International Journal of Cell Biology and autophagosome membranes are composed of mem- branes derived from rough ER. ER and Golgi complex- derived vesicles could be delivered to the lysosome/vacuole by a pathway that is diﬀerent from canonical autophagy. Under certain conditions, these two organelles might not require sequestration by a double membrane for delivery and subsequent degradation. Furthermore, it has been reported that one of the Atg proteins, Atg9, a membrane protein with unknown function, shuttled between the trans-Golgi network and late endosomes. Bax-interacting factor 1 (Bif- 1) binds the proapoptotic Bcl-2 family protein Bax. Bif-1 overexpression promotes Bax activation and apoptosis. Bif-1 also regulates the ﬁssion of Golgi membranes and the traf- ﬁcking of Atg9 from the Golgi complex to autophagosomes during starvation [46]. Therefore, isolation membranes and autophagosome membranes may be made up of membranes from several diﬀerent sources, including the Golgi; however, there is little evidence that the Golgi complex contributes directly to the formation of the autophagic membrane. 8. Mitochondria and Autophagy Mitochondria are key players in several cancer cellular func- tions, including growth, division, energy metabolism, and apoptosis, and play a central role in cell survival and death. Mitochondria also play an important role in the formation of autophagosome and its subsequent docking and fusion with lysosome and could contribute to the increased autophagy and autophagic ﬂux in metastatic cancer. The ability of tumor mitochondria to increase autophagy may help cancer cells to fulﬁll high anabolic needs during rapid growth; however, mitochondrial functions (such as respiration) can block autophagic cell death [47]. Autophagy often occurs when the mitochondria fail to maintain ATP levels, during starvation, or when the mitochondria are damaged. A process known as mitophagy selectively eliminates the dam- aged mitochondria. Furthermore, blocking mitochondria- mediated apoptosis through the elimination of mouse Bax and Bak (Bak1) expression promotes autophagy-induced cell death. Arsenic-trioxide- (As2O3-) induced cell death in human malignant glioma cell lines was accompanied by involvement of an autophagy-speciﬁc marker, LC3, and damage to mitochondrial membrane integrity. Arsenic trioxide induces the mitochondrial localization of the pro- cell-death Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3), which deter- mines the on/oﬀ state of the mitochondrial permeability transition (MPT) pore [48]. The reciprocal relationship between mitochondrial metabolism and the activity of the rapamycin-sensitive pathway has been described before [49]. Mitochondria dysfunction leads to inhibition of mTOR, and inactivation of p70S6K (ribosomal protein S6 kinase) results in dephosphorylation of Bad, potentially leading to increased interaction between Bad and Bcl-xL or Bcl-2, to mitochondrial damage and cell death [50]. In addition, the promotion of MPT also contributed to increased autophagy. These results suggested that mitochondria from a highly metastatic breast cancer cell line can promote homeostatic 5 autophagy of cancer by opening low-conductance MPT pores. The activation of autophagy by tumor mitochondria was reduced by cyclosporine A, which is an inhibitor that blocks the low-conductance state of MPT pores [47]. ROS could be the mediator leading to the loss of mitochondria membrane potential, possibly by triggering the opening of the MPT pore that subsequently leads to cell death. ROS were recently shown to activate starvation-induced autophagy, antibacterial autophagy, and autophagic cell death. Short- chain fatty acids were shown to induce ROS production, which led to AMPK activation and consequential mTOR inhibition [51]. Current ﬁndings implicate ROS in the regu- lation of autophagy through distinct mechanisms, depending on the cell type and stimulation conditions; conversely, autophagy can also suppress ROS production. 9. Experimental Manipulation of Autophagy For a better understanding of the regulation of autophagy, many studies have used chemical modulators of autophagy: inhibitors and inducers. The diﬀerent modulators are an important tool in studying the functions of autophagy, although it is diﬃcult to deﬁne the mechanism closely. Appropriate modiﬁcation of autophagy, that is, inhibi- tion of cytoprotective autophagy or promotion of cyto- killing autophagy could mitigate the cytotoxicity caused by anticancer therapy in tumor cells. 9.1. ProAutophagics (Table 1) 9.1.1. Beclin 1. The mechanism by which a small molecule Bcl-2/Bcl-xL antagonist ABT-737 can potentiate autophagy may be related to its ability to competitively disrupt the binding of Bcl-2/Bcl-xL to the autophagic protein Beclin 1 (Atg6) [52]. Treatment with fenretinide (RT-101), a synthetic derivative of retinoic acid, resulted in an increase in Beclin 1 expression, the conversion to the form LC3-II form, and its shift from diﬀuse to punctate staining and ﬁnally an increase in lysosomes/autophagosomes [53]. In addi- tion, HIF-1α is required for fenretinide-induced protective autophagy under hypoxia. The inositol 1,4,5-trisphosphate receptor (IP3R) antagonist xestospongin B induces autophagy by disrupting a molecular complex formed by IP3R and Beclin 1. Xestospongin B-induced autophagy was inhibited by overexpression of the IP3R ligand-binding domain, which co-immunoprecipitated with Beclin 1 [54]. Autophagy can be generally induced via several distinct pathways, such as inhibition of mTOR or activation of Beclin 1. Okadaic acid, a protein phosphatase 2A inhibitor, increased both the mTOR and Beclin 1 pathways simultaneously, which suggests that autophagy in okadaic acid-treated neurons is induced mainly via the Beclin 1 pathway, and less so via mTOR inhibition [55]. 9.1.2. DNA Damage. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chem- icals, and endogenous byproducts of metabolism, including ROS, can cause alterations in DNA structure (DNA damage).