Article pubs.acs.org/jnp
Bisleuconothine A Induces Autophagosome Formation by Interfering with AKT-mTOR Signaling Pathway Chin Piow Wong, Ari Seki, Kaori Horiguchi, Tomokazu Shoji, Takashi Arai, Alfarius Eko Nugroho, Yusuke Hirasawa, Fumiaki Sato, Toshio Kaneda, and Hiroshi Morita* Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41 Shinagawa-ku, Tokyo 142-8501, Japan S Supporting Information *
ABSTRACT: We have previously reported that bisleuconothine A (BisA), a novel bisindole alkaloid isolated from Leuconotis grif f ithii, showed cytostatic activity in several cell lines. In this report, the mechanism of BisA-induced cytostatic activity was investigated in detail using A549 cells. Bis-A did not cause apoptosis, as indicated by analysis of annexin V and propidium iodide staining. Expression of all tested apoptosis-related proteins was also unaffected by Bis-A treatment. Bis-A was found to increase LC3 lipidation in MCF7 cells as well as A549 cells, suggesting that Bis-A cytostatic activity may be due to induction of autophagy. Subsequent investigation via Western blotting and immunofluorescence staining indicated that Bis-A induced formation but prevented degradation of autophagosomes. Mechanistic studies showed that Bis-A downregulated phosphorylation of protein kinase B (AKT) and its downstream kinase, PRAS40, which is an mTOR repressor. Moreover, phosphorylation of p70S6K, an mTOR-dependent kinase, was also down-regulated. Down-regulation of these kinases suggests that the increase in LC3 lipidation may be due to mTOR deactivation. Thus, the cytostatic activity shown by Bis-A may be attributed to its induction of autophagosome formation. The Bis-A-induced autophagosome formation was suggested to be caused by its interference with the AKT-mTOR signaling pathway.
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INTRODUCTION Autophagy is a catabolic process that degrades and recycles unnecessary cellular components. Autophagy is derived from the Greek words “auto”, meaning self, and “phagein”, meaning eat, which when combined together mean self-eating.1 The autophagy process (autophagy flux) is dependent on the formation of a double-membrane vesicle that “consumes” cytoplasmic constituents. This vesicle, termed the autophagosome, later fuses with a lysosome, forming an autolysosome, which breaks down unnecessary cytoplasmic constituents.2 Many factors can trigger the onset of autophagy such as oxidative stress, endoplasmic reticulum stress, organelle damage, and misfolded protein aggregates.3 Autophagy is a homeostatic process that degrades and recycles cytoplasmic constituents to maintain cellular biosynthesis during metabolic stress and nutrient deprivation.4 Autophagy can function as a tumor suppressor by preventing stressed or damaged cells from becoming tumor cells by acting as a cell growth inhibition mechanism during metabolic stress and cell damage.5,6 However, some studies also suggested that tumors exploit autophagy to promote its proliferation. Through autophagy, tumor cells are able to sustain cellular biosynthesis by recycling unnecessary cellular components to provide resources for cell growth and proliferation.5−7 In our continuous quest to identify novel bioactive compounds of plant origin, many compounds with biological © XXXX American Chemical Society and American Society of Pharmacognosy
activities have been identified such as cytostatic, vasorelaxation, antilipid droplet accumulation, osteoclast differentiation inhibition, and inhibition of nitric oxide.8−14 We have previously isolated two novel bisindole alkaloids from the leaves of Leuconotis grif f ithii Hk.f of the Apocynaceae family, leucoridine A N-oxide and bisleucocurine A, which possess antiplasmodial and cytotoxic activity, respectively.15,16 Additionally, investigation of L. grif f ithii bark led to the isolation of a novel bisindole alkaloid, bisleuconothine A (Bis-A) (Figure 1),17 and
Figure 1. Structure of bisleuconotine A (Bis-A), an eburnane− aspidosperma-type skeleton bisindole alkaloid isolated from the bark of Leuconotis grif f ithii. Received: March 24, 2015
A
DOI: 10.1021/acs.jnatprod.5b00258 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 2. Cytostatic activity of Bis-A. (A) MTT assay on Bis-A and positive control, vinblastine. (B) Oxidative effect of Bis-A. A549 cells preloaded with dichlorodihydrofluorescein diacetate (20 μM; H2DCFDA) for 1 h (4 × 105 cells/dish) were treated with Bis-A (6 and 24 μM) for another hour. Hydrogen peroxide (100 μM) was used as a positive control. The x-axis represents the fluorescence intensity of intracellular dichlorodihydrofluorescein (H2DCF). (C−E) A549 cell line (5 × 105 cells/dish) was treated with vehicle (C), vinblastine 100 nM (D), and Bis-A 24 μM (E) for 24 h. Treated cells were then stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI).
