Highly potent antiausterity agents from Callistemon species from Egypt and their mechanism of action against the PANC-1 human pancreatic cancer cell line

概要

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 extract
Callistemon 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 on comparison 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 a 1′′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 extract
C. subulatus is a small shrub cultivated in Egypt for its abundant crimson flowers. No available data 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 Compounds
The isolated compounds were tested for their antiausterity activity against the PANC-1 human pancreatic 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. Both compounds 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.

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