Role of nano-DIM on glioblastoma

1. Arijit Bhowmik

2. • The epidermal growth factor receptor (EGFR)
belongs to the ErbB family of receptor tyrosine
kinases (RTK)
• These trans-membrane proteins are activated
following binding with peptide growth factors
of the EGF-family of proteins
• Binding of the protein to a ligand induces
receptor dimerization and tyrosine
autophosphorylation and leads to cell proliferation
• Amplification of the EGFR gene and mutations
of the EGFR tyrosine kinase domain have been
demonstrated to occur in cancer patients.
Figure source : Bessman et al. Nature Structural & Molecular Biology
19, 1–3 (2012)

4. Figure Source: Wikipedia
EGFR Pathway….

5. Scope of Breast Cancer
- Most common cancer (31%) in
women.
- Incidence – 110/10K people
- Life time risk is 13.48% (1 in 7)
- 0.6% of all cases are in men

6. • EGFR overexpression in breast cancer is associated with large tumor size, poor
differentiation, and poor clinical outcomes.
• Treatment of patients with high EGFR level has been challenging not only because of
the aggressive behaviour of these diseases but also because of the lack of established
clinically relevant treatment targets.
• The role of EGFR in breast cancer has been scrutinized, and several therapies that target
EGFR, including gefitinib, cetuximab, lapatinib, and others, have been developed.
• However, results of clinical studies of EGFR-targeted therapy in breast cancer have been
disappointing.

7. Anti-proliferative effects of indole derivatives
a) 1,1',2,2'-tetramethyl-
3,3'-diindolylmethane
(TetraMDIM),
b) 2,2'-dimethyl-3,3'-
diindolylmethane (DMDIM),
c) 1,1'-dimethyl-5,5'-
dimethoxy-3,3'-
diindolylmethane
(DMDMODIM),
d) 5,5'-dimethoxy-3,3'-
diindolylmethane
(DMODIM) and
e) 2,2'-diphenyl-3,3'-
diindolylmethane (DPDIM)
Bhowmik et.al; Plos one

8. Cytotoxicity study of DPDIM
Bhowmik et.al; Plos one

9. Evaluation of the inhibitory effect of DPDIM on EGFR pathway and induction of
mitochondrial cytochrome c release
Bhowmik et.al; Plos one

10. Activation of mitochondrial caspases and induction of apoptosis in DPDIM treated
breast cancer cells
Bhowmik et.al; Plos one

11. Binding analysis of DPDIM with EGFR.
Bhowmik et.al; Plos one

12. In silico comparison of DPDIM binding with EGFR, HER2, HER3 and other
known EGFR inhibitors
Bhowmik et.al; Plos one

13. Inhibition of EGF induced EGFR activation, cell viability and colony formation
of MCF7 cells by DPDIM
Bhowmik et.al; Plos one

14. Inhibition of phosphorylation of constitutively active EGFR (EGFRvIII) and
regulation of cell viability in EGFRvIII overexpressed cells by DPDIM
Bhowmik et.al; Plos one

15. Reduction of breast tumor growth by DPDIM in animal model
Bhowmik et.al; Plos one

16. In vivo determination of DPDIM induced EGFR pathway regulation
directed to apoptosis
Bhowmik et.al; Plos one

17. In summary ………
 Indole Derivative DPDIM Inhibits Proliferation and Survival of Cancer Cells.
 DPDIM is a Non-cytotoxic Compound.
 Regulation of EGFR Pathway by DPDIM Leads to Mitochondrial Cyt c Release
in Breast Cancer Cells.
 DPDIM Induces apoptosis Through Activation of the Mitochondrial
Caspase Cascade.
 DPDIM Inhibits Growth and Induces Apoptosis in DMBA Induced
Breast Tumors also.
 Thus, DPDIM emerges as a promising anti-cancer agent showing high efficacy in
breast cancer cells and the breast tumor model

18. Scope of Brain Cancer
- Incidence of primary brain tumors
(benign or malignant) 12.8/100,000
- 10%–15% of cancer patients develop
brain metastases
- Brain cancer is associated with high
mortality rate
- Glioma is the most common type of
brain cancer
- Overall survival rate for patients with
high grade glioma remains low—around
8%
- Till date, malignant glioma has no
effective therapy

19. Source : Oncomine Database
Incidence of malignant primary brain tumor by age and histology

20. The major cause of drug failure : Blood Brain Barrier
Figure source : ABhowmik et al.,BMRI

21. 0
10
20
30
40
50
60
70
80
0 20 40 60
%
cell
viability
Compound dose (µM)
DIM/DBTRG
IC50 value – 20µM
0
20
40
60
80
0 20 40 60
%
cell
viability
Compound dose (µM)
DIM/U87MG
IC50 value - 24µM
3,3′-Diindolylmethane
0
20
40
60
80
100
120
0 20 40 60
%
viability
Compound dose (µM)
DIM/C6
IC50 value – 28 µM
Background…….

22. Drawbacks of native DIM
Mass spectrometry of rat brain extract

23. 3,3′-diindolylmethane(DIM)-induced apoptosis in ovarian cancer cells is
mediated by inhibition of EGFR-ERK pathway; PK. Kandala, KC. Pramanik, S
Reddy Boreddy, and SK. Srivastava; Cancer Res ; April 15, 2010 70; 3781
Blocking Epidermal Growth Factor Receptor Activation by 3,3′-
Diindolylmethane Suppresses Ovarian Tumor Growth In Vitro and In
Vivo; PK. Kandala, SE. Wright and SK. Srivastava; JPET ; April 2012 vol.
341 no. 124-32
Some reports of DIM targeting EGFR………..

