Apoptosis of SiHa and HCT116 cancer cell-lines by Scorpaenopsis venosa toxic extracts

Indian Journal of Geo-Marine Sciences Vol. 45(1), January, 2016, pp. 155-160

Apoptosis of SiHa and HCT116 cancer cell-lines by Scorpaenopsis venosa toxic extracts

R. Sasikala & Vinoth S. Ravindran^*#

Suganthi Devadason Marine Research Institute, 44, Beach Road, Tuticorin-628 001, India.

#Present address: Centre for Coastal and Marine Research (CCMR),37A/1, Third Mile Tuticorin- 628 008,India [*E-mail: ccmr.tuticorin@gmail.com]

Received 07 February 2014; revised 22 April 2014

Spine-venom gland and gonad of the scorpion fish Scorpaenopsis venosa was excised and extracted with Phosphate Buffer Saline. Crude protein was purified using DEAE cellulose anion exchange column chromatography. Protein content of the purified fractions of spine venom gland ranged from 0.053 to 0.076 mg.ml^-1. Whereas, the protein content of the purified gonad extracts ranged from 0.056 to 0.076 mg.ml^-1. Crude as well as the partially purified proteins had considerable haemolytic activity in chicken and goat erythrocytes and it was found to be dose dependent. Fractionated extracts of the spine-venom gland induced cytotoxicity in Human cervical cancer cell line (SiHa) and Human colon cancer cell line (HCT 116). Purified fractions of spine-venom gland showed maximum apoptosis (59/100) in chromatin condensation assay against SiHa and HCT 116 (20.5/100). SDS-PAGE analysis of spine-venom gland fractionated protein yielded three bands at 71.2, 51 and 34 kDa and that of gonad yielded two bands one each at 40 and 35kDa. The FTIR analysis of the purified extracts showed peak values aligned as halide, amine, ether and alcohol.

[Keywords: Chromatin condensation, FTIR, Hemolysis, Human cervical cancer cells (SiHa), Human colon cancer cells (HCT), MTT, SDS-PAGE].

Introduction

Nature has been a relevant resource for the discovery of anticancer compounds. More than 60 % of the anticancer drugs commercially available are of natural origin¹. The discovery of the anti-cancer compound 1-beta-D- arabinofuranosylcytosine (ARA-C) marked the beginning of bioprospecting for anti-cancer compounds from the marine environment². Ecteinascidin-743 (ET-743) (Trabectedin, Yondelis

TM

) from the colonial tunicate Ecteinascidia turbinata has been commercialized³. In 1998, Novartis Pharma AG licensed Discodermolide extracted from the deep- sea sponge Discodermia sp. for development as a candidate agent for treatment of cancers⁴.

Venomous creatures have long been studied as potential source of pharmacological compounds. More than 1200 species of marine fishes are known to be venomous⁵. comparatively less research has been undertaken on venomous fishes⁶ particularly due to the difficulty in collecting the venom and the marked instability

7,8,9

. Hence the venom of marine animals, particularly fishes remains a largely untapped source of novel compounds. Therefore, the present investigation with S. venosa extracts was undertaken to elucidate the hemolytic activity in chicken and goat erythrocytes,

cytotoxicity and anticancer activity in Human cervical cancer cell line (SiHa) and Human colon cancer cell lines (HCT116). The structural elucidation of the most active fractions was done using Fourier Transform Infrared Spectroscopy (FTIR) and MW characterization using SDS-PAGE.

Materials and Methods

Scorpaenopsis venosa¹⁰ was collected from Kanyakumari coast, Tamil Nadu, West Coast of India, brought live to the laboratory and identified.

