Liang-Liang Bai

Saxifragifolin D induces the interplay between apoptosis and autophagy in breast cancer cells through ROS-dependent endoplasmic reticulum stress

Breast cancer is the leading cause of cancer death among females, and novel chemotherapeutic drugs for treating breast cancer are needed urgently. Saxifragifolin D (SD) was isolated by our group from Androsace umbellata which is commonly used to treat solid tumor. In this study, we evaluated its growth inhibitory effect on breast cancer cells and explored the underlying molecular mechanisms. Our results showed that SD inhibited the growth of both MCF-7 and MDA-MB-231 cells significantly. Mechanistic studies demonstrated that SD induced apoptosis through mitochondrial apoptotic pathway. Evidence of SD-induced autophagy included the occurrence of autophagic vacuoles, up-regulation of LC3-II, Beclin1 and Vps34. Inhibition of autophagy by bafilomycin A1 or Beclin1 siRNA pretreatment decreased the ratio of apoptosis, indicating that autophagy induction contributes to apoptosis and is required for the latter. SD was also found to induce endoplasmic reticulum stress, accompanied by ROS production, increase of intracellular calcium and up-regulation of Bip, IRE1α and XBP-1s. Inhibition of endoplasmic reticulum stress by N-acetyl-l-cysteine, tauroursodeoxycholic acid or IRE1α siRNA pretreatment could suppress both apoptosis and autophagy. Besides, increases in CHOP, calnexin, calpain, p-JNK and p-Bcl-2 were followed by subsequent dissociation of Beclin1 from Bcl-2, further suggesting endoplasmic reticulum stress to be the common signaling pathway shared by SD-induced apoptosis and autophagy. In conclusion, SD inhibits breast cancer cell growth and induces interplay between apoptosis and autophagy through ROS-mediated endoplasmic reticulum stress. It will provide molecular bases for developing SD into a drug candidate for the treatment of breast cancer.

Virosaines A and B, two new birdcage-shaped Securinega alkaloids with an unprecedented skeleton from Flueggea virosa.

Two new Securinega alkaloids, virosaines A (1) and B (2), were isolated from the twigs and leaves of Flueggea virosa. The structures and absolute configurations were elucidated by means of NMR, X-ray diffraction, and CD analyses. Compounds 1 and 2 represent the first examples of Securinega alkaloids bearing a 7-oxa-1-azabicyclo[3.2.1]octane ring system, whose plausible biogenetic pathways were also proposed.

Isocoumarins from American cockroach (Periplaneta americana) and their cytotoxic activities.

Four new isocoumarins (1-4), along with three known ones (5-7), were isolated from the 70% ethanol extract of the whole body of the traditional Chinese insect medicine, American cockroach (Periplaneta americana). The structures with absolute configurations of new compounds were elucidated by extensive spectroscopic methods in combination with X-ray diffraction experiment and CD analyses. Compounds 3-5 showed significant cytotoxic activities in HepG2 and MCF-7 cells with IC50 values in the ranges 6.41-23.91 μM and 6.67-39.07 μM, respectively.

Ilelic acids A and B, two unusual triterpenes with a seven-membered ring from Ilex latifolia.

Two unusual triterpenes, ilelic acids A (1) and B (2), together with their biosynthetic related compounds ilelic acids C (3) and D (4) were isolated from the leaves of Ilex latifolia. Their structures with absolute configurations were elucidated by spectroscopic analysis and modified Moshers method. The plausible biogenetic pathway of 1 and 2 is proposed. These triterpenes exhibited a potent inhibitory effect on MCF-7 and MDA-MB-231 cells.

Arenobufagin intercalates with DNA leading to G 2 cell cycle arrest via ATM/ATR pathway

Arenobufagin, a representative bufadienolide, is the major active component in the traditional Chinese medicine Chansu. It possesses significant antineoplastic activity in vitro. Although bufadienolide has been found to disrupt the cell cycle, the underlying mechanisms of this disruption are not defined. Here, we reported that arenobufagin blocked the transition from G2 to M phase of cell cycle through inhibiting the activation of CDK1-Cyclin B1 complex; The tumor suppressor p53 contributed to sustaining arrest at the G2 phase of the cell cycle in hepatocellular carcinoma (HCC) cells. Moreover, arenobufagin caused double-strand DNA breaks (DSBs) and triggered the DNA damage response (DDR), partly via the ATM/ATR-Chk1/Chk2-Cdc25C signaling pathway. Importantly, we used a synthetic biotinylated arenobufagin-conjugated chemical probe in live cells to show that arenobufagin accumulated mainly in the nucleus. The microscopic thermodynamic parameters measured using isothermal titration calorimetry (ITC) also demonstrated that arenobufagin directly bound to DNA in vitro. The hypochromicity in the UV-visible absorption spectrum, the significant changes in the circular dichroism (CD) spectrum of DNA, and the distinct quenching in the fluorescence intensity of the ethidium bromide (EB)-DNA system before and after arenobufagin treatment indicated that arenobufagin bound to DNA in vitro by intercalation. Molecular modeling suggested arenobufagin intercalated with DNA via hydrogen bonds between arenobufagin and GT base pairs. Collectively, these data provide novel insights into arenobufagin-induced cell cycle disruption that are valuable for the further discussion and investigation of the use of arenobufagin in clinical anticancer chemotherapy.

