Triphala, an Ayurvedic Formulation, Inhibits Cancer Cell Proliferation by Perturbing Microtubule Assembly Dynamics

Introduction: Triphala (Trl) is a traditional Ayurvedic herbal formulation consisting of fruits of Emblica officinalis, Terminalia bellirica, and Terminalia chebula. It has been widely in use for treating digestive, respiratory, and nervous systems disorders. We studied effects of Trl on tubulin, a target protein for several anticancer drugs, and systematically elucidated a possible, tubulin-targeted antiproliferative mechanism of action of Trl.

Materials and methods: After assessing its antiproliferative and anti-clonogenic potential using SRB and clonogenic assay, respectively, we studied the effects of Trl on tubulin using tryptophan quenching assay, anilinonaphthalene sulfonate binding assay (ANS assay), and spectropolarimetry. Further, using sedimentation analysis, the effect of Trl on tubulin polymerization was examined. The intracellular manifestations of Trl-tubulin interactions were examined using immunofluorescent microscopy and western blot analysis. Cell death was studied by annexin V staining.

Results and discussion: Trl inhibited proliferation of HeLa, SIHA, PANC-1, MCF-7, MDA-MB-231, HOP62, A549 and PC-3 cancer cell lines in microgram quantities (< 200 μg/mL) and strongly suppressed the clonogenicity of HeLa cells. A concentration-dependent quenching of intrinsic tryptophan fluorescence of tubulin indicated binding of Trl to tubulin. Trl caused considerable reduction in the far UV-CD spectrum of tubulin indicating disruption of the secondary conformation of the protein. It disrupted tubulin without considerably inhibiting in vitro tubulin assembly. As indicated in ANS assay, Trl did not severely damage tubulin. It, as well, did not show gross inhibition of microtubule assembly. In cells, Trl didn’t inflict severe, direct damage to the microtubules. However, the microtubules reassembled after cold-induced disassembly showed defective, shrunken networks in the presence of Trl, suggesting that Trl prevents normal nucleation and reassembly of cellular microtubules. Further, Trl induced considerable microtubule acetylation indicating persistent suppression of microtubule dynamics. The formulation induced apoptosis at a concentration that is sufficient to perturb tubulin and damage microtubules.

Conclusion: Our results indicate the capacity of the chemical constituents of Trl to inhibit proliferation of a variety of cancer cells by disrupting the tubulin structure and to microtubule dynamicity. Trl, therefore, may be investigated further for its tubulin-targeted anticancer potential.