(−)-Liriopein B Suppresses Breast Cancer Progression via Inhibition of Multiple Kinases

Numerous breast cancer patients who achieve an initial response to HER-targeted therapy rapidly develop resistance within one year, leading to treatment failure. Observations from clinical samples indicate that such resistance correlates with an increase in Src, EGFR, and PI3K/Akt activities and a decrease in PTEN activity. Furthermore, Akt survival signaling activation is also found in tumors treated by toxic chemotherapeutic agents. Because cotreatment with a PI3K inhibitor is a promising strategy to delay acquired resistance by preventing secondary gene activation, we therefore investigated the effects of a newly identified compound, (−)-Liriopein B (LB), on PI3K/Akt signaling activity in breast cancer cells. Our results showed that nontoxic doses of LB are able to inhibit AKT activation in both luminal-like MCF-7 and basal-like MDA-MB-231 breast cancer cells. Low doses of LB also inhibited cell migration, invasion, and cancer-stem cell sphere formation. Suppression of EGF-induced EGFR and ERK1/2 activation by LB might contribute in part to retardation of cancer progression. Furthermore, LB increases sensitivity of MDA-MB-231 cells to gefitinib in vitro, suggesting that EGFR may not be the only target of LB. Finally, a small scale in vitro kinase assay screen demonstrated that LB has a potent inhibitory effect on multiple kinases, including PI3K, Src, EGFR, Tie2, lck, lyn, RTK5, FGFR1, Abl, and Flt. In conclusion, this study demonstrates for the first time that the compound LB improves tumor therapeutic efficacy and suggests LB as a promising candidate for studying new leads in the development of kinase inhibitors

Isolation of Phorbol Esters from <i>Euphorbia grandicornis</i> and Evaluation of Protein Kinase C- and Human Platelet-Activating Effects of Euphorbiaceae Diterpenes

Human platelets contain conventional (α and β) and novel isoforms of PKC (δ and θ), and PKC activation can result in platelet aggregation and secretion reaction that are important for thrombus formation. Several tumor-promoting Euphorbiaceae diterpenes are known to act as direct activators of PKC, but many types of such diterpenes have not been studied as platelet stimulators. In the present study, two new and five known phorbol esters were isolated from <i>Euphorbia grandicornis</i>. Two of the isolated phorbol esters together with compounds representing ingenane, jatrophane, and myrsinane structural types were studied on PKC activation and platelet stimulation. The investigated phorbol esters and ingenane esters induced blood platelet aggregation and ATP secretion. PKC activation was demonstrated by inducing membrane translocation of PKCs, phosphorylation of PKC substrates, and activation of PKC signaling pathways. The PKC-activating effect of the compounds correlated well with their efficacy to cause platelet stimulation. Moreover, by using an isoform-specific PKC inhibitor, it was found that besides conventional PKCs novel PKCs also play a positive role in platelet activation caused by phorbol/ingenane esters, especially in regulating platelet aggregation. The present results suggest that platelets afford a useful model for studying PKC activators of natural origin or their chemical derivatives

Effect of withanolide on the IKK/NF-κB pathway.

<p>(A) Withanolides induce IKK degradation and inhibit NF-κB activation. MDA-MB-231 cells were treated with WA (5 µM), HW (20 µM), AA (20 µM), PH (20 µM) or GM (10 µM) for 12 h. After treatment, cells were harvested and analyzed for the proteins involved in the NF-κB pathway by Western blotting. (B) Withanolides decrease anti-apoptotic proteins that are regulated by NF-κB. Cells were treated with withanolides for 48 h. After treatment, cells were harvested and analyzed for Bcl-2, Bcl-xL, and c-FLIP by Western blotting. (C) NAC prevents IKK degradation and NF-κB inhibition by WA. Cells were treated with WA (5 µM) or GM (10 µM) in the absence or presence of NAC (2.5 mM) for 12 h. After treatment, cells were harvested and subjected to Western blotting.</p

Withanolides cause thiol oxidation and aggregation of Hsp90 in MDA-MB-231 cells.

<p>(A) Cells were treated with WA (5 µM), TA (5 µM), HW (20 µM), AA (20 µM) or PH (20 µM) for 12 h. The Triton-insoluble fractions of cell lysates were prepared as described under “Experimental Procedures.” The protein levels of the disulfide-linked-Hsp 90 were analyzed by nonreducing SDS-PAGE. (B, C) The thiol-reducing agent NAC prevents the degradation of Hsp90 client proteins and cell death caused by withanolides. MDA-MB-231 cells were pretreated with or without NAC (2.5 mM) for 1 h before challenged with withanolides for 12 h (B) or 48 h (C). The protein levels of heat shock proteins and client proteins were analyzed by western blot (B). The viability of MDA-MB-231 cells was determined by the MTT assay (C). Results are presented as means ± S.E.M. (n = 3). **<i>P</i><0.01, ***<i>P</i><0.001 as compared with each group without NAC pretreated.</p

Effect of the withanolides on ER-positive human breast cancer cells.

