Apoptosis Of Hepatocarcinoma Cells Induced By PEGFP-anti-AFP Vector And Arsenic Trioxide

Background and objectiveHepatocellular carcinoma (HCC) is one of the most common malignancies in the world. The significant features of hepatocellular carcinoma are high cancer incidence, malignancy degree, mortality and metastasis. There is still no effective methods for treatment. With the development of molecular biology technology, immunological technology and cellular signaling transduction technology, more and more biological methods are used in treatment of cancer. One method is inducing tumor cell apoptosis.In the 1700's, arsenic trioxide was found to be a valuable therapeutic tool for patients with acute promyelocytic leukemia. Now arsenic trioxide is used as an medicinal agent for many malignancies. But few researchs focus on comparing effects of arsenic trioxide on hepatocellular carcinoma cell line with different characters.a-fetoprotein (AFP) is serves as a diagnostic tumor-specific marker in majority of hepatocellular carcinoma cases.As an immune suppressive protein, AFP can inhibit the immune reaction and enhancethe tumor growth. AFP has crucial function on the development of hepatocellular carcinoma, so gene therapy targeting on a-fetoprotein is a hot point in associated research.This study used AFP positive cell lines HepG2 and Hep3B, AFP negative cell line SMMC-7721 and normal cell line Changliver as research objects, observed and compared effects of arsenic trioxide on these cells. We constructed recombine antisense-AFP gene vector. When transfected the vector into different cells, we detected the reduce of AFP gene. Then we treated cells combined with arsenic trioxide and antisense-AFP gene vector and observed the apoptosis. This study tried to find a new strategy for cancer treatment by combination of chemical drug and gene therapy. Methods1. Studies on arsenic trioxide induced different cells apoptosis1) Cell viability was detected by MTT assay;2) Nuclear change was detected by Giemsa staining;3) Nuclear condensation was detected by Hochest 33342 staining;4) Cell cycle change and apoptosis was analyzed by FACSCalibur;5) The expression of NF-κB were detected by immunocytochemistry;2. Studies on transfection pEGFP-anti-AFP vector into hepatocarcinoma cells1) The construction of pEGFP-anti-AFP vector;2) pEGFP-anti-AFP vector was transfected into different cells using Lipofectamine TM 2000;3) The expression of intracellular AFP mRNA was detected by RT-PCR;4) The expression of AFP protein was detected by immunocytochemistry;5) Cell viability was detected by MTT assay;6) Cell cycle change and apoptosis was analyzed by FACSCalibur;3. Studies on combination of pEGFP-anti-AFP vector and arsenic trioxide in inducing apoptosis of different cells1) Cell viability was detected by MTT assay;2) Cell cycle change and apoptosis was analyzed by FACSCalibur;Results1,ATO has obvious inhibition effects on HepG2 and Hep3B AFP positive hepatocarcinoma cells, but not on SMMC-7721 AFP negative hepatocarcinoma cells and Changliver normal liver cells2,After HepG2 and Hep3B cells were treated with ATO, the cells exhibited special morphologies of apoptosis, such as cell shrinkage, smaller cell size, concentration of vesicles on cell surface, and appearance of apoptotic bodies. Giemsa and Hochest33324 staining indicated that the nucleus condensed and the chromatin cleaved.3,After treated with ATO, the percentage of HepG2 and Hep3B cells obviously increased in G0/G1 phase, while the percentage of SMMC-7721 cells slightly increased in G2/M phase. However, the percentage of Changliver cells in different phases has little changed.4,After treated with ATO, NF-κB protein expression was increased.5,RT-PCR results showed the level of AFP mRNA increased in ATO-treated HepG2 and Hep3B cells.Immunocytochemistry results showed the level of AFP increased in ATO-treated HepG2 and Hep3B cells.6,pEGFP-anti-AFP vector has obvious inhibition effects on HepG2 and Hep3B AFP positive hepatocarcinoma cells. When pEGFP-anti-AFP vector treated on HepG2 cells, the quantity of apoptosis cells was increased.7,When combination of pEGFP-anti-AFP vector and arsenic trioxide treated on HepG2 and Hep3B AFP positive hepatocarcinoma cells, the quantity of apoptosis cells was increased, but not on SMMC-7721 AFP negative hepatocarcinoma cells and Changliver normal liver cells.8,After treated with pEGFP-anti-AFP vector and ATO, the percentage of HepG2 cells obviously increased in G0/G1 phase and G2/M phase.Conclusion1. ATO has obviously inhibition effects on HepG2 and Hep3B AFP positive hepatocarcinoma cells, but not on SMMC-7721 AFP negative hepatocarcinoma cells and Changliver normal liver cells.2. After treated with ATO, the percentage of HepG2 and Hep3B cells obviously increased in G0/G1 phase, while the percentage of SMMC-7721 cells slightly increased in G2/M phase. However, the percentage of Changliver cells in different phases little changed.3. After treated with ATO, NF-κB protein expression was increased.4. Antisense-AFP gene vector has obviously inhibition effects on AFP positive hepatocarcinoma cells.5. When combination of pEGFP-anti-AFP vector and arsenic trioxide treated on HepG2 and Hep3B AFP positive hepatocarcinoma cells, the quantity of apoptosis cells was increased, but not on SMMC-7721 AFP negative hepatocarcinoma cells and Changliver normal liver cells.6. After treated with pEGFP-anti-AFP vector and ATO, the percentage of HepG2 cells obviously increased in G0/G1 phase and G2/M phase.