‫‪Administrative Information‬‬
‫المعلومات االدارية‬
‫المرجع‪:‬‬
‫عنوان المشروع (عربي وأجنبي) ‪Project Title -‬‬
‫‪Pro-angiogenic Factors and Hetero-cellular Interaction in Cancer Cell Extravasation: Molecular‬‬
‫‪and Functional studies.‬‬
‫العوامل المؤيدة لتبرعم األوعية الدموية وتفاعل واتصاالت خاليا من نماذج مختلفة في تسرب الخلية السرطانية‪ :‬دراسات‬
‫جزيئية ووظيفية‪.‬‬
‫الباحث الرئيسي ‪Principal Investigator -‬‬
‫االسم‬
‫‪Name‬‬
‫المؤسسة‬
‫‪Institution‬‬
‫كاظم عبد‬
‫كلية العلوم‪,‬‬
‫الحسين زيبارة‬
‫الجامعة اللبنلنية‬
‫الوظيفية‬
‫‪Post‬‬
‫‪Professor,‬‬
‫‪Full-Time‬‬
‫العنوان‬
‫‪Address‬‬
‫كلية العلوم‪,‬‬
‫رقم الهاتف‬
‫العنوان االلكتروني‬
‫‪Telephone‬‬
‫‪e-mail‬‬
‫‪70-939994 kzibara@ul.edu.lb‬‬
‫الجامعة اللبنلنية‬
‫الباحثون المشاركون ‪Co-Workers -‬‬
‫االسم‬
‫‪Name‬‬
‫الدكتور مروان‬
‫الصبّان‬
‫المؤسسة‬
‫‪Institution‬‬
‫كلية الطب‪ ،‬الجامعة‬
‫األميركية في بيروت‬
‫المدة التعاقدية للمشروع‪): Duration -‬‬
‫‪Page 1 of 5‬‬
‫العنوان االلكتروني‬
‫‪e-mail‬‬
‫‪me00@aub.edu.lb‬‬
‫)‪ (2 years‬سنتين‪ 2010 :‬و ‪2011‬‬
‫برنامج دعم البحوث العلمية ‪2013‬‬
‫‪Grant Program for Scientific Research in Lebanon – 2013‬‬
‫‪Programme de subvention à la recherche scientifique au Liban – 2013‬‬
‫العلمية‬
‫ المعلومات‬- Scientific Information
ّ
Objectives - ‫الهدف‬
To investigate the efficacy of Avastin (anti-VEGF) and Oleamide (anti-gap junction) therapy on
the extravasation of hematological and solid cancer cells in NSG animal models.
1- Cell culturing for 2 cancer cell lines (MDA-MB-231 & HUT-102) was done during
the first and second year, each time cells were needed to inoculate the mice.
2- Mice were bought (NOD-SCID & NSG) and were bred in a transgenic animal care
unit at the AUB in collaboration with Prof. Marwan El-Sabban. This was established
in the first 6 months of the study.
3- In addition, two in vivo cancer metastasis models were established during the first
and second year of the study.
4- Taking care of required practical activities in the animal care facility (cages, food,
water), cleaning, autoclaving has been done throughout first and second year of the
study.
5- Treatment with anti-angiogenic and anti-gap junctions was done during the first and
second year of the study.
6- Sampling of various organs of the two cancer models, at different time points, were
done, before and after treatment with various drugs, throughout first and second
year of the study.
Outputs:
1- Data has been generated during the first and second year and during the 1-year
extension.
2- Analysis of cell culturing, cell invasiveness and microscopy was done at the end of
first year.
3- Analyses of results concerning survival curves, as well as drug treatments, were done
by Kaplan-Meier using SPSS and GraphPad Sigma softwares during the first and the
second year and during the 1-year extension.
4- Analysis of results concerning drug treatments was during the second year.
5- The preparation of the final progress report was done after the end of the 1-year
extension after the end of the second year.
