CRISPR-mediated upregulation of DR5 and downregulation of cFLIP synergistically sensitize HeLa cells to TRAIL-mediated apoptosis

Naresh Poondla, Arun Pandian Chandrasekaran, Klaus Heese, Kye-Seong Kim, Ramakrishna Suresh

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received attention as an anticancer therapy because it mediates apoptosis of several cancer cell types but not normal human cell types. In this study, we implemented genome editing techniques to upregulate DR5 and downregulate cFLIP in HeLa cells to stimulate TRAIL-induced apoptosis. We designed and validated sgRNAs to enrich the endogenous level of DR5 by dead Cas9 (dCas9). Similarly, we designed two sgRNAs to disrupt the cFLIP gene by CRISPR/Cas9. We analyzed the effect of TRAIL on tumor cells by co-transfecting HeLa cells with the best combinations of sgRNAs regulating DR5 and cFLIP genes. TRAIL-induced apoptosis in HeLa cells was evaluated by the γH2AX foci formation assay to check for double-strand break and propidium iodide and Annexin V staining to quantify apoptotic cells. Viable cells were identified by CCK-8 assay, and cleaved-PARP level was evaluated by Western blot. This is the first study to demonstrate that genome editing techniques can be used as an effective combinatorial treatment strategy to induce apoptosis of cancer cells. In particular, enhancement of DR5 expression and inhibition of cFLIP expression by genome editing had a synergistic effect of inhibiting proliferation and inducing apoptosis in TRAIL-resistant HeLa cells. These results suggest that combinatorial treatment strategies mediated by the CRISPR/Cas9 system may be effective for design of other human TRAIL-resistant cell types.

Original languageEnglish
Pages (from-to)60-65
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume512
Issue number1
DOIs
StatePublished - 2019 Apr 23

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Clustered Regularly Interspaced Short Palindromic Repeats
HeLa Cells
Up-Regulation
Down-Regulation
Apoptosis
Genes
Cells
Assays
Sincalide
Propidium
Annexin A5
Neoplasms
Tumors
Tumor Necrosis Factor-alpha
Ligands
Western Blotting
Staining and Labeling

Keywords

  • Anticancer therapy
  • DR5
  • Genome editing
  • Tumor necrosis factor-related apoptosis-inducing ligand
  • cFLIP

Cite this

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title = "CRISPR-mediated upregulation of DR5 and downregulation of cFLIP synergistically sensitize HeLa cells to TRAIL-mediated apoptosis",
abstract = "Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received attention as an anticancer therapy because it mediates apoptosis of several cancer cell types but not normal human cell types. In this study, we implemented genome editing techniques to upregulate DR5 and downregulate cFLIP in HeLa cells to stimulate TRAIL-induced apoptosis. We designed and validated sgRNAs to enrich the endogenous level of DR5 by dead Cas9 (dCas9). Similarly, we designed two sgRNAs to disrupt the cFLIP gene by CRISPR/Cas9. We analyzed the effect of TRAIL on tumor cells by co-transfecting HeLa cells with the best combinations of sgRNAs regulating DR5 and cFLIP genes. TRAIL-induced apoptosis in HeLa cells was evaluated by the γH2AX foci formation assay to check for double-strand break and propidium iodide and Annexin V staining to quantify apoptotic cells. Viable cells were identified by CCK-8 assay, and cleaved-PARP level was evaluated by Western blot. This is the first study to demonstrate that genome editing techniques can be used as an effective combinatorial treatment strategy to induce apoptosis of cancer cells. In particular, enhancement of DR5 expression and inhibition of cFLIP expression by genome editing had a synergistic effect of inhibiting proliferation and inducing apoptosis in TRAIL-resistant HeLa cells. These results suggest that combinatorial treatment strategies mediated by the CRISPR/Cas9 system may be effective for design of other human TRAIL-resistant cell types.",
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CRISPR-mediated upregulation of DR5 and downregulation of cFLIP synergistically sensitize HeLa cells to TRAIL-mediated apoptosis. / Poondla, Naresh; Chandrasekaran, Arun Pandian; Heese, Klaus; Kim, Kye-Seong; Suresh, Ramakrishna.

In: Biochemical and Biophysical Research Communications, Vol. 512, No. 1, 23.04.2019, p. 60-65.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - CRISPR-mediated upregulation of DR5 and downregulation of cFLIP synergistically sensitize HeLa cells to TRAIL-mediated apoptosis

AU - Poondla, Naresh

AU - Chandrasekaran, Arun Pandian

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AU - Kim, Kye-Seong

AU - Suresh, Ramakrishna

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