Hemistepsin a Induces Apoptosis of Hepatocellular Carcinoma Cells by Downregulating STAT3.

Hemistepta lyrata (Bunge) Bunge is a biennial medicinal plant possessing beneficial effects including anti-inflammation, and hemistepsin A (HsA) isolated from H. lyrata has been known as a hepatoprotective sesquiterpene lactone. In this report, we explored the cytotoxic effects of H. lyrata on hepatocellular carcinoma (HCC) cells and investigated the associated bioactive compounds and their relevant mechanisms. From the viability results of HCC cells treated with various H. lyrata extracts, HsA was identified as the major compound contributing to the H. lyrata-mediated cytotoxicity. HsA increased expression of cleaved PARP and cells with Sub-G1 phase, Annexin V binding, and TUNEL staining, which imply HsA induces apoptosis. In addition, HsA provoked oxidative stress by decreasing the reduced glutathione/oxidized glutathione ratio and accumulating reactive oxygen species and glutathione-protein adducts. Moreover, HsA inhibited the transactivation of signal transducer and activator of transcription 3 (STAT3) by its dephosphorylation at Y705 and glutathione conjugation. Stable expression of a constitutive active mutant of STAT3 prevented the reduction of cell viability by HsA. Finally, HsA enhanced the sensitivity of sorafenib-mediated cytotoxicity by exaggerating oxidative stress and Y705 dephosphorylation of STAT3. Therefore, HsA will be a promising candidate to induce apoptosis of HCC cells via downregulating STAT3 and sensitizing conventional chemotherapeutic agents.

Hemistepsin A alleviates liver fibrosis by inducing apoptosis of activated hepatic stellate cells via inhibition of nuclear factor-κB and Akt.

Hemistepsin A (HsA), isolated from Hemistepta lyrata (Bunge) Bunge, has the ability to ameliorate hepatitis in mice. However, the effects of H. lyrata and HsA on other types of liver disease have not been explored. In this report, we investigated the effects of H. lyrata and HsA on liver fibrosis and the underlying molecular mechanisms in activated hepatic stellate cells (HSCs). Based on cell viability-guided isolation, we found HsA was the major natural product responsible for H. lyrata-mediated cytotoxicity in LX-2 cells. HsA significantly decreased the viability of LX-2 cells and primary activated HSCs, increased the binding of Annexin V, and altered the expression of apoptosis-related proteins, suggesting that HsA induces apoptosis in activated HSCs. HsA reduced the phosphorylation of IKKε and the transactivation of nuclear factor-κB (NF-κB). Moreover, HsA decreased the phosphorylation of Akt and its downstream signaling molecules. Transfection experiments suggested that inhibition of NF-κB or Akt is essential for HsA-induced apoptosis of HSCs. In a CCl4-induced liver fibrosis model, HsA administration significantly decreased ALT and AST activities. Furthermore, HsA attenuated CCl4-mediated collagen deposits and profibrogenic genes expression in hepatic tissue. Thus, HsA may serve as a natural product for managing liver fibrosis through inhibition of NF-κB/Akt-dependent signaling.

Hemistepsin A Inhibits Cell Proliferation and Induces G0/G1-Phase Arrest, Cellular Senescence and Apoptosis Via the AMPK and p53/p21 Signals in Human Hepatocellular Carcinoma.

Hemistepsin A (HsA), a natural sesquiterpene lactone isolated from Hemistepta lyrata, has been known as a wide range of anti-tumor effects. The aim of this study was to determine whether HsA suppresses hepatocellular carcinoma (HCC) and to figure out the cellular signaling pathways involved in the anti-HCC activities by experiments using the Huh7 cells (a human HCC cell line) and a xenograft HCC model. In this study, HsA completely inhibited HCC cell proliferation, presumably because it induced G0/G1 cell cycle arrest and mitochondrial-related apoptosis. HsA up-regulated p53, p21, cleaved caspase-3 and cleaved PARP (poly (ADP-ribose) polymerase), but reduced cyclin D, CDK6 and Bcl-2 expressions, and it disrupted mitochondrial membrane potential (ΔΨm). Moreover, phosphorylation of AMP-activated protein kinase (AMPK) was increased by HsA as did the resveratrol and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, positive controls). Inhibition of AMPK by using compound C, a competent inhibitor of AMPK, attenuated the loss of ΔΨm, p53 up-regulation and cellular senescence. The efficacy of HsA to reduce HCC cell proliferation, compared to that of other known anti-HCC agents, appears to be similar or slightly better. The anti-tumor effect of HsA was also determined in mice, showing reduced growth of xenografted tumors with no weight loss. Overall, the results suggest that HsA should be considered as a candidate anti-HCC drug.