6- Three articles were prepared in this period, one accepted, one under revision and one
is being sent at the beginning of May 2013.
2013 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2013
Programme de subvention à la recherche scientifique au Liban – 2013
Page 2 of 5
Achievements -‫أالنجازات المحققة‬
I-
In vitro study
1- Growth curves of MDA-MB-231 and HuT-102 cell lines.
2- Metastatic mRNA expression profile of MDA-MB-231.
3- Effect of Av/OL and DCQ on the MDA-231 cell morphology.
4- Cytotoxity assay of Av/OL and DCQ on MDA-231 and HuT-102 cells.
5- Effect of Av/OL and DCQ on MDA-231 & HuT-102 cell proliferation
6- Effect of Av/OL and DCQ on MDA-MB-231 cell cycle.
7- Effect of Av/OL on homo- and hetero-cellular communication, by dye transfer assay.
8- Effect of Av/OL on the transcriptional expression of metastatic (VEGF, HIF-1, and
CXCR4) and connexin markers (Cx26 and Cx43) of MDA-MB-231 cells, by real-time PCR.
9- Effect of Av/OL on Cx43, MMP2 and MMP9 protein expression levels in MDA-MB-231
cells, by western-blot and Gelatin Zymography.
II-
In vivo study
1- NOD-SCID & NSG mice breeding.
2- Effect of Av/OL and DCQ on toxicity to the mice.
3- Effect of Av/OL and DCQ on survival studies of MDA-MB-231 (sub-dermal injections).
4- Effect of Av/OL and DCQ on survival studies of MDA-MB-231 (intravenous injections).
5- Effect of Av/OL on survival studies of HuT-102 (intra-peritoneal injections only).
6- Effect of Av/OL and DCQ on tumor volume measurement of subdermally injected mice.
7- Effect of Av/OL and DCQ on metastasis to the lungs and livers of MDA-MB-231 subdermally injected NSG mice.
8- Effect of Av/OL on metastasis to the lungs and livers of MDA-MB-231 intra-venously
injected NSG mice.
12345-
I took 3 M2R student from UL during this project
An article was submitted at the end of December 2012.
An article is being submitted during May 2013
An article was accepted in 2012, we acknowledged CNRS-L.
An abstract was sent for Oral presentation to the IGJC 2013 (International Gap Junction
Conference) that will be held at South Carolina, USA in July 13-18
PROBLEMS:
Analysis of the in vivo data took a lot of time, which explains the delay of 1 year that we had
in this project. In addition, we waited for the Software to do the cell cycle analysis which also
took some time to learn how to use it because of its sophistication
Setting up of the animal work was very complicated to put in place, which delayed the project.
Perspectives - ‫آفاق البحث‬
This project will be continued in the near future, especially on HUT-102 cells, another paper
will be prepared based on the results in hematological malignancies.
2013 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2013
Programme de subvention à la recherche scientifique au Liban – 2013
Page 3 of 5
Publications & Communications - ‫المنشورات والمساهمات في المؤتمرات‬
1- Zibara K, Awada Z, El-Saghir J, Dib L, El-Ghadban S, and El-Sabban M.
Anti-angiogenesis therapy and gap junction inhibition reduce MDA-MB-231 breast cancer
cell invasion and metastasis in vitro and in vivo.
Is BEING Sent for publication during beginning of May 2013.
2- Ghattass K, El-Sitt S, Zibara K, Rayes S, Haddadin MMJ, El-Sabban M, Gali-Muhtasib H.
DCQ is a hypoxia-activated drug that induces ROS-mediated apoptosis and reduces MDAMB-231 breast cancer metastasis in vitro and in vivo.
Antioxidants and Redox Signaling, Sent for publication in December 2012.
3- Zibara K, Hamdan R, Dib R, Sindet-Pedersen S, Kharfan-Dabaja M, Bazarbachi A, ElSabban M.
