Smokeless Tobacco Extract (STE)-Induced Toxicity in Mammalian Cells is Mediated by the Disruption of Cellular Microtubule Network: A Key Mechanism of Cytotoxicity

Abstract

Smokeless tobacco usage is a growing public health problem worldwide. The molecular mechanism(s) underlying smokeless tobacco associated tissue damage remain largely unidentified. In the present study we have tried to explore the effects of aqueous extract of smokeless tobacco (STE) on tubulin-microtubule, the major cytoskeleton protein that maintains cells morphology and participates in cell division. Exposure to STE resulted in dose-dependent cytotoxicity in a variety of mammalian transformed cell lines such as human lung epithelial cells A549, human liver epithelial cells HepG2, and mouse squamous epithelial cells HCC7, as well as non-tumorogenic human peripheral blood mononuclear cells PBMC. Cellular morphology of STE-treated cells was altered and the associated disruption of microtubule network indicates that STE targets tubulin-microtubule system in both cell lines. Furthermore it was also observed that STE-treatment resulted in the selective degradation of cellular tubulin, whereas actin remains unaltered. In vitro, polymerization of purified tubulin was inhibited by STE with the IC50 value∼150 µg/ml and this is associated with the loss of reactive cysteine residues of tubulin. Application of thiol-based antioxidant N-acetyl cysteine (NAC) significantly abrogates STE-mediated microtubule damage and associated cytotoxicity in both A549 and HepG2 cells. These results suggest that microtubule damage is one of the key mechanisms of STE-induced cytotoxity in mammalian cells.

Global methylation profiles in buccal cells of long-term smokers and moist snuff consumers

“…The buccal cell methylation data from MSC suggests that microtubules, cell cycle and cell death are impacted in that cohort. This result is consistent with studies using smokeless tobacco extract, where toxicity was mediated by disruption of the microtubule network (Das et al 2013). Based on the few loci detected in the MSR methylation signature, which are exclusive to MSC, it is possible TR methylation signature is predominantly driven by changes from combustible tobacco use.…”

Dietary flavonoid tangeretin induces reprogramming of epithelial to mesenchymal transition in prostate cancer cells by targeting the PI3K/Akt/mTOR signaling pathway

“…Cultured prostate cancer cells (1x10 4 cells/ml) were treated with different concentrations of tangeretin (0, 25, 50 µM) for 24, 48 and 72 h. Following treatment, MTT solution (0.5 mg/ml; Thermo Fisher Scientific, Inc.) was added to each well followed by 100 µl of isopropanol (10%)/PBS and the resultant purple-blue formazan complex was measured using a Varian Cary 50MPR microplate reader (Akribis Scientific, Knutsford, UK) at an absorbance 570 nm, as described previously (26).…”

Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

“…There have been reports of cytotoxicity caused by smokeless tobacco products such as gutkha (Avti et al, 2010), khaini (Das et al, 2013), Sudanese toombak (Costea et al, 2010), American moist snuff (Misra et al, 2014), Swedish moist snuff (Coggins et al, 2012, Costea et al, 2010, Kentucky reference moist smokeless tobacco product (Lombard et al, 2010) and commercial chewing tobacco (Coppe et al, 2008). The present study is the first assessment of cytotoxicity induced by tobacco constituents of pituri as a means of investigating its potential carcinogenicity.…”

“…There have been reports of cytotoxicity caused by Indian manufactured smokeless tobacco such as gutka (Avti et al, 2010) and khaini (Das et al, 2013). Limited data is available on the toxicity of homemade traditional or indigenous smokeless tobacco products used around the world.…”

“…The sterile filtrate was then lyophilized and stored at -80°C to be used for chemical analysis and preparing stock concentrations in sterile media as required for experiments (Das et al, 2013). All containers and solutions used in the extraction steps were sterilised prior to use in order to prevent contamination.…”