production, hplc analysis, and in situ apoptotic activities of...

Production, HPLC Analysis, and In Situ Apoptotic Activities of Swainsonine

Toward Lepidopteran, Sf-21 Cell Line

Digar Singh and Gurvinder KaurDept. of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781 039, Assam, India

DOI 10.1002/btpr.1943Published online July 25, 2014 in Wiley Online Library (wileyonlinelibrary.com)

Swainsonine, a secondary metabolite from Metarhizium anisopliae has been extensivelystudied in the complementary areas of therapeutics and toxicology. This work aims todevelop a simple UV-HPLC method of analyses for swainsonine in Metarhizium fermentationbroth and to explore its in situ entomotoxic activities. The partially purified broth was quan-titatively analyzed using middle UV (205 nm)-reverse phase HPLC method with differentmobile phases and gradient programmes. Swainsonine was eluted as single peak at (te)6.0–6.9 min with average concentration of 4.04 6 0.52 lg/mL using optimal mobile phase(0.1% trifluoroacetic acid in water and acetonitrile). The mass spectrometry analysis furtherindicated the characteristic MS1 species for swainsonine, [M1H]1 174.30 in correspondingHPLC peaks. The antiproliferative effects of swainsonine on lepidopteran, Sf-21 cellswere determined through 3-(4, 5-dimethylthia-zol-2-yl)22, 5-diphenyl tetrazolium bromide(IC50 standard 5 3.90 lM and IC50 purified 5 5.27 lM) and trypan blue dye exclusion(IC50 standard 5 6.91 lM and IC50 purified 5 8.67 lM) assays. The fluorescence activated cellsorting evaluation of Sf-21 cells showed nearly 35% and 42% of population in variousapoptotic stages at 36 h, when treated with standard and purified swainsonine, respectively.The morphodimensional field emission scanning electron and atomic force microscopic anal-yses further confirmed the characteristic apoptotic features like membrane blebbings, rup-tures and volume shrinkage in the lepidopteran cells after 24–36 h of post-treatmentincubation. The study describes the potential entomotoxic activities of swainsonine and itsrole in the virulence of Metarhizium spp. VC 2014 American Institute of Chemical EngineersBiotechnol. Prog., 30:1196–1205, 2014Keywords: swainsonine, middle-UV HPLC, entomotoxicity, apoptosis, ultra-morphodimensions

Introduction

Metarhizium spp. has been extensively studied for its inva-sive entomopathogenic activities. The various strains areknown to produce an array of bioactive compounds essentialto its virulence such as chitinases,1 destruxins,2 cytochala-sins, viridoxins, fungerins, metacytofilin, and swainsonine3.Swainsonine is a trihydroxy indolizidine alkaloid with poten-tial anticancer, antiproliferative, and immunosuppressiveactivities.4

The extraction of polyhydroxy alkaloids with polar sol-vents (ethanol, methanol, or water) results in coextraction ofbroth components like glucose, amino acids, and other suchhydrophilic metabolites.5 There are numerous other methodsreported for analysis of swainsonine including enzymatic,6

liquid-chromatographic using evaporative light scattering,7

pulsed amperometric,8 and mass-based detectors.9 However,the above discussed methods are very expensive and gener-ally inaccessible. Although, the readily available UV-HPLCsuffers some limitations on account of the chromophoregroups in alkaloids, it has the best combination of sensitivity,

linearity, and reliability. Moreover, alkaloids with weak orno chromophore groups can also be analyzed at middle-UV(MUV) range �200 nm. However, few mobile phase impur-ities may interfere with the signals, which needs to be prop-erly corrected.10

Swainsonine is a well characterized Golgi a-mannosidase-II inhibitor that blocks the synthesis of complex type oligo-saccharides in N-glycan biosynthesis pathway, necessary forcancer and insect cell progressions.11,12 The antiproliferativeactivity of swainsonine is also coupled with cytotoxicity,inhibition of metastasis, immunomodulation, and collusiveenhancement in the activity of chemotherapeutic drugsagainst cancer progressions.13 Although many studies havedescribed the therapeutic potentials of swainsonine, itsentomotoxic applications on account of entomopathogenic(Metarhizium) origin are still unexplored. The secondarymetabolites from entomopathogenic fungus Beauveria bassi-ana are recently been described to induce apoptotic effectsin certain pest species, both in situ and ex situ.14 The ento-motoxic effects of various fungal lectins of Rhizoctonia sp.and Nomuraea15 conditioned media16 against lepidopterans,have revealed the entirely new paradigm of modern biopesti-cides and their role in environment, health and agriculture.The lepidopteran cell line, Sf-21 is very often used to assessthe toxicological aspects of the novel compounds with

