Androstane Derivatives Induce Apoptotic Death in ...

Supplementary Material

Androstane Derivatives Induce Apoptotic Death in MDA-MB-231 breast cancer cells Dimitar S. Jakimov a 1, Vesna V. Kojić a, Lidija D. Aleksić a, Gordana M. Bogdanović a, Jovana J. Ajduković b, Evgenija A. Djurendić b, Katarina M. Penov Gaši b, Marija N. Sakač b, Suzana S. Jovanović-Šanta b a

b

Oncology Institute of Vojvodina, Put Doktora Goldmana 4, 21204 Sremska Kamenica, Serbia Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia

Content: EXPERIMENTAL METHODS ................................................................................................. 2 Cell culturing and chemicals ............................................................................................................... 2 Colorimetric MTT assay ..................................................................................................................... 2 Cell cycle analysis by flow cytometry ................................................................................................ 3 Measuring of apoptosis and necrosis by flow cytometry .................................................................... 3 Morphological study of apoptosis ....................................................................................................... 3 Western Blot analysis.......................................................................................................................... 3 Data analysis ....................................................................................................................................... 4

RESULTS ................................................................................................................................... 5 In Vitro cytotoxicity results................................................................................................................. 5 Cytotoxicity on MCF-7 cell line during 24 h exposure ....................................................................... 5 Cytotoxicity on MCF-7 cell line during 48 h exposure ....................................................................... 6 Cytotoxicity on MCF-7 cell line during 72 h exposure ....................................................................... 7 Cytotoxicity on MDA-MB-231 cell line during 24 h exposure ........................................................... 8 Cytotoxicity on MDA-MB-231 cell line during 48 h exposure ........................................................... 9 Cytotoxicity on MDA-MB-231 cell line during 72 h exposure ......................................................... 10 Cytotoxicity on PC3 cell line during 48 h exposure .......................................................................... 11 Cytotoxicity on HeLa cell line during 48 h exposure ........................................................................ 12 Cytotoxicity on HT-29 cell line during 48 h exposure ...................................................................... 13 Cytotoxicity on A549 cell line during 48 h exposure ........................................................................ 14 Cytotoxicity on MRC-5 cell line during 48 h exposure ..................................................................... 15 The IC50 values .................................................................................................................................. 17

Results of flow cytometry analysis ................................................................................................... 19 Analysis of cell cycle distribution by flow cytometry ....................................................................... 19 Modulation of the cell cycle .............................................................................................................. 24 Analysis of apoptosis by flow cytometry........................................................................................... 25

Results of apoptotic morphology screening ...................................................................................... 31 Photographs of native cells in cultures and stained specimens .......................................................... 31 Detected morphological changes ....................................................................................................... 35

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Corresponding author: Tel.: +381 21 4805577; fax: +381 21 6613741. E-mail address: [email protected] (Dimitar S. Jakimov).

Experimental methods Cell culturing and chemicals Stock solutions of the investigated steroidal compounds (0.1 M) were prepared and diluted to various concentrations with serum-free culture medium. All other chemicals used in this study, if not specified, were commercial products of a reagent grade. Cells were cultured in flasks (25 cm2, 10 mL, Costar) in DMEM (Dulbecco's Modified Eagle's Medium, Sigma) with 4.5% of glucose and 2 mM L-glutamine, supplemented with 10% (v/v) fetal calf serum (FCS, Sigma), and antibiotics antimicotics solution (Sigma), and incubated at 37ºC, with absolute humidity and 5% CO2 atmosphere in total darkness (incubator HeraCell, Heraeus). Cells were sub-cultured twice a week and a single-cell suspension was obtained using 0.1% trypsin in EDTA (Serva). Cells were harvested in logarithmic phase of growth. Exponentially growing viable cells were used throughout the experiments. Cell viability, determined by trypan blue dye exclusion test (DET) was >90%. For in vitro antiproliferative assays cell lines were seeded in flat base 96 well microplates (Sarstedt) with a density of 5103 cells in 90 µL volume of medium per well, in quadruplicate for each sample. Seeded plates were left in incubator, allowing the cells to attach to the bottom. After 24 h, all tested compounds were added to the corresponding wells with experimental cultures in 10 µL volumes of appropriate concentrations in order to obtain required final concentration of the compounds in cultures. Cells were further incubated under the same conditions. Total incubation time of control and samples treated with investigated compounds was 24, 48 or 72 hours. The MDA-MB-231 cells for analysis of cell cycle modulation and apoptosis induction experiments were grown in flasks under similar conditions. Flasks were seeded with 5105 cells per 10 mL medium volume and kept in incubator overnight allowing the cells to adhere to the flask surface. After initial incubation of 24 h, compounds 1 – 9 and formestane were added to the experimental cultures in 1 mL volume with proper concentration to set their final concentration in cultures equal to the IC50 values estimated earlier by MTT assay. Total incubation time with steroidal compounds was 48 h or 72 h. Cultures were terminated after treatment and cells were separated from flasks by trypsinization (0.1% trypsin in EDTA solution), pelleted by centrifugation 10 min / 200g (800 rpm), and counted in a 0.1% trypan blue stain (DET). Colorimetric MTT assay The assay is based on the cleavage of the tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] to formazan by mitochondrial dehydrogenases in viable cells. Sterile, 5 mg/mL MTT solution (Sigma), prepared just before the application, was added to each well (10 L per well) and the incubation continued for next 3 hours. The precipitated formazan crystals were solubilized adding 100 μL of acidified (0.04 mol/L HCl) 2-propanol to each well. The absorbance was read after 5 minutes incubation at room temperature on a microplate reader (Multiskan Accent, Thermo Labsystems) at 540 nm test wavelength and a reference wavelength of 690 nm. Wells containing cells without tested compounds were used as a control. Wells containing only complete medium were used as blank. The absorbance (A) of control samples was at least three times greater than the mean A value of blanks. The cytotoxicity (antiproliferative activity) of compounds trough the administrated concentration array was expressed as a percentage, according to the formula: CI [%] = (1 – As / Ac) x 100 2

