5.3 Results
5.3.4 Danazol abolishes tumorogenic (PMA) specific phosphorylation signal in MDAMB-231 Cells: We further asked whether Danazol has the potential to disrupt carcinogenic PMA specific
Chapter 5|133 Figure 5.11: CGA (Chlorogenic acid), β-Gly (β-Glycyrrhetinic acid) and GA (Gallic acid) cause prolonged translocation of PKC-α from the cytosol to the plasma membrane as revealed by immune- blotting. (A) Cells were stimulated with CGA (75 μg/ml) for 30 mins, 60 mins and 120 mins to study time- dependent translocation of PKC-α to the plasma membrane. (B) Cells were stimulated with β-Gly (10 μg/ml) for 60 mins, 120 mins and 240 mins to study time-dependent translocation of PKC-α to the plasma membrane in MDAMB-231 cells. (C) Cells were stimulated with GA (2.5 μg/ml) for 30 mins, 60 mins, 120 mins, 180 mins, 240 mins and 480 mins respectively to study time-dependent translocation of PKC-α to the plasma membrane in MDAMB-231 cells on stimulation with GA.
gave 23 and 20 phosphorylated proteins in lysate and membrane fractions respectively (Figure 5.12).
Cell lysate of PMA stimulated cells gave phosphorylated proteins in the molecular weight range of 13-156 kDa (Figure 5.12). In the presence of Danazol, PMA stimulated cells gave 17 and 19 phosphorylated proteins in lysate and membrane fractions respectively (Figure 5.12). Danazol was specifically targeting phosphorylated proteins with a native molecular weight of 156.3, 137.9, 120.7, 82 and 11.7 kDa (Figure 5.12, denoted by "*"). These phosphorylated bands were common in cells stimulated with PMA or Danazol, and PMA didn’t show these bands in presence of Danazol. This is probably due to competitive inhibition or feedback inhibition via over-activation. There were additional bands appearing in all the three treatments (PMA, Danazol or PMA in the presence of
Figure 5.12: MDAMB-231 cells respond to Danazol and Danazol treatment affects PMA induced cell signaling in cancer cells. MDAMB-231 cells were stimulated with PMA (100ng/ml), Danazol (70µg/ml) or PMA (100ng/ml) in presence of Danazol (100µg/ml) for 30mins and phosphorylated proteins were identified by immunoblotting as described in Chapter 2 (section 2.12). The developed blot was analyzed by PyElph 1.4 and a mock blot representing pattern of phosphorylated protein in each treatment is given. The details of molecular weight of each phosphorylated protein in each treatment is yet to be identified. The bands in Danazol lane are marked with "*" to denote phosphorylated protein disappeared in PMA stimulated cells due to Danazol whereas bands are marked with "#" to denote phosphorylated protein appearing probably due to stress in cells.
Chapter 5|135 danazol) to cancer cells, and probably represent stress linked signalling molecules (Figure 5.12, denoted by "#"). These results are initial observations that Danazol was affecting PMA induced phosphorylation signal in MDAMB-231 cells but an in-depth phospho-proteomics study is required to confirm the findings.
5.3.5 PKC-directed molecules induce apoptosis to kill breast cancer cells: MDAMB-231 breast cancer cells were treated with Danazol (70 µg/ml) in serum-free media for 24 hrs. Apoptosis was assessed by Acridine Orange & Propidium Iodide (AO/PI) staining as described in detail in Chapter 2 (Section 2.5). Quadrant analysis of the cell populations for appearance of healthy (lower left), early apoptotic (lower right), late apoptotic (upper right) and necrotic (upper left) phases was performed.
Untreated cells showed mostly healthy cells (~83 ± 2.48%) which was evident from a lower intake of propidium iodide into their genomic DNA. Danazol treated cells showed a lesser number of healthy cells with a concomitant increase of cells in the apoptotic (early and late) or death phases (Figure 5.13- A). At 70 µg/ml, it exhibited the proportion of cells in the early apoptotic (9.8 ± 0.82%) & late apoptotic (57.5 ± 3.13%) phases and only 30 ± 1.62% healthy cells (Figure 5.13-A). The flow cytometric analysis indicates that the cell-death caused by Danazol might be following apoptotic rather than necrotic pathways. Likewise, MDAMB-231 cells were also treated either with Flunarizine (5.9 μg/ml) or Cinnarizine (54 μg/ml) for a period of 24 hrs. Apoptosis was assessed by flow cytometry by AO/PI method as described in Chapter 2 (Section 2.5). AO/PI staining indicated that a significant
Figure 5.13: Danazol, Flunarizine and Cinnarizine causes cell death in cancer cell due to induction of apoptosis: MDAMB-231 cells were treatment with (A) Danazol (70 μg/ml) (B) Flunarizine (5.9 μg/ml) &
Cinnarizine (54 μg/ml) in serum-free media for 24 hrs and stained with Acridine orange (AO)/Propidium iodide (PI) as given in Chapter 2 (Section 2.5). Cells treated with serum-free media served as control.