D. Singh and G. Kaur contributed equally to the work.Correspondence concerning this article should be addressed to:

G. Kaur at [email protected].

1196 VC 2014 American Institute of Chemical Engineers

entomopathogenic origin on account of its marked sensitivitytoward the higher expression of characteristic apoptoticgenes viz., Sf-procaspases and Sf-caspases.16 This study isfocused on the development of simple and sensitiveUV-HPLC analytical method for swainsonine and to deter-mine its in situ entomotoxic mechanism of action againstlepidopteran cell line.

Materials and Methods

Microorganism and cultivation conditions

Metarhizium anisopliae ARSEF 1724 was procured fromARS Collection of Entomopathogenic Fungal Cultures(ARSEF) USDA, Ithaca, New York and maintained inSabauraud dextrose agar slants at 4�C.

Standards, chemicals, and reagents

Swainsonine standards were purchased from Sigma-Aldrich.The 1 mg of standard swainsonine crystals were dissolved in1 mL of HPLC grade water to prepare stock (5.78 mM) andworking (1 lg/mL 5 5.78 lM) standard solutions as per theexperimental requirements. All standards were then stored at2–8�C for 3–6 months. All HPLC grade solvents, for example,acetonitrile, methanol, and trifluoroacetic acid (TFA) wereobtained from Merck, Germany. The assay enzymes “a-D-mannosidase” from jack bean, the substrate “p-nitrophenyl-a-D-mannopyranoside,” and L-glutathione reduced were allobtained from Sigma-Aldrich. The cell culture-related materi-als were obtained from Sigma-Aldrich. The ingredientsrequired for culture maintenance and swainsonine productionmedia were all purchased from Hi-Media, India.

Swainsonine production, solvent extraction, andenzymatic quantification

Swainsonine production was carried out in 250 mL Erlen-meyer flasks in 10 replicates using complex oatmeal based-genetic algorithm optimized medium.17 The culture wasmaintained at 28�C, 180 rpm, and initial pH of 5.5 in 250 mLErlenmeyer flask. Swainsonine was solvent extracted from the72 h fermentation broth using acetone based cold precipitationmethod.9 The swainsonine titers in the partially purified brothsamples were determined using a-mannosidase inhibitionassay.6

Experimental and blank samples in HPLC calibration

Experimental sample (extracted swainsonine) was purifiedfrom 72 h Metarhizium fermentation broth using acetone pre-cipitation method.9 The HPLC grade water was maintainedas blank for chromatographic runs. The low range (2–8 lg/mL)HPLC standard curve was prepared using swainsonine standardsolutions in triplicates.

Analytical HPLC instrumentation andchromatographic conditions

The partially purified broth fractions were analyzed quali-tatively and quantitatively for swainsonine titers using theVarian Prostar HPLC system. The system was equipped witha binary pump, UV-visible detector, and a 20 lL injectionloop. Hypersil BDS RP-C18 column (Thermo, Waltham,MA) of dimensions 250 3 4.6 mm, 5 A� particle sizes was

used for chromatographic separation. The eluted sampleswere detected at MUV wavelength, 205 nm.18 Samples(crude and standard) were filtered through 0.22 lm cellulosenitrate membrane prior to their injection into the analyticalcolumn. Three different mobile phases (pH 5 5.5), (1) 5%methanol in ammonium acetate buffer (Solvent-A) and water(Solvent-B), (2) Acetonitrile (Solvent-A) and water (Solvent-B), and (3) Acetonitrile (solvent-A) and 0.1% TFA in water(Solvent-B), were evaluated for chromatographic resolutionof swainsonine. The gradient programme employed for HPLCoperations involved: 100% of Solvent-B from 0–3 min (flowrate 1 mL/min) and 100% of solvent-A from 3 to 10 min(flow rates 0.5–0.25 mL/min). The partially purified broth wassubjected to preparatory liquid chromatography-mass spec-trometry (LC-MS) procedure for higher recovery of purifiedswainsonine as described in earlier reports.9

