The antitumor potential of cartilage has been investigated extensively in laboratory and animal studies. Some of these studies have assessed the toxicity of cartilage products toward cancer cells in vitro .[1-5]Powdered Cartilage Products
In one study, cells from 22 freshly isolated human tumors (nine ovary, three lung, two brain, two breast, and one each of sarcoma, melanoma, colon, pancreas, cervix, and testis) and three human cultured cell lines (breast cancer, colon cancer, and myeloma) were treated with Catrix, which is a commercially available powdered preparation of bovine (cow) cartilage.[1,3,4] In the study, the growth of all three cultured cell lines and cells from approximately 70% of the tumor specimens were inhibited by 50% or more when Catrix was used at high concentrations (1–5 mg /mL of culture fluid). However, it is unclear whether the inhibitory effect of Catrix in this study was specific to the growth of cancer cells because the preparation’s effect on the growth of normal cells was not tested. In addition, the cytotoxic component of Catrix has not been identified, and it has not been shown that equivalent inhibitory concentrations of this component can be achieved in the bloodstreams of patients who may be treated with either injected or oral formulations of this product. (Refer to the Human/Clinical Studies section of this summary for more information.)
A commercially available preparation of powdered shark cartilage (no brand name given) was reported to have no effect on the growth of human astrocytoma cells in vitro. The shark cartilage product tested in this study, however, was examined at only one concentration (0.75 mg/mL).
The immune system –stimulating potential of cartilage has also been investigated in laboratory and animal studies. In one study, Catrix was shown to stimulate the production of antibodies by mouse B cells (B lymphocytes) both in vitro and in vivo . However, increased antibody production in vivo was observed only when Catrix was administered by intraperitoneal or intravenous injection. It was not observed when oral formulations of Catrix were used. In most experiments, the proliferation of mouse B cells (i.e., normal, nonmalignant cells) in vitro was increasingly inhibited as the concentration of Catrix was increased (tested concentration range, 1–20 mg/mL). Catrix has also been reported to stimulate the activity of mouse macrophages in vivo, but results demonstrating this effect have not been published.
The effects of shark cartilage on the immune system were also reported in two studies that used the same purified protein fraction that had exhibited the most immunostimulatory effects when tested.[7,8] One study explored the effects of this fraction on tumor immune response by observing the infiltration of this fraction on CD4 and CD8 lymphocytes in a murine tumor model. An increase in the ratio of CD4/CD8 lymphocytes was seen in tumor-infiltrating lymphocytes but not in peripheral blood lymphocytes. The second study exploring immune system response measured antibody response, cytotoxic assay, lymphocyte transformation, and intratumor T-cell ratio in mice. The fraction exhibited the ability to augment delayed-type hypersensitivity response against sheep red blood cells in mice and to decrease the cytotoxic activity of natural killer cells. In addition, this fraction showed a strong inhibitory effect on human brain microvascular endothelial cell proliferation and migration in the fibrin matrix.
Additional in vivo studies of the antitumor potential of shark cartilage have been published in the peer-reviewed scientific literature.[9-11] In one study, oral administration of powdered shark cartilage (no brand name given) was shown to inhibit chemically induced angiogenesis in the mesenteric membrane of rats. In another study, oral administration of powdered shark cartilage (no brand name given) was shown to reduce the growth of GS-9L gliosarcomas in rats. It was reported in a third study that oral administration of two powdered shark cartilage products, Sharkilage and MIA Shark Powder, did not inhibit the growth or the metastasis of SCCVII squamous cell carcinomas in mice.
A large number of laboratory and animal studies concerning the antiangiogenic potential of cartilage have been published.[2,9,12-32] Overall, these studies have revealed the presence of at least three angiogenesis inhibitors in bovine cartilage [13,14,16-18,21,23,33] and at least two in shark cartilage.[2,9,25,26]Aqueous Extracts of Cartilage
A liquid (i.e., aqueous) extract of shark cartilage called AE-941/Neovastat has also been reported to inhibit the growth of a variety of cancer cell types in vitro. These results have not been published in a peer-reviewed scientific journal and are not consistent with other results obtained by the same group of investigators.[27,34]
Three angiogenesis inhibitors in bovine cartilage have been very well characterized.[13,14,16-18,21,23,33] They are relatively small proteins with molecular masses that range from 23,000 to 28,000.[13,14,16,23] These proteins, called cartilage-derived inhibitor (CDI), cartilage-derived antitumor factor (CATF), and cartilage-derived collagenase inhibitor (CDCI) by the researchers who purified them,[13,14,21] have been shown to block endothelial cell proliferation in vitro and new blood vessel formation in the chorioallantoic membrane of chicken embryos.[14,16-18,21,23,33] Two of the proteins (CDI and CDCI) have been shown to inhibit matrix metalloproteinase activity in vitro,[13,14,16,18] and one (CDI) has been shown to inhibit endothelial cell migration in vitro.[14,16] These proteins do not block the proliferation of normal cells or of tumor cells in vitro.[14,16,17,21,33] When the amino acid sequences of CDI, CATF, and CDCI were determined, it was discovered that they were the same as those of proteins known otherwise as tissue inhibitor of matrix metalloproteinases 1 (TIMP-1), chondromodulin I, and TIMP-2, respectively.[13,14,18,23,33]
A possible fourth angiogenesis inhibitor in bovine cartilage has been purified not from cartilage but from the culture fluid of bovine chondrocytes grown in the laboratory. This inhibitor, which has been named chondrocyte-derived inhibitor (ChDI), is a protein that has a molecular mass of approximately 36,000. It has been reported that ChDI and CDI/TIMP-1 have similar antiangiogenic activities,[15,16,33] but the relationship between these proteins is unclear because amino acid sequence information for ChDI is not available. Thus, whether CDI/TIMP-1 is a breakdown product of ChDI or whether ChDI is truly the fourth angiogenesis inhibitor identified in bovine cartilage is unknown.
