RA-XII, a bicyclic hexapeptidic glucoside isolated from Rubia yunnanensis Diels, exerts antitumor activity by inhibiting protective autophagy and activating Akt-mTOR pathway in colorectal cancer cells.

The roots of Rubia yunnanensis Diels (Chinese name 'Xiao-Hong-Shen'), a traditional Chinese medicine native to Yunnan province (China), have a long history of use for treating several diseases, such as tuberculosis, rheumatism and cancers. A bicyclic hexapeptidic glucoside named RA-XII was isolated from R. yunnanensis, which has been reported to exert anti-inflammatory and antitumor activities. This study was designed to investigate the antitumor activity and potential mechanism of RA-XII on colorectal cancer (CRC) cell lines. Sulforhodamine B assay, clonogenic assay and cell cycle analysis were conducted to assess the anti-proliferative activity of RA-XII on CRC cells. GFP-LC3B plasmid transfection, MDC and AO staining assays, cathepsin activity assay, and siRNAs against several genes were used to investigate the effect of RA-XII on autophagy. Western blotting was used to examine the expression levels of proteins associated with cell cycle arrest, apoptosis and autophagy. Human CRC xenograft-bearing BALB/c nude mice were used to evaluate the antitumor effect of RA-XII in vivo. RA-XII showed favorable antineoplastic activity in SW620 and HT29 cells in vitro and in vivo. RA-XII did not induce apoptosis indicated by no obvious changes on mitochondrial membrane potential and apoptosis-related marker proteins in SW620 or HT29 cells. Treatment of RA-XII inhibited the formation of autophagosomes, which is implied by the GFP-LC3 fluorescent dots, MDC-stained autophagic vesicles and LC3 protein expression. It was indicated that RA-XII suppressed autophagy by regulating several signaling pathways including mTOR and NF-κB pathways. Pharmacological or genetic inhibition of autophagy could enhance the cytotoxicity of RA-XII while autophagy inducer could rescue RA-XII-induced cell death. Besides, RA-XII could increase the susceptibility of CRC cells to bortezomib. Our study demonstrated that RA-XII exerted antitumor activity independent of apoptosis, and suppressed protective autophagy by regulating mTOR and NF-κB pathways in SW620 and HT29 cell lines, which suggested that RA-XII is a key active ingredient for the cancer treatment of Rubia yunnanensis and possesses a promising prospect as an autophagy inhibitor for CRC therapy.

Wang J ，Wang J ，Li L ，Feng L ，Wang YR ，Wang Z ，Tan NH ... -  《-》

Rubioncolin C, a natural naphthohydroquinone dimer isolated from Rubia yunnanensis, inhibits the proliferation and metastasis by inducing ROS-mediated apoptotic and autophagic cell death in triple-negative breast cancer cells.

Rubia yunnanensis Diels is a traditional Chinese medicine that has diverse pharmacological activities, including antituberculosis, antirheumatism and anticancers. Rubioncolin C (RC), a natural naphthohydroquinone dimer isolated from the roots and rhizomes of R. yunnanensis Diels, has shown potent antitumor activity. However, the antitumor activity and its potential mechanism of RC in triple-negative breast cancer (TNBC) cell lines remained unclear. This study was aim to investigate the anti-proliferation and anti-metastasis activity as well as the potential mechanism of RC on triple-negative breast cancer cells in vitro and in vivo. The sulforhodamine B assay, colony formation assay and cell cycle analysis were used to determine the anti-proliferative activity of RC on TNBC. The anti-metastatic activity in vitro of RC was detected through the scratch wound assay, cell migration and invasion assays and gelatin zymography. The flow cytometry, JC-1, GFP-LC3B plasmid transfection, MDC, Lysotracker red and Carboxy-H2DCFDA, DHE, and MitoSOX™ Red staining were performed to investigate the effect of RC on apoptosis, autophagy and ROS level. The apoptosis inhibitor, autophagy inhibitors and ROS inhibitors were used to further verify the antitumor mechanism of RC. The protein levels related with cell cycle, apoptosis, and autophagy were examined with western blotting. In addition, the anti-tumor activity of RC in vivo was assessed in an experimental metastatic model. In the present study, RC suppressed the proliferation of TNBC cells in a time- and dose-dependent manner via regulating cell cycle. Further experiments showed that RC inhibited the migration and invasion of TNBC cells by downregulating MMPs and inhibiting EMT. Moreover, we demonstrated that RC induced obviously apoptotic and autophagic cell death, activated MAPK signaling pathway and inhibited mTOR/Akt/p70S6K and NF-κB signaling pathways. Furthermore, the excessive ROS was produced after treatment with RC. The antioxygen NAC and GSH could rescue the cell viability and reestablish the ability of cell metastasis, and inhibit the RC-induced apoptosis and autophagy. In a mice lung metastasis model of breast cancer, RC inhibited lung metastasis, and induced autophagy and apoptosis. These findings clarified the antitumor mechanism of RC on TNBC cell lines and suggested that RC is a key active ingredient for the cancer treatment of R. yunnanensis, which would help RC develop as a new potential chemotherapeutic agent for TNBC treatment.

