The Preliminary Research Of The Subcellular Location And Transport Mechanism Of Tyroserleutide In The Human Hepatocellular Carcinoma BEL-7402 Cells

Objective:To explore the location of antitumor tripeptide compound tyroserleutide (YSL) in the human hepatocellular carcinoma BEL-7402 cells and its preliminary mechanism of transport mediated by human proton dependent peptide transporter.Methods:1. Establishing a synthetic method of fluorescent analogue of YSL and purified it by native PAGE.2. The purity and the stability of the fluorescent analogue of YSL were assessed by capillary electrophoresis and fluorescent spectrophotometric assay; The biochemical activity of the fluorescent analogue of YSL was evaluated through the excretion of LDH of the injured human hepatocellular carcinoma BEL-7402 cells by speptrophotometric assay.3. The subcellular distribution of the fluorescent analogue of YSL in the human hepatocellular carcinoma BEL-7402 cells and whether the fluorescent analogue of YSL were colocalized with the mitochondria of the human hepatocellular carcinoma BEL-7402 cells were observe by laser scanning confocal microscopy.4. The effect of YSL on the membrane fluidity and plasma membrane potential of human hepatocellular carcinoma BEL-7402 cells in vitro were assessed by fluorescent polarization assay and flow cytometry to provide the evidence for the style of transporting into the tumor cells.5. The effect of YSL on the mitochondrial membrane potential and mitochondrial swelling of human hepatocellular carcinoma BEL-7402 cells were measured by spectrophotometric assay to observe whether YSL has the direct effect on the mitochondria of the tumor cells.6. The expression and the subcellular location of PEPT1 in the human hepatocellular carcinoma BEL-7402 cells were observed by fluorescent immunoassay.7. The influence of GLY-GLN, GLY-SARCOSINE and GLY-GLY-GLY on the effect of the proliferative inhibition rate of YSL to the human hepatocellular carcinoma BEL-7402 cells was assessed by MTS method; The influence of GLY-GLN, GLY-SARCOSINE and GLY-GLY-GLY on the the YSL uptake of the human hepatocellular carcinoma BEL-7402 cells was observed by inverted fluorescent microscopy.8. The effect of YSL on PEPT1 mRNA of human hepatocellular carcinoma BEL-7402 cells was observed by Real-time quantitative PCR.Results:1. The fluorescent material 5(6)-TAMRASE can conjugate with YSL stably. The fluorescent analogue of YSL can be purified by 20% native PAGE.2. The purified fluorescent analogue of YSL was stable. There was no separation between the 5(6)-TAMRASE and YSL when it was stored at-20℃avoiding from light for 2 weeks and both of the fluorescent intensities and the spectrum had no great changes taken place. There was no significant decrease of the activity of LDH excreted by human hepatocelluar carcinoma BEL-7402 cells between the tumor cells labeled YSL treated and unlabeled YSL treated (P>0.05). The biological activity of the YSL had no obviously changes labeled before and after.3. YSL fluorescent analogue can enter the human hepatocellular carcinoma BEL-7402 cells when they were incubated with the cells and distributed in the cells intensely. The distribution pattern of fluorescent analogue of YSL was different from that of the same doze of fluorescent material 5(6)-TAMRASE. YSL fluorescent analogue mainly located at the cytoplasm of the human heaptocarcinoma BEL-7402 cells and colocalization with the mitochondria.4. YSL (1.6mg/ml) can decrease the membrane microviscosity and increase the membrane fluidity of the human heaptocellular carcinoma BEL-7402 cells in vitro. YSL can decrease the plasma membrane potential of the human heaptocellular carcinoma BEL-7402 cells in vitro and lead to the depolarizing of the cells.5. YSL can decrease the isolated mitochondrial potential of the human heaptocellular carcinoma BEL-7402 cells, influence the permeability of the mitchondria and caused the mitochondrial swelling when it was incubated with the isolated mitochondria of the tumor cells in vitro.6. The PEPT1 protein can express in the human heaptocellular carcinoma BEL-7402 cells and mainly located at the membrane and cytoplasm of the tumor cells.7. The substrate of PEPT1 GLY-SARCOSINE,GLY-GLN and GLY-GLY-GLY can weaken the inhibition effect of YSL on human hepatocellular carcinoma BEL-7402 cells in vitro and inhibited the uptake of YSL by human hepatocellular carcinoma BEL-7402 cells in vitro respectively.8. YSL (1.6mg/ml) can upregulate the PEPT1 rnRNA expression of the human hepatocellular carcinoma BEL-7402 cells in vitro.Conclusion:YSL can be labeled with fluorescent material 5(6)-TAMRASE stably and the antitumor biological activity of YSL was matained without any changes. We speculate that YSL can enter the human hepatocellular carcinoma BEL-7402 cells and located at the plasma to kill the tumor cells. PEPT1 in the human hepatocellular carcinoma BEL-7402 cells maybe involved in the transport process of YSL across the BEL-7402 cells membrane. One of the antitumor mechanisms of YSL when they were transported into the tumor cells was realized by injuring the mitochondria of the tumor cells.