As indicated previously, shark cartilage, like bovine cartilage, contains more than one type of angiogenesis inhibitor. One shark cartilage inhibitor, named U-995, reportedly contains two small proteins, one with a molecular mass of approximately 14,000 and the other with a molecular mass of approximately 10,000. Both proteins have shown antiangiogenic activity when tested individually. The exact relationship between these two proteins and their relationship to the larger bovine angiogenesis inhibitors are not known because amino acid sequence information for U-995 is not available. U-995 has been reported to inhibit endothelial cell proliferation, endothelial cell migration, matrix metalloproteinase activity in vitro, and the formation of new blood vessels in the chorioallantoic membrane of chicken embryos. It does not appear to inhibit the proliferation of other types of normal cells or of cancer cells in vitro. Intraperitoneal but not oral administration of U-995 has been shown to inhibit the growth of mouse sarcoma-180 tumors implanted subcutaneously on the backs of mice and the formation of lung metastases of mouse B16-F10 melanoma cells injected into the tail veins of mice.
The second angiogenesis inhibitor identified in shark cartilage appears to have been studied independently by three groups of investigators.[2,26,35] This inhibitor, which was named SCF2 by one of the groups, is a proteoglycan that has a molecular mass of about 10,000. Proteoglycans are combinations of glycosaminoglycans and protein. The principal glycosaminoglycan in SCF2 is keratan sulfate. SCF2 has been shown to block endothelial cell proliferation in vitro,[2,26,35] the formation of new blood vessels in the chorioallantoic membrane of chicken embryos,[2,26] and tumor-induced angiogenesis in the corneas of rabbits.[2,26]
Other studies have demonstrated that AE-941/Neovastat, the previously mentioned aqueous extract of shark cartilage, has antiangiogenic activity,[12,27,28,34,36-39] but the molecular basis for this activity has not been defined. Therefore, whether AE-941/Neovastat contains U-995 and/or SCF2 or some other angiogenesis inhibitor is not known. It has been reported that AE-941/Neovastat inhibits endothelial cell proliferation and matrix metalloproteinase activity in vitro and the formation of new blood vessels in the chorioallantoic membrane of chicken embryos.[12,27,31] In addition, AE-941/Neovastat has been shown to induce endothelial cell apoptosis by activating caspases, enzymes important in the promotion and regulation of apoptosis.[32,34,38] It also appears to inhibit the action of vascular endothelial growth factor, thus interfering with the communication between tumor cells and nearby blood vessels.[28,34,37,38] AE-941/Neovastat may also inhibit angiogenesis through promotion of tissue plasminogen activator (tPA) activity. Neovastat stimulates tPA expression in endothelial cells through an increase in the transcription of the tPA gene. This transcriptional activation is associated with activation of c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-kappa B) signaling pathways to an extent similar to tumor necrosis factor-alpha (TNF-alpha). Furthermore, AE-941/Neovastat has been reported to inhibit the growth of DA3 mammary adenocarcinoma cells and the metastasis of Lewis lung carcinoma cells in vivo in mice.[5,27,34,41] In the Lewis lung carcinoma experiments, AE-941/Neovastat enhanced the antimetastatic effect of the chemotherapy drug cisplatin.[5,27,34,41] All the aspects of preclinical development have been reviewed.
The cartilage-derived antiangiogenic substance troponin I (TnI) has been isolated from human cartilage and has been produced by the cloning and expression of cDNA of human cartilage. It has been shown to specifically inhibit angiogenesis in vivo and in vitro and tumor metastasis in vivo. The active site of Tnl has been located in the amino acid residues of 96 to 116. The synthetic peptide Glu94-Leu123 (pTnl) has been shown to be a potent inhibitor of endothelial cell tube formation and endothelial cell division and to inhibit pancreatic cancer metastases in an in vivo liver metastases model.References
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