...

1. Medroxyprogesterone Acetate in Glial
Tumor Treatment. Our Experimental Results
Neuroacademy Group

2. What were our results regarding the influence of
progestins on glial tumor growth?

3. Soft Agar Colony Assay
The soft agar colony formation
assay is a method used to confirm
cellular anchorage-independent
growth in vitro, which illustrates the
tumorigenic potential of
transformed cells and the tumor
suppressive effects of novel
treatment strategies.

4. 3H-Thymidine Labeling Index: Demonstrating S-phase
fraction in monolayer cultures

7. What were our results regarding the influence of
progestins on glial tumor growth?

10. XTT Cell Proliferation Assay
Our Current Unpublished Results Regarding MPA Actions
on Glial Tumor Cells

11. MPA testing on XTT cell respiration-based growth tests in glial
tumor cell lines
U251MG and U87MG human glial tumor cell
lines were treated by two-fold increasing doses
of MPA, which ranged from 3.25 uM to 104 uM
(1.25 to 40 ug/ml), respectively.
For each treatment, cells in six separate wells
of a 24-well plate were used. They were treated
with various doses of MPA, which was dissolved
in same amount of (5 ul) ethanol in 1ml DMEM.
Controls were treated with the ethanol vehicle.

12. MPA testing on XTT cell respiration-based growth tests in glial
tumor cell lines
Cells were treated with MPA either (a) only
following 24 hours of plating, or (b) both after
24-, and 72 hours of incubation (media for
controls were replenished at the treatment
periods).
XTT assay was performed following 96 hours of
drug treatment.

13. MPA Effects on U251MG Cell Growth
U251 Cytotoxicity (XTT)
-35.00%
-15.00%
5.00%
25.00%
45.00%
65.00%
85.00%
105.00%
1
MPA Doses
P1
P2
P3
P4
P5
P6
U251 Cytotoxicity (XTT)
-15.00%
5.00%
25.00%
45.00%
65.00%
85.00%
105.00%
MPA Doses
P1
P2
P3
P4
P5
P6

14. MPA Effects on U251MG Cell Growth
In U251 cells (a) P5 and P6 treatments showed
effectiveness, with very high significance values
(p<0.00001).
In (b) P3 dose also showed high significant
efficacy (p<0.00001), as compared to control.
Slight increases in growth at P1-P2 doses did
not show statistical difference (p>0.05).

15. MPA Effects on U87 Cell Growth
U87-Cytotoxicity (XTT)
-35.00%
-15.00%
5.00%
25.00%
45.00%
65.00%
85.00%
105.00%
MPA Doses
P1
P2
P3
P4
P5
P6
U87-Cytotoxicity (XTT)
-35%
-15%
5%
25%
45%
65%
85%
105%
MPA Doses
P1
P2
P3
P4
P5
P6

16. MPA Effects on U871MG Cell Growth
In U87 cells (a) P5 and P6 treatments showed
effectiveness, with very high significance values
(p<0.00001).
In (b) P4 dose also showed similar significant
efficacy (p<0.00001), when compared to
control. In both (a) and (b) slight increases in
growth P1-P3 doses did not show statistical
difference (p>0.05).

17. 3-Dimensional Tumor Spheroid Models To Test Anticancer Drug
Efficacies Regarding Cell Growth, Metabolism and Invasion

18. Differential Growth and Invasion Patterns of Glial Tumor
Spheroids

19. MPA tests in C6 glioma spheroid model
Effects of MPA and/or 5-FU were studied by
measuring C6-glioma spheroid growth and
invasion. MPA doses were for h- 104 uM, m-
52 uM, and l- 26 uM, respectively. 5-FU was
used at a 200 uM level.
Spheroids were incubated throughout 8 days
in the same conditions in type I collagen
(Vitrogen ®), and both the drugs and feeding
media were replenished every 72-hours

20. C6 Spheroid growth
-20
-10
0
10
20
30
40
50
60
1 2 3 4 5 6 7 8
Days
%Changeindiameter
Con
Con-et
MPA-h
MPA-m
MPA-l
Flu
MPA-h+Flu
C6 Spheroid Invasion Distance
0
500
1000
1500
2000
2500
3000
1 2 3 4 5 6 7 8
Days
uM
Con
Con-et
MPA-h
MPA-m
MPA-l
Flu
MPA-h+Flu

21. MPA suppresses C6 glioma spheroid growth
After an incubation of 8th days, control
spheroids had a 44% percent increase in
diameter, whereas MPA treated spheroids had
increased their diameter by only 24%, 14%
and 10% percent in the presence of 26, 52
and uM MPA, respectively.
The differences were statistically significant at
a dose dependent manner (p<0.01 for 26 uM
and p<0.005 for 104 uM).

