Effects for Sequential Treatment of siAkt and Paclitaxel on Gastric Cancer Cell Lines

Abstract

Real-time screening of cellular response on the drugs could provide valuable insights for the early detection of therapeutic efficiency and the evaluation of disease progression. Cancer cells have the ability to vary widely in response to stress in a manner to adjust the signaling pathway to promote the survival or having a resistance to stimulation. Cell-based label-free technologies using electronic impedance sensor have strategies for constructing the signature profiles of each cells. To achieve exquisite sensitivity to substantially change of live-cell response have an important role that predict the potential of therapeutic effects. In this study, we use an impedance-based real-time cell analysis system to investigate dynamic phenotypes of cells described as a cellular index value. We show that gastric cancer cells generated characteristic kinetic patterns that corresponded to the treatment order of therapeutics. The kinetic feature of the cells offers insightful information that cannot be acquired from a conventional single end-point assay. Furthermore, we employ a 'sequential treatment strategy' to increase cytotoxic effects with minimizing the use of chemotherapeutics. Specifically, treatment of paclitaxel (PTX) after down-regulating Akt gene expression using RNAi reduces the cell proliferation and increases apoptosis. We propose that the sequential treatment may exhibit more effective approach rather than traditional combination therapy. Moreover, the dynamic monitoring of cell-drug interaction enables us to obtain a better understanding of the temporal effects in vitro.

Keywords: Akt; gastric cancer; paclitaxel (PTX); real-time cell analysis (RTCA); sequential treatment, small interfering RNA (siRNA)..

Figure 1

Chemo-sensitivity of PTX on gastric cancer cell lines. (A) Cellular microscopic images for MKN28 and MKN45 cells at 24 h after addition of PTX. Scale bars mean 50 μm. (B) Proliferation profiles for MKN28 (left) and MKN45 (right) cells obtained by the RTCA after 24 h from PTX treatments (0 - 10⁴ nM, 1:10 serial dilutions). All graphs represent three independent experiments and with standard deviations (n = 3). (C) Cell viabilities for MKN28 and MKN45 cells calculated from (B) at 24 h from PTX treatment.

Figure 2

Effects of siAkt on gastric cancer cell lines. Relative Akt mRNA expression levels as a function of siScr and siAkt in a dose-dependent manner for (A) MKN28 and (B) MKN45 cells. Proliferation profiles monitored by RTCA for (C) MKN28 and (D) MKN45 cells treated with 50 nM of scrambled siRNA (siScr) and 50, 100, and 200 nM of siAkt, respectively. The normalized cell index was calculated every 2 h. All graphs represent three independent experiments and with standard deviations (n = 3). *p<0.01.

Figure 3

Sequential treatment of siAkt and PTX against MKN28 cells. (A) The index for sequential treatment of siAkt and PTX; DMSO treatment as a control (NT, ●), siAkt transfection (siAkt only, ○), simultaneous treatment of siAkt and PTX (siAkt & PTX, ▼), siAkt transfection after PTX treatment in sequential manner (PTX→siAkt, △), and PTX treatment after siAkt transfection in sequential manner (siAkt→PTX, ■). (B) Cellular microscopic images for MKN28 cells after the sequential treatment of siAkt and PTX at the indicated treatment time; △t = 12, 24, and 48 h. (C) Normalized proliferation profiles of MKN28 cells after the sequential treatment of siAkt and PTX. (D) ΔNormalized cell index calculated from (C). Non-treatment condition was used as a control. *p<0.01.

Figure 4

Sequential treatment of siAkt and PTX against MKN45 cells. (A) The index for sequential treatment of siAkt and PTX; DMSO treatment as a control (NT, ●), siAkt transfection (siAkt only, ○), simultaneous treatment of siAkt and PTX (siAkt & PTX, ▼), siAkt transfection after PTX treatment in sequential manner (PTX→siAkt, △), and PTX treatment after siAkt transfection in sequential manner (siAkt→PTX, ■). (B) Cellular microscopic images for MKN45 cells after the sequential treatment of siAkt and PTX at 24 h of treatment time; 100 (left column) and 200 nM (right column) of siAkt. (C) Normalized proliferation profiles of MKN45 cells after the sequential treatment of siAkt and PTX; 100 (left) and 200 nM (right) of siAkt. (D) ΔNormalized cell index calculated from (C). Non-treatment condition was used as a control. *p<0.01.

Figure 5

Apoptosis-related gene expressions after the sequential treatment of siAkt and PTX on gastric cancer cell lines. (A) The index for sequential treatment of siAkt and PTX; DMSO treatment as a control (NT, ●), siAkt transfection (siAkt only, ○), simultaneous treatment of siAkt and PTX (siAkt & PTX, ▼), siAkt transfection after PTX treatment in sequential manner (PTX→siAkt, △), and PTX treatment after siAkt transfection in sequential manner (siAkt→PTX, ■). Total RNA was extracted from the cells at 24 h of treatment time. The RNA samples were subjected to qRT-PCR to analyze apoptosis-associated genes (Akt, Bcl-xL, Bcl-2, Bad, and Caspase-3) of interest in (B) MKN28 and (C) MKN45 cells. *p<0.01.