Background
Breast cancer is the most common cancer which causes significant morbidity and mortality among women worldwide [
1]. In India, over 80,000 new cases of breast cancer diagnosed and metastasis is one of the leading cause of death [
2]. In recent years, due to lack of medical facilities for early detection, therapeutic strategies for treatment and side effects due to pharmacological compounds have encompassed the need for new therapies mostly from natural sources for long-term cancer prevention and treatment [
3‐
5].
Snake venom is a rich source of many proteins, peptides, macromolecules and many cytotoxins, neurotoxins, cardiotoxins, myotoxins, dendrotoxins, haemotoxins, fibrinolytic enzymes, Phospholipase A2 etc. [
6]. Calmette et al. [
7] in 1933 first reported that snake venom has anticancer activity and thereafter a lot of components from different snake venoms have been identified as showing therapeutic properties. A group of polypeptides (60–70 amino acid residues) called cytotoxins or cardiotoxins present in an elapid family of snakes have a wide variety of pharmaceutical actions and have the tendency to damage a wide variety of cells including cancerous cells [
8,
9].
A number of compounds from venomous animals, such as snakes, scorpions, spiders, toads, frogs, bees, caterpillars, insects, wasps, centipedes and ants have been isolated and showed therapeutic applications [
10‐
13]. For example, a compound TM601, modified form of the peptide Chlorotoxin (CTX) purified from
Leiurus quinquestriatus scorpion venom, has been shown to block chloride channel specifically on glioma cell surface and also shown potent pleiotropic anti-angiogenic effect, which is currently under phase-II clinical trials [
14,
15].
NN-32 is a 6.7 KDa proteinous toxin isolated from Indian spectacled cobra venom, which showed antioxidant and antitumor properties against EAC bearing BALB/c mice [
16]. The present study reports the cytotoxic activity of a toxin NN-32 present in
Naja naja venom against human breast cancer cell lines.
Methods
Chemicals
Carboxymethyl cellulose (CM Cellulose), Dulbecco’s modified eagle medium (DMEM), Fetal bovine serum (FBS), Penicillin, Streptomycin and Trypsin-EDTA Solution were purchased from Himedia (India). Thiazolyl Blue Tetrazolium Bromide (MTT), Dimethyl Sulfoxide (DMSO), Neural Red (NR), Formaldehyde, β-Nicotinamide adenine dinucleotide reduced dipotassium salt (β-NADH), Trypan Blue and Triton X-100 dye solution were purchased from Sigma-Aldrich (USA).
Snake venom collection and ethics approval
Lyophilized
Naja naja [
17] venom was purchased from Calcutta Snake Park, Kolkata, India and stored at 4 °C till further use. Venom concentration was expressed in terms of dry weight/protein equivalent. The study protocol was approved by the Institutional Biosafety Committee (IBSC) of Savitribai Phule Pune University, Pune, India.
Purification of NN-32
A 250 mg of Lyophilized whole venom of
Naja naja was dissolved in 5 ml of de-ionized water and given a heat treatment for 30 min at 60
oc in a water bath followed by centrifugation at 2500 rpm for 20 min. 50 mg of the supernatant was loaded onto a CM-cellulose column (100 × 20 mm) which was equilibrated with 0.02 M phosphate buffer (pH 7.2). A total of 42 fractions (each of 5 ml volume) were collected using the stepwise gradient of sodium chloride (0.02 M – 1 M in phosphate buffer, pH 7.2) with a constant elution rate of 30 ml/min at room temperature. Protein content in the fractions was estimated by Lawry’s method [
18]. All the fractions were checked for their cytotoxic activity against MCF-7 cells. The fraction which was showing cytotoxic activity was further purified by Reverse phase HPLC (Shimadzu LC-2010HT, Japan) using C18 column (4.6 × 250 mm) (Waters, USA) equilibrated with 0.1% Trifluoroacetic acid (TFA) in water and eluted with a linear gradient of 100% acetonitrile in 0.1% TFA at a flow rate of 1 ml/min. The HPLC profile of the fraction was monitored at 280 nm for 60 min using Shimadzu Prominence UV/Vis detector (SPD-20A).
Characterization of NN-32
MALDI-MS (Applied Biosystems, 4700 Proteomics Analyzer 170) was performed to determine the mass of the fraction protein. Mass spectrometric spectra were obtained using MALDI-TOF system. MS/MS spectra were searched using the Mascot database search engine against the NCBInr protein database.
