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Cytotoxic Effect of Microbial BiosurfactantsAgainst Human Embryonic Kidney Cancerous Cell:HEK-293 and Their Possible Role in Apoptosis

Arun Kumar Pradhan & Nilotpala Pradhan &

Purusottam Mohapatra & Chanakya Nath Kundu &

Prasanna Kumar Panda & Barada Kanta Mishra

Received: 1 May 2014 /Accepted: 15 August 2014# Springer Science+Business Media New York 2014

Abstract Two different microbial biosurfactants S9BS and CHBS were isolated fromLysinibacillus fusiformis S9 and Bacillus tequilensis CH. Cytotoxicity effect of thesebiosurfactants on human embryonic kidney cancerous cell (HEK-293) were studied with thehelp of 3-(4,5-dimethylthiazol-2yl-)-2, 5-diphenyl tetrazolium bromide (MTT) assay andmorphological changes were observed under inverted microscope. The biosurfactants exhib-ited positive cytotoxic effect on HEK-293 cell line. It was found that LC50 of S9BS and CHBSwere 75 and 100 μg ml−1, respectively. Further cell cycle and apoptosis analysis ofbiosurfactant-treated HEK-293 cell line were done by FACS. In this study, cytotoxic effectof glycolipid biosurfactant against HEK-293 cell lines is reported for the first time. Mechanismtowards increased membrane permeability of biosurfactant-treated cancer cell may be theincorporation of its lipid moiety into the plasma membrane leading to formation of poresand membrane disruption. Hence, these microbial biosurfactants can prove to be significantbiomolecule for cancer treatment.

Keywords Biosurfactants . Cytotoxic effect . HEK-293 cell . Apoptosis

Appl Biochem BiotechnolDOI 10.1007/s12010-014-1168-8

A. K. Pradhan (*) : N. PradhanAcademy of Scientific and Innovative Research, CSIR—Institute of Minerals and Materials Technology,Bhubaneswar 751013, Indiae-mail: [email protected]

A. K. Pradhan : N. Pradhan : P. K. PandaBioresources Engineering Department, CSIR—Institute of Minerals and Materials Technology,Bhubaneswar 751013, India

B. K. MishraCSIR—Institute of Minerals and Materials Technology, Bhubaneswar 751013, India

P. Mohapatra : C. N. KunduKIIT School of Biotechnology, KIIT University, Odisha, India

Introduction

Biosurfactants are surface active molecules with hydrophilic and hydrophobic moieties pro-duced by many microbes and plants. Ron and Rosenberg [1] emphasized the importance ofdifferent types of biosurfactants produced by various microbes in diverse ecological niche fortheir survival. By chemical nature, the biosurfactants can be glycolipid, lipopeptide,polysaccharide-fatty acid complex, neutral lipid, polysaccharide-protein complex, etc. Theyare reported to be useful in applications like bioremediation of soil, oil removal, antimicrobialactivity, pathogenic biofilm inhibition, hydrocarbon degradation, metal extraction, etc. [2–7].By virtue of its diverse applications, biosurfactant may become one of the essential compo-nents of pharmaceutical, cosmetic, petroleum oil, food industry, etc. in the near future.

Cancer is one of the most perilous areas in the biomedical field towards which intense andswift research is being focused. In the recent past, cytotoxicity of biosurfactants on different celllines has been gaining much importance. Cao et al. [8–10] reported the anticancer and apoptoticproperties of surfactin (lipopeptide) against human breast cancer MCF-7 cells. Antitumorproperty of viscosin produced by Pseudomonas libanensis M9-3 on prostate cancer cell linePC-3M is reported [11]. Cytotoxic effect of lipopeptide biosurfactant on human colon cancer celllines HCT-15 (LC50 80 μg ml−1) and HT-29 (LC50 120 μg ml−1) is also reported [12]. Apoptosisin H7402 human liver cancer cells was induced by sophorolipid [13]. Renal cell carcinoma(RCC) and transitional cell carcinoma of the kidney are the seventh and ninth most commoncancer among men and women, respectively, in the USA [14]. Jemal et al. reported that 4 % ofthose diagnosed with cancer in the USA and 2 % death of the total population are due to kidneymalignant tumor [15]. Hence, utilizing the cytotoxic property of biosurfactant in treatment ofcancer in kidney may prove significant in advancement in the field of cancer therapy.

