Functional Evaluation of a Fluorescent Schweinfurthin: Mechanism of Cytotoxicity and Intracellular Quantification

Abstract

Schweinfurthins are potent inhibitors of cancer cell growth, especially against human central nervous system tumor lines such as SF-295 cells. However, the mechanisms through which these compounds impede cell growth are not fully understood.In an effort to understand the basis for the effects of schweinfurthins, we present a fluorescent schweinfurthin, 3-deoxyschweinfurthin B-like p-nitro-bis-stilbene (3dSB-PNBS), which displays biological activity similar to that of 3-deoxyschweinfurthin B (3dSB). These two schweinfurthins retain the unique differential activity of the natural schweinfurthins, as evidenced by the spindle-like morphological changes induced in SF-295 cells and the unaltered appearance of human lung carcinoma A549 cells. We demonstrate that incubation with 3dSB or 3dSB-PNBS results in cleavage of poly-ADP-ribose polymerase (PARP) and caspase-9, both markers of apoptosis. Coincubation of 3dSB or 3dSB-PNBS with the caspase-9 inhibitor (Z)-Leu-Glu(O-methyl)-His-Asp(O-methyl)-fluoromethylketone prevents PARP cleavage. Therapeutic agents that induce apoptosis often activate cellular stress pathways. A marker for multiple stress pathways is the phosphorylation of eukaryotic initiation factor 2␣, which is phosphorylated in response to 3dSB and 3dSB-PNBS treatment. Glucose-regulated protein 78 and protein disulfide isomerase, both endoplasmic reticulum chaperones, are up-regulated with schweinfurthin exposure. Using the fluorescent properties of 3dSB-PNBS and dimethoxyphenyl-p-nitro-bis-stilbene (DMP-PNBS), a control compound, we show that the intracellular levels of 3dSB-PNBS are higher than those of Rhodamine 123 or DMP-PNBS in SF-295 and A549 cells.

3‐Deoxyschweinfurthin B Lowers Cholesterol Levels by Decreasing Synthesis and Increasing Export in Cultured Cancer Cell Lines

“…In the A549 cells, 3dSB incubation leads to APO-A1 specific cholesterol efflux further emphasizing the differences between sensitive and insensitive cell lines. We have previously noted that 3-dSB induces ERstress in the SF-295 cell line, as demonstrated by increasing GRP78 expression [39]. Increased ER-stress has been shown to reduce the cellular level of ABCA1 in hepatocellular carcinoma HepG2 cells, and to cause a reduction in cholesterol efflux [38,40].…”

“…After indicated incubation conditions, cells were collected and lysed in buffer containing (0.15 M NaCl, 0.05 M Tris HCl, 1 % w/v sodium deoxycholate, 1 % w/v SDS, 1 % w/v TX-100, and 1 mM EDTA, and protease inhibitor cocktail, Sigma-Aldrich). Lysate was incubated on ice for 30 min prior to passage through a 27-G needle [27]. Lysate was centrifuged at 14,000×g for 15 min.…”

Schweinfurthins: Lipid Modulators with Promising Anticancer Activity

“…Synthesis of the natural products and development of synthetic analogs opened the door for explorations into the mechanism of schweinfurthin action and initial studies delved into the nature of schweinfurthin cytotoxicity. For sensitive cell lines, schweinfurthin treatment results in a cytotoxic cellular response that is both dose and time dependent (Kuder et al, ). In the sensitive SF‐295 cells, caspase‐9 and PARP cleavage are observed 24 and 36 h, respectively, after treatment with 1 μM of the schweinfurthin analog 3dSB.…”

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

“…4G), confirming an ER stress induction effect by SB after a 9 h treatment in cells. 7 Notably, a higher drug concentration was required to induce significant p-EIF2a up-regulation than to inhibit AKT phosphorylation in the same WSU-DLCL2 cells (Figs. 1F, 4G), suggesting that induction of ER stress contributed to inhibition of AKT activation by schweinfurthins.…”

“…[4][5][6] However, this effort has been hampered by a lack of understanding of mechanism. Recently, schweinfurthin A (SA) was shown to interfere with phosphorylation of Rho in glioma cells 5 and to induce expression of glucose-related protein 78 and phosphorylation of eukaryotic initiation factor 2a, 7 suggesting regulatory roles in signaling and endoplasmic reticulum (ER) induction by schweinfurthins. Interestingly, OSBP and ORP4L, 2 members of the oxysterol-binding protein related family proteins (ORPs) were reported as potential targets for SA and for a few other natural compounds that showed a similar anti-proliferative pattern in the NCI-60 cancer cell panel.…”