Mass spectrometry instrumentation and analysisof HPLC fractions

Electrospray ionization-time of flight mass spectrometry(ESI-TOF MS) detection was carried out on Waters quadru-pole time-of-flight premier mass spectrometer with microchan-nel plate detector (Waters) in positive ion mode. TheESI-TOF MS ion source parameters were adjusted for sourceand desolvation gas temperatures at 100�C and 250�C, respec-tively. The sampling, capillary, and extraction cone voltageswere set at 35 V, 3 KV, and 3 V, respectively, with ion guideat 1 V. The flow injection rate was fixed at 20 lL/min.The MS data was obtained in full scan mode (mass range100–1,000 amu) with MassLynx analytical software.

Cell culture and treatments

An early passage Spodoptera frugiperda, ovarian cells(Sf221) were procured from National Centre for Cell Scien-ces Pune, India. These cells were cultured routinely in T-25and T-75 flasks (BD biosciences) using TNM-FH mediumsupplemented with 10% heat inactivated fetal bovine serum.The cells were maintained at 28�C in a non-humidified andnon CO2 incubator.

Cytotoxicity assays

The standard and purified swainsonine samples weretested for their cytotoxic effects on Sf-21 cells at variableconcentrations (2.0–10.0 lM) for 36 h with appropriate sol-vent controls. A 100 lL of 1 3 105 cells/mL were seededinto 96 well microtiter plates with 100 lL of swainsonine ofvarying concentrations in complete growth media. Thetreated and control cells were analyzed for swainsonineinduced cytotoxic effects as follows;

Cell Viability Assay. Cell viability was determined by acolorimetric 3-(4, 5-dimethylthia-zol-2-yl)22, 5-diphenyltetrazolium bromide (MTT) assay. The experimental designand data analysis were performed according to the modifiedprotocol.9

Cell Death Assay. The cytotoxic effect of swainsonineon Sf-21 cells was determined using the trypan blue (TB)dye exclusion assay. At the stipulated time point, the cellsuspension was treated with TB dye (1:1) and counted underthe inverted microscope using a hemocytometer. The cyto-toxic effects of swainsonine were expressed as the mean

Biotechnol. Prog., 2014, Vol. 30, No. 5 1197

percentage of dead cells in each treatment group from threeindependent experiments.

Fluorescence activated cell sorting analysis for apoptosis

Swainsonine (standard and purified) treated Sf-21 cellswere fluorescence activated cell sorting (FACS) analyzedusing AnnexinV–FITC and propidium iodide (PI) apoptosisdetection kit (Sigma-Aldrich) following the manufacturer’s

instruction. The control and swainsonine-treated (IC50 con-centrations of standard and purified), 1 3 105 of Sf-21 cells/mL were analyzed at 12 h interval for 36 h. The cells werethen harvested, washed in PBS, and incubated with fluores-cent dyes mixture - Annexin V-FITC (5 lL) and PI (10 lL)in 13 binding buffer (250 lL) for 10 min under dark androom temperature conditions. The stained cells were ana-lyzed using FACS Calibur instrument (BD Biosciences)equipped with CellQuest 3.3 software at 12 h interval. The

Figure 1. HPLC chromatogram for partially purified broth samples for chromatographic resolution of major peaks with mobile phase(a) 5 % methanol in ammonium acetate buffer (Solvent-A) and water (Solvent-B), (b) Acetonitrile (Solvent-A) and water(Solvent-B), and (c) Acetonitrile (solvent-A) and 0.1 % TFA in water (Solvent-B), at MUV detection wavelength of 205 nm.

1198 Biotechnol. Prog., 2014, Vol. 30, No. 5

fluorophore excitation was performed at wavelength of488 nm with emission filters FL-1 (515–535 nm, green) andFL-3 (635–665 nm, red). A minimum of 50 3 103 cells(events) were analyzed for statistical data analysis. The earlyapoptotic cells stained with FITC (green fluorescence) andthe late apoptotic cells stained with both FITC and PI (red-green fluorescence) are represented in the lower right andupper right quadrants, respectively, in the FACS histogram.