In the present study, cytotoxic effect of two purified biosurfactants (S9BS and CHBS)against human embryonic kidney cell lines (HEK-293) was determined and compared. Therole of apoptosis towards biosurfactant-induced cytotoxicity was studied. For the first time,this study reports the antitumor and apoptotic property of glycolipid type of biosurfactantagainst HEK-293 cell line.

Materials and Methods

Microbial Biosurfactant

S9BS and CHBS are glycolipid and lipopeptide type of microbial biosurfactants isolated fromLysinibacillus fusiformis S9 and Bacillus tequilensis CH respectively and are previously char-acterized [6, 7]. For the cytotoxicity assay of S9BS and CHBS, human embryonic kidney cellline HEK-293 was used; the stepwise procedure used for the experimentation is given in Fig. 1.

HEK-293 Cell Line Culture

Modified Eagle’s Minimum Essential Medium (MEMEM) [16] was prepared by adding thefollowing components: D-MEM high glucose (435 ml), fetal bovine serum (50 ml), 100×stock Pen/Strep (5 ml), and 200 mM L-glutamine (5 ml).

For reviving the cell lines, old media was discarded, and the cell layers were washed with0.25 % (w/v) Trypsin-0.53 mM EDTA solution to eliminate the generated trypsin inhibitor.Trypsin-EDTA solution (2.0 to 3.0 ml) was added to flask and incubated for 15 min at 37 °C.MEMEM growth medium (6.0 to 8.0 ml) was added, and cells were aspirated by gently

Appl Biochem Biotechnol

pipetting. Appropriate aliquots of the cell suspension were added to new culture vessels andincubated at 37 °C.

MTT Cytotoxicity Assay

About 9,000 to 10,000 HEK-293 cells were plated in 96-well flat bottom tissue culture platesin duplicates. These cells were exposed to increasing concentration of biosurfactants (S9BSand CHBS) separately after the incubation period of 24 h. Then, the culture media wasremoved, and cells were treated with 100 μl of 0.05 % 3-(4,5-dimethylthiazol-2yl-)-2, 5-diphenyl tetrazolium bromide (MTT) reagent [8–10, 17, 18]. To allow the formation of purpleformazan crystals, cells were incubated at 37 °C overnight. The cells of each well were againincubated for 1 h in the dark at room temperature with 100 μl of NP-40 detergent solution todissolve formazan crystals. The intensity of developed color was measured spectrophotomet-rically at 570 nm using a microplate reader (ELx800 Absorbance Microplate Reader, BioTek,Winooski, USA). Obtained data were represented graphically to calculate LC50 value (con-centration of biosurfactant at which 50 % of cell growth inhibition occurs in comparison tocontrol) of each biosurfactant.

Morphological Change Under Inverted Microscope

To observe the morphological changes in untreated and treated cancerous cell line, micro-scopic study was done. Approximately 1×106 cells ml−1 of HEK-293 cell line was plated in25-ml cell culture flask (containing MEMEM). One of the cell line flask was treated with75 μg ml−1 S9BS (LC50 value), and the other flask was treated with 100 μg ml−1 CHBS (LC50

value). The treated cell lines were then allowed to grow in a humidified CO2 incubator (RPMI-1640, Sigma, MO, USA) at 37 °C for 24 h. Untreated cell lines were taken as negative control.The morphology of cell lines was compared under inverted microscope (Eclipse TS100/TS100F, Nikon, Japan) [16, 19–21].

Cell Cycle Arrest and Apoptosis Analysis by FACS

HEK-293 cell line (1×106) was grown on 60-mm tissue culture discs, and after attaining 60–70 % confluency, cells were exposed to different concentrations (0, 50, 100, and 200 μg ml−1)

Seven day old culture

BS extraction using Chloroform: Methanol (2:1) solvent system

Concentration of extract in rotary evaporator

Purification with the help of TLC

Wash and resuspend in water

Centrifugation to get crude BS as pellet

Centrifugation to obtain cell free supernatant

Acidification for crude biosurfactant precipitation

Partial characterization by:

TLC NMRFTIR

Fig. 1 Schematic representationof biosurfactant isolation, purifica-tion, and partial characterization


of bioactive compounds CH and S9 for 24 h. After the incubation period, the cells weretrypsinized and washed twice with 1× PBS containing 0.05 % RNase-A. Cells were resus-pended in 0.1 mL of propidium iodide (10 μg ml−1 in 1× PBS) solution and incubated for30 min in the dark at room temperature. The stained cells were then analyzed by FlowCytometry (FACS Canto-II, Becton and Dickinson, CA) with event count of 10,000 cellsper sample. By using FACS Diva software (Becton and Dickinson, CA), DNA content of thecells at different phases of cell cycle was determined [22–25].

