Preclinical Evaluation of the Supercritical Extract of Azadirachta Indica (Neem) Leaves in Vitro and in Vivo on Inhibition of Prostate Cancer Tumor Growth

Published OnlineFirst March 27, 2014; DOI: 10.1158/1535-7163.MCT-13-0699

Molecular Cancer Small Molecule Therapeutics Therapeutics

Preclinical Evaluation of the Supercritical Extract of Azadirachta Indica (Neem) Leaves In Vitro and In Vivo on Inhibition of Prostate Cancer Tumor Growth

Qiang Wu1, Manish Kohli2, H. Robert. Bergen III3, John C. Cheville4, R. Jeffrey Karnes1, Hong Cao3, Charles Y.F. Young1, Donald J. Tindall1, Mark A. McNiven3, and Krishna Vanaja Donkena1

Abstract Azadirachta indica, commonly known as neem, has gained worldwide prominence because of its medical properties, namely antitumor, antiviral, anti-inflammatory, antihyperglycemic, antifungal, and antibacterial activities. Despite these promising results, gaps remain in our understanding of the molecular mechanism of action of neem compounds and their potential for use in clinical trials. We investigated supercritical extract of neem leaves (SENL) for the following: molecular targets in vitro, in vivo efficacy to inhibit tumor growth, and bioactive compounds that exert antitumor activity. Treatment of LNCaP-luc2 prostate cancer cells with SENL suppressed dihydrotestosterone-induced androgen receptor and prostate-specific antigen levels. SENL inhibited integrin b1, calreticulin, and focal adhesion kinase activation in LNCaP-luc2 and PC3 prostate cancer cells. Oral administration of SENL significantly reduced LNCaP-luc2 xenograft tumor growth in mice with the formation of hyalinized fibrous tumor tissue, reduction in the prostate-specific antigen, and increase in AKR1C2 levels. To identify the active anticancer compounds, we fractionated SENL by high-pressure liquid chromatography and evaluated 16 peaks for cytotoxic activity. Four of the 16 peaks exhibited significant cytotoxic activity against prostate cancer cells. Mass spectrometry of the isolated peaks suggested the compounds with cytotoxic activity were nimbandiol, nimbolide, 20,30-dihydronimbolide, and 28-deoxonimbolide. Analysis of tumor tissue and plasma samples from mice treated with SENL indicated 28-deoxonimbolide and nimbolide as the bioactive compounds. Overall, our data revealed the bioactive compounds in SENL and suggested that the anticancer activity could be mediated through alteration in androgen receptor and calreticulin levels in prostate cancer. Mol Cancer Ther; 13(5); 1067–77. 2014 AACR.

Introduction stage after the failure of androgen deprivation controls Prostate cancer is the most frequently diagnosed disease, but many of these therapies are short lived (4, 5). malignancy among men in Western society (1). Tumor This is presumably because the cancer cells develop development and progression involve multiple cellular resistance. Moreover, many therapeutics exhibit chemo- processes, including cell transformation, deregulation of toxic clinical effects (6). Despite the incorporation of new programmatic cell death, proliferation, invasion, angio- chemotherapies and novel hormonal regimens in pros- genesis, and metastasis (2). Targeting a single molecule tate cancer therapeutics (7), only an incremental increase for the treatment of cancer has shown limited promise in the response rate and median overall survival for because of the diversity of deregulated pathways in treated patients has been achieved at best, highlighting a cancer (3). The initial effect of the drugs currently need for continued expansion into investigating newer approved by the U.S. Food and Drug Administration therapeutic approaches to improve upon the medication (e.g., abiraterone and docetaxel) for castrate-resistant selection process in this tumor type. An approach to overcoming such a problem is the development of new agents that can be used in combination with existing Authors' Afﬁliations: Departments of 1Urology, 2Oncology, and 3Bio- chemotherapeutic agents yield a better result than that chemistry and Molecular Biology; and 4Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota achieved with a single chemotherapeutic agent (8). Accumulating evidence suggests that many natural pro- Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). ducts including extracts and isolated chemicals have the potential to interact with multiple targets in the network Corresponding Author: Krishna Vanaja Donkena, Department of Urology, Mayo Clinic, Guggenheim 5-01B, 200 First Street SW, Rochester, MN of pathways that support numerous molecular cascades 55905. Phone: 507-284-4226; Fax: 507-284-3757; E-mail: involved in controlling the progression of cancer (9). [email protected] Therefore, a systemic study of natural products is need- doi: 10.1158/1535-7163.MCT-13-0699 ed to deﬁne their antitumor effects and understand their 2014 American Association for Cancer Research. mechanisms of action for developing new treatments.