Extraction of spine- venom gland - Fishes were chilled at -20˚C for 10-20 min and then decapitated; the dorsal spines were cut approximately 3-5 mm from their base along with the venom gland. Spines were homogenized in 0.9% NaCl. Supernatant was centrifuged for 15 min at 6000g to remove debris. The resulting supernatant was designated as Scorpaenopsis venosa spine-venom gland extract. All steps were performed in a cold room at 4-10˚C. Freshly extracted soluble fraction was used for all experiments ¹¹.Extraction of gonad- The gonad of S. venosa was dissected and the extract was prepared by squeezing the sand-free specimens in triple distilled water. Resultant solution was filtered and it was again squeezed with Phosphate

Buffer Saline (PBS). Supernatant so obtained was lyophilized (Labcono Freeze Dry System) and stored at -20 ^o C until further use.

Partial purification of the crude extract using column chromatography -Partial purification of the crude extracts of spine-venom gland and gonad were carried out using DEAE cellulose anion exchange chromatography ¹². Four fractions each of spine-venom gland and gonad extracts were collected in a step-wise gradient (0.2, 0.5, 0.8 and 1M) with Nacl and stored at - 20C for further use.

Protein estimation- Protein estimation¹³ was done using Bovine Serum Albumin as standard.

The absorbance was read spectrophotometrically at 280 nm.

Anticancer assays

Hemolytic activity- Citrated chicken and goat whole blood was used within 24 h after bleeding ¹⁴. 1% RBC suspension was prepared ¹⁵. 1 ml of 1% RBC was incubated for 6 h with crude and purified fractions (250, 500, 750, and 1000 µg/ml) of spine-venom gland and gonad extracts at 37°C. Total lysis of erythrocyte suspension was obtained by incubating the cells with 0.1 % v/v Tween 20. In order to evaluate the degree of spontaneous lysis, tubes containing exclusively the red blood cell suspension in D-PBS were set.

For each concentration and control, the experiments were set in triplicate. The absorbance at 405/540 nm was measured with a spectrophotometer (ELICO SL-164). Haemolytic levels were expressed by percentage of haemolysis, calculated as the ratio between the value measured for each sample and that registered for the total haemolysis.

MTT cell line toxicity assay-The glasswares used in the experiment were of neutral glass of Borosil make and non-toxic to the cell cultures.

They were soaked overnight in mild HCl solution.

Sterile disposable plasticwares such as tissue culture flasks, petridishes, and multiwall plates of tissue culture grade (Tarsons, India) were used for the experiment.

Human colon cancer cell line (HCT116) and Human cervical cancer cell line (SiHa) were sub-cultured into a fresh flask containing Eagles Minimum Essential Medium (EMEM) with 10%

fetal bovine serum (FBS). The cells were rinsed in serum free medium and trypsin (0.1%) was added to remove the cells from the substratum.When the monolayer had detached from the flask, the activity of trypsin was stopped by adding serum- containing medium (serum contains the trypsin inhibitor α-1 antitrypsin). The cells were counted and the flask concentration was

adjusted to 10⁴-10⁵ cells /ml and seeded on to a 96 well microtitre plate at the rate of 200µl/well and incubated at 37ºC.

When the cells reached 80% confluence, they were used for cytotoxicity assay with toxins of the target species. Crude samples were diluted in EMEM at the rate of 1 mg/ml and serial ten- fold dilutions were made in sterile tubes with 2%

fetal calf serum. Dilutions without the toxins served as the control. Plates were incubated at 37ºC for 48 h. The cells were observed under an inverted microscope for changes in their morphology.

The toxin treated plates after 48 h of incubation were taken for MTT (3,[4,5- dimethylthiazole-2,4]-2-5-diphenyl tetrazolium bromide) cell viability test. MTT stock solution was prepared (1 mg/ml) and filtrates were sterilized in 0.22µ membrane filter. MTT stock solution (50µl) was added to each well. Plates were incubated in a humidified incubator at 37ºC for 4 h. Medium was replaced with 200µl DMSO and incubated for 10 min on a micro shaker to dissolve the formazan crystals of MTT.