wherein Ac is the absorbance of the control sample, and As the absorbance of the experimental samples. Mean values, standard deviations (SD) and coefficients of variation (CV) were calculated for each concentration. The CV in both experimental and control samples never exceeded 15%, and for most of the samples were below 10%. Cell cycle analysis by flow cytometry Following 48 and 72 hours of treatment with the compounds, cell fixation was performed with 70% ethanol, 30 minutes on ice. After centrifugation, ribonuclease A (RNase A, 100 units mL−1, Sigma Aldrich) and PI (400 mg mL−1) were added to each sample. Cell suspensions were incubated in the dark at room temperature for 30 minutes. Each sample was then filtered through a 35 μm grid before analysis on the flow cytometer. The cell excitation wavelength was 488 – 514 nm, and the fluorescence emission of PI was approximately 610 nm. The FL2 parameter of BD FACSCalibur E440 flow cytometer was used for one parametric analysis. In each analysis, 20 thousand events were recorded and the percentages of the cells in the different cell-cycle phases were calculated by using BD CellQuest Pro software.

Measuring of apoptosis and necrosis by flow cytometry Cells from each sample were collected by centrifugation for 5 min on 800 rpm, (Megafuge 1.0 R, Heraeus, Thermo Fisher Scientific) and pellets were re-suspended in 1 mL of phosphate buffer (PBS, pH 7.2). The cells were washed twice with cold PBS and then re-suspended in binding buffer to reach the concentration of 1106 cells/mL. The cell suspensions in volume of 100 µL were transferred to 5 mL culture tubes and mixed with 5 µL of both Annexin V and propidium iodide. The cells were gently vortexed and incubated for 15 min at 25°C in the dark. After incubation, 400 µL of binding buffer was added to each tube and suspensions were analyzed after 1 h on BD FACSCalibur E440 (Becton Dickinson) flow cytometer and Cell Quest computer software.

Morphological study of apoptosis The same cell harvesting methodology was used as for flow cytometry analysis. Slide preparation process was conducted in aseptic conditions. All processing manipulations on cell suspensions (hypotonic treatment for adequate cell spreading, fixation and centrifugation) were adapted to preserve the cell cytoplasm. Staining of specimens (aged for 24 h) was performed using 2% Giemsa stain (Merck) in potassium phosphate buffer. Stained cells were observed and analyzed by microscope Olympus BX51. Microscope slides were coded to exclude biases. Documentation images of native cells in culture and stained specimens were captured with Canon 1100D and Olympus CAMEDIA C3040 digital cameras attached to the microscopes, inverted and normal, respectively.

Western Blot analysis The protein concentration in MDA-MB-231 cell lysate was determined by Bradford protein assay in a 96 well microplate (Multiskan Accent spectrophotometer, Thermo Labsystems) using bovine serum albumin as a standard. Molecular mass markers for proteins were obtained from Amersham Biosciences. For Western blot, 50 μg of proteins per sample were separated by SDSPAGE vertical electrophoresis (Hoefer MiniVE), transferred to a polyvinylidene difluoride 3

(PVDF) membrane Hybond-P (Amersham Biosciences) and blotted with monoclonal primary antibodies against proteins of the apoptotic pathway. Mouse monoclonal antibodies against human Bcl-2 and caspase-3 were obtained from R&D Systems, BAX (BCL-2-associated X protein) and Actin (which was used as internal control) were purchased from Calbiochem. Rabbit anti- poly (ADP-ribose) polymerase (PARP) was purchased from Santa Cruz Biotechnology. Blots were developed with an enhanced chemiluminescence (ECL Plus, Amersham Biosciences) detection kit including peroxidase-labeled donkey anti-rabbit and sheep anti-mouse secondary antibodies used for detection of proteins. Chemiluminescent signals were recorded on Hyperfilms (Amersham Biosciences), which were processed with Kodak EXOMAT II developer reagents and photographed on a negatoscope with Canon 1100D camera on mini-tripod.