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Leaves from Azadirachta indica, commonly known as 49C with a dry ice and acetone bath to collect volatile neem, have been used widely in Southeast Asia for treat- oil. The process yielded approximately 5 g of the extract. ment of many diseases, including cancer (10–16). An aliquot of the extract was dissolved in dimethyl sulf- Although neem leaves have been used for treatment of oxide (DMSO) plus ethanol to make a stock solution of 200 many diseases, bioactive neem compounds and their mg/mL, as described previously (21). The final concentra- molecular basis of action have not been well studied. In tion of DMSO in the culture medium never exceeded this study, we investigated the anticancer activity of 0.01%. The effect of the extract on cell viability and HPLC supercritical extract of neem leaves (SENL) in prostate profiles described below was assessed to standardize the cancer. Supercritical fluid extraction with carbon dioxide method of extraction. We obtained consistent results with minimizes thermal and chemical degradation of com- different lots of the extract. pounds (17). The relatively low temperature of the process and the stability of CO2 allow most compounds to be Cell line and cell culture extracted with little damage or denaturing. The extraction LNCaP-luc2, originated from lymph node-derived process facilitates the collection of volatile oils from the LNCaP, is a luciferase-expressing, androgen-dependent processing material. Volatile oils reduce acute and chronic prostate cancer cell line stably transfected with the firefly inflammation and, more importantly, facilitate the luciferase gene (luc2). It was purchased from Caliper Life absorption of other bioactive compounds in humans Sciences. PC3, a bone-derived prostate cancer cell line, (18). Compounds obtained by supercritical fluid extrac- was purchased from the American Type Cell Collection tion are very stable and can be used directly in experi- (ATCC). LNCaP-luc2’s parental cell line was obtained mental diet supplementation (19, 20). from ATCC by the Caliper Life Sciences. LNCaP-luc2 and Our study was designed to evaluate the efficacy of PC3 cell lines were obtained in 2012, expanded and frozen SENL in prostate cancer and identify the compounds with down in several aliquots. Each aliquot was thawed and high antitumor activity. SENL inhibits LNCaP-luc2 and used for no more than 6 months. ATCC uses Promega PC3 prostate cancer growth. Our results demonstrated PowerPlex system to authenticate their cell lines. These that dihydrotesterone (DHT)-induced androgen receptor cell lines were not re-authenticated by our laboratory. (AR) and prostate-specific antigen (PSA) levels are sup- LNCaP-luc2 cells were cultured in RPMI 1640 medium, pressed in LNCaP-luc2 cells with SENL treatment. Oral and PC3 cells were cultured in Dulbecco’s modified Eagle administration of SENL dramatically reduced LNCaP- medium media as described previously (22). luc2 prostate cancer xenograft tumor growth in nude mice. SENL downregulates calreticulin (CALR) levels and Cell growth assay in vitro and flow cytometry inhibits focal adhesion kinase (FAK) activation in LNCaP- To determine cell growth, LNCaP-luc2 and PC3 cells luc2 and PC3 prostate cancer cells. We fractionated SENL were seeded at a density of 3 103 and 1.5 103 per well, by high-pressure liquid chromatography (HPLC) and as described (21). Cells were treated with 5 to 25 mg/mL tested the individual fractions for cytotoxic activity. HPLC of SENL, serial dilutions of HPLC fractions, 0.468 to 15.0 fractions were analyzed on a mass selective detector time- mg/mL, or the vehicle (ethanol þ DMSO) as control for 24 of-flight (MSD-TOF) system to identify the compounds hours. Cell medium was replenished and cell growth was that exhibit anticancer activity. In this study, we have determined by MTS-formazan reduction using CellTiter shown for the first time molecular targets of SENL, eval- 96 Aqueous One Solution Cell Proliferation Assay (Pro- uated the efficacy to inhibit tumor growth, and identified mega), as described (21, 23). To evaluate the mechanism of bioactive compounds of anticancer activity. cell death, LNCaP-luc2 and PC3 cells were treated with m IC50 concentrations of SENL (12 and 15 g/mL, respectively) and stained with annexin-V FITC and propidium Materials and Methods iodide (PI) according to the dead cell apoptosis kit pro- Supercritical extraction of neem leaves tocol (Life Technologies), then analyzed by flow-cytome- Supercritical extract of fresh neem leaves was prepared try (FACSCalibur, BD) as described (24). in our laboratory using the Speed SFE-2 System (Applied Separations). Fresh neem leaves were purchased from PSA and DHT determination Neem Tree Farms, a US Department of Agriculture-cer- LNCaP-luc2 cells were treated with SENL (12 mg/mL) tified organic neem farm. Supercritical fluid grade carbon in the presence or absence of 10 nmol/L DHT (Sigma- dioxide was obtained from Praxair, Inc. Supercritical CO2 Aldrich). PSA assay was performed using the superna- is a fluid state of CO2 that is held at or above its critical tants collected from LNCaP-luc2 cells after 24 hours of temperature (31.1C) and critical pressure (72.9 atm/7.39 treatment. PSA secretion was determined by ELISA PSA MPa). Neem leaves of the same age were washed with [Human] ELISA Kit; Abnova. DHT measurements were distilled water and air-dried, and 100 g of the pulverized performed in the LNCaP-luc2 cells after 8 and 24 hours of leaves were used for extraction using 300-mL vessel. The SENL treatment. Total proteins were extracted using following extraction parameters were used: 9,000 psi and RIPA buffer as described (21). Deuterated stable isotope 50C for 1-hour static and 2-hour dynamic flow of liquid (d4-DHT) was added to the protein extract as internal CO2 at 3 L/min. The collection glass vial was cooled to standard. DHT levels were measured by conventional

