Study On Growth Inhibitory Effects Of Heparanase Antisense Oligodeoxynucleotide In Malignant Melanoma

Heparanase is a recently cloned endo-β-D-glucuronidase that catalyzesthe hydrolytic cleavage of theβ-1, 4-glycosidic bond between aD-glucuronate and a D-glucosamine in heparan sulfate. It has beenconsidered to play an important role in tumor invasion and hematogenousmetastasis by degrading heparan sulphate proteoglycan and destroying theextra-cellular matrix. Pathological increases in heparanase mRNAexpression and heparanase protein levels have been reported in gastriccancer, liver cancer, pancreatic cancer, colon carcinoma, ovarian cancer,prostatic carcinoma, lymphoma and melanoma, and have been confirmedto be associated with the tumor invasion, metastatic potential and thepatient's poor prognosis. Recent studies have also found that increasedheparanase expression is implicated in the growth, proliferation andapoptosis in several malignant tumors, including ovarian cancer, gastriccancer, et al. To date, however, no research reports have been conducted onthe relationship between heparanase activity and the tumor growth inmalignant melanoma. Moreover, the study on growth inhibitory effectscaused by antisense oligodeoxynucleotide (ASODN) against heparanase mRNA gene in melanoma A375 cells and transplanted melanoma have notbeen reported.Melanoma is a common malignancy of melanocytes locatedpredominantly in the skin, accounts for 1～2% in all malignancies. Datashowed that the average survival time of patients with advanced melanomais 6～10 months. In recent years, the incidence of melanoma has beensignificantly increased. In 2006, melanoma is the seventh most commoncancer in the United States; 40～60 individuals per 100,000 people inAustralia were new melanoma cases per year; From 1981 to 2000, thestatistics of China also showed that there was an increasing trend in skinmelanoma, with the average annual growth rate of 3.9%. Although a largenumber of clinical and experimental studies about melanoma therapy havebeen performed, but its efficacy has not shown significant improvement.With the rapid development of gene therapy recently, ASODNtechnique has been used extensively in the study of regulation geneexpression and anti-tumor growth, and has been proven to be a neweffective method in the treatment of malignant tumor.To investigate the relationship between heparanase gene expressionand the tumor growth in malignant melanoma, the in vitro and in vivoexperiment in this study have been employed, by using artificial design andsynthesis ASODN directed against human heparanase mRNAs, cell culture,liposome transfection, transplanted tumor experiment in nude mice,RT-PCR, in situ hybridization, immunohistochemistry methods, todetermine the inhibition of heparanase ASODN on tumor growth inmalignant melanoma. Three parts were included in the present study.Part 1Expression of heparanase mRNA and protein in malignant melanomaObjective:To evaluate the difference of heparanase mRNA and protein expression between benign and malignant tumors in melanocytes, and therole of heparanase in the development of malignant melanoma.Methods:1. In situ hybridization and immunohistochemistry(SP, AEC stain)were respectively employed to detect the expression of heparanase mRNAand protein in 30 cases of malignant melanoma, A375 cell lines, 30 casesof melanocytic nevi and 15 cases of normal skin samples.2. Theχ² test was used to analyze the data with SPSS10.0 software(α=0.05). P value of less than 0.05 was considered significant.Results:1. Heparanase mRNA expression in tissue and A375 cells: Thepositive signal was seen in 18 cases (60.00%, 18/30) of malignantmelanoma, A375 cells and 1 case (3.33%, 1/30) of melanocytic nevi, therewas no positive signal in normal skin samples. The intensity of signal wasstrong in malignant melanoma and A375 cells, but weak in melanocyticnevi. There was significant difference in positive rate of heparanase mRNAexpression between malignant