Microscopic analysis

An approximate 5 3 105 cells (control and treated) wereseeded in each well of six-well culture plates in duplicateand treated with appropriate concentration of swainsonine(IC50 concentrations of standard and purified). The cellswere observed under inverted light microscope (Nikon TS100-F, Japan) at regular time intervals, centrifuged (300g),and finally washed twice with PBS to proceed further ineach case as follows;

Field Emission-Scanning Electron Microscopy. Theultra-morphology of the swainsonine treated and control cellswere examined using field emission scanning electronmicroscopy (Carl Zeiss, Germany). The samples for fieldemission-scanning electron microscopy (FE-SEM) analysiswere prepared using the earlier reported methods.19 Thesamples were finally sputter-coated with gold film using asputter coater (SC7620“Mini,” Polaron Sputter Coater,Quorum Technologies, England) before FE-SEM analysis.

Atomic Force Microscopy. The multimode atomic forcemicroscopy (AFM) equipped with ambient air scanningprobe microscope (Agilent Technologies 5500) was used forthe ultra-morphodimensional analysis of swainsonine treatedand control cells. Images were scanned using the Picoscan25 software in non contact mode. Topography AFM imageswere collected in tapping mode using the rms amplitude ofthe cantilever as feedback signal operating in a saline liquidbuffer. The silicon nitride probes were used with nominal

Figure 2. HPLC chromatograms and corresponding mass spectrum (inset) for the major peaks collected (a) swainsonine standard(8 lg/mL) eluted at flow rates, 1 mL/min from 0 to 5 min and 0.5 mL/min from 5 to 10 min (b) partially purified brothfractions eluted at flow rates, 1 mL/min from 0 to 5 min and 0.5 mL/min from 5 to 10 min, and (c) partially purifiedbroth fraction eluted at flow rates, 1 mL/min from 0 to 5 min and 0.25 mL/min from 5 to 10 min, at MUV detectionwavelength of 205 nm.


spring constant around 2.5 N/m (NT-MDT, Russia) and can-tilever length of 120 lm. The cantilever resonance frequencywas about 30 kHz. The rms free amplitude of the cantileverwas on the order of 15 nm and the relative set-point above95% of the free amplitude. Images were recorded with aslow scan rate (below 1 Hz) and a resolution of 512 3 512pixel per image was chosen.

Statistical analysis

One-way ANOVA followed by Dunnett’s test of pairwisemultiple comparisons was used for MTT and TB dye exclu-sion cell death data analysis. Cell viability plots were fitted inSPSS version 11 (Science, Chicago, IL) with an exponentialdecay-linear combination equation to generate graphs. Eachdata point represented mean of multiple wells. The P� 0.05was considered to be statistically significant in all cases.

Results

Production and partial purification of swainsonine

The Metarhizium fermentation broth (72 h) was subjectedto acetone-methanol cold extraction procedure whichresulted in 2.5 fold purification of swainsonine. The shakeflask production of swainsonine was determined to be3.87 6 0.45 lg/mL using a-mannosidase inhibition assays.

Quantitative HPLC analysis of swainsonine

The HPLC analysis of swainsonine was performed at anoptimized MUV- detection wavelength of 205 nm. The threedifferent mobile phases with gradient proportions (program)were optimized for maximum peak resolution and minimalsignal-to-noise ratio for chromatographic analysis of swain-sonine (Figure 1). The optimal results were obtained withgradient elution of swainsonine between te 5 5.6–6.4 min,using- acetonitrile (solvent-A) and 0.1% TFA in water (Sol-vent-B) as mobile phase (Figure 1c). The chromatographicseparation of swainsonine was further calibrated by reducingthe flow rates to 0.25 mL/min from 5.0 to 10.0 min, to

resolve the two separate peaks between te 5 5.2–7.0 min.Hence, two separate peaks were resolved at te 5 6.0–6.9 min(comparable to the elution time for swainsonine standard)and an unknown peak between 7.2 and 8.2 min (Figure 2).The corresponding peak fractions were further characterizedusing mass spectrometry analysis, with corresponding MS1fraction -[M]1 173.04, [M1H]1 174.30 and [M12H]1

175.04 for swainsonine (insets of Figures 2a–c). The HPLCquantification of swainsonine was performed using the stand-ard curve between swainsonine concentrations (lg/mL) andpeak area (mAU.min) with regression equation

Y54:50X144:94

where Y is peak area for swainsonine, X is swainsonine con-centration with correlation coefficient R of 0.98 (data notshown). The average swainsonine concentration was deter-mined to be 3.98 6 0.43 lg/mL. The preparatory LC-MSanalysis of partially purified broth resulted in swainsoninefractions of high purity for all the cell culture treatments(data not shown).