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liquid chromatography on a multiplexed LC System and heparinized tubes; plasma was separated and stored at analyzed on a tandem mass spectrometer equipped with 80C until used for analysis. The mice were sacrificed by an electrospray ion as described (25). CO2 inhalation; xenograft tumor tissues and the organs, including heart, lungs, liver, kidneys, and spleen, were Western blot analysis and immunofluorescence excised for fixation and hematoxylin and eosin staining or For Western blot analysis, LNCaP-luc2 and PC3 prostate stored frozen at 80C for analysis of SENL compounds. cancer cells were treated with SENL (12 and 15 mg/mL, We performed scoring of 2 sections of tumor tissues from respectively) for 8 and 24 hours in the presence and each of 8 mice in both untreated control and SENL treated absence of 10 nmol/L DHT or 50 mmol/L MG132 group for tumor fibrosis, apoptosis, necrosis, and mitotic (S2619; Selleckchem). Total proteins were extracted using activity on a scale of 0 to 3 (0, negative; 1, mild; 2, RIPA buffer, immunoblotting was performed, as des- moderate; and 3, strong). Immunohistochemistry staining cribed in ref. 26. The primary antibodies for androgen was performed using primary antibodies AKR1C2 receptor (SC-816; Santa Cruz Biotechnology), phospho- (ab170613; Abcam) and PSA (NCL-PSA-431; Leica Bio- AR (S81) (07-135; Millipore), (phospho-FAK (Y-397) systems) as described (28). Brown color was considered (ab4803; Abcam), FAK (ab131435; Abcam), integrin b1 positive staining for antibodies, and nuclear staining was (4706; Cell Signaling), CALR (2891; Cell Signaling), Rab5 denoted by blue color. (2143; Cell Signaling), and glyceraldehyde-3-phosphate For analysis of SENL compounds in plasma and tissues, dehydrogenase (GAPDH; sc-137179; Santa Cruz Biotech- we initially performed spike and recovery experiments nology, Inc.) were purchased from companies. For immu- using human plasma samples to obtain maximum recov- nofluorescence, LNCaP-luc2 and PC3 cells were grown on ery rates for selection of a suitable method for extraction. coverslips at a density of 4 104 and 3 104 cell per well, Extraction of spiked SENL from human plasma treated respectively. After treatment of cells with SENL for 24 with methanol and chloroform in 1:3:1 ratio yielded hours, fixation, permeabilization, and immunofluore- maximum recovery. Plasma and tumor tissue samples sence of the cells were performed, as described (27). collected from SENL-treated mice were analyzed for bio- Primary antibodies FAK rabbit polyclonal antibody availability of SENL compounds, and the profiles were (ab131435; Abcam) and integrin b1 mouse monoclonal compared with the vehicle-treated group as a control. antibody (610467; BD Biosciences) and secondary anti- Mouse plasma or tumor tissues were treated with meth- bodies red, Cy5-labeled goat antirabbit IgG (Alexa Fluor anol and chloroform in 1:3:1 ratio for deprotonation; after 594; Life Technologies Corp.) and green, FITC-labeled incubation at room temperature for 5 minutes, the mixture goat antimouse IgG (Alexa Fluor 488; Life Technologies was centrifuged at 3,500 rpm for 15 minutes. The extraction Corp.) were used for immunostaining. Nuclei were procedure was performed in glass tubes. The supernatant stained with 40,60-diamidino-2-phenylindole (DAPI), and was evaporated using a centrifugal evaporator at 4C, confocal images were taken on a laser scanning micro- reconstituted in methanol, and analyzed by HPLC and scope (LSM 780; Carl Zeiss Microscopy GmbH) at 100 electrospray ionization mass spectrometry (ESI-MS), as magnification. described below. HPLC profiles indicate that the variations in the extraction recoveries for mouse plasma are within Xenograft animal model 10% compared with the human plasma. All the procedures involving animals were reviewed and approved by the Mayo Clinic Institutional Animal HPLC analysis Care and Use Committee. Male 5-week-old athymic nu/nu Because SENL contains a mixture of compounds, active mice (Charles River Laboratories) were implanted subcu- anticancer compounds present in the SENL were sepa- taneously in the flank with LNCaP-luc2 cells (2 106 rated by HPLC to evaluate the effect on growth inhibitory cells/100 mL/mouse) suspended in 50% basement mem- activity (Shimadzu LC-10AT vp Pump). Separation was brane matrix (Matrigel; BD Biosciences) in RPMI medium. performed on a core-shell silica column (250 4.6 mm, Tumor dimensions were measured 2 to 3 times per week, particle size 5 mm, pore size 100 A) (Kinetex C18; Phenom- and volume was calculated as length width height enex). Column temperature was maintained at 40C. The 0.52. Once tumors reached 30 mm3, the mice were ran- autosampler injected 20 mL of sample. Mobile phase A was domly assigned to 3 groups of 8 each. Group 1 received 100% water and phase B was 100% methanol. A flow rate vehicle (olive oil; Sigma-O1514); group 2 received SENL, of 1.0 mL/min, starting with a 50-minute linear gradient 100 mg/kg body weight; and group 3 SENL, 200 mg/kg from 50% to 100% B, 50 to 56 minutes with 100% B, and 56 body weight. Animals in each group received the same to 58 minutes to 10% B, and a total run time of 60 minutes, volume of vehicle (olive oil) or SENL in 100 mL of olive oil was used with UV detection at 254 nm. Methanol and orally by gavage, 6 days per week for 9 weeks. Tumor water used for analysis were HPLC grade obtained from growth, tumor imaging, and body weights were deter- Sigma-Aldrich. mined, as described (21). At the end of the study, 2 hours before the mice were euthanized, they were given either ESI-MS analysis vehicle or SENL according to their groups, and blood was The fractions collected from HPLC were separated on a collected from each mouse via retro-orbital plexus into reversed-phase analytical column (2.1 30 mm, 3.5 mm;