Data analysis All manual data analysis and statistical processing were done in Microsoft Office Excel program.

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Results In Vitro cytotoxicity results Graphics represent data obtained from in vitro MTT assay and a median effect analysis used to calculate IC50 values of the investigated compounds 1 – 9 and reference compounds (R) formestane (For) and Doxorubicin (Dox). Coefficients of variation (CV) are given as error bars.

Cytotoxicity on MCF-7 cell line during 24 h exposure

Mean value: CV:

3.84 9.87

18.54 15.01 33.45 37.93 6.57 5.81 10.17 7.96

Mean value: CV:

2.81 3.74

5.02 3.28

28.74 12.46 43.21 4.61 6.33 10.00

Mean value: 13.58 23.37 27.40 19.71 16.20 CV: 9.29 6.69 6.68 3.14 4.02

Mean value: CV:

1.78 5.93

8.58 5.11

38.01 30.31 52.33 14.36 6.85 4.53

Mean value: 10.49 17.59 24.95 26.95 55.81 CV: 4.48 11.25 8.46 9.34 8.44

Mean value: 26.95 33.86 35.08 34.51 55.95 CV: 9.62 7.10 14.74 4.70 8.11

Mean value: 11.31 12.47 23.50 29.90 26.82 CV: 4.55 7.27 6.83 7.31 8.62

Mean value: 16.87 24.98 36.17 27.83 31.84 CV: 8.87 9.03 7.01 9.81 8.89

Mean value: CV:

2.75 3.00

7.07 5.21

3.26 8.75

5.66 6.81

15.82 9.21

Mean value For: 1.43 3.88 3.06 3.23 18.40 CV: 6.998 8.386 3.744 9.827 5.125 Mean value Dox: 27.37 40.18 50.39 60.97 68.53 CV: 6.56 8.18 7.61 3.06 5.78

5


Mean value: 16.05 23.20 24.76 32.62 39.66 CV: 4.76 2.68 5.11 7.78 7.96

Mean value: CV:

4.30 4.14

6.00 5.24

33.82 14.99 44.79 3.69 6.33 10.00

Mean value: 30.23 39.96 44.59 29.12 22.55 CV: 7.58 8.29 7.36 6.84 7.54

Mean value: CV:

9.35 4.04

10.34 28.90 51.93 65.53 3.12 7.96 5.69 10.68

Mean value: CV:

7.89 4.26

16.45 24.69 42.34 55.08 11.44 2.83 7.56 8.44

Mean value: 22.47 32.77 44.44 53.10 64.92 CV: 4.06 3.37 8.24 9.25 13.30

Mean value: CV:

9.98 4.55

11.15 22.38 25.18 25.66 7.27 6.83 12.18 8.62

Mean value: 15.59 23.77 35.09 26.75 30.76 CV: 8.87 9.03 7.64 9.81 8.89

Mean value: CV:

3.12 3.45

7.38 4.59

6.78 4.58

8.30 6.10

21.25 7.71


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Mean value: 11.61 16.64 13.71 18.87 20.90 CV: 11.69 3.86 5.67 6.40 4.68

Mean value: CV:

2.36 4.42

6.73 4.75

21.11 31.95 24.43 6.71 9.61 7.57

Mean value: CV:

9.97 5.05

Mean value: 10.75 16.22 41.36 51.90 62.11 CV: 6.92 1.38 4.21 6.72 6.48

Mean value: 18.20 33.06 32.58 37.16 61.19 CV: 9.55 7.02 6.77 8.93 1.40

Mean value: 27.65 28.82 45.03 49.36 62.25 CV: 7.05 6.21 5.73 5.11 6.58

Mean value: 25.93 33.65 25.20 36.71 52.92 CV: 10.75 7.25 9.40 9.45 8.21

Mean value: 15.79 15.45 24.02 26.35 54.00 CV: 6.04 2.70 10.04 10.67 9.00

Mean value: CV:

9.51 8.81

12.22 19.75 34.11 5.57 9.54 6.75

9.44 2.60

14.47 20.25 26.83 25.09 10.11 13.22 9.42 3.64


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Cytotoxicity on MDA-MB-231 cell line during 24 h exposure

Mean value: 11.29 CV: 6.76

8.91 6.44

19.29 29.97 49.76 9.12 12.48 12.56

Mean value: 23.00 18.98 31.75 39.07 41.27 CV: 5.18 7.67 8.69 5.70 5.30

Mean value: CV:

