VDAC1 ARE THE symbiont CALCIUM CHANNELS in MCF-7 cell lines targets

1. Autocrine Human Growth hormone
dependent calcium in Oligomerization of
VDAC1 on Carcinoma
D.Meera
Research Scholar
Under the Supervision of:
Prof. Ch. Venkata Ramana Devi
Dept of Biochemistry,
University of Science College, Hyderabad

2. • Introduction
• Applications
• Material and methods
• Expected Outcome
• Reference

3. Cancer
• Cancer is an abnormal growth of cells which tend to
proliferate in an uncontrolled way eventually
metastasize in some cases
• The word Cancer describes many categories for
example: carcinoma, sarcoma
• > 100 different types of cancer

4. •Breast cancer is one of the most common cancers
diagnosed in women.
• Approximately two-thirds of all breast cancers diagnosed
are classified as hormone dependent, which indicates that
hormones are the key factors that drive the growth of these
breast cancers.
•Ovarian and pituitary hormones play a major role in the
growth and development of normal mammary glands and
breast cancer.
• In particular, the effect of the ovarian hormone estrogen
has received much attention in regard to breast cancer.
•Pituitary hormones prolactin and growth hormone have
also been associated with breast cancer.
•Although the role of these pituitary hormones in breast
cancers has been studied, it has not been investigated
extensively.

5. • Breast cancer is the most common cancer and the leading
cause of mortality among women worldwide (1).
• Cancer cells are characterized by their state of higher
proliferation compared with normal cells (2).
• This requires reprogramming of cell metabolism and is thus
associated with the mitochondria, the site of ATP generation
(3).
• Therefore, mitochondrial bioenergetics, biosynthesis and
signaling are involved in tumorigenesis (4).

6. Proliferation of normal and cancer cell

7. Endocrine Mediators of Breast Cancer
• Estrogen
• Progesterone
• Growth hormone
• Prolactin

8. Growth Hormone (GH)
• The major hormone
regulating somatic growth
(somatotropin).
• GH secretion is under
control of two
hypothalamic factors,
GHRH and somatostatin.
• There is an overall
increase in GH levels
during puberty.

9. Role of calcium
• Calcium performs a number of basic functions in your body.
• Your body uses 99 percent of its calcium to keep your bones
and teeth strong, thereby supporting skeletal structure and
function.
• The rest of the calcium in your body plays key roles in cell
signaling, blood clotting, muscle contraction and nerve
function

10. Role of Growth hormone
• The breast is a multi-hormone target organ that undergoes
changes throughout the reproductive phases under the
influence of various hormones.
• It is also a well-known fact that hormones influence both the
development and growth of breast cancers.
• Global breast cancer incidence is on the rise, with nearly 1.7
million women being diagnosed with the disease and 0.5
million women succumbing to the disease annually.
• One out of eight women is expected to develop breast cancer
during their lifetime in the developed countries.
• A majority of the breast cancers develop sporadically.

11. VDCA1
• Voltage-dependent anion channels (VDACs) are situated in the
outer membrane of the mitochondria and serve as
gatekeepers that control metabolite and ion exchange
between the cytosol and mitochondria.
• VDAC1 is one of the most studied members of the VDAC
protein family and is overexpressed in multiple types of
cancer.
• However, the specific biological function and regulatory
mechanism of VDAC1 in breast cancer remains unclear.

12. Pathways depicting GH
Hypothesis
GH GHR JAK Ras Raf MEK ERK1/2
P
Tie Ang2
HIF-1 SP-1
ANGIOGENESIS
VEGF R2 VEGF
VEGF-A
• The Ras-MEK-ERK pathway stimulates through phosphorylation of the transcription
factor Sp1 and HIF-1 subunit and their recruitment to the VEGF-A promoter.
•Ang-2 is an antagonist ligand for Tie-2 in endothelial cells and like VEGF-A, is induced
under low oxygen condition through the HIF-1 
P

13. OBJECTIVES
• To explain the Role of autocrine growth hormone
in the breast cancer and disease progression.
• To demonstrate the role of Calcium in prognosis
of breast cancer.
• To demonstrate the role of calcium in regulation
of oligomerisation and cancer.
• To demonstrate VDAC1 expression in
mitochondria of MCF-7 cell lines and apoptosis.

