Leucas aspera Nanomedicine Shows Superior Toxicityand Cell Migration Retarded in Prostate Cancer Cells

Anjusha Mohan1 & Shantikumar V. Nair1 &

Vinoth-Kumar Lakshmanan1,2

Received: 2 June 2016 /Accepted: 10 October 2016 /Published online: 3 November 2016# Springer Science+Business Media New York 2016

Abstract Prostate cancer is one of the most common malignancies among menworldwide. The main aim of the present work was to clarify the advantages of ananoformulation of ayurvedic herbal plants. Specifically, we assessed the improvedanticancer activity of Leucas aspera nanoparticles compared with methanolic crudeextract in PC3 prostate cancer cells and normal cells. L. aspera is a plant that is usedin ayurveda due to the antirheumatic, antipyretic, anti-inflammatory, antibacterial,anticancer, and cytotoxic activities. Nanoparticles of L. aspera were prepared fromplant methanolic extracts. Cytotoxic effect was studied in the normal and prostatecancer cells. Size and morphology of the formulated nanoparticles was assessed usingdynamic light scattering and scanning electron microscopy. In vitro cytotoxicity ofL. aspera nanoparticles for PC3 cells was concentration- and time-dependent. In vitrohemolysis assay, cellular uptake studies, cell aggregation studies, and cell migrationassay established the anticancerous activity of L. aspera in prostate cancer.

Keywords Leucas aspera . PC3 . Cytotoxicity . Prostate cancer . Colony assay . Therapy

Appl Biochem Biotechnol (2017) 181:1388–1400DOI 10.1007/s12010-016-2291-5

* Vinoth-Kumar Lakshmananvinoth@jnu.ac.kr; http://www.jnu.ac.kr

Anjusha Mohanresearchadmin@aims.amrita.edu

Shantikumar V. Nairshantinair@aims.amrita.edu

1 Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences andResearch Centre, Amrita Vishwa Vidyapeetham University, Kochi Campus, Kochi, Kerala 682041,India

2 Department of Biomedical Sciences, Chonnam National University Medical School, ChonnamNational University, 160 Baeksuh-Roh, Dong-Gu, Gwangju 61469, Republic of Korea

Introduction

Prostate cancer (PCa) is one of the most common age-related cancers affecting men.Prostate specific antigen (PSA) testing is the method that helps to diagnose 70 % ofPCa cases at an early curable stage [1]. In 2015, the American Cancer Societyestimated that 27,540 American men will die annually from PCa due to the progres-sion of localized diseases into metastatic, castration-resistant PCa (CRPC) [2]. PCa istypically classified in three stages—low-risk, intermediate-risk, and high-risk—due toPSA level, tumor grade, and the extent of primary tumor in the prostate gland [3].Low-risk or early-stage disease can be cured with radiotherapy or surgery; cure inhigh-risk disease was reported as about 25 % in one study [4]. Many therapeuticstudies show PCa affects patients who are resistant to hormonal therapies that restrainthe signaling of androgen receptors; the inhibited signaling drives tumor developmentand progression. Targeted therapy that induces cellular plasticity can prevent PCaprogression [5]. The main advantage for the slowed or halted progression of diseaseis for early stage diagnosis that often occurs in men in their sixth or seventh decadeof life [6, 7]. Clinical strategies like Gleason grade, serum PSA, TNM stage, surgicalmargin status, performance status, hemoglobin, and weight loss have improved theidentification of high- and low-risk stage PCa, and are useful in predicting whichtherapy is most suitable for patients [8].

Ayurvedic medicinal plants have long been used to treat many diseases including cancer. Ofthe approximately 20,000 known medicinal plants, 90 % are found wild in different climaticregions and 15 % grow in India [9]. Plant secondary metabolites like tannins, terpenoids,alkaloids, flavanoids, steroids, glycosides phenols, and volatile oils have antibacterial, antimi-crobial, cytotoxic, and other effects [10]. This therapeutic potential of the bioavailable materialhas driven the biosynthesis of nanoparticles of plant extracts [11–13]. Particle size reduction ofayurvedic preparations to the nano-scale enhances the therapeutic efficacy of plants [14–18]and different nanomedicines efficacy has been explored against prostate cancer cells [19–22].