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Zorbax Eclipse 300SB-C18;) on a MSD-TOF instrument LNCaP-luc2 cells were treated with proteasome inhib- (Agilent Technologies) in a positive-mode ESI, as des- itor MG132 and SENL. Immunoblotting suggests that cribed in ref. 21. The instrument was operated with mobile SENL suppresses androgen receptor levels in the pres- phase A as water and phase B as methanol, with a gradient ence of MG132 (Fig. 2B). 50% B to 100% B over 10 minutes, 100% B for 5 minutes and 5 minutes to equilibrate, and a flow rate of 300 mL/min. SENL inhibits the formation of focal adhesions in The scan range for acquisition was 300 to 1,000 counts LNCaP-luc2 and PC3 prostate cancer cells versus mass-to-charge (m/z) range; scan rate: 1 spectra/s; In our previous study (23), we showed inhibition of gas temperature 325C; gas flow 7 L/min; Nebulizer: 40 migration, and angiogenesis of human umbilical vein psi; Capillary voltage: 3,800 V; Fragmentor: 140 V; Skim- endothelial cells after treatment with ethanol extraction mer: 65 V; Octapol: 750 V. Nimbolide, purchased from of neem leaves. FAK, which is involved in endothelial BioVision Inc., and 20,30-dehydrosalannol, purchased cell proliferation, migration, and survival, is upregu- from Sigma-Aldrich, were used as standards. lated in many cancers (29). To determine whether growth inhibition of prostate cancer cells is mediated Statistical analysis through FAK modulation, we assessed FAK activation Data are presented as the mean SD for the indicated signaling in prostate cancer cells after SENL treatment. number of separate experiments. Statistical significance LNCaP-luc2 and PC3 cells were examined for integrin was tested by using the Student t test, one-way ANOVA, b1 and FAK activation (30). Western blot analysis Fisher exact test, and Kruskal–Wallis nonparametric showed that the levels of integrin b1 and phosphory- ANOVA based on ranks with a Dunn multiple compar- lation of FAK at Y-397 were reduced with SENL treat- ison test used to compare the different experimental ment, suggesting that FAK signaling was inhibited (Fig. groups. P value less than 0.05 was considered significant. 2C). Immunofluorescence for integrin b1andFAKfur- Fifty percent inhibition concentration (IC50) values were ther demonstrated the suppression of formation of the calculated by probit regression. focal adhesions after SENL treatment in LNCaP-luc2 and PC3 cells (Fig. 3). Because integrin and FAK changes may be affected by alterations in the calci- Results um-signaling protein CALR and membrane traffic-reg- SENL suppresses cell growth, induces apoptosis, and ulating protein Rab5, we examined the SENL effects on inhibits DHT-induced androgen receptor and PSA these 2 proteins. SENL downregulated CALR levels in levels in prostate cancer cells both LNCaP-luc2 and PC3 cells, whereas modest inhi- To assess anticancer activity, we investigated the cell bition of Rab5 was observed in LNCaP-luc2 compared death effects induced by SENL in LNCaP-luc2 and PC3 with PC3 cells (Fig. 2C). prostate cancer cell lines. The viability of cells treated with SENL was measured by MTS assay (23). Exposure SENL induces hyalinization of tumor tissue and of cells to SENL for 24 hours exhibited a dose-depen- inhibits the growth of human LNCaP-luc2 prostate dent inhibition of LNCaP-luc2 and PC3 cell growth. cancer xenografts in nude mice with reduction in the m LNCaP-luc2 cells exhibited IC50 of 12 g/mL, whereas PSA, and increase in the AKR1C2 levels m PC3 cells had IC50 of 15 g/mL(Fig.1A).Todetermine We next evaluated whether administration of SENL via the mechanism of death, apoptosis was evaluated with gavage exhibited anticancer activity in vivo in a xenograft annexin V/propidium iodide staining and flow cyto- tumor model. Male nude mice bearing LNCaP-luc2 xeno- metry. SENL induces apoptosis of LNCaP-luc2 and PC3 grafts were treated with SENL or vehicle control. At the cells (Fig. 1B). We next investigated the effects of SENL start of the treatments, mean LNCaP-luc2 tumor volumes on the expression levels of PSA and DHT levels in were similar in the 3 groups. Treatment with SENL sig- LNCaP-luc2 cells. PSA levels in the cell supernatants nificantly suppressed tumor growth. By 9 weeks, mean were analyzed by ELISA (21), and intracellular DHT tumor volume in the SENL-treated group (200 mg/kg levels were analyzed by liquid chromatography/mass body weight) was 189 mm3 compared with 712 mm3 in the spectrometry (LC/MS; ref. 25). Treatment with DHT control group (P 0.05; Fig. 4A and B). At the end of the induced PSA expression, whereas SENL significantly study, a significant decrease was found in tumor weights reduced DHT-induced PSA and intracellular DHT with all SENL treatments (Fig. 4C). There was no signif- levels in LNCaP-luc2 cells (Fig. 1C and D). To ascertain icant change in body weight in any of the groups after if the effect was mediated by androgen receptor regu- treatment, which suggests that oral administration of lation, Western blot analysis was performed to monitor SENL causes no major toxic effects in mice (Fig. 4D). the expression levels of androgen receptor after SENL Histologic examination revealed hyalinization and apo- treatment. DHT-induced androgen receptor Ser-81 ptosis of the tumor tissues in 7 of 8 mice treated with SENL phosphorylation and total androgen receptor expres- (200 mg/kg body weight; Fig. 5). The group mean score sion was significantly inhibited in LNCaP-luc2 cells for fibrosis, apoptosis, necrosis, and mitotic activity in afterSENLtreatment(Fig.2A).Furthermore,todelin- SENL-treated xenograft tumor tissues was 3, 2, 0, and 1, eate the mechanism of androgen receptor degradation, respectively, whereas in the vehicle control group was 0, 1,