22. 5-FU & MPA strongly reduce C6 glioma spheroid growth
In 5-FU treated spheroids there was even a
reduction of diameter by 3.2%, which was
further reduced to 17% with 104 uM MPA.
Reductions by MPA or 5-FU occurred with
significance of p<0.0001 in comparison to
control.

23. MPA & 5-FU reduces C6 glioma spheroid invasion at the 8th day
Invasion Distance From The Edge of
C6-Spheroids (µm)
0
500
1000
1500
2000
2500
3000
3500
4000
Con Con-et MPAh MPAm MPAl Flu MPAh+Flu

24. MPA reduces invasion rate of C6 glioma spheroids
After eight days C6 tumor cells had invaded
2700 microns on average, which was reduced
to 1520, 860 and 560 microns in the presence
of 26, 52 and 104 uM MPA, respectively
(p<0.05 for the highest dose). 5-FU treated
spheroids were only able to invade 630
microns on average, which was further
reduced to 530 microns with 104 uM MPA.

25. MPA testing in human U251 glioma spheroid model
MPA effects were also studies on growth and
invasion of human U251 spheroids.
One day after spheroid implantation (“day-1”),
spheroid diameters and invasion distances were
measured. Drugs were given at day-1 and every
48 hours thereafter in new media.
Growth values represent one day later than the
invasion data to determine changes as
percentage values.

26. Growth and Invasion of human U251 glioma spheroids
with low (52 um) and high dose (104 uM) MPA
-5.00%
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
Day2 Day3 Day4 Day5 Day6 Day7 Day8
Control
MPAhigh
MPAlow
0
100
200
300
400
500
600
700
800
900
Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8
Control
MPAhigh
MPAlow

27. Progesterone, pregnancy and their impact on growth of
central nervous system neoplasias:
Growth and Invasion of human U251 glioma spheroids
Growth: High dose MPA (104 uM) was
statistically effective (p=0.022) in reducing
growth.
Invasion: Both low (26 uM) and high doses of
MPA were effective in reducing invasion with
significance of p<0.05 and p<0.000001,
respectively. High dose of MPA was significantly
more effective than its low dose (p<0.05).

28. Dexamethasone and MPA interactions with PI3-Kinase Pathway
in regard to spheroid growth and invasion
Dexamethasone is a steroid like MPA and it is
widely used to treat brain tumor edema. Both
MPA and dexamethasone were shown to
suppress tumor invasion via blocking MMP-9.
Both progesterone and dexamethasone were
also shown to modulate PI-3-Kinase pathway,
which involves in invasion and apoptosis
resistance of glial tumors.

29. PI3 Kinase as a protumorigenic pathway

30. Kubiatowski et al. have found that blocking PI-
3-Kinase pathway via LY294002 and
wortmannin reduces glioma invasion. J
Neurosurg. 95:480-8, 2001
Hence, we wanted to elucidate, if MPA or
dexamethasone induced changes in glioma
growth and invasion can be modified by
pharmacological inhibitors of the PI-3-Kinase.

31. Pharmacological Inhibitors of PI3-Kinase:
LY294002 and Wortmannin
LY294002 Wortmannin

32. LY294002 & MPA and human U251 glioma spheroid
growth and invasion
Modulation of U251 human spheroid growth
(a) and (b) invasion was studied with
medium (52 uM) dose of MPA in the presence
and absence of high (112 uM) or low (1.4 uM)
dose of LY294002. All agents were replenished
every two days in fresh medium, and the
treatment began at the day of spheroid
implantation.

33. LY294002 & MPA and human U251 glioma spheroids
-5.00%
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
Day2 Day3 Day4 Day5 Day6 Day7 Day8
Control
MPA
MPA+LY-h
MPA+LY-l
LY-h
LY-l
Modulation of U251 spheroid invasion by MPA
and/or high or low dose of LY
0
100
200
300
400
500
600
700
800
1 2 3 4 5 6 7
Control
MPA
MPA+LY-h
MPA+LY-l
LY-h
LY-l

34. LY294002 & MPA and human U251 glioma spheroids
– Effects regarding growth
a) At the end of 8th
day incubation in type I
collagen, MPA significantly (p<0.05) reduced
spheroid growth, which was not altered
either by high or low dose of LY294002
(p>0.05 for both doses). Peculiarly, low
dose of LY294002 has significantly reduced
spheroid growth (p<0.05), but not its high
dose.

35. LY294002 & MPA and human U251 glioma spheroids
– Effects regarding invasion
b) MPA was efficient (p<0.005) in reducing
spheroid invasion, which was only augmented
by high (p<0.005), but not low dose of
LY294002. LY294002 efficiently reduced
invasion with both low (p<0.005) and high
dose (p<0.00001) application, and the dose
effect was significant (p<0.005).

36. Wortmannin & MPA and human U251 glioma spheroids
Modulation of U251 human spheroid growth
(a) and (b) invasion was studied with
medium (52 uM) dose of MPA in the presence
and absence of high (400 nM) or low (10 nM)
dose of Wortmannin. All agents were
replenished every two days in fresh medium,
and the treatment began at the day of
spheroid implantation.

