Ancistrolikokine E3, a 5,8â²-Coupled ... - ACS Publications

Article Cite This: J. Nat. Prod. 2018, 81, 2282−2291

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Ancistrolikokine E3, a 5,8′-Coupled Naphthylisoquinoline Alkaloid, Eliminates the Tolerance of Cancer Cells to Nutrition Starvation by Inhibition of the Akt/mTOR/Autophagy Signaling Pathway Suresh Awale,*,† Dya Fita Dibwe,† Chandrasekar Balachandran,† Shaimaa Fayez,‡ Doris Feineis,‡ Blaise Kimbadi Lombe,‡,§ and Gerhard Bringmann*,‡ †

Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan Institute of Organic Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany § Faculté des Sciences, Université de Kinshasa, B.P. 202, Kinshasa XI, Democratic Republic of the Congo J. Nat. Prod. 2018.81:2282-2291. Downloaded from pubs.acs.org by UNIV OF WINNIPEG on 10/26/18. For personal use only.

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ABSTRACT: PANC-1 human pancreatic cancer cells are characterized by their ability to proliferate aggressively under hypovascular and hypoxic conditions in the tumor microenvironment, displaying a remarkable tolerance to nutrition starvation. The antiausterity strategy is a new approach in anticancer drug discovery aiming at the identification of potent agents that inhibit preferentially the survival of tumor cells during a limited supply of nutrients and oxygen. The new 5,8′-coupled naphthyldihydroisoquinoline alkaloid ancistrolikokine E3 (4), isolated from the Congolese liana Ancistrocladus likoko, showed potent preferential cytotoxicity against PANC-1 cells under nutrient-deprived conditions, with a PC50 value of 2.5 μM, without exhibiting toxicity in normal, nutrient-rich medium. The compound was found to induce dramatic alterations in cell morphology, leading to cell death. Moreover, it inhibited significantly PANC-1 cell migration and colony formation in a concentration-dependent manner. This study on 4 provides the first live evidence of the effect of a naphthyldihydroisoquinoline alkaloid against PANC-1 cells in nutrient-deprived medium. Mechanistic investigations conducted suggest that compound 4 is a potent inhibitor of the activation of the Akt/mTOR pathway. Furthermore, it inhibited the expression levels of the key autophagy regulators Atg5, Atg12, Beclin-1, LC3-I, and LC3-II. The results demonstrated that ancistrolikokine E3 (4) is a potent early-stage inhibitor of the autophagy pathway in PANC-1 human pancreatic cancer cells. Ancistrolikokine E3 (4) and related naphthylisoquinoline alkaloids are promising potential lead compounds for anticancer drug development based on the antiausterity strategy.

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novel therapeutic strategies to target pancreatic cancer are urgently required. Human pancreatic cancer cells are known to proliferate rapidly and aggressively, causing a chronic deficiency of essential nutrients and oxygen due to their high metabolic need, combined with inefficient vascular supply.11,12 Thus, in contrast to normal tissues, the microenvironment of pancreatic tumors is characterized by low levels of nutrients and oxygen. The ability of pancreatic cancer cells to tolerate these extreme hypovascular and hypoxic conditions, by displaying a remarkable resistance to starvation, is referred to as “austerity”.13−15 The search for potent new agents that preferentially affect tumor cells under nutrient deprivation has emerged as the “antiausterity approach” in anticancer drug discovery.16−18 Based on this concept, phytochemical investigations on Asian medicinal plants have already provided several promising candidates,16−21 with the most important

ancreatic cancer is one of the most common lethal malignancies worldwide, with a 5-year survival rate of less than 5%.1,2 Over the past decades, the incidence of this devastating disorder has dramatically increased in industrialized nations, in particular in the United States,3 Japan,4 and European countries,5 with a strong rise of mortality rates6 for both men and women. While for many solid tumors, such as melanoma, colon, or breast malignancies, important improvements in diagnosis and treatment have been achieved during the past years,7,8 the prognosis for patients with pancreatic cancer still remains extremely poor.1,2 Current therapeutic interventions,1,2,9 such as surgical resection, radiation, and immuno- or chemotherapy, suffer from the fact that the disease is usually diagnosed at an advanced and, in most cases, metastatic stage. Moreover, conventional and targeted therapeutic protocols clinically used for the treatment of pancreatic cancer often fail to be effective due to intrinsic (de novo) or acquired (therapy-induced) drug resistance and because of impaired drug delivery pathways.1,2,10 Therefore, © 2018 American Chemical Society and American Society of Pharmacognosy

