Studies Of Somatostatin Analogues And Methylated Oligonucleotide Reverse The Multidrug Resistance In Hepatomacellular Carcinoma

Backgroud and ObjectiveHepatocellular carcinoma (HCC) is one of the most common malignant tumors in our country. HCC is highly resistant to cytotoxic agents in clinic. The early resistant at the beginning of chemotherapy is the main cause of chemotherapy failure. In general, HCC cells may become cross-resistant to a whole range of drugs with different structures and function after exposure to a single cytotoxic agents, which is called multidrug resistance (MDR). It was suggested that P-glycoprotein (Pgp) and multidrug resistance-associated protein₂ (MRP₂), the members of the ATP-binding cassette (ABC) transporters family associating with MDR, were overexpression in HCC cells.This situation has not only motivated attempts to develop novel cytotoxic agents, but also stimulated research innovative noncytotoxic approaches to cancer treatment. Somatostatin analogues have been found to exhibit antiproliferated activity to non-neuroendocrine neoplasm by binding to somatostatin receptor both in vitro and in vivo. Up to now, there are 5 somatostatin receptor subtypes. Octreotide has high binding affinity for somatostatin receptor subtype SSTR₂, SSTR₃ and SSTR₅.However, the effect of octreotide on chemosensitivity of cytotoxic agents is less reported, and the mechanism remains unknown. This study was designed to investigate the expression of SSTR2 and SSTR3, and the changes of expression of Pgp and MRP2 after exposure to octreotide in HCC cells, to evaluate the antiproliferated activity of octreotide and the effect on chemosensitivity of cytotoxic agents in vitro. To investigate the relation between chemosensitivity of cytotoxic agents increased by octreotide and the expression of Pgp and MRP2.A accumulating evidence showed that DNA hypermethylation, especially in its promoter region, was associated with transcriptional silencing. This study is to evaluate the inhibiting effect of methylated oligonucleotide (MON) on gene transcription and the increasing chemosensitivity of HCC cells to cytotoxic agents.Methods1. A single-cell suspension of HepG2 cells with the concentration of 5 X 105 /ml was prepared for seeding. The cell growth inhibition was evaluated by tetrazolium (MTT) assay and the 50% inhibitory concentration (IC50) of epirubincin, hydroxyl-camptothecin, carboplatin and 5-fluorouracil were calculated by Bliss's software. RT-PCR was used to detect the mRNA expression of Pgp, MRP2, SSTR2, and SSTR3. Fluorescence-activated cell sorting (FACS) was used to detect the protein expression of Pgp and MRP2 in HepG2 cells.2. HepG2 cells were divided into five groups, control group and four experimental grpups with different concentrations of octreotide (0.2 u g/mk 0.4 u g/mk 0.8 u g/mk 1.6 u g/ml). Cytotoxic agents were added into experimental groups. RT-PCR and FACS were used to analyse the expression of Pgp and MRP2. Cytotoxicity was determined by MTT assay.3. Methylated oligonucleotide (MON) complementary to human MRP2 promoter region was designed. The non-methylated oligonucleotide (NON) carried same nucleotide acid sequence with MON, but the cytosines were not methylated. HepG2 cells were divided into three groups, control group, NON group and MON group, the later two groups were transfected with liposome-encapsulatedoligonucleotide. RT-PCR and FACS were used to analyse the expression of the MRP2. Cytotoxicity was determined by MTT assay.We incubated HepG2 cells with 0.8 u mol/L MON for 48 hrs. Cells were collected and then seeded in a new plate without further exposure to MON.Results1. Cell growth inhibition and IC50 changes: Octreotide inhibited the growth of HepG2 cells in a dose-dependent manner in vitro. The IC50 of epirubincin, hydroxyl-camptothecin, carboplatin and 5-fluorouracil were significantly reduced after combine with moderate or high dose octreotide (0.4 u g/mh 0.8 u g/ml, 1.6 u g/ml) (P < 0.05). There was synergistic cytotoxic effect between sub-cytotoxic dose octreotide (0.2 u g/ml) and vary concentration cytotoxic agents, and combination drug index were less than 1. The IC50 of cytotoxic agents negative linear correlated with concentration of octreotide (r=-0.956, -0.957, -0.958, -0.987, P<0.05).2. SSTR2 and SSTR3 were expressed in HepG2 cells.3. After exposed to octreotide for 24 hrs, the density of Pgp and MRP2 mRNA decreased gradually with different octreotide concentration in a dose-dependent manner (P<0.05). After 48 hrs, the expression of Pgp and MRP2 were not reduced significantly compared with 24 hrs (P> 0.05).4. After exposed to different concentration of octreotide for 24 h, the positive rates of Pgp were 12.69%, 8.68%, 4.01% and 3.65% by FACS respectively. The positive rates of MRP2 were 22.46%, 15.44%, 12.23% and 5.47%. The positive rates of Pgp and MRP2 were reduced significantly in a dose-dependent manner (P< 0.05). The positive rates of Pgp and MRP2 were 13.38% and 26.92% in control group.5. The IC50 of epirubincin, hydroxyl-camptothecin and carboplatin were significantly reduced after combine with moderate or high dose MON (0.4 u mol/L, 0.8 u mol/L, 1.6 U mol/L) in a dose-dependent manner (P<0.05), whereas NON had no effect. The IC50 of cytotoxic agents negative linear correlated with concentration of MON (r=-0.852, -0.914, -0.86, P<0.05).6. HepG2 cells expressed MRP2 highly. After transfected with different concentration of MON, the density of MRP2 mRNA decreased gradually with increase of MON concentration by RT-PCR (P< 0.05), while NON had no effect.7. The positive rates of MRP2 were 18.0%, 9.15%, 5.73%, 3.73% and 2.56% after transfected with different concentration of MON for 24 hrs respectively, and they were reduced significantly compared with control cells whose was 24.5% (P< 0.05). It was 22.3% in NON group.8. The inhibiting effect of MON on MRP2 could last 96 hrs. The positive rates of MRP2 were 4.26% and 19.86% for 96 h and 144 h respectively.Conclusions1. HepG2 cells is highly resistant to cytotoxic agents. The overexpression of Pgp and MRP2 is one of the main cause of inherent multidrug resistance in HCC.2. SSTR2 and SSTR3 are expressed in HepG2 cells and octreotide exhibits antiproliferated activity by binding to SSTR.3. Combinations of cytotoxic agents with octreotide have synergistic cytotoxic effect, and the IC50 of cytotoxic agents negative linear correlate with concentration of octreotide.4. The synergistic cytotoxic effect between cytotoxic agents and octreotide relates to reduction of the expression of Pgp and MRP2.5. Our data suggest that MON induces MRP2 DNA methylation by binding to MRP2 DNA target sequences, results in inhibiting MRP2 gene transcription. Methylation of MRP2 induced by MON can be maintained for 96 hrs.6. The IC50 of epirubincin, hydroxyl-camptothecin, carboplatin are reduced significantly in the cells transfected by MON, and negative linear correlate with concentration of MON.Meanings1. Our study firstly suggested there was synergistic cytotoxic effect between cytotoxic agents and octreotide, which might relate to the reduction of the expression...