three novel indole alkaloids, leucomidines A−C.18 Although leucomidines A−C are not bioactive, Bis-A, a bisindole alkaloid connecting the C-16 of an eburnane skeleton to C-12′ of an aspidosperma skeleton (Figure 1), was found to have cytostatic activity.17 Many autophagy-inducing compounds have also been reported to induce apoptosis. In the current study, Bis-A was found to induce autophagosome formation without apoptosis, even at high concentrations. Thus, the mechanism of Bis-Ainduced autophagosome formation was investigated in a more detailed manner using the A549 cell line.
death, as confirmed from microscopic observations. A known cytotoxic bisindole alkaloid, vinblastine, was used as a positive control. Treatment with vinblastine for 48 h also showed a concentration-dependent reduction of succinate dehydrogenase activity. Microscopic observation showed a significant number of dead cells. Oxidative Effect of Bis-A. Defined as the imbalance between generation and elimination of reactive oxygen species, oxidative stress occurs when the balance leads to accumulation of reactive oxygen species to a point of disturbing the normal redox state of cells.19 Hence, oxidative stress can play a major part in causing cytostatic or cytotoxic activity. Moderate oxidative stress is known to cause apoptosis, while severe oxidative stress can cause necrosis.20 Many anticancer drugs are known to cause oxidative stress. The possibility of Bis-A achieving cytostatic activity via oxidative stress was investigated. Bis-A-treated A549 cells were preloaded with dichlorodihydrofluorescein diacetate (H2DCFDA) for 1 h prior to flow cytometry analysis. The fluorescent intensity of Bis-A (6 μM)-treated cells did not shift from that of preloaded control (Figure 2B). This indicated that a low concentration of Bis-A (6 μM) did not result in generation of intracellular reactive oxygen species (ROS). The histogram of cells treated with a high concentration of Bis-A (24 μM) shifted slightly to the right, suggesting a slight increase of ROS generation. Hydrogen peroxide at 100 μM was used as a positive control for the generation of ROS. Correlations between oxidative stress and the occurrence of apoptosis and autophagy have been reported by other researchers.21−24 Thus,
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RESULTS AND DISCUSSION Cytostatic Activity of Bis-A. Bis-A was previously shown to be cytostatic to the human lung adenocarcinoma epithelial cell line, A549; the human breast adenocarcinoma cell line, MCF7; the human colorectal cancer cell line, HCT116; and the human promyelocytic leukemia cell line, HL-60.17 Thus, the cytostatic activity of Bis-A was studied in more detail using the A549 cell line. The MTT assay showed that A549 cells treated with Bis-A resulted in a concentration-dependent reduction of succinate dehydrogenase activity, reaching a plateau of maximum reduction at 24 μM, which indicated possible cytostatic or cytotoxic activity (Figure 2A). The IC50 value of Bis-A was determined at approximately 8 μM (IC50 value of previous article was at 6 μM).17 Furthermore, no significant cell death was observed microscopically (data not shown), suggesting BisA to have cytostatic activity. However, treatment of Bis-A at higher concentration (24 μM) for 48 h slightly increased cell B
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it is suggested that Bis-A does not cause apoptosis even at a high concentration (24 μM). Bis-A Induces Autophagosome Formation. The involvement of autophagy in cytostatic activity induced by Bis-A was investigated. Autophagy begins with the formation of the autophagosome, a double membrane vesicle that “eats” cytoplasmic constituents. The formed autophagosome then fuses with a lysosome, forming the autolysosome, which breaks down cytoplasmic constituents contained within. One of the hallmarks of autophagy is LC3 lipidation, a conversion process of cytosolic-associated light chain 3 (LC3-I) to a lipidated form (LC3-II), which is a component of the autophagosome’s double membrane.2 Therefore, by detecting LC3 lipidation, the possibility of Bis-A inducing autophagy can be ascertained. A549 cells treated with Bis-A showed a time-dependent increase of LC3 lipidation (Figure 4A). The increase of LC3-II from 4 to 24 h was accompanied by a decrease of LC3-I