Received: August 30, 2018 Published: October 10, 2018 2282

DOI: 10.1021/acs.jnatprod.8b00733 J. Nat. Prod. 2018, 81, 2282−2291

Journal of Natural Products

Article

one being arctigenin,15,22 which has successfully passed an early phase II human clinical trial in the National Cancer Center Hospital East (Kashiwa, Japan), revealing a significant survival benefit for patients in an advanced stage of pancreatic cancer, without showing toxic side effects.23 More recently, we have reported on the isolation and biotesting of mono- and dimeric naphthylisoquinoline alkaloids from Congolese Ancistrocladus plants, such as jozilebomine B (1),24 ancistrobonsoline A1 (2),25 and ancistrolikokine H2 (3)26 (Figure 1), which displayed strong

cytotoxicity against PANC-1 cells in nutrient-deprived medium (NDM), with no toxicity under normal, nutrient-rich conditions (Dulbecco’s modified Eagle’s medium, DMEM), exhibiting a PC50 value of 2.5 μM (i.e., the concentration at which 50% of the pancreatic cancer cells were killed in NDM without displaying toxicity in DMEM). The compound most efficiently inhibited cell migration and colony formation; thus investigations on its mode of action were performed.

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RESULTS AND DISCUSSION Isolation and Structural Elucidation of Ancistrolikokine E3 (4). Ground material of air-dried twigs of A. likoko27 were extracted exhaustively with CH2Cl2−MeOH (1:1, v/v) at room temperature. The crude extract was subjected to liquid− liquid separation, followed by column chromatography (CC) on silica gel, and the resolved metabolites were finally purified by preparative reversed-phase HPLC, thus permitting isolation of a new metabolite, obtained as a yellow, amorphous solid. According to the HRESIMS and 13C NMR data, it displayed a molecular formula of C25H27NO4, which, together with the UV spectrum, suggested it to be a naphthyldihydroisoquinoline alkaloid. The 1H NMR spectrum revealed the presence of the spin system of a substituted naphthalene with four aromatic methines, H-1′ (δH 6.57), H-3′ (δH 6.80), H-6′ (δH 6.78), and H-7′ (δH 7.05), a methoxy function, OCH3-4′ (δH,C 4.08, 56.9), and an aromatic methyl group, CH3-2′ (δH,C 2.29, 22.1). Furthermore, another spin system was observed, corresponding to a dihydroisoquinoline subunit, with a shielded aromatic proton at δH 6.82 (H-7), an unshielded aliphatic methine at δH 3.78 (H-3), two diastereotopic protons, Heq-4 (δH 2.69) and Hax-4 (δH 2.26), two methoxy groups, OCH3-6 (δH,C 3.82, 56.8) and OCH3-8 (δH,C 4.14, 57.0), and two methyl groups, CH3-1 (δH,C 2.80, 24.9) and CH3-3 (δH,C 1.19, 17.8). The constitution of the isolated compound was additionally supported by two NOESY correlation sequences, {H-1′ ↔ CH3-2′ ↔ H-3′ ↔ OCH3-4′} and { CH3-1 ↔ OCH3-8 ↔ H-7 ↔ OCH3-6} (Figure 2A), thus clearly establishing the biaryl axis to be located at C-8′ in the naphthalene part and at C-5 in the dihydroisoquinoline moiety. The 5,8′-coupling site was also corroborated by HMBC correlations from H-7 and H-7′ to C-5 (δC 123.7) and by those from both H-1′ and H-6′ to C8′ (δC 123.2) (Figure 2A). The absolute configuration at the stereocenter in the dihydroisoquinoline moiety was determined by rutheniummediated oxidative degradation with subsequent GC-MSD analysis of the chiral degradation products. This efficient analytical method was developed by us earlier.28 The exclusive formation of the R-enantiomer of 3-aminobutyric acid clearly established the alkaloid to be R-configured at C-3. The absolute configuration at the biaryl axis was deduced to be P, by NOESY interactions between H-4eq and H-7′, on one hand, and between H-4ax and H-1′, on the other (Figure 2B). This was in agreement with the ECD spectrum of the compound (Figure 2C), which was virtually opposite to that of the known25 structurally closely related, but M-configured alkaloid ancistrobonsoline A (2) (see structures in Figure 1). Thus, the new alkaloid possesses the stereostructure 4 as outlined in Figure 1. It resembles the molecular framework of the co-occurring ancistrolikokines E and E2,26 previously isolated from A. likoko as minor constituents. Compared with the latter two compounds, the new metabolite shows a higher degree of O-methylation in the dihydroisoquinoline portion. In continuation of this series of 5,8′-linked naphthyldihydroiso-

Figure 1. Mono- and dimeric naphthylisoquinoline alkaloids with antiausterity activities against PANC-1 human pancreatic cancer cells, jozilebomine B (1), ancistrobonsoline A1 (2), ancistrolikokine H2 (3), and the highly potent new agent ancistrolikokine E3 (4), for the latter its discovery and biotesting are presented in the present report.

