the compounds also showed anti-inflammation effect in the A␤42 induced IL-1␤ assay in THP-1 cells. CPT and its analogs showed

slight effect on Notch signaling with the IC50 in the range of 1∼10 ␮M, but there are about 1000 fold higher compare to the concentrations of A␤ inhibition. CPT is a cytotoxic quinoline alkaloid which inhibits the DNA enzyme topoisomerase I (topo I). It was discovered in 1966 by M. E. Wall and M. C. Wani in systematic screening of natural products for anticancer drugs. It was isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China used as a cancer treatment in traditional Chinese medicine [31]. CPT showed remarkable anticancer activity in preliminary clinical trials but also low solubility and (high) adverse drug reaction. Numerous CPT and various derivatives were synthesized to increase the biological activity of the chemical. Topotecan and irinotecan, two CPT analogues, have been approved and are used in cancer chemotherapy [32]. Most promisingly, Pascual’s group reported that CPT activated endogenous p53 to decrease the intracellular levels of A␤PP by affecting Sp1 binding function in murine N2a␤ neuroblastoma cells and SHSY5Y cells.

J. Wang et al. / Camptothecin Reduces Production of Aβ and IL-1β

They suggested p53, the tumor suppressor could play two different and opposite functions in the neurodegenerative processes that characterize AD [24]. Results obtained from in vivo and in vitro models have previously demonstrated that p53 plays an important role in the neuronal death induced by A␤ in AD brains. It has been described that A␤42 activates the p53 promoter, thus inducing p53-dependent apoptosis and neurotoxicity [33, 34]. Interestingly, wild-type A␤PP appears to prevent neuronal apoptosis by controlling p53 activation [35]. Only when amyloidogenic fragments were generated, it contributes to post-mitotic neuron death [36]. We hypothesized that CPT and its analogs activated p53 signaling and inhibited A␤PP expression to reduce A␤ production. Activated microglial cells produce a wide spectrum of pro-inflammatory and cytotoxic factors, including TNF-␣ and IL-1␤. IL-1␤ known as a driving force in inflammatory process promotes the cascade of glial cell reactions [37]. It can promote the synthesis and processing of ␤-amyloid precursor protein and may therefore increase A␤ production and deposition in plaques and cause further glial activation [38]. In addition, IL-1␤ activates astrocytes and induces expression of several cytotoxic factors and/or A␤binding proteins, such as nitric oxide, IL-6, TNF-␣, ␣1antichymotrypsin, and apolipoprotein E [39]. More importantly, IL-1␤ induces S100␤ (a neurite growthpromoting cytokine) expression in reactive astrocytes, which might be directly responsible for dystrophic neurite growth near A␤ deposits [40]. Therefore, a pharmacological inhibition of microglial and IL1␤ production may contribute to neuroprotection. In our study, we found that CPT and its analogs pretreatment dose-dependently reduced oligomeric A␤1-42-induced increase in the levels of IL-1␤, suggesting that CPT and its analogs may serve as an anti-inflammatory drug to reduce neuronal injury. Furthermore, the inhibitory effect of CPT and its analogs on microglia activation is not due to its direct toxicity on these cells, since the doses of these compounds we used (1 ␮M to 100 nM) did not affect microglial cell viability and induce apoptosis. Harmine, a ␤-carboline alkaloid, possessed anticancer [41] and anti-nociceptive effects [42]. It is a high affinity inhibitor of the dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) protein. DYRK1A plays an important role in the phosphorylation of tau protein on multiple sites associated with tau pathology in Down’s syndrome and AD. Pharmacological inhibition of this kinase may provide an opportunity to intervene therapeutically to alter the

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onset or progression of tau pathology in AD [43, 44]. In our study, harmine showed potential inhibition of A␤42 and A␤40 production with IC50 of 750 nM and 3.3 ␮M, respectively. In the A␤ AlphaLISA screen, Cinobufagin (CBG) showed potent inhibition of A␤42 and A␤40 level with IC50 99 nM and 120 nM, respectively. However, it had no cytotoxicity at 100 ␮M and did not induce apoptosis at 10 ␮M. Resibufogenin showed inhibition of A␤42 and A␤40 with IC50 3 ␮M and 3.5 ␮M, respectively, but no cytotoxicity and apoptosis effect at 100 ␮M and 10 ␮M, respectively. CBG, a major bioactive component of the traditional Chinese medicine Chansu, is one of the most intensively investigated bufadienolides because of its natural abundance (approximately 4–6% dry weight) and potent biological activities, such as its cardiotonic, blood pressure-stimulating, local anesthetic, antimicrobial, and anticancer activities [45–47]. A recent study investigated the effects of two bufadienolides, RBG and CBG, on the outward delayed rectifier potassium current (IK) and outward transient potassium current (IA) in rat hippocampal neurons. RBG inhibited both IK and IA, whereas CBG inhibited IK without noticeable effect on IA. Moreover, at 1 ␮M concentration both RBG and CBG could alter channel kinetics and gating properties of IK. These findings suggested that IK is probably a target of bufadienolides in central nervous system [48]. Roburic acid is a component of Huo-Lou-Xiao-Lin Dan (HLXL), which is a botanical dietary supplement used to treat arthritis and related disorders. Breemen suggested the anti-inflammation of HLXL was possibly induced by the inhibition effect on COX1/COX2 activity by Roburic acid with IC50 of 5 ␮M/ 9 ␮M [49]. In our study, Roburic acid specific inhibited A␤42 with IC50 of 1.8 ␮M, however, the IC50 of A␤40 was 36 ␮M. Unfortunately, it showed potent activity in apoptosis induction with 100% inhibition at 1 ␮M. We hypothesized that the A␤ lowering effect partially depends on the apoptosis caused cell function decreasing. Digoxin is a purified cardiac glycoside and extracted from the, inhibited A␤ with the IC50 equal to ∼100 nM. The interest in digoxin has recently increased due to the expanding knowledge regarding endogenous cardiac glycosides and a potential oncological application of this drug, as reported digoxin administration was followed by an increase in the free hydrogen sulfide (H2 S) concentrations in mouse brain, heart, and kidney tissues [50]. H2 S, a crucial co-modulator of various physiological processes, is involved in the regulation of vascular tone, myocardial contractility, neurotransmission, and insulin secretion.

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H2 S deficiency was observed in various animal models of arterial and pulmonary hypertension, AD, gastric mucosal injury, and liver cirrhosis [51].

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CONCLUSIONS [8]

Above all, tremendous progress has been made in developing strategies to treat AD. Some of these strategies include anti-inflammatory, anti-amyloid, anti-oxidant, and pro-cholinergic medicines. Currently available FDA-approved drugs treat AD symptomatically and provide temporary relief from dementia. However, these drugs are frequently associated with adverse drug effects and do not cure the disease by modifying its pathology. There remains an urgent need for developing alternative approaches to AD therapeutic applications. The compounds selected from the in-house natural products library would be a promising alternative drug/tool in AD therapeutic study.

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ACKNOWLEDGMENTS [12]

This work was supported by the New Century Excellent Talents in University (NCET-12-0976), the National Science Foundation of China (No. 81073006), “Eleventh-Five Years” Supporting Programs from the Ministry of Science and Technology of China (No. 2008BAI51B01), Program for Changjiang Scholars and Innovative Research Teams in Universities (No. IRT0868). Authors’ disclosures available online (http://www.jalz.com/disclosures/view.php?id=2391).

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