A. B.
C.
D.
proportion of treated cells are shifted to the right corner with a significant proportion of late apoptotic cells in Flunarizine (35.09 ± 4.51%) as well as Cinnarizine treated (30.93 ± 3.57%) cells (Figure 5.13- B). There was also a significant proportion of early apoptotic cells in Flunarizine treated (24.27 ± 1.81
%) and Cinnarizine treated (27.46 ± 2.34%) cells.
Likewise, phytochemicals were also evaluated for their ability to induce apoptosis in cancer cells. MDAMB-231 cells were treated individually with Chlorogenic acid (75 μg/ml), β- Glycyrrhetinic acid (10 µg/ml) and Gallic acid (2.5 µg/ml) for a period of 24 hrs. In Chlorogenic acid treated cells, the proportion of cells in early apoptotic was 0.3±0.12%, late apoptotic was 44.2±3.56
% and necrotic was 25.9± 1.66% (Figure 5.14-A). Whereas in untreated cells, the proportion of cells in early apoptotic was 8.3±1.52%, late apoptotic was 17.1±2.85% and necrotic was 13.8±0.48%.
Chlorogenic acid treated cells showed quite lesser number of healthy cells (29.6±2.33%), with a significant increase of cells in the apoptotic (early and late) or death phases (Figure 5.14-A). Similarly, in β-Glycyrrhetinic acid, Epigallocatechin gallate and Gallic acid treatments, there was a significant shift of healthy cells to the early apoptotic and late apoptotic phases in comparison to untreated cells.
Figure 5.14: Evidence of apoptotis as a mode of death in CGA (Chlorogenic acid), β-Gly (β- Glycyrrhetinic acid) and GA (Gallic acid) treated breast cancer cells. MDAMB-231 cells were treated with CGA (75 μg/ml), β-Gly (10 μg/ml) and GA (2.5 μg/ml) in serum-free media for 24 hrs and stained with acridine orange (AO)/Propidium iodide (PI) as described in Chapter 2 (Section 2.5). Cells treated with serum- free media served as control.
Chapter 5|137 In β-Glycyrrhetinic acid treated cells, 12.73 ± 1.63% and 56.57 ± 5.21% cells were in the early and late apoptotic phases respectively while those statistics in case of Epigallocatechin gallate treated cells were 33.85 ± 4.26% and 31.74 ± 2.95% respectively. Similarly, in Gallic acid treated cells 12.23 ± 1.45%, and 51.22 ± 6.21% cells were in the early and late apoptotic phases respectively (Figure 5.14- B).
We also explored the alkyl cinnamates mediated cell death of breast cancer cells and the underlying molecular mechanism (necrotic or apoptotic). MDAMB-231 breast cancer cells were treated with the different alkyl cinnamates in serum-free medium for a period of 24 hours at 37ºC at their respective IC50 concentration. The differential population of healthy, apoptotic (early or late), necrotic and dead cells were identified by Acridine orange & Propidium iodide (AO/PI) double staining method described in Chapter 2 (Section 2.5). Untreated cells showed mostly healthy cells (70
± 2.69%), and this was evident from a lower intake of propidium iodide into their genomic DNA.
Alkyl cinnamate treated cells showed a lesser number of healthy cells with a concomitant increase of cells in the apoptotic (early and late) or death phases (Figure 5.15). DM 2-8 was the most potent molecule and exhibited the highest proportion of cells in the necrotic phase (39.66 ± 3.72%) and 24.68
± 2.54% in the late apoptotic phase. It was followed by DM 2-4 and DM 2-3 which showed late apoptotic cells at 38.04 ± 3.63% and 35.69 ± 2.92 % respectively. Thus, all the candidate molecule treatment pushed a significant fraction of cells towards apoptosis. The flow cytometric analysis indicates that the cell-death caused by alkyl cinnamates might be following apoptotic rather than necrotic pathways.
Figure 5.15: Alkyl cinnamates cause death of MDAMB-231 cells following apoptosis. MDAMB-231 treated with different alkyl cinnamates and distribution of healthy, dead, apoptotic and necrotic cells. Cells were treated alkyl cinnamates (IC50 for 24 h), stained with acridine orange and propidium iodide and analyzed by flow cytometry.
5.3.6 PKC directed molecules induce degradation of genomic DNA during apoptosis of