6.98 5.58

13.42 17.86 33.64 53.99 8.44 9.81 7.21 9.64

Mean value: 14.99 23.82 35.99 36.08 50.73 CV: 9.88 8.23 5.84 8.89 8.45

Mean value: 21.43 32.90 36.22 47.19 49.17 CV: 9.66 12.53 11.18 10.13 7.14

Mean value: 21.64 28.40 39.35 46.07 52.06 CV: 7.50 11.71 8.41 10.37 9.23

Mean value: 13.89 20.23 29.59 44.76 55.79 CV: 8.67 6.34 11.74 6.10 7.49

Mean value: 10.81 30.95 21.18 43.14 59.98 CV: 7.09 13.90 9.02 11.69 2.81


9.52 2.26

15.65 37.57 25.80 5.16 11.60 7.67


8


Mean value: 17.91 17.49 47.44 56.24 60.69 CV: 6.56 6.56 3.33 5.89 4.87

Mean value: 25.03 24.00 48.06 56.95 61.24 CV: 4.58 3.27 9.89 4.56 5.16

Mean value: CV:

4.72 3.51

Mean value: 17.12 36.16 41.96 51.49 79.69 CV: 6.77 7.22 7.75 7.00 8.49

Mean value: 32.05 35.80 52.17 53.82 56.61 CV: 3.87 7.25 8.03 9.39 5.84

Mean value: 36.28 38.24 53.79 55.52 65.66 CV: 5.00 7.17 4.95 10.13 8.60

Mean value: 26.17 35.00 38.94 49.65 77.10 CV: 3.39 7.32 8.00 6.22 9.00

Mean value: 20.31 37.42 41.43 50.58 69.95 CV: 3.21 6.93 8.21 7.35 9.65

Mean value: CV:

9.32 5.89

32.51 51.79 52.43 61.13 5.35 5.09 6.57 7.56

32.06 50.22 50.46 63.01 9.17 9.24 2.81 6.49


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Mean value: 17.33 20.11 50.27 64.74 66.48 CV: 2.93 3.52 8.52 8.05 9.75

Mean value: 23.33 27.13 50.60 63.24 67.39 CV: 7.95 5.41 5.19 8.63 10.97

Mean value: 11.38 31.85 52.85 55.24 68.62 CV: 2.39 4.04 6.35 3.62 5.62

Mean value: 22.70 44.50 54.60 63.48 78.54 CV: 9.33 5.52 7.30 4.82 7.85

Mean value: 41.32 42.40 52.65 54.73 58.74 CV: 7.97 3.91 4.07 3.83 3.62

Mean value: 32.95 39.35 59.01 64.15 66.00 CV: 8.27 3.79 2.97 8.18 4.59

Mean value: 31.78 35.73 46.35 59.83 66.62 CV: 10.87 8.21 7.72 6.36 8.84

Mean value: 27.12 35.36 47.22 59.98 69.28 CV: 8.66 7.00 2.80 6.67 3.14

Mean value: 12.20 23.11 55.59 60.76 65.71 CV: 9.86 7.56 6.65 7.43 6.83

Mean value For: CV: Mean value Dox: CV:

26.77 37.95 44.77 44.76 54.94 14.03 8.38 6.04 4.51 5.08 34.53 46.44 71.66 85.61 89.76 10.47 7.69 8.11 8.85 9.47

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Cytotoxicity on PC3 cell line during 48 h exposure

Mean value: CV:

9.47 5.96

17.17 37.24 33.72 59.00 2.95 6.18 5.22 10.67

Mean value: 13.24 22.07 32.32 40.22 62.88 CV: 5.09 5.80 3.30 5.36 7.36

Mean value: 13.68 19.69 25.36 47.19 58.76 CV: 4.60 6.40 3.95 4.61 11.59

Mean value: 13.31 15.65 35.39 49.12 57.77 CV: 3.58 4.30 5.88 5.76 10.00

Mean value: 10.81 18.14 28.58 50.83 58.06 CV: 8.97 5.92 8.53 7.46 6.05

Mean value: 20.72 17.73 37.50 43.80 60.29 CV: 7.22 2.45 4.13 6.53 9.93

Mean value: 20.09 27.57 23.19 45.38 63.29 CV: 7.78 5.97 5.83 8.20 7.90

Mean value: 16.09 25.39 31.16 43.29 57.50 CV: 8.41 5.30 6.98 10.84 9.09

Mean value: CV:

7.52 4.01

29.11 45.54 48.14 57.05 6.64 6.49 9.71 4.89


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Cytotoxicity on HeLa cell line during 48 h exposure

