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Nano-DIM.ppt

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Arijit Bhowmik
Arijit Bhowmik

The document discusses research on developing a nanoparticle drug delivery system for treating brain cancer. It summarizes that a compound called 3,3'-Diindolylmethane (DIM) shows anti-cancer effects but cannot cross the blood-brain barrier. The researchers encapsulated DIM in nanoparticles and modified the nanoparticle surface with a peptide that targets the somatostatin receptor SSTR2, which is expressed on brain cancer cells and blood vessels. In vivo tests in a rat brain tumor model showed the peptide-tagged DIM nanoparticles reduced tumor growth by regulating the EGFR pathway and inducing apoptosis. The nanoparticle formulation helped deliver an effective dose of DIM to the brain and showed potential for treating brain cancer.

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Arijit Bhowmik
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, 13 (2012)
Nano-DIM.ppt
Figure Source: Wikipedia EGFR 永温岳鞄敬温霞.
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
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.
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
Cytotoxicity study of DPDIM Bhowmik et.al; Plos one
Evaluation of the inhibitory effect of DPDIM on EGFR pathway and induction of mitochondrial cytochrome c release Bhowmik et.al; Plos one
Activation of mitochondrial caspases and induction of apoptosis in DPDIM treated breast cancer cells Bhowmik et.al; Plos one
Binding analysis of DPDIM with EGFR. Bhowmik et.al; Plos one
In silico comparison of DPDIM binding with EGFR, HER2, HER3 and other known EGFR inhibitors Bhowmik et.al; Plos one
Inhibition of EGF induced EGFR activation, cell viability and colony formation of MCF7 cells by DPDIM Bhowmik et.al; Plos one
Inhibition of phosphorylation of constitutively active EGFR (EGFRvIII) and regulation of cell viability in EGFRvIII overexpressed cells by DPDIM Bhowmik et.al; Plos one
Reduction of breast tumor growth by DPDIM in animal model Bhowmik et.al; Plos one
In vivo determination of DPDIM induced EGFR pathway regulation directed to apoptosis Bhowmik et.al; Plos one
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
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 lowaround 8% - Till date, malignant glioma has no effective therapy
Source : Oncomine Database Incidence of malignant primary brain tumor by age and histology
The major cause of drug failure : Blood Brain Barrier Figure source : ABhowmik et al.,BMRI
0 10 20 30 40 50 60 70 80 0 20 40 60 % cell viability Compound dose (袖M) DIM/DBTRG IC50 value 20袖M 0 20 40 60 80 0 20 40 60 % cell viability Compound dose (袖M) DIM/U87MG IC50 value - 24袖M 3,3-Diindolylmethane 0 20 40 60 80 100 120 0 20 40 60 % viability Compound dose (袖M) DIM/C6 IC50 value 28 袖M Background.
Drawbacks of native DIM Mass spectrometry of rat brain extract
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..
Basic characterization of Poly(Lactic-co-Glycolic Acid) (PLGA) encapsulated DIM
Confirmation of encapsulation, release and binding of nanoparticles DAPI NP+FITC Merged Z-scanning
Control Treated Cellular internalization of DIM released from nanoparticle
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
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
Designing and in silico characterization of SSTR2 peptide.
In silico screening..
Simulation study of SSTR2 peptide
HPLC MASS SPEC Synthesis of peptide. Biotin- AGCRNYFWKSFSSC FITC- AGCRNYFWKSFSSC TAMRA- AGCRNYFWKSFSSC Synthesis scheme
FACS analysis: Binding efficiency Peptide SSTR2 DAPI Control Treated (1nM peptide) Treated (10nM peptide) Peptide specificity checking and binding with SSTR2
% 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 袖M Designed SSTR2 peptide is an inert peptide
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
Study of apoptotic effect of DIM nanoparticle having surface modification with peptide
Primary endothelial cell (ICAM1 staining) C6 astrocytes In vitro validation of drug passaging through BBB TEER value : 200 to 350 立 cm2
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
In vivo validation of internalization of DIM in rat brain by ESI-Mass spectrophotometry Native DIM
Glioma sample of peptide tagged nanotized DIM treated rat
Bioavailability and toxicity analysis of DIM nanoparticle
Nano-DIM.ppt
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
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
Nano-DIM.ppt
SSTR2 Low and high grade brain tumors Indole compound EGFR Nanoparticle based modification and peptide tagging Peptide BBB In Summary
Nano-DIM.ppt
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 receptorpositive 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 2Protein 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 硫-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. Publications
Nano-DIM.ppt

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Nano-DIM.ppt

  • 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, 13 (2012)
  • 4. Figure Source: Wikipedia EGFR 永温岳鞄敬温霞.
  • 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 lowaround 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 (袖M) DIM/DBTRG IC50 value 20袖M 0 20 40 60 80 0 20 40 60 % cell viability Compound dose (袖M) DIM/U87MG IC50 value - 24袖M 3,3-Diindolylmethane 0 20 40 60 80 100 120 0 20 40 60 % viability Compound dose (袖M) DIM/C6 IC50 value 28 袖M 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.
  • 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 袖M 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 立 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 receptorpositive 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 2Protein 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 硫-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. Publications