Effect of polyclonal antisera to recombinant tNOX protein on the growth of transformed cells

Abstract

Previous reports have described a tumor-associated NADH oxidase (tNOX) and its continuous activation in transformed culture cells. Certain anticancer drugs have been shown to inhibit preferentially both the tNOX activity and the growth of transformed culture cells and the cytotoxicity is associated with the induction of apoptosis. To investigate the biological function of tNOX protein, we have raised polyclonal antisera against bacterial expressed tNOX protein and the antisera are able to recognize protein bands in transformed cells but not the non-transformed cells tested. With tNOX antisera treatment, the survival in transformed cell lines is decreased but not the non-transformed cells. In addition, tNOX antisera-induced cytotoxicity is accompanied by the induction of apoptosis. However, slightly higher amount of PARP cleavage and activation of caspase-9 are observed in tNOX antisera treated HCT116 cells. Further experiments have demonstrated the activation of JNK and phosphorylation of p53 by treatment. In addition, tNOX antisera treatment leads to an impressive increase in reactive oxygen species in COS cells but not the control sera. Our data suggest that (a) tNOX antisera treatment may inhibit the growth of transformed cells by inducing apoptosis and (b) the apoptotic mechanism might be through modulating ROS production and JNK pathway.

Stress‐induced down‐regulation of tumor‐associated NADH oxidase during apoptosis in transformed cells

“…2 A). Using an antiserum that recognizes multiple tNOX forms [13], we showed that the increase in PARP cleavage occurred concomitantly with a decrease in tNOX levels (Fig. 2B); moreover, this EGCg‐induced reduction in tNOX occurred in a time‐dependent manner (Fig.…”

“…Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from GIBCO/BRL Life Technologies (Grand Island, NY). Antisera to tNOX were generated as described previously [13]. The anti‐PARP, anti‐phosphorylated p53, and anti‐rabbit IgG antibodies were from Cell Signaling Technology Inc. (Beverly, MA); the anti‐actin antibody was from Chemicon International Inc. (Tamekula, CA); the anti‐tubulin antibody was from Abcam Inc. (Cambridge, MA).…”

Down-Regulation of Tumor-Associated NADH Oxidase, tNOX (ENOX2), Enhances Capsaicin-Induced Inhibition of Gastric Cancer Cell Growth

“…Dihexyloxocarbocyanine iodide (DiOC6(3)) was purchased from Calbiochem (San Diego, CA, USA). Antisera to tNOX used in Western blot analyses were generated as described previously [29] and the tNOX protein band recognized by this antisera was also identified by the commercial available anti-tNOX polyclonal antibody (Protein Tech Group, Inc. Chicago, IL) [39]. The anti-p53 and anti-mouse IgG antibodies as well as other chemicals were purchased from the Sigma Chemical Company (St. Louis, MO, USA), unless otherwise specified.…”

“…The NADH oxidase activity initially identified in rat liver plasma membranes is responsive to growth factors and hormones (CNOX; ENOX1), whereas that from rat hepatoma is constitutive active [28] and has since been identified in various cancer cell lines [18,[29][30][31] and in the sera of cancer patients (tNOX; ENOX2) [32,33]. In fact, tNOX protein was originally purified from pooled cancer patient sera [34], suggesting its clinical relevance.…”

Trans-Plasma Membrane Electron Transport in Mammals: Functional Significance in Health and Disease

“…Mouse embryonic fibroblasts derived from transgenic mice overexpressing a truncated form of ENOX2 showed accelerated growth rates with respect to wild-type fibroblasts and enhanced invasiveness (315). In contrast, decreased cell growth and migration, as well as increased cell death, are observed when the function of ENOX2 is impaired, either by inhibiting the catalytic activity with (3)-epigallocatechin-3-gallate, capsaicin, phenoxodiol, or anti-ENOX2 antibodies (47,72,306,315) or by down-modulating its expression by RNA interference (173). Morré and colleagues also viewed ENOX2 as both (i) a noninvasive, circulating tumor marker (based on their description of several cancer-type specific ENOX2 isoforms) (119,307) and (ii) a potential antitumoral drug target, since this enzyme is blocked by quinone site inhibitors, which also exhibit anticancer activity (54,119,208).…”