antiausterity activities against PANC-1 human pancreatic cancer cells inducing morphological alterations such as membrane rupture and disintegration of cell organelles. Some of the alkaloids, among them jozilebomine B (1), significantly inhibited colony formation of PANC-1 cells, even when the cells were exposed to noncytotoxic concentrations of the agent for a short time. In this paper, we report on the isolation and structural elucidation of ancistrolikokine E3 (4), a new 5,8′-coupled naphthylisoquinoline alkaloid discovered in the twigs of Ancistrocladus likoko J. Léonard (Ancistrocladaceae), and on the investigations of its antiausterity potential. A. likoko is a tropical liana endemic to the evergreen rainforests and swamp regions of the north-central area of the Democratic Republic of the Congo.27 The new alkaloid 4 showed potent preferential 2283



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Figure 3. Preferential cytotoxic activity of ancistrolikokine E3 (4) against the PANC-1 human pancreatic cancer cell line in nutrientdeprived medium (NDM) and Dulbecco’s modified Eagle’s medium (DMEM).

in NDM in a concentration-dependent manner, displaying a PC50 value of 2.5 μM (i.e., the concentration at which 50% of the tumor cells were preferentially killed in NDM without exhibiting cytotoxicity in DMEM). Due to its potent activity to retard the survival of PANC-1 tumor cells comparable to that of arctigenin15 (PC50 0.83 μM), which was used as the positive control in this study, the new alkaloid 4 was classified as a rewarding candidate for further studies on its properties as a potential antiausterity agent. Morphological Changes of PANC-1 Tumor Cells. Ancistrolikokine E3 (4) was studied further for its effects on the cell morphology and apoptosis of PANC-1 cells under nutrient-deprived conditions using the ethidium bromide (EB)/acridine orange (AO) double-staining fluorescence assay. PANC-1 cells treated with 1.25, 2.5, and 5 μM compound 4 and untreated PANC-1 cells, serving as the control, were incubated in NDM for 24 h and then stained with the EB/AO reagents. Both compounds AO and EB are DNA-intercalating agents. AO is a cell-membrane-permeable dye emitting bright green fluorescence in living cells, while EB is only capable of penetrating the membrane of dead or dying cells, staining them red when intercalated into double-strand DNA. During cell death, through apoptosis or necrosis, the integrity of the cell membrane is loosened, and both reagents EB and AO penetrate into the cell. During late apoptosis and necrosis, AO and EB infiltrate the cells and then emit orange fluorescence. As shown in Figure 4 (left), untreated PANC-1 cells (the control) only emitted bright green fluorescence in AO/EB staining, displaying intact cell morphology and chromatin organization. Treatment with ancistrolikokine E3 (4), however, led to a concentration-dependent increase of EBstained cells emitting red fluorescence and displaying an apparent alteration of cell morphology (Figure 4, center). Incubation of PANC-1 cells with 5 μM compound 4 even resulted in a red EB fluorescence exclusively, thus revealing necrotic-type cell death, as characterized by rounded cell morphology, membrane blebs, rupture of the cell membrane, and leakage of cellular contents into the culture medium. Inhibition of PANC-1 Cell Migration. One of the most frequently encountered failures in pancreatic cancer treatment results from the distant-organ metastases at the very early stages of diagnosis, making surgery almost impossible.29 Cancer metastasis starts with cell migration, followed by mesenchymal transition, intravasation into blood vessels, and extravasation to distant organs.30 Therefore, inhibitors of cancer cell migration have a potential of suppressing the

Figure 2. (A) Selected 1H and 13C NMR data (δ in ppm) of ancistrolikokine E3 (4), as well as the decisive HMBC (single red arrows) and NOE (double blue arrows) interactions. (B) NOESY correlations evidencing the configuration of 4 at the biaryl axis relative to the stereogenic center at C-3. (C) Assignment of the absolute axial configuration of 4, by comparison of its ECD spectrum with that of the known,25 structurally related, yet oppositely configured alkaloid ancistrobonsoline A (2).

quinoline alkaloids with an R-configuration at C-3 and a Pconfiguration at the biaryl axis,26 the new metabolite was named ancistrolikokine E3. Preferential Cytotoxicity of Ancistrolikokine E3 (4) toward PANC-1 Tumor Cells. Human PANC-1 pancreatic cancer cells show a high tolerance toward nutrient starvation and possess the ability to survive for over 3 days even in the complete absence of essential nutrients such as glucose, amino acids, and serum.13 Following the antiausterity strategy,13,17 the new alkaloid ancistrolikokine E3 (4) was investigated for its preferential cytotoxic activity against the PANC-1 tumor cell line in nutrient-deprived medium versus normal, nutrient-rich medium (DMEM). As outlined in Figure 3, the compound showed potent preferential cytotoxicity against PANC-1 cells 2284



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Figure 4. Morphological changes of PANC-1 cells induced by ancistrolikokine E3 (4) in comparison to untreated cells (i.e., the control) in nutrient-deprived medium (NDM). PANC-1 tumor cells were treated with compound 4 at the indicated concentrations in NDM in a 12-well plate and incubated for 24 h. Cells were stained with ethidium bromide (EB) and acridine orange (AO) and photographed under fluorescence (red and green) and phase contrast modes using an EVOS FL digital microscope.