Mean value: 18.33 28.25 33.32 39.66 62.14 CV: 4.23 7.79 6.88 5.85 6.50

Mean value: 14.78 27.55 33.52 42.91 60.52 CV: 5.92 6.46 6.13 6.03 7.89

Mean value: 19.71 23.27 39.14 48.55 51.71 CV: 1.98 8.21 10.90 12.33 7.70

Mean value: 25.80 31.30 44.24 46.21 54.14 CV: 9.15 5.64 6.60 7.79 9.65

Mean value: 22.54 26.50 40.94 46.96 56.66 CV: 9.19 11.23 7.99 12.50 5.48

Mean value: 10.79 12.73 30.97 44.89 57.75 CV: 11.48 5.43 4.44 6.60 11.89

Mean value: 23.22 34.54 39.89 47.54 53.66 CV: 11.16 5.53 7.59 11.49 8.90

Mean value: 31.64 34.12 37.53 51.93 58.57 CV: 11.44 8.52 5.45 9.04 13.98

Mean value: 22.50 28.30 33.33 54.86 62.98 CV: 7.60 7.92 4.48 11.93 12.88

Mean value For: CV: Mean value Dox: CV:

17.22 23.34 52.57 64.23 71.74 11.69 2.78 4.05 10.77 3.34 22.80 34.16 48.88 69.24 62.14 10.53 6.92 9.40 3.79 7.46

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Cytotoxicity on HT-29 cell line during 48 h exposure

Mean value: 23.09 29.96 36.41 62.25 59.95 CV: 12.55 6.37 9.71 6.39 6.10

Mean value: 31.38 43.66 42.65 54.96 62.84 CV: 2.23 10.26 7.65 10.34 4.50

Mean value: 21.88 30.72 41.41 48.41 55.22 CV: 9.24 10.02 7.51 4.78 8.21

Mean value: 22.67 31.20 41.29 50.30 58.39 CV: 13.34 3.08 8.01 3.20 12.03

Mean value: 41.57 38.55 44.74 52.84 71.60 CV: 5.80 4.03 7.57 11.79 8.07

Mean value: 14.19 19.14 18.54 20.82 31.98 CV: 10.71 8.73 12.21 11.53 13.14

Mean value: 37.61 43.03 39.81 25.48 25.47 CV: 8.48 12.29 8.08 11.46 11.14

Mean value: 15.02 30.87 35.17 44.55 51.85 CV: 10.61 8.99 8.73 7.62 9.40

Mean value: 33.20 40.23 42.30 42.93 58.70 CV: 13.10 4.61 9.27 10.56 6.09


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Cytotoxicity on A549 cell line during 48 h exposure

Mean value: CV:

2.28 4.13


12.82 13.40 21.37 35.99 9.91 6.49 11.35 7.09

Mean value: CV:

7.02 6.78

Mean value: 15.09 42.25 38.67 45.76 55.95 CV: 7.29 6.37 6.38 9.96 12.33

Mean value: 15.20 17.42 CV: 8.03 9.62

7.35 6.10

31.59 35.98 10.87 9.25

Mean value: CV:

Mean value: CV:

3.22 7.41

8.47 7.83

21.75 8.76

7.06 4.57

28.09 4.44

Mean value: 36.42 41.41 49.94 43.49 55.98 CV: 6.56 3.78 10.57 5.78 6.69

0.77 4.15

1.89 6.33

9.10 8.37

10.90 38.14 40.39 6.99 6.02 10.70

6.26 17.08 11.10 11.23 11.00 10.49

4.21 1.95

Mean value: CV:

3.02 6.89

2.60 3.96

12.47 30.83 36.95 47.93 9.96 8.83 8.99 9.69

2.93 5.02

8.36 11.47


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Cytotoxicity on MRC-5 cell line during 48 h exposure

Mean value: 14.58 20.23 24.07 27.83 27.74 CV: 7.86 3.62 5.44 10.11 4.42

Mean value: CV:

Mean value: CV:

23.40 21.55 24.72 23.07 6.23 5.50 3.31 6.74

Mean value: 10.31 21.30 24.83 15.49 CV: 10.15 9.45 4.63 9.34

7.32 9.46

Mean value: 12.25 15.18 26.30 22.96 27.10 CV: 4.18 5.44 8.10 7.23 6.52

7.89 6.99


22.69 27.19 28.24 3.83 6.46 5.67

7.04 8.77

24.73 21.12 20.82 29.76 5.69 6.62 0.57 6.34

9.19 8.10

Mean value: 21.64 20.62 22.19 26.17 28.99 CV: 4.93 9.85 6.81 5.14 10.43

Mean value: 8.18 12.41 16.68 8.86 11.24 CV: 10.13 5.93 13.37 10.82 7.96

Mean value: CV:

4.97 5.24

15.95 11.58 11.92 8.12 9.17 4.77 12.43 13.21


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Figure S-1: Comparison of the cytotoxicity of compounds 4, 5 and 6 after 48 h treatment. Compounds were active on both breast cancer cell lines MCF-7 and MDA-MB-231 and antiproliferative effects were mostly linear and dose-dependent. Derivatives were inactive on MRC5 cells.

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The IC50 values Table S-1: Colored presentation of the IC50 values (50% inhibitory concentration) [µM] of tested steroidal compounds 1 – 9 and reference compounds formestane (For) and Doxorubicin (Dox) determined by median effect analysis.

