Two major proteolytic systems for the clearance of proteins are conserved in eukaryotic cells: the first is the UPS and the second is autophagy. Both UPS and autophagy are involved in most aspects of normal physiology and development. They are also implicated in multiple pathological states, such as cancer, neurodegeneration and aging. Although UPS and autophagy are generally thought to be independent from each other, recent investigations now support that the two proteolytic systems are functionally linked, and autophagy is activated and plays a compensatory role when UPS function is impaired
[12–20]. Autophagy is frequently activated in cancer cells in response to chemo- or radiotherapy
[33, 34]. The contribution of autophagy to cell death induced by therapy generally remains controversial as autophagy protects some cancers against chemotherapy yet sensitizes others to chemotherapy-mediated cytotoxicity
[33, 34]. In the current study, we confirmed that proteasome inhibitors activated autophagy in ovarian cancer cells, as evidenced by accumulation of acidic vacuoles, increase in LC3-II transition. However, suppression of autophagy at the early stage by knockdown of Atg7, as well as at the late stage by cotreatment with chroroquine or bafilomycin A1 demonstrated little effects on cytotoxicity of ovarian cancer cells mediated by proteasome inhibition. The different role of autophagy in chemotherapy-induced cytotoxicity might represent cell-specific and/or stress-specific response. The dual roles of autophagy in survival and cell death require further clarification.
Beclin 1, the mammalian homologue of the yeast Atg6 was initially identified as a Bcl2-interacting tumor suppressor
. It is now known that Beclin 1 cooperates with several cofactors to activate lipid kinase PI3KC3, which is essential for recruitment of other Atg proteins to form autophagic vacuoles or autophagosomes
. However, several recent studies have demonstrated that some stimuli can also induce PI3KC3 and Beclin 1-independent autophagy, so named as non-canonical autophagy
[26–30]. For example, resveratrol, Parkinsonian neurotoxin MPP+ and a small compound targeting the BH3 binding groove of Bcl-XL has been shown to activate autophagy in a Beclin 1-independent manner in breast cancer MCF7 cells, neuroblastoma cells and HeLa cells, respectively
[26–30]. In the current study, for the first time, we reported that proteasome inhibitors elicited PI3KC3 and Beclin 1-independent autophagy in ovarian cancer cells, as evidenced by neither PI3Ks inhibitor wortmannin or 3-MA, nor shRNA against Beclin 1 could block accumulation of acidic vacuoles and increase in LC3-II transition induced by proteasome inhibitors. The mechanisms by which autophagosome formation can bypass the Beclin 1-PI3KC3 pathway remain to be clarified in the future.
Genetic analysis has revealed that Beclin 1 is implicated in tumorigenesis and plays a role in cellular proliferation
[8, 22, 36, 37]. It has been reported that overexpression of Beclin 1 activates autophagy and reduces the tumorigenetic potential of breast cancer cells
. In addition, overexpression of Beclin 1 has been shown to enhance the sensitivity of cervix and gastric cancer cells to chemotherapeutic drugs
[38, 39]. On the contrary, heterozygous disruption of Beclin 1 in mice increases cellular proliferation and results in spontaneous malignancies
. Consistent with previous reports
[38, 39], in the current study, flowcytometry analysis and caspase 3 activity assay indicated that a greater in crease in apoptosis was observed in Beclin 1-transfected cells than the mock-transfected cells. Controversially, Beclin 1 knockdown has been shown to promote apoptosis induced by doxorubicin in HepG2 cells
. These reports therefore suggest that Beclin 1 may modulate apoptosis in cell-specific and stimuli-specific patterns. It has been generally believed that Beclin 1 functions as a haploinsufficient tumor suppressor via autophagy activation
[22, 41]. However, in the current study, we found that proteasome inhibitors activated autophagy in a Beclin 1-independent manner. In addition, suppression of autophagy both at the early stage and at the late stage had no obvious effects on cytotoxicity mediated by proteasome inhibitors. On the contrary, Beclin 1 overexpression enhanced responsiveness of ovarian cancer cells to proteasome inhibitors-mediated cytotoxicity, indicating that Beclin 1 exerts autophagy-independent tumor suppressive effect in ovarian cancer cells upon exposure to proteasome inhibitors. Therefore, mechanisms underlying enhancing effects of Beclin 1 on chemosensitivity may be multifactorial, and the mechanisms by which Beclin 1 sensitizes ovarian caner cells to proteasome inhibition require further investigation.