Sorensens’s glycine buffer (25µl; 0.1M glycine and 0.1 M NaCl) was added to each well. The plates were then read at 570 nm aginst a reference of 690 nm in an ELISA reader. Plates were read within 30 min after the addition of DMSO.

Samples were tested thrice for confirmation.

DNA Fragmentation studies by Hoechst staining- The human colon cancer cell line (HCT116) and cervical cancer cell line (SiHa) were cultured in 15 cm cell culture plates in Dulbecco’s Modified Eagle’s Medium (GIBCO™

DMEM) supplemented with L-glutamine, 1%

streptomycin and 10% heat inactivated fetal bovine serum (FBS). Cultures were incubated in a humidified incubator in 5 % CO2 at 37°C. SiHa and HCT116 cells were freezed, revived, seeded and those with 70% confluence were treated against 100 μl.ml^-1 purified extracts of spine- venom gland and gonad extracts in 96 well plates to induce apoptosis. Cell counts were performed using a phase contrast microscope.

After treatment of cell lines (HCT116 &

SiHa) with purified extracts (spine-venom gland and gonad) 60 µL of DMEM media was removed from the wells and same volume of diluted dye solution (5 µg.ml^-1Hoechst stock in HBSS) was added to the wells. Plate was incubated at 37°C / 5% CO2 for 20 min. Upon image analysis using Simple PCI (Compix Inc., Imaging Systems, and Sewickle) fluorescent microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan),three independent images were taken in each well containing about

50-100 cells, for each fraction at 100X magnification. For each image, the number of cells containing condensed chromatin was made visible through Hoechst staining. Bright fluorescent blue dots were counted as condensed chromatin (apoptosised cells). Apoptosised cells counted were tabulated for each fraction and were subsequently compared to the untreated control.

Final results were calculated and presented as chromatin condensation percentages for each fraction compared to the control.

Characterization using SDS-PAGE- One dimension Sodium Dodecyl Sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) was carried out¹⁶ to elucidate the MW of proteins.

FTIR analysis- The most active DEAE cellulose column fractions (spine-venom gland and gonad) in hemolytic and cancer cell line assays were analyzed qualitatively for the active groups by Fourier Transform Infra Red (FTIR) spectrometry ¹⁷.

Statistical analysis-The data were analyzed statistically, standard deviation and mean values were quoted ¹⁸.

Results & Discussion

Lyophilized extracts (crude and partially purified) of spine-venom gland and gonads of S.

venosa yielded a total amount of 5.4 g of crude venom from 500 g excised spine- venom gland.

30 g of gonad extract yielded 3.09 g of lyophilized protein^. When the crude was fractionated with DEAE-cellulose the protein content in crude spine-venom gland and gonad extracts were 4.35 and 0.9 mg.ml^-1 respectively.

Protein in purified fractions ranged between 0.053 and 0.076 mg.ml^-1 for spine-venom gland extract and 0.056 and 0.076 mg.ml^-1 for gonad extracts (Table I).

Table I: Protein content of crude and purified extracts of S.

venosa (mg.ml^-1)

Part/organ extract Crude/Fractions

Crude I II III IV

Spine-venom gland 4.35

± 0.157

0.063

± 0.002

0.065

± 0.002

0.076

± 0.003

0.053

± 0.002

Gonad 0.9

± 0.020

0.076

± 0.011

0.068

± 0.005

0.056

± 0.003

0.061

± 0.005

In an earlier study ¹⁹, the protein in venom gland extract of S. horrida purified with DEAE ²⁰ was 1 mg.ml^-1.

Hemolytic assay- Hemolysis was found to be dose dependent (Table II). Haemolysis produced by spine-venom gland crude extract ranged from 62 to 70% for chicken erythrocytes and 49 to 76% in goat erythrocytes.

