Figure 1.
SRL histochemistry to normal and cancerous human breast tissues.
SRL histochemistry was performed in human breast (normal, primary cancer and metastatic) tissue samples. SRL shows weak binding to normal human breast tissues but strong binding to primary cancer and metastatic breast tissues. All the images were obtained with 100X magnification. Arrows point to SRL binding to apical surface of the secretory gland epithelia. Representative images of both Haematoxylin-Eosin and Biotin-SRL staining are shown. SRL binding was evaluated through optical analysis by measuring the mean area of stained cells scored arbitrarily as intense (+++), moderate (++), light (+) and no staining (−).
Figure 2.
SRL inhibits proliferation of human breast cancer cells.
(A). SRL causes dose-dependent inhibition of human breast cancer MCF-7 and ZR-75 cell proliferation. The cells were incubated with or without different concentrations of SRL for 72 h. SRL-mediated cell growth inhibition is prevented by the presence of TF-expressing glycoprotein: MCF-7and ZR-75 cells were incubated with or without 20 µg/ml SRL in the presence of 100 µg/ml asialo bovine submaxillarymucin (aBSM) for 72 h. SRL caused time-dependent inhibition of MCF-7 cell proliferation. (B). The MCF-7 cells were incubated with or without different concentrations of SRL, BSA (40 µg/ml) and TBS for 24, 48 and 72 h before cell proliferation was assessed. Data represent Mean ±SD of triplicate determinations from three different assessments. *p<0.05; ***p<0.001.
Figure 3.
Effect of SRL on proliferation of non-tumorigenic and normal human breast epithelial cells.
(A and B) Non-tumorigenic MCF-10A and normal HMECs were incubated with different concentrations of SRL in serum free media for different time intervals (24, 48 and 72 h for MCF-10A) and 48 h (HMECs). Cell proliferation was measured using Calcein AM and MTT for MCF-10A and HMEC respectively. The experiments were carried out in triplicate, and the data were expressed as Mean ± SD percentage of control. *p<0.05; ***p<0.001.
Figure 4.
Sepharose-conjugated SRL induced growth inhibition of breast cancer cells.
(A) SRL shows strong binding to MCF-7 cells and very weak binding to (B) HMEC. (C) MCF-7 cells were incubated without or with SRL or Sepharose-SRL (C-SRL), at equivalent concentrations to SRL with 128 and 256 hemagglutination units (HAU) activity for 72 h before the cell proliferation was assessed by Calcein AM. Data represent Mean ±SD of triplicateexperiments. *** p<0.0001 when compared to control.
Figure 5.
(A) Annexin V cell surface binding in MCF-7 cells after incubation with SRL (20 µg/ml) for 24, 36 or 48 h. Percentages of cells in each quadrant are shown as inserts. The graph indicates percentage of the cell population in different phases. (B) Effect of SRL on cell cycle. MCF-7 cells were exposed to SRL (20 µg/ml) for 24, 36 and 48 h before the cellswere stained with propidium iodide and DNA content was measured by flow cytometry. The graph indicates percentage of cells in subG1, G1, S, and G2–M phases of the cell cycle.
Figure 6.
SRL-mediated inhibition of cell proliferation is associated with induction of apoptosis.
(A) MCF-7 cells were incubated without or with SRL (20 µg/ml) in the presence or absence of inhibitors (50 µM) to caspase-8 (z-IETD), caspase- 9 (z-LEHD) or pan-caspases (z-VAD-(OMe) for 48 h before the caspase 3/7 activity was assessed. (B) MCF-7 cells were incubated with or without SRL (20 µg/mL) in the presence or absence 50 µM pan-caspase inhibitor z-VAD-(OMe) for 48 h before the viable cells were assessed using Calcein AM. *P<0.05, **P<0.01, ***P<0.001. (C) MCF-10A and MCF-7 cells were incubated without or with SRL (20 µg/ml) for 48 h before the caspase 3/7 activity was assessed by caspase 3/7 Glo assay. P = 0.5847 and ***P<0.001 respectively for MCF-10A and MCF-7 cells.
Figure 7.
SRL induced apoptosis involves alteration of regulators.
(A) Immunohistochemistry of MCF-7 cells after treatment with SRL. MCF-7 cells were grown on cover slips and incubated with 20 µg/ml SRL for 36 h followed by staining with FITC-SRL, Anti-active caspase-3 primary antibody followed by pycoerythrin labelled secondary antibody and images were observed by fluorescence microscopy. (B) SRL-induced apoptosis in MCF-7 cells involves alteration of regulators in both the extrinsic and intrinsic apoptosis pathways. MCF-7 cells were incubated with SRL (20 µg/ml) for different time intervals. At specific time intervals whole cell protein was obtained, separated by electrophoresis, blotted on to Immobilon polyvinylidene difluoride membrane and probed with antibodies against FasL, FADD, tBID, PARP, pro-caspase-8, -9,-3 or active caspase-8, -9,-3. The bands were visualized using chemiluminescence kit. The blots were stripped and reprobed for β-actin which was used as loading control.