Method validations for MUV-HPLC analysis ofswainsonine

The optimized MUV-HPLC method was further validatedfor certain validation criteria viz., linearity, precision, limitsof detection (LOD), accuracy, and method robustness.

Linearity. The method showed a significant linear rela-tionship between the middle UV (205 nm) absorption(mAU) and swainsonine concentrations (2–8 lg/mL). A linearregression curve was observed with correlation coefficientR 5 0.98 in triplicate HPLC runs.

Precision. The intra-run precision was determined by theevaluation of relative standard deviation (RSD) value for theconsecutive chromatographic runs for swainsonine (8 lg/mL)in triplicates. The RSD values for the peak area and retentiontime were 3.72% and 2.28% (n 5 3), respectively.

LOD. The LOD is defined as the lowest concentration ofswainsonine that gave an average signal to noise ratiogreater than 3 over the three replicate HPLC runs. The LODfor swainsonine was 0.50 lg/mL.

Method Accuracy (Inter- and Intra-Method). The inter-method accuracy for the MUV-HPLC method was evaluatedby comparing the swainsonine concentration determinedfrom the enzymatic assay (mannosidase inhibition assay)method, with the low RSD value of 1.98%. The intra-method accuracy was alternatively assessed by spiking thebroth samples with known titers of standard swainsonine (5lg/mL) and their subsequent quantitative MUV-HPLC analy-sis. The observed HPLC determined concentration was9.23 6 0.22 lg/mL (triplicate runs) against the expected con-centration of 8.98 lg/mL (5 lg/mL added 1 3.98 lg/mL inbroth) with 105% calculated recovery.

Stability. The stability of method was examined by theanalysis of swainsonine samples (standard and purified)stored at 4�C for 3 months. There was no significant differ-ence observed in the average elution time, peak area, andabsorbance units for swainsonine.

Cytotoxicity analysis of swainsonine with Sf-21 cells

The in situ cytotoxic evaluation of swainsonine towardSf-21 cells was performed with MTT-cell viability and

Figure 3. MTT cell proliferation and TB dye exclusion celldeath assays for Sf-21 cells measured at 36 h withdifferent doses of standard and purified swainsonine.One-way ANOVA at *P< 0.001 and Dunnett’s pairwise multiple comparisons with control groups indi-cated that swainsonine affected Sf-21 cell prolifera-tion and cell death in a dose dependent fashion.


TB-cell death assays. The MTT determined IC50 valuesdetermined for standard and preparatory-LC purified frac-tions were 3.90 lM and 5.27 lM, respectively. However, theTB-assay showed slightly higher IC50 values of 6.91 lM and8.67 lM, respectively, for standard and purified swainsonine(Figure 3). All cytotoxicity evaluations were expressed forthe data observed at 36 h of post treatment incubation.

FACS analysis of swainsonine induced apoptosisin Sf-21 cells

The control and swainsonine-treated Sf-21 cells were ana-lyzed for 36 h post-treatment incubation at 12 h interval.The FACS quadrant statistics showed the swainsonine-treated Sf221cells in various stages of cell death only after24 and 36 h of incubations. The standard swainsonine-treated Sf-21 cells showed �28.04% (apoptotic) - 1.47%(necrotic) and 24.05% (apoptotic) - 11.46% (necrotic) of the

cell population in various death phases at 24 and 36 h,respectively. However, the higher rates of cell damage wereobserved upon treatment with purified swainsonine with�11.38% (apoptotic) - 1.47% (necrotic) at 24 h and 25.26%(apoptotic) - 17.15% (necrotic) of the cell population at 36h, respectively, in different phases of cell death (Figure 4).