PC3

HeLa

HT-29

MRC-5

K562

A549

Compounds

Cell lines

24h

48h

72h

24h

48h

72h

48h

48h

48h

48h

48h

48h

1

>100

>100

>100

>100

6.428

2.721

41.289

23.966

5.288

N/A (>100)

>100

>100

2

>100

>100

>100

>100

4.560

1.971

22.385

21.071

2.459

>100

>100

>100

3

>100

N/A (>100)

>100

>100

5.183

2.730

28.206

32.731

17.413

N/A (>100)

N/A (>100)

>100

4

86.623

12.324

9.502

>100

2.166

0.615

19.375

22.376

10.458

>100

13.716

>100

5

>100

52.701

32.012

64.358

3.822

1.016

20.619

18.690

1.044

N/A (>100)

6

>100

4.113

6.640

39.041

1.031

0.560

21.372

29.739

>100

>100

30.741

7

>100

>100

>100

38.278

2.496

1.609

22.527

24.254

N/A (>100)

>100

27.307 12.738

8

>100

>100

>100

33.025

3.461

1.595

33.155

10.246

45.831

>100

>100

>100

9

>100

>100

>100

>100

5.182

2.825

11.503

7.820

16.428

>100

75.566

>100

For

>100

>100

>100

80.998

53.294

19.611

45.648

1.900

>100

>100

>100

>100

Dox

1.121

0.620

0.294

0.260

0.173

0.091

89.900

1.678

0.095

0.110

0.424

7.521

MCF-7

MDA-MB-231

52.439 16.865

>100

Color legend Very strong cytotoxicity (IC50 ≤ 1 µM/ml) Strong cytotoxicity (IC50 ≤ 10 µM/ml) Cytotoxic (IC50 ≤ 25 µM/ml) Moderate cytotoxicity (IC50 ≤ 50 µM/ml) Weak cytotoxicity (IC50 ≤ 100 µM/ml) Considered as non-cytotoxic (IC50 > 100µM/ml) IC50 value irrelevant due nonlinear dose dependence or hormetic effect

17

Figure S-2: Comparison of the IC50 values of steroidal derivatives and reference compounds on MCF-7 (ER+) and MDA-MB-231 (ER-) cell lines.

18

Results of flow cytometry analysis Analysis of cell cycle distribution by flow cytometry Marks M1, M2, M3 and M4 on the histograms correspond to G0/G1, S, G2/M and subG1 phases of cell cycle.

