The present report describes the mechanism through which guggulsterone induces apoptosis in tumor cells. Guggulsterone inhibited proliferation of a wide variety of tumor cells, including ones resistant to gleevac, dexamethasone, and doxorubicin. It arrested cells at S-phase through the down-regulation of cyclin D1 and cdc2 and upregulation of p21, and p27, and it inhibited expression of the antiapoptotic genes Bfl-1, xIAP, cFLIP, cMyc, Bcl-2, Bcl-XL, survivin, c-myc, and COX-2. Guggulsterone increased the number of apoptotic cells as demonstrated by annexin V staining, TUNEL, and live-dead assays. Further, it activated caspase-8, induced Bid cleavage, caused mitochondrial cytochrome c release, and induced caspase-3 activation and PARP cleavage. We also found that guggulsterone induced activation of JNK that was mediated through MKK4. Finally, suppression of JNK activation by the specific JNK inhibitor SP600125 or deletion of the MKK4 gene blocked guggulsterone-induced apoptosis.
Our results suggest that guggulsterone blocked proliferation of tumor cells by arresting the cells in S-phase of the cell cycle. Cyclin D1 and cdc2 are required for the progression of cells from the G1 phase to the S-phase of the cell cycle [22
], and guggulsterone suppressed the expression of both of them in a time-dependent manner in U937 cells. The expression of the cyclin-dependent kinase inhibitors p21 WAF1/CIP1
and p27, which block cell cycle progression by inhibiting the activity of cyclin/Cdk2 complexes, was also upregulated by guggulsterone.
Our results indicate that guggulsterone treatment downregulates the expression of antiapoptotic gene products Bfl-1, xIAP, cFLIP, cMyc, Bcl-2, Bcl-Xl, survivin, c-myc and COX-2. These observations are in agreement with a report in which guggulsterone was shown to downregulate the expression of Bcl-2 and Bcl-XL in prostate cancer cell lines [11
]. Our study also demonstrates that guggulsterone induces apoptosis in part through the activation of caspase-8. However, the mechanism by which guggulsterone activates caspase-8 is not clear. Several reports indicate that auto-activation induced by oligomerization can activate caspase-8 [40
]. Thus, it may be possible that guggulsterone induces the oligomerization of caspase-8. Activation of caspase 8 cleaves Bid that then translocates to the mitochondria and stimulates the release of cytochrome c, which in turn activates caspase 9. Our results demonstrate that guggulsterone induced Bid cleavage followed by the release of cytochrome c
and the activation of caspase-3 and PARP cleavage. Thus, one possible mechanism by which guggulsterone induces apoptosis is through changes in the mitochondrial membrane potential, which would lead to the release of cytochrome c
from the mitochondria, leading to sequential activation of caspase-9 and caspase-3. These observations are in agreement with a report in which guggulsterones were shown to induce a loss in mitochondrial membrane potential, leading to apoptosis [12
Interestingly, we found that normal human fibroblast cells are relatively resistant to growth inhibition by guggulsterone in comparison to tumor cells. These observations are in agreement with previous reports that guggulsterone inhibits proliferation of PC-3 cells, whereas the normal prostate epithelial cell line is resistant to guggulsterone [11
The pharmacological activity of guggulsterone has been suggested to be mediated by the nuclear hormone receptor FXR. Guggulsterone has been shown to be an antagonist to ligand for FXR. Previous studies have shown that FXR agonists enhance apoptosis in ovarian cancer cells [42
] and vascular smooth muscle cells [43
]. FXR is expressed at a higher level in ductal epithelial cells of normal breast and infiltrating ductal carcinoma cells in breast cancer. FXR was also present in the human breast carcinoma cells, MCF-7 and MDA-MB-468. Activation of FXR by high concentrations of ligands induced apoptosis in breast cancer cells [44
]. A recent study by De Gottardi et al. shows that overexpression of bile acid receptor FXR in Barrett's esophagus enhances apoptosis by guggulsterone in vitro [45
]. Thus, it is possible that this steroid acts through the bile acid receptor.
Our results indicate that besides downregulating antiapoptotic gene products, guggulsterone also mediates its effects through downregulation of Akt pathway. This pathway is closely linked with cell survival [46
]. We examined the effect of guggulsterone treatment on Akt phosphorylation and found that guggulsterone suppressed the phosphorylation of Akt at both Serine 473 and Threonine 308 residues. Guggulsterone also suppressed the phosphorylation of the upstream kinase PDK1 at Serine 241. The phosphorylation of the p85 subunit of another upsteam kinase, PI3K was also suppressed upon guggulsterone treatment. Suppression of Akt by guggulsterone led to the suppression of phosphorylation of GSK3β, the substrate of Akt. We also found that guggulsterone activated JNK and that suppression of JNK by its specific inhibitor abolished the activation of caspase 3, PARP cleavage, and cell proliferation. The activation of JNK requires the activation of an upstream kinase MKK4 [21
]. Using MKK4-gene deleted murine embryonic fibroblast cells, we found that guggulsterone activated JNK in wild type cells but not in MKK4-deficient mutant cells, thus suggesting that the activation of JNK by guggulsterone requires MKK4. The lack of JNK activation in mutated cells correlated with suppression of guggulsterone-induced apoptosis; again suggesting the critical role of JNK. These results are in agreement with reports that JNK is required for apoptosis induced by TNFα [47
], FasL [48
], and TRAIL [49
]. JNK activation is also needed for apoptosis induced by chemotherapeutic agents [51
]. Whether the mechanism of action of guggulsterone is different in various cell types is possible but unlikely as we found that GS activated JNK in both leukemic as well as in epithelial cells.
Guggulsterone treatment suppressed the activation of Akt, the prosurvival signal but induced the JNK activation. The results suggest that suppression of Akt and activation of JNK by guggulsterone are related to each other. Guggulsterone did not induce JNK activation in IKKβ-deficient and p65-deficient murine embryonic fibroblasts, thereby, suggesting that activation of JNK is mechanistically related to NF-κB activation, which has antiapoptotic role.
Because of lack of any known toxicity, guggulsterone should be further explored for its anticancer potential. Whether the concentrations used in our studies can be achieved in vivo, is unclear at present. No data is at present available on the pharmacokinetics, pharmacodynamics, and bioavailability of guggulsterone in animals or human. The hypolipidemic and antoxidant effects have been reported in human with as little as 50 mg of guggulipids, administered twice daily for 24 weeks [52
]. How does these doses compare with studies performed here, is not clear. Overall, our results indicate that guggulsterone inhibits the growth of wide variety of cells and induces apoptosis through downregulation of antiapoptotic gene products, modulation of cell cycle proteins, activation of caspases, inhibition of Akt and activation of JNK.