富山大学・富医薬博甲第345号・Ahmed Mohammed Abdel Latif Mohammed Tawila・2020/09/28・★論文非公開★富山大

TAWILA Ahmed Mohammed Abdel Latif Mohammed

University of Toyama Repository

     あはめど もはめど あぶでる らちふ もはめど   たびら 氏 名   Ahmed Mohammed Abdel Latif Mohammed Tawila  学 位 の 種 類  博士（薬科学）  学 位 記 番 号  富医薬博甲第 345号  学位授与年月日  令和２年９月 28日  学位授与の要件  富山大学学位規則第 3条第 3項該当  教 育 部 名    富山大学大学院医学薬学教育部 博士後期課程 薬科学専攻  学位論文題目          論文審査委員   （主査）   教 授 松谷 裕二 （副査）   教 授 森田 洋行 （指導教員） （副査）   准教授 渡辺 志朗 （副査）   准教授 Suresh Awale   Highly potent antiausterity agents from Callistemon species from Egypt and their mechanism of action against the PANC-1 human pancreatic cancer cell line （エジプト産のカリステモン種からの強力な栄養飢餓耐性解除抗がん物質の探索とそれらのヒト膵臓がん細胞株 PANC-1 に対する作用機序）  Highly potent antiausterity agents from Callistemon species from Egypt and their mechanism of action against the PANC-1 human pancreatic cancer cell line Ahmed Mohammed Tawila Natural Drug Discovery Lab  Institute of Natural Medicine University of Toyama Pancreatic cancer is a major leading cause of mortality with the lowest five-year survival of 9%. It was ranked as the third and fifth leading cause of cancer-related death in Japan in the year 2019, with an annual mortality rate of nearly 35,000 persons. The majority of pancreatic cancer patients at the time of diagnosis possess metastatic colonies in the duodenum, stomach, and liver, making corrective surgery almost impossible. The prognostic outcome of chemotherapeutic agents in pancreatic cancer is poor due to its intrinsic resistance to almost all agents in clinical use. Therefore, there is a desperate need to find effective new chemotherapeutic agents for pancreatic cancer.   Cancer cells grow and proliferate in an aggressive manner, which requires an excessive nutrient supply for the cells. The majority of tumors form new blood vessels within their microenvironment for the effective supply of nutrients, a process termed as ‘angiogenesis’. Unlike other tumors, pancreatic tumors have poor blood supply. However, pancreatic tumor cells can overcome these harsh conditions of hypoxia and starvation through modulation of their energy metabolism, which lead to acquiring tolerance to nutrition starvation, a phenomenon termed as "austerity". The search for new agents that inhibit the ability of pancreatic cancer cells to tolerate nutrition starvations is a promising approach in anticancer drug discovery.  In the present study, plant extracts from Egypt were screened for their preferential cytotoxic activity against the PANC-1 human pancreatic cancer cells under nutrient-deprived conditions. Among these, Callistemon citrinus and Callistemon subulatus (Myrtaceae) showed strong preferential cytotoxicity. The phytochemical investigation of the active extracts was carried out in order to identify the active constituents responsible for the preferential cytotoxic activities.  1. Chemical constituents of Callistemon citrinus extractCallistemon citrinus is a small tree native to Australia and cultivated in Egypt as ornamentals.Traditionally, it is used for treating hemorrhoids, gastrointestinal, and respiratory disorders. The phytochemical investigation of C. citrinus leaf extract resulted in the isolation of seven new compounds, callistrilones L−Q (1−6) and epicallistrilone Q (7), together with twenty-two previously reported compounds.1-4 The 1H NMR spectrum of 1 displayed a pair of signals in a ratio of 4:1, suggesting the possibility of the presence of two isomers. Further purification of the compound by semi-preparative HPLC resulted in isolation of two peaks. Each peak displayed a pair of signals in 1H NMR spectrum which resemble d the original mixture. Repeated HPLC separation of these two peaks resulted in a reproducible chromatogram of two peaks