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120 ABControl SENL (12 hours) SENL (24 hours) PC3 100 LNCaP-luc2

60 FL1-H:Annexin V-FITC FL1-H:Annexin V-FITC FL1-H:Annexin V-FITC 35 (% control) 40 30 LNCaP-luc2

Relative cell viability Relative PC3 25 20 20 15 0 10 0 5 10 15 20 25 5 SENL (μg/mL) 0

Apoptosis proportion (%) Control SENL (12 hours) SENL (24 hours) C D 500 450 8 hours 90 400 80 24 hours 350 70 60 300 50 250 40 200 30 (pg/mg protein) PSA (ng/mL) 150 20

10 DHT levels Intracellular 100 0 50

DHT (10 nmol/L) − − + + 0 DHT (10 nmol/L) − − + + − + − + SENL SENL −−+ +

Figure 1. SENL suppressed growth, induced apoptosis, and inhibited PSA and DHT levels in prostate cancer cells. A, the viability of LNCaP-luc2 and PC3 prostate cancer cells treated with SENL was evaluated by using the MTS assay after 24 hours of treatment. B, LNCaP-luc2 and PC3 prostate cancer cells treated with SENL (12 and 15 mg/mL, respectively) for 12 and 24 hours. Apoptotic changes in plasma membrane were analyzed by concurrent staining with annexin V-FITC and propidium iodide and the ﬂuorescence was measured by ﬂow cytometry. Representative FACS scan of PC3 cells from one experiment is depicted. Bar diagram represents the percentage of apoptotic proportion of cells in LNCaP-luc2 and PC3 cells. C, LNCaP-luc2 cells were treated with SENL (12 mg/mL) in the presence and absence of 10 nmol/L DHT for 24 hours. Cell supernatants were analyzed for PSA levels by ELISA. D, LNCaP-luc2 cells were treated with SENL (12 mg/mL) in the presence and absence of 10 nmol/L DHT for 8 and 24 hours. Intracellular DHT levels were measured by LC/MS. All experiments were performed in triplicate; data are expressed as the mean SD of the triplicate determinations of a representative experiment. , P < 0.05. Vehicle-treated cells were used as control.

3, and 3 respectively (0 ¼ none, 1 ¼ mild, 2 ¼ moderate, Separation of SENL compounds by HPLC and and 3 ¼ strong). More fibrosis and apoptotic activity evaluation of cytotoxic activity on prostate cancer shows more hyalinized connective tissue and regression cells in vitro of tumor in the SENL-treated mice. High mitotic activity Chromatographic separation of compounds in SENL and necrosis indicates more proliferative tumors in con- was performed using HPLC column. A methanol gradient trol mice. Immunohistochemistry analysis of SENL-trea- was used for fractionation. The HPLC profile of the SENL is ted mice tumor tissues revealed reduction in the PSA and illustrated in Supplementary Fig. S1. UV absorption of the increase in the AKR1C2 levels (Fig. 5). These data dem- compounds at 254 nm showed a total of 16 peaks. Fractions onstrate that oral administration of SENL caused regres- 1 to 11 eluted with the 70% to 80% methanol gradient at sion of the tumor tissue and inhibited tumor growth by retention time of 22 to 34 minutes were colorless, whereas promoting hyalinization and apoptosis. There was no fractions 12 to 16 eluted with the 100% methanol at 50 to 56 significant change in the histology of the heart, lungs, minutes were yellow. The fractions were concentrated at liver, kidneys, and spleen after 9 weeks of SENL treatment 4C using a centrifugal evaporator, and equal weight compared with the control group, which indicates that fractions were reconstituted in ethanol. Serial dilutions of SENL has no adverse effects on these vital organs. the fractions 0.468 to 15 mg/mL were used to determine the