expression. The increase of LC3 lipidation suggested formation of autophagosomes and the possibility of autophagy induction by Bis-A. Everolimus (EVERO; rapamycin derivative and known inducer of autophagy) at 5 μM was used as a positive control (Figure 4A).25 The same experiment was also performed with MCF7 cells. The results showed that MCF7 cells have a similar pattern of LC3 lipidation to Bis-A-treated A549 cells (Figure 4A). Together, these data suggest that Bis-A might promote an autophagic process, which was not cell line specific. For further interpretation of the effects of Bis-A, autophagic flux (the process of autophagy induction and autophagosome formation, maturation, fusion with lysosomes, and degradation)26 was investigated. SQSTM1/sequestome 1 (p62) is a substrate that is incorporated into autophagosomes and is degraded after fusion with lysosomes. The expression of p62 is generally known to be reduced during autophagy, making it a common marker to measure autophagy flux.27−30 Although Bis-A treatment resulted in increased conversion of LC3-II from LC3-I, p62 was unexpectedly increased (Figure 4B, lanes 1 and 2). These effects were similar to that of bafilomycin A (BAF), which is known to interrupt autophagy flux by inhibiting the fusion of autophagosomes and lysosomes (Figure 4B, lanes 2 and 4). On the other hand, as a result of autophagy induction, EVERO did not elevate expression of p62; however LC3 lipidation was increased (Figure 4B, lane 3). These data suggested that Bis-A induced formation but prevented degradation of autophagosomes. Furthermore, the effect of Bis-A on LC3 and p62 distribution in autophagic flux was investigated by immunofluorescence staining. LC3 and p62 were used as a marker for autophagosome puncta, while lysosome-associated membrane protein 2 (LAMP-2) was a common marker for lysosome puncta.31 Compared to untreated cells, cells treated with Bis-A showed a significant increase of puncta with LC3 and p62, and morphological changes and localization of puncta with LAMP2 were also observed (Figure 4C). Consistent with Western blot data (Figure 4B), increased puncta with LC3 and p62 pointed to formation of autophagosomes. In EVERO-treated cells, only a slight increase in puncta with LC3 and p62 was observed, implying weaker autophagosome formation compared to Bis-A. On a further note, BAF also increased puncta with LC3 and p62. Interestingly, a merged image of LC3 and LAMP2 showed that autophagosomes induced by Bis-A (green puncta) did not fuse with lysosomes (red puncta) to form autolysosomes (yellow puncta). Autophagosome and lysosome puncta also
for the next course of studies, an investigation was carried out to determine whether Bis-A cytostatic activity is due to apoptotic and/or autophagic properties. Apoptosis-Inducing Activity of Bis-A. The possibility of apoptotic cell death induced by Bis-A was investigated using flow cytometry. Samples were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) and subjected to analysis. The distribution of untreated control cells to the lower left quadrant indicated the cells are healthy and no significant induction of apoptosis (Figure 2C). In the sample treated with vinblastine (100 nM) used as positive control, approximately 30% of the cells were distributed to the upper left and upper right quadrants, indicating that vinblastine-treated cells undergo early apoptosis and late apoptosis phase, respectively (Figure 2D). However, the distributions of Bis-A-treated cells were similar to that of the untreated control (Figure 2E). These results indicated that Bis-A does not cause apoptosis even at high concentration (24 μM). The nonapoptotic activity of Bis-A was confirmed by investigating the expression of apoptosis-related proteins via Western blotting. All tested apoptosis-related proteins (Bcl-2 family proteins: Bcl-xL, Mcl-1, Bcl-2, Bax-1, Bid, Puma, BimEL, and Bik; and tumor suppressor: p53) (Figure 3) were
Figure 3. Analysis of Bis-A apoptosis-inducing activity. Western blot analysis on total cell lysate harvested from A549 cells at 0, 6, 12, and 24 h. The activity of Bis-A at 24 μM on apoptosis-related proteins, Bcl2 subfamily proteins (Bcl-xL, Mcl-1, and Bcl-2), Bax subfamily protein (Bax1), BH3-only proteins (Bid, PUMA, Bim, and Bik), and tumor suppressor (p53) level was investigated.
