Tachyplesin activates the classic complement pathway to kill tumor cells.

Tachyplesin is a small, cationic peptide that possesses antitumor properties. However, little is known about its action mechanism. We used phage display to identify a protein that interacted with tachyplesin and isolated a sequence corresponding to the collagen-like domain of C1q, a key component in the complement pathway. Their interaction was subsequently confirmed by both ELISA and affinity precipitation. Tachyplesin seemed to activate the classic complement cascade because it triggered several downstream events, including the cleavage and deposition of C4 and C3 and the formation of C5b-9. When TSU tumor cells were treated with tachyplesin in the presence of serum, activated C4b and C3b could be detected on tumor cells by flow cytometry, Western blotting, and confocal microscopy. However, this effect was blocked when the tumor cells were treated with hyaluronidase or a large excess of hyaluronan, indicating that hyaluronan or related glycosaminoglycans were involved in this process. Treatment of cells with tachyplesin and serum increased in membrane permeability as indicated by the ability of FITC-dextran to enter the cytoplasm. Finally, the combination of tachyplesin and human serum markedly inhibited the proliferation and caused death of TSU cells, and these effects were attenuated if the serum was heat-inactivated or if hyaluronidase was added. Taken together, these observations suggest that tachyplesin binds to both hyaluronan on the cell surface and C1q in the serum and activates the classic complement cascade, which damages the integrity of the membranes of the tumor cells resulting in their death.

Hypoxia-induced alterations in hyaluronan and hyaluronidase.

Hyaluronan (HA), a large negatively-charged polysaccharide, is a major component of vessel basal membrane. HA is expressed by a variety of cells, including tumor and endothelial cells. We hypothesized that HA could be up-regulated by hypoxia to enhance vessel formation. To determine the effect of hypoxia on the production of HA, tumor cells were treated with either media alone (control) or a hypoxia inducer (CoCl or NaN3) for 24 h. The level of HA in the media was then measured by ELISA. The results showed that both CoCl and NaN3 induced the production of HA. Since the low molecular weight form of HA (SMW) possesses pro-angiogenic properties, we investigated whether hypoxia-induced HA can be processed into SMW. Under hypoxic conditions, the activity of hyaluronidase, the enzyme responsible for degrading HA, was measured by an ELISA-like assay. The activity of hyaluronidase was shown to be up-regulated by hypoxia and, further, could carry out the function of processing HA into SMW. In addition, the hypoxic areas of tumor tissues were stained strongly with biotinylated HA-binding proteins, indicating that the level of HA was high compared to the oxic areas. This study demonstrates that hypoxia can stimulate the production of HA and the activity of hyaluronidase, which may promote angiogenesis as a compensation mechanism for hypoxia.

[Expression of PH20 in primary and metastatic breast cancer and its pathological significance].

OBJECTIVE: To evaluate the expression pattern of PH20 in primary and metastatic breast cancer and its relationship to tumor metastatic potential. METHODS: Anti-PH20 antibody was synthesized by injection of conjugated human PH20 peptides into rabbits. Immunohistochemical study was performed on 53 cases of human breast cancer. Western blot was used to detect PH20 expression in 5 cases of breast cancer with available fresh tissue. Two oligonucleotide probes were prepared for in-situ hybridization using breast tissue microarray. RESULTS: Normal breast tissue did not express PH20 (0/3), while 58.4% (31/53) of breast cancer cases did. The highest expression rate was found in metastatic foci in regional lymph nodes (83.3%), followed by primary breast cancer tissue in cases with lymph node secondaries (70.8%). The breast cancer cases with no any metastasis had an expression rate of 48.2%. The immunohistochemical staining results were further confirmed by Western blotting. In-situ hybridization showed PH20 RNA in 75% of the breast cancer tissue (21/28). Two of the 17 cases of normal breast tissue showed weak expression in some ductolobular units. CONCLUSIONS: The expression of PH20 has a positive correlation with metastatic potential in breast cancer. It is possible that PH20 may play an important role in the invasive growth and metastasis of breast cancer cells, via mechanisms such as digestion of surrounding stromal tissue and release of FGF-2.

Hyaluronan-binding peptide can inhibit tumor growth by interacting with Bcl-2.