37. Wortmannin & MPA and human U251 glioma spheroids
Modulation of U251 spheroid growth with MPA
and/or high or low doses of Wortmannin
-15.00%
-10.00%
-5.00%
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
Day2 Day3 Day4 Day5 Day6 Day7 Day8
Control
MPA
MPA+W-h
MPA+W-l
W-h
W-l
Modulation of U251 spheroid invasion byMPA
and/or high or low dose of LY
0
100
200
300
400
500
600
700
800
1 2 3 4 5 6 7
Control
MPA
MPA+LY-h
MPA+LY-l
LY-h
LY-l

38. Wortmannin & MPA and human U251 glioma spheroids
– Effects regarding growth
a) At the end of 8th
day incubation in type I
collagen, MPA significantly (p<0.05) reduced
spheroid growth, which was also significantly
augmented by high dose Wortmannin
(p=0.023). Combined treatment of MPA with
high dose wortmannin reduced spheroid
growth significantly more than the
combination of MPA with low dose
wortmannin (p<0.001).

39. Wortmannin & MPA and human U251 glioma spheroids
– Effects regarding invasion
b) MPA was efficient (p<0.005) in reducing
spheroid invasion, however either low or high
dose wortmannin did not significantly alter
MPA mediated invasion inhibition, despite
they were alone potent inhibitors of invasion
(p<0.00001).

40. LY294OO2 at low dose blocks Dex induced-inhibitions on
glioma growth and invasion
Modulation of U251 spheroid growth with
Dexamethasone and/or high or low dose of
LY294002
0
5
10
15
20
25
30
Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8
Con
Dex
LY-h
LY-l
Dex+LY-h
Dex+LY-l
Modulation of U251 spheroid invasion with
Dexamethasone and/or high or low dose of
LY294002
0
100
200
300
400
500
600
700
800
900
Day2 Day3 Day4 Day5 Day6 Day7 Day8 Day9
Con
Dex
LY-h
LY-l
Dex+LY-h
Dex+LY-l

41. Wortmannin at low dose blocks Dex induced-inhibitions on
glioma growth and invasion
Modulation of U251 spheroid growth with
Dexamethasone and/or high and low dose of
Wortmannin
0
5
10
15
20
25
30
Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8
Con
Dex
Wort-h
Wort-l
Dex+Wort-h
Dex+Wort-l
Modulation of U251 spheroid invasion with
Dexamethasone and/or high or low dose of
Wortmannin
0
100
200
300
400
500
600
700
800
900
Day2 Day3 Day4 Day5 Day6 Day7 Day8 Day9
Con
Dex
Wort-h
Wort-l
Dex+Wort-h
Dex+Wort-l

42. Janus faces of PI3 Kinase Pathway in Tumor Behavior
Esteve et al have found that
blocking PI-3-Kinase pathway via
wortmannin or via expressing the
dominant negative form of PI3-
Kinase further enhanced IL-1 or
TNF-alpha induced MMP-9
synthesis in C6 glioma cells.
Biochem Biophys Res Commun 296: 864-9,
2002
Ma et al demonstrated that androgen receptor
induced adhesiveness and inhibition of prostate
cancer invasion involves activation of PI3-Kinase/akt
pathway. Cancer Lett 351: 64-71, 2014

43. Studies With Tibolone
Tibolone itself has weak activity in body, it is
converted to active metabolites in gut. In brain and
bones it exerts estrogenic activity, leading positive
effects on mood/cognition and preventing bone loss.
In endometrium, its progestin-like effects dominate
leading less/absent carcinogenicity on endometrium
in comparison to hormon replacement therapy with
combined equine estrogens and progesterone.

44. Tibolone and MPA – Comparison of Cell Counts in C6
glioma monolayer culture after 24 hours

45. Tibolone and Blockage of Colony Growth:
1- Con; 2- 0,1; 3-1; 4-10; 5: 100 ug/ml

46. All dosages
were 100
ug/ml. Counts
were obtained
with Thoma
chamber

47. 3D-Spheroid BrdU Assay
BrdU incorporates into the newly
synthesized DNA of replicating cells,
substituting for thymidine during
DNA replication. Antibodies specific
for BrdU can then be used to detect
the incorporated chemical, thus
measuring S-phase fraction. BrdU
differs from thymidine in that BrdU
substitutes a Br for thymidine's
CH3 group.

48. C6 glioma 3D-
spheroid
S-phase fraction
as assessed by
BrdU-labeling.
Results were
separated
according to the
spheroid size.

50. Clonogenic
inhibitons
obtained in C6
glioma cells
in soft agar assay

51. Medroxyprogesterone reduces intracranially implanted
and X-gal labelled C6 glioma (50 mg/kg for 3 weeks)