1β-OH-arenobufagin induces mitochondrial apoptosis in hepatocellular carcinoma through the suppression of mTOR signaling pathway.

Chansu, dried secretions from Bufonidae, has long been used for cancer treatment as a traditional Chinese medicine. In searching for effective anti-hepatoma agents from Chansu, our preliminary drug screening found that a bufadienolide, namely 1β-hydroxyl-arenobufagin (1β-OH-ABF), displays anti-hepatoma activities. However, the anti-hepatoma effects and molecular mechanisms of 1β-OH-ABF have not been defined. To evaluate the anti-hepatoma activity of 1β-OH-ABF against liver cancer Hep3B and HepG2 cells in vitro and in vivo, as well as explore the underlying mechanisms. The anti-proliferative effects of 1β-OH-ABF on liver cancer Hep3B, HepG2, HuH7, SK-HEP-1 and normal hepatocyte LO2 cells were examined by MTT assay and colony formation assay. Hoechst 33258 staining and Annexin V-FITC/PI staining assay were used to analyze apoptosis induced by 1β-OH-ABF. The collapse of the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining assay. Western blotting was used to examine the expression levels of targeted proteins. The role of mTOR in 1β-OH-ABF-induced apoptosis was investigated using small interfering RNA (siRNA) transfection. Zebrafish xenograft model was established to evaluate the anti-hepatoma effects of 1β-OH-ABF in vivo. We found that 1β-OH-ABF inhibits the proliferation of Hep3B, HepG2, HuH7, SK-HEP-1 cells but has little cytotoxicity towards LO2 cells. 1β-OH-ABF induces mitochondria dysfunction and triggers mitochondria apoptotic pathway, which is accompanied by the loss of ΔΨm, upregulation and translocation of Bax, as well as cleavages of caspase-9, caspase-3 and PARP. Mechanistically, 1β-OH-ABF markedly decreases the expression level of p-AKT/AKT and p-mTOR (Ser2248 and Ser2481)/mTOR in a time-dependent manner. Inhibition of mTOR by siRNA strengthens 1β-OH-ABF-mediated apoptosis. Critically, 1β-OH-ABF shows a marked in vivo anti-hepatoma effect on human Hep3B cell xenografts in zebrafish model. 1β-OH-ABF induces mitochondrial apoptosis through the suppression of mTOR signaling in vitro and in vivo, indicating that 1β-OH-ABF may serve as a potential agent for the treatment of liver cancer.

Deng LJ ，Lei YH ，Quan JY ，Li BJ ，Zhang DM ，Tian HY ，Chen Y ，Zhang EX ，Chen L ，Ye WC ，Ning WM ，Yu LZ ，Liu JS ... -  《-》

Arenobufagin, a natural bufadienolide from toad venom, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/mTOR pathway.

Zhang DM ，Liu JS ，Deng LJ ，Chen MF ，Yiu A ，Cao HH ，Tian HY ，Fung KP ，Kurihara H ，Pan JX ，Ye WC ... -  《-》

Aloperine induces apoptosis and G2/M cell cycle arrest in hepatocellular carcinoma cells through the PI3K/Akt signaling pathway.

Hepatocellular carcinoma (HCC) ranks third among the most common causes of cancer-related deaths worldwide. The chemotherapy for HCC is still insufficient, so far. In searching for effective anti-HCC agents from traditional Chinese medicine, we discovered that aloperine (ALO), a quinolizidine alkaloid from Sophora alopecuroides L., exerts anti-HCC activities. However, the effects of ALO on HCC have been rarely studied, and its underlying mechanisms remain unknown. This study aims to evaluate the anti-HCC activities of ALO and explore its underlying mechanisms. MTT assay and colony formation assay were used to investigate the anti-proliferative effects of ALO on human HCC Hep3B and Huh7 cells. Hoechst 33258 staining was used to observe the morphological changes of cells after ALO treatment. Flow cytometry was used to analyze apoptosis induction, the collapse of the mitochondrial membrane potential and cell cycle distribution. Western blotting was used to examine the expression levels of proteins associated with apoptosis and cell cycle arrest, and key proteins in the PI3K/Akt signaling pathway. Small interfering RNA (siRNA) transfection was used to investigate the role of Akt in ALO-induced apoptosis and cell cycle arrest. Zebrafish tumor model was used to evaluate the anti-HCC effects of ALO in vivo. ALO inhibited the proliferation of Hep3B and Huh7 cells. ALO induced apoptosis in HCC cells, which was accompanied by the loss of mitochondrial potential, the release of cytochrome c into cytosol, as well as the increased cleavages of caspase-9, caspase-3 and PARP. Moreover, ALO induced G2/M cell cycle arrest by downregulating the expression levels of cdc25C, cdc2 and cyclin B1. In addition, ALO inhibited activation of the PI3K/Akt signaling pathway by decreasing the expression levels of p110α, p85, Akt and p-Akt (Ser473). Further study showed that inhibition of Akt by siRNA augmented ALO-mediated apoptosis and G2/M cell cycle arrest in HCC cells. Critically, ALO inhibited the growth of Huh7 cells in vivo. We first demonstrated that ALO induced apoptosis and G2/M cell cycle arrest in HCC cells through inhibition of the PI3K/Akt signaling pathway. This study provides a rationale for ALO as a potential chemotherapeutic agent for HCC.