melanoma and melanocytic nevi(χ1²=22.259, P=0.000), malignant melanoma and normal skin samples(χ²=25.714, P=0.000).2. Heparanase protein expression in tissue and A375 cells: Thepositive staining for heparanase antibody was seen in 19 case (63.33%,19/30) of malignant melanoma, A375 cells, and 2 cases (6.67%, 2/30) ofmelanocytic nevi, there was no positive signal in normal skin samples. Theintensity of staining was strong in malignant melanoma and A375 cells, butweak in melanocytic nevi. There was significant difference in positive rateof heparanase protein expression between malignant melanoma andmelanocytic nevi (χ1²=21.172, P=0.000), malignant melanoma and normalskin samples (χ1²=27.805, P=-0.000).3. Correlation between heparanase mRNA and protein expression in malignant melanoma: Positive expression of heparanase mRNA andprotein was simultaneously seen in 18 cases (60.00%), negative expressionin 11 cases (36.67%). The concordance rate of heparanase mRNA andprotein expression was 96.67 % (29/30).Part 2Inhibition of heparanase ASODN on A375 cells proliferation in vitroObjective:To investigate the effects of heparanase ASODN on the proliferationand heparanase mRNA and protein expression in A375 cells, and screenthe most effective heparanase ASODN sequence in vitro.Methods:1.4 sequences of ASODN (ASODN-t₁, ASODN-t₂, ASODN-t₃ andASODN-t₄) against human heparanase mRNAs and 1 sequence ofheparanase N-ODN were synthesized, prepared in the concentration of10μmol/L, 20μmol/L and 30μmol/L, and transfected into melanoma A375cells by cationic liposomes. The control group was incubated in RPMI1640 Medium alone.2. The morphological change and growth of A375 cells in all groupswere observed by inverted microscope. The viability of the cells wasdetermined by the trypan blue exclusion test.3. The heparanase mRNA and protein expression in 30μmol/LASODN-t₂ group, N-ODN group and control group were detected by insitu hybridization and immunohistochemistry (SP, DAB stain).4. Data were expressed as Mean±SD. The ANOVA and q-test wasused to analyze the data with SPSS10.0 software (α=0.05). P value of lessthan 0.05 was considered significant.Results:1.Effects of heparanase ASODN on the growth and morphology inA375 cells: When observed by inverted microscope, the cells in the control group growed thrively, and polygonal cells arranged closely in "pavedstone" style. HE staining showed cells in the control group were variablesizes and polygonal, with a increased nuclear-cytoplasmic ratio, deeplystained nucleus and mitosis. Compared with those in control and N-ODNgroup, the growth of A375 cells in different ASODN groups was slow, butno apparently morphological change was seen.2. Effects of heparanase ASODN on A375 cells proliferation: Therewas significant difference between the cell numbers in all groups(F=86.390, P=0.000). The cell numbers in 10μmol/L, 20μmol/L,30μmol/L ASODNs groups were all significantly lower than those incontrol group (P all=0.000). The cell numbers in all 10μmol/L, 20μmol/L,30μmol/L ASODNs groups showed significant difference (P all=0.000)each other. The cell number in 30μmol/L ASODN-t2 group weresignificantly lower than those in the other ASODN groups (P all＜0.000).30μmol/L ASODN-t2 was the most effective sequence in the present study.3. Effects of heparanase ASODN on heparanase mRNA expression inA375 cells: The results in situ hybridization for heparanase mRNAexpression found that the positive signal was confined to the cytoplasma ina blue granular staining pattern. The intensity of signal was weak in 30μmol/L ASODN-t2 group, but strong in N-ODN group as well as in controlgroup. The scores were 1.250±0.500 in ASODN-t2 group, 4.000±0.817 inN-ODN group and 4.250±0.500 in control group, respectively. One-wayANOVA showed significant differences among the 3 groups (F=28.500,P=0.000). The scores in N-ODN group and control group weresignificantly higher than that of 30μmol/L ASODN-t2 group (P all=0.000).4. Effects of heparanase ASODN on heparanase