Biomedical subjects

Thomas C Chen

Publications and source records attributed to Thomas C Chen.

23 records · Page 2Linked to original sources

PubMed2003

Combination therapy with irinotecan and protein kinase C inhibitors in malignant glioma.

The topoisomerase-I inhibitor irinotecan (CPT-11) is currently used in Phase I/II trials for the treatment of patients with recurrent malignant gliomas. Protein kinase C (PKC) inhibitors such as high-dose tamoxifen and hypericin also have been used in the treatment of malignant gliomas. The current study examined the role of PKC inhibitors as chemosensitizers for CPT-11 and their proposed mechanism of action. Two glioma cell lines (A-172 and U-87) and one primary glioma cell culture (LA-567) were used. Proliferation ((3)H-thymidine) and cytotoxicity (methylthiotetrazole) studies were performed using CPT-11 (0-100 microM) alone, 7-ethyl-10-hydroxy camptothecin (SN-38) (0-1000 nM) alone or in the presence of a PKC inhibitor, tamoxifen (10 microM), hypericin (10 microM), calphositin C (400 nM), or staurosporine (10 nM). The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) assay was used to determine apoptosis as the mechanism of cytotoxicity; alterations in bcl-2 and bax expression were determined using Western blot analysis. Conversion of CPT-11 to SN-38 by glioma cells was determined using high-performance liquid chromatography (HPLC) analysis. Increasing CPT-11 and SN-38 concentrations induced cytotoxic morphologic changes, decreased proliferation, and increased cytotoxicity on all glioma cell lines tested. These changes were increased in the presence of a PKC inhibitor. The mechanism of the cytotoxicity was determined to be apoptosis by the TUNEL assay. The combination of a PKC inhibitor with CPT-11 or SN-38 led to decreased expression of the antiapoptotic protein bcl-2, and increased expression of the proapoptotic protein bax. HPLC analysis demonstrated conversion of CPT-11 to SN-38 by glioma cells. A combination of CPT-11 or SN-38 with a PKC inhibitor was found to lead to a decrease in proliferation and an increase in apoptosis in malignant glioma cells. The induction of apoptosis was secondary to a decrease in bcl-2 and an increase in bax expression. Glioma cells are capable of converting CPT-11 to SN-38 by intrinsic tumor carboxylesterases.

Anthracenes↗

PubMed2003

Highly efficient and tumor-restricted gene transfer to malignant gliomas by replication-competent retroviral vectors.

The first large randomized phase III trial in gene therapy demonstrated no improvement in the survival of patients injected with packaging cells that produced conventional replication-defective retroviral vectors carrying the herpes simplex virus thymidine kinase gene, a disappointing result that was attributed to extremely poor levels of transduction efficiency. To circumvent this problem, we have developed a modified replication-competent retrovirus (RCR) that is capable of transducing human glioma cell lines A-172, U-87, T-98G, U-373, and U-138 and rat glioma cell lines C6 and 9L, over multiple infection cycles in vitro, resulting in a tremendous enhancement in transduction efficiency over conventional replication-defective retroviral vectors at the same dose. Whereas the transduction efficiency of conventional retroviral vectors injected into preestablished subcutaneous U-87 tumors at a dose of 1.0 x 10(5) transducing units (TU) was only 0.2% at 6 weeks postinjection, the same dose of RCR vector resulted in up to 97.2% transduction. When RCR vectors at a dose of 1.0 x 10(4) TU were injected into preestablished intracranial U-87 tumors, transduction efficiency at 2 and 3 weeks was 74 and 98.1%, respectively. Notably, however, intracranial injection of RCR vectors did not result in detectable infection of normal brain cells. Furthermore, using a sensitive polymerase chain reaction assay, no detectable RCR signal could be observed in any extracerebral tissues, including lung, liver, kidney, upper gastrointestinal tract (esophagus and stomach), lower gastrointestinal tract (colon and small intestine), skin, spleen, and bone marrow. Treatment of U-87 intracranial gliomas with RCR vectors carrying the yeast cytosine deaminase suicide gene followed by 5-fluorocytosine prodrug administration resulted in 100% survival over a 60-day follow-up period, compared with 0% survival of control groups receiving vector alone or prodrug alone. Our results demonstrate that RCR vectors can achieve therapeutically significant levels of transduction in malignant human gliomas, and that RCR vector spread after intratumoral injection is restricted to the tumor itself.