process of tumor metastasis formation.31 Thus, the ability of 4 to inhibit PANC-1 cell migration was investigated further using a wound-healing assay. Ancistrolikokine E3 (4) was exposed to PANC-1 cells at a noncytotoxic concentration in a standard nutrient-rich medium (DMEM) against the monolayer of PANC-1 cell scratch to study more closely whether 4 is able to suppress the PANC-1 cell mobility and to inhibit wound healing when tumor cells are exposed to the compound for 48 h. PANC-1 human pancreatic cancer cells (5 × 105 cells/well) were seeded on a 12-well plate to form a monolayer of 90% confluence in standard growth medium (DMEM). A scratch wound of ca. 600 μm was made in the center of each well using the tip of a 25 μL micropipet. The cells were then incubated for 48 h in either DMEM alone (the control) or DMEM with 5.0 or 10.0 μM ancistrolikokine E3 (4). At the end of the experiment, the wound areas in each well were photographed in the phase-contrast mode (Figure 5A) and the wound lengths were compared with those of the control. The average value calculated for the wound length of the control significantly decreased by 73%. The exposure of the tumor cells to compound 4, however, substantially inhibited the wound closure in a concentration-dependent manner. The average wound lengths of PANC-1 cell monolayer exposed to 5.0 and 10.0 μM ancistrolikokine E3 (4) were found to be reduced to only 58% and 11%, respectively, when compared to initial wound length at T0 (Figure 5B). Inhibition of Colony Formation. During cancer invasion and metastasis, the invading cancer cells adapt to foreign tissue microenvironments and form small colonies of cancer cells

(micrometastases), which then grow into large tumors. This phenomenon is termed “colonization”.32 Most pancreatic tumor patients quickly develop liver metastases soon after the time of diagnosis.33 In the present investigation, the effect of ancistrolikokine E3 (4) against PANC-1 cell colony formation was thus further investigated. For this purpose, PANC-1 cells were exposed to 4 at concentrations of 2.5, 5.0, and 10.0 μM (noncytotoxic concentrations) in DMEM for 24 h. The medium was then replaced with fresh DMEM and placed in a CO2 incubator to allow colony formation for 12 days. As shown in Figure 6, in the absence of compound 4 (the control), PANC-1 tumor cells grew aggressively to form a large number of colonies occupying 86% of the total well area. When exposed to ancistrolikokine E3 (4) at a noncytotoxic concentration for 24 h in DMEM, however, colony formation was found to be inhibited significantly in a concentrationdependent manner. Most definitively, compound 4 already reduced colony formation strongly at the lowest concentration of 2.5 μM used in this study, causing a drastic reduction (up to 90%) of colony formation, since only 8% of the well area colonies were detected compared to 86% of the well area showing colony formation in the untreated control. Exposure to 5 and 10 μM 4 even resulted in a 100% inhibition of colony formation (Figure 6). Live Evidence of the Anticancer Potential of Ancistrolikokine E3 (4). The promising antiproliferative and antimetastatic activities of the new alkaloid 4 presented in this paper suggested this naphthyldihydroisoquinoline to be a rewarding candidate for further studies regarding its 2285



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Figure 5. Ancistrolikokine E3 (4) suppresses the migration of PANC-1 cells in a concentration-dependent manner in the wound-healing assay when subjected for 48 h. (A) The broken white line indicates the wound length at the start of the experiment. The assay was repeated three times, and the representative images are shown. (B) Quantification of wound healing by measuring the wound length. Data are means ± SD. ***p < 0.001 vs control.

anticancer potential against human pancreatic cancer. Therefore, in order to disclose detailed events of PANC-1 cell death induced by compound 4 in a real-time frame, a time-lapse live imaging experiment was performed to observe the effect of 4 on pancreatic tumor cells. For this purpose, PANC-1 cells were treated with 10 μM compound 4 in NDM and incubated in a stage-top incubator at 37 °C in an atmosphere of 5% CO2. The images were captured every 10 min in the phase-contrast mode on an EVOS FL digital cell imaging system for 24 h. As seen in Movie 1, treatment with ancistrolikokine E3 (4) inhibited the cell mobility within 90 min and induced morphological alteration of PANC-1 cells after 7 h, leading to cell death after 13 h, finally causing complete cell death within 20 h (Movie 1). PANC-1 cells cultured under the same conditions in NDM, yet without compound 4 (control), survived until the end of the experiment (24 h) (Figure 4). Thus, this study provided the first live evidence of the anticancer therapeutic potential of the 5,8′-coupled new naphthyldihydroisoquinoline alkaloid ancistrolikokine E3 (4). Ancistrolikokine E3 (4) Induces PANC-1 Cell Death without the Signs of a Classic Apoptotic Pathway. Apoptosis is one of the most common pathways of programmed cell death.34 Accordingly, most of the chemotherapeutic agents against cancer are investigated for their ability to induce apoptosis. Cells undergoing apoptosis show characteristic morphological features such as membrane blebbing, chromatin fragmentation, and formation of apoptotic bodies.34 To study more closely whether cell death induced by

Figure 6. Effect of ancistrolikokine E3 (4) on colony formation by PANC-1 cells. (A) PANC-1 cell colonies treated with different concentrations of compound 4. (B) Graph showing mean values of the area occupied by PANC-1 cell colonies (three replications). ****p < 0.0001, *p < 0.05 when compared with the untreated control group.

