Enforced expression of METCAM/MUC18 increases tumorigenesis of human prostate cancer LNCaP cells in nude mice.

PURPOSE: Metastasis cell adhesion molecule/MUC18, a cell adhesion molecule in the Ig-like gene super family, is a key determinant in prostate cancer cell progression. However, the mechanisms by which human metastasis cell adhesion molecule/MUC18 stimulates progression are poorly understood. To investigate this and determine whether human metastasis cell adhesion molecule/MUC18 may act as a possible tumor progression gene, we studied the effect of its enforced expression on LNCaP cell tumorigenesis. MATERIALS AND METHODS: We subcutaneously co-injected a metastasis cell adhesion molecule/MUC18 expressing LNCaP clone and control clones/cells with Matrigel™ into nude mice, observed tumor formation of these cells and measured tumors at different times. To understand the mechanisms we also determined the expression of several downstream key effectors of metastasis cell adhesion molecule/MUC18 in subcutaneous tumors and compared them to those in previously obtained orthotopic (prostatic) tumors. RESULTS: Tumors derived from human metastasis cell adhesion molecule/MUC18 expressing LNCaP clones/cells appeared about 18 days earlier than the empty vector transfected clone/cells. Enforced expression of human metastasis cell adhesion molecule/MUC18 also increased tumor take 2-fold, tumorigenicity 10 to 12-fold and final tumor weight 5-fold. Enforced expression appeared to render the cells with increased levels of the proliferation indexes Ki67 and proliferating cell nuclear antigen, the survival index phospho-AKT, and the angiogenesis indexes vascular endothelial growth factor, vascular endothelial growth factor receptor 2 and CD31. However, it did not significantly render the cells with altered levels of various apoptosis indexes. CONCLUSIONS: Enforced expression of human metastasis cell adhesion molecule/MUC18 increases prostate tumorigenesis in vivo and may affect the process by increasing proliferation, up-regulating the AKT survival pathway, and augmenting the angiogenic ability of prostate cancer cells.

Enforced expression of MCAM/MUC18 increases in vitro motility and invasiveness and in vivo metastasis of two mouse melanoma K1735 sublines in a syngeneic mouse model.

Human MCAM/MUC18 has been shown to increase metastasis of human melanoma cells in xenograft mouse systems. To be more relevant to understanding the progression of clinical melanoma and for designing better preclinical therapeutic trials, it is highly desirable to establish a syngeneic mouse model for studying the mechanisms of MCAM/MUC18-mediated tumorigenesis and metastasis of melanoma cells. To reach this goal, we transfected the mouse MCAM/MUC18 (moMCAM/MUC18) cDNA into two MCAM/MUC18-minus, low-metastatic mouse melanoma K1735 sublines, K1735-10 (tumor(-)/met(low)) and K1735-3 (tumor(+)/met(low)), and selected for G418-resistant clones, which expressed different levels of moMCAM/MUC18, and used for testing the effect of MCAM/MUC18 overexpression on their in vitro growth rate, motility, and invasiveness and in vivo subcutaneous tumor growth and pulmonary metastasis in syngeneic mice. Enforced expression of moMCAM/MUC18 did not significantly affect in vitro growth rate, but it increased the in vitro motility and invasiveness of clones derived from both sublines. Ectopic expression of moMCAM/MUC18 did not alter the nontumorigenicity of the K1735-10 clones per cells nor significantly affect the subcutaneous tumor growth of the K1735-3 clones per cells. The moMCAM/MUC18-expressing K1735-10 clones were able to establish only microscopic lung modules in 86% of the mice. In contrast, the moMCAM/MUC18-expressing K1735-3 clones could induce numerous large lung nodules (3-4 mm in diameter) in all the mice. We concluded that increased moMCAM/MUC18 expression in the two K1735 sublines minimally affected their tumorigenicity, but it augmented their in vitro motility and invasiveness and increased their pulmonary metastasis in the syngeneic C3H mice.

Increased expression of MUC18 correlates with the metastatic progression of mouse prostate adenocarcinoma in the TRAMP model.

