Examination of resistance of lung adenocarcinoma cells to cisplatin by technetium-99m methoxyisobutyl isonitrile

Abstract

To study whether technetium-99m methoxyisobutyl isonitrile ((99m)Tc-MIBI) can be used to examine the drug resistance of lung adenocarcinoma cells and to explore the efficiency of gensenoside Rh2 in reversing the resistance of adenocarcinoma cells to cisplatin (DDP). Human lung adenocarcinoma cells of the line A549 sensitive to DDP and drug-resistant lung adenocarcinoma cells of the line A549DDP were cultured. DDP and gensenoside (Rh2) of different concentrations were added. Mthoxyisobutylisnitrile (MTT) method was used to test the inhibit concentration (IC) of DDP and Rh2 to the cells. The IC(50) of DDP to these 2 kinds of cells and its low-efficiency inhibition concentration (< or = IC(20)) to A549DDP cells, and the IC(5) of Rh2 to A549DDP cells were calculated. < or = IC20 was regarded as the low-efficiency concentration of DDP to A549DDP cells and IC(5) was regarded as the in-toxic concentration of Rh2 to A549DDP cells. A549DDP cells were divided into 4 groups: control group, added with normal saline; DDP group, added with DDP at the low-efficiency concentration; Rh2 group, added with in-toxic Rh2; and DDP + Rh2 group, added with DDP at the low-efficiency concentration and Rh2 at the in-toxic concentration. Cell apoptosis was detected by fluorescence microscopy and flow cytometry. Forty-seven hours after the stimulation by different drugs (99m)Tc-MIBI solution was added and 1 hour later the radioactivity of the cells was detected by gamma-counter. Twenty-four nude mice were divided into 4 equal groups: A549 group, inoculated with A549 cells and normal saline intraperitoneally; control group, inoculated with A549DPP cells and normal saline intraperitoneally; DDP group, inoculated with A549DDP cells and low-efficient DDP intraperitoneally; and Rh2 + DDP group, inoculated with A549DDP cells and low-efficient DDP and in-toxic Rh2intraperitoneally. The growth of tumor and survival of mice were observed. Before the inoculation of tumor cells, 4 mice were randomly selected to undergo single photons emission computed tomography (SPECT). Two months after the inoculation SPECT was performed on all mice. By the end of experiment all the mice were killed and their tumors underwent pathological examination. The IC(50) of DDP was 24 microM to A549 cells and 325 microM to A549DDP cells, with a resistance index of 13.54. When the concentration of Rh2 was < or = 10 microM there was no evident toxicity to A549DDP cells. The inhibition rate of 100 microM DDP to the A549DDP cells was 12%. After the cells were treated by 10 microM Rh2 and 100 microM DDP, the IC(50) of DDP to A549DDP cells was decreased to 94 microM; compared with the cells treated by 100 microM DDP alone, the reverse resistance of the latter was 3.5 times that of the former. Fluorescence microscopy showed that fluorescence was distributed uniformly in the nuclei of A549DD cells in the Rh2 group, DDP group, and the control group, and fluorescence were conglomerated like grain in the nuclei and apoptotic little substance appeared in the Rh2 + DDP group. The apoptotic rates of the control group, Rh2 group, DDP group, and DDP + Rh2 group were 6.1% +/- 1.0%, 5.9% +/- 1.1%, 8.2% +/- 1.0%, and 59.5% +/- 1.2% with a significant difference between the DDP + Rh2 group and control group (P < 0.01). There was no evident apoptotic apex in the control group, Rh2 group and DDP group, whereas there was distinct apoptotic apex in the Rh2 + DDP group. The radioactivity of (99m)Tc-MIBI could be incepted by the 4 groups. The radioactivity of the DDP + Rh2 group was significantly lower than that of the control group (P < 0.05) and there were no significant difference in radioactivity between the other 3 groups and the control group (all P > 0.05). The radioactivity of the A549 cells was significantly higher than that of the A549DDP cells (P < 0.01). Dense (99m)Tc-MIBI image of tumor could be seen in the A549 group mice, control group mice, and DDP group mice, the latter 2 groups with lighter images. No tumor image was seen in the Rh2 + DDP group mice. The R or R' value in the A549 group mice was remarkably higher than those in the control group and DDP group mice (both P < 0.05). (99m)Tc-MIBI can be used to examine the resistance of lung adenocarcinoma A549DDP cells. Gensenoside Rh2 of in-toxic concentration can reverse the resistance of lung adenocarcinoma A549DDP cells to cisplatin.