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pamoate, kigamicin D, and grandifloracin have been found to be potent inhibitors of Akt.14−16 The activation of Akt involves phosphorylation of the two key regulatory sites, Thr-308 and Ser-473. Upon activation, it transduces signals to the downstream substrates mTOR (the mammalian target of rapamycin) and regulates various intracellular processes in cancer cells, such as inhibition of apoptosis, regulation of cell survival, tumor progression, and metastasis.38 Deregulation of multiple elements of the Akt/ mTOR pathway has been reported in many types of cancers, e.g., in melanoma, where alterations in major components of this pathway significantly affected tumor progression.37,39 As a consequence, Akt/mTOR is an attractive therapeutic target in the search for new anticancer agents. Therefore, the effect of the new alkaloid 4 on the activation of Akt/mTOR in PANC-1 cells was investigated by Western blot analysis. As shown in Figure 8, there was a significant increase in phosphorylation of

ancistrolikokine E3 (4) involved apoptosis, the Hoechst 33342 staining assay was performed. For this purpose, PANC-1 cells were treated with 5 μM 4 in NDM and incubated for 24 h. The cells were then stained with Hoechst 33342 and visualized by fluorescence microscopy. The treated cells revealed total cell death with membrane blebs and leaking of cellular contents into the medium (Figure 7A). Hoechst 33342 stained cells,

Figure 7. Assessment of apoptosis as a possible factor involved in PANC-1 cell death induced by ancistrolikokine E3 (4): (A) Hoechst 33342 staining intact nucleus; (B) Western blot against Bcl-2, Bcl-xL, and caspase 3.

Figure 8. Effects of ancistrolikokine E3 (4) against key proteins involved in Akt/mTOR and in the autophagy signaling pathway.

however, showed intact nuclei and lacked the classical signs of apoptosis such as chromatin fragmentation and formation of apoptotic bodies (Figure 7A). These findings indicated that apoptosis is not involved in PANC-1 cell death induced by ancistrolikokine E3 (4). To confirm this assumption, a Western blot analysis was performed against key proteins involved in apoptosis. Mechanistically, one of the remarkable features of apoptosis is the cleavage of caspase-3 into active cleaved caspase-3, while Bcl-2 and Bcl-xL proteins negatively regulate apoptosis and function as antiapoptotic proteins. Treatment of PANC-1 cells with compound 4 for 6 h inhibited Bcl-2, Bcl-xL, and caspase-3 in a concentration-dependent manner (Figure 7B). Interestingly, it did not induce the formation of active cleaved caspase3. The outcome of these investigations also suggested that PANC-1 cell death induced by ancistrolikokine E3 (4) does not involve the caspase-dependent apoptotic signaling cascade. Ancistrolikokine E3 (4) Induces PANC-1 Cell Death by Inhibition of Akt/mTOR and the Autophagy Signaling Pathway. Akt is a serine/threonine kinase that is activated in the majority of human tumors and regulates tumor cell functions such as cell survival, proliferation, metabolism, angiogenesis, migration, and invasion.35 Akt is also activated during nutrition starvation and confers tolerance to nutrition starvation.13 Therefore, targeting the Akt signaling pathway is one of the primary therapeutic strategies in cancer therapy.36,37 Previously, antiausterity agents such as arctigenin, pyrvinium

Akt and mTOR within 6 h in the control cells cultured in nutrient-deprived medium compared to DMEM. Treatment with 4, however, inhibited total Akt, p-Akt, mTOR, p-mTOR, and GβL in a concentration-dependent manner. Significant inhibition was observed at the alkaloid concentrations of 20 and 40 μM, leading to an inhibition of p-Akt of 65% (in the case of 20 μM 4) and of even 99% (in the case of 40 μM 4) in comparison to the control. Similar results were also obtained for total Akt, giving rise to an inhibition of 48% after treatment of the cells with 20 μM 4 and of 86% after application of 40 μM compound 4, as compared to the control. In a similar manner, a concentration of 40 μM 4 inhibited the expression of p-mTOR and mTOR by 92% and 100%, respectively. In DMEM, however, no apparent changes in the Akt/mTOR proteins were observed. These data strongly suggest that inhibition of the Akt/mTOR signaling pathway significantly contributed to the preferential cytotoxicity of ancistrolikokine E3 (4) toward PANC-1 cells in NDM. These results prompted an in-depth investigation as to whether the inhibition of the Akt/mTOR pathway by ancistrolikokine E3 (4) affects the downstream autophagy signaling pathway. Autophagy is a natural process of cellular recycling. During autophagy, unwanted proteins and damaged mitochondria are engulfed by autophagosomes and digested in lysosomes to refuel the cells with essential amino acids, thus maintaining cellular metabolism.40 Autophagy can be activated by many factors, particularly in the case of extreme nutrient 2287