Leucas aspera is an ayurvedic medicinal plant from the Lamiaceae family. It is commonlyknown as Thumbai and is found throughout India from the Himalayas to Ceylon. The plant isused traditionally as an antipyretic and insecticide; flowers have aperient, diaphoretic, andinsecticide activities, and the leaves are useful in relief of chronic rheumatism, psoriasis, snakebites, and other chronic skin eruptions [23]. A preliminary phytochemical study reported thatthe methanolic extract of L. aspera is enriched in glycosides, alkaloids, flavonoids, phenols,steroids, and tannins and has antifungal, antioxidant, antimicrobial, anti-inflammatory, andcytotoxic activities [24]. Phytochemicals increase the cytotoxicity of cancer chemotherapeuticdrugs [25, 26]. Flavonoids and phenolic compounds of L. aspera are efficient scavengers offree radical responsible for oxidative stress [27].

Different parts of the plant are active against different diseases. The ethanolic extract ofL. aspera flowers is active against ulcers in rats [28]. L. aspera aerial extract shows higherantitumor activity, free radical scavenging, neoangiogenesis inhibition, and macrophage stim-ulation compared with the standard drug 5-flourouracil in a mouse model of Dalton’slymphoma [29]. L. aspera root extract has antinociceptive, antioxidant, and cytotoxic activities[30].

Enhancing the anticancer activity of L. aspera extract is a worthwhile aim. The presentstudy was undertaken to assess a novel method utilizing nanoparticles and compared theactivity with crude extract in prostate cancer cells.

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Materials and Methods

Materials and Chemicals

L. aspera plants were collected from the Perumbavoor area in Ernakulam District,Kerala. The methanol extraction solvent was purchased from MERCK (India). Com-mercially acquired PC3 human prostate cancer cells (NCCS pune), Dulbecco-s mini-mum essential medium (DMEM), fetal bovine serum (FBS) were supplied byinvitrogen, and penicillin were used in cell culture. Phosphate buffered saline (PBS)was used to wash cells and remove dead cells and nutrient-exhausted medium andTrypsin for cell detachment. Tryphan was used for cell visualization using a hemo-cytometer. PC3 cells were maintained in culture flasks and well plates at 37 °C, 5 %CO2, and 85 % relative humidity for the following studies. The cell maintained in anincubator that contains.

Preparation of L. aspera Methanolic Extract and Nanoparticles

Methanolic extract of whole L. aspera was prepared. Freshly harvested plants were washedand cut into smaller pieces and dried in shade at room temperature. The samples werepowdered using a mortar and pestle. Each powdered sample was extracted using 300 mLusing soxhlet apparatus at 65 °C in 6 days. The methanol content of the extract was evaporatedusing an IKA evaporation cylinder at 55 °C after proper cooling of the extract. L. asperawholeplant extract nanoparticles were prepared by the precipitation method. Ten milligrams of themethanolic extract of L. aspera was dissolved in 1 mL of methanol using a rotary mixer. Thedissolved sample was added dropwise to 1 mL of Milli-Q water with constant stirring. HCLwas used for pH stability. The solution was centrifuged and the recovered sample waslyophilized.

Size and Surface Morphology

Dynamic light scattering (DLS) is a characteristic method used to measure averageparticle size. In DLS, the polydispersity index of the nanoparticles is measuring as theintensity of the light scattered as particles undergo Brownian motion. The size of theparticles and intensity changes are related. Nanoformulation was prepared as describedabove and material recovered after centrifugation was collected. The material wasresuspended in water and serially diluted for size, surface charge, and stabilityanalyses using a Malvern Zetasizer NanoZS. Scanning electron microscopy (SEM)was used for high-resolution surface structure examination of nanoparticles. Thediluted sample was spread over double-sided carbon tape on conductive aluminumstubs and dried. SEM was then conducted.

