Human breast cancer cells, MCF-7 was purchased from NCCS (Pune, India). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Col I, FN, LN, dimethyl sulfoxide (DMSO), 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI), annexin V staining kit, acridine orange, ethidium bromide, RIPA buffer, protein A beads and agarose were purchased from Sigma Chemical, Inc. (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM), Fetal bovine serum (FBS), trypsin and antibiotic-antimycotic (100X) solution were obtained from Himedia Laboratories (Mumbai, India). Caspase 9 assay kit and mitochondria fractionation kit were purchased from Biovision (Milpitas, CA, USA). Anchorage independent plate (polyHEMA coated) was purchased from Cell Biolabs (San Diego, CA, USA). Protein estimation kit (Bradford method) was purchased from Thermo Scientific (Rockford, IL, USA). Primary antibodies against Bax, Bad, FAK, pFAK, α5 integrin, and HRP conjugated anti mouse and anti rabbit secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ras activation assay kit, and antibodies against MMP-9, MMP-2, FN, Col I, LN, Akt, pAkt, PI3K, pPI3K, JNK, pJNK, P³⁸MAPK, pP³⁸MAPK, actin and GAPDH were bought from Cell Signaling Technology (Beverly, CA, USA). Chemiluminescence western blot developing kit was from Bio-Rad (Hercules, CA, USA). All other reagents used in the study were of analytical grade or high quality available in the market.

Morus alba leaf lectin was purified from M. alba leaves by ammonium sulfate fractionation followed by immobilized metal ion affinity chromatography and anion exchange chromatography on DEAE [23]. The activity of MLL was measured using hemagglutination assay and the purity of the lectin was analyzed by silver stained SDS-PAGE (results are given as Supplementary Figure S1).

Breast cancer (MCF-7) cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml of antibiotic/antimycotic solution and incubated at 37°C in a humidified 5% CO₂ atmosphere. At 70% confluency, the cells were subcultured and the doubling time was found to be ∼30 h. After subculturing the cells were reseeded in 96 well culture plates and treated with varying concentrations of MLL (2, 5, and 10 μg/ml) for different time intervals (4, 8, 12, 24, and 48 h). MTT assay was done to measure the cytotoxicity and the results were expressed as the percentage of viable cells by measuring the absorbance at 560 nm of the dissolved formazan crystals produced under each condition (Ahmad et al., 2012). Also we have confirmed the anoikis induction caused by MLL by seeding equal number (2 × 10⁴) of cells (control as well as MLL treated) on anchorage independent plate first for 24 h and then transfered to normal plate for 4 h to check the attachment. Cell viability was checked by MTT assay.

Morphological analysis as well as the detachment of cells at different time points, under control and MLL treated conditions were done using the phase contrast microscope Nikon Eclipse TS-100 (Nikon Instruments Inc., Melville, NY, USA). The detached cells (after 24 h treatment) were reseeded in culture dishes coated with different matrix proteins (Col I, FN, and LN) and observed under the microscope for re-adherence and growth. Matrix proteins were coated on the culture dishes at a concentration of 50 μg/ml before seeding the cells and polylysine coated plates served as the control.

Nuclear fragmentation assay was done using agarose gel electrophoresis and DAPI staining. Intact and fragmented DNA from control and detached cells (after 24 h treatment) were isolated using apoptotic ladder kit (G-Biosciences, USA) and electrophoresed on agarose gel incorporated with ethidium bromide and visualized using gel doc (Bio-Rad, Hercules, CA, USA).

4′, 6-diamidino-2-phenylindole dihydrochloride staining was used to study the nuclear fragmentation within the cells (Tanious et al., 1992). Control as well as the detached cells (after 24 h treatment) were stained with DAPI (300 nM) and subjected to fluorescent imaging (BD Pathway^TM Bioimager system, USA).

Acridine orange/Ethidium bromide staining was used to check the membrane integrity during apoptosis (Cohen, 1993). 25 μl of cell suspension from control and detached cells after 24 h treatment (1 × 10⁴ cells/ml) was mixed with 1 μl acridine orange/ethidium bromide solution (one part each of 100 μg/ml of acridine orange and 100 μg/ml of ethidium bromide in PBS) for 5 min. Cells were observed and images were taken using fluorescent microscope (BD Pathway^TM Bioimager system, USA).

