Zyflamend^®-mediated inhibition of human prostate cancer PC3 cell proliferation: Effects on 12-LOX and Rb protein phosphorylation

Abstract

The multiherb anti-inflammatory product Zyflamend was investigated for its antiproliferative effects on PC3 human prostate cancer cells and eicosanoid metabolism in this prostate cancer cell line. Zyflamend produced a concentration-dependent inhibition of cloned COX-1, COX-2, and 5-LOX enzyme activities, with inhibition of 5-HETE production being greater than that of PGE 2 formation. Applied to intact PC3 cells, Zyflamend was found to be most potent against 12-LOX, followed by 5-LOX and then COX activities. The concentration-dependent inhibition of PC3 cell proliferation was associated with a selective G 2 /M arrest of the cell cycle and induction of apoptosis, as evidenced by flow cytometric staining of PC3 cells with annexin V. Zyflamend also produced a concentration-dependent down-regulation of 5-LOX and 12-LOX expression. Determination of cell signal transduction proteins demonstrated that Zyflamend produced an increase in p21 phosphorylation but down-regulated phosphorylation of retinoblastoma (Rb) protein. The decrease in pRb protein was shown to be due to 12-LOX inhibition and a decline in 12-HETE levels in the cells. Replenishing 12-HETE in Zyflamend-treated cells overcame the ability of this multiple herb product to inhibit cell proliferation, and concordantly, 12-HETE blocked Zyflamend's ability to down-regulate phosphorylation of Rb protein.We conclude that the effective control of human prostate cancer cell proliferation with Zyflamend is multi-mechanistic but, in part, involves regulation of aberrant tumor cell eicosanoid metabolism, especially on 5-and 12-LOX, as well as restoration of Rb tumor suppressor protein function through regulation of its phosphorylation status.

Zyflamend, a polyherbal mixture, inhibits lipogenesis and mTORC1 signalling via activation of AMPK

“…Our data pertaining to Zyflamend and several studies from other laboratories (Bemis et al, 2005;Burke et al, 2015;Capodice et al, 2009;Ekmekcioglu et al, 2011;Huang, Chen, et al, 2011;Huang et al, 2014;Kim et al, 2012;Kunnumakkara et al, 2012;Mohebati et al, 2012;Rafailov et al, 2007;Sandur et al, 2007;Yan et al, 2012;Yang et al, 2007Yang et al, , 2008 reveal diverse mechanisms of action, possibly explaining its robust effects using in vivo models of tumorigenesis at oral doses that mimic human intakes (Huang, Chen, et al, 2011;Kunnumakkara et al, 2012). Because herbal extracts are composed of multiple compounds, it is difficult to directly extrapolate a standard concentration.…”

“…Its original formulation almost 2 decades ago was based on those herbs that had antioxidant and antiinflammatory properties. Its subsequent use in experimental investigations in an assortment of cancer models has drawn attention by a variety of laboratories interested in the role of natural products in health and disease (Bemis et al, 2005;Burke et al, 2015;Capodice et al, 2009;Ekmekcioglu et al, 2011;Huang, Chen, et al, 2011;Huang et al, 2014;Kim et al, 2012;Kunnumakkara et al, 2012;Mohebati et al, 2012;Rafailov et al, 2007;Sandur et al, 2007;Yan et al, 2012;Yang et al, 2007Yang et al, , 2008. There are always concerns regarding reproducibility and quality control of commercial products.…”

“…Group comparisons, AICAR or Zyflamend versus control,* p < 0.05. Huang et al, 2014;Kim et al, 2012;Kunnumakkara et al, 2012;Mohebati et al, 2012;Rafailov et al, 2007;Sandur et al, 2007;Yan et al, 2012;Yang et al, 2007Yang et al, , 2008. The best combinations of extracts within this formulation have not been systematically determined because it would be infeasible to reproduce the results from 1028 possible combinations.…”

“…To date, there have been 14 research articles investigating the anti-cancer effects of this blend, covering in vitro, preclinical and clinical studies involving the prostate, bone, skin, kidney, breast and pancreas (Bemis, Capodice, Anastasiadis, Katz, & Buttyan, 2005;Burke et al, 2015;Capodice et al, 2009;Ekmekcioglu et al, 2011;Huang, Chen, et al, 2011;Huang et al, 2014;Kim et al, 2012;Kunnumakkara et al, 2012;Mohebati et al, 2012;Rafailov, Cammack, Stone, & Katz, 2007;Sandur et al, 2007;Yan, Xie, Capodice, & Katz, 2012;Yang et al, 2007Yang et al, , 2008, with prostate being the predominant focus. It has been reported that a number of its constituents act synergistically such that physiological doses are possible (Zhao, Collier, Huang, & Whelan, 2014) and multiple mechanisms of action have been linked in a variety of cancer cell lines by down regulating the androgen receptor (Huang, Chen, et al, 2011;Huang et al, 2014;Yan et al, 2012), insulin-like growth factor-1R (Huang, Chen, et al, 2011;, class I and II HDACs (Huang, Chen, et al, 2011;Huang et al, 2014), COX-1 and COX-2 activities (Bemis et al, 2005;Ekmekcioglu et al, 2011;Kunnumakkara et al, 2012), 12-LOX and Rb protein phosphorylation (Yang et al, 2007), and the up regulation of tumour suppressor genes such as p21 and p27 (Bemis et al, 2005;Huang et al, 2014). Zyflamend has also been shown to modulate NF-κB activation (Burke et al, 2015;Kunnumakkara et al, 2012;Sandur et al, 2007), iNOS (Ekmekcioglu et al, 2011), apoptotic pathways (Ekmekcioglu et al, 2011;…”

Comparative Analysis of Peritoneum and Tumor Eicosanoids and Pathways in Advanced Ovarian Cancer

“…These studies have shown significant differences in patterns of eicosanoid metabolism between different types of malignant tissues; for example, it has been shown that colon cancer is regulated in part by the relative expression of 13-S-hydroxyoctadecadienoic acid (13-S-HODE; ref. 14), that glioblastomas invariably overexpress 5-LOX, 6 and that prostate cancer is connected to an age-associated decline in the expression of 15-hydroxyeicosatetraenoic acid (15-HETE), a tumor suppressor (15), as well as up-regulation of 12-LOX which, in turn, inhibits Rb tumor suppressor activity (16).…”