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Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors with poor survival. It is urgent to search for new efficient drugs with good stability and safety for clinical therapy. This study aims to identify potential anticancer drugs from a compound library consisting of 429 natural products. Echinatin, a compound isolated from the Chinese herb Glycyrrhiza uralensis Fisch, was found to markedly induce apoptosis and inhibit proliferation and colony-formation ability in ESCC. Confocal fluorescence microscopy data showed that echinatin significantly induced autophagy in ESCC cells, and autophagy inhibitor bafilomycinA1 attenuated the suppressive effects of echinatin on cell viability and apoptosis. Mechanistically, RNA sequencing coupled with bioinformatics analysis and a series of functional assays revealed that echinatin induced apoptosis and autophagy through inactivation of AKT/mTOR signaling pathway, whereas constitutive activation of AKT significantly abrogated these effects. Furthermore, we demonstrated that echinatin had a significant antitumor effect in the tumor xenograft model and markedly suppressed cell migration and invasion abilities of ESCC cells in a dose-dependent manner. Our findings provide the first evidence that echinatin could be a novel therapeutic strategy for treating ESCC.

Gambogic acid (GA) has been shown to inhibit cancer cell proliferation, induce apoptosis, and enhance reactive oxygen species accumulation. However, whether GA could improve multidrug resistance through modulating autophagy has never been explored. We demonstrated that the combination of GA and cisplatin (CDDP) resulted in a stronger growth inhibition effect on A549 and NCI-H460 cells using the MTT assay. Furthermore, treatment with GA significantly increased autophagy in these cells. More importantly, GA-induced cell death could be largely abolished by 3-methyladenine (3-MA) or chloroquine (CQ) treatment, suggesting that GA-induced cell death was dependent on autophagy. Western blot analysis showed that GA treatment suppressed the activation of Akt, mTOR, and S6. In addition, using a GA and rapamycin combination induced more cell death compared to either GA or rapamycin alone. In summary, GA may have utility as an adjunct therapy for non-small cell lung cancer (NSCLC) patients through autophagy-dependent cell death, even when cancer cells have developed resistance to apoptosis.

Plant lectin, a class of highly diverse non-immune origin and carbohydrate-binding proteins, has been reported to specially induce cancer cell through programmed cell death (PCD) pathways (apoptosis and/or autophagy), shedding lights on screening promising anti-cancer candidate agent for further therapeutic trials. However, the complicated molecular mechanisms by which plant lectins induced the programmed death of tumor cells, have not yet been fully clarified. Here, we summarized a novel model, based on vast amount of research, by which plant lectins eliminate various types of cancer cells via three major pathways, including a) direct ribosome inactivating, b) endocytosis-dependent mitochondrial dysfunction and c) sugar-containing receptors binding. A better understanding of the role of plant lectins played and further elucidation of the strategies targeting PCD would provide a new clue for the applications and modifications of plant lectin as a potential anti-cancer agent from bench to clinic.

Timosaponin A‑III (TAIII) is a saponin isolated from anemarrhena asphodeloides and possesses the inhibitory effect on proliferation of multiple tumor cells. In the present study, the antitumor effect of TAIII and its underlying molecular mechanisms were investigated in vitro in T‑cell acute lymphoblastic leukemia (T‑ALL) Jurkat cells. The results demonstrated that TAIII inhibits the viability of Jurkat cells in a time‑ and dose‑dependent manner, and induces apoptosis of Jurkat cells in a dose‑dependent manner. Transmission electron microscopy demonstrated the formation of numerous autophagosomes in TAIII‑treated Jurkat cells. Furthermore, monodansylcadaverine (MDC)‑labeled autophagic vacuoles were observed following TAIII treatment by an inverted fluorescence microscope and MDC accumulation increased notably in TAIII treatment groups in a concentration‑dependent manner. B‑cell lymphoma‑2 (Bcl‑2)‑associated X (Bax) was upregulated while Bcl‑2 was reduced following TAIII treatment, indicating that the pro‑apoptotic mechanism of TAIII may be associated with upregulation of Bax. Further investigation revealed that TAIII promotes the expression of autophagy‑associated proteins Beclin 1 and LC3‑II, and inhibits the phosphoinositide 3‑kinase/Akt/mechanistic target of rapamycin kinase pathway. The present study revealed that the antitumor activity of TAIII was primarily achieved by the induction of cell apoptosis and autophagy, indicating a promising potential as a novel effective reagent against T‑ALL.

