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Inhibitory Effect and Mechanism of P-5m Octapeptide on Hepatocarcinoma Metastasis

Author: HanXiao
Tutor: ZhuXun
School: Jilin University
Course: Immunology
Keywords: hepatocarcinoma tumor metastasis P-5m octapeptide matrixmetalloproteinase-2 5-fluorouracil
CLC: R735.7
Type: PhD thesis
Year: 2012
Downloads: 108
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Abstract

In recent years, increasing attention has been paid to synthetic antitumor peptides.Modern chemotherapy based on synthetic oligopeptides has the potential to provide aneffective anti-metastasis treatment for cancer patients whilst minimizing severeside-effects.P-5m, an octapeptide derived from domain5of HKa, was initially found to inhibitthe invasion and migration of melanoma cells. The high metastatic potential ofmelanoma cells was prevented by the HGK motif in the P-5m peptide in vitro and in anexperimental lung metastasis model, suggesting that P-5m may play an important rolein the regulation of tumor metastasis. Although it is quite clear that P-5m may inhibitthe invasion of various cancer cells in vitro and melanoma pulmonary metastasis inanimal models in vivo, the precise impact of P-5m on metastasis of human cancercells in an in vivo model is still uncertain. In the present study, we aimed to measurethe effect of P-5m on tumor metastasis of a HCCLM3human hepatocarcinoma cell lineand in a nude mouse model of hepatocellular carcinoma and to detect the underlyingmechanisms of any inhibitory effects. We also want to determine if P-5m couldincrease the sensitivity of H22cells to5-Fu chemotherapeutic, in vivo. Our study willprovide fundamental basis for R&D of this drug.1. P-5m inhibits the migration and invasion of HCCLM3cellsCancer cell migration and invasion are two important steps in cancer metastasis.We examined the effects of P-5m peptide on cell migration and invasion by tumorcells. An in vitro migration assay was used to investigate the inhibitory effect of P-5m.The quantitative assessment showed that the wound area of10and100μM of P-5mgroup were5%(p>0.05) and20%more than the control group, respectively. At48h,the wound area of10and100μM of P-5m group were11.8%and19%more than thecontrol group, respectively. Furthermore, the effect of P-5m on invasion by HCCLM3cells and cells of anadditional human hepatocarcinoma cell line (SMMC-7721) was determined in atranswell chamber and basement membrane matrigel invasion assay. Quantitative dataderived from three independent experiments shows that P-5m effectively inhibited thecell invasion in this assay. Reductions were concentration-dependent: at the lower dose(10μM) of P-5m, reductions were23.2%and26.8%, for SMMC-7721and HCCLM3cells, respectively; at the higher dose (100μM) of P-5m, reductions were46.4%and40.3%, for SMMC-7721and HCCLM3cells, respectively. In addition, HCCLM3cellsexhibited a more noticeable invasive ability, with the trans-filter cell number2timeshigher than SMMC-7721cells.2. Effect of P-5m on the protein level of MMP-2in HCCLM3cellsIncreased expression and activity of MMP-2has been well characterized inhuman HCC. To determine whether P-5m inhibits the MMP-2activity of HCCLM3cells, the cancer cells were treated with various concentration of P-5m in serum-freemedia. Because the expression of MMP-2in HCCLM3cells peaked at36h afterincubation, this time-point was selected as optimal for further experiments. Theconditioned media were analyzed by gelatin zymography. P-5m treatment reducedMMP-2release in a dose-dependent manner. Densitometric analysis of thezymograms showed reductions of95.6%,68.2%and47.6%compared to control at1,10, and100μM of P-5m, respectively.In addition, the cell samples were also lysed to detect MMP-2expression bywestern blotting. Densitometric analysis of the MMP-2levels in western blots showedreductions of84.2%,46.4%and33.8%compared to control at1,10, and100μM ofP-5m, respectively. Thus, changes in production of MMP-2measured intracellularlyand extracellularly by western blotting and gelatin zymography respectively, wereconcordant.3. P-5m suppresses pulmonary metastasis of hepatocellular carcinoma in vivoWe further examined the therapeutic efficacy of P-5m against tumor metastasis in anorthotopic hepatocellular carcinoma animal model. In the present study, we usedHCCLM3to establish a nude mouse orthotopic implantation model of humanhepatocellular carcinoma which may more effectively mimic natural human biologic behavior. During the experiment,5-Fu-treated mice showed serious sighs of toxicity.The metastasis nodules in the lung were counted and verified by HE staining. Therewas