Combination Treatment of Sorafenib and Bufalin Induces Apoptosis in NCI-H292 Human Lung Cancer CellsIn Vitro

Abstract

Background/Aim: Lung cancer notably contributes to tumor-associated mortality worldwide, and standard chemotherapy is used for lung cancer patients. However, its therapeutic efficacy remains unsatisfactory. This study aimed to evaluate the effects and molecular mechanisms of sorafenib and bufalin combination therapy on lung cancer cells in vitro. Materials and Methods: NCI-H292 cells were treated with sorafenib, bufalin, and sorafenib in combination with bufalin. Cell viability, ROS production, Ca 2+ release, and mitochondrial membrane potential were examined by flow cytometric assay. Annexin V/PI staining and chromatin condensation were examined by the apoptosis assays. Finally the molecular mechanism of apoptosis-associated protein expression was investigated by western blotting. Results: NCI-H292 cells treated with sorafenib in combination with bufalin showed significantly decreased viability, enhanced cellular apoptosis, and DNA condensation when compared to that with sorafenib or bufalin alone. Moreover, the combination treatment exhibited higher reactive oxygen species (ROS) production and lower mitochondrial membrane potential (ΔΨm). The combined treatment resulted in higher expression of SOD but lower catalase compared to sorafenib treatment alone. Compared to sorafenib or bufalin treatment alone, the combination treatment resulted in lower Bcl-2 expression but higher Bax, Bad, APAF-1, caspase-3, and caspase-9. Conclusion: Sorafenib in combination with bufalin shows more potent cytotoxic effects and cell apoptosis than sorafenib or bufalin treatment alone in NCI-H292 cells. The combined treatment significantly enhanced apoptotic cell death in NCI-H292 lung cancer cells by activating ROS-, mitochondria-, and caspase-signaling pathways in vitro.Cancer affects human health globally with its incidence being the most severe public issue of the 21 st century. The global number of lung cancer deaths remains high yearly (1). Lung cancers are mainly divided into two subtypes, including nonsmall cell lung cancer (NSCLC) and small cell lung cancer (SCLC). However, the NSCLC subtype occupies about 80-85% of lung cancers (2). Currently, patients undergo surgery, radiation, chemotherapy, and targeted therapy as the most commonly used strategies for lung cancer. Yet clinical outcomes of current therapies remain unsatisfactory: the 5year survival rate of lung cancer patients is less than 15% (3), and that of patients with metastatic disease is less than 10% 582 This article is freely accessible online.

Phenethyl isothiocyanate and irinotecan synergistically induce cell apoptosis in colon cancer HCT 116 cells in vitro

“…HCT 116 cells were maintained in 12‐well plates at a density of 1 × 10 5 cells/well for 24 h and treated with DMSO (vehicle control), 10 μM IRI, 10 μM PEITC, or 10 μM of IRI combined with 10 μM of PEITC for 48 h. After being harvested from each treatment, cell suspensions were resuspended in 500 μL of DCFH‐DA, Fluo‐3/AM (2.5 μg/mL), or DiOC 6 (4 μmol/L) for ROS, intracellular Ca 2+ level, or ΔΨ m level, respectively. Finally, all samples were further analyzed by flow cytometry as described previously 28,29 …”

“…Finally, each blot was incubated with a secondary antibody, horseradish peroxidase (HRP)‐linked anti‐mouse IgG antibody (#7076, dilution 1:10 000), or HRP‐linked anti‐rabbit IgG antibody (#7074, dilution 1:10 000; Cell Signaling Technology, Inc.). The signals of protein expressions were detected by chemiluminescence kits as the guideline (Merck KGaA) and quantitative analysis of each immunoreactive blot as previously described 28 …”

“…Finally, all samples were further analyzed by flow cytometry as described previously. 28,29 2.6 | Caspase-3, -8, and -9 activity assays HCT 116 cells (1 Â 10 5 cells/well) were cultured in 12-well plates overnight and then were incubated with DMSO (vehicle control), 10 μM IRI, 10 μM PEITC, and 10 μM of IRI combined with 10 μM of PEITC for 48 h. Cells were detached, collected, and individually resuspended in 25 μL of 10 μM substrate solution containing PhiPhiLux-G1D2, CaspaLux8-L1D2, and CaspaLux9-M1D2 that were substrates of caspase-3, -8, and -9, respectively, and maintained at 37 C for 60 min. Finally, cells were measured for individual caspase-3, -8, and -9 activities by flow cytometry as described previously.…”

“…Western blot analysis was performed using published procedures as described previously. 28 Briefly, cells were collected from the treatment of DMSO (vehicle control), IRI, PEITC, or IRI combined with PEITC for 48 h, followed by using a PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Inc., Seoul, Korea) to extract the total proteins. Subsequently, the whole-cell lysates (total proteins) were quantified by Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA).…”

“…Subsequently, the membrane was incubated with primary antibodies at 4 C overnight, and quantitative analysis of each immunoreactive blot as previously described. 28…”