unaffected by treatment with 24 μM Bis-A. However, cells treated with 100 nM vinblastine showed decreased Bcl-xL, Mcl1, and BimEL expression 24 h after treatment. On the other hand, the expression of p53 increased 24 h after treatment with vinblastine (Figure 3). Collectively, the decrease in Bcl-xL, Mcl1, and BimEL expression and increase in p53 expression indicated that vinblastine causes apoptosis. The expression of apoptosis-regulating proteins in cells treated with a high concentration of Bis-A did not change under the same experimental conditions as in vinblastine-treated cells. Thus, C
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Figure 4. (A) Effect of Bis-A at 6 μM on LC3 lipidation investigated using A549 and MCF7 cell lines. The expression level of LC3-I and LC3-II of total cell lysates harvested from A549 and MCF7 cells at 0, 4, 8, 12, or 24 h was investigated. EVERO at 5 μM was used as a positive control for autophagy induction. (B) Autophagy flux studied by monitoring LC3 and p62 expression. LC3 and p62 turnover was also investigated by addition of BAF. A549 cells were treated with Bis-A (6 μM) and BAF (400 nM) for 6 h. Cells were cultured in serum-free medium for 1 h prior to addition of Bis-A and BAF. EVERO at 5 μM was used as a positive control for autophagy induction. (C) Effect of Bis-A on formation of autophagic punctation D
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Figure 4. continued in A549 cells investigated using fluorescence microscopy. Cells were cultured in serum-free medium for 1 h prior to treatment with Bis-A (6 μM), EVERO (5 μM), and BAF (400 nM) for 6 h. Cells were labeled with LC3, LAMP2, and p62. Scale bar = 20 μm.
not. This down-regulation suggested that Bis-A inactivates mTOR via suppression of PRAS40 phosphorylation. Furthermore, Bis-A also down-regulated the phosphorylation of AKT thr308 from 3 to 24 h after treatment. Phosphorylation of AKT at ser473 was down-regulated from 3 to 6 h. However, the phosphorylation of PDK1 at ser241 was unaffected by Bis-A. These results suggested that Bis-A induces autophagosome formation by interfering with the AKT-mTOR signaling pathway. Many alkaloids are known to possess autophagy- and apoptosis-inducing activity. These alkaloids were reported to induce autophagy via different mechanisms. Harmol, a βcarboline alkaloid, was reported to induce autophagy via activation of the extracellular-signal-regulated kinase (ERK1/2) pathway. Additionally in the same article, harmol also showed no effect on the AKT-mTOR pathway.43 Evodiamine, an indoloquinazoline alkaloid, induces calcium/JNK-mediated autophagy. Apart from autophagy, evodiamine was also reported to induce apoptosis in human glioblastoma cells.44 Piperlongumine promotes autophagy by interfering with AKT phosphorylation and subsequently inhibits mTOR activation. However, piperlongumine is also reported to cause apoptosis.45 Bis-A, formed from eburnane and aspidosperma skeletons, is categorized as a dimeric alkaloid. Similar to monomeric alkaloids, dimeric alkaloids are also known to have cytotoxic activity. Vinblastine, a dimeric alkaloid generated from combining catharanthine and vindoline, had been reported to induce both autophagy and apoptosis.46,47 Similar to Bis-A, mekongenines C−F are also eburnane−aspidosperma-type bisindole alkaloids. Mekongenines C−F isolated from Bousigonia mekongensis were reported to have cytotoxic activity, but their mechanism of action is yet to be elucidated.48 The effect of Bis-A in inducing autophagosome formation without apoptosis is noteworthy because many alkaloids were reported to either induce apoptosis independently or induce apoptosis and autophagy mutually. However, a clear explanation as to how Bis-A induces autophagosome formation devoid of apoptosis is yet to be found. This is due to the fact that interferences of AKT-mTOR signaling can have both proapoptotic and anti-apoptotic roles.49,50 In this study, Bis-A not only inhibited the AKT-mTOR signaling pathway but might also modulate lysosome function due to its effect in elevating p62 expression and changing the distribution of LAMP2. The relation between the inhibition of the AKT-mTOR signal by Bis-A and its action on lysosomes is presently not clear. Elevation of p62 was reported in the condition where AKT phosphorylation was inhibited by knocking out the tuberous sclerosis complex (TSC), a downstream substrate of AKT.51 In the future, it is necessary to further investigate the action of Bis-A on lysosomes, in which the inhibition of AKT phosphorylation might explain the increase in the p62 level.