Annexin V staining was done to detect the number of live, necrotic and apoptotic cells as per the instructions from the manufacturer (Sigma Chemical, Inc. St. Louis, MO, USA). The detached cells (after 24 h treatment) along with control were treated with 50 μl of double label staining solution (AnnCy3 and 6CFDA). After 10 min incubation, washed with PBS and examined the cells under fluorescent microscope (BD Pathway^TM Bioimager system, USA).

Measurement of the Caspase 9 activity in the cell lysates of control and detached cells (after 24 h treatment) was done using fluorimetric assay kit by following the instructions from the manufacturer (Biovision). The assay was based on measuring the absorption maxima of the cleaved product AFC (7-amino-4-trifluoromethyl coumarin) at 505 nm from the substrate LEHD-AFC on caspase 9 action. Results were expressed as relative units in terms of fluorescence.

Matrix metalloproteinase activity was studied using gelatin zymography in cell culture supernatants from control and 24 h MLL treated cells (Kiran et al., 2006). The medium was subjected to gelatin impregnated polyacrylamide gel electrophoresis. After electrophoresis, the gel was washed with 2.5% Triton X-100 solution and incubated in 50 mM Tris–HCl containing 5 mM CaCl₂, 0.2 M NaCl at pH 7.5 for 24 h. The gel was stained with Coomassie brilliant blue (0.25%) followed by destaining. White bands of enzyme digested regions were visualized against a dark blue background.

Measurement of the active form of Ras was done using assay kit by following the instructions from the manufacturer (Cell Signaling Technology). It helped to measure the activation of Ras-GTPase in the cell by immunoprecipitating the active GTP bound Ras with glutathione resin and then determined by western blot analysis using the monoclonal antibody against Ras.

Effect of MLL on matrix proteins production was measured using ELISA (Engvall and Perlman, 1971). The medium was collected from control as well as the cells treated with MLL (5 and 10 μg/ml) for 24 h. ELISA was carried out using specific primary antibody (1:500 dilution) and HRP conjugated secondary antibody (1:1000 dilution) and the color was developed using o-dianisidine as the substrate. The concentrations of the antigens were estimated by measuring the absorbance of the colored HRP product spectrophotometrically at 490 nm in a microplate reader (Biotek Synergy 4, Winooski, VT, USA). The same protocol was used for determining the activity of Bax in the mitochondrial fraction of control as well as detached cells using anti Bax antibody.

Co-immunoprecipitation was done to study the interaction of integrin and FAK under normal as well as the 24 h MLL treated condition. Briefly, 200 μl of the cell extracts were incubated overnight at 4°C with primary antibody against α5 integrin. Then protein A agarose beads were added (20 μl of 50% bead slurry) and incubated with mild rocking for 3 h at 4°C. The tubes were centrifuged for 30 s at 4°C and washed the pellet five times with 500 μl of 1X cell lysis buffer. The pellets were resuspended in 20 μl 2X SDS sample buffer, mixed well and spun for 30 s. The samples were heated to 95°C for 5 min and centrifuged for 1 min at 14,000 × g. The input, immunoprecipitate and flow through fractions were loaded on SDS-PAGE gel and were analyzed by western blotting using anti-FAK and anti-integrin antibodies.

Whole cell extracts were prepared in RIPA buffer containing protease inhibitor cocktail. Estimation of total protein was done using Bradford method. Mitochondrial and cytoplasmic fractions were separated by using standard kit by following the manufacturer’s instructions. An equivalent amount of protein (∼40 μg/well) from control as well as treated cells (24 h) were electrophoresed in SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with 5% skim milk and probed with respective primary antibodies. After washing, HRP conjugated secondary antibodies were added and incubated. Protein bands were developed by chemiluminescent method. Images of interested proteins bands were captured and analyzed using ChemiDoc MP^TM System with Image Lab^TM Software, Bio-Rad (Hercules, CA, USA).

Minimum three independent experiments were performed for all assays. Results were expressed as mean ± SD of three experiments and data were analyzed using one way ANOVA using SPSS statistical analysis program. p-values of < 0.05 were considered as statistically significant.