Li L ，Wang J ，Feng L ，Fan J ，Wang J ，Tan N ，Wang Z ... -  《-》

Natural Cyclopeptide RA-XII, a New Autophagy Inhibitor, Suppresses Protective Autophagy for Enhancing Apoptosis through AMPK/mTOR/P70S6K Pathways in HepG2 Cells.

Song L ，Wang Z ，Wang Y ，Guo D ，Yang J ，Chen L ，Tan N ... -  《MOLECULES》

NF-κB and JNK mediated apoptosis and G(0)/G(1) arrest of HeLa cells induced by rubiarbonol G, an arborinane-type triterpenoid from Rubia yunnanensis.

Rubia yunnanensis is a medicinal plant mainly grown in Yunnan province in Southwest China, and its root named "Xiaohongshen" has been used as a herb in Yunnan for the treatment of cancers. Three major types of chemical components, Rubiaceae-type cyclopeptides, quinones, and triterpenoids, were identified from R. yunnanensis, in which some of compounds including rubiarbonol G (RG), a unique arboriane-type triterpenoid, showed cytotoxicity on cancer cells. But the cytotoxic mechanism of RG has not been reported. To investigate the cytotoxic mechanism of RG on cancer cells. RG was evaluated its cytotoxicity on 7 cancer cell lines by the SRB assay, and detected the effect on apoptosis and cell cycle arrest by Annexin V-FITC/PI apoptosis assay and DNA contents analysis. The expression and activity of apoptosis and cell cycle related proteins were also investigated by western blot and caspase activity assay. Furthermore, the effect of RG on NF-κB signaling was also tested by luciferase assay, western blot, and immunofluorescence staining. RG showed potent cytotoxicity on 7 human cancer cell lines, whose activity was attributed to apoptosis induction and G0/G1 arrest in HeLa cells. Results from the mechanism study showed that RG promoted the activation of ERK1/2 and JNK pathway in MAPK family, which in turn increased the expression of p53, thereby triggering the G0/G1 arrest through p53/p21/cyclin D1 signaling. Moreover, RG-mediated JNK activation down-regulated the expression of the anti-apoptotic protein Bcl-2, which caused the release of cytochrome c to the cytosol and activated the cleavage of caspase cascade and poly(ADP-ribose) polymerase, thereby inducing apoptosis in HeLa cells. In addition, RG was also found to inhibit the activation of NF-κB signaling by down-regulating the expression and attenuating the translocation to nucleus of NF-κB p65, by which the down-stream p53, cyclin D1, Bcl-2, and caspases were regulated, thereby triggering apoptosis and G0/G1 arrest in HeLa cells. These results indicated that RG induces mitochondria-mediated apoptosis and G0/G1 cell cycle arrest by activation of JNK signaling as well as inactivation of NF-κB pathway in HeLa cells, which suggests that RG is one of the key active ingredients accounting for the anti-tumor effect of R. yunnanensis.

Zeng GZ ，Wang Z ，Zhao LM ，Fan JT ，Tan NH ... -  《-》

Inhibition of fatty acid synthesis arrests colorectal neoplasm growth and metastasis: Anti-cancer therapeutical effects of natural cyclopeptide RA-XII.

Emerging evidence has shown that metabolism, in particular the synthesis of fatty acids, has great significance for growth and metastasis of colorectal neoplasm. The previous results showed that RA-XII, a natural cyclopeptide isolated from Rubia yunnanensis, inhibits tumor growth and metastasis by AMPK/mTOR/P70S6K pathway and PI3K/AKT/NF-κB pathway. But if or not lipid metabolism involves the antitumor mechanism of RA-XII is not clear. Herein the results indicated that RA-XII reduced the cell motility by decreasing the expressions of β-catenin and β-catenin dependent proteins CD44 and MMP7 in HCT116 cells. Then RA-XII effectively reduced fatty acids levels by decreasing the expression of SREBP-1 and inhibiting the expressions of de novo fatty acid synthesis proteins FASN and SCD. Moreover the decreased cell motility caused by RA-XII was attenuated with the SREBP-1 knockdown. In addition, the in vivo experiments also demonstrated that RA-XII inhibited tumor growth and metastasis via restraining lipogenesis in colorectal neoplasm mouse models. Taken together, these results indicated that RA-XII suppressed the colorectal neoplasm growth and metastasis by inhibition of lipogenesis depended on SREBP-1 suppression.

Wang Y ，Guo D ，He J ，Song L ，Chen H ，Zhang Z ，Tan N ... -  《-》