Abstract
Objective Ultrasound-targeted microbubble destruction (UTMD) technique has recently been developed as a nonviral delivery of gene therapy. This study aimed at investigating the survival and apoptosis of ovarian cancer cell line OVCA-433 by inhibiting Livin expression through ultrasound-targeted microbubble destruction. Methods We synthesized a targeted microbubble agent for UTMD-mediated shRNA against Livin gene in human ovarian cancer OVCA-433 cells. Lipid microbubbles were conjugated with a luteinizing hormone-releasing hormone analog (LHRHa) by an avidin-biotin linkage to target the ovarian cancer OVCA-433 cells expressing LHRH receptors. The microbubbles were mixed with the recombinant plasmid harboring shRNA-Livin. shRNA-Livin was transfected into OVCA-433 cells upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm2) for 8 s. Cell survival was measured by the MTT assay, cell apoptosis by flow cytometry using annexin V/PI double staining, and cell ultrastructure by using the transmission electron microscope. The mRNA and protein expression levels of caspase-3 and caspase-8 were detected by RT-qPCR and western blotting. Results UTMD-mediated delivery of shRNA-Livin remarkably reduced the survival of OVCA-433 cells but promoted the apoptosis compared with shRNA-Livin alone, shRNA-Livin plus nontargeted microbubbles, and shRNA-Livin plus LHRHa-conjugated microbubbles containing shRNA-Livin with or without exposure to ultrasound pulses. It was also found that UTMD-mediated delivery of shRNA-Livin notably declined the mRNA and protein expression levels of caspase-3 and caspase-8 in OVCA-433 cells compared with shRNA-Livin alone, shRNA-Livin plus nontargeted microbubbles, and shRNA-Livin plus LHRHa-conjugated microbubbles containing shRNA-Livin with or without exposure to ultrasound pulses. Conclusion Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles can enhance the transfection efficiency of shRNA-Livin in ovarian cancer cells.
Highlights
Ovarian cancer usually originates from the fallopian tube rather than the ovary
OVCA-433 cells were assigned into blank, shRNA-Livin, shRNA-Livin-Nontargeted Lipid Microbubbles (NMBs), shRNA-Livin-Targeted Lipid Microbubbles (TMBs), shRNA-Livin + US, shRNA-Livin-NMB + US, and shRNA-Livin-TMB + US groups. e concentration of lipid microbubbles was 0.6 × 108/mL
Compared with the blank group, the other 6 groups showed remarkable increases in OVCA-433 cell apoptosis rate (P < 0.05), and the shRNA-Livin-TMB + US group indicated much higher cell apoptosis rate compared to the shRNA-Livin, shRNA-Livin-NMB, shRNA-Livin-TMB, shRNA-Livin + US, and shRNA-Livin-NMB + US groups (P < 0.01, Figure 1). ese data suggested that Ultrasound-targeted microbubble destruction (UTMD) enhanced the promotion of shRNA-Livin on OVCA-433 cell apoptosis
Summary
Ovarian cancer usually originates from the fallopian tube rather than the ovary. It refers to the epithelial cancer of the ovary or fallopian tube, as well as the histologically similar primary peritoneal cancer [1]. It has been reported that the people with ovarian cancer were with 47.4% five-year survival [4]. E prevalence of ovarian cancer is positively correlated with age. Women under 40 years of age are more likely to have high probability of ovarian germ cell tumor rather than ovarian cancer. Ovarian cancer is common among women over the age of 40 especially in developed countries, which is the second most common malignancy after breast cancer among this group [5, 6]
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