Effect of trichostatin A on anti HepG2 liver carcinoma cells: inhibition of HDAC activity and activation of Wnt/ß-Catenin signaling.

PURPOSE: To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms. MATERIALS AND METHODS: HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of ß-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for ß-catenin, HDAC1and HDAC3 was tested by q-PCR. ß-Catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit. RESULTS: Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/ G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was 6.22±0.25%, which increased to 7.17±0.20% and 18.1±0.42% in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of ß-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of ß-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of ß-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased. CONCLUSIONS: TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/ß-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.

Ginsenoside Rg1 induces apoptosis through inhibition of the EpoR-mediated JAK2/STAT5 signalling pathway in the TF-1/ Epo human leukemia cell line.

Ginsenoside Rg1 is one effective anticancer and antioxidant constituent of total saponins of Panax ginseng (TSPG), which has been shown to have various pharmacological effects. Our previous study demonstrated that Rg1 had anti-tumor activity in K562 leukemia cells. The aim of this study was designed to investigate whether Rg1 could induce apoptosis in TF-1/Epo cells and further to explore the underlying molecular mechanisms. Here we found that Rg1 could inhibit TF-1/Epo cell proliferation and induce cell apoptosis in vitro in a concentration and time dependent manner. It also suppressed the expression of EpoR on the surface membrane and inhibited JAK2/STAT5 pathway activity. Rg1 induced up-regulation of Bax, cleaved caspase-3 and C-PAPR protein and down-regulation of Bcl-2 and AG490, a JAK2 specific inhibitor, could enhance the effects of Rg1. Our studies showed that EpoR-mediated JAK2/STAT5 signaling played a key role in Rg1-induced apoptosis in TF-1/Epo cells. These results may provide new insights of Rg1 protective roles in the prevention a nd treatment of leukemia.

Down-regulation of phosphoglucose isomerase/autocrine motility factor enhances gensenoside Rh2 pharmacological action on leukemia KG1α cells.

AIMS AND BACKGROUND: Ginsenoside Rh2, which exerts the potent anticancer action both in vitro and in vivo, is one of the most well characterized ginsenosides extracted from ginseng. Although its effects on cancer are significant, the underlying mechanisms remain unknown. In this study, we sought to elucidate possible links between ginsenoside Rh2 and phosphoglucose isomerase/autocrine motility factor (PGI/AMF). METHODS: KG1α, a leukemia cell line highly expressing PGI/AMF was assessed by western blot analysis and reverse transcription- PCR (RT-PCR) assay after transfection of a small interfering (si)-RNA to silence PGI/AMF. The effect of PGI/ AMF on proliferation was measured by typan blue assay and antibody array. A cell counting kit (CCK)-8 and flow cytometry (FCM) were adopted to investigate the effects of Rh2 on PGI/AMF. The relationships between PGI/AMF and Rh2 associated with Akt, mTOR, Raptor, Rag were detected by western blot analysis. RESULTS: KG1α cells expressed PGI/AMF and its down-regulation significantly inhibited proliferation. The antibody array indicated that the probable mechanism was reduced expression of PARP, State1, SAPK/JNK and Erk1/2, while those of PRAS40 and p38 were up-regulated. Silencing of PGI/AMF enhanced the sensibility of KG1α to Rh2 by suppressing the expression of mTOR, Raptor and Akt. CONCLUSION: These results suggested that ginsenoside Rh2 suppressed the proliferation of KG1α, the same as down-regulation of PGI/AMF. Down-regulation of PGI/ AMF enhanced the pharmacological effects of ginsenoside Rh2 on KG1α by reducing Akt/mTOR signaling.