Morphological analysis of swainsonine inducedapoptosis in Sf-21 cells

The morphological analysis of swainsonine treated cellsshowed characteristic apoptotic features at 24–36 h of posttreatment incubation. The FE-SEM examination of thetreated cells revealed the characteristic membrane blebbing,apoptotic bodies, condensed cytoplasm, and cell lysis at 36 h(Figure 5). The cell membrane probation using AFMrevealed the marked abrasions on cell surface and significantreduction in cell dimensions. The average height and average

Figure 4. FACS analysis of swainsonine (standard and purified) induced apoptosis in Sf-21 cells for 36 h of post-treatment incuba-tions. The data was representative for three different experiments at P < 0.05.


size (diameter) of swainsonine-treated Sf-21 cell populationwas reduced significantly by 4 lm in length and diameter(height and size were from 13 to 8 lm and 6 to 4 lm,respectively) as shown in Figure 6. The ultramicroscopicexaminations of swainsonine-treated cell morphologiesshowed severely damaged and punctured cell membranescompared to the control cells.

Discussion

Swainsonine production was preliminarily reported fromMetarhizium strains in the complex oatmeal based, GA-optimized production media under shake flask conditions.17

The alkaloid production was carried out under shake flaskconditions to perform the analytical and in situ studies. Thisstudy describes the simple and sensitive method for theHPLC analysis of swainsonine at MUV range of 205 nm.The three different mobile phases and a gradient HPLC pro-gram was optimized for the best resolution of swainsoninepeak in chromatograms. A number of chromatographic tech-niques including LC,7,8 gas chromatography (GC),5 and ionexchange chromatography (IEC)20 have been reported forthe analysis of swainsonine. However, these methods withappreciable sensitivity are either expensive (LCMS), requiresthe complex derivatization steps (GC) or destructive (GCand IEC). In contrast, the UV-HPLC method offers a simple,rapid, and high throughput alternative excluding the complex

procedures of sample preparation and derivatization in a nondestructive manner. Natural products with double bonds orunpaired electrons can be detected at short UV wavelengthsof �200 nm.10 Hence, a MUV-HPLC method at detectionwavelength (205 nm) was considered in the present study forthe analysis of swainsonine in Metarhizium broth. The par-tially purified broth fractions were HPLC analyzed using dif-ferent mobile phases (solvent systems) and gradient runswith variable flow rates (Figure 1). The mobile phase (1) 5%methanol in ammonium acetate buffer (Solvent-A) and water(Solvent-B), resulted in a major peak between 2.12 and 2.65min with two minor peaks thereafter between elution time5.2–5.8 min (Figure 1a). The elution of the major peakbefore 3 min of initial run of water (solvent-B) might haveresulted in the poor chromatographic resolution of swainso-nine due to its extreme hydrophilicity and polarity as alsoreported by Stockigt et al.21 Therefore, swainsonine elutionwas further optimized using mobile phases (2) Acetonitrile(Solvent-A) and water (Solvent-B), and (3) Acetonitrile (sol-vent-A) and 0.1% TFA in water (Solvent-B), which resulteda major chromatographic peak between elution time 5.59–6.13 min (Figure 1b) and 5.85–6.23 min (Figure 1c), respec-tively. The chromatographic separation of major peaks inpartially purified broth fractions was further confirmed withstandard swainsonine runs with elution time 5.59–6.5 min(Figure 2a) followed by ESI-TOF-MS analysis of the HPLCcollected fractions (Figure 2). The major chromatographicpeaks in broth fractions were further resolved by reducing

Figure 5. Ultra-morphological FE-SEM analysis of the control and swainsonine treated (standard and purified), Sf-21 cells at 0 h,24 h, and 36 h post-treatment incubations at 15 KX magnification.


the flow rates to 0.25 min between te 5 and 10 min (Figure2c). The MS characterization of the HPLC fractions providedthe sensitive analysis method for swainsonine in Meta-rhizium fermentation broth. The characteristic MS1 [M1H]1

174 was observed in both standard and purified samples,confirming the presence of swainsonine.

The standard and preparative LC- purified swainsoninefractions were further tested for their entomotoxic activity.Swainsonine has recently been reported to exhibit apoptoticactivities in human lung cancer (A-549) and esophagealsquamous cell lines through mitochondrial mediated apopto-tic pathways.4,22 To the best of our knowledge, the in situ orin vivo entomotoxic properties of swainsonine have not beenreported previously. This study describes the first systematiccharacterization of the in situ induction of cell death mecha-nism in lepidopteran cells (Sf221) by swainsonine treatment.The cell viability and cell death assays for swainsoninetreated Sf-21 cells showed a significant decrease in both theparameters at as early as 12 h to the maximum of 36 h. Thisfurther suggested the role of secondary metabolites in theentomopathogenic virulence of Metarhizium sp under ex situ

conditions. The data demonstrated the profound efficacy ofswainsonine (both standard and purified) at micromolar con-centrations with approximately 60% of treated cell popula-tion lost viability in a dose and time-dependent manner. Theentomotoxic and antiproliferative effects of swainsoninewere further studied in the aspect of apoptosis induction inSf-21 cells.

