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Initial figures for Fig

Posted on October 24, 2024 by president2010

Initial figures for Fig. of this study are available on request. Abstract Background Merr. is usually a multipurpose coastal tree, the potential medicinal effects of which have been studied, including malignancy suppression. Here, we present evidence that this ethanol extract of Merr. (eGSM) induces autophagy in human lung adenocarcinoma cells. Methods Two different human lung adenocarcinoma cell lines, A549 and SNU2292, were treated with varying amounts of eGSM. Cytotoxicity elicited by eGSM was assessed by MTT assay and PARP degradation. Autophagy Cish3 in A549 and SNU2292 was determined by western blotting for AMPK, mTOR, ULK1, and LC3. Genetic deletion of AMPK in HEK293 cells was carried out by CRISPR. Results eGSM elicited cytotoxicity, but not apoptosis, in A549 and SNU2292 cells. eGSM increased LC3-II production in both A549 and, more extensively, SNU2292, suggesting that eGSM induces autophagy. In A549, eGSM activated AMPK, an essential autophagy activator, but not suppressed mTOR, an autophagy blocker, suggesting that eGSM induces autophagy by primarily activating the AMPK pathway in A549. By contrast, eGSM suppressed mTOR activity without activating AMPK in SNU2292, suggesting that eGSM induces autophagy by mainly suppressing mTOR in SNU2292. In HEK293 cells lacking AMPK expression, eGSM increased LC3-II production, confirming that this autophagy induced by eGSM can occur without the AMPK pathway. Conclusion Our findings suggest that eGSM induces autophagy by activating AMPK or suppressing mTOR pathways, depending on cell types. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-021-03454-4. Merr., Human lung carcinoma cells, mTOR, AMPK, Autophagy Introduction Merr. is usually a coastal tree species found in Japan, China, Taiwan, India, Sri Lanka, and the Philippines [1]. Merr. has been known to contain numerous chemical constituents regulating bacterial infection, inflammation, and malignancy [2]. For example, garcinielliptones were reported to inhibit the release of -glucuronidase and lysozyme [3]. They also suppress superoxide formation from activated neutrophils and peripheral mast cells [4]. Xanthone isolated from your herb is usually cytotoxic to malignancy cells [5], and benzophenonoids exhibit cytotoxicity to A549 non-small lung carcinoma cell, DU145 prostate carcinoma cell, and KB nasopharyngeal carcinoma cells [6]. These results suggest that the potential pharmacological significance of the herb is usually high. However, the use of herb extract as an herbal remedy appears relatively limited. A recent statement showed that this ethanol extract of the leaf of has anti-inflammatory activity [7]. The leaf extract reduced nitric oxide production, cyclooxygenase-2, and proinflammatory cytokines in RAW264.7 cells stimulated with lipopolysaccharide. These results are in accord with the reports that this herb contains numerous constituents contributing to anti-inflammatory effects. Given the cytotoxicity of some constituents of the herb to several malignancy cells, it would be worth exploring the herb extract as a potential herbal remedy to treat Afloqualone malignancy. Cytotoxicity could trigger autophagy, a cellular system that enables cells to cope with a constantly changing environment [8]. Autophagy is now considered a process of homeostasis that involves the digestion of self-proteins and organelles, by which cells can deal with numerous environmental challenges such as starvation [9] and infectious and other diseases [10]. Detailed molecular processes for autophagy are well-documented [11]. Mechanistically, autophagy is usually primarily regulated by two essential kinases, the mechanistic target of rapamycin (mTOR) and the AMP-activated protein kinase (AMPK). In a metabolically favorable environment, mTOR becomes phosphorylated at Ser2448 and active [12]. The activated mTOR senses energy-rich environmental cues, prompting anabolism and suppressing autophagy by phosphorylating ULK1 at S757 [13]. On the other hand, in a metabolically adverse Afloqualone environment where glucose or amino acids are limited and catabolism is required for cell survival [14], AMPK becomes activated and increases autophagy while suppressing mTOR activity, resulting in enhanced catabolism [15]. When sensing the lack of ATP, AMPK phosphorylates several serine residues in ULK1 [16], including S317 and S777 [17]. ULK1 phosphorylated at these Afloqualone sites triggers autophagosome formation, making autophagy start [14]. Concurrently, AMPK suppresses mTOR activity to stop anabolism. For suppressing mTOR activity, AMPK phosphorylates and activates TSC2, an mTOR upstream regulator, while phosphorylating and inactivating RAPTOR, a subunit of mTORC1 [13]. Phosphorylating both TSC2 and RAPTOR contributes to the decrease of mTOR activity. Reduced mTOR activity results in decreased phosphorylation of ULK1 at S757 [17]. Phosphorylation at S757 in ULK1 is known as inactivating phosphorylation because it blocks autophagosome formation [17]. Once ULK1 becomes active, as indicated by phosphorylation at S317 and but not at S757, autophagy process initiates to form autophagosomes [18]. During autophagosome formation, microtubule-associated protein 1A/1B-light chain 3 (LC3) in the cytosol is usually truncated to LC3-I and subsequently conjugated with phosphatidylethanolamine to form LC3-II.

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