Virtual Library

Start Your Search

S. Hsu



Author of

  • +

    MINI 37 - SCLC Therapy (ID 165)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Small Cell Lung Cancer
    • Presentations: 1
    • +

      MINI37.01 - Chk1 Inhibition Enhances Cisplatin Cytotoxicity Regardless of p53 Status in Human Small Cell Lung Cancer Cells (ID 947)

      18:30 - 20:00  |  Author(s): S. Hsu

      • Abstract
      • Presentation
      • Slides

      Background:
      Small cell lung cancer (SCLC) has a poor prognosis and harbors complex genetic alterations including frequent loss-of-function mutations of p53 and Rb, which impair the G1/S checkpoint control. Checkpoint Kinase 1 (Chk1) is a vital serine/threonine specific protein kinase responsible for halting the cell cycle in check after DNA damage. With abrogation of Chk1-mediated cell cycle checkpoint control, cancer cells may enter mitosis with extensive DNA damage leading to mitotic catastrophe and apoptotic cell death. Previous in vitro studies showed that p53 deficient cancer cells benefit from Chk1 inhibition. Here we demonstrate that a combination of Chk1 inhibition and cisplatin causes more growth inhibition and caspase activation in SCLC cell lines compared to cisplatin alone, regardless of p53 status.

      Methods:
      Chk1 inhibition was achieved by siRNA knockdown (Qiagen) and AZD7762 (Selleckchem) in p53 mutant SCLC cell lines (GLC4, NCI-H82) and p53 intact SCLC cell lines (NCI-H128, NCI-H209). Cell viability was measured by Cell-Titer Glo assay (Promega) after 72hrs of drug treatment. Synergism was defined by combination index (CI)>1 using the Chou-Talalay method. Cell cycle analysis was performed by PI staining and detected by FACS. Western blotting and immunofluorescent staining were used to evaluate caspase activation and other signaling proteins.

      Results:
      SCLC cell lines were treated with cisplatin 24hrs at each IC50 dosage after Chk1 siRNA transfection. In GLC4 after 2.5uM cisplatin treatment, cell viabilities of control siRNA-treated and Chk1 siRNA-treated cells were 28% and 10.6% (p=0.006, by paired t-test), respectively. Similar significant reduction of cell viability was observed in 1uM cisplatin-treated NCI-H82 cells (44.6% vs. 29.7%; p=0.0632) and in 3uM cisplatin-treated NCI-H128 cells (62.5% vs. 45.3%; p=0.0155), respectively. More cleaved caspase-2 and caspase-3 were noted in Chk1 knockdown plus cisplatin-treated GLC4 cells than in cisplatin alone. The IC50 (72hrs) of single agent AZD7762 (Chk1 inhibitor) treatment was 240nM, 211nM, 266nM and 215nM in GLC4, NCI-H82, NCI-H128 and NCI-H209 respectively. The combination indexes of AZD7762 and cisplatin (both given at around IC50s) calculated by Chou-Talalay method indicated synergism in all these 4 cell lines. Cell cycle analysis revealed that AZD7762 abrogated cisplatin-induced G2/M arrest in GLC4 and G1 arrest in NCI-H128. Inhibition Chk1 by AZD7762 was associated with reduction of CDC25C and CDC2 phosphorylation. Phospho-Histone H3 (mitotic marker) was increased in AZD7762 and cisplatin combined treatment compared to cisplatin alone in a p53 independent fashion. Intriguingly, inhibition of Chk1 by AZD7762 alone in GLC4 cells activated caspase-2.

      Conclusion:
      Chk1 inhibition both by siRNA knockdown and AZD7762 enhances cisplatin cytotoxicity. The synergism was primarily due to increased apoptosis and abolished cell cycle arrest. Although p53 is frequently mutated in SCLC, growth inhibition was seen in a p53 independent manner. In GLC4, single agent AZD7762 treatment can cause caspase-2 activation through an as yet unidentified mechanism. Our findings suggest that Chk1 is a potential therapeutic target in small cell lung cancer and is synergistic with chemotherapy. The effects of Chk1 inhibitor and its combination with chemotherapy agents in SCLC animal models are currently underway.

      Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.