Abstract
Camptothecin-20(s)-O-[N-(3’α,12’α-dihydroxy-24’-carbonyl-5’β-cholan)]-lysine (B2) is a novel camptothecin analogue. Our previous study had shown that it displayed higher cytoxicity activity towards hepatocellular carcinoma SMMC-7721 cells than camptothecin (CPT) in vitro. In this paper, the underlying mechanism of anti-proliferation of B2 towards SMMC-7721 cells was further examined. Cell growth inhibition of B2 was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; morphological changes were observed under Laser Scanning Confocal Microscope (LSCM); cell cycle distribution, apoptotic population, changes in mitochondrial membrane potential, intracellular calcium concentration and reactive oxygen species (ROS) production were determined by flow cytometry (FCM). Activities of caspase-3 and caspase-9 were measured, and the expression level of Bcl-2 and Bax proteins were analyzed by Western blot. The results suggested that B2 inhibited SMMC-7721 cell growth by causing cell cycle arrest at the S and G2/M phases, and induced apoptosis involving a mitochondrial pathway. B2 appears to cause a high induction of apoptosis on SMMC-7721 cells in vitro, which suggests it might be a potential drug for cancer therapy.
Highlights
Hepatocelluar carcinoma (HCC) is one of the most common malignancies in the World
The anti-proliferative activity of B2 was determined in SMMC-7721 cells by the MTT assay
In order to further explore the effects of B2 treatment on the cells, morphological observation was performed. Both the control cells and cells treated with 0.5 nM B2 for 48 h were viewed under the Laser Scanning Confocal Microscope (LSCM) (Figure 3)
Summary
According to the World Health Organization, 42% of HCC cases occur in China each year [1] It caused by unlimited proliferation and migration of cancer cells, and frequently is diagnosed as one of common solid tumors in the liver [2]. Some of the analogues showed stronger cytotoxicity to human liver cancer cells and low toxicity to normal liver cell compared to that of CPT. Among these derivatives, camptothecin-20(s)-O[N-(3’α,12’α-dihydroxy-24’-carbonyl-5’β-cholan)]-lysine (B2, Figure 1) demonstrated super cytotoxicity towards the human hepatocarcinoma cancer SMMC-7721 cell line than CPT, and competition assays indicated that bile acids played an important role to carry B2 into the bile acid–positive cancer cells. The present study was carried out to evaluate the effects of B2 on human hepatocarcinoma cancer cells proliferative using MTT assay, cell cycle distribution, the various factors of apoptosis, Bcl-2 and Bax protein expression
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