A Gelatinases-targeting scFv-based Fusion Protein Shows Enhanced Antitumour Activity with Endostar against Hepatoma.

Gelatinases play important roles in tumour invasion and metastasis and are thus considered promising targets for cancer therapy. In this study, a new single-chain variable fragment (scFv)-based fusion protein Fv-LDP, composed of the anti-gelatinases scFv and lidamycin apoprotein (LDP), was prepared, and its combination with angiogenesis inhibitor Endostar was then investigated. The fusion protein Fv-LDP specifically bound to various tumour cells, and its binding capability to human pulmonary giant cell carcinoma (PG) cells was higher than that of LDP. Fv-LDP inhibited the expression and secretion of gelatinases and could be internalized into tumour cells via endocytosis. Fv-LDP also suppressed the growth of human hepatoma cells and murine hepatoma 22 transplanted in Kunming mice in various degrees. In addition, Endostar could enhance the synergistic or additive inhibition of Fv-LDP on the growth, migration or invasion of human hepatoma cells shown by a colony formation assay and a transwell-based migration or invasion assay, respectively. In vivo, Fv-LDP/Endostar combination showed a significantly synergistic effect on the growth of a human hepatoma xenograft, with an inhibition rate of 80.8% compared with the Fv-LDP (44.1%) or Endostar (8.9%)-treated group. The above-mentioned results indicate that the fusion protein Fv-LDP is effective against transplantable hepatoma in mice and human hepatoma xenografts in athymic mice. Moreover, Endostar can potentiate the inhibition effect of Fv-LDP on the growth of human hepatoma cells and xenografts. These data will provide a new combined strategy for improving the therapeutic efficacy of treatments for hepatoma or other gelatinase-overexpressing tumours.

[Inhibitory effect of antisense human telomerase reverse transcriptase(hTERT) on telomerase activity of human pulmonary giant cell carcinoma cell line(PLA-801D)].

BACKGROUND & OBJECTIVE: Telomerase has been thought to play an important role in the carcinogenesis in recent years. Human telomerase reverse transcriptase (hTERT) is a limiting component for telomerase activity. This study was designed to explore the effect of transfection of the full-length cDNA of antisense hTERT on the malignant phenotype of human pulmonary giant cell carcinoma cell line (PLA-801D) and its potential role in the gene therapy for cancers. METHODS: An antisense hTERT cDNA eukaryotic expression vector pcDNA3.1(-)-hTERT including the full length of hTERT cDNA sequence was constructed using recombinant DNA technique and transfected into human pulmonary giant cell carcinoma cells (PLA-801D) with liposome. The effect of antisense hTERT on the cellular proliferation capacity of PLA-801D cells was analyzed by the growth curve. The expression of hTERT mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). The telomerase activity was determined by telomeric-repeat amplification protocol enzyme-linked immunoassay (TRAP-ELISA). RESULTS: Antisense pcDNA3.1 (-)-hTERT eukaryotic expression have been constructed and was successfully transfected into the PLA-801D cells. The growth speed of PLA-801D transfected with antisense hTERT was significantly inhibited compared with the control cells, and the hTERT mRNA expression was inhibited, the relatively expression was only 15.7% of control cells, and telomerase activity was down-regulated about 82.4%. CONCLUSION: Full-length antisense hTERT cDNA can suppress hTERT mRNA expression and telomerase activity, and restrict the growth speed of tumor cells.

[Inhibition of hTR activity for suppression of lung cancer cell proliferation using antisense phosphorothioate oligonucleotides].

OBJECTIVE: Telomerase activity was measured in 8 lung tumor cell lines and transfected antisense phosphorothiate oligodeoxynucleotides (anti-PS-ODN) of hTR as therapeutic agents into target cell LTEP-a-2 to investigate the inhibitory effect on telomerase activity and tumor cell growth. METHODS: 1. Telomerase activity assay in 8 human lung tumor cell lines using telomerase PCR ELISA. 2. Synthesized anti-PS-ODNs of hTR and random PS-ODN transfected with Clonfectin into LTEP-a-2 cell lines for 72 hr. 3. Measure telomerase activity by telomerase PCR ELISA, SDH activity by MTT assay and cell growth. RESULTS: Eight cell lines showed positive expression of telomerase activity. Various anti-PS-ODNs could inhibit telomerase activity, SDH activity and cell growth. The inhibition became more marked with the increase of anti-PS-ODNs concentrations. Concentrations of 5 - 40 micromol/L anti-PS-ODNs of hTR specifically reduced the telomerase activity by 25.7% - 84.0%. PS-ODNs of anti-hTR were also able to reduce SDH activity by 19.4% - 74.7% at 5 - 40 micromol/L. The dose of 5 - 40 micromol/L PS-ODNs of anti-hTR had the ability to inhibit cell growth by 14.8% - 72.5%. The results indicate that random sequence of PS-ODN(9) failed to inhibit telomerase, SDH activity and cell growth. A statistically significant difference exists between random PS-ODN and three anti-PS-ODNs (P < 0.01). CONCLUSIONS: Human lung tumor cell lines express high telomerase activity. PS-ODNs of anti-hTR had the ability to inhibit telomerase and reduce LTEP-a-2 cell growth and metabolism.