Fourier Transform Infrared Spectroscopy (FTIR) and Thermal GravimetricAnalysis (TGA)

FTIR was used to determine functional groups present in nanoparticles at differenttemperatures. Dried crude extract and prepared nanoparticles (4 mg) obtained afterlyophilization of L. aspera plants was loaded in the FTIR apparatus. Scan range was

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400 to 4000 cm-1 and the peak values were recorded. Thermal stability of 9 mg oflyophilized nanoparticles was analyzed by TGA a temperatures ranging from 35–700°.

In Vitro Analysis of Cell Proliferation

Analysis of cell proliferation and cytotoxicity was carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay. The yellow-coloredtetrazolium salt is reduced to purple formazan crystals by mitochondrial reductase enzyme inmetabolically active cells. PC3 cells were seeded in 96 well plates at a seeding density of4 × 105 cells per well. The cells were treated with varying concentration of the free drugs or thenano-formulations and incubated for 48 h. Cells treated with 1 % Triton X-100 served as thenegative control and untreated cells were the positive control. After incubation, 10 uL of MTTand 90 uL medium was added to each well, and the plate was incubated at 37 °C in ahumidified incubator for 4 h. of solubilization buffer (100 uL) was added to each well todissolve the purple formazan crystals into a colored solution. The plates were incubated at37 °C for 1 h. Absorbance was then measured on a PowerWave XS microplate reader at 570and 660 nm. Percentage cell viability was calculated as:

Cell viability ¼ Absorbance of sample

Absorbance of negative control� 100

Cellular Uptake

Cellular uptake of prepared nanoparticles into PC3 cells was determined by fluorescentmicroscopy. L. aspera nanoparticles were prepared with 5 μL Rhodamine dye, washed severaltimes, and lyophilized. Coverslips were etched in concentrated HCL and dried. The cells werecultured on an etched cover slip in a 24-well plate with a seeding density of 15,000 cells perwell. After 24 h, 0.25 mg/mL L. aspera and 500 μL of medium containing penestrep wasincubated for 1 h prior to use for cell treatment. After 48 h, the wells were washed with PBSand the cells were fixed with 300 μL 4 % paraformaldehyde for 20 min. After two PBSwashes, cells were exposed to 4′,6-diamidino-2-phenylindole (DAPI) for 5 min to stain cellnuclei. After two PBS washes, each cell preparation was dried overnight in the dark. The slideswere then transferred and mounted on a glass slide using DPX (sigma). After complete drying,the prepared slides were viewed by fluorescent microscopy.

Blood Cell Compatibility Assay

Hemolysis assay was used to determine the compatibility of red blood cells to the nanopar-ticles. Nanoparticle-induce rupture of erythrocytes and release of hemoglobin into the sur-rounding fluids was assessed by a hemolysis experiment. Fresh blood (10 mL) was collected ina vial containing 1.5 mL of acid citrate dextrose. To 450 μL of the blood, 50 μL of each of thedifferent concentrations (0.25, 0.125, and 0.0625 mg/mL) of the prepared nanoparticles andcrude was added. Blood samples treated with 1 % (v/v) Triton X-100 and 0.9 % saline preparedsimilarly was used as positive and negative control, respectively. All the samples wereincubated for 3 h in an incubator at 37 °C. The samples were centrifuged at 4500 rpm for10 min to obtain the plasma, which would be red in color if hemolysis had occurred. Aliquots

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(50 μL) of plasma were collected and 450 μL of 0.01 % Na2CO3 was added and mixed slowly.Aliquots (200 μL) of the samples were dispensed into wells of a 96-well plate and theabsorbance was read at 380, 415, and 450 nm. Plasma hemoglobin (Hb) concentration wasquantified and percentage of hemolysis occurred was calculated as:

