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

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Apoptosis

• Programmed cell death
• Homeostasis

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www.people.bath.ac.uk/pr1cemb/apo2.png
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What is cancer?
www. research.nki.nl/agamilab/images/3_2.png
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Malignant pleural mesothelioma

• Rare
• Rapid local progression
• Late metastases
• Poor prognosis
• Fatal

www.mesothelioma-health.org
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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

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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

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Apoptosis, survivin and mesothelioma?

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

inhibits
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Antisurvivin oligonucleotide

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

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Hypothesis

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

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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

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Results
Chunyao Xia et al, Molecular Cancer Therapeutics,
1, 687694, (2002).
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cDNA Microarray Hybridization

• Over expression of Bcl2 and survivin in
  mesothelioma cells.
• Bcl2 5.1 folds
• Survivin 4.6 folds

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RT-PCR Western Blotting

• a. Survivin expression in mesothelioma cell
  lines.
• b. Survivin expression in freshly resected human
  mesothelioma tissues.

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Treatment of cells with Antisense and Control
Oligonucleotides
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Dose-dependent Analysis

• Dose-response curve of the effect of antisurvivin
  oligonucleotides on the growth and viability of
  H28 cells.

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Caspase-3 Activity Assay

• Apoptosis is induced due to increased caspase-3
  activity.

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Apoptosis Analysis by Flow Cytometry

• High of cell death seen with antisurvivin
  oligonucleotides.

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Western blot analysis of survivin expression

• Time dependent analysis of survivin expression.

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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

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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.

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Relevance to Therapy

• ?

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Reference

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

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Special Thanks

• Dr.Friesen

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Questions

• ?