In some apoptosis-resistant cancer cells, autophagy can also enhance the efficacy of anti-cancer drugs through autophagy-mediated mechanisms of cell death. Many novel anti-cancer compounds have been isolated from natural products; therefore, we worked to discover natural, anti-cancer small-molecule enhancers of autophagy. Here, we have identified a group of natural alkaloid small-molecules that function as novel autophagic enhancers. These alkaloids, including liensinine, isoliensinine, dauricine and cepharanthine, stimulated AMPK-mTOR dependent induction of autophagy and autophagic cell death in a panel of apoptosis-resistant cells. Taken together, our work provides novel insights into the biological functions, mechanisms and potential therapeutic values of alkaloids for the induction of autophagy. Autophagy is usually a cellular degradation process that involves the delivery of cytoplasmic cargos, such as aged proteins, mis-folded proteins or damaged organelles, for lysosomal degradation following sequestration in double-membrane vesicles (autophagosomes). Autophagy occurs at a low basal level in cells, turning over proteins and organelles to maintain homeostasis. However, upon conditions of cellular stress, such as nutrient deprivation, oxidative stress, contamination or accumulation TMB-PS of protein aggregates, autophagy begins with membrane isolation and growth to form autophagosomes that sequester all unwanted cytoplasmic materials. Following fusion of the autophagosome with the lysosome to form an autolysosome, the engulfed materials are degraded to recycle intracellular nutrients and energy1. Impairment of autophagy and the age-related decline of autophagic function can lead to the pathogenesis of cancers2. Developing mechanisms to circumvent the common problem of chemoresistance in cancer cells to improve the efficacy of anti-cancer TMB-PS therapies is usually highly desirable. Autophagy, a process that restores metabolic homeostasis through the catabolic lysis of excessive proteins or injured organelles, is considered a potential target for cancer therapy by way of either its pro-death or pro-survival mechanisms3. For example, autophagic dysfunction is usually associated with DNA damage, chromosome instability4, and increased incidence of malignancies5. Moreover, enhancers of autophagy may play a protective role in cancer therapy by promoting autophagic cell death in tumours or by augmenting the efficacy of chemotherapeutic brokers6. Several clinically approved and experimental antitumor brokers have been shown to induce autophagy-mediated cell death in various types of cancer cells7,8. Although autophagy may also promote tumour growth by providing energy to poorly-vascularised cancer cells under hypoxic conditions or nutritional deprivation, autophagy-blocking molecules could be used in combination with chemotherapeutic brokers to improve their therapeutic efficacy7. Recently, natural compounds from flavonoids, ginsenosides, naphthoquinones and alkaloids have been found to exhibit anti-cancer effects through the modulation of autophagy. For example, plant flavonoids, such as wogonin and luteolin, have been shown cancer cell death through inhibition of autophagy9,10,11. Ginsenosides such as F212 have also been shown to exhibit anti-cancer effects through the modulation of autophagy. Naphthazarin, a naphthoquinone compound, is usually a microtubule depolymerising agent that induces cell death by activating apoptosis and autophagy13, and plumbagin induces G2-M arrest and autophagic cell death by inhibiting the AKT/mTOR (mammalian target of rapamycin) pathway in breast malignancy cells14. Alkaloids isolated from plants used in Chinese herbal medicine are an important source for drug discovery15. The alkaloid berberine exhibits its anti-cancer effects by inducing autophagic cell CLG4B death and mitochondrial apoptosis in liver cancers16, whereas tetrandrine acts as an enhancer of autophagy that induces early G1 arrest in colon carcinoma cells17. Additionally, camptothecin and vinblastine are chemotherapeutic drugs that have been approved for clinical use18,19,20,21. Therefore, in this study we set out to identify novel enhancers of autophagy from five primary categories of compounds: flavonoids, flavanols, ginsenosides, naphthoquinone and alkaloids. These compounds may exert putative anti-cancer effects through the modulation of autophagic pathways. Using bioactivity-guided screening of selected compounds isolated from natural products, we have identified a group of alkaloids, including liensinine, isoliensinine, dauricine and cepharanthine, that function as novel inducers of autophagy. Here, we present evidence that isoliensinine, dauricine and cepharanthine induce mTOR-dependent autophagy and autophagic cell death in a panel of apoptosis-resistant TMB-PS cells. Taken together, our work provides novel insights into the autophagic effects of selected alkaloids and their potential uses in anti-tumour therapy. Results Alkaloid compounds induce formation of GFP-LC3 puncta in multiple cancer cells An increasing number of studies have identified natural compounds from flavonoids, ginsenosides, naphthoquinones and alkaloids as autophagy modulators with potential therapeutic uses in cancers9,14,16. In the current study, we aimed to identify novel inducers of autophagy from five groups of compounds: the flavonoids, flavanols, ginsenosides, naphthoquinones and alkaloids (Table 1). To verify whether the selected compounds were capable of TMB-PS inducing autophagy, we adopted the HeLa human cervical cancer cell line as a model for autophagy detection because it provided a discrete compartment for accurate immunofluorescence imaging analysis22. Previously, we successfully exhibited the autophagic effect of a triterpenoid compound, saikosaponin-d, using HeLa cells23. Here, to determine the optimal concentrations of compounds required for induction of autophagy, we first evaluated the cytotoxicity of each compound. Then, all.