Microtubules and Actin Cytoskeleton of Cryptococcus neoformans as Targets for Anticancer Agents to Potentiate a Novel Approach for New Antifungals

Abstract

Background: We investigated the targeting of microtubules (MT) and F-actin cytoskeleton (AC) of the human pathogenic yeast Cryptococcus neoformans with agents for cancer therapy, in order to examine whether this yeast cytoskeleton could become a new antifungal target for the inhibition of cell division. Methods: Cells treated with 10 cytoskeleton inhibitors in yeast extract peptone dextrose medium were investigated by phase-contrast and fluorescence microscopy, and growth inhibition was estimated by cell counts using a Bürker chamber and measuring absorbance for 6 days. Results: Docetaxel, paclitaxel, vinblastine sulfate salt, cytochalasin D and chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] did not inhibit proliferation. The MT inhibitors methyl benzimidazole-2-ylcarbamate (BCM), nocodazole, thiabendazole (TBZ) and vincristine (VINC) disrupted MT and inhibited mitoses, but anucleated buds emerged on cells that increased in size, vacuolated and seemed to die after 2 days. The response of the cells to the presence of the actin inhibitor latrunculin A (LA) included the disappearance of actin patches, actin cables and actin rings; this arrested budding and cell division. However, in 3-4 days, resistant budding cells appeared in all 5 inhibitors. Disruption of the MT and AC and inhibition of cell division and budding persisted only when the MT and AC inhibitors were combined, i.e. VINC + LA, BCM + LA or TBZ + LA. Conclusion: The MT and AC of C. neoformans are new antifungal targets for the persistent inhibition of cell division by combined F-actin and MT inhibitors.

Fenbendazole Controls In Vitro Growth, Virulence Potential, and Animal Infection in the Cryptococcus Model

“…In addition, the effects of fenbendazole on ␤-tubulin staining were apparently more drastic in C. gattii R265 than in C. neoformans H99, as concluded from the weaker signals of ␤-tubulin staining in the former species. These results were similar to those obtained with the control drug vinblastine, which binds tubulin and inhibits the assembly of microtubules (26), indicating that the mechanism of antifungal activity of fenbendazole against Cryptococcus is similar to that described for human parasites.…”

Lysosome Inhibitors Enhance the Chemotherapeutic Activity of Doxorubicin in HepG2 Cells

“…Cells were washed 3 times with PBS and incubated for a further 30 min at room temperature with fluorophore-conjugated secondary antibodies diluted in PBS/bovine serum albumin [17]. Coverslips were mounted in Mowiol, allowed to dry, and analyzed using an Olympus FV1000 confocal microscope with the DT72 camera and FV10-ASW 3.0 viewer software [18]. …”