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AB8 hours 24 hours Figure 2. Suppression of AR, CALR, integrin-b1 expression, and AR AR FAK activation in prostate cancer p-AR cells. A, LNCaP-luc2 cells were GAPDH treated with SENL (12 mg/mL) in (Ser81) the presence and absence of 10 GAPDH nmol/L DHT for 8 and 24 hours, SENL and AR protein levels were Control MG132 measured with specific antibodies SENL DHT SENL DHT Control Control MG132+SENL by Western blot analysis. B, DHT+SENL DHT+SENL LNCaP-luc2 cells treated with SENL (12 mg/mL) in the presence and absence of 50 mmol/L MG132 C LNCaP-luc2 PC-3 and SENL for 8 hours. AR protein p-FAK levels were measured with (Y-397) specific antibody by Western blot Total-FAK analysis. C, LNCaP-luc2 and PC3 prostate cancer cells treated with Integrin-β1 SENL (12 and 15 mg/mL, respectively) for 24 hours. Protein Calreticulin levels were measured with specific antibodies by Western Rab5 blot analysis. All experiments were performed in triplicate; vehicle- treated cells were used as control. GAPDH GAPDH was the loading control and the representative blot is shown. SENL SENL Control Control

efﬁcacy to inhibit cell growth. Cell viability was measured exhibited some reduction in the viability of cells, fractions by MTS assay after treatment of PC3 prostate cancer cells 2, 3, and 5 exhibited a stronger dose-dependent inhibition for 24 hours with the isolated fractions. Vehicle-treated of cell viability. The IC50 concentration to inhibit cell cells were included as controls. Although fractions 2 to 11 growth for fractions 2, 3, and 5 were less than 1.85 mg/mL.

LNCaP-luc2 PC3 AB Figure 3. Inhibition of FAK and integrin b1 expression in prostate cancer cells. LNCaP-luc2 and PC3 cells were treated with SENL for 24 hours, and immunoﬂuoresence was performed for FAK and integrin b1 localization. Cy5- labeled secondary antibody was

Vehicle control Vehicle used for FAK staining, FITC- labeled secondary antibody was used for integrin b1 costaining, and the nuclei were stained with DAPI. Confocal images were taken on

Treatment CD LSM 780 at 100 magniﬁcation. A and B, LNCaP-luc2 and PC3 cells treated with the vehicle control show extensions of the focal adhesions and integrin b1 expression at the focal adhesions. C and D, treatment with SENL

SENL caused cell rounding, with signiﬁcant reduction in FAK and integrin b1 expression. Arrows, FAK expression. Scale bar, 10 mm.

Red = FAK, Green = Integrin b1, Blue = DAPI

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SENL SENL Control AB200 mg/kg bw 100 mg/kg bw vehicle Figure 4. SENL inhibited the growth 1,000 of LNCaP-luc2 prostate cancer ) Vehicle 3 xenografts in nude mice. A, 800 bioluminescence imaging of mice SENL 100 mg/kg bw implanted with LNCaP-luc2 600 SENL 200 mg/kg bw tumors. Group 1 animals received vehicle control (olive oil) orally. 400 Group 2 and 3 animals were administered SENL, 100 or 200 mg/kg body weight (bw), 6 days a 200 week. A representative image of (mm volume Tumor the mice from each group at the 0 end of 9 weeks of treatment is 23456789 shown. B, tumor volume changes Weeks after inoculation of mice treated by oral gavage with 30 vehicle, and SENL, 100 or SENL CD3.5 200 mg/kg body weight, for 9 3 Vehicle 25 weeks. C, tumor weight of the mice SENL 100 mg/kg bw 2.5 20 after 9 weeks of treatment. D, body SENL 200 mg/kg bw 2 Vehicle weight of mice over the treatment 15 period. Data represent mean SD 1.5 SENL 100 mg/kg bw 10 of tumor volume, tumor weight, SENL 200 mg/kg bw 1 (g) Body weight and body weight changes of 8 (g) weight Tumor 5 mice per group. , P < 0.05. 0.5 0 0 23456789 Treatment Weeks after inoculation

Fractions 6 to 11 exhibited an IC50 concentration in the Identiﬁcation of the compounds in SENL by ESI-MS range of 7.5 to 15.0 mg/mL. Our results revealed fractions 2, analysis 3, and 5 were the most active fractions to inhibit prostate ESI-MS analysis was performed on the described HPLC cancer cell growth (Supplementary Fig. S2). fractions to identify potential active anticancer compounds

Figure 5. Histologic changes of Control SENL LNCaP-luc2 tumor tissues of mice AB treated with SENL, 200 mg/kg body weight. At the end of 9 weeks, xenograft tumor tissues were collected and stained with hematoxylin and eosin. Two H&E sections of tumor tissue from each of 8 mice in a group were examined for histologic changes. A, tumor tissue of vehicle-treated mouse showing mitotic ﬁgures (thick CD arrows), and apoptosis (thin arrows). B, tumor tissue of a SENL-treated mice shows nests of tumor cells separated by hyalinization. C and D, PSA immunohistochemistry analysis for PSA expression in the tumor tissues of vehicle- and SENL- treated mice. E and F, EF immunohistochemistry analysis for AKR1C2 expression in the tumor tissues of vehicle- and SENL-treated mice. Arrows, positive staining for PSA and AKR1C2 AKR1C2 expression. Original magniﬁcation, 400.