Liu JS ，Huo CY ，Cao HH ，Fan CL ，Hu JY ，Deng LJ ，Lu ZB ，Yang HY ，Yu LZ ，Mo ZX ，Yu ZL ... -  《-》

The lipid homeostasis regulation study of arenobufagin in zebrafish HepG2 xenograft model and HepG2 cells using integrated lipidomics-proteomics approach.

Arenobufagin (ArBu) is an important anti-tumor ingredient of Chan'su which has long been used as traditional Chinese medicine in clinic for tumor therapy in China. The purpose of our study is to investigate the lipid homeostasis regulation effects of ArBu on zebrafish model of liver cancer and hepatoma cells, and to provide a reference for further clarifying its active mechanisms. The zebrafish xenograft model was established by injecting HepG2 cells stained with CM-Dil red fluorescent dye. Both the xenograft model and HepG2 cells were used to evaluate the anti-hepatoma activity of ArBu. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was the main method to study lipidomics, proteomics and the semiquantification of endogenous metabolites. Bioinformatics was used as an assistant tool to further explore the antitumor mechanism of ArBu. The lipidomics analysis revealed that ArBu caused differential lipids changes in a dose-dependent manner, including PCs, PEs, TGs, SMs, DGs, Cer and PA. PCs, PEs, SMs and TGs were markedly altered in both two models. The influence of glycerophospholipid metabolism was the major and commonly affected pathway. Notably, DGs and Cer were significantly changed only in HepG2 cells. Furthermore, the proteomics research in HepG2 cells fished the target proteins related to lipid homeostasis abnormalities and tumor suppression. ArBu reduced the expression of 65 differential proteins associated with the lipid metabolism, apoptosis and autophagy, such as LCLAT1, STAT3, TSPO and RPS27. Meanwhile, 7 amino acids of 29 determined metabolites were significantly changed, including tyrosine, glutamate, glutamine, leucine, threonine, arginine and isoleucine. ArBu has a significant anti-hepatoma effect in vitro and a therapeutic effect on zebrafish xenograft model. It regulated the lipid homeostasis. Activated SM synthase and arginine deiminase, inhibited sphingomyelinase, amino acid supply and JAK-STAT3 signaling pathway, and the affected glycerophospholipid metabolism might explain these results.

Zhao LJ ，Zhao HY ，Wei XL ，Guo FF ，Wei JY ，Wang HJ ，Yang J ，Yang ZG ，Si N ，Bian BL ... -  《-》

Bufotalin from Venenum Bufonis inhibits growth of multidrug resistant HepG2 cells through G2/M cell cycle arrest and apoptosis.

Venenum Bufonis, a traditional Chinese medicine, is widely used in the treatment of liver cancer in modern Chinese medical practices. In our search for anti-hepatoma constituents in Venenum Bufonis, bufotalin, bufalin, telocinobufagin and cinobufagin were obtained. Bufotalin was the most potent active compound among these four bufadienolides, and it exerted stronger inhibitory effect on the viability of doxorubicin-induced multidrug resistant liver cancer cells (R-HepG2) than that of their parent cells HepG2. Structure-activity relationship analysis indicated that the acetyl group linked to C-16 of bufadienolides might be useful for increasing anti-hepatoma activity. Further mechanistic studies revealed that bufotalin treatment induced cell cycle arrest at G(2)/M phase through down-regulation of Aurora A, CDC25, CDK1, cyclin A and cyclin B1, as well as up-regulation of p53 and p21. Bufotalin treatment also induced apoptosis which was accompanied by decrease in mitochondrial membrane potential, increases in intracellular calcium level and reactive oxygen species production, activations of caspase-9 and -3, cleavage of poly ADP-ribose polymerase (PARP) as well as changes in the expressions of bcl-2 and bax. It was also found that the inhibition of Akt expression and phosphorylation was involved in apoptosis induction, and specific Akt inhibitor LY294002 or siRNA targeting Akt can synergistically enhanced bufotalin-induced apoptosis. In vivo study showed that bufotalin significantly inhibited the growth of xenografted R-HepG2 cells, without body weight loss or marked toxicity towards the spleen. These results indicate that bufotalin has a promising potential to become a novel anti-cancer agent for the treatment of liver cancer with multidrug resistance.

Zhang DM ，Liu JS ，Tang MK ，Yiu A ，Cao HH ，Jiang L ，Chan JY ，Tian HY ，Fung KP ，Ye WC ... -  《-》