Phenoxodiol Treatment Alters the Subsequent Response of ENOX2 (tNOX) and Growth of HeLa Cells to Paclitaxel and Cisplatin

Abstract

Phenoxodiol is an experimental anticancer drug under development as a chemosensitizer intended to reverse multidrug resistance mechanisms in ovarian and prostate cancer cells to most standard cytotoxics. The putative molecular target of phenoxodiol is a cell-surface, tumor-specific NADH oxidase, ENOX2 (tNOX), with phenoxodiol having no apparent effect on the constitutive form of this enzyme ENOX1 (CNOX). Using ENOX2 as the target, this study was conducted to explore the temporal relationship between phenoxodiol and paclitaxel or cisplatin in achieving chemosensitization in HeLa cells which are relatively resistant to both paclitaxel and cisplatin. Sequential addition of phenoxodiol and paclitaxel or phenoxodiol and cisplatin showed greater inhibition of HeLa cell ENOX1 activity and growth compared to adding the drugs simultaneously or individually. In parallel, a similar chemosensitizing response of phenoxodiol for cisplatin was observed. ENOX1 was not affected and trans-platinum had no effect. With spent media from phenoxodiol-treated cells sensitivity was enhanced to both paclitaxel and cisplatin if the cells were first pretreated with phenoxodiol. Similar results were obtained with ENOX2-enriched preparations stripped from the surfaces of phenoxodiol-treated cells. In keeping with a speculative prion model, it seems as though the ENOX2 "remembers" the phenoxodiol and "teaches" other ENOX2 molecules to respond to paclitaxel and cisplatin as if phenoxodiol were still present.

tNOX (ENOX2) Target for Chemosensitization-Low-Dose Responses in the Hormetic Concentration Range

“…5). As we have observed previously [27] , activities of tNOX in the assay were inhibited by the addition of 10 µM phenoxodiol but returned to the original activity level after phenoxodiol was removed. Paclitaxel (1 µM) addition 72 min after washing cells (231.5 min from beginning of the experiment) inhibited tNOX activity but did not affect the activity of CNOX (activity of this protein remained constant after cells were washed).…”

“…The above findings were reproduced in growth experiments for both CP-70 Table 2 and HeLa cells [27] . For CP-70 cells, phenoxodiol at 0.5 or 1 µM followed by 1 µM cisplatin gave augmented growth inhibition only with sequential application where the phenoxodiol was removed prior to the addition of cisplatin (compare with Fig.…”

“…Inhibitions with sulforaphane pretreatment were significantly greater than inhibitions for co-administration (p<0.04 for HeLa and p<0.01 for CP-70) Sulforaphane chemosensitization of response to cisplatin correlated with altered drug responsiveness of tNOX: In previous studies, the phenoxodiol chemosensitization of response of CP-70 and HeLa cells to cisplatin and/or paclitaxel was traced to a increased drug responsiveness of cell surface tNOX in response to the phenoxodiol. [27] The procedure for assignment of drug inhibitions specifically to tNOX The supernatant was discarded. The cells were washed through two more centrifugation resuspension cycles (total elapsed time 50 min), resuspended in fresh assay medium containing substrate and the assay was continued for 60 min after which 1 µM paclitaxel was added and the assay was continued for an additional 60 min.…”

“…Phenoxodiol and possibly also sulforaphane seem to have that capability. In fact, some degree of chemosensitization is achieved by merely adding back tNOX proteins that have been exposed to phenoxodiol [27] . In plants an ECTO-NOX activity is activated by the synthetic plant growth auxin regulator, 2,4-dichlorophenoxyacetic acid (2,4-D) [36] .…”

The effects of phenoxodiol on the cell cycle of prostate cancer cell lines

“…In this study, we explored the ability of phenoxodiol to impact the cell cycle of prostate cancer cells and investigate the underlying signalling pathways c-Myc, Cyclin-D1, Ki-67 and p21 WAF1 . Phenoxodiol, [2H-1-benzopyran-7-0, 1,3-(4-hydroxyphenyl)], is a synthetic isoflavone molecule first isolated from soy beans and now currently undergoing Phase III clinical trials for the treatment of platinum and taxane refractory ovarian cancer [ 9 , 10 ]. We examined three cell lines: LNCaP cells are responsive to 5-alpha-dihydrotestosterone (DHT), which means they contain a functioning androgen receptor (AR) and are indicative of an early stage prostate cancer cell line even though they are metastatic in nature.…”