1. В.A.Рихтер
Пептид молока человека – лактаптин, как потенциальный противораковый препарат
Москва, 2013

2. Lactaptin purification from human milk
In vivo study (clinical trial)
disadvantages
APOPTIN
Multiple myeloma
TRAIL-based proteins (anti-TRAIL-receptor mAb and recombinant TRAIL)
Mapatumumab: non-Hodgkin’s
lymphoma, colorectal, ovarian,
prostate cancer;
Lexatumumab: advanced solid tumors.
Narrow range of constitutively TRAIL-sensitive tumors
ER-specific mAb
Herceptin- Breast cancer
Rituximab- B-cell lymphoma
ER-dependent spectrum of tumors
MDA-7
Advanced carcinomas, melanomas
Resistance of pancreatic and colorectal cancer
Alfa-lactalbumin +5 molecules of oleic acid (HAMLET, BAMLET)
Papillomatous skin tumor, bladded cancer, glioma
Restricted spectrum of tumors (need for local administration)

3. Human k-casein, lactaptin and recombinant analogue RL2
GGSHHHHHH
Lactaptin
8.6 kDa
NH2
182
COOH 23 57
134
Human kappa-casein
YYGTNLYQRRPAIAINNPYVPRTYYANPAVVRPHAQIPQRQYLPNSHPPTVVRRPNLHPSFIAIPPKKIQDKIII

4. RL2 cytotoxicity in vitro
Cultured human cells
Primary human cells

5. Sensitivity of mice cancer cells to RL2
Cell line
IC50, μg/mL
hepatoma 22
100
MX-82
hepatoma A1 (HA1) 8
L-1210
180
MX-7
170
Ehrlich carcinoma
Lewis carcinoma
190

6. Study design Tumor cells inoculation Tumor growth RL2 therapy
Control group
RL2 therapy
i.v. injection
s.p. injection

7. The dose effect of RL2 on the growth rate of HA-1 tumors* **
Statistical differences in mean tumor weight between control and experimental groups are indicated by
* for p < 0.01;
** for p < 0.05 (statistically significant differences between groups).

8. Course-dependent antitumor activity of RL2** *
RL2 delays the formation of primary HA1 tumor

9. Metastasis suppression effect of RL2
control
RL2 therapy
P<0,001
A/Sn female mice were s.c. injected with 5x105 hepatoma HA-1 cells. RL2 (1 mg) was injected i.v. 5 times.
Liver metestases

10. Postsurgical metastasis model
A/Sn mice were i.v. injected with 2x105 hepatoma HA1 cells. Two days later RL2 (1 mg) was injected i.v. for 4 days.

11. RL2RL2 inhibition of MDA-MB-231 tumor growth in SCID mice

12. RL2 enhances antitumor effect of cyclophosphamide
A/Sn female mice (5 in group) were i.p. inoculated with 2x106 hepatoma HA-1 cells.
Next day mice were i.p. treated with RL2 (0.5 mg/dose) and cyclophosphamide (30 mg/kg). Therapeutic course consisted of 5 injections.

13. RL2 suppresses tumor growth and metastases: comparison with TNFa
- Tumor cell inoculation
- Repetitive RL2/TNFa injections
- Tumor growth and metastases progression
- Surgical removal of the tumor
- Survival upon metastases response

14. what is the mechanism of action
LACTAPTIN
- induces apoptosis in cancer cells in vitro
- Inhibits tumor growth in vivo
what is the mechanism of action

15. Accumulation and distribution of RL2-Rh conjugate into MCF-7 and MSC cells
Fluorescent microscopy of DAPI stained cells

16. Confocal microscopy of MCF-7 cells with RL2-Rh conjugate
Staining
DAPI (nucleus)
Cell Tracker Green (cytoplasm)
RL2-Rhodamin

17. Effect of RL2 on mitochondria membrane potential (HA1 cells)
Study design
Effect of RL2 on mitochondria membrane potential (HA1 cells)
- control
- RL2
control
Cell number
+ RL2
JC-1 staining

18. Mitochondrial apoptotic pathway activation by RL2
O h
4 h
48 h
A - control; B - RL2-treated cells.
The apoptotic changes in MCF-7 cells after RL2 treatment (4h) were detected as a shift of Δψ
Early apoptotic transition of TMP was analyzed by flow cytometry of DiOC6/PI stained MCF-7 cells

19. RL2 activates procaspases 8 and 9 in MCF-7 cells

20. Caspase 7 activity in MCF-7 cells
control
RL2
Western blot analysis of caspase 7 activity
1- mass marker
2- control cells
3- RL2-treated cells
Population with active caspase 7

21. Transcriptional response of MCF-7 cells on RL2 treatment
Functional groups of genes
p-value
Gene number
Centromeric regions
1.21E-05 11
Microtubule cytoskeleton organization
3.06E-04 14
Actin cytoskeleton organization
3.83E-03
Regulation of mitotic cell cycle
1.53E-03
DNA damage response, signal transduction of p53 class mediator
9.40E-03 6
Data of НТ-12 (Illumina)-analysis

22. RL2 interacts with cell proteins α-and β-chains of tubulin,
Identification by SwissProt database
Affine chromatography of МСF-7 cells lysates
electrophoresis
trypsinolysis
α-and β-chains of tubulin,
α-actinin-1

23. Apoptosis pathway inducing by RL2

24. Work was supported by: RFBR10-04-01109-а; 11-04-12100; 13-04-01313
Our team:
Olga Koval
Alexandr Fomin
Miraslava Potapenko
Elena Kuligina
Dmitry Semenov
Vivarium ICG SB RAS
Vasily Kaledin
Valery Nikolin
Histology
Eugeny Nikitenko
Work was supported by:
RFBR а; ;
FTP № and 16.N

25. Expression of apoptosis-related molecules
The expression levels of apoptosis-related molecules, such as Bcl-2, p53 and MDM2 in RL2-treated MDA-MB-231 cells were analyzed by Western blot.
We observed that during RL2 treatment p53-cleaved protein level decreased and p53 held in active form. Active p53 able to decreases Bcl-2 protein level that can be seen in the blot. Moreover, analysis of MDM2 level in MDA-MB-231 cells shows the appearance of MDM2 cleaved form. Together, these data could indicate that RL2-dependent apoptosis in MDA-MB-231 cells was accompanied by p53 stabilization and Bcl-2 depletion.