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10).45 Immunoblot analysis revealed that 4 inhibited the expression of all the key regulators of autophagy, Beclin-1, Atg5, Atg12, LC3-I, and LC3-II, in a concentration-dependent manner (Figure 8). These results suggested strongly that ancistrolikokine E3 (4) is a potent inhibitor of autophagy. Autophagy inhibitors can be classified as early- or late-stage inhibitors. Early stage inhibitors such as 3-methyladenine (3MA) interfere during the recruitment of membranes and inhibit the autophagosome formation. On the other hand, latestage inhibitors, such as chloroquine (CQ), prevent the acidification of lysosomes and impair autophagosome degradation. Therefore, to understand the stage at which the alkaloid 4 interferes with the autophagy pathway, a cotreatment experiment with 3-MA and CQ was carried out to study more closely the expression of the key autophagy marker LC3. As shown in Figure 9A, treatment of PANC-1 cells with CQ led to a nearly 2-fold increase in the accumulation of LC3-II in NDM (control). 3-MA, on the other hand, inhibited the expression of LC3-II by a factor of nearly 2. Treatment with 40 μM compound 4 was found to significantly inhibit the expression of both LC3-I and LC3-II. This was further confirmed by the combination of 4 with CQ, which also resulted in a nearly complete inhibition of the expression of LC3-II compared to the effects induced by CQ alone. These data suggested strongly that compound 4 inhibits autophagy at an early stage and interferes with membrane nucleation and autophagosome formation. Inhibition of autophagy by 4 was also confirmed by fluorescence microscopy using staining experiments with monodansylcadaverine dye (MDC), a specific marker for autolysosomes. PANC-1 cells were treated with MDC and with or without 4 and incubated for 6 h. The cells were then visualized under the fluorescence and phasecontrast mode in EVOS FL. As shown in Figure 9B, after treatment of the cells with 4 (and, for the control, without 4), nutrition starvation led to the intense blue fluorescence of MDC in the control. Treatment with compound 4, however, resulted in a quite weak fluorescence, thus suggesting ancistrolikokine E3 (4) to be a potent early-stage inhibitor of the autophagy pathway (Figure 10). In conclusion, a new structurally unique naphthylisoquinoline, ancistrolikokine E3 (4), was isolated from the twigs of the Congolese liana A. likoko. It belongs to the very rare subclass of 5,8′-coupled naphthyldihydroisoquinolines with the R-configuration at C-3. It showed a potent, highly selective preferential cytotoxic activity against human PANC-1 pancreatic cancer cells in nutrient-deprived medium, without causing any noticeable toxicity under normal, nutrient-rich conditions. The new alkaloid significantly inhibited cancer cell migration and colony formation and, therefore, has a pronounced antimetastatic and anti-invasion potential. Mechanistically, ancistrolikokine E3 (4) strongly inhibited the activation of the Akt/mTOR pathway. The compound is also a potent inhibitor of early-stage autophagy and thus restrains the autophagy-mediated cell survival pathway in pancreatic cancer cells. The results of the present investigation suggest that naphthylisoquinoline alkaloids in generaland ancistrolikokine E3 (4) in particularmay be considered as promising new lead structures for anticancer drug development based on the antiausterity strategy.

deprivation, so that it secures energy production and promotes cell proliferation and, thus, confers tolerance to starvation. Therefore, inhibition of the autophagy pathway may lead to increased cancer cell death in the tumor microenvironment. Autophagy is, however, known to play a dual role in the tumor. Thus, apart from its role in promoting tumor cell survival, it can, when hyperactivated during nutrition starvation, also act as an alternative mechanism of cancer cell death leading to tumor suppression, by preventing the accumulation of dysfunctional or unnecessary organelles and proteins.41−43 (+)-Grandifloracin, an antiausterity agent from Uvaria dac, is one such example that induces hyperactivation of autophagy, thus causing PANC-1 cancer cell death under nutrition starvation conditions.16 Therefore, an immunoblot analysis was carried out to investigate the possible involvement of ancistrolikokine E3 (4) in interfering with the autophagy pathway leading to PANC-1 cell death. For this purpose, expression of the key regulators of autophagy, microtubuleassociated protein-light chain 3 (LC3), Beclin-1, Atg5, and Atg12, were examined. The process of autophagy involves a series of sequential steps:44 formation of phagophores (vesicle nucleation step), engulfment of organelles and cytoplasmic materials by the phagophores to form autophagosomes (vesicle elongation and completion steps), fusion of autophagosomes with lysosomes to form autolysosomes (docking and fusion steps), and the degradation of autolysosomes to release nutrients in the cytosol to recycle (vesicle breakdown and degradation step). During autophagy, Beclin-1 is involved in the vesicle nucleation step, while LC3-II, Atg5, and Atg12 are essential components for autophagosome formation (Figure

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Figure 9. Ancistrolikokine E3 (4) inhibits autophagy markers. (A) Comparison of the effects of 4 (40 μM) with those of chloroquine (CQ) and 3-methyladenine (3-MA). (B) Monodansylcadaverine (MDC) staining as a marker of autophagy.