remain separated in BAF-only-treated cells (Figure 4C). Collectively, the data from immunofluorescence staining and Western blot suggested that Bis-A induced autophagosome formation but prevented the fusion of autophagosomes and lysosomes through an unknown mechanism. Regulation of AKT-mTOR Pathway by Bis-A. It is well established that the AKT-mTOR pathway plays a major role in autophagy suppression. The mammalian target of rapamycin (mTOR) is a critical modulator of autophagy, particularly the lipidation of LC3. Activated mTOR suppresses autophagy, while its inactivation promotes autophagy.25,32−36 Activation of mTOR, an upstream regulator of LC3 lipidation, was investigated using A549 cells to elucidate the mechanism of action of Bis-A in inducing autophagosome formation. Bis-A does not affect the phosphorylation of mTOR at ser2448, as shown by Western blot analysis. Treatment with EVERO (positive control) at 5 μM was shown to effectively down-regulate phosphorylation of mTOR at ser2448 from 3 to 24 h (Figure 5). However, both Bis-A and EVERO were shown
Figure 5. Expression of AKT-mTOR pathway related proteins investigated in A549 total cell lysate harvested at 3, 6, 12, or 24 h after treatment with 6 μM Bis-A. EVERO at 5 μM was used as a positive control for mTOR deactivation. Phosphorylation of mTOR at ser2448 was unaffected by Bis-A as compared to EVERO.
to down-regulate the phosphorylation of p70S6K at thr389, a mTOR-dependent kinase.37,38 These results suggested that BisA may inactivate mTOR via a different mechanism from EVERO. The proline-rich Akt substrate of 40 kDa (PRAS40) is a repressor of mTOR. PRAS40 association with mTOR results in mTOR inactivation, while disassociation results in mTOR activation. The association and disassociation mechanism is regulated through AKT-dependent PRAS40 phosphorylation.39−42 Thus, the phosphorylation of PRAS40 at thr246 was investigated. Treatment with Bis-A down-regulated phosphorylation of PRAS40 thr246 from 3 to 12 h (Figure 5), while EVERO did
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CONCLUSIONS In conclusion, a bisindole alkaloid with an eburnane− aspidosperma dimer skeleton was found to induce autophagosome formation, which precedes autophagy. The induction of E
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Cell Culture. The human lung adenocarcinoma epithelial cell line A549 and human breast adenocarcinoma cell line MCF7 (Riken Cell Bank, Ibaraki, Japan) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, St. Louis, MO, USA), and the human promyelocytic leukemia cell line, HL-60, was maintained in RPMI1640. Growth media were supplemented with 10% FBS (fetal bovine serum, Cell Culture Bioscience, Tokyo, Japan) and 1% penicillin (10 000 U/mL)−streptomycin (10 mg/L). Cytostatic Activity. Cytostatic activity of Bis-A and vinblastine (positive control) was also evaluated indirectly via the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which is based on mitochondrial succinate dehydrogenase activity and confirmed via microscopic observation. At the end of incubation, 15 μL of MTT (Sigma, USA) at 5 mg/mL was added to each of the wells. The cultures were incubated for another 3 h before the media was removed. After the removal of the media, 100 μL of dimethyl sulfoxide was added to each well to dissolve formed formazan. The optical density was measured using a microplate reader (Bio-Rad, Irvine, CA, USA) at 550 nm with a reference wavelength of 700 nm. Flow Cytometry. The oxidative effect of Bis-A was measured via flow cytometry. H2DCFDA (20 μM) was added to A549 cells (4 × 105 cells/dish) and preincubated for 1 h. Bis-A (24 μM) and hydrogen peroxide (100 μM) (positive control) were then added to the A549 cells, and the mixture was incubated in the dark at 37 °C for another 1 h. Before flow cytometric analysis, cells were washed and resuspended in FACS buffer with 0.3% BSA and analyzed by using BD Biosciences FACSCalibur flow cytometer. The cytostatic effect of Bis-A was also analyzed using flow cytometry. A549 cells (5 × 105 cells/dish) were incubated with BisA (24 μM) or vinblastine (100 nM; positive control) for 24 h. At the end of the incubation the cells were washed twice with phosphatebuffered saline (PBS) and stained with FITC conjugate annexin V and propodium iodide in FACS buffer with 0.3% BSA. Before flow cytometric analysis, cells were washed and resuspended in FACS buffer with 0.3% BSA and analyzed by using a BD Biosciences FACSCalibur flow cytometer. Western Blotting. Total cellular protein and nuclear protein were prepared from A549 and MCF7 cell lines at various time points. Total protein concentration was quantified using the BCA protein assay kit (ThermoScientific, Rockford, IL, USA). Proteins were resolved using SDS-polyacrylamide gel