8%, 12. 1%, 45. 0%, 65. 6% in SW620 and 2. 6%, 10. 0%, 22. 7%, 30. 6% in MDA MB 231 after treatment with various concentra tions of hirsutanol A for 72 h. These data indicated that hirsutanol A could induce apoptosis in a dose dependent manner. Furthermore, with Western blot ana lysis we Vandetanib hypothyroidism found that pro caspase 3 was cleaved to form a 17KDa fragment and PARP was cleaved into an 89KD fragment. These results suggest that hirsuta nol A significantly induced apoptosis in SW620 and MDA MB 231 cells. Hirsutanol A induced mitochondrial independent accumulation of intrinsic ROS Previous studies had confirmed that hirsutanol A could induce autophagical cell death by causing an accumula tion of ROS level in human hepatocellular carcinoma cells.

As reactive o ygen species mainly include hydrogen pero ide H2O2 and supero ide anion radical O, in the present study, the effect of hirsutanol A on cellular supero ide and hydrogen pero ide level was measured in SW620 cells and MDA MB 231 cells. The level of supero ide and hydrogen pero ide in cancer cells were analyzed by flow cytometry using DHE and CM H2DCF DA as fluorescent probe. There was no significant change in DHE fluorescence after treat ment with hirsutanol A for 3h but a remarkable increase of CM H2DCF DA fluorescence in a dose dependent fashion, suggesting that the ROS induced by hirsutanol A were mainly hydrogen pero ide instead of supero ide. Since accumulation of ROS was mainly caused by the increase of mitochondrial respira tory chain production and decrease of capability for scavenging ROS by the redo system, we thereby investi gated whether hirsutanol A induced increase of ROS was related to mitochondria.

C6F cells, a clone of rho 0 cells derived from HL 60 cells and parental HL 60, were used to detect whether hirsutanol A induced accumulation of ROS production was mitochondrial respiratory chain related. Results showed that both the parental HL 60 and roh 0 cells were highly sensitive to hirsutanol A. C2, C8 cells and parental Raji cells also showed similar effect after treatment with hirsutanol A, which suggested that the accumulation of ROS production were mitochondrial respiratory chain independent. Preventing ROS accumulation by antio idant agent NAC reduced hirsutanol A induced apoptosis E cessive ROS could lead to mitochondrial membrane damage, the release of cytochrome c from mitochondrial and cell apoptosis.

The evidences of apoptosis and up regulation of ROS levels in cells treated with hirsutanol A prompted us to investigate whether up regulation of ROS would resulted in apoptosis. The increase of ROS levels in hirsutanol A treated cancer cells was prevented by pre incubation with NAC for 1h. Cell growth inhib ition was analyzed Dacomitinib using MTT assay and Anne inV positive cells were detected by Anne in V PI double staining assay. The results showed that hirsutanol A induced Anne inV positive cells and growth inhibition were significantly selleckbio reduced.