at similar retention time, suggesting the presence of this compound as a mixture of two equilibrating conformational isomers. Callistrilone L (1) displayed two different sets of NOESY correlations around C-7 suggesting a change in interproton distance due the presence of the two conformations. The absolute configuration was assigned using theoretical calculations of ECD spectrum and the specific optical rotation.1 Compounds 2−5 were also isolated as mixture of conformational isomers for each. However, 2−5 differ mainly from 1 in the way of attachment of the α-phellandrene to the acylated phloroglucinol ring. In callistrilone L (1), the α-phellandrene is attached to C-4′ and C-5′ while in 2−5, α-phellandrene is attached to C-5′ and C-6′ through a dihydrofuran ring. The absolute configuration of 2 and 3 was determined based oncomparison of their ECD spectra to that of callistrilone E (8), while the absolute configuration of callistrilone O (4) and P (5) was deduced by ECD calculation.1,2 Callistrilone Q (6) and epicallistrilone Q (7) represents unusual callistrilone structures as α-phellandrene is only attached to C-5′. Theoretical calculations of ECD spectra of 6 and 7 suggested that both compounds possess a1′′R,4′′R,6′′R configuration. However, ECD was indecisive in determination of the absolute configuration of C-7. Therefore, theoretical calculations of specific optical rotations were employed to remove the ambiguities regarding the absolute configuration at C-7 for compounds 6 and 7.4 2. Chemical constituents of Callistemon subulatus extractC. subulatus is a small shrub cultivated in Egypt for its abundant crimson flowers. No availabledata was traced concerning chemical constituents or a traditional use as medicine. The phytochemical investigation of C. subulatus leaves extract resulted in the isolation of one new compound named subulatone (30), together with fourteen previously reported compounds belonging to seven meroterpenoids, three flavonoids, one lignan and three triterpenes. The 1H NMR of the new compound 30 displayed the presence of six methyl groups and an olefinic methine. The 13C NMR showed the presence of two olefinic carbons, six methyls, two quaternary carbons and an oxygenated carbon. Analysis of HMBC correlations suggested that 30 is a 5-(2-hydroxypropan-2-yl)-2,2,6,6-tetramethylcyclohex-4-ene-1,3-dione.5  3. Antiausterity activity of the Isolated CompoundsThe isolated compounds were tested for their antiausterity activity against the PANC-1 humanpancreatic cancer cell line in standard nutrient-rich medium (DMEM) and nutrient-deprived medium (NDM). Among the tested compounds, all callistrilones displayed unprecedented antiausterity activities that supersedes the positive control with PC50 values of less than 100 nM. Of these, callistrilone L (1) and O (4) induced alterations in PANC-1 cell morphology under nutrient-deprived conditions.1,2 Pancreatic cancer remains challenging due to it is remarkable metastatic properties as it migrates to distant organs in human body forming secondary tumors. Therefore, the effect of callistrilones L (1) and O (4) against PANC-1 tumor cell migration and colony formation was investigated. Bothcompounds strongly suppressed PANC-1 cell migration and colony formation under nutrient rich conditions (DMEM) in real-time.1,2 Mechanistically, 1 was found to inhibit the key survival protein Akt and mTOR expression and phosphorylation and inhibited autophagy pathway.1  The active triterpene, 3β-hydroxy-urs-11-en-13(28)-olide (22) was able to induce dramatic changes in PANC-1 cell morphology after 12 h and plasma blebbing after 14 h leading to cell death in a real-time experiment. It also displayed antimetastatic properties under DMEM conditions.  