Hemolysis %ð Þ ¼ plasma Hb content in sample=total Hb contentð Þ � 100

Isolation of Blood Cells

Ten milliliters of fresh blood was collected in a vial containing 1.5 mL of acid citrate dextrose.Leukocytes were isolated first from the blood. Five milliliters of blood was transferred to a50 mL Falcon tube followed by addition of 5 mL RPMI 1640 medium. The suspension wasgently mixed by inversion. The diluted blood was gently overlaid on Ficoll-Paque (GEHealthcare) in a 1:3 ratio. The material was centrifuged at 400 g for 30 min at 18–20 °C.The upper Ficoll layer was removed and the buffy coated layer containing leukocytes at theinterface were carefully collected. Fresh blood (6.5 mL) was added to another Falcon tube andcentrifuged at 2500 rpm for 10 min at 12–22 °C. The upper layer containing platelet-richplasma (PRP) was collected. After separation of PRP, 2 mL normal saline was added toremaining RBC-containing pellet and the suspension was washed by centrifugation at 400 gfor 5 min at 22–24 °C. The supernatant was removed the step was repeated two more times.RBCs were collected and diluted with normal saline in a 1:4 ratio.

Blood Cell Aggregation Treatments

Isolated cell fractions (RBCs, white blood cells [WBC], and PRP) were treated with 0.25 mg/mL concentration of L.aspera nanoparticles and crude extracts. Concentrated samples of0.25 mg/mL were prepared using normal saline. Crude extract contained 0.4 %dimethylsulfoxide (DMSO). Ninety microliters isolated blood and 10 μl of a prepared samplewere mixed. Blood cells treated with Triton X-100 or saline, respectively. Each sample was for

Fig. 1 Image of L. aspera plantextract after vaporization

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30 min at 37 °C. A drop was dispensed on a glass slide, covered with a coverslip, and viewedby phase contrast microscopy.

Scratch-Wound Assay

Cell migration assay (scratch assay) was used to determine the effect of nanoparticles in inhibitingthe cell migration process of PC3 cancer cells. PC3Cells were seeded in wells of a 6-well plate at2 × 105 cells/well and allowed to reach 80% confluency. A scratchwas drawn on the cell monolayerusing a 200μl pipette tip. The plate was washed twice with PBS to remove floating cells and debris.The adherent cells were treatedwith 0.0625 or 0.25mg/mLof crude extract and the nanoformulationof the methanolic extract of L. aspera. Positive control was treated with medium alone. The woundwas photographed at 0, 4, 8, 24, 30, and 48 h using amicroscope fittedwith digital camera. Areawascalculated from a randomly selected field using ImageJ software and wound recovery by migratingcells was determined in percentage of wound closure by following equation:

wound closure %ð Þ ¼ Af=A0ð Þ � 100

where Af is the area at the end point and A0 is the area at the time zero.

Fig. 2 a DLS histogram showing the size distribution of L. aspera nanoparticles. The average size ranged from200–400 nm. b SEM image of surface morphology of L. aspera nanoparticles

Fig. 3 a FTIR data of L. aspera crude extract and nanoparticles. b TGA analysis of L. aspera crude extract andnanoparticles

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Results and Discussion

Plant Extraction and Nanoparticle Formulation

The main aim of this study was to develop a nanoformulation of L. aspera to enhancethe efficacy of PCa treatment. Figure 1 displays part of the novel process.

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Fig. 4 a Images of PC3 cells treated for 48 h with 0.0625, 0.125, and 0.25 mg/mL of L. aspera crude extract orextract and nanoparticles. (b and c) Cytoxicity assay analysis of L. aspera nanoparticles and crude extract at 24and 48 h

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Nanoparticle Characterization

DLS analysis examined the size distribution of L. aspera nanoparticles. The peakvalue represents 220 nm range in 100 % intensity. The average size of nanoparticlesrange from 200 to 400 nm (Fig. 2a). Zetapotential distribution of the nanoparticleswas −22 mv. SEM revealed the spherical shape of the nanoparticles (Fig. 2b) aspherical shape.

FTIR and TGA

Comparison of the FTIR spectra of nanoparticles appended methanolic extract andextract alone did not show appreciable differences (Fig. 3a). Significant losses ofcomponents were evident in both spectra, perhaps reflecting bondstretching in thefunctional groups. The functional groups represented phytochemical compounds in-cluding glycosides, alkaloids, flavonoids, phenols, steroids, and tannins. TGA analysisconfirmed the thermal stability of both preparations. Crude extract degraded fasterthan nanoparticles.