protein expression inA375 cells: The positive staining for heparanase antibody was seen in all 3groups. Heparanase positive staining was confined to cytoplasma in a red granular staining pattern.The intensity of staining was weak in 30μmol/LASODN-t2 group, but strong in N-ODN group as well as in control group.The scores were 1.500±0.527 in 30μmol/L ASODN-t2 group, 4.500±0.577in N-ODN group and 5.000±0.817 in control group, respectively. One-wayANOVA showed significant differences among the 3 groups (F=32.250,P=0.000). The scores of N-ODN group and control were significantlyhigher than that of 30μmol/L ASODN-t2 group (P all=0.000).Part 3Growth inhibition of heparanase ASODN on the transplantedmelanoma in nude miceObjective:To establish the model of human malignant melanoma transplantedsubcutaneously in nude mice, and investigate the effects of heparanaseASODN on tumor growth, expression of heparanas mRNA and protein aswell as the expression of VEGF and bFGF protein in human melanomatransplanted subcutaneously in nude mice.Methods:1.25 female BALB/c nude mice were divided randomly into 5 groups,each group contained 5 mice.The mice were injected subcu- taneouslywith 10μmol/L,20μmol/L,30μmol/L ASODN-t₂ and 30μmol/L N-ODNtransfected cells, respectively. Cells cultured in RPMI 1640 Medium alonewas used as control group. The size of tumors was measured with verniercaliper. The histological changes were observed after 6 weeks.2. Heparanase mRNA expression was determined by RT-PCR and insitu hybridization, and heparanase protein was determined by immuno-histochemistry (SP, DAB stain) in 5 groups.3. VEGF and bFGF protein in 30μmol/L ASODN-t₂ group,30μmol/LN-ODN group and control group were determined by immuno-histochemistry (SP, DAB stain). 4. Data were expressed as Mean±SD. The ANOVA and q-test wasused to analyse the data with SPSS10.0 software (α=0.05). P value of lessthan 0.05 was considered significant.Results:1. Establishment of the animal model in nude mice with A375 cellstransfected by heparanase ASODN: By means of subcutaneous injectionwith A375 cells, transplanted melanoma was obtained in all 25 nude mice,the achievement ratio of heterogenic transplantation was 100%.2. Histological changes in transplanted melanoma in nude mice: 6weeks later, HE staining showed various sizes of cell mass and focalnecrosis were observed under light microscopy.The sizes of necrosis weresmall in ASODN-t₂ groups, and large in control group and N-ODN group.3. Effects of heparanase ASODN on tumor growth in transplantedmelanoma in nude mice: 6 weeks After injection, one-way ANOVAshowed significant differences among the 5 groups in transplanted tumorvolumes (F=8.228, P=0.000).There were significant differences betweenall ASODN -t₂ groups and control group, all ASODN-t₂ groups andN-ODN group, 30μmol/L ASODN-t₂ group and the other ASODN-t₂ groupin the transplanted tumors volume (P all＜0.05); However, there were nosignificant differences between control group and N-ODN group,10μmol/L ASODN-t₂ group and 20μmol/L ASODN-t₂ group (P all＞0.05).4. Effects of heparanase ASODN on heparanase mRNA expression intransplanted melanoma in nude mice:RT-PCR results indicated that positive expression of heparanasemRNA was obtained in 5 groups. The expression intensity was weak in allASODN-t₂ groups, and strong in N-ODN group as well as control group.One-way ANOVA showed significant differences among the 5 groups inrelative amount of heparanase mRNA (F=90.392, P=0.000). There was nosignificant difference between control and N-ODN group (P＞0.05). In situ hybridization results found that the positive signal wasconfined to the cytoplasma in a blue granular staining pattern. Theintensity of signal was weak in all ASODN-t₂ groups, and strong inN-ODN group as well as control group. One-way ANOVA showedsignificant differences among the 5 groups in scores of heparanase mRNAexpression (F=90.392, P=0.000). There was significant difference betweenall ASODN-t₂ groups and control group, all