Previous studies have indicated that proteins that bind hyaluronan can also inhibit the growth of tumor cells. To determine if synthetic peptides also possessed these properties, we tested a series of polypeptides containing structural motifs from different proteins for their ability to bind [(3)H]hyaluronan, and identified one compound termed P4 that had a particularly strong interaction. Further studies revealed that P4 also inhibited the growth of tumor cells in tissue culture as well as on the chorioallantoic membranes of chicken embryos. In addition, expression vectors for P4 caused tumor cells to grow slower in nude mice and reduced their vascularization. The P4 peptide also inhibited VEGF-induced angiogenesis in the chorioallantoic membranes of chicken embryos. Studies on cultured cells indicated that P4 induced apoptosis, which was blocked by a pan-caspase inhibitor. Confocal microscopy revealed that shortly after its uptake, P4 became associated with mitochondria. Immunoprecipitation indicated that P4 could bind to Bcl-2 and Bcl-x(L), which are associated with mitochondria and regulate apoptosis. This was also supported by the fact that P4 induced the release of cytochrome c from preparations of mitochondria. Taken together, these results suggest that P4 binds to Bcl-2 and related proteins and this activates the apoptotic cascade.

CD44-hyaluronic acid interactions mediate shear-resistant binding of lymphocytes to dermal endothelium in acute cutaneous GVHD.

For circulating lymphocytes to migrate to inflammatory sites, they must first adhere to the target tissue endothelium with sufficient strength to overcome the shear forces of blood flow. We previously reported that dermal papillary vessels in acute graft-versus-host disease (aGVHD) support shear-resistant lymphocyte adherence. We now identify the relevant adhesion molecule(s) directing this binding, showing that interactions between lymphocyte CD44 and hyaluronic acid (HA) expressed on dermal vessels in aGVHD alone confer this shear-resistant attachment. Native HA deposits on vascular endothelium support lymphocyte adherence, whereas HA immobilized on plastic does not. HA expressed at dermal endothelium in aGVHD is thus specialized to support lymphocyte adherence under flow conditions, and CD44-HA interactions may contribute to lymphocytotropism to skin in aGVHD.

Extracellular matrix protein 1 (ECM1) is over-expressed in malignant epithelial tumors.

The extracellular matrix protein 1 (ECM1) is a secreted protein that has been implicated with cell proliferation, angiogenesis and differentiation. In the present study, we used immunohistochemical staining to examine the expression of ECM1 in a panel of human tumors and found that it was closely correlated with some types of tumors including: invasive breast ductal carcinoma (83%), esophageal squamous carcinoma (73%), gastric cancer (88%) and colorectal cancer (78%). Significantly, ECM1expression was correlated with the metastatic properties of the tumors. Primary breast cancers that had formed metastases were 76% positive while those that had not metastasized were only 33% positive. ECM1 expression was also correlated with PCNA a marker for proliferation, but not with CD34, a marker for endothelial cells. These results indicate that ECM1 tends to be preferentially expressed by metastatic epithelial tumors.

A peptide with three hyaluronan binding motifs inhibits tumor growth and induces apoptosis.

A number of hyaluronan (HA) binding proteins such as soluble CD44, receptor for hyaluronan-mediated motility (RHAMM), and metastatin inhibit tumor growth and metastasis. To determine whether the HA binding motif is the element responsible for the antitumor effect of this family of proteins, we examined the biological activity of a 42-amino acid peptide (designated as BH-P) that contains three HA binding motifs [B(X(7))B] from human brain HA binding protein. In initial experiments with cultured cells, we found that synthetic BH-P inhibited the proliferation and colony formation of tumor cells. It also blocked the growth of tumors on the chorioallantoic membranes of 10-day chicken embryos. In addition, MDA-435 melanoma cells that had been transfected with an expression vector for BH-P grew at a slower rate in nude mice than the vector-alone transfectants. Final studies revealed that the BH-P could activate caspase-8, caspase-3, and poly(ADP-ribose) polymerase and trigger the apoptosis of tumor cells. Taken together, these results suggest that the HA binding motif that is present in HA binding proteins may be responsible for the antitumor effect exerted by the members of this family.

Triptolide inhibits the growth and metastasis of solid tumors.

Triptolide (TPL), a diterpenoid triepoxide purified from the Chinese herb Tripterygium wilfordii Hook F, was tested for its antitumor properties in several model systems. In vitro, TPL inhibited the proliferation and colony formation of tumor cells at extremely low concentrations (2-10 ng/ml) and was more potent than Taxol. Likewise, in vivo, treatment of mice with TPL for 2-3 weeks inhibited the growth of xenografts formed by four different tumor cell lines (B16 melanoma, MDA-435 breast cancer, TSU bladder cancer, and MGC80-3 gastric carcinoma), indicating that TPL has a broad spectrum of activity against tumors that contain both wild-type and mutant forms of p53. In addition, TPL inhibited experimental metastasis of B16F10 cells to the lungs and spleens of mice. The antitumor effect of TPL was comparable or superior with that of conventional antitumor drugs, such as Adriamycin, mitomycin, and cisplatin. Importantly, tumor cells that were resistant to Taxol attributable to the overexpression of the multidrug resistant gene 1 were still sensitive to the effects of TPL. Studies on cultured tumor cells revealed that TPL induced apoptosis and reduced the expression of several molecules that regulate the cell cycle. Taken together, these results suggest that TPL has several attractive features as a new antitumor agent.