Hellebrigenin induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells through inhibition of Akt

Hellebrigenin, one of bufadienolides belonging to cardioactive steroids, was found in skin secretions of toads and plants of Helleborus and Kalanchoe genera. In searching for natural constituents with anti-hepatoma activities, we found that hellebrigenin, isolated from traditional Chinese medicine Venenum Bufonis, potently reduced the viability and colony formation of human hepatocellular carcinoma cells HepG2, and went on to explore the underlying molecular mechanisms. Our results demonstrated that hellebrigenin triggered DNA damage through DNA double-stranded breaks and subsequently induced cell cycle G2/M arrest associated with up-regulation of p-ATM (Ser(1981)), p-Chk2 (Tyr(68)), p-CDK1 (Tyr(15)) and Cyclin B1, and down-regulation of p-CDC25C (Ser(216)). It was also found that hellebrigenin induced mitochondrial apoptosis, characterized by Bax translocation to mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c into cytosol and sequential activation of caspases and PARP. In addition, Akt expression and phosphorylation were inhibited by hellebrigenin, whereas Akt silencing with siRNA significantly blocked cell cycle arrest but enhanced apoptosis induced by hellebrigenin. Activation of Akt by human insulin-like growth factor I (hIGF-I) could obviously attenuate hellebrigenin-induced cell death. In summary, our study is the first to report the efficacy of hellebrigenin against HepG2 and elucidated its molecular mechanisms including DNA damage, mitochondria collapse, cell cycle arrest and apoptosis, which will contribute to the development of hellebrigenin into a chemotherapeutic agent in the treatment of liver cancer.

Isolation, chemotaxonomic significance and cytotoxic effects of quassinoids from Brucea javanica.

A new quassinoid, bruceene A (1) along with seventeen known quassinoids (2-18) was isolated from the fruits of Brucea javanica. The structure of 1 was elucidated by extensive spectroscopic methods, and was further confirmed by single-crystal X-ray diffraction analysis. Isolation of similar quassinoids 1-3 as those in genus Ailanthus from genus Brucea, indicated the close chemotaxonomic relationship between these two genera, which further supported the phylogenetic study by DNA analysis. Compounds 5, 7, 10 and 12 with a 3-hydroxy-3-en-2-one moiety showed potent inhibitory activities against the MCF-7 and MDA-MB-231 cells with IC50 values in the ranges 0.063-0.182 μM and 0.081-0.238 μM, respectively; while glycosidation at 3-OH significantly decreased the cytotoxicity. It was also found that the most potent compound 7 induced apoptosis in MCF-7 cells via the intrinsic mitochondrial apoptotic pathway.

Elaboration of thorough simplified vinca alkaloids as antimitotic agents based on pharmacophore similarity

Thorough simplification of vinca alkaloids based on pharmacophore similarity has been conducted. A concise process for the syntheses of target compounds was successfully developed with yields from poor to excellent (19-98%). Cell growth inhibitory activities of these synthesized compounds were evaluated in five cancer cell lines including MCF-7, MDA-MB-231, HepG2, HepG2/ADM and K562. Almost all compounds exhibited moderate antitumor activity with optimal IC50 value of 0.89 ± 0.07 μM in MCF-7 cells. Investigation of structure-activity relationship (SAR) indicates that electron-withdraw substituents on the ring contribute to the enhancement of the antitumor activities. The simplified vinca alkaloids are confirmed as antimitotic agents, which inhibit the polymerization of tubulin just like vinblastine.

Abstract 990: Saxifragifolin D induces autophagy via activating IRE1α/ JNK pathway in breast cancer cells

Background and Objective: Saxifragifolin D (SD) was isolated from Androsace umbellata by our group. It has been reported to inhibit some types of cancer cells, such as Hep3B, HepG2, SK-OV-3, A549, HCT-115 and multidrug resistante cell line HepG2/ADM. However, the growth inhibitory effect of SD on breast cancer cells and the mechanisms is still unclear. In this study, we explored the underlying molecular mechanisms of SD-induced autophagy in breast cancer cells. Methods and Results: The autophagosomes, double membrane vacuolar structures, were observed in SD-treated MCF-7 and MDA-MB-231 cells by transmission electronic microscopy. And the autophagic vacuoles labeled by MDC were also increased in a time-dependent manner significantly. Besides, the autophagic ratio, quantified by using Cyto-ID® detection kit, was enhanced in a dose-dependent manner in these two cells. What9s more, the marker of autophagy, LC3, was activated in MCF-7 and MDA-MB-231 cells treated with SD showed by western blot assay, further confirmed autophagy induced by SD. We also found that IRE1α which associated with endoplasmic reticulum (ER) stress was activated, and the JNK and Bcl-2 were phosphorylated in SD-treated MCF-7 and MDA-MB-231 cells by western blot assay. Accordingly, the up-regulation of Beclin1, Vps34, Atg5 and Atg16L were also detected in these two cells. Furthermore, suppression of autophagy by pretreating with Beclin1 siRNA or bafilomycin A1 decreased apoptosis and cell death, and resulted in decreases in expression of LC3- II and cleaved PARP. It9s worth noting that inhibition of ER stress by IRE1α siRNA or TUDCA suppress autophagy and apoptosis, and the down-expression of LC3- II and cleaved PARP were measured. Conclusion: SD induces autophagy associated with IRE1α/ JNK pathway in breast cancer cells and these findings will provide molecular bases for molecular mechanistic studies of SD-treated breast cancer cells. Key words: Saxifragifolin D, autophagy, breast cancer. Citation Format: Liang-Liang Bai, Jun-Min Shi, Min-Feng Chen, Dong-Mei Zhang, Ying Wang, Wen-Cai Ye. Saxifragifolin D induces autophagy via activating IRE1α/ JNK pathway in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 990. doi:10.1158/1538-7445.AM2015-990