Triptolide, an extract of Tripterygium wilfordii
Hook F, has been widely used in the People’s Republic of China for the treatment of inflammatory and autoimmune diseases for centuries. In addition to its anti-inflammatory and immunosuppressive effects, anticancer effects have also been reported in recent years.10
Triptolide can covalently interact with and inhibit the general transcription factor TFIIH component XPB, explaining its transcriptional effects.8
The expression of metalloproteinase 10 (ADAM10) is increased in several tumors, including leukemia, and is involved in malignant cell growth and cancer progression. ADAM10 is a novel target of triptolide; inhibition of ADAM10 by triptolide might be another mechanism by which triptolide inhibits tumorigenesis.34
Polycystin-2 (PC2) has been identified as a candidate target for the therapeutic action of triptolide and an association between PC2 expression and sensitivity to triptolide-induced growth arrest/cell death has been observed.35
Triptolide has been reported to be the first known inhibitor of dCTP pyrophosphatase 1 (DCTPP1).36
DCTTP1 is postulated to be a gatekeeper enzyme, protecting RNA or DNA against incorporation of noncanonical nucleotide triphosphates. However, it is also possible that the antitumor effects of triptolide may be explained by some other, yet unidentified, mechanisms. The fact that multiple molecular targets participate in triptolide’s pharmacological functions support the notion that triptolide can be an effective anticancer agent against cells and tumors that lack caspase-3.
Among the various in vitro cell line models available for breast cancer, the MCF-7 cell line presents distinctive properties that may help shed new light on the mechanism of action of triptolide. In particular, MCF-7 is an estrogen receptor-positive cell line that lacks caspase-3 and beclin-1;37
thus, it represents a cell model with compromised apoptotic machinery and low autophagic activity that might influence cellular response to anticancer drug treatment. We propose that the deletion of caspase-3 in tumors or cells will not limit triptolide’s usefulness because other mechanisms are at work, such as autophagy. Triptolide induces apoptosis in various cancer cells and, thus far, very little is known about the effect of triptolide on MCF-7 cells, except that triptolide exposure results in an increase in p53 expression,33
a decrease in estrogen receptor-α expression,33
a decrease in ADAM10 expression,34
and focal adhesion kinase cleavage.30
A previous study suggested that triptolide may regulate lysosomal-mediated apoptosis in MCF-7 breast cancer cells.39
In the present study, we demonstrate that triptolide’s cytotoxicity is dose- and time-dependent in MCF-7 cells, as examined by morphology (), Hoechst staining (), MTT assay (), and western blot analysis (). Our MTT data are in agreement with a previous report by Liu et al.33
Regarding triptolide-induced morphology, MCF-7 cells displayed less cytoplasmic blebbing (), and the pattern of pro-apoptotic protein expression levels () was similar to that of staurosporine-treated MCF-7 cells.32
PARP and procaspase-9 and -7 protein levels were downregulated in experimental cells in a dose-dependent manner, and cleaved PARP, caspase-9, and caspase-7 protein levels increased in a dose-dependent manner (). Active caspase-9 is an initiator protease that complexes with Apaf-1 and cytochrome c to form the apoptosome.40
Caspase-7 has been shown to cleave PARP-1 in apoptotic MCF-7 cells,41
while a more recent study revealed that caspase-7 uses an exosite to promote PARP proteolysis.42
Therefore, we concluded that MCF-7 cell death as a result of triptolide exposure is atypical apoptosis.
Before the discovery of the caspase family of proteases, most cell deaths were considered to be lysosomal43
or “type II,”15
requiring activation of the lysosomal compartment. In insects, cell death at metamorphosis is typically autophagic, and blocking autophagy is a pupariation lethal.45
This autophagic type of cell death may also be used in situations in which conventional apoptosis is blocked or limited by mutation or other controls, as in MCF7 cells, which lack caspase-3, or in which massive cell death overwhelms the ability of phagocytes to clear the terrain.46
Zakeri et al reported that in metamorphosing, secretory cells and under conditions where the majority of cells die, the bulk of the cytoplasm is consumed by expansion of the lysosomal system well before nuclear collapse is manifested.16
In the current study, we demonstrate that triptolide induces programmed cell death in MCF-7 cells that is presumably characterized by a leakage of lysosomal enzymes into the cytosol.
We report here that triptolide induces lysosomal-mediated apoptosis in MCF-7 cells. illustrates the activity of AP Halaby et al previously used AP to monitor apoptosis in degenerating M. sexta
labial glands during the larval to pupal metamorphosis.19
Others have used this enzyme to measure cell death in Drosophila
salivary glands as well as insect intersegmental muscles.47
AP activity was elevated after 24 and 72 hours in experimental cells (). These results are supported by our previous study examining AP activity in the labial gland of Manduca
during the larval to pupal molt.48
The relatively high levels of AP activity in control cells at 72 hours may be explained by the fact that activities of specific lysosomal hydrolases are higher in tumor cells than in the corresponding normal cells.49
These results suggests that triptolide activates lysosomes. Next, experimental cells stained with the vital dye acridine orange displayed neutral cytosolic staining, which did not occur in control cells ( and ). Acridine orange concentrated in lysosomes emits a granular red fluorescence, whereas, in the cytosol, it emits a diffuse green fluorescence.50
These data suggest that triptolide mediated a reduction in red fluorescence while an increased diffuse cytosolic green fluorescence indicated a relocation of acridine orange from the lysosomes to the cytosol following a change in lysosome permeability. This provides supporting evidence for our hypothesis that triptolide modulates LMP. Similarly, exposure of cells to triptolide resulted in an increase in the accumulation of the lysosomotropic probe LysoTracker Green ().
LysoTracker Green staining also demonstrated that triptolide-treated cells displayed diffuse and intense staining, whereas staining was punctate and reduced in control cells ().
Finally, expression levels of cathepsin B in MCF-7 cytosolic fractions supported susceptibility to lysosomal permeabilization. Elevated cathepsin B was detected at the early stage of cell death (3 hours post-triptolide-treatment). Decreased expression of cathepsin B was detected in control cells (). Altogether, these results suggest that triptolide affects LMP. The resulting change in lysosomal membrane permeability in turn leads to leakage of the lysosomal hydrolases into the cytosol, where they can trigger the apoptotic cascade by activating proteins such as Bid.23
This notion, that lysosomal rupture may be an upstream event during some instances of apoptosis, is supported by and is also reinforced by work from others.51
These findings suggest that lysosomal destabilization might play an integral part in programmed cell death via the intrinsic pathway.
Increasing evidence suggests that lysosomes are important mediators of programmed cell death. In autophagic cell death, lysosomes fuse with autophagosomes to form autophagolysosomes, by which their contents are degraded.54
In apoptosis, cathepsins are released from lysosomes into the cytoplasm and trigger a cascade of intracellular degradation.53
The involvement of lysosomes in both programmed cell death pathways55
may suggest an involvement of cathepsins as a functional link between apoptosis and autophagy. This hypothesis was confirmed by results of a recent study showing that inhibition of cathepsins by E64d, an inhibitor of papain-like cysteine proteases, resulted in a significant reduction of apoptotic cell numbers accompanied by an increase in autophagosome formation in MCF-7 cells exposed to camptothecin.58