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Sigma Xi Presentation
- 1. Melissa Carmichael Mentor: Dr. Merideth Krevosky
- 3. Surgery Platinum- Based Chemotherapy Recurrence Platinum Resistance Platinum Sensitivity
- 4. Withaferin A ~ WA • The main phytoconstituent of the Indian medicinal plant Withania somnifera. • Anticancer activity ~ induces apoptosis. • Fewer adverse side effects. • Few reports focus on the role of Hsps or resistance to cisplatin.
- 5. Hanahan andWeinberg, The Hallmarks of Cancer. Cell (100) 1, 7 January 2000 • Cancer cells acquire one or more hallmarks allowing them to replicate uncontrollably, generate their own blood supply, or metastasize to other organs. • One specific hallmark that often underlies recurrence and resistance to chemotherapy is the ability to evade apoptosis; also known as programmed cell death or cell suicide. • Evading Apoptosis is the hallmark on which my project focuses.
- 6. Apoptosis: Two Pathways by which Cells Undergo Programmed Cell Death • Apoptosis is a tightly regulated molecular pathway ultimately resulting in cell suicide. • Apoptosis is induced by two pathways, the extrinsic and the intrinsic pathway. • Both pathways converge on the activation of caspase-3 that leads to the disassembly of the cell.
- 7. Cisplatin-Sensitive OVCAR8 Control Cells Cisplatin-Sensitive OVCAR8 Cells Undergoing Apoptosis PARP Tubulin - + Tubulin Caspase-3 - + Immunoblot AnalysisMorphological AnalysisAnnexin Staining
- 9. Heat Shock Protein expression in cancer cells confers resistance to apoptosis and promotes a more aggressive tumor phenotype.
- 11. Cisplatin-Resistant OVCAR8R Cisplatin-Sensitive OVCAR8 OVCAR8R OVCAR8 B-Crystallin Hsp27 Vimentin Tubulin Caspase-3 OVCAR8R Cells Express Increased Small Hsps and Vimentin and Decreased Caspase-3
- 12. Treated OVCAR8 and OVCAR8R cells with WA for 24hrs to induce Apoptosis C WA Morphology Annexin Immunoblot Analysis Question: Is there a differential death response to treatment with WA? Negative Control 0.5µM WA 1.0µM WA 2.5µM WA 5.0µM WA 7.5µM WA
- 13. Morphological Studies Cisplatin-Sensitive OVCAR8 Cisplatin-Resistant OVCAR8R Cisplatin-Sensitive OVCAR8 cells undergo WA-induced apoptosis more readily than the Cisplatin- Resistant OVCAR8R cells.
- 17. WA (𝜇M)
- 18. Question: Does Alpha-B Crystallin play a role in conferring resistance to apoptosis? To Silence 𝛂B Crystallin Expression in Cisplatin-Resistant OVCAR8R Cells Utilized CRISPR/Cas9 System with a small guide RNA directed to Alpha-B Crystallin Once Clones Created and Selected: Challenge cells + WA HDR NHEJ
- 19. OVCAR8R cells were co-transfected with a Alpha-B Crystallin CRISPR/Cas9 plasmid and a HDR plasmid containing a puromycin resistance gene.
- 20. Puromycin Death Curve Clone Maintenance Clones were selected, expanded, and analyzed by Western Blot 15 successful clones were identified Two clones were selected for the continuation of the study Prior to transfection a puromycin death curve was performed to determine the optimal working concentration for the target cell line (OVCAR8R). Mammalian cell lines recommended working concentration range for puromycin is 0.5 – 10µg/mL. 0.5µg/mL was determined to be the optimal working concentration for the OVCAR8R cell line.
- 22. Cisplatin-Resistant OVCAR8R Cisplatin-Sensitive OVCAR8 Cr𝛂B1A Cr𝛂B7A Morphological studies show the 𝛂B-Crystallin knockout clones return to the cisplatin-sensitive cell morphology and lose the morphology they had prior to selection which was that of the cisplatin-resistant cells.
- 23. PARP Caspase-3 0.5 1.0 2.5 -C 5.0 7.5 Cr𝛂B1A Tubulin 0.5 1.0 2.5 -C 5.0 7.5 Cr𝛂B7A PARP Caspase-3 Tubulin PARP Caspase-3 Tubulin OVCAR8R 0.5 1.0 2.5 -C 5.0 PARP Caspase-3 Tubulin OVCAR8 0.5 1.0 2.5 -C 5.0 WA induced cleavage of PARP and Caspase-3 in the 𝛂B-Crystallin knockout clones more closely resembles that of the Cisplatin-Sensitive cell line rather than that of the Cisplatin-Resistant cell line from which they were created.
- 24. Overexpress 𝛂B Crystallin in Cisplatin-Sensitive OVCAR8 Cells: Transfect cells with wild-type Alpha-B Transfect cells with mutant Alpha-B Challenge cells with WA
Editor's Notes
- Add puryomcin article + CRISPR articles