Animals↗

PubMed2002

Tamoxifen-induced growth arrest and apoptosis in pituitary tumor cells in vitro via a protein kinase C-independent pathway.

Protein kinase C (PKC), a kinase family involved in cell signal transduction, is overexpressed in most pituitary adenoma cells. We studied the effect of tamoxifen, an estrogen receptor antagonist and also a protein kinase inhibitor, on pituitary tumor cell proliferation and the induction of apoptosis; and we compared its effects with those of another PKC inhibitor, staurosporine. Tamoxifen induced growth arrest and apoptosis in a mouse pituitary adenoma cell line, AtT20, and in low-passage human primary pituitary tumor cell cultures. Staurosporine also inhibited pituitary tumor cell growth. PKC activity in AtT20 cells was inhibited by staurosporine and by prolonged treatment with phorbol myristate acetate, which down-regulates PKC activity, but not by tamoxifen, at the dosages used to induce apoptosis. Our findings suggest that tamoxifen induces apoptosis in AtT20 cells independent of a classical PKC isozyme pathway.

Adenoma↗

PubMedDate not supplied

Functional effect of contortrostatin, a snake venom disintegrin, on human glioma cell invasion in vitro.

The metastatic spread of cancer is a complex process that involves the combination of different cellular actions including cell adhesion to the extracellular matrix (ECM), breakdown of the ECM by specific matrix-degrading proteinases, and active cell locomotion. Contortrostatin (CN), a homodimeric snake venom disintegrin, has previously been demonstrated to be effective in blocking vitronectin/fibronectin-dependent adhesion and invasion of T98G human glioblastoma cells through Matrigel using in vitro studies. However, it is not known at what step of the invasion process CN exerts its inhibitory effect. In the present report, CN is shown to decrease invasion of various glioma cell lines through Matrigel affecting neither cell adhesion, nor cell viability. While CN had no effect on cell binding to laminin and type IV collagen, it blocked adhesion of alphav beta3-positive, but not alphav beta3-negative cells, to vitronectin and fibronectin. Furthermore, members of the matrix metalloproteinase (MMP) family and their physiological inhibitors, and of the plasminogen activator (PA)/plasmin system were demonstrated not to be involved in CN-induced loss of glioma cell invasiveness. Instead, CN inhibited active locomotion of cells on Matrigel. These data suggest that CN-mediated inhibition of glioma cell invasion through Matrigel is a direct result of impaired cell motility. Moreover, use of several glioma cell lines and integrin antibodies strongly indicates the versatility of CN in inhibiting the invasion process based on the ability of CN to interact with different integrins, including alphav beta3, alphav beta5, and alpha5beta1.

Blotting, Western↗

PubMedDate not supplied

The type IV phosphodiesterase inhibitor rolipram induces expression of the cell cycle inhibitors p21(Cip1) and p27(Kip1), resulting in growth inhibition, increased differentiation, and subsequent apoptosis of malignant A-172 glioma cells.

Upregulation of the cAMP/protein kinase A (PKA) pathway has been shown to result in decreased proliferation, increased differentiation, and subsequent apoptosis of malignant glioma cells. Conventional cAMP analogs, however, are difficult to use in a clinical setting. Therefore, we investigated the effects of rolipram, a drug that has undergone clinical trials as an antidepressant and has also been proposed as a treatment for multiple sclerosis. Rolipram acts as a specific inhibitor of type IV phosphodiesterase (PDE4), leading to increased intracellular levels of cAMP. We report that the inhibition of PDE4 by rolipram results in the activation of the cAMP/PKA pathway, with potent stimulation of a reporter gene containing a cAMP-responsive element in its promoter region. Further, treatment of the human glioma cell line A-172 with rolipram results in increased expression of the cell cycle inhibitors p21(Cip1) and p27(KiP1), and decreased activity of cdk2, a cyclin-dependent kinase essential for cell cycle progression. As a result, the proliferation of A-172 cells is inhibited, with induction of a Gl block. Eventually, rolipram-treated A-172 cells undergo differentiation, which is followed by apoptotic cell death. As we observe this effect with other glioma cell cultures as well, our results suggest that rolipram could prove useful as a novel differentiating agent with both cytostatic and cytotoxic potential in the treatment of malignant gliomas.

3',5'-Cyclic-AMP Phosphodiesterases↗