Cell culture
Human Breast cancer cell lines (MCF-7 and MDA-MB-231) along with Human normal breast epithelial cell line (MCF-10A) were purchased from National Facility for Animal Tissue and Cell Culture, Pune, India. They were cultured in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 units/ml) and streptomycin (10 mg/ml). Cells were grown to sub confluence at 37 °C in a humidified atmosphere of 5% CO2.
MTT assay
MCF-7, MDA-MB-231 and MCF-10A cells were seeded at densities of 1 × 104/well into 96-well plates and incubated at 37 °C for 24 h under 5% CO2. The cells were then treated with different concentrations of NN-32 (0.125–16 μg/ml) and doxorubicin (0.5–5 μM) as the positive control. After 48 h of incubation, 20 μl of MTT solution (5 mg/ml) was added into each well and incubated for 4 h. The medium was discarded and the formazan precipitate was dissolved in DMSO. The absorbance of the mixtures was determined using a microtiter plate reader at 570 nm and the cell viability expressed as percentage inhibition relative to controls. All experiments were performed in triplicates. The IC50 was generated for each cell line from the dose response curve.
Anti-proliferation assay
MCF-7 and MDA-MB-231 cells were seeded at densities of 1 × 104 cells/well into 6-well plates and allowed to incubate for cell attachment for 24 h. These cells were then exposed to 5, 10 and 15 μg/ml concentrations of NN-32 and the plates incubated at 37 °C under 5% CO2, for 24, 48, and 72 h. At the end of the incubation periods, the medium was removed and washed with cold PBS followed by the addition of 1 ml of 0.05% trypsin-EDTA. The plates were then incubated for 15 min at 37 °C and after the majority of the cells had detached from the plate, they were harvested by spinning the suspension for 10 min at 1000 rpm using Eppendorf Centrifuge 5810 R (Hamburg, Germany) and the supernatant was discarded. 20 μl of the cell pellet were re suspended in 20 μl of 0.4% trypan blue solution. The dye-excluding viable cells were counted microscopically using a haemocytometer and expressed as percent of control cells that were still viable.
Neutral red uptake assay
The cells were seeded in 96-well plates and incubated overnight under 5% CO
2 at 37 °C until they reached 60% confluence. The medium was then discarded and replaced with 200 μl of fresh growth medium containing the same concentrations of the NN-32 as that used in the MTT assay. Untreated cells under the same conditions were used as controls. The plates were incubated at 37 °C for 24, 48 and 72 h under 5% CO2 and the cells were then washed three times with 200 μl of PBS. The plates were further incubated at 25 °C for 3 h in medium containing 200 μl NR solutions, and the cells subsequently washed to remove the NR solution. Cells were then exposed to fixing solution consisting of 1% CaCl
2 and 0.5% formaldehyde in milli-Q water for 2 min followed by two washes with 1% acetic acid and 50% ethanol in milli-Q water. The plates were incubated for 10 min and then read in a micro plate reader at 540 nm [
19].
Lactate dehydrogenase release assay
The cells were seeded in 96-well plates in 100 μl of media and then treated with different concentrations of NN-32 (0.125–16 μg/mL). The permeability of the cell membrane of MCF-7 and MDA-MB-231 cell lines after treatment with NN-32 was determined by LDH release assay [
19].
Statistical analysis
For the number of experiments indicated, data are shown as mean ± SD. The paired Student’s t test was performed to evaluate two independent groups of samples. In all analyses, p < 0.05 was taken as statistically significant.
Discussion
Since long, natural products from floral and faunal origin have been used for therapeutic purposes and around 87% of the human diseases are treated with these natural products and their related drugs [
20].
Cardiotoxin-3 (CTX-3), a basic polypeptide of 60 amino acid residues from
Naja naja atra venom induced apoptotic cell death accompanied by upgradation of both bax & endonuclease G and down regulation of bcl-x in K562 cells [
21]. drCT-1 a heat stable, 7.2 kDa protein toxin from Indian Russell’s viper venom is supposed to possess anti-proliferative, cytotoxic and apoptotic activity on EAC mice and human leukemic cells (U937/K562) [
22].
Salmosin, a disintegrin isolated from Korean snake venom, efficiently suppressed the growth of the metastatic tumor as well as the solid tumor in mice. Also, LAAO isolated from
Agkistrodon acutus snake venom arrest tumor cells at a sub-G1 phase of cell cycle and induced apoptosis via fas pathway in A549 cells [
23].