14. Materials and Methods
Cell transfection
MTT Assay
Wound
healing assay
Cell Cycle
analysis
Western Blot
MTT assay
MTT assay
Cell culture

15. • The present study will be investigated on the biological role
of VDAC1 in breast cancer cells using an MTS assay.
• The association of clinicopathological features with VDAC1 in
breast cancer will be analyzed by Gene Expression Profiling
Interactive Analysis.
• The regulatory mechanism of VDAC1 will be determined by
cell transfection, western blot analysis, reverse transcription-
quantitative (q)PCR analysis.

16. Cell lines and transfection
• Mammary carcinoma cell lines MCF-7 were obtained from the
American Type Culture Collection (ATCC)
• Stably transfected with either the wild-type or translation-deficient
mutant hGH gene using Lipofectamine 2000 transfection reagent
(Life Science Technologies, Carlsbad, CA, USA).
• hGH-MCF-7 and MUT-MCF-7 cells will be cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), penicillin (100 units/ml), and streptomycin
(100 mg/ml) at 37 °C in a humidified 5% CO2 incubator.
• Observed under GFP fluorescent microscopy .
• At 24 h post-transfection, cells will be collected for further analysis.
• The transfection efficiency will be determined by western blot
analysis.

17. Apoptosis analysis
• MCF-7 (2 × 105), untreated or treated with BAPTA-AM, ionomycin or
other reagents, were analyzed for apoptotic cell death using
propidium iodide (PI), annexin V-fluorescein isothiocyanate (FITC)
and flow cytometer analysis.
• Collected cells (1500 × g for 10 min), washed, and resuspended in
200 μl binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl,
and 2.5 mM CaCl2).
• Annexin V–FITC will be added according to the recommended
protocol, and the cells incubated in the dark for 15 min.
• Cells were then washed once with binding buffer and resuspended
in 200 μl binding buffer, to which PI was added immediately before
flow cytometry analysis.
• Results are recorded on a dot plot, and analyzed by the FACS
Calibur flow cytometer software (BD Biosciences).

18. Expected Outcome
•Several clinical observations and experimental studies indicate that
pituitary hormones, including growth hormone, play a role in the
development of human breast cancer.
•Some studies suggest that calcium may help reduce the risk of certain
cancers, such as breast and colorectal cancer, but more research is
needed.
• In this regard, there is no data available with the combination, our aim is
to investigate the role of calcium in Autocrine Human Growth hormone
dependent activation and Epithelial mesenchymal transistion of mammary
carcinoma.
•VDAC1 has been identified as being critical for cancer cell survival.
•Investigating the specific regulatory mechanism of VDAC1 in cells will not
only improve the understanding of the tumorigenic process of cancer
cells, but may also provide novel therapeutic strategies for the treatment
of breast cancer.

19. References
• Goll DE, Thompson VF, Li H, et al. The calpain system.
Physiol Rev. 2003; 83:731–801. [PubMed: 12843408] ** A
very complete review of the calpain system and its complexity
• Schád E, Farkas A, Jékely G, et al. A novel human small
subunit of calpains. Biochem J. 2002; 362:383–388. [PubMed:
11853546]
• Kulkarni S, Goll DE, Fox JEB. Calpain cleaves RhoA
generating a dominant-negative form that inhibits integrin-
induced actin filament assembly and cell spreading. J Biol
Chem. 2002; 277:24435–24441. [PubMed: 11964413] .
• Sorimachi H, Ishiura S, Suzuki K. Structure and 12.
physiological function of calpains. Biochem J. 1997; 328 :721-
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