In order to study the effect of MLL on detachment induced cell death in MCF-7 cells, confluent monolayers of cells were treated with varying concentrations (2, 5, and 10 μg/ml) of MLL for different time intervals (4, 8, 12, 24, and 48 h) and observed for detachment of cells from the culture dishes. Morphological observations (Figure 1A) indicated that the detachment of cells started during 8 h of incubation with 10 μg/ml MLL treatment. During 12 h, the detachment started with 5 μg/ml as well similar to that of 10 μg/ml MLL treatment. During 24 and 48 h, significant number of cells was detached even at 2 μg/ml concentration. Also, we have observed the percentage of viable cells remained in the plate after MLL treatment by MTT assay. More than 70% of cells were detached from the plates during 24 and 48 h in all the concentrations of MLL treatment (Figure 1B). We have also studied whether these detached cells can re-adhere and proliferate when sufficient conditions are given. For this, the detached cells were collected and reseeded on culture plates coated with different matrix proteins (Col I, FN, and LN and polylysine coated plates served as the control). The plates were kept under standard culture condition for 24 h and observed under microscope. After 24 h also the cells were floating and not able to adhere and grow even if they were provided with different matrix support for attachment. We have seeded the trypsinised cells (from the control plate) in different matrix coated plates as experimental control which got adhere to the plate. The results are given in Figure 1C. Anoikis induction by MLL was also confirmed by first seeding the cells (equal number of cells with and without MLL) on anchorage independent plate for first 24 h and subsequently in normal plate for 4 h. Here the control cells were able to attach and re-grow in the normal plate, but the cells treated with MLL could not. The cell viability in the normal plates were measured by MTT assay and the results are given in Figure 1D. So the results indicated that the MLL caused the detachment of cells from the plate and failed to re-adhere and grow further.

Further to this, we have done experiments (DNA fragmentation, membrane integrity measurement, phosphatidyl serine translocation, activity of caspase 9, Bax and Bad) to confirm the programmed death in the detached cells. Significant fragmentation of the nuclear DNA was observed in DAPI staining (Figure 2A) as well as agarose gel electrophoresis (Figure 2B) in detached cells compared to control. The percentage of cells with fragmented nucleus was significantly increased in a concentration dependent manner in the detached cells compared to control cells (shown as bar diagram). The integrity of the cell membrane was checked by acridine orange/ethidium bromide staining. Because of the loss of membrane integrity in apoptotic cells, the detached cells appeared as orange (colocalization of ethidium bromide along with acridine orange) compared to control cells where the cells gave bright green fluorescence (only acridine orange has taken up by the cells). The percentage of EtBr incorporation was measured and plotted as a bar diagram which indicated that the MLL treated cells, the membrane damage was significant. The results are given in Figure 2C. Phosphatidyl serine translocation to the outer membrane is an indication of apoptosis. Quantitative fluorescence staining using annexin V in control as well as detached cells gave the percentage of live (green fluorescent), apoptotic (orange fluorescent), and necrotic cells (red fluorescent). The results in Figure 2D indicated that the apoptotic cells were significantly more in detached cells on treatment with MLL. Bax and Bad are two proapoptotic BCl2 family protein often getting activated during apoptosis. Mitochondrial translocation of Bax from cytosol and oligomerisation of Bad in the mitochondrial membrane are responsible for the intrinsic apoptotic pathway. Western blot analysis (Figure 2E) showed an upregulation of both these proteins in detached cells compared to control in the mitochondrial fractions. Bar diagram indicated the relative intensity of each band in western blot analysis. Here we have used HSP-60 as the mitochondrial marker protein to show the change in expression was in mitochondria. The activity of caspase 9, the major caspase involved in the intrinsic apoptotic pathway was also upregulated in the detached cells (Figure 2F). We have also studied the activity of Bax in the mitochondrial fractions separated from control as well as detached cells by indirect ELISA. The results given in Figure 2G showed the upregulation of active Bax in the mitochondria in the detached cells which is also a positive indication of apoptosis. These results indicated that MLL promoted detachment induced cell death (anoikis) in MCF-7 cells in a time and concentration dependent manner.