electrophoresis. Resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Hybond − P, GE Healthcare, Buckinghamshire, United Kingdom). Primary antibodies were prepared by diluting with BlockAce. Primary antibodies used in this study were β-actin (Sigma-Aldrich); phospho-mTOR (ser2448), mTOR (7C10), phospho-AKT (thr308), phospho-AKT (ser473), AKT (C67E7), phosphop70S6K (thr389), p70S6K (49D7), phospho-PDK1 (ser241), PDK1, PRAS40 (thr246), PRAS40 (D23C7), PUMA, Bim, Bik, and LC3B (Cell Signaling Technology, CA, USA); Bax1, Bcl-xL, Bcl-2, Mcl-1, Bid, and p53 (BD Biosciences, San Jose, CA, USA); and p62 (PROGEN Biotechnik GmbH, Heidelberg, Germany). Horseradish peroxidaselinked secondary antibodies were used in this study (Amersham, GE Healthcare). Fluorescence was detected using an Immunostar LD kit (WAKO, Osaka, Japan) and viewed with a BIAquire 285II imaging system. Immunofluorescence Staining. A549 cells were cultured on chamber slides, fixed with ice-cold methanol for 15 min at −20 °C, and subsequently blocked with Immunoblock (DS Pharma Biomedical, Osaka, Japan). Primary antibodies used for staining are LC3B (Cell Signaling Technology), LAMP2 (Abcam, Cambridge, UK), and p62 (PROGEN Biotechnik GmbH, Heidelberg, Germany). Second antibodies used are Alexa Fluor 488 anti-rabbit IgG, Alexa Fluor 546 anti-mouse IgG, and Alexa Fluor 647 anti-guinea pig IgG (Invitrogen, Carlsbad, CA, USA), respectively. Secondary antibody stained slides were sealed with VECTASHILD antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). All images were acquired using a confocal laser microscope (FV1200 IX83, Olympus, Tokyo, Japan).
Figure 6. Autophagy is preceded by the conversion of LC3-II from LC3-I, a component of the autophagosome double membrane. In a condition that is favorable for cell growth, the lipidation of LC3 is inhibited by mTOR, effectively preventing autophagosome formation. Activation of mTOR is regulated by the upstream kinase AKT, which relays information from the membrane-bound receptor in response to the presence of growth factors. Growth factors activate AKT via phosphorylation at thr308 and ser473 by PDK1 and PDK2/mTORC2, respectively. Activated AKT relays its information to PRAS40 by phosphorylating it at thr246. Nonphosphorylated PRAS40 functions as an mTOR repressor. Phosphorylation of PRAS40 causes its disassociation from mTOR, activating mTOR and subsequently inhibiting autophagy under conditions favorable for cell growth. BisA deactivates mTOR by preventing the disassociation of PRAS40 from mTOR. Bis-A prevents the disassociation of PRAS40 by inhibiting the phosphorylation of AKT, the upstream kinase of PRAS40. Deactivation of mTOR subsequently led to LC3 lipidation and subsequent formation of autophagosomes.
autophagosome formation may explain the previously reported Bis-A cytostatic activity. Bis-A up-regulation of LC3 lipidation may be achieved by mTOR inactivation. The phosphorylation of PRAS40, an mTOR repressor, was suppressed by Bis-A. This result suggests that Bis-A may inactivate mTOR via suppression of PRAS40 phosphorylation. Furthermore, the phosphorylation of AKT, an upstream regulator of PRAS40 phosphorylation, was also down-regulated by Bis-A. Together these findings suggested that Bis-A induces autophagosome formation by interfering with the AKT-mTOR signaling pathway. Although Bis-A was suggested to induce formation of autophagosomes, Bis-A also interrupts the autophagic flux by preventing fusion of autophagosomes and lysosomes through an unknown mechanism. Further investigations are required to unravel this unknown mechanism.
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EXPERIMENTAL SECTION
Plant Material. Leuconotis grif f ithii Hk.f was collected from Mersing, Malaysia, in 2001. The botanical identification was made by Mr. Teo Leong Eng, Faculty of Science, University of Malaya. The voucher specimen (Herbarium No. KL 4976) was deposited at the Herbarium of the Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia. Isolation and Purification. Bisleuconothine A from L grif fithii bark was isolated according to the methods described previously.17 Purification of isolated Bis-A was done by recrystallization and confirmed using high-performance liquid chromatography (HPLC). Bis-A used in present study is approximately 97% pure, as confirmed by HPLC data (Figure S1, Supporting Information). F
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ASSOCIATED CONTENT
S Supporting Information *
HPLC data on the purity of Bis-A. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00258.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +813-5498 5778. Fax: +813-5498 5778. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant from the Open Research Center Project.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.5b00258 J. Nat. Prod. XXXX, XXX, XXX−XXX