<p>(A) Withanolides reduce the viability of MCF-7 cells. MCF-7 cells were treated with the indicated concentrations of WA, HW, AA, or PH for 48 h, and cell viability was determined by the MTT assay. (B) Withanolides induce caspase-7 activation and PARP cleavage. MCF-7 cells were treated with the withanolides for 48 h. After treatment, cells were harvested and analyzed for caspase-7 and PARP by Western blot. (C) Withanolides induce degradation of Hsp90 client proteins and induction of Hsp70. MCF-7 cells were treated with the withanolides or GM for 12 h. Levels of Hsp90 client proteins and Hsp70 were determined by Western blot. (D) Withanolides induce IKK degradation and inhibit NF-κB activation. MCF-7 cells were treated with the withanolides or GM for 12 h. After treatment, cells were harvested and analyzed for the proteins involved in the NF-κB pathway by Western blot.</p

Chemical structures of the withanolide compounds.

<p>Chemical structures of the withanolide compounds.</p

Anti-inflammatory and Cytotoxic Neoflavonoids and Benzofurans from <i>Pterocarpus santalinus</i>

Five new benzofurans, pterolinuses A−E (1−5), six new neoflavonoids, pterolinuses F−J (8−13), and five known compounds (6, 7, 14−16) were isolated from an extract of Pterocarpus santalinus heartwood. All new structures were elucidated by spectroscopic methods, and configurations were confirmed by CD spectral data and optical rotation values. The isolates were evaluated for anti-inflammatory and cytotoxic activities. Six compounds (1, 2, 4, 6, 7, and 15) showed significant inhibition in at least one anti-inflammatory assay. Compound 2 showed the best selective effect against superoxide anion generation in human neutrophils with, an IC50 value of 0.19 μg/mL, and was 6.2-fold more potent than the positive control LY294002. Compound 14 showed the highest cytotoxicity against Ca9-22 cancer cells, with an IC50 value of 0.46 μg/mL

Cytotoxic Triterpenoids from the Leaves of <i>Microtropis fokienensis</i>

Five new triterpenoids, microfokienoxanes A−D (1−4) and 3β,28-dihydroxy-11α-methoxyurs-12-ene (5), were isolated and identified from the leaves of Microtropis fokienensis, along with nine known compounds. The structures of the new compounds were elucidated by spectroscopic methods. The compounds obtained in this investigation were evaluated against a small panel of human cancer cell lines for cytotoxicity. Only compounds 3 and 5 exhibited cytotoxicity (IC50 ≤ 5 μg/mL) for one or more cell lines

(−)-Liriopein B Suppresses Breast Cancer Progression via Inhibition of Multiple Kinases

Numerous breast cancer patients who achieve an initial response to HER-targeted therapy rapidly develop resistance within one year, leading to treatment failure. Observations from clinical samples indicate that such resistance correlates with an increase in Src, EGFR, and PI3K/Akt activities and a decrease in PTEN activity. Furthermore, Akt survival signaling activation is also found in tumors treated by toxic chemotherapeutic agents. Because cotreatment with a PI3K inhibitor is a promising strategy to delay acquired resistance by preventing secondary gene activation, we therefore investigated the effects of a newly identified compound, (−)-Liriopein B (LB), on PI3K/Akt signaling activity in breast cancer cells. Our results showed that nontoxic doses of LB are able to inhibit AKT activation in both luminal-like MCF-7 and basal-like MDA-MB-231 breast cancer cells. Low doses of LB also inhibited cell migration, invasion, and cancer-stem cell sphere formation. Suppression of EGF-induced EGFR and ERK1/2 activation by LB might contribute in part to retardation of cancer progression. Furthermore, LB increases sensitivity of MDA-MB-231 cells to gefitinib in vitro, suggesting that EGFR may not be the only target of LB. Finally, a small scale in vitro kinase assay screen demonstrated that LB has a potent inhibitory effect on multiple kinases, including PI3K, Src, EGFR, Tie2, lck, lyn, RTK5, FGFR1, Abl, and Flt. In conclusion, this study demonstrates for the first time that the compound LB improves tumor therapeutic efficacy and suggests LB as a promising candidate for studying new leads in the development of kinase inhibitors

Effect of the withanolides on the viability of MDA-MB-231 cells.

<p>(A) MDA-MB-231 cells were treated with the indicated concentrations of various withanolide compounds for 48 h, and cell viability was determined by the MTT assay. (B) The IC₅₀ values of the withanolides on the viability of MDA-MB-231 cells. Results are presented as means ± S.E.M. (n = 3).</p