The FACS quadrant statistic analysis of the swainsonine(standard and purified) treated cells indicated a fairly signifi-cant number of cells in both the early and late apoptoticphases between 24 and 36 h of post-treatment incubation,further confirming the apoptotic triggered cell death mecha-nism (Figure 4). The treated cells might have undergoneeither apoptotic or necrotic or a combination of both, whichcan be evaluated satisfactorily by the morphological, dimen-sional and mechanical examination using ultramicroscopictechniques like FE-SEM and AFM. The morphologicalparameters of the treated cells viz., cell membrane damage,stiffness, rigidness and volume shrinkage could be correlatedwell with in situ cytotoxicity of an inhibitory or cytotoxiccompound.23 The FE-SEM imaging of swainsonine treated

Figure 6. Ultra-morphodimensional AFM analysis of control and swainsonine (standard and purified) treated Sf-21 cells after 36 h ofpost-treatment incubation. The full scan 2-D image (first column), 3-D image (second column), and average height-size curve(third column), for Sf-21 cells. The color gradients light to dark indicates the average height of cells under scan correspond-ing to their higher to lower topographies, respectively.


Sf-21 cells showed the disrupted cell membrane with charac-teristic cytoplasmic blebbings and apertures contrary to thecontrol cells after 24–36 h of incubation (Figure 5). Thenecrosis is characterized by the cytosolic swelling whereasapoptosis is marked by the cell shrinkage causing detectablechanges in cell dimensions.24 The AFM mediated morphodi-mensional analysis of the swainsonine treated cells indicateda significant decrease in average height and size suggestingthe apoptotic mode of cell death (Figure 6). The FE-SEMand AFM analysis together showed the enhanced roughnessand decreased dimensions (height and volume) of the swain-sonine treated Sf-21 cells compared to the control cells,which were significantly altered with post treatment incuba-tion hours (Figures 5 and 6).

The MTT and TB determined IC50 values were signifi-cantly lower for standard swainsonine compared to that forthe purified one, which infers the higher antiproliferativesensitivity of lepidopteran cell toward the pure swainsonine.However, the observed cytotoxicity index based on FACSand ultramicroscopic evaluations was higher for the Meta-rhizium extracted LC- purified samples than the standardcompound. This enhanced virulence could potentially beattributed to the synergistic toxicity of sister compoundsinflicting the physicomechanical injuries to the treated cells.Hence, the work further unravels the selectivity of in vitrobiomarkers in similar cytotoxicity studies.

Conclusions

In summary, this study suggested an efficient method forthe qualitative and quantitative MUV-HPLC analysis ofswainsonine in Metarhizium fermentation broth. The standardand preparatory LC- purified swainsonine fractions were fur-ther investigated for their in situ entomotoxic applicationsagainst Sf-21 cells which suggested an apoptotic mechanismof cell death after 24–36 h of post-treatment. The identifica-tion of swainsonine as an apoptotic agent in Lepidopteran,Sf-21 cells further signified the role of secondary metabolitesin the virulence of entomopathogenic fungi.

Acknowledgments

The authors thank the Department of Biotechnology,Indian Institute of Technology Guwahati, India for providinginstrumental and lab facilities. They gratefully acknowledgethe ARS Collection of Entomopathogenic Fungal Cultures(ARSEF) USDA, Ithaca, New York for providing the fungalstrain and NCCS, Pune, India for providing the insect cellline. The authors are also grateful to Central InstrumentsFacility (CIF), Indian Institute of Technology Guwahati forESI-TOF MS, FE-SEM and AFM analyses. They thank toThe Council of Scientific and Industrial research (CSIR),PUSA, New Delhi, India, for providing the research fellow-ship. The contents of this manuscript have not been previ-ously copyrighted or published. The contents of thismanuscript are not under consideration for publication else-where. There are no directly related manuscripts or abstracts,published or unpublished, by any author(s) of this paper.The authors have no conflict of interest.

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Manuscript received Jan. 21, 2014, and revision received Mar. 26,

2014.


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