Crototoxin 2, a disintegrin isolated from
Crotalus atrox induced cancer cell migration and lung tumor colonization in BALB/c mice [
24]. VRCTC-310 is a natural product by combining two purified snake venom, a three protein fraction from
Crotalus durissus terrificus venom and
Naja naja atra, exerted an inhibitory effect on human and murine cell lines. In a phase-I study, 15 patients with refractory malignancies were given intramuscular injection daily for 30 days continuously to evaluate the tolerated dose (MTD), safety profile and pharmaceutical data. MCD was found out to be 0.017 mg/kg and recommended for phase-II studies [
25]. Cytotoxin-II isolated from Caspian cobra (
Naja Naja oxiana) showed potent anticancer effects in a breast carcinoma cell line via induction of apoptosis through lysosomal damage, production of intracellular ROS, mitochondrial damage and activation of caspases [
26].
The present study showed that NN-32 isolated from
Naja naja (Indian spectacled cobra) venom showed significant cytotoxicity against MCF-7 and MDA-MB-231 in both dose and time dependent manner, and considerably less towards normal breast cells (MCF-10A). The molecular mass of NN-32 was 6.7 kDa and N-terminal amino acid sequence for first 10 amino acid was LKCNKLVPLF [
16].
MS/MS spectra of NN-32 searched against the NCBInr protein database using Mascot database search engine showed homology with Cytotoxin 2 (accession no. P01440), Cytotoxin 2a (accession no. P86538), Cytotoxin 3 (accession no. P24780) from Naja naja; Cytotoxin 1 (accession no. P01447), Cytotoxin 2 (accession no. P01445), Cytotoxin 3 (accession no. P01446) from Naja kaouthia and Cytotoxin 6 (accession no. P073858) from Naja sputatrix.
NKCT1 is a protein toxin isolated from
Naja Koauthia venom after conjugation with gold nanoparticle i.e. GNP-NKCT1 showed anticancer effect both in vivo and in-vitro in EAC cells. GNP-NKCT1 induced caspase dependent apoptosis pathway in EAC cells and induced the late apoptotic stage and arrested cell cycle division at G0/G1 stage [
27].
NN-32 showed antitumor and antioxidant properties against EAC bearing BALB/c mice [
16]. NN-32 also showed anticancer activity in human leukemic U937 cells by promoting apoptosis, arresting cell cycle, suppressing vascular endothelial growth factor and matrix metalloproteinase activities [
28].
The major anticancer mechanism activated by snake venom toxins is by mitochondria-dependent cell death pathway. Taiwan cobra cardiotoxin-III induced apoptosis by loss of mitochondrial membrane potential, release of cytochrome c, activation of caspase 9 and caspase 3, and altered expression of Bcl-2 family proteins [
29,
30].
NN-32 showed cytotoxicity on EAC cells and it upregulated expression of proapoptotic proteins Bax and downregulated antiapoptotic protein Bcl-2 expression [
16]. Increase in Bax: Bcl-2 ration results in series of events that lead to the conversion of procaspase 9 to active caspase 9. Caspase 9 is an effective downstream molecule of the mitochondrial pathway of apoptosis and will lead to the conversion of active caspase 3 from procaspase 3, which further trigger a cascade of intracellular events leading to programmed cell death [
16]. Increase in expression of caspase 3 and caspase 9 in EAC cells after treatment suggested that NN-32 might have a role in the intrinsic pathway of apoptosis [
16].
Conclusion
The MTT, Antiproliferation, Neural red uptake and LDH release assay used to evaluate the cytotoxicity of NN-32 toxin purified from Naja naja venom on MCF-7 and MDA-MB-231 cell lines showed that the protein toxin NN-32 is significantly cytotoxic to these cell lines in a both dose and time dependent manner, and considerably less towards normal breast cells (MCF-10A). This finding highlights the potential of NN-32 toxin in the treatment of Breast cancer. However, detailed investigation of complete sequence, structure and molecular mechanism of action for this activity is required to potentiate the use this NN-32 toxin as a therapeutic agent for cancer treatment.
Acknowledgements
We are grateful to Prof. Ameeta Ravikumar for valuable suggestions, Mr. Prashant Gaikwad for assistance in the purification of NN-32 and to the Head, Department of Zoology for providing laboratory facilities.