Activation of MMP-2 by human GCT23 giant cell tumour cells induced by osteopontin, bone sialoprotein and GRGDSP peptides is RGD and cell shape change dependent.

We show that osteopontin (OPN), bone sialoprotein (BSP) and GRGDSP peptides, in solution, induce activation of metalloproteinase-2 (MMP-2) secreted by human GCT23 giant cell tumour cells. Activation of MMP-2 is RGD sequence dependent, possibly involves anti-alphaVbeta3 integrins, is preceded by a change from spread to rounded cell morphology and is mimicked by the actin depolymerising agent cytochalasin B. Cells that had spread on OPN, BSP and GRGDSP substrata failed to activate MMP-2, but subsequent addition of soluble GRGDSP induced rounding and MMP-2 activation. Activation induced by GRGDSP and cytochalasin B was cell mediated, inhibited by EDTA, tissue inhibitor of metalloproteinase-2 (TIMP-2) and carboxyl terminal MMP-2 consistent with a role for membrane type (MT)-MMP but did not involve urokinase, plasmin or thrombin activity. Activation induced by GRGDSP and cytochalasin B, but not cell rounding, was inhibited by herbimycin A, cycloheximide and actinomycin D, suggesting a role for tyrosine kinases, protein and RNA synthesis, but was not associated with changes in mRNA for MT-MMP-1, MMP-1, MMP-2, TIMP-1 or TIMP-2. GRGDSP and cytochalasin B enhanced levels of membrane-associated pro- and active form MMP-1 and MMP-2 but not MT-MMP-1, stimulated cell surface MMP-1 staining and induced that of MT-MMP-1, MMP-2 and TIMP-2. This was consistent with the possible relocation of constitutive MT-MMP-1 to the cell surface as a prerequisite for subsequent cell surface MMP-2/TIMP-2/MT-MMP-1 complex formation and to the potential induction of conditions favourable for reciprocal cell surface MMP-1/MMP-2 activation. Our data provide a novel insight into interactions between RGD containing bone matrices, GCT cells and MMPs of potential relevance to GCT pathology.

[Zymographic analysis of MMPs in human carcinoma cell lines with different metastasis potential].

OBJECTIVE: To investigate the correlation between matrix metalloproteinases (MMPs) activities and metastatic potential of several groups of human carcinoma cells with different metastatic potential. METHODS: Several groups of human carcinoma cell lines (human lung carcinoma, human prostatic carcinoma and melanoma) with different metastatic potential were selected. By using cell culture, collection and concentration of conditioned media and zymographic analysis method the difference of MMPs production and activity among those cell lines were detected. RESULTS: The MMPs production capabilities of carcinoma cells rose following the increase of their invasive and metastatic potentials: that of PG is much higher than PAa's, and BE1 is higher also than CL3 and LH7. Advanced stage melanoma cell WM983a and WM451 product MMP-9, but primary stage cells don't. There are MMP-9 in the conditioned medium of prostatic carcinoma cell PC-3M, the metastatic clone of PC-3 which don't express MMP-9. CONCLUSION: The expression of MMPs especially MMP-9 of carcinoma cells is closely correlated to the metastatic and invasive potential.

[A study on the correlation between the activity of 72,000 type IV collagenase and the metastatic potential of cancer cells].

OBJECTIVE: To evaluate the enzyme activity of 72,000 type IV collagenase and its relationship with the metastatic potential of cancer cells. METHODS: The levels of secreted 72,000 type IV collagenase in the conditioned media of five human cancer cell lines with different metastatic potential and a normal lung fibroblast strain treated with cancer cell culture media were examined by gelatin zymography and densitometric analysis. RESULTS: The levels of 72,000 type IV collagenase secreted by cancer cells with high metastatic potential (PG, WM451 and WM983a) were higher than those secreted by cancer cells with low metastatic potential (PAa and WM35). In the conditioned media of fibroblasts which were treated with the culture media of PG and WM451, enhanced levels of activation were observed. CONCLUSION: The secretion of 72,000 type IV collagenase is closely correlated to the metastatic potential of cancer cells. The cancer cells with high metastatic potential may possibly through certain soluble mediators stimulate normal fibroblasts to activate 72,000 type IV procollagenase.