Control sample 48 h

72 h

Marker Label Events % Gated % Total X Mean

Marker Label Left, Right % Gated % Total X Mean

All

17886

100.00

89.43

253.33

All

0, 1023

100.00

74.16

258.03

M1

9311

51.60

46.55

199.23

M1

170, 237

54.01

40.43

202.28

G0/G1

M2

7010

38.84

35.05

302.98

M2

237, 380

33.68

25.21

309.48

S

M3

1351

7.48

6.75

391.59

M3

380, 447

10.37

7.76

400.68

G2/M

M4

374

2.07

1.87

171.30

M4

108, 170

1.94

1.45

162.46

SubG1

Cell cycle phase

Formestane 48 h

72 h



All

15023

100.00

75.11

246.27

All

0, 1023

100.00

83.12

261.75

M1

10417

68.30

52.09

206.98

M1

170, 237

51.13

42.87

205.31

G0/G1

M2

2764

18.12

13.82

296.21

M2

237, 380

36.67

30.74

305.91

S

M3

1968

12.90

9.84

389.35

M3

380, 447

10.39

8.71

402.20

G2/M

M4

104

0.68

0.52

140.92

M4

108, 170

1.81

1.52

157.63

SubG1

Cell cycle phase

19

Compound 1 48 h

72 h



All

18030

100.00

90.15

243.21

All

0, 1023

100.00

80.40

263.72

M1

9092

49.93

45.46

189.10

M1

170, 237

53.32

43.26

208.40

G0/G1

M2

6326

34.74

31.63

272.81

M2

237, 380

34.11

27.67

304.95

S

M3

2516

13.81

12.58

371.16

M3

380, 447

11.91

9.66

401.70

G2/M

M4

277

1.52

1.39

168.76

M4

108, 170

0.65

0.53

155.47

SubG1

Cell cycle phase

Compound 2 48 h

72 h



All

18101

100.00

90.50

254.75

All

0, 1023

100.00

93.28

242.74

M1

10010

54.76

50.05

202.67

M1

170, 237

51.96

48.95

197.14

G0/G1

M2

6252

34.20

31.26

292.42

M2

237, 380

35.72

33.66

306.22

S

M3

1946

10.65

9.73

395.12

M3

380, 447

4.74

4.47

395.08

G2/M

M4

70

0.39

0.35

170.59

M4

108, 170

7.58

7.15

160.03

SubG1

Cell cycle phase

20

Compound 3 48 h

72 h



All

16774

100.00

83.87

253.82

All

0, 1023

100.00

84.15

258.17

M1

8024

47.32

40.12

197.76

M1

170, 237

52.83

44.87

202.35

G0/G1

M2

7403

43.65

37.02

293.74

M2

237, 380

37.19

31.59

308.63

S

M3

1283

7.57

6.42

391.71

M3

380, 447

8.54

7.26

401.17

G2/M

M4

246

1.45

1.23

171.18

M4

108, 170

1.44

1.22

160.43

SubG1

Cell cycle phase

Compound 4 48 h

72 h



All

17376

100.00

86.88

241.73

All

0, 1023

100.00

72.77

256.07

M1

9663

54.87

48.32

192.26

M1

170, 237

51.05

37.54

200.58

G0/G1

M2

6705

38.08

33.52

310.16

M2

237, 380

36.01

26.48

308.96

S

M3

477

2.71

2.38

386.67

M3

380, 447

8.83

6.49

401.16

G2/M

M4

765

4.34

3.82

171.66

M4

108, 170

4.12

3.02

163.36

SubG1

Cell cycle phase

21

Compound 5 48 h

72 h



All

15774

100.00

78.87

250.04

All

0, 1023

100.00

84.16

249.74

M1

6913

43.18

34.56

192.94

M1

170, 237

48.58

41.39

198.89

G0/G1

M2

6352

39.67

31.76

278.68

M2

237, 380

35.99

30.67

307.88

S

M3

2038

12.73

10.19

378.76

M3

380, 447

7.34

6.26

396.95

G2/M

M4

708

4.42

3.54

171.01

M4

108, 170

8.09

6.90

160.63

SubG1

Cell cycle phase

Compound 6 48 h

72 h



All

15988

100.00

79.94

232.58

All

0, 1023

100.00

79.12

268.17

M1

8590

52.70

42.95

187.87

M1

170, 237

48.29

38.58

208.96

G0/G1

M2

5257

32.25

26.28

286.99

M2

237, 380

39.13

31.27

303.11

S

M3

1211

7.42

6.05

370.17

M3

380, 447

11.89

9.50

401.31

G2/M

M4

1244

7.63

6.22

171.40

M4

108, 170

0.68

0.54

157.29

SubG1

Cell cycle phase

22

Compound 7 48 h

72 h



All

15317

100.00

76.59

247.62

All

0, 1023

100.00

68.42

265.57

M1

7738

49.80

38.69

195.05

M1

170, 237

47.65

32.92

205.43

G0/G1

M2

6586

42.39

32.93

306.25

M2

237, 380

37.79

26.11

305.86

S

M3

496

3.19

2.48

388.70

M3

380, 464

11.94

8.25

401.70

G2/M

M4

717

4.61

3.58

171.32

M4

108, 170

2.61

1.81

156.97

SubG1

Cell cycle phase

Compound 8 48 h

72 h



All

17887

100.00

89.44

242.87

All

0, 1023

100.00

86.27

257.04

M1

10073

56.74

50.37

196.89

M1

170, 237

51.19

44.53

206.70

G0/G1

M2

4983

28.07

24.92

286.49

M2

237, 380

37.29

32.44

301.25

S

M3

2168

12.21

10.84

379.13

M3

380, 447

8.58

7.46

399.72

G2/M

M4

528

2.97

2.64

137.64

M4

108, 170

2.95

2.56

156.79

SubG1

Cell cycle phase

23

Compound 9 48 h

72 h



All

16876

100.00

84.38

243.53

All

0, 1023

100.00

73.57

258.65

M1

9518

56.73

47.59

195.93

M1

170, 237

53.50

39.69

202.64

G0/G1

M2

4767

28.42

23.84

287.80

M2

237, 380

33.55

24.89

309.27

S

M3

2094

12.48

10.47

378.08

M3

380, 447

10.65

7.90

401.78

G2/M

M4

397

2.36

1.98

136.47

M4

108, 170

2.29

1.70

158.46

SubG1

Cell cycle phase

Modulation of the cell cycle

Figure S-3: Modulation of the cell cycle phase’s distribution of MDA-MB-231 cells caused by steroidal compounds in comparison to the control sample (100%).

24

Analysis of apoptosis by flow cytometry Dot plots and pie diagrams on the right represent portions of apoptotic (lower and upper right quadrant for early and late apoptosis – LR and UR, respectively), necrotic (upper left quadrant UL) or live cells (lower left quadrant - LL) detected with Annexin V flowcytometric test.