EXPERIMENTAL SECTION

General Experimental Procedures. All spectroscopic and chiroptical measurements were performed on the same devices 2288



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Figure 10. Proposed mechanism of action of ancistrolikokine E3 (4) against PANC-1 human pancreatic cancer cells. following the same protocols as previously reported.26 Briefly, the optical rotation and the UV, CD, and IR spectra were recorded on a JASCO P-1020 polarimeter and a Shimadzu UV-1800, a JASCO J715, and a JASCO FT/IR-410 spectrophotometer, respectively. NMR spectra were measured on a Bruker DMX 600 instrument and referenced to methanol-d4 (δH 3.31 and δC 49.15 ppm). HRESIMS data were obtained using a Bruker micrOTOF instrument in the positive ion mode. Preparative HPLC separations were carried out on a JASCO System (PU-1580 Plus) operating with a UV/vis detector (JASCO MD-2010 Plus diode array detector). GC-MSD analysis was carried out on a Shimadzu GC-MS-QP 2010 instrument. All organic solvents were of analytical grade quality. Ultrapure water was obtained from an Elga Purelab Classic system. Plant Material. Twigs of Ancistrocladus likoko were collected by Prof. V. Mudogo (August 1997), in the rainforest close to Yangambi, Democratic Republic of the Congo. A voucher specimen (No. 16) has been preserved at the Herbarium Bringmann, University of Würzburg, Germany. Extraction and Isolation. Air-dried twigs of A. likoko (200 g) were powdered and extracted with CH2Cl2−MeOH (1:1, v/v) at room temperature. The crude extract was concentrated in vacuo to give ca. 22.1 g of a solid residue, which was dissolved in MeOH−H2O (9:1, v/v) and purified by liquid/liquid partition with n-hexane. The alkaloid-rich fraction thus obtained (ca. 7.8 g) was subjected to CC on silica gel using a gradient of CH2Cl2−MeOH (90:10 to 55:45), giving rise to 17 fractions. Fraction 10 (1.07 g) was further resolved by CC on silica gel with n-hexane−CH2Cl2 and CH2Cl2−MeOH (90:10 to 55:45) as the eluents, furnishing 24 subfractions (F101− F1024). Purification of F1021 by preparative HPLC on a Waters SymmetryPrep C18 column (19 × 300 mm, 7 μm), flow rate 10 mL min−1, with the mobile phases (A) 90% H2O with 10% CH3CN (0.05% trifluoroacetic acid) and (B) 90% CH3CN with 10% H2O (0.05% trifluoroacetic acid) under gradient conditions (0 min 20% B, 10 min 45% B, 20 min 54% B) yielded pure ancistrolikokine E3 (4) (12.5 mg) (retention time 15.0 min). Ancistrolikokine E3 (4): yellow, amorphous solid; [α]25 D +6.9 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 231 (3.4) nm; ECD (c 0.1, MeOH) λmax (Δε) 190 (−3.0), 198 (−0.14), 210 (+0.6), 227 (−0.9), 240 (+0.2), 261 (−0.2), 312 (−0.1), 350 (+0.03) nm; IR (ATR) νmax 3389, 2951, 1673, 1578, 1432, 1196, 1174, 1129, 1084, 830, and 720 cm−1; 1H NMR (600 MHz, methanol-d4) δ 1.19 (3H, d, J = 6.7 Hz, CH3-3), 2.26 (1H, dd, J = 13.6, 20.4 Hz, Hax-4), 2.29 (3H, s, CH3-2′), 2.69 (1H, dd, J = 5.4, 16.8 Hz, Heq-4), 2.80 (3H, s, CH3-1), 3.78 (1H, m, H-3), 3.82 (3H, s, OCH3-6), 4.08 (3H, s, OCH3-4′), 4.14 (3H, s, OCH3-8), 6.57 (1H, s, H-1′), 6.78 (1H, d, J = 7.8 Hz, H-6′), 6.80 (1H, s, H-3′), 6.82 (1H, s, H-7), 7.05 (1H, d, J = 7.8 Hz, H-7′); 13C NMR (150 MHz, methanol-d4) δ 17.8, 22.1, 24.9, 32.6, 48.3, 56.8, 56.9, 57.0, 95.8, 107.7, 108.9, 110.3, 114.8, 118.5, 123.2, 123.7, 130.9,