Haemolysis produced by gonad extracts ranged from 65 to 78% in chicken erythrocytes and 59 to 87% in goat erythrocytes. Likewise, purified gonad extracts produced 42 to 78% lysis in chicken erythrocytes and 51 to 81% lysis in goat erythrocytes. Similarly purified spine-venom gland extracts produced 42 to 70% haemolysis in chicken erythrocytes and 53 to 88% in goat erythrocytes.

Table II: Haemolysis by crude and purified extracts of S.

venosa

Part/

organ extract

Crude/

Fraction

Protein content in (µg)

Haemolysis (%) Chicken

Spine- venom

gland

Crude

FractionI

FractionII

FractionIII

FractionIV 250 500 750 1000

250 500 750 1000

250 500 750 1000

250 500 750 1000

250 500 750 1000

62.7±1.2 67.7±0.8 69.5±0.7 70.3±0.6 42.8±1.5 57.7±0.8 65.5±076 67.3±0.8 57.7±1.2 65.7±0.7 61.5±0.7 63.0±0.4 52.6±1.1 61.7±0.8 68.5±0.7 70.1±0.8 52.7±1.2 67.8±0.7 70.5±0.9 68.3±0.6

49.8±0..8 56.9±0.4 59.7±0.23 76.4±0.25 53.7±1.2 68.9±0.8 73.6±0.7 72.9±0.9 56.0±1.2 68.7±0.9 72.5±0.7 75.3±0.6 52.7±1.3 68.9±0.9 73.8±0.7 81±0.57 61.7±1.3 74.7±0.8 80.8±0.7 88.3±1.7

Gonad

Crude

FractionI

FractionII

FractionIII

FractionIV 250 500 750 1000

250 500 750 1000

250 500 750 1000

250 500 750 1000

250 500 750 1000

65.7±1.2 67.7±0.68 75.5±0.77 78.3±0.6 52.7±0.7 64.7±0.8 74.7±0.45 76.5±0.63 42.7±1.4 76.7±0.82 71.5±0.6 72.1±0.68 52.7±1.21 57.7±0.18 67.3±0.75 74.3±0.62 52.7±0..27 55.2±0.66 45.5±0.73 67.3±0.5

51.3±1.6 67.9±0.80

77.5±0.7 77.3±0.8 51.7±1.9 69.7±0.8 71.5±0.7 81.2±0.56

72.7±1.4 75.7±0.8 76.5±0.72 77.3±0.65 72.7±1.42 74.7±0.78 78.5±0.17 79.6±0.75 67.7±1.5 75.7±0.8 81.4±0.7 79.3±0.96

The non-enzymic toxic protein, SNTX from S.

horrida¹⁷ has been reported to induce potent hemolytic activity through the formation of pores in the cell membrane. Similar reports of hemolysis by ovarian protein of fishes,Protonibea diacanthus, Otolithoides biauritus and Muraenesox talabonoides has been reported²¹. The venom of the catfish Plotosus canius and Heteropneustes fossilis both produce haemolysis.

Weeverfish venoms also possess haemolytic activity. Haemolytic activity of Trachinus draco is very potent on rabbit erythrocytes, less potent on rat erythrocytes, weakly effective against mouse and bovine erythrocytes, and completely

ineffective against human, guinea pig and chicken erythrocytes, as is the lethal toxin, Dracotoxin²². Pterois volitans venom has also been found to possess haemolytic activity which is highly effective against rabbit erythrocytes²³. The up-side down catfish, Synodontis guttata produces haemolysis in sheep erythrocytes²⁴, and the venom of the bullrout, Notesthes robusta demonstrated haemolytic activity against human erythrocytes²⁵. Like in the present study, the venom of Thalassophryne nattereri shows potent haemolytic activity against mouse erythrocytes, as well as other cell types²⁶. TLY, a cytolysin extracted from Synareceia trachynis was found to be most active against rabbit erythrocytes, followed by dog, rat, pig, and guinea pig erythrocytes, with negligible activity against sheep, cow, human, monkey, mouse, goat, horse or cat erythrocytes²⁷. Verrucotoxin, extracted from Synanceia verrucosa is also potently haemolytic for rabbit erythrocytes²⁸. The above findings are in accordance with the present study.