Anoikis is a type of cell death induced by improper cell-ECM contacts and in this process, ECM proteins and ECM degrading enzymes have a major role to play. We have analyzed the effect of MLL on the production of two major matrix degrading enzymes MMP-2 and MMP-9 by gelatin zymography (Figure 3A) and western blot analysis (Figure 3B). The results indicated that there was an upregulation of MMP-9 activity in MLL treated condition compared to control, whereas the activity of MMP-2 was unaltered in the same experimental conditions. Also, we have checked the production of three important matrix proteins Col I, FN, and LN in control and MLL treated conditions using indirect ELISA. The level of Col I and LN was minimal and unaltered in control as well as MLL treated cells. The level of FN was significantly high in control cells and the level was reduced to more than 50% upon treatment with MLL (Figure 3C). The downregulation in the production in FN was also confirmed by western blot analysis (Figure 3D).

The downstream signaling mediates the interaction of cells with ECM through the transmembrane receptor protein integrin. Since we have found that the level of FN was significantly reduced upon MLL treatment, we were interested in studying the level of integrin which interacts with FN. Since the α5 subunit of integrin is involved in the FN mediated signaling, we have studied its level using western blot analysis. The results in Figure 4A indicated a downregulation of α5 integrin upon treatment with MLL compared to control. The next effector molecule in this signaling cascade is the FAK and the phosphorylation of FAK achieves further signaling. We have studied the level of FAK and its phosphorylated form by western blot analysis. The results (Figure 4A) indicated that there was a significant downregulation in the phosphorylation of FAK in MLL treated cells compared to control. We have studied the interaction between FAK and integrin by co-immunoprecipitation. First immunoprecipitation was done in the cell lysates (from control, MLL 5 μg/ml and MLL 10 μg/ml) with integrin antibody and the input, immunoprecipitate and flow through fractions were electrophoresed and immunoblotted with FAK and integrin antibody. In this experiment in control IP, we got a band when blotted with FAK indicated the association between FAK and integrin. The IP bands were stronger compared to input band indicating that the IP worked. But in the treated cell lysates, FAK bands were absent in IP but were detected in the flow through indicating the FAK-integrin interactions were not there. When blotted with integrin antibody, intense bands (compared to input) of equal intensity were observed in the IP of control as well as treated conditions. Nothing we could detect in flow through. These results are given in Figure 4B. Second immunoprecipitation was done with a control antibody (other than integrin, but from same species) and repeated the western blot using FAK and integrin antibody. Here in the immunoprecipitate could not detect any band but were detected in the flow through. This indicted that the IP band came in the previous experiment in the control was due to the interaction between FAK and integrin and not because of the binding to the FC binding sites of the antibody used. These experiment data we have included as a Supplementary Figure S2 in the manuscript.

So the decreased level of α5 integrin, reduced pFAK level and diminished interaction between integrin and FAK indicated that MLL inhibited the integrin mediated downstream signaling through FAK.

Ras superfamily of proteins belong to the class small GTPase are involved in switching on the genes that induce cell growth and proliferation and overexpression of Ras activity leads to cancer. The increased FAK signaling may be mediated through increased Ras activity. In this context, we have studied the effect of MLL on Ras activity (active GTP bound Ras) and Ras expression (western blot analysis of total cell lysate using anti ras antibody) in MCF-7 cells. The results in Figure 5 indicated a downregulation in the activity of Ras, upon treatment with MLL when compared to control cells but the protein expression remains the same.

PI3Ks are the heterodimeric lipid kinases involved in cell survival, growth, adhesion, transformation and apoptosis. It is the next effector molecule in the FAK signaling cascade and phosphorylated activation of PI3K resulted in the phosphorylation of Akt resulted in cell growth by preventing apoptosis. We have studied the expression of PI3K, pPI3K, Akt, and pAkt in MCF-7 cells treated with MLL. Significant reduction in the expression of pPI3K and pAkt was found when treated with MLL compared to control (Figure 6). p38 MAPK and JNK are belonging to the mitogen activated protein kinases induced by stress stimuli and have the significant role in inducing apoptosis. We have studied the effect of MLL on these two protein kinases and the results indicated that there was an upregulation in the phosphorylation of p38 MAPK, but no change in the expression of JNK/pJNK (Figure 6).