Abstract. Background: LY52 is a caffeoyl pyrrolidine derivative designed to fit and extend into the active pocket of matrix metalloproteinase (MMP). In this study, the effects of LY52 on MMP-2 and MMP-9 activities and tumor invasion and metastasis were examined. Materials and Methods: MMP expression in SKOV3 cells was analyzed by gelatin zymography. The anti-invasion and anti-metastasis abilities of LY52 were evaluated with penetration of SKOV3 cells through Matrigelcoated membrane in vitro and pulmonary metastasis of Lewis lung carcinoma in mice, respectively. Results: LY52 significantly blocked the proteolytic activity of gelatinase. Gelatin zymography revealed that MMP-2 and MMP-9 expressions in SKOV3 cells were reduced in the presence of LY52. LY52 also suppressed SKOV3 cell invasion in vitro. Furthermore, a significant inhibition of pulmonary metastasis of Lewis lung carcinoma cells was observed in LY52-administrated mice. Conclusion: LY52 might suppress invasion and metastasis of carcinoma cells via inhibition of MMP-2 and MMP-9 proteolytic activities.

Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedion) is a naturally occurring substance and was the first antineoplaston in the human body to be chemically identified. The effect of antineoplaston A10 on human hepatocellular carcinoma cell lines HepG2 and HLE has been examined. Antineoplaston A10 displayed anti-proliferative action inhibiting cell growth in a dose-and time-dependent manner in vitro as measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Incubation with antineoplaston A10 for 48 h induced apoptotic events such as a typical apoptotic morphology, formation of a characteristic ladder pattern of DNA migration and accumulation of sub-G1 phase cells. Next, hepatoma xenografts in nude mice were employed to study the antitumor effects of antineoplaston A10 in vivo. Oral administration of antineoplaston A10 delayed the growth of HepG2 and HLE cells in the mice without a reduction in body weight. A higher proportion of apoptotic cells in xenografts was observed by means of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. In addition, the level of expression of apoptotic marker p53 increased while that of anti-apoptotic protein bcl-2 decreased, as evaluated with immunohistochemical staining in the xenografts. These results suggested that antineoplaston A10 may inhibit the growth of human hepatoma cells through the induction of apoptosis.

N3-o-toluyl-fluorouracil (TFU), the pro-drug of 5-fluorouracil (5-FU), is the metabolite of N1-acetyl-N3-o-toluyl-fluorouracil (atofluding). We aimed to evaluate the efficacy of TFU as a precursor of 5-FU on the growth inhibition of human gastric carcinoma cell lines SGC-7901 and MKN-45. Growth of SGC-7901 and MKN-45 cells was remarkably suppressed by treatment with TFU in the presence of liver microsomal enzymes in vitro, suggesting that TFU may be converted to 5-FU by the enzymes. Similar treatment of TFU induced apoptosis of the cells, which was deduced from typical apoptotic features such as morphology, the formation of characteristic ladder pattern of DNAmigration and the accumulation of sub-G1 phase. Cancer cells xenografts in nude mice were employed to evaluate the efficacy of TFU in vivo. Growth of human gastric carcinoma cells was significantly delayed by oral administration of TFU with low side effects. Apoptosis in xenografts was also observed by means of TUNEL staining method. These results suggest that the treatment of TFU in the presence of liver microsomal enzymes and the oral administration of TFU in mice induced anti-proliferation and apoptosis in gastric carcinoma cells. This suggests that TFU may be a promising pro-drug of 5-FU for cancer treatments.

SIP-SII is the sulfated S. maindroni ink polysaccharide (SIP) isolated from cuttlefish Sepiella maindroni. SIP-SII weakly inhibited tumor cell growth without cytotoxicity in vitro assay. Herein, we examined the effects of SIP-SII on the expression of matrix metalloproteinase MMP-2 and MMP-9 as well as tumor cell invasion and migration. SIP-SII (0.8e500 mg/ml) significantly decreased the expression of MMP-2 activity in human ovarian carcinoma cells SKOV3 as evidenced by the gelatin zymography analysis. No significant decrease of MMP-9 was detected in the cell line after SIP-SII treatment. The expression of MMP-2 was also evaluated using Western blot analysis. The results showed that SIP-SII inhibited the expression of MMP-2 in SKOV3 and human umbilical vein vascular endothelial cells ECV304 after 24 h incubation. Furthermore, the activity of invasion and migration of SKOV3 and ECV304 cells were measured. SIP-SII displayed an inhibitory effect on the penetration of SKOV3 cells through Matrigel-coated membrane in transwell chamber. A significant inhibition of ECV304 cell migration was observed in the presence of SIP-SII. These results suggest that SIP-SII might suppress invasion and migration of carcinoma cells via inhibition of MMP-2 proteolytic activity.

Matrix metalloproteinase-2 (MMP-2) and MMP-9 have been associated with the ability of tumor cells to metastasize due to their capacity to degrade type IV collagen, the main component of basement membrane, and to their elevated expression in malignant tumors. (S)-methyl 6-(benzyloxycarbonylamino)-2-(2-(S)-2,6-dioxo-3-(3,4,5-trimethoxybenzamido) piperidin-1-yl)acetamido)hexanoate (CH1104I) is a galloyl cyclic-imide derivative designed to fit and extend into the S1' active pocket of MMP-2 and MMP-9. We aimed to evaluate the efficacy of CH1104I as a candidate compound for anti-invasion and anti-metastasis of tumor cells. CH1104I significantly blocked gelatinase activity as evidenced by a decrease in the degradation of succinylated gelatin. Gelatin zymography analysis showed that the compound (7-210 μM) inhibited the activity of MMP-2 and MMP-9 produced by human ovarian carcinoma SKOV3 cells. Inhibition of MMP-2 and MMP-9 expression was also observed using the assays of immunocytochemical staining and Western blot analysis. The results showed that CH1104I suppressed the expression of zymogens and active-MMP-2 and MMP-9. The effects of CH1104I on invasion and migration of SKOV3 cells were then measured. CH1104I displayed an inhibitory effect on the penetration of SKOV3 cells through Matrigel-coated membrane in transwell chamber. Furthermore, Lewis lung carcinoma (LLC) model was employed to evaluate the efficacy of CH1104I in vivo. A significant inhibition of pulmonary metastasis of carcinoma cells was observed in CH1104I-administrated mice (25-100 mg/kg). These results suggest that CH1104I is a potential MMP-2 and MMP-9 inhibitor that may effectively suppress tumor invasion and metastasis.