MTT Cell line toxicity assay- The results indicate cytotoxicity of purified S. venosa spine- venom gland and gonad extracts to possess dose dependent cell lytic activity on SiHa and HCT 116 cell lines. The results are tabulated in Table.III.

Table III: Effect of crude protein of the S. venosa on MTT cell viability test in human cell lines (HCT116

& SiHa)

Level of dilution

Level of inhibition (%) of S. venosa spine venom gland extract

Level of inhibition (%) of S. venosa

gonad extract HCT cell

line

SiHa cell line

HCT cell line

SiHa cell line

10⁰ 81.5 95.81 71.23 80.36

10^-1 80.23 92.55 69.58 78.23

10^-2 77.26 90.12 65.20 65.24

10^-3 72.18 89.30 64.85 61.28

10^-4 69.44 84.56 62.58 60.89

10^-5 63.81 79.88 56.80 58.94

10^-6 58.38 75.42 46.39 56.37

10^-7 52.10 71.21 38.69 52.46

Inhibition by spine-venom gland extracts varied from 95.81 to 71.21 for SiHa cell lines and 81.5 to 52.10 % for HCT 116 cell lines. Apoptosis by gonad extracts varied from 80.36 to 52.46 for SiHa cell lines and 71.23 to 38.69 for HCT 116 cell lines. Likewise in a similar experiment ¹¹ 10 µg venom of the Weever fish Scatophagus argus showed 29% cell viability against human cervical cancer cell line (HeLa) which had comparatively higher activity than present observation of 55.2%cell viability at 10µg concentration.

HeLa and endothelial cell lines treated with 3 and 10 µg concentrations of Thalassophryne nattereri venom showed apoptotic function characterized by reduced cell size and intense fluorescence of condensed nuclear chromatin⁶. Also it has been reported⁶ that the venom of fishes viz., Gymnapistes marmoratus, Pterois volitans and Synanceja trachynis produced apoptosis on cultured murine cortical cells.

Anticancer activity- The apoptosis of the cells after addition of crude and partially purified spine-venom gland and gonad extracts are presented in Table IV. Chromatin condensation in the SiHa cells was high when compared to that of HCT116 cells. Likewise, the purified fractions of spine-venom gland produced more chromatin condensed cells than purified gonad extracts (Table IV). Also, maximum apoptosis (59/100) against cervical cancer cells (SiHa) and Human Colon cancer cells (HCT116) (20.5) were exhibited by fraction II of spine-venom gland purified proteins.

Table IV: Chromatin condensation in cell lines (HCT116 &

SiHa) produced by crude and purified fractions of S.venosa

Part/

organ extract

Crude/

Fraction

Protein content (mg/ml)

% of Condensed Chromatin

HCT SiHa

Venom gland

Fraction I Fraction II Fraction III Fraction IV

0.061 0.059 0.081 0.056

15±2.8 20.5±0.7 20.3±0.76 15.5±6.36

48.5±13.1 59±14.1 21.5±0.7 7.52±20.5

Gonad

Fraction I Fraction II Fraction III

0.049 0.059 0.051

5±6.36 14.5±7.4 12.38±6.3

2.5±6.36 18±4.1 31.2±3.54

Similarly, anticancer activity of ovarian protein of fishes viz., Protonibea diacanthus, Otolithoides biauritus and Muraenesox talabonoides on L929 and P388 cell lines has been reported. Changes in the electrophysiological properties²¹ of three breast cancer cell lines exposed to different concentrations of Tetrodotoxin (TTX) from puffer fish is reported ²⁹.The apoptosis is defined as the programmed cell death and has been demonstrated with lots of natural products.

