Enhancement of anti-tumor effects of 5-fluorouracil on hepatocellular carcinoma by low-intensity ultrasound.
BACKGROUND
Hepatocellular carcinoma (HCC) accounts for 75% of liver cancers and is the second most lethal cancer, associated with its multiple etiologies, poor prognosis and resistance to chemotherapy drugs. Chemotherapy treatment on HCC suffers low efficacy of drug uptake and can produce a range of side effects. Here we report an investigation on the effect of a combined treatment on human hepatocellular carcinoma BEL-7402 cells using low-intensity ultrasound (US) and 5-fluorouracil (5-FU).


METHODS
The uptake of 5-FU was measured by the high-performance liquid chromatography (HPLC). DNA damage was detected by the comet assay. MTT assay was used to examine cell viability. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) were respectively detected by the fluorescent probes DCFH-DA or JC-1. Endogenous apoptosis-associated proteins were analyzed by the western blot and immunohistochemistry. Histopathological changes were evaluated by the hematoxylin and eosin (H&E) staining. Cell apoptosis was evaluated by the TUNEL and flow cytometry assays. Cell proliferation was measured using the immunohistochemical staining of PCNA.


RESULTS
Our results showed that low-intensity US (1.1 MHz, 1.0 W/cm2, 10% duty cycle) significantly enhanced the uptake of 5-FU, 5-FU-mediated DNA damage and reactive oxygen species (ROS) generation. The increased ROS production up-regulated the p53 protein level, which led to the up-regulation of Bax and down-regulation of Bcl-2. The enhancement of ROS generation and the activation of the apoptosis-associated proteins further triggered the collapse of mitochondrial membrane potential, released cytochrome c from mitochondria into cytosol and activated the mitochondria-caspase pathway, and cell apoptosis. Such enhanced effects could be partially blocked by the ROS scavenger N-acetylcysteine (NAC). Overall, low-intensity US combined with 5-FU led to an effective inhibition of tumor growth and prolonged overall survival of BEL-7402 HCC-bearing nude mice by more than 15% compared with 5-FU treatment alone.


CONCLUSIONS
Our results showed that low-intensity ultrasound combined with 5-FU produced much enhanced synergistic anti-tumor effects via enhanced ROS production in treating HCC.
Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin, 150080, China.
Zheng
Z
Hu
Department of Biochemistry and Molecular Biology, Harbin Medical University, Harbin, 150086, China.
Guixiang
G
Lv
Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin, 150080, China.
Yongning
Y
Li
Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin, 150080, China.
Enze
E
Li
Department of Biochemistry and Molecular Biology, Harbin Medical University, Harbin, 150086, China.
Haixia
H
Li
Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin, 150080, China.
Qi
Q
Zhou
Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin, 150080, China.
Bin
B
Yang
Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin, 150080, China. dzk@psu.edu.|Department of Mathematics, and Materials Research Institute, The Pennsylvania State University, University Park, 16802, PA, USA. dzk@psu.edu.
Wenwu
W
Cao
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage* / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000628:therapy*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003131:Combined Modality Therapy; D015536:Down-Regulation; D005472:Fluorouracil / Q000008:administration & dosage* / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000628:therapy*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D017382:Reactive Oxygen Species / Q000378:metabolism; D016896:Treatment Outcome; D014464:Ultrasonic Therapy / Q000379:methods*; D015854:Up-Regulation; D023041:Xenograft Model Antitumor Assays
D000964:Antimetabolites, Antineoplastic; D017382:Reactive Oxygen Species; D005472:Fluorouracil
5-fluorouracil; Anti-tumor effects; Hepatocellular carcinoma; Low-intensity ultrasound; Reactive oxygen species