Control sample 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

138 255 4265 61

2.92 5.40 90.39 1.29

1.38 2.55 42.65 0.61

UR LR LL UL

118 256 3860 49

2.76 5.98 90.12 1.14

1.18 2.56 38.6 0.49

Legend: Late apoptosis Early apoptosis Live cells Necrosis

Formestane 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

72 76 2241 265

2.71 2.86 84.45 9.98

0.72 0.76 22.41 2.65

UR LR LL UL

1044 209 3719 528

18.98 3.8 67.62 9.6

10.44 2.09 37.19 5.28


25

Compound 1 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

380 123 2525 445

10.94 3.54 72.71 12.81

3.80 1.23 25.25 4.45

UR LR LL UL

1016 105 2045 717

26.17 2.7 52.67 18.46

10.16 1.05 20.45 7.17


Compound 2 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

449 218 2517 322

12.81 6.22 71.79 9.18

4.49 2.18 25.17 3.22

UR LR LL UL

890 191 4281 459

15.29 3.28 73.54 7.89

8.9 1.91 42.81 4.59


26

Compound 3 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

525 215 2316 243

15.91 6.52 70.20 7.37

5.25 2.15 23.16 2.43

UR LR LL UL

983 85 3398 1229

17.26 1.49 59.67 21.58

9.83 0.85 33.98 12.29


Compound 4 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

180 91 2122 278

6.74 3.41 79.45 10.40

1.80 0.91 21.22 2.78

UR LR LL UL

1356 95 1448 280

42.65 2.99 45.55 8.81

13.56 0.95 14.48 2.8


27

Compound 5 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

306 198 3454 348

7.11 4.60 80.21 8.08

3.06 1.98 34.54 3.48

UR LR LL UL

841 272 4562 188

14.34 4.64 77.81 3.21

8.41 2.72 45.62 1.88


Compound 6 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

257 85 2675 457

7.40 2.45 77.00 13.15

2.57 0.85 26.75 4.57

UR LR LL UL

1034 163 1345 606

32.85 5.18 42.73 19.24

10.34 1.63 13.45 6.06


28

Compound 7 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

197 186 5582 163

3.21 3.04 91.09 2.66

1.97 1.86 55.82 1.63

UR LR LL UL

1159 557 2115 294

28.1 13.5 51.27 7.13

11.59 5.57 21.15 2.94


Compound 8 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

231 253 2907 424

6.06 6.63 76.20 11.11

2.31 2.53 29.07 4.24

UR LR LL UL

85 6 4691 902

1.5 0.11 82.53 15.86

0.85 0.06 46.91 9.02


29

Compound 9 48h

72h

Quad Label

Events

% Gated

% Total

Quad Label

Events

% Gated

% Total

UR LR LL UL

137 273 1426 408

6.11 12.17 63.55 18.17

1.37 2.73 14.26 4.08

UR LR LL UL

861 97 3549 865

16.03 1.81 66.06 16.1

8.61 0.97 35.49 8.65


30

Results of apoptotic morphology screening Photographs of native cells in cultures and stained specimens Morphology of MDA-MB-231 cells (a – f). First two columns from left contain photographs of native cells in culture photographed with a Canon 1100D camera attached to a Reichart BioStar inverted microscope with 20x (left column) and 40x magnification objectives (middle column). Stained slides (column to the right) were photographed with an Olympus Camedia 3040 digital camera attached to an Olympus BX51 microscope with 10x magnification objective, after staining with Giemsa. First rows of images (a – c) contain images taken after 48 h of incubation and second rows (d – f) represents images of samples treated for 72 h.

Control sample

Formestane

31

Compound 1

Compound 2

Compound 3

32

Compound 4

Compound 5

Compound 6

33

Compound 7

Compound 8

Compound 9

34

Detected morphological changes

Figure S-4: Cells in different stages of apoptosis in treated cultures are easily distinguishable. Most cells had normal morphology (a). Nuclear condensation is evident in cells (dark, condensed and rounded nuclei), as well as vacuolated cytoplasm (b – e). Degradation of nuclei and cytoplasm are present (f – k). Formation of apoptotic bodies are also evident (l).

Table S-2: Percentage of apoptotic cells (mean values of two independent analysis) estimated by visual observation of the cell morphology of MDA-MB-231 cell line after 48 hand 72 h treatment with equitoxic doses of steroidal derivatives 1 – 9 and formestane. Ctrl.

For

1

2

3

4

5

6

7

8

9

Nn Na Nt

1019 77 1096

1028 120 1148

1009 275 1284

1024 143 1167

1212 227 1439

1012 131 1143

1027 104 1131

1056 150 1206

1065 124 1189

1025 141 1166

1004 115 1119

Pa

7.0% 10.5% 21.4% 12.3% 15.8% 11.5% 9.2% 12.4% 10.4% 12.1% 10.3%

Parameter

48 h

72 h Nn Na Nt

1048 43 1091

1048 159 1207

1066 211 1277

1041 68 1109

1034 74 1108

1008 109 1117

1020 178 1198

1023 96 1119

1035 47 1082

1027 49 1076

1061 71 1132

Pa

3.9% 13.2% 16.5% 6.1%

6.7%

9.8% 14.9% 8.6%

4.3%

4.6%

6.3%

Nn - Number of cells with normal morphology; Na - Number of cells with apoptotic morphology; Nt Total number of cells examined; Pa - The percentage of apoptotic cells

35