Cytotoxic activity of NN-32 toxin from Indian spectacled cobra venom on human breast cancer cell lines

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).

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.

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).

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.

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.

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.

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.

The cells were seeded in 96-well plates and incubated overnight under 5% CO₂ 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% CaCl₂ 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].

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].

50 mg of venom applied on Ion exchange chromatography column (100 × 20 mm) resolve into several peaks (Fig. 1a). Among all the fractions, fraction number 35 was eluted with 0.5 M NaCl showed cytotoxicity against MCF-7 cell line. This fraction was further resolved through RP-HPLC using C18 column (4.6 × 250 mm), produced a single peak with a retention time of 3.6 mins (Fig. 1b).

The molecular mass of NN-32 determined by MALDI-MS was found to be 6.7 kDa (Fig. 2). MS/MS spectra of NN-32 were searched against the NCBInr protein database using the Mascot database search engine showed the list of toxins having homology with NN-32 (Table 1).

Cytotoxic activity of NN-32 toxin was determined against two human breast cancer cell lines of which, one is estrogen receptor positive (ER+) MCF-7 cells and another is estrogen receptor negative (ER-) MDA-MB-231 cells. The percent inhibition of growth of these two breast cancer cells after treatment with the NN-32 toxin and Doxorubicin after 48 h was determined. The response of MCF-7 and MDA-MB-231 cells to increasing concentration of NN-32 toxin and Doxorubicin are shown in Figs. 3 and 4 respectively. The results showed the tendency of both cell lines to inhibit sharply upon treatment with low NN-32 and Doxorubicin concentrations, with narrowing response intensity as the concentration of NN-32 and Doxorubicin were increased. The IC₅₀ values of Doxorubicin after 48 h of treatment was found out to be 4.1 μM and 15.1 μM for MCF-7 and MDA-MB-231 cell lines, while IC₅₀ values of NN-32 toxin after 48 h treatment ranged between 2.5 and 6.7 μg/ml for MCF-7 and MDA-MB-231 cell lines respectively. The IC₅₀ value of Doxorubicin and NN-32 after 48 h of treatment was found out to be 39.6 μM and 25 μg/ml respectively for the normal MCF-10A cells, is 10 times higher than that of the MCF-7 cells (Fig. 5). The NN-32 showed lower IC₅₀ values for the cancer cells compared to the normal breast cells, suggesting that the toxin could have great potentials as an anti-cancer agent.

The Anti-proliferative effects of the NN-32 toxin on MCF-7 and MDA-MB-231 cells were illustrated in Fig. 6. The percent survival of the MCF-7 cells after 24, 48 and 72 h of incubation with 10 μg/ml of NN-32 were 89, 68 and 58% respectively. However, MDA-MB-231 cells similarly treated with the NN-32 did not show much reduction in viability as the MCF-7 cells with values 87, 83 and 64% respectively. This suggested that the NN-32 could have a greater effect on the viability of MCF-7 than MDA-MB-231 cells.

Neutral Red uptake assay was performed to determine the lysosomal activity of MCF-7 and MDA-MB-231 cells treated with the NN-32 toxin showed a significant decrease in lysosomal activity in a dose-dependent manner (Fig. 7). This NR uptake assay showed the lower sensitivity than that of MTT assay, this may be because of the lower number of lysosomes in the breast cancer cell lines. Neutral red is a positively charged dye that diffuses through the cellular membrane of viable cells and accumulates in the lysosomes, thus the intensity of its staining is directly proportional to a number of viable cells.

LDH release in culture media is one of the indicators of cell death. Thus LDH release assay was performed to determine the cytotoxic activity of NN-32 toxin on human breast cancer cell lines. The LDH release curves of MCF-7 and MDA-MB-231 cell lines treated with different concentration of the NN-32 toxin showed that the cytotoxic effect of the NN-32 was concentration dependent (Fig. 8). The percent LDH release from MCF-7 cell lines after 72 h exposure to 0.25, 0.5, 1 and 2 μg/ml NN-32 toxin were 52, 61, 67 and 74% respectively. This effect is greater than those observed for the same concentration of NN-32 on MDA-MB-231 cell lines with 36, 46, 59 and 66% respectively after 72 h. However, a higher concentration of NN-32 toxin produced ever higher LDH release in both MCF-7 and MDA-MB-231 cells.