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Table 1. Fractionation of SENL was performed In our initial study, we evaluated the antitumor activity and of ethanol extracts of neem leaves in prostate cancer using a Kinetex C18 column on a Shimadzu models both in vitro and in vivo (21, 23). To obtain higher HPLC system extraction efficiencies of bioactive compounds, we adopted a supercritical extraction method (17). The IC Fraction 50 no. Major compounds concentration of ethanol extracts of neem leaves to inhibit PC3 cells is 25 mg/mL, whereas for SENL is 15 mg/mL. 2 Nimbandiol and nimbolide Compared with the ethanol extract of neem leaves, the 320,30-Dihydronimbolide current method of supercritical extraction showed 4 Nimbolide, 20,30-dihydronimbolide, and improved extraction efficacy. The research described in desacetylnimbin this communication is novel for the following reasons: 5 Desacetylnimbin and 28-deoxonimbolide first, it focuses on a molecular mechanism for anticancer 6 28-Deoxonimbolide and desacetylSalannin activity of the SENL. Second, it utilizes a well-established 7 DesacetylSalannin and salannin prostate cancer model for demonstration of the in vivo NOTE: Major compounds identified by mass-spectrometric efficacy. Third, it reveals several important active anti- analysis in each HPLC fraction are shown. cancer compounds in the SENL, some of which can be detected in plasma and tissues after treatment. Our data show that androgen-dependent LNCaP-luc2 in the SENL. The major compounds identified by mass cells were slightly more sensitive to SENL treatment spectrometric analysis are shown in Table 1. Mass spectro- compared with androgen-independent PC3 cells (Fig. metricanalysisofSENLfractions2,3,4,5,6,7,9,11,12,and 1A and B). This could be because of the differential 14 obtained from HPLC and the standards nimbolide and expression of genes in the cell lines (32). DHT is the most 20,30-dehydrosalannol are shown in Supplementary Fig. S3 effective androgen to activate androgen receptor func- to S6. We did not perform mass spectra for fractions 1, 8, 10, tions (33). SENL treatment significantly decreased the 13, 15, and 16 because they have not shown significant expression of DHT-induced PSA and intracellular DHT cytotoxic activity. The structures of the compounds iden- levels in LNCaP cells (Fig. 1C and D). To address the role tified in the mass spectra are presented in Supplementary of SENL in suppression of PSA and DHT levels, we Fig. S7. analyzed the modulation of DHT-induced androgen receptor expression levels. SENL significantly inhibited Bioavailability of SENL compounds in plasma and DHT-induced androgen receptor Ser 81 phosphorylation tumor tissues of mice and total androgen receptor levels in LNCaP-luc2 cells We examined the availability of SENL compounds in (Fig. 2A). To delineate the mechanism for suppression of the plasma and xenograft prostate tumor tissues of the androgen receptor levels through proteasome degrada- treated mice. The extraction recoveries for SENL with tion, we analyzed the expression of androgen receptor in methanol and chloroform were 82% to 92%. The bioavail- the presence of proteasome inhibitor MG132 (26). SENL ability of SENL compounds was assessed by ESI-MS treatment reduced androgen receptor levels in the pres- analysis of plasma and tumor tissues of SENL treated ence of MG132 (Fig. 2B), which indicates that proteasome (200 mg/kg body weight) and vehicle-treated mice. Plas- degradation pathway may not play a major role in the ma and tumor tissues pooled from 4 mice of each group SENL effects on the androgen receptor. were extracted with methanol and chloroform. HPLC was We showed previously that ethanol extracts from neem performed as described above with the plasma and tumor leaves induces AKR1C2 in prostate cancer cells and sup- tissue extracts; fraction 1 and fraction 2 was collected from presses DHT levels in xenograft prostate tumor tissues of 20 to 25 minutes and 25.01 to 30 minutes, respectively. mice (21). AKR1C2 plays a prominent role in DHT catab- Both the fractions from plasma and tissues were analyzed olism (34). We speculate that increase in the AKR1C2 for identification of the compounds by mass spectrometry levels with SENL treatment induces degradation of DHT. in duplicate. Tumor tissues and plasma of mice treated Reduction in the DHT levels affects ligand induced sta- with SENL suggested the presence of both 28-deoxonim- bility of the androgen receptor levels. The effects of SENL bolide and the sodium salt of nimbolide (Supplementary on the inhibition of androgen-dependent LNCaP-luc2 cell Fig. S8). Plasma and tumor tissues of vehicle treated group growth could be partially mediated by downregulation of have not showed any trace of UV absorption and mass the androgen-regulated pathways; however, the inhibi- spectrometric data for nimbolide and 28-deoxonimbolide. tion of androgen receptor–negative PC3 cell growth indi- Overall, these results indicate that 28-deoxonimbolide cates that androgen receptor–independent mechanisms and nimbolide are the most active bioavailable com- may be affected by SENL treatment. pounds for antitumor activity. Many studies have reported FAK as a positive regulator of tumor progression (35, 36). FAK is the primary enzyme Discussion involved in the engagement of integrins and assembly of Natural products are an important source of potential focal adhesion (29). Phosphorylation of FAK at Y-397 has cancer chemotherapeutic and antimetastatic agents (31). been shown to be elevated in tumor metastasis (37). We