24. Basic characterization of Poly(Lactic-co-Glycolic Acid) (PLGA) encapsulated DIM

25. Confirmation of encapsulation, release and binding of nanoparticles
DAPI NP+FITC Merged
Z-scanning

26. Control Treated
Cellular internalization of DIM released from nanoparticle

27. 0
10
20
30
40
50
60
70
80
90
0 100 200 300 400 500
%
cell
viability
Compound dose (nM)
IC50 value – 390 nM
Effective growth inhibiting and apoptotic response of nanotized DIM
C C
T T
0 hr 0 hr
48 hr 48 hr
C C
0 day
0 day
4th day
4th day
T T C T
** No cell death in HEK293 cells

28. EGFR
SSTR2
SSTR2
EGFR
Why SSTR2?
Validation of EGFR and SSTR2 expression in Human and Rat glioma samples
Human
glioma
Rat
glioma
r= + 0.1504
r= + 0.2901

29. Designing and in silico characterization of SSTR2 peptide.

30. In silico screening…..

31. Simulation study of SSTR2 peptide

32. HPLC
MASS SPEC
Synthesis of peptide…….
Biotin- AGCRNYFWKSFSSC
FITC- AGCRNYFWKSFSSC
TAMRA- AGCRNYFWKSFSSC
Synthesis scheme

33. FACS analysis: Binding efficiency
Peptide SSTR2 DAPI
Control
Treated (1nM peptide)
Treated (10nM peptide)
Peptide specificity checking and binding with SSTR2

34. %
viability
Peptide dose
Peptide dose
%
viability
44
42
42
44
38
38
21
43
P ERK 1/2
ERK 1/2
P p38MAPK
p38MAPK
p21
Actin
C 1 10 10
1 1 10
pM nM µM
Designed SSTR2 peptide is an inert peptide

35. Confirmation of peptide coupling with nanoparticles
EDC (1-Ethyl-3-(3-dimethylaminopropyl)-
carbodiimide)- NHS (N-hydroxysuccinimide)
Coupling of nanoparticle and peptide
PLGA nanoparticle
DIM
SSTR2 peptide

36. Study of apoptotic effect of DIM nanoparticle having surface modification with peptide

37. Primary endothelial cell
(ICAM1 staining)
C6 astrocytes
In vitro validation of drug passaging through BBB
TEER value :
200 to 350 Ω cm2

38. T
C
In vivo validation of drug passaging through BBB
1
2
3
5
4
6
7
1
2
4
7
3
5
6
1 – Brain, 2- Heart, 3- Lungs, 4-Liver, 5- Spleen, 6- Pancreas , 7- Kidney

39. In vivo validation of internalization of DIM in rat brain by ESI-Mass spectrophotometry
Native DIM

40. Glioma sample of peptide tagged nanotized DIM treated rat

41. Bioavailability and toxicity analysis of DIM nanoparticle

43. Untreated Treated Untreated Treated
0
10
20
30
40
untreated treated
Days
of
survivability
of
SD
rats
Nanotized DIM treatment Groups
*
Reduction of brain tumor growth by peptide tagged nanotized DIM in animal model

44. P-EGFR
P-AKT
P-ERK
P-STAT3
Bcl-XL
p21
Untreated tumor Treated tumor Untreated tumor Treated tumor
Regulation of EGFR pathway members by peptide tagged nanotized DIM in vivo

46. SSTR2
Low and high
grade brain
tumors
Indole
compound
EGFR
Nanoparticle based
modification and peptide
tagging
Peptide
BBB
In Summary……

48. 1.A Bhowmik, N Das, U Pal, M Mandal, S Bhattacharya, M Sarkar et al.,
“2, 2′-diphenyl-3, 3′-diindolylmethane: a potent compound induces apoptosis in breast cancer
cells by inhibiting EGFR pathway,” PloS one, 8 (3), e59798, 2013.
2.A Bhowmik, R Khan, MK Ghosh, “Blood Brain Barrier: A Challenge for Effectual
Therapy of Brain Tumors,” BioMed Research International, vol. 2015, Article ID 320941,
20 pages, 2015.
3. K De, A Bhowmik, A Behera, I Banerjee, MK Ghosh, M Misra, “Synthesis, radiolabeling, and
preclinical evaluation of a new octreotide analog for somatostatin receptor‐positive tumor
scintigraphy,” Journal of Peptide Science, 18 (12), 720-730, 2012.
4. I Paul, SF Ahmed, A Bhowmik, S Deb, MK Ghosh, “The ubiquitin ligase CHIP regulates c-Myc
stability and transcriptional activity,” Oncogene, 32 (10), 1284-1295, 2013.
5. T Mandal, A Bhowmik, A Chatterjee, U Chatterjee, S Chatterjee et al., “Reduced phosphorylation
of Stat3 at Ser-727 mediated by casein kinase 2—Protein phosphatase 2A enhances Stat3
Tyr-705 induced tumorigenic potential of glioma cells,” Cellular signalling, 26 (8), 1725-1734, 2014.
6. KKN Guturi, M Sarkar, A Bhowmik, N Das, MK Ghosh, “ DEAD-box protein p68 is regulated
by β-catenin/transcription factor 4 to maintain a positive feedback loop in control of breast cancer
progression,” Breast Cancer Research 16 (6), 496, 2014
7. A Ghosh , A Bhowmik, S Bhandary , S Putatunda, A Laskar et al., Synthesis and antitumorigenic activities
of nanoencapsulated nifetepimine: A promising approach in treating triple negative breast carcinoma.
Nanomedicine: Nanotechnology, Biology, and Medicine. 2016, In press.
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