Induction of hyaluronan synthase 2 by human chorionic gonadotropin in mural granulosa cells of equine preovulatory follicles.

In contrast to other species, the preovulatory rise in gonadotropins in mares causes a remarkable expansion of the entire granulosa cell layer in vivo, suggesting that hyaluronan (HA) synthesis may be regulated in mural granulosa cells in this species. The objectives of this study were to clone and characterize equine hyaluronan synthase 2 (HAS2) and investigate the regulation of its transcript and of HA synthesis in equine follicles during human chorionic gonadotropin (hCG)- induced ovulation. Results showed that the equine HAS2 cDNA contains a 5'-untranslated region of 436 bp, an open reading frame of 1659 bp, and a 3'-untranslated region of 707 bp. The open reading frame encodes a 552-amino acid protein that is highly conserved (98-99% identity), compared with other known mammalian homologs. The regulation of HAS2 mRNA was studied in equine follicles isolated during estrus between 0 and 39 h after an ovulatory dose of hCG and in corpora lutea obtained on d 8 of the estrous cycle. Results from semiquantitative RT-PCR/Southern blotting analyses revealed a transient induction of HAS2 during the ovulatory process. Levels of HAS2 transcripts were undetectable in follicles before hCG treatment (0 h), increased markedly after gonadotropin treatment (P < 0.05), but returned to undetectable levels in corpora lutea. Analyses performed on isolated preparations of theca interna and granulosa cells showed that the granulosa cell layer was the predominant site of HAS2 expression. An immunohistochemical approach showed that this induction of HAS2 transcript was accompanied by a dramatic increase in HA production after hCG treatment. The isolation and characterization of a 1.8-kb fragment of genomic sequence located immediately upstream of equine HAS2, and comparison with corresponding human and mouse genomic regions identified several conserved putative cis-acting elements. Thus, this study describes the primary structure of equine HAS2, demonstrates for the first time the regulation of HAS2 in mural granulosa cells during the ovulatory process in vivo and identifies a valuable model in which to study the molecular control of HAS2 gene expression.

[Promotion of growth of human breast cancer cells MDA231 by human sperm membrane-bound hyaluronidase: an experimental study].

OBJECTIVE: To study the mechanism how human sperm membrane-bound hyaluronidase (PH20) promotes the gowth of human breast cancer. METHODS: Full-length cDNA of human PH20 was transfected into human breast cancer cell line MDA231. The transfectant MDA231-PH20 was then implanted into the chorio-allantoic membrane (CAM) of chicken embryo to form a tumor. Four days after implantation, the tumors were resected to be weighed. The angiogenesis in tumor tissue was examined by immunohistochemistry. Trans-well cell culture was used to study the effect of MDA231-PH20 on the growth of adult bovine aortic endothelial cells (ABAE). The expression of fibroblast growth factor-2 (FGF-2) in the tumor cells was investigated by Western blotting. ELISA was used to examine the secretion of FGF-2 and hyaluronic acid. The same amount of empty vector pcDNA3, instead of PH20, was transfected into human breast cancer cell line MDA231 as control group. RESULTS: The average weight of tumor four days after implantation was 44.7 mg +/- 10.2 mg in the MDA231-PH20 group, and was 21.3 mg +/- 2.8 mg in the control group (t = 2.418, P = 0.038). Neogenetic vessels increased remarkably in MDA231-PH20 tumor tissues. The expression of FGF-2 protein was much higher in MDA231-PH20 cells. The FGF content and HA secretion were higher in the MDA231-PH20 group than in the control group (8.10 pg/ml +/- 1.56 pg/ml vs. 3.94 pg/ml +/- 0.82 pg/ml, and 1 220 ng/ml +/- 254 ng/ml vs. 462 ng/ml +/- 96 ng/ml, all P < 0.01). The growth of ABAE cells was significantly accelerated after co-culture with MDA231-PH20 transfectant. CONCLUSION: PH20 may promote the growth of human breast cancer by accelerating the release of FGF-2 from tumor cells, decomposing HA into small fragments, and promoting angiogenesis.