Fig. 5 Images of cellular uptake of L. aspera nanoparticles in PC3 cells. Magnifications are 20× and 60×

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Anticancer Activity of L. aspera Nanoparticles versus Crude Extract in PC3 Cells

To determine the anticancer efficacy of L. aspera nanoparticles compared with crudeextract in PC3 PCa cells, the seeded PC3cells were treated with different concentra-tion of nanoparticles PC3for 24 and 48 h. The stock solution of nanoparticles crudeextract was serially diluted to reach concentrations of 1, 0.5, 0.25, and 0.125 mg/mL.After 48 h, cells were treated with a combination of nanoparticles and DMSO.Nanoparticles were cytotoxic in a concentration-dependent manner (Figs. 4a, b).

Cellular Uptake

The cellular uptake of prepared nanoparticles was observed using fluorescent microscopy.L. aspera extract showed slight autofluorescence when hodamine dye was incubated withextract after nanoparticle preparation. Excess dye in the pellet was removed by PBS wash. PC3cells were treated with 0.25 mg/mL nanoparticles and compared with the positive control.Microscopy images showed that prepared nanoparticles were easily taken up by the cellswithout any dose dependence (Fig. 5).

Fig. 6 Images of hemocompatibility of L. aspera. a Photographic of hemolysis assay. b Graphical image of thesame experiment

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Blood Cell Compatibility

Compatibility of L. aspera nanoparticles in different concentrations was evaluatedagainst the controls. Figure 6a plasma separation from whole blood after thetreatment protocol. The percentage of hemolysis after 3 h incubation with 0.25,0.125, and 0.0625 mg/mL L. aspera nanoparticles and crude extract is shown inFig. 6b.

Fig. 7 Images of blood cell aggregation studies. a Isolation of leukocytes by buffy coated layer. bPRP, RBC’s, and WBC’s were treated with 0.25 mg/mL nanoparticles Nps and crude extract whencombined with control

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Blood Cell Aggregation

RBCs, leukocytes, and PRP were isolated from blood and treated. Cell fractions treated withcrude extract appeared crowded compared with fractions exposed to nanoparticles (Fig. 7).

Antimigratory Effect of L. aspera Treated PC3 Cells

The antimigratory effect of L. aspera nanoparticles and crude extract was higher. After 30 hincubation, cells were fully confluent. The cell morphology was changed with evidence of

Fig. 8 a Photographs of migration of L. aspera crude and nanoparticle treated PC3 cells compared with positivecontrol. b Graph of percent wound closure

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stress visible after 24 h incubation (Fig. 8a). After 30 h, cells treated with L. aspera nanopar-ticles had died and detached from the plate at the time of PBS wash. Therefore, the area ofwound was increased and the graph expresses negative value at 30 h incubation. At prolongedincubation, wound closure was <30 %. L. aspera treated cells migrated 60 % less than controlafter 30 h incubation (Fig. 8b).

Conclusions

Stable nanoparticles of L. aspera were prepared by nanoprecipitation with methanol. Charac-terization of the nanoparticles was done. FTIR values represented the functional groups of thenanoparticles and crude extract and TGA analysis data showed the thermal stability ofL. aspera nanoparticles were higher than crude extract. The anticancer activity or cytotoxicityof L. aspera nanoparticles was studied in PC3 cancer cells. Nanoparticles induced significantcytotoxicity compared with crude extract. Uptake of L. aspera nanoparticles by PC3 cells.Hemolysis was not evident in L. aspera treated blood, but was in Triton X-100 treated blood.Treated PRP, RBCs, andWBCs did not aggregate. Nanoparticles retarded cell migration. It canbe concluded that the nanoparticles containing L. aspera extract are more cytotoxic to PC3cells compared to crude extract.

Acknowledgments Vinoth-Kumar Lakshmanan is grateful to Department of Science and Technology, India, forproviding the fund and guidelines to this work. Authors also acknowledge the financial support in the form of aM-Tech grant from Department of Science and Technology. We thank lab members K.S. Snima, PratheekshaPillai, Sajin Ravi and Cochin University of Science and Technology for help in guidelines, and the SEM, DLS,and TGA procedures.

Compliance with Ethical Standards

Conflict of Interest The authors declare that they have no conflict of interest.

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