Acellular Bone Marrow Extracts Significantly Enhance Engraftment Levels of Human
Hematopoietic Stem Cells in Mouse Xeno-Transplantation Models.
PLoS ONE, 2012, 7(7): e40140. doi:10.1371/journal.pone.0040140
4- M2R training report (for the Masters Program of Genomics and Health at the Lebanese
University, UL) for a student, Mayyasa Rammah. Report was submitted to UL
committee in September 2010
5- M2R training report (for the Masters Program of Genomics and Health at the Lebanese
University, UL) for a student, Tahani Zein-Eddine. Report was submitted to UL
committee in July 2011
6- M2R training report (for the Masters Program of Genomics and Health at the Lebanese
University, UL) for a student, Zahra’a Awada. Report was submitted to UL committee
in September 2012
7- Abstract & Poster presentation for the 1st Annual AUB Biomedical Research Day, AUB,
February 2011. (See attached abstract)
8- Abstract & Poster presentation for the 3rd Annual AUB Biomedical Research Day, AUB,
February 2013. (See attached abstract)
9- Abstract for Oral presentation was submitted to the IGJC 2013 (International Gap Junction
Conference) that will be held at South Carolina, USA in July 13-18.
2013 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2013
Programme de subvention à la recherche scientifique au Liban – 2013
Page 4 of 5
Abstract - ‫موجز عن نتائج البحث‬
Metastatic spread of cancer cells is the most devastating aspect of malignancy. Cancer cells
secrete VEGF, which plays a key role in their growth, invasion, extravasation and metastasis.
Direct cancer cell-endothelial cell interaction, mediated by gap junctions, is of critical
importance in the process of extravasation. In this study, we evaluated the anti-angiogenic, antitumor and anti-metastatic activities of avastin (Av), an anti-VEGF antibody; and oleamide
(OL), a gap junction inhibitor, using MDA-MB-231 human breast cancer cells in vitro and a
xeno-graft murine model in vivo. The effect of Av, OL or their combination on proliferation,
cell cycle, migration, invasion, homo and hetero-cellular communication was studied. The
expression of several metastasis markers including VEGF, HIF1α, CXCR4, Cx26, Cx43, and
MMPs, were evaluated at the transcriptional and protein levels. In addition, the in vivo antitumor effect and survival studies of Av/OL were determined by subcutaneous and intravenous
injections of MDA-MB-231 cells in NSG mice. The effect of Av/OL on pulmonary and hepatic
metastases was also assessed. Results showed that Av/OL significantly decreased proliferation,
induced cell cycle arrest and decreased migration and invasion of MDA-MB-231 cells in vitro.
In addition, Av/OL significantly decreased homo and hetero-cellular communication interaction
between MDA-MDA and MDA-ECV cells, respectively. The transcriptional expression levels
of several factors including VEGF, HIF1α, CXCR4, Cx26, Cx43, and MMPs decreased by
more than 2-fold upon Av/OL treatment in vitro. On the other hand, avastin, but not oleamide,
reduced tumor size of NSG mice injected subdermally (s.d.) with MDA-MB-231 cells, which
was also associated with increased survival. Furthermore, both avastin and oleamide
significantly increased the survival rate of intravenously (i.v.) injected mice. Finally, Av or
Av+OL, and to a lesser extent OL, reduced pulmonary and hepatic metastatic foci in s.d. or i.v.
injected mice in vivo. In conclusion, while avastin has anti-angiogenic, anti-tumor and antimetastatic activities, oleamide has anti-metastatic activity, presumably at the extravasation
level, providing further evidence for the role of GJIC in the accomplishment of cancer cell
extravasation.
‫توقيع الباحث‬
Kazem Zibara
2013 ‫برنامج دعم البحوث العلمية‬
Grant Program for Scientific Research in Lebanon – 2013
Programme de subvention à la recherche scientifique au Liban – 2013
Page 5 of 5