Pristimerin, a naturally occurring triterpenoid, has been shown to cause cytotoxicity in several cancer cell lines. However, the mechanism for the cytotoxic effect of pristimerin was never explored. In the present study, human breast cancer MDA-MB-231 cells treated with pristimerin (1 and 3 μmol/L) showed rapid induction of apoptosis, as indicated by caspase activation, DNA fragmentation, and morphologic changes. Pretreatment of a pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) completely prevented pristimerin-induced apoptosis. Treatment of tumor cells with pristimerin resulted in a rapid release of cytochrome c from mitochondria, which preceded caspase activation and the decrease of mitochondrial membrane potential. In addition, neither benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone nor permeability transition pore inhibitor cyclosporin A markedly prevented pristimerin-induced mitochondrial cytochrome c release. Pristimerin did not significantly alter the protein level of Bcl-2 family members (Bcl-2, Bcl-X_L, and Bax), nor did it induce Bax translocation. Moreover, Bcl-2 overexpression fails to prevent pristimerin-induced apoptosis. The generation of reactive oxygen species in MDA-MB-231 cells was also not affected by pristimerin. In a cell-free system, pristimerin induced cytochrome c release from isolated mitochondria. Taken together, these results suggested that pristimerin is a novel mitochondria-targeted compound and may be further evaluated as a chemotherapeutic agent for human cancer.

Withanolides are a large group of steroidal lactones found in Solanaceae plants that exhibit potential anticancer activities. We have previously demonstrated that a withanolide, tubocapsenolide A, induced cycle arrest and apoptosis in human breast cancer cells, which was associated with the inhibition of heat shock protein 90 (Hsp90). To investigate whether other withanolides are also capable of inhibiting Hsp90 and to analyze the structure-activity relationships, nine withanolides with different structural properties were tested in human breast cancer cells MDA-MB-231 and MCF-7 in the present study. Our data show that the 2,3-unsaturated double bond-containing withanolides inhibited Hsp90 function, as evidenced by selective depletion of Hsp90 client proteins and induction of Hsp70. The inhibitory effect of the withanolides on Hsp90 chaperone activity was further confirmed using in vivo heat shock luciferase activity recovery assays. Importantly, Hsp90 inhibition by the withanolides was correlated with their ability to induce cancer cell death. In addition, the withanolides reduced constitutive NF-κB activation by depleting IκB kinase complex (IKK) through inhibition of Hsp90. In estrogen receptor (ER)-positive MCF-7 cells, the withanolides also reduced the expression of ER, and this may be partly due to Hsp90 inhibition. Taken together, our results suggest that Hsp90 inhibition is a general feature of cytotoxic withanolides and plays an important role in their anticancer activity.

BACKGROUND AND PURPOSE Activation of human platelets by thrombin is mediated predominately through two proteinase‐activated receptors (PARs), PAR1 and PAR4. Phosphatidylinositol 3‐kinase (PI3K) inhibition leads to reversible PAR1‐mediated platelet aggregation, but has no effect on the stability of platelet aggregation induced by thrombin. In the present study, the molecular mechanisms underlying this difference were investigated.EXPERIMENTAL APPROACH The functions of PI3K and PAR4 were assessed using specific inhibitors and aggregometry. The duration of platelet glycoprotein (GP) IIb/IIIa exposure was determined by flow cytometry with the antibody PAC‐1. Western blotting and fluo‐3 was used to evaluate the activation of Akt and protein kinase C (PKC) and intracellular Ca²⁺ mobilization respectively.KEY RESULTS When PAR4 function was inhibited either by the PAR4 antagonist YD‐3 [1‐benzyl‐3‐(ethoxycarbonylphenyl)‐indazole] or by receptor desensitization, the PI3K inhibitor wortmannin turned thrombin‐elicited platelet aggregation from an irreversible event to a reversible event. Moreover, wortmannin plus YD‐3 markedly accelerated the inactivation of GPIIb/IIIa in thrombin‐stimulated platelets. The aggregation‐reversing activity mainly resulted from inhibition of both PI3K‐dependent PKC activation and PAR4‐mediated sustained intracellular Ca²⁺ rises. Blockade of ADP P2Y₁₂ receptor with 2‐methylthioadenosine 5′‐monophosphate triethylammonium salt mimicked the inhibitory effect of wortmannin on PI3K‐dependent PKC activation and its ability to reverse PAR1‐activating peptide‐induced platelet aggregation. Co‐administration of 2‐methylthioadenosine 5′‐monophosphate triethylammonium salt with YD‐3 also decreased the stability of thrombin‐induced platelet aggregation.CONCLUSIONS AND IMPLICATIONS These results suggest that PAR4 acts in parallel with the P2Y₁₂/PI3K pathway to stabilize platelet aggregates, and provide new insights into the mechanisms of thrombus stabilization and potential applications for antithrombotic therapy.

Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A₂ mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y₁₂ receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.