Apoptosis in Mesothelioma Cells by Antisurvivin Oligonucleotides - PowerPoint PPT Presentation

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Apoptosis in Mesothelioma Cells by Antisurvivin Oligonucleotides

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Malignant Pleural Mesothelioma. Survivin. Antisurvivin oligonucleotides. Hypothesis. Methods ... Malignant pleural mesothelioma. Rare. Rapid local progression ... – PowerPoint PPT presentation

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Title: Apoptosis in Mesothelioma Cells by Antisurvivin Oligonucleotides


1
Apoptosis in Mesothelioma Cells by Antisurvivin
Oligonucleotides
  • Nucleic
    Acids

  • 440.02

  • Swathi Gannavaram

2
Overview
  • Apoptosis
  • Malignant Pleural Mesothelioma
  • Survivin
  • Antisurvivin oligonucleotides
  • Hypothesis
  • Methods
  • Results
  • Discussion
  • Conclusion

3
Apoptosis
  • Programmed cell death
  • Homeostasis

http//comchem.edugrid.ac.in/news/discovery/images
/8438wnews3a_art.gif
www.people.bath.ac.uk/pr1cemb/apo2.png
4
What is cancer?
www. research.nki.nl/agamilab/images/3_2.png
5
Malignant pleural mesothelioma
  • Rare
  • Rapid local progression
  • Late metastases
  • Poor prognosis
  • Fatal

www.mesothelioma-health.org
6
Other cancers Vs Mesothelioma
  • Mutations in p53,Ras and pRB genes
  • Uncommon
  • homozygous deletion of INK4a/ARF locus
  • Loss of p14ARF
  • Increase of MDM2
  • Functional Inactivation of p53

7
Survivin
  • inhibitors of apoptosis protein(IAP) gene family
  • Survivin-S 5-ATGGGTGCCCCGACGTTG-3
  • Survivin-A 5-AGAGGCCTCAATCCATGG-3
  • Internal control L19
  • L19-S 5-GAAATCGCCAATGCCAACT-3
  • L19-A 5-TCTTAGACCTGCGAGCCTCA-3

8
Apoptosis, survivin and mesothelioma?
  • Overexpression of survivin in human mesothelioma
    cells makes them resistant to apoptosis and
    insensitive to chemotherapy.
  • Survivin Apoptosis
    mesothelioma

inhibits
9
Antisurvivin oligonucleotide
  • Targets 232-251 nucleotides of survivin mRNA
  • 5-CCCAGCCTTCCAGCTCCTTG-3
  • Control has reverse sequence
    5-GTTCCTCGACCTTCCGACCC-3

10
Hypothesis
  • Survivin overexpressed in mesothelioma cells
  • Inhibits apoptosis
  • Antisurvivin oligonucleotides used to inhibit
    survivin promoting apoptosis

11
Materials and Methods
  • Tumor tissues and Cell lines
  • RNA isolation
  • cDNA Microarray Hybridization
  • RT-PCR
  • Western Blotting
  • Antisurvivin and Control Oligonucleotides
  • Treatment of cells with Antisense and Control
    Oligonucleotides
  • Dose-dependent Analysis
  • Caspase-3 Activity Assay
  • Annexin V Apoptosis Analysis

12
Results
Chunyao Xia et al, Molecular Cancer Therapeutics,
1, 687694, (2002).
13
cDNA Microarray Hybridization
  • Over expression of Bcl2 and survivin in
    mesothelioma cells.
  • Bcl2 5.1 folds
  • Survivin 4.6 folds

14
RT-PCR Western Blotting
  • a. Survivin expression in mesothelioma cell
    lines.
  • b. Survivin expression in freshly resected human
    mesothelioma tissues.

15
Treatment of cells with Antisense and Control
Oligonucleotides
16
Dose-dependent Analysis
  • Dose-response curve of the effect of antisurvivin
    oligonucleotides on the growth and viability of
    H28 cells.

17
Caspase-3 Activity Assay
  • Apoptosis is induced due to increased caspase-3
    activity.

18
Apoptosis Analysis by Flow Cytometry
  • High of cell death seen with antisurvivin
    oligonucleotides.

19
Western blot analysis of survivin expression
  • Time dependent analysis of survivin expression.

20
Discussion
  • Bcl2 (?) 3 of 14 cases of mesothelioma
    and 7.5 of 174 gastric carcinomas.
  • Target for antisense oligonucleotide gene therapy
    survivin(?)
  • 7 out of 8 cell lines and 12/12 fresh
    mesothelioma tissues are found to be survivin
    positive
  • Data obtained

21
Conclusions
  • Survivin is over expressed in mesothelioma cell
    lines and fresh human mesothelioma tissues.
  • Antisurvivin oligonucleotides induce apoptosis by
    down regulating survivin and thus sensitizing the
    cells to chemotherapeutic agents.

22
Relevance to Therapy
  • ?

23
Reference
  • Chunyao Xia et al., Induction of Apoptosis in
    Mesothelioma Cells by Antisurvivin
    Oligonucleotides. Molecular Cancer Therapeutics,
    1, 687694, (2002).

24
Special Thanks
  • Dr.Friesen

25
Questions
  • ?
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