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Antitumor Effects of Supercritical Extract of Neem

demonstrated that activation of FAK at Y-397 and integrin and nimbolide as the bioavailable compound after SENL b1 levels were reduced in SENL-treated cells (Fig. 2C). treatment (Supplementary Fig. S8). We anticipate that Further evidence of inactivation of FAK by SENL is SENL can mediate antitumor activity in vivo by modulat- demonstrated by our immunofluorescence staining, ing multiple pathways in tumor development and pro- which reveals inhibition in the formation of focal adhe- gression. We speculate that neem, with its low risk of sions in LNCaP-luc2 and PC3 cells (Fig. 3). These data toxicity, can be used safely for prostate cancer prevention indicate that SENL may contribute to antimigration, anti- and treatment trials. invasion, and growth inhibitory effects by suppressing Terpenoids constitute the largest class of natural com- integrin and FAK signaling. pounds for drug discovery (44–46). More than 70 terpe- CALR is a multicompartmental protein, which regu- noids have been identified in the neem plant (47, 48). To our lates many important cellular responses (38, 39). Elevated knowledge, nimbolide is the only neem terpenoid reported CALR expression promotes the migration and invasion of to have anticancer activity (49, 50). In addition to nimbolide, cancer cells (40). SENL treatment decreased CALR expres- our data reveal that the anticancer activity of other terpe- sion in LNCaP-luc2 and PC3 cells (Fig. 2C). We speculate noids from neem leaves, which includes nimbandiol, 20,30- that suppression of CALR expression with SENL partially dihydronimbolide, and 28-deoxynimbolide in prostate contributes to the inhibition of focal adhesions in prostate cancer cells (Table 1 and Supplementary Fig. S2). Therefore, cancer cells. The formation of endoplasmic reticulum these compounds can be further exploited to study their tubules in vitro requires a Rab family GTPase, Rab-5 anticancer efficacy. In summary, we have shown that (41). SENL did not substantially alter Rab-5 in LNCaP- terpenoids in SENL are active compounds for inhibition luc2 cells, whereas it decreased Rab-5 significantly in PC3 of cancer growth. Anticancer activities of SENL are medi- cells (Fig. 2C). Although the exact mechanism of SENL on ated through regulation of CALR, androgen receptor, and inhibition of prostate cancer cells is not completely under- FAK levels. We also demonstrated the in vivo therapeutic stood, it seems to entail FAK inactivation, androgen potential of SENL in a preclinical prostate cancer model. receptor downregulation, and DHT degradation path- Further studies are required to elucidate the mechanism of ways. We postulate that SENL effects are mediated action of isolated neem compounds on inhibition of tumor through CALR for the regulation of FAK required for progression individually and in combinations. tumor cell migration and metastasis. We also demonstrated that oral administration of SENL Disclosure of Potential Conflicts of Interest suppressed xenograft tumor growth. There were no sig- No potential conflicts of interest were disclosed. nificant changes in the body weight in the SENL-treated groups compared with the control group, which confirms Authors' Contributions Conception and design: M. Kohli, J.C. Cheville, C.Y.F. Young, K.V. that SENL at both 100 and 200 mg/kg body weight has no Donkena adverse physiologic effects (Fig. 4). The most significant Development of methodology: Q. Wu, H.R. Bergen III, J.C. Cheville, M.A. histologic change observed after SENL treatment in mice McNiven, K.V. Donkena Acquisition of data (provided animals, acquired and managed patients, was the formation of hyalinized tumor tissue in more than provided facilities, etc.): Q. Wu, H.R. Bergen III, K.V. Donkena 80% of the mice (200 mg/kg body weight; Fig. 5). In our Analysis and interpretation of data (e.g., statistical analysis, biostatis- previous study, we observed a similar change in the C4-2B tics, computational analysis): Q. Wu, H.R. Bergen III, K.V. Donkena Writing, review, and/or revision of the manuscript: Q. Wu, H.R. Bergen and PC3 xenograft tumor tissues of mice-treated intra- III, J.C. Cheville, C.Y.F. Young, D.J. Tindall, K.V. Donkena peritoneally with ethanol extract of neem leaves (23). Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): R.J. Karnes, K.V. Donkena Fibrous tissue formation is an indicator of decreased Study supervision: K.V. Donkena tumor invasiveness and improved tumor regression (42, 43). This histologic feature of hyalinization confirms Grant Support the tumor regression with SENL treatment. We have This work is supported by the grants awarded to K.V. Donkena by shown that SENL treatment of prostate cancer cells in American Cancer Society RSG-09-175-01-CCE and U.S. Department of vitro Defense W81XWH-09-1-0216. reduces PSA (Fig. 1C) and increases AKR1C2 levels The costs of publication of this article were defrayed in part by the (21). Immunohistochemistry analysis of SENL treated payment of page charges. This article must therefore be hereby marked mouse tumor tissues revealed reduction in the PSA and advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. increase in AKR1C2 levels, which further supports our in vitro findings (Fig. 5). Mass spectrometric analysis of Received August 20, 2013; revised January 31, 2014; accepted February tumor tissues and plasma suggests 28-deoxonimbolide 12, 2014; published OnlineFirst March 27, 2014.

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Preclinical Evaluation of the Supercritical Extract of Azadirachta Indica (Neem) Leaves In Vitro and In Vivo on Inhibition of Prostate Cancer Tumor Growth

Qiang Wu, Manish Kohli, H. Robert. Bergen III, et al.

Mol Cancer Ther 2014;13:1067-1077. Published OnlineFirst March 27, 2014.

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