Results and Discussion

MTT Cytotoxicity Assay

MTT cytotoxicity assay clearly showed that the intensity of the purple color decreased onincreasing the concentration of biosurfactants (Fig. 2a), which highlights the inhibitory effectof biosurfactants on HEK-293 cell line. The cell line viability graph (Fig. 2b) indicated that theviability percentage is less than 50 % at a concentration of 75 μg ml−1 for S9BS and100 μg ml−1 for CHBS. Hence, 75 and 100 μg ml−1 were considered as the LC50 values forS9BS and CHBS, respectively. MTT assay result indicated that the percentage of cell viabilitywas inversely proportional to the concentration of biosurfactant. Hence, MTT assay confirmsthe cytotoxic effect of the isolated biosurfactants on HEK-293 cells with CHBShaving higher LC50 than S9BS. The reported literature also showed similar results,but with different cell lines. Cao et al. [8] reported that LC50 of lipopeptidebiosurfactant produced by Bacillus natto TK-1 was 93.8 μg ml−1 for HEK-293 cellline which is almost parallel to the CHBS lipopeptide results obtained in this study(LC50 is 100 μg ml−1). Sivapathasekaran et al. [12] reported that lipopeptide producedby Bacillus circulans DMS-2 had cytotoxic effect against human colon cancer celllines HCT-15 (LC50=80 μg ml−1) and HT-29 (LC50=120 μg ml−1).

0

20

40

60

80

100

120

0 50 100 150 200

% V

iab

ilit

y

Concentration (µg ml-1)

HEK-293 cell

S9

CH

b

Control

5 µg/ml

25 µg/ml

50 µg/ml

75 µg/ml

100 µg/ml

125 µg/ml

150 µg/ml

S9BS CHBSa

Fig. 2 MTT cytotoxicity assay: a decreased purple color on increasing concentration of both type ofbiosurfactants indicating the death of cells; b viability graph of MTT assay showing LC50 values of eachbiosurfactants


Morphological Study Using Inverted Microscope

The significant morphological differences were observed between the biosurfactant-treatedand biosurfactant-untreated cell lines when viewed under the inverted microscope (Fig. 3).Huge rounding structures with sporadic distribution (indicated by arrows) and loss of adhesionwere observed in biosurfactant-treated cell line flasks (Fig. 3b, c) whereas cells in untreatedflask were found to be regular in shape and size with few rounding cells (Fig. 3a). Thisobservation indicates the possibility of apoptosis in biosurfactant-treated cell lines. To furthersubstantiate this observation (the role of apoptosis in cell toxicity), FACS study wasperformed.

Role of Apoptosis in Biosurfactant-Induced Cytotoxicity of HEK-293 Cells

To validate the role of apoptosis in biosurfactant-induced cytotoxicity, FACS was performed toconfirm the presence of the formed apoptotic bodies. As reported in literature at the later stageof apoptosis, endonucleases break the linkers between the nucleosomes, one of the units ofchromatin organization. Large number of small DNA fragments (oligomers) accumulates inthe cell. If cells are fixed in ethanol and subsequently rehydrated, some of the lower molecularweight DNA leaches out, lowering the DNA content. Cells may be observed as a ‘sub-G1’peak in a DNA histogram [9, 10, 13, 26].

The FACS results of treated HEK-293 cell line showed that the percentage of sub-G1 cellsincreased with the increment of S9BS and CHBS concentrations (Figs. 4a–d and 5a–d). S9BS,at a concentration of 50 μg ml−1, resulted in 38.7 % cells being apoptotic (sub-G1), whereas ata concentration of 100 μg ml−1, 93.9 % of cells were apoptotic (Table 1). This results clearlyindicated that LC50 value of S9BS for HEK-293 cell line is between 50 and 100 μg ml−1 whichwas similar to that observed in the MTT assay (LC50 value for S9BS is 75 μg ml−1).

On the other hand, 54.3 % of cells were apoptotic after treatment with 100 μg ml−1 CHBS(Table 1). It indicates that the LC50 value of CHBS on HEK-293 cell line is 100 μg ml−1 whichconfirms the MTT assay result.