Here, the obtained results can be compared with activity exhibited by other marine natural products. HeLa cell cycle was strongly affected by the fish P. lineatus toxin in S-phase at 48 hrs.

Similar level S phase cell cycle arrest was exhibited by venom of scorpionfish Hypodytes rubripinnis and stonefish Synanceia verrucosa which strongly affected the cell cycle of splenocytes and murine P388 leukemic cells³⁰.

In a similar study, the toxin of the jelly fish Chiropsalamus quadrigatus produced apoptosis with a IC50 value with 49.8 ± 3.6 µg.ml^-1 for ECV304 (transformed vascular endothelial cell) cells and 80.3 ± 1.3 µg.ml^-1 for U251 (human malignant glioma) cells and difference in the activity of jellyfish toxins was attributed to the sensitivity of cell line ³¹, which is found to have very low cytotoxic activity than this study.

Similarly, fish venom of Thalassophryne nattereri has shown similar level of apoptotic population on endothelial cell lines³² and Gymnapistes marmoratus, Pterois volitans and Synanceja trachynis venom on cultured murine cortical cells³³.

SDS-PAGE- The analysis yielded three bands for spine-venom gland purified extract at 71.2, 51 and 34 k Da. Two bands for gonad purified extracts at 40 and 35kDa. Earlier ²⁰, two bands one at 71 and other at 79 k Da has been reported from S. horrida purified extracts. The extracts have been reported to show potent hemolytic activity in rat, guinea pig and rabbit erythrocytes. Present study has also shown hemolysis in chicken and goat erythrocytes.

FTIR Analysis-The peak values of FTIR analysis are shown in Table V. The analysis of spectrum of fraction II of spine-venom gland which shows maximum anticancer activty (Table 5) revealed the following groups. The peak value of 393.45cmˉ¹ is due to the presence of C-I. The peak value at 1242.07cmˉ¹ is charecteristic of C- O group. The other peak value at 3396.41 cmˉ¹ is R-OH group. This is the functional group of alcohol. Siganls at 1056.92 cmˉ¹ is due to the presence of C-N. Peak at 1375.15cmˉ¹ is due to the group of R-S(=O)2-R′. It is the functional group of ether. Peak value at 1641.31cmˉ¹ indicate the presence of RON=O.

Table V: FTIR Analysis of Active Fractions of S.venosa

Active Fraction Peak values in cmˉ¹

Specific context

Anticancer Assay (Fraction II of spine-

venom gland )

378.02 393.45 403.09 1056.92 1242.07 1375.15 1641.31 2088.76 3396.41

C-I C-I C-I C-N C-O R-S(=O)2-OR'

RO-N=O Nil ROH Haemolytic Assay

(Fraction I of gonad)

387.67 412.74 432.03 478.31 1083.92

1244 1639.38 2084.91 2362.64 3394.48

C-I C-I C-I C-I C-N C-O C=C Nil RNH3+,R2NH2+,R3NH+

R-OH

Spectral analysis of fraction I of gonad showing maximum haemolytic activity revealed the following groups. Peak at 387.67 cmˉ¹ region is charecteristic of C-I group. The peak at 1244 cmˉ¹ region is due to C-N. The other peak at 2362.64 cmˉ¹ is charecteristic of the N-H group.

The is the functional group of amines. FTIR has been used extensively to study structure of proteins and lipids ^{34, 35}. FTIR has been earlier used as a tool to study alteration of proteins, lipids, and nucleic acids due to intoxication in fishes ³⁶.

Conclusion

The present study indicates that the spine- venom gland and gonad proteins of the scorpion fish S. venosa possess good cytolytic activity as evidenced by the haemolytic activity in goat and chicken erythrocytes and cytotoxic effect as evidenced by the MTT and chromatin condensation assays on Human colon Cancer cells (HCT116) and cervical cancer cells (SiHa).

Further research in this area including clinical studies could lead to the formulation of a new therapeutic anti-cancer drug.

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