Liver cancer is a progressive, irreversible and aggressive malignant disease, which has no effective chemotherapeutic drugs. RA-XII, a natural cyclopeptide isolated from the traditional Chinese medicine Rubia yunnanensis, exerts anti-cancer and anti-inflammatory activities. This work aimed to investigate the effects of RA-XII on a hepatic tumor and its underlying mechanisms in human hepatoma HepG2 cells. The results showed that RA-XII effectively inhibited the proliferation of HepG2 cells. Consistently, RA-XII significantly induced apoptosis in HepG2 cells by decreasing the expression of caspase 3, 8, 9, and promoting the Cleavage of PARP. Moreover, RA-XII-induced apoptosis was attenuated in the presence of apoptosis inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl keton, suggesting that RA-XII induced apoptosis-cell-death in HepG2 cells. Furthermore, autophagy-related proteins and mRNA levels were dramatically reduced after RA-XII treatment. Meanwhile, we observed that autophagy inhibitor chloroquine could enhance apoptosis in RA-XII-treated HepG2 cells, indicating that autophagy played a protective role in HepG2 cells and RA-XII might inhibit protective autophagy. Further analysis showed that RA-XII inhibited AMPK phosphorylation and led to the mTOR/P70S6K pathway activation, suggesting that RA-XII inhibited autophagy through AMPK/mTOR/P70S6K pathways. This study demonstrated that RA-XII promoted apoptosis and inhibited protective autophagy through AMPK/mTOR/P70S6K pathways in HepG2 cells. In conclusion, these findings suggest that RA-XII might potentially be a candidate as an autophagy inhibitor agent for further therapy of liver cancer.

The Hippo pathway restricts organ size during development and its inactivation plays a crucial role in cancer. Yes-associated protein (YAP) and its paralog transcriptional coactivator with PSD-95/Dlg/ZO-1 (PDZ)-binding motif (TAZ) are transcription co-activators and effectors of the Hippo pathway mediating aberrant enlargement of organs and tumor growth upon Hippo pathway inactivation. It has been demonstrated that genetic inactivation of YAP could be an effective approach to inhibit tumorigenesis. In order to identify pharmacological inhibitors of YAP, we screened a library of 52,683 compounds using a YAP-specific reporter assay. In this screen we identified cyclopeptide RA-V (deoxybouvardin) as a specific inhibitor of YAP and TAZ but not other reporters. Unexpectedly, later experiments demonstrated that RA-V represses the protein but not mRNA levels of YAP target genes. Nevertheless, RA-V strongly blocks liver enlargement induced by Mst1/2 knockout. Furthermore, RA-V not only inhibits liver tumorigenesis induced by YAP activation, but also induces regression of established tumors. We found that RA-V inhibits dedifferentiation and proliferation, while inducing apoptosis of hepatocytes. Furthermore, RA-V also induces apoptosis and inhibits proliferation of macrophages in the microenvironment, which are essential for YAP-induced tumorigenesis. RA-V is thus a drug candidate for cancers involving YAP/TAZ activation.

Lipogenesis plays a critical role in the growth and metastasis of tumors, which is becoming an attractive target for anti-tumor drugs. RA-XII, one of the cyclopeptide glycosides isolated from Rubia yunnanensis, exerts anti-tumor effects on liver cancer. However, the underlying mechanisms are not clear. In the present study, the effects of RA-XII on lipogenesis were evaluated and the underlying mechanisms were investigated. The results indicated that RA-XII strongly inhibited tumor growth and lipogenesis (triglycerides and lipid droplets) in HepG2 cells, and the expression of key factors involved in lipogenesis (SREBP, SCD, FASN) was also obviously downregulated. Further investigation showed that the anti-tumor effects of RA-XII were attenuated by SREBP knockdown. Moreover, RA-XII downregulated the expression of SREBP cleavage-activating protein (SCAP), an upstream regulator of SREBP, and siRNA of SCAP prevented its restrained effects on tumor growth and lipogenesis. In addition, the in vivo experiment showed that RA-XII strongly restrained the lipogenesis and growth of liver tumor in nude mice xenograft model. Taken together, these results indicate that RA-XII suppresses the liver cancer growth by inhibition of lipogenesis via SCAP-dependent SREBP suppression. The findings reveal the potentials of RA-XII to be used in a novel therapeutic approach for treating liver cancer.

Naphthohydroquinone dimers isolated from Rubia plants have garnered more attention due to their distinctive chemical structures and intriguing bioactivities. In our previous studies, we obtained ten naphthohydroquinone dimers containing seven novel ones and found that most of them possessed anti-tumor activities, especially rubioncolin C. However, the underlying mechanism remains unknown. In this study, we focused on rubioncolin C and found that it could inhibit the growth of cancer cell lines with IC₅₀ values between 1.14 and 9.93 μM. Further experiments demonstrated that rubioncolin C induced apoptotic and autophagic cell death and inhibited the Akt/mTOR/P70S6K signaling pathway in HCT116 and HepG2 cells. Moreover, we observed that rubioncolin C inhibited the TNF-α- and LPS-induced NF-κB activation upstream of the p65 protein, which contributed to rubioncolin C-induced cell death. Rubioncolin C could also prevent LPS-induced endotoxin shock in vivo. Moreover, rubioncolin C suppressed tumor growth through inducing apoptosis and autophagy and inactivating NF-κB in vivo. These findings clarify the anti-tumor mechanism of rubioncolin C using biochemical techniques and pharmacological models and might contribute to the future development of rubioncolin C as a new therapeutic agent for treating cancer.