Hypothesized Mechanism of Biosurfactant-Induced Cytotoxicity

The above study confirms the cytotoxic effect of biosurfactants on HEK-293 cells by means ofapoptosis, but insight into the exact molecular mechanism of cytotoxic induction causingapoptosis requires a more detailed investigation. Numerous studies have reported that

HEK-293 + 100 µg ml -1CHBSHEK-293 (control) HEK-293 + 75 µg ml-1 S9BS

a cb

Fig. 3 Morphological changes in untreated and treated cell line as observed under inverted microscope: anegative control (untreated cell line) showing regular-shaped cells and very few rounding structures; b, cbiosurfactant-treated cell lines showing huge number of rounding cells and sporadical distribution (indicatedwith arrows)


biosurfactants being amphiphilic modulate the physicochemical properties of plasma mem-brane by partitioning into them causing either membrane solubilization or hemolysis. Zaragoza

DNA content DNA content

DNA contentDNA content

0 µg ml-1 (Control)

100 µg ml-1 200 µg ml-1

50 µg ml-1

a b

c d

Fig. 4 a–d Cell cycle regulation and apoptosis were analyzed by FACS in HEK 293 cell line treated withdifferent concentrations of S9BS

DNA content

DNA content DNA content

DNA content

a b

c d

0 µg ml-1 (Control)

100 µg ml-1 200 µg ml-1

50 µg ml-1

Fig. 5 a–d Cell cycle regulation and apoptosis were analyzed by FACS in HEK 293 cell line treated withdifferent concentration of CHBS


et al. [27] investigated the molecular mechanism of cell toxicity by trehalose lipid, a glycolipidtype of biosurfactant using fluorescent probe assays and concluded that the trehalose lipidincorporates into the membrane by flip-flop and perturbs the lateral domains leading to theformation of pores by which leakage occurs damaging the integrity of the cell. Later, anothermolecular mechanism of cell toxicity by the trehalose lipid was identified wherein hemolysisof human erythrocytes occurred by a colloid-osmotic mechanism which is also most likely dueto formation of pores in the membrane [27]. Recently, Janek et al. [28] reported thatpseudofactin II (a lipopeptide type of biosurfactant) induces apoptosis in melanoma A 375cell lines by specific interaction with plasma membrane causing loss of cell integrity. Hence,the primary cause of cell toxicity is probably the interaction/incorporation of the biosurfactantin the plasma membrane leading to formation of pore-triggering apoptosis.

Table 1 Percentage of HEK-293 cells in different cell cycle phases with increasing biosurfactant concentration

Concentration of biosurfactants (μg ml−1) Percentage of HEK-293 cells in different cell cycle phases

Sub-G1 G1 S G2/M

S9BS

0 2.4 57.2 25.7 13.9

50 38.7 30.6 18.8 10.5

100 93.9 4.8 1.0 0.1

200 97.6 1.9 0.3 0.0

CHBS

0 2.7 58.0 27.6 11.1

50 21.5 45.0 27.7 5.2

100 54.3 24.8 18.7 1.8

200 95.5 3.6 0.8 0.00

Cancer cell

Integral Plasma Membrane

Permeabilized Plasma Membrane

Apoptosis of cancer cell leading to cell lysis

Incorporation of biosurfactant

Peptide/Glycan moiety

Lipid moiety

Fig. 6 Hypothesized mechanism showing increased membrane permeability of cancer cell by biosurfactants


Since the nature of the biosurfactants included in the present study are glycolipid (S9BS)and lipopeptide (CHBS), both having a lipid moiety, its likely that these too get incorporatedinto the plasma membrane. This results in increased cell permeability and destruction of themembrane integrity-triggering apoptosis. Figure 6 provides a visual insight into this molecularmechanism schematically.

Conclusion

Cytotoxic effect of two microbial biosurfactants (S9BS and CHBS) was studied on HEK-293cell line. It was found that cytotoxic effect of glycolipid biosurfactant S9BS with LC50 about75 μg ml−1 was more than that of lipopeptide biosurfactant CHBS (LC50 100 μg ml−1) onHEK-293 cell line. The result of this study suggests that S9BS and CHBS can be furtherstudied for their potential anticancer properties. Further understanding into the exact molecularmechanism of biosurfactant-induced cytotoxicity will offer an innovative path towards cancertherapy in biomedical field.

Acknowledgments One of the authors (AKP) would like to express his sincere thanks to the Council ofScientific and Industrial Research (CSIR), India for the award of Senior Research Fellowship. The authors arethankful to Jacintha Esther for the editorial assistance in manuscript preparation.

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