PURPOSE: The transgenic adenocarcinoma mouse prostate (TRAMP) model is a paradigm that closely mimics the progression of clinical prostate cancer. We have previously reported that MUC18, a cell adhesion molecule in the Ig gene superfamily, is a marker as well as an important mediator for the metastatic potential of human prostate cancer cells. In this study we investigated the possible correlation of increased MUC18 expression with the malignant progression of prostate cancer in the TRAMP model. MATERIALS AND METHODS: We used immunohistochemistry, Western blot and reverse transcriptase-polymerase chain reaction analyses to determine MUC18 expression in the prostate gland of 178 to 282-day-old TRAMP positive males with a prostate tumor size of 0.4 to 12.7 gm. Eight normal prostates, 10 prostates with high grade prostatic intraepithelial neoplasia (PIN), 24 prostates with primary prostate cancer, 10 metastatic lesions from 50 pure C57BL/6 TRAMP mice (Wu colony) and 2 normal prostates, 2 prostates with high grade PIN, 6 prostates with primary prostate cancer and 4 metastatic lesions from 10 [C57BL/6 TRAMP x FVB] F1 mice (NMG colony) were used. RESULTS: We found that mouse MUC18 was expressed in all (100%) high grade PIN, adenocarcinomas and metastatic lesions. All mice bearing primary prostate tumors had prostate cancer metastatic to the peri-aortic lymph nodes and some had it to other organs (liver, lung, kidney, testes, seminal vesicles and abdominal cavity). In contrast, prostates from 10 nontransgenic littermates did not have detectable MUC18 expression. CONCLUSIONS: MUC18 expression is up-regulated in the TRAMP model and it correlates with the malignant progression of mouse prostate adenocarcinoma in this transgenic model. This further strengthens the hypothesis that MUC18 has an important role in increasing the metastatic potential of prostate cancer cells.

Ectopical expression of human MUC18 increases metastasis of human prostate cancer cells.

MUC18, a cell adhesion molecule (CAM), has been reported to be a diagnostic marker for the early detection of the metastatic potential of prostate cancers as well as implicated to be an important determinant for mediating the tumorigenesis and metastasis of prostate cancer. To test the hypothesis, we further investigated the possible role of MUC18 in the malignant progression of human prostate cancer. The human MUC18-minus, non-metastatic human prostate cancer LNCaP cells were transfected with the human cytomegalovirus immediate-early gene (HCMV-IE) promoter-driven human MUC18 (huMUC18) cDNA. The G418-resistant (G418R)-LNCaP clones that expressed a high level of huMUC18 were selected and used for testing the effect of huMUC18 expression on the in vitro growth, motility, and invasiveness as well as on the in vivo metastasis (via orthotopical injection) in a xenograft nude mouse model. HuMUC18 expression increased by four- to fivefold of in vitro motility and invasiveness of LNCaP cells. Anti-huMUC18 antibody significantly inhibited the in vitro motility and invasiveness of huMUC18-expressing LNCaP clones, but not the control clones. We suggest that huMUC18 expression is responsible for increasing these behaviors of LNCaP cells. HuMUC18 expression also directly increased the in vivo metastatic abilities of the LNCaP cells from the prostate gland to multiple distant organs. Western blot and immunohistochemistry analyses showed that the prostatic tumors as well as metastatic lesions expressed high levels of MUC18, indicating that they originated from the injected huMUC18-expressing LNCaP cells. We therefore conclude that HuMUC18 is an important determinant in increasing metastasis of human prostate cancer LNCaP cells to distant organs in a nude mouse model.

Protection of retinal pigment epithelial cells from oxidative damage by oltipraz, a cancer chemopreventive agent.

OBJECTIVE: To determine whether oltipraz (4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione) protects against oxidative injury in cultured human retinal pigment epithelial (hRPE) cells. METHODS: Primary cultured hRPE cells were incubated with various concentrations of oltipraz followed by treatment with the chemical oxidant tert-butylhydroperoxide (tBH). Cell viability was assessed by release of lactate dehydrogenase (LDH) and cleavage of WST-1. Intracellular and mitochondrial levels of glutathione (GSH) were measured by HPLC. Glutathione S-transferase (GST), NADPH-quinone reductase (NQR), and glutathione peroxidase (GPx) were measured by specific enzyme activity assays. RESULTS: Treatment of hRPE cells with oltipraz inhibited tBH-induced cell death in a concentration-dependent manner with significant inhibition at 50 micro M. Olitpraz (50 micro M) increased GSH levels in hRPE cells by approximately 18% and in hRPE mitochondrial fractions by approximately 50% after 24 hours of exposure. Treatment with oltipraz increased GST and NQR activities by approximately 21% and 11%, respectively. CONCLUSIONS: Oltipraz protects hRPE cells against tBH induced injury. The mechanism of protection is likely to include increased cellular and mitochondrial GSH levels and induction of detoxification enzymes, including GST and NQR. Dietary supplementation with oltipraz or other dithiolethiones may help protect the hRPE against oxidant induced injury.