Anticancer Activity Of 9-Nitrocamptothecin Liposomes On Hepatocellular Carcinoma

Nowadays, the treatment of malignant tumors remains a worldwide problem. Besides surgical operation, the chemotherapy is the most important approach of cancer treatment. However, the high drug toxicity is a barrier to treatment because adverse effects limit drug dosage that can be administered. This is best exemplified by cytotoxic drugs. Usually, cytotoxic cancer drugs are very effective in vitro, but in human clinical use the drugs act indiscriminately on both tumorous and healthy tissues. Adverse effects can be both serious and unpleasant and range from nausea and hair loss to neutropenia and kidney failure. One of the many challenges in chemotherapy is how to deliver an effective dose of a cytotoxic agent to the tumor site, while at the same time minimizing unintended harmful side effects.The use of drug delivery systems (DDS), especially the nano drug delivery system (NDDS) provides a new approach for delivery of chemotherapeutics. NDDS can improve the pharmacological properties of traditional chemotherapeutics by altering drug pharmacokinetics and biodistribution.The compound camptothecin (CPT) is an alkaloid which extracted from the stem wood of the Chinese tree Camptotheca acuminate. The discovery of CPT as an anticancer drug has added an entirely new dimension to the field of chemotherapy in 1960s.9-nitrocamptothecin (9NC) is a water insoluble analogue of camptothecin. 9NC has shown a strong antitumour activity against solid tumour xenografts. The antitumor activity of 9NC closely depended on its structure:the lactone form of 9NC was important to its antitumor activity. However, the maintenance of the lactone form is a challenge for the clinical application of 9NC. The applications of 9NC were also limited due to its poor solubility, instability and low oral bioavailability.Objective:In order to maintain the antitumor activity and improve the water solubility of 9NC for tumor treatment by intravenous administration, a novel liposome system containing 9NC was constructed via entrapped 9NC in liposomes with classic film-ultrasonic method.In this study, we test the anticancer properties and mechanisms of free 9NC and 9NC loaded liposomes (9NC-LP). We accomplished further in vitro and in vivo studies of them and examined the possible molecular mechanisms responsible for its anticancer activity.Methods:1. The effects of the test compounds on HepG2, Bel-7402, Hep 3B, SMMC-7721 and L02 cells viability were determined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The effects of 9NC and 9NC-LP on cell proliferation were determined using cell proliferation ELISA assay (BrdU (colorimetric); Roche Diagnostics). After that, IC50 of drugs was calculated by SPSS 13.02. To measure the cell cycle and Sub-G1 peak after cells incubated with compounds. Cells were exposed to 9NC and 9NC-LP for 24 h,48h and 72h, followed by incubation with RNase and staining with propidium iodide. The DNA content was determined by FACSCalibur flow cytometer. To measure the cell apoptosis after cells incubated with compounds. Cells were staining with Annexin V-PE/7-AAD after exposure of 9NC and 9NC-LP and were analyzed with a Becton Dickinson FACSCalibur instrument.3. Human hepatocellular cancer xenograft tumor model was established via injecting s.c. HepG2 cells into the backs area of the nude mice.9NC and 9NC-LP were administrated intravenously (i.v.) via tail vein with saline. Tumor size was determined every other day. All animals were monitored for activity, physical condition, body weight, and tumor growth.4. The levels of cell cycle related protein and cell apoptosis related protein in cell lysates and tissue homogenates were assessed by western blot.Results:1. Both 9NC and 9NC-LP can inhibit the hepatocellular carcinoma cell growth in vitro and in vivo. The results showed that inhibitory effects on the cell viability and proliferation have a cell type dependent. In general, for both drugs, the order of inhibitory was HepG2> Bel-7402> SMMC-7721> L02> Hep3B. meanwhile, inhibitory effects of compounds also show a time-and dose-dependent manner.2. The FACS results show that both 9NC and 9NC-LP can cause apoptosis and arrest of cell cycle. Cell cycle phase is alternation according to the compound concentration and incubation time in all test cell lines. S phase and G2/M phase delay was observed in all cell lines. Results also show both the compounds induced apoptosis in a dose-dependent manner in all tested cell line.3. In vivo,9NC-LP show a more potent inhibitory effect on HepG2 xenograft tumor model than 9NC after administrated via intravenous; while 9NC show more side effects than 9NC-LP, such as vomiting, diarrhoea and severe haemorrhagic cystitis.4. In vitro, expression of cell cycle-related and apoptosis-related proteins was regulated by compounds in HepG2 and L02 cells, however, this modification was not found in Hep3B cells. In vivo, the patterns of protein expression in the treated xenograft tumors were similar with those observed in vitro.Conclusions and Innovation:1. Incorporation with liposomes did not reduce the anticancer activity of 9NC. Anticancer activity of 9NC-LP show a time-and dose-dependent in vitro and in vivo which means it is consistent with the general characteristics of chemotherapeutics.2. Both the most common causes of cell growth inhibition-cell cycle arrest and apoptosis-were involved in the effect of 9NC-LP. Apoptosis was observed only at higher concentration and longer incubation time than cell cycle arrest which indicate cell cycle arrest is predominantly in the effect. And p53 may be one of the important regulators in 9NC-LP caused cell death.3. More importantly,9NC-LP has a more therapy effects and fewer side effects than 9NC in vivo which means 9NC-LP can improve the pharmacological properties of 9NC.9NC-LP is a promising compound for cancer therapy via intravenous administration.