ASODN-t₂ groups and N-ODNgroup, 30μmol/L ASODN-t₂ group and the other ASODN-t₂ group (P all＜0.05).However, there was no significant difference between control groupand N-ODN group, 10μmol/L ASODN-t₂ group and 20μmol/L ASODN-t₂group (P all＞0.05).5. Effects of heparanase ASODN on heparanase protein expression intransplanted melanoma in nude mice: The positive staining for heparanaseantibody was obtained in 5 groups. Heparanase positive staining wasconfined to the cytoplasma in a yellow-brown granular staining pattern.The intensity of staining was weak in all ASODN-t₂ groups, and strong inN-ODN group as well as control group. One-way ANOVA showedsignificant differences among the 5 groups in scores of heparanase proteinexpression (F=76.257, P=0.000). There was significant difference betweenall ASODN-t₂ groups and control group, all ASODN-t₂ groups and N-ODNgroup, 30μmol/L ASODN-t₂ group and the other ASODN-t₂ group (P all＜0.05); However, there was no significant difference between controlgroup and N-ODN group, 10μmol/L ASODN-t₂ group and 20μmol/LASODN-t₂ group(P all＞0.05).6. Effects of heparanase ASODN on VEGF protein expression intransplanted melanoma in nude mice: The positive staining for VEGFantibody was observed in all 3 groups. The positive staining was confinedto the cytoplasma in a yellow-brown granular staining pattern. Theintensity of staining was weak in 30μmol/L ASODN-t₂ groups, and strong in N-ODN group as well as control group. One-way ANOVA showedsignificant differences among the three groups in scores of VEGF proteinexpression (F=25.194, P＜0.001).7. Effects of heparanase ASODN on bFGF protein expression intransplanted melanoma in nude mice: The positive staining for bFGFantibody was observed in all 3 groups. The positive staining was confinedto the cytoplasma in a yellow-brown granular staining pattern.The intensityof staining was weak in 30μmol/L ASODN-t₂ groups, and strong inN-ODN group as well as control group. One-way ANOVA showedsignificant differences among the 3 groups in scores of bFGF proteinexpression (F=14.807, P＜0.005).Conclusions:1. There are high expressions of heparanase mRNA and protein inmelanoma. Moreover, the. positive expression rate in melanoma issignificantly higher than that in melanocytic nevi and normal skin. Thesefingdings suggest heparanase may play an important role in thedevelopment of melanoma.2. High expressions of heparanase mRNA and protein in A375 cellsare firstly observed in this study. These data indicate that cell line A375can be used as a target cell line to investigate the relation betweenmelanoma and heparanase.3. The proliferation of melanoma A375 cells cultured in vitro can beinhibited by Heparanase ASODN in a concentration-dependent manner.These results suggest that heparanase ASODN have an anti-melanomaactivity in vitro.4. The expressions of heparanase mRNA and protein in melanomaA375 cells can be down-regulated by Heparanase ASODN. These findingsindicate that the inhibitory effect on proliferation of melanoma A375 cellscan derive from the down-regulation of heparanase mRNA and protein expressions among them.5. For the first time, Animal transplanted melanoma model in nudemice is successfully established by subcutaneous injection with A375 cellstransfected by heparanase ASODN liposome.6. It is firstly proved in the present study that heparanase ASODNcan markedly inhibit the growth of transplanted melanoma anddown-regulate the expression of heparanase mRNA and protein in nudemice. This result suggest heparanase ASODN have an anti-melanomaactivity in vivo.7. It is firstly found in this study that heparanase ASODN candown-regulate VEGF and bFGF protein expressions in transplantedmelanoma in nude mice. This finding indicate that heparanase ASODNmay have anti-vessel growth activity in melanoma.8. Anti-melanoma mechanism of heparanase ASODN is associatedwith not only the inhibitory effects on proliferation of melanoma cells, butalso the inhibitory effects on growth of tumor vessels.