136.9, 138.0, 141.7, 156.1, 158.1, 166.3, 168.7, 175.8; HRESIMS m/z 406.2033 [M + H]+ (calcd for C25H28NO4, 406.2018). Oxidative Degradation. This experiment was carried out following a well-established protocol, as described earlier.28 Preferential Cytotoxicity Assay against PANC-1 Cells. Ancistrolikokine E3 (4) was evaluated for its preferential cytotoxicity against PANC-1 human pancreatic cancer cells by a procedure described previously.14,24 Morphological Assessment of Cancer Cells. PANC-1 cells (2 × 105 cells/well) were seeded in DMEM in a 12-well plate dish and incubated for 24 h to allow cell attachment. The cells were then washed twice with phosphate-buffered saline (PBS), followed by treatment with ancistrolikokine E3 (4) in NDM or, in the case of the control cells, without any agent, just in NDM. Both the treated and the untreated (control) cells were incubated for 24 h and then treated with EB/AO reagent, and cell morphology was captured using an Evos FL digital microscope (20× objective) with phase-contrast and fluorescence modes. Wound-Healing Assay. PANC-1 cells (5 × 105 cells/well) were seeded in a 12-well plate dish and allowed to attach for 24 h. The monolayers were then incised with a micropipet tip in the central area of the culture to create a wound (600 μM in length). Cells were then gently washed with PBS and treated with medium containing ancistrolikokine E3 (4) at the concentrations of 10, 5, 2.5, or 0 μM (control). For each tested concentration, three replicates were carried out. Photographs were taken in the phase-contrast mode by an inverted Nikon Eclipse TS100 microscope (4× objective) after 48 h of treatment. Images were processed using ImageJ software. Measurements of wound lengths were carried out at 10 different points, covering all the smallest and largest distances that the PANC-1 cells had migrated, using ImageJ, and the data were processed with GraphPad Prism 6 software. Colony Formation Assay. PANC-1 cells (5 × 102 cells/well) were seeded in a 12-well plate dish in DMEM (1 mL/well) and incubated for 24 h for cell attachment. The medium was then changed to DMEM containing ancistrolikokine E3 (4) at 10, 5, 2.5, or 0 μM (control) in DMEM. PANC-1 tumor cells were allowed to be exposed for 24 h, with three replicates in each group. The cells were then washed twice with PBS, and the medium was replaced by fresh DMEM (2 mL) without any test compound. Then the cells were allowed to grow for 12 days. On the last day, the cells were washed with PBS, fixed with 4% formaldehyde, and stained with crystal violet for 10 min. The colony area measurement and data analysis were performed as described previously.41 Time-Lapse Imaging. PANC-1 cells (3 × 106) in 35 mm cell culture dishes were treated with 10 μM ancistrolikokine E3 (4) in the nutrient-deprived medium and subjected to time-lapse imaging at 10 2289



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min intervals for 24 h (145 time-lapse images) on an EVOS FL cell imaging system as described previously.18 Hoechst 33342 Staining Assay. PANC-1 cells (2 × 105) were plated in 35 mm culture dishes and incubated for 24 h to allow cell attachment. The cells were then washed twice with PBS, treated with 5 μM ancistrolikokine E3 (4) in nutrient-deprived medium, and allowed to incubate for a further 24 h. The cells were then stained with NuncBlue Live Ready Probe (Hoechst 33342, Thermo Fisher) by adding two drops directly to the culture medium. The cells were incubated for 5 min, and images were captured using an Evos FL digital microscope (20× objective) with phase-contrast and fluorescence modes. Western Blot Analysis. PANC-1 cells in NDM or DMEM were exposed to ancistrolikokine E3 (4) at different concentrations for 6 h, and Western blotting was carried out as described previously.16,18

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00733. 1D and 2D NMR, HRESIMS, IR, and ECD spectra of 4, including GC-MSD chromatograms from oxidative degradation (PDF) Images from colony formation and time-lapse experiments (PDF) W Web-Enhanced Feature *

A time-lapse video showing the detailed events upon exposure to 10 μM ancistrolikokine E3 (4) against PANC-1 cells in nutrient-deprived medium with one frame per 10 min for 24 h is available as a video file in the HTML version of the paper. The video was acquired with a 20× objective on an EVOS FL digital imaging system.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (S. Awale): [email protected]. Tel: +81-76434-7640. Fax: +81-76-434-7640. *E-mail (G. Bringmann): [email protected]. de. Tel: +49-931-318-5323. Fax: +49-931-318-4755. ORCID

Suresh Awale: 0000-0002-5299-193X Gerhard Bringmann: 0000-0002-3583-5935 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Japanese Society for the Promotion of Science (JSPS), Japan, Kakenhi (16K08319; grant to S.A.), the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG, Br 699/14-2, grant to G.B.), and the German Academic Exchange Service (Deutscher Akademischer Austauschdienst, DAAD, grants to S.F. and B.K.L.). We acknowledge experimental assistance from Dr. M. Büchner and Mrs. J. Adelmann (MS), Dr. Grüne and Mrs. P. Altenberger (NMR), and Mrs. M. Michel (oxidative degradation). We thank Prof. V. Mudogo for the collection of the plant material.

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REFERENCES

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