The withanolid compounds (Tubocapsanolide A, B, and C) were purify from Tubocapsium anomalum and shown with cell growth inhibitory property. However, the molecular mechanisms of withanolide type compounds on the cell have not been fully clarified. Tubocapsanolide A, B, and C (TA, TB, and TC) of antiproliferative activity on human colorectal adenocarcinoma cells, HT29 and HCT116, were tested and the inhibitory concentration of 50% cell viability (IC50) for these compounds was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. We found that TA has the best inhibitory effect in these compounds. Therefore we utilized TA to study the mechanism of cell toxicity. The caspase 3 and PARP cleavage experiment results indicated that TA induced apoptosis was time and dose dependent. Cells with functional p53 (HCT116) are more sensitive to TA compared to the mutant p53 cells (HT29). With low dose TA treatment, HCT116 and HT29 are arrested at G1 and G2/M phase respectively. We also found that TA induced sub-G1 accumulation and reactive oxygen species (ROS) release with flow cytometry analysis. Pretreatment of N-Acetyl-L-cysteine (NAC), an antioxidant agent, can reverse the antiproliferation effect by TA. Our results indicated that TA can induce cell apoptosis and intracellular ROS generation. The ROS triggers cells damage and decreases the mitochondria membrane potential and thus induce cell apoptosis.

In spite of recent therapeutic advances, multiple myeloma (MM) remains a malignancy with very low curability. This has been partly attributed to the existence of a drug-resistant subpopulation known as cancer stem cells (CSCs). MM-CSCs are equipped with the necessary tools that render them highly resistant to virtually all conventional therapies. In this study, the growth inhibitory effects of withanolide D (WND), a steroidal lactone isolated from Withania somnifera, on drug-sensitive tumoral plasma cells and drug-resistant MM cells have been investigated. In MTT/XTT assays, WND exhibited similar cytostatic effects between drug-resistant and drug-sensitive cell lines in the nM range. WND also induced cell death and apoptosis in MM-CSCs and RPMI 8226 cells, as examined by the calcein/ethidium homodimer and annexin V/propidium iodide stainings, respectively. To determine whether P-glycoprotein (P-gp) efflux affected the cytostatic activity of WND, P-gp was inhibited with verapamil and results indicated that the WND cytostatic effect in MM-CSCs was independent of P-gp efflux. Furthermore, WND did not increase the accumulation of the fluorescent P-gp substrate rhodamine 123 in MM-CSCs, suggesting that WND may not inhibit P-gp at the tested relevant doses. Therefore, the WND-induced cytostatic effect may be independent of P-gp efflux. These findings warrant further investigation of WND in MM-CSC animal models.