Abstract
Targeting autophagy may serve as a promising strategy for cancer therapy. Ganoderma lucidum polysaccharide (GLP) has been shown to exert promising anti-cancer effects. However, the underlying mechanisms remain elusive. Whether GLP regulates autophagy in cancer has never been reported. In this study, GLP induced the initiation of autophagy in colorectal cancer (CRC) HT-29 and HCT116 cells, as evidenced by enhanced level of LC3-II protein, GFP-LC3 puncta, and increased formation of double membrane vacuoles. However, GLP treatment caused marked increase of p62 expression. Addition of late stage autophagy inhibitor, chloroquine (CQ), further enhanced LC3-II and p62 level, as well as increased autophagosome accumulation, suggesting a blockage of autophagic flux by GLP in CRC cells. We then found GLP blocked autophagosome and lysosome fusion as determined by mRFP-GFP-LC3 colocalization analysis. Mechanistic study revealed that GLP-induced disruption of autophagosome-lysosome fusion is due to reduced lysosome acidification and lysosomal cathepsin activities. Cell viability and flow cytometry assays revealed that GLP-induced autophagosome accumulation is responsible for GLP-induced apoptosis in CRC cells. In line with this, inhibition of autophagy initiation by 3-methyladenine (3-MA), an early stage autophagy inhibitor, attenuated GLP-induced apoptosis. In contrast, suppression of autophagy at late stage by CQ enhanced the anti-cancer effect of GLP. Furthermore, we demonstrated that GLP-induced autophagosome accumulation and apoptosis is mediated via MAPK/ERK activation. Finally, GLP inhibited tumor growth and also inhibited autophagic flux in vivo. These results unveil new molecular mechanism underlying anti-cancer effects of GLP, suggesting that GLP is a potent autophagy inhibitor and might be useful in anticancer therapy.
Highlights
Colorectal cancer (CRC) is the third most common malignancy and the third leading cause of cancer-related death in the USA1
In order to examine the effect of Ganoderma lucidum polysaccharide (GLP) on autophagy, we evaluated the distribution pattern of GFP-LC3 in CRC cells transiently expressing GFP-LC3, reminiscent of autophagosome formation[19]
The cytoplasmic form LC3-I is modified to LC3-II, the amount of LC3-II increases with the formation of autophagosomes[19]
Summary
Colorectal cancer (CRC) is the third most common malignancy and the third leading cause of cancer-related death in the USA1. A substantial number of preclinical and clinical studies have reported that G. lucidum has numerous pharmacological effects, including antioxidant, hypoglycemic, immune-regulatory, anti-diabetic, and Official journal of the Cell Death Differentiation Association. Pan et al Cell Death and Disease (2019)10:456 anti-cancerous[5,6,7,8,9,10]. Many studies have demonstrated that GLP is one of the main bioactive components responsible for anti-cancer effects of G. lucidum[3,5]. We showed that GLP extracted from the sporoderm-broken spores of G. lucidum significantly inhibited cell proliferation and induced apoptosis in colorectal and prostate cancer cells[11,12]. The molecular mechanisms underlying the anti-cancer effects of GLP remain unclear
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