Further investigation of the mechanism of action suggested that besides inhibiting Akt/mTOR pathway, it blocks the unfolded protein response in PANC-1 cells, leading to apoptosis under glucose deprivation.3   Conclusion Phytochemical investigation of the active Callistemon extracts of led to the isolation of thirty-six compounds. Among these, seven new structurally challenging meroterpenoids named callistrilones L−Q (1−6) and epicallistrilone Q (7) from C. citrinus and a new compound from C. subulatus named subulatone (30). The absolute configuration for the isolated callistrilone compounds was determined based on theoretical calculations of the ECD, and specific optical rotation. The isolated callistrilone compounds showed unprecedented preferential cytotoxic activities against PANC-1 human pancreatic cancer cells at the sub-nanomolar level. Callistrilones L (1) and O (4) inhibited PANC-1 cells tumor migration and colony formation in DMEM, suggesting that these compounds are the most potent antiausterity agents with antimetastatic properties. Further investigation of the mechanism of action of callistrilone L (1) suggested that its activity is mediated via suppression of Akt/mTOR signaling cascade and autophagy pathway.  References 1- Ahmed M. Tawila, Sijia Sun, Min Jo Kim, Ashraf M. Omar, Dya Fita Dibwe, Jun-ya Ueda,Naoki Toyooka and Suresh Awale.Highly potent antiausterity agents from Callistemon citrinus and their mechanism of action againstthe PANC-1 human pancreatic cancer cell line. J. Nat. Prod. 2020,https://doi.org/10.1021/jnatprod.0c00330.2- Ahmed M. Tawila, Sijia Sun, Min Jo Kim, Ashraf M. Omar, Dya Fita Dibwe, Jun-ya Ueda,Naoki Toyooka and Suresh Awale.Chemical constituents of Callistemon citrinus from Egypt and their antiausterity activity againstPANC-1 pancreatic cell line. Bioorg. Med. Chem. Lett. 2020,https://doi.org/10.1016/j.bmcl.2020.127352.3- Ahmed M. Tawila, Sijia Sun, Min Jo Kim, Ashraf M. Omar, Dya Fita Dibwe, and Suresh Awale.A triterpene lactone from Callistemon citrinus inhibits the PANC-1 human pancreatic cancer cellsviability via suppression of unfolded protein response. Chem. Biodivers.https://doi.org/10.1002/cbdv.2020004954- Ahmed M. Tawila, Ashraf M. Omar, Sijia Sun, Min Jo Kim, and Suresh Awale.Chiral discrimination of new callistrilone anomers from Callistemon citrinus and theirantiausterity activity against the PANC-1 human pancreatic cancer cell line. In preparation.5- Ahmed M. Tawila, Sijia Sun, Min Jo Kim, Ashraf M. Omar, and Suresh Awale.Chemical constituents of Callistemon subulatus and their antiausterity activity against thePANC-1 human pancreatic cancer cell line. In preparation. Chart 1. Compounds isolated from C. citrinus Figure 2. 1H NMR and NOESY correlations observed for conformers I and II of callistrilone L (1).   Figure 3. Experimental and calculated ECD spectra of compound 1. Chart 2. Compounds isolated from C. subulatus Figure 1. COSY (bold green lines) and key HMBC (red arrows) correlations in 1−3. Figure 4. Comparison of the experimental ECD spectra of compounds 2 and 3 with that of the known compound 8.  Figure 5. PANC-1 cell death induced by callistrilone L (1) in nutrient-deprived medium (NDM). Figure 7. Effect of callistrilone L (1) on PANC-1 cells colony formation. Figure 8. Effect of callistrilone L (1) on key proteins involved in PI3K/Akt/mTOR/autophagy signaling under NDM in PANC-1 cells.  Figure 6. Callistrilone O (4) suppresses the migration of PANC-1 cells in a wound-healing assay in real time. 

エジプト産のカリステモン種からの強力な栄養飢餓耐性解除抗がん物質の探索とそれらのヒト膵臓がん細胞株PANC-1に対する作用機序

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エジプト産のカリステモン種からの強力な栄養飢餓耐性解除抗がん物質の探索とそれらのヒト膵臓がん細胞株PANC-1に対する作用機序

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