significant suppression of pulmonary metastasis in animals treated with50μg/kgP-5m therapy compared with the control group (p <0.05). Although500μg/kg P-5mtreatment caused some reduction in lung metastasis, this was not statistically significant(p>0.05).4. P-5m inhibits MMP-2expression in orthotopic hepatoma animal modelImmunohistochemical analysis showed that MMP-2was present in all tumorsamples from primary nodules, although differences in staining intensity were evidentbetween groups. MMP-2staining seemed to be more abundant in control group micethan either P-5m groups. Western blotting further confirmed that the50μg/kg P-5mdose, but not the500μg/kg dose, significantly decreased expression of MMP-2by46.7%in hepatoma tissue from mice. This suggests that the P-5m peptide suppressedmetastasis, at least in part through down-regulation of MMP-2, which is a crucialmolecule in tumor metastasis.To study the molecular mechanisms through which P-5m regulates hepatocellularcarcinoma cells motility, we compared expression of several genes in P-5m (50μg/kg)treated and control mice. The quantitative real-time PCR used contains24unique genesrelated to degradation of ECM, adhesion molecules, metastasis-related markers,apoptosis-related factors, cell cycle regulators, immune-regulating factors togetherwith other genes. Six genes were up-regulated in xenografts, namely TIMP-1, TIMP-2,CASP-3, MTSS-1, TGF-β1and IL-6. The up-regulation of the former five genes wasapproximately3-fold, but expression of IL-6was increased13-fold relative to thecontrol. Decreased expression of MMP-2in tumors was paralleled by the elevatedmRNA levels of metalloproteinase inhibitor TIMP-1and TIMP-2. These data furthersupported our hypothesis that the effect of P-5m on HCC is mediated throughregulating expression of MMP-2.5. Therapeutic effect of P-5m and/or5-Fu on mice bearing ascetic H22cellsIntraperitoneal chemotherapy could be a more optimal method in the treatment ofmalignant ascited, because it could provide high drug concentrations in ascetic fluidwith fewer systemic adverse effects. In the present study, a hepatocarcinoma H22 malignant ascites model was established in Kunming mice. The results showed that allgroups of mice besides high-dose of P-5m group had a reduction in body wight whencompared to the model control group. In line with this finding, a significant reduction inascites volume was observed in mice that were treated with P-5m plus5-Fu, whereasmedium-dose of P-5m had similar effect. We observed a significant reduction in totalH22asciteic cell count in mice from the combination group.All groups of drugs besides high-dose of P-5m group prolonged the survival time ofmice bearing ascetic H22cells when compared to the model control group. The mediansurvival time in mice in the combination group was52.8days, which corresponds to anincrease of305%compared to17.3day survival observed in mice in the model controlgroup.6. Effect of P-5m and/or5-Fu on the H22cells cell cycle and peritoneal capillarypermeability of the miceWe observed a significant reduction in the percentage of cells in the G0-G1phase in both the low-dose and medium-dose of P-5m groups, and5-Fu could inducethe S phase arrest of H22cells. Evans blue dye was used to evaluate the peritonealcapillary permeability of the mice. Briefly, mice were injected via the caudal veinwith8%Evans blue, the ascites were collected and the light aborption at thewavelength of580nm with a spectrophotometer.5-Fu had an inhibitory effect onpetironeal Evans blue concentrations. The data suggested P-5m enhance theanti-tumor effect of5-Fu on H22bearing mice, and antagonize its toxicity markedly.In conclusion, P-5m, an octapeptide derived from the D5domain of HKa, showed asignificant inhibitory effect on the metastatasis by the HCCLM3cell line in vitro and invivo. This effect appeared to occur, at least in part, through regulation of MMP-2expression. We also observed significant reduction in ascites volume and cell count inmice that were treated with P-5m plus5-Fu. The median survival time in mice in thecombination group was prolonged. We observed a significant reduction in total H22asciteic cell count in mice from the combination group. P-5m plus5-Fu could inducethe cell cycle arrest and inhibit the peritoneal capillary permeability of the mice. P-5min combination with5-Fu may be potentially useful in R&D of anti-metastatic drug andcancer intraperitoneal chemotherapy.5-Fu could induce the S phase arrest of H22cells.

Inhibitory Effect and Mechanism of P-5m Octapeptide on Hepatocarcinoma Metastasis

Abstract

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