Kaempferol belongs to the flavonoid family and has been used in traditional folk medicine. Here, we investigated the antitumor effects of kaempferol on cell cycle arrest and autophagic cell death in SK-HEP-1 human hepatic cancer cells. Kaempferol decreased cell viability as determined by MTT assays and induced a G2/M phase cell cycle arrest in a concentration-dependent manner. Kaempferol did not induce DNA fragmentation, apoptotic bodies or caspase-3 activity in SK-HEP-1 cells as determined by DNA gel electrophoresis, DAPI staining and caspase-3 activity assays, respectively. In contrast, kaempferol is involved in the autophagic process. Double-membrane vacuoles, lysosomal compartments, acidic vesicular organelles and cleavage of microtubule-associated protein 1 light chain 3 (LC3) were observed by transmission electron microscopy, LysoΤracker red staining, GFP-fluorescent LC3 assays and acridine orange staining, respectively. In SK-HEP-1 cells, kaempferol increased the protein levels of p-AMPK, LC3-II, Atg 5, Atg 7, Atg 12 and beclin 1 as well as inhibited the protein levels of CDK1, cyclin B, p-AKT and p-mTOR. Taken together, CDK1/cyclin B expression and the AMPK and AKT signaling pathways contributed to kaempferol-induced G2/M cell cycle arrest and autophagic cell death in SK-HEP-1 human hepatic cancer cells. These results suggest that kaempferol may be useful for long-term cancer prevention.

Bufalin is the major component of Chan-Su (a traditional Chinese medicine, TCM) extracts from the venom of Bufo bufo gargarizan. In the present study, we investigated the pharmacological mechanisms of cell cycle arrest and autophagic cell death induced by bufalin in SK-HEP-1 human hepatocellular carcinoma cells in vitro. Bufalin inhibited cell survival by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. In addition, bufalin induced G2/M phase arrest by reducing CDK1 activity. Bufalin triggered DNA fragmentation and apoptotic cell death in SK-HEP-1 cells by DNA gel electrophoresis, TUNEL and caspase-3 activity assay, while bufalin induced autophagic cell death by double-membrane vacuoles (transmission electron microscopy, TEM), acidic vesicular organelles (acridine orange staining) and cleavage of microtubule-associated protein 1 light chain 3 (LC3). Protein expression levels of cyclin A and B, CDK1, phospho-CDK1 (Thr161), Cdc25c, phospho-Cdc25c (Ser198), phospho-AKT (Thr308), phospho-AKT (Ser473), phospho‑mTOR (Ser2481) were downregulated. In contrast, protein expression levels of the Chk1, Wee1, LC3-II, Beclin-1, Atg 5, Atg 7 and Atg 12 were upregulated in SK-HEP-1 cells after bufalin treatment. Inhibition of autophagy by 3-methyladenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) or bafilomycin A1 (an inhibitor of the vacuolar proton pump of lysosomes and endosomes) reduced the effect of bufalin on cell viability and enhanced the effect of bufalin on apoptosis. In conclusion, bufalin triggered autophagic cell death and G2/M phase arrest through the AKT/mTOR signaling pathway in SK-HEP-1 cells. Our findings showed that bufalin may be potentially efficacious in the treatment of human hepatocellular carcinoma.

Salivary adenoid cystic carcinoma is an epithelial tumor in the head and neck region. Despite its slow growth, patients with salivary adenoid cystic carcinoma exhibit poor long term survival because of a high rate of distant metastasis. Lung and bone are common distant metastasis sites. Zoledronic acid, a third generation bisphosphonate, has been used for tumor-induced osteolysis due to bone metastasis and has direct antitumor activity in several human neoplasms. Here, we observed that zoledronic acid inhibited salivary adenoid cystic carcinoma cell line SACC-83 xenograft tumor growth in nude mice. In vitro, zoledronic acid induced apoptosis and reduced clonogenic survival in SACC-83. Flow cytometry and western blotting indicated that the cell cycle was arrested at G0/G1. Zoledronic acid treatment upregulated reactive oxygen species as well as the autophagy marker protein LC-3B. Reactive oxygen species scavenger N-acetylcysteine and autophagy antagonist 3-methyladenine decreased zoledronic acid-induced apoptosis and increased clonogenic survival. Silencing of the autophagy related gene Beclin-1 also decreased zoledronic acid-induced apoptosis and inhibition of clonogenic formation. In addition, isobolographic analysis revealed synergistic effects on apoptosis when zoledronic acid and paclitaxel/cisplatin were combined. Taken together, our results suggest that zoledronic acid induced apoptosis and reduced clonogenic survival via upregulation of reactive oxygen species and autophagy in the SACC-83 cell line. Thus, zoledronic acid should be considered a promising drug for the treatment of salivary adenoid cystic carcinoma.