The Tumor Proteasome Is a Primary Target for the Natural Anticancer Compound Withaferin A Isolated from “Indian Winter Cherry”

Abstract

Withaferin A (WA) is a steroidal lactone purified from medicinal plant "Indian Winter Cherry" that is widely researched for its variety of properties, including antitumor effects. However, the primary molecular target of WA is unknown. By chemical structure analysis, we hypothesized that Withaferin A might be a natural proteasome inhibitor. Computational modeling studies consistently predict that C 1 and C 24 of WA are highly susceptible toward a nucleophilic attack by the hydroxyl group of N-terminal threonine of the proteasomal chymotrypsin subunit ␤5. Furthermore, WA potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC 50 ϭ 4.5 M) and 26S proteasome in human prostate cancer cultures (at 5-10 M) and xenografts (4 -8 mg/kg/day). Inhibition of prostate tumor cellular proteasome activity in cultures and in vivo by WA results in accumulation of ubiquitinated proteins and three proteasome target proteins (Bax, p27, and IB-␣) accompanied by androgen receptor protein suppression (in androgen-dependent LNCaP cells) and apoptosis induction. Treatment of WA under conditions of the aromatic ketone reduction, or reduced form of Celastrol, had significantly decreased the proteasomeinhibitory and apoptosis-inducing activities. Treatment of human prostate PC-3 xenografts with WA for 24 days resulted in 70% inhibition of tumor growth in nude mice, associated with 56% inhibition of the tumor tissue proteasomal chymotrypsinlike activity. Our results demonstrate that the tumor proteasome ␤5 subunit is the primary target of WA, and inhibition of the proteasomal chymotrypsin-like activity by WA in vivo is responsible for, or contributes to, the antitumor effect of this ancient medicinal compound.

Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells

“…Indeed, it has been widely reported that ER stress is implicated in antitumoral effects of proteasome inhibitors. [34][35][36] Consistent with previous research, 21,22 the present study demonstrated that WA inhibited proteasome activity and accumulation of ubiquitinated proteins in PC cells. Subsequent investigations revealed that CHX attenuated WA-induced cell death, indicating that proteotoxicity is important for the effect of WA.…”

“…21 Thus, we sought to determine whether the incomplete autophagy induced by WA was associated with proteasome inhibition. As shown in Fig.…”

“…19,20 WA targets the tumor proteasome, which leads to ubiquitinated protein accumulation and induction of ER stress in human cancer cells. 21,22 WA also has effects on autophagy, although the role of autophagy in the anticancer effects of WA remains to be determined. 23,24 In this study, the effect of WA on protein homeostasis in PC cells was examined.…”

Combination of withaferin A and X-ray irradiation enhances apoptosis in U937 cells

“…Wit A, a steroidal lactone isolated from medicinal plant 'Indian Winter Cherry', has anti-inflammatory and anti-tumor effect (Yang et al, 2007). Several studies have reported that Wit A can induce cancer cell death (Mohan et al, 2004;Sharada et al, 1996).…”

Withaferin a synergistically enhances the effects of paclitaxel against lung cancer.

“…For example, in a study conducted in nude mice, intraperitoneal administration of WFA [4-8 mg/kg/day] for 24 days resulted in over 70% inhibition of PC3 subcutaneous tumor growth [57].…”