The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells that Leads to Paraptosis-like Cell Death

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuoli-zation was observed in tumour cells, but not in other organs. This study shows that the antiprolif-erative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo

Erratum: Synthesis of Usnic Acid Derivatives and Evaluation of Their Antiproliferative Activity against Cancer Cells (Journal of Natural Products (2019) 82:7 (1768-1778) DOI: 10.1021/acs.jnatprod.8b00980)

The (+)-enantiomer of compound 1 was incorrectly assigned as the S-configuration where it is in fact the Rconfiguration. As a consequence, compounds 2b, 3b, 3d, 3f, 3h, 3j, 3l, and 3n are also the R-configuration. Page 1769: The table within Scheme 2 should be substituted with the following:. The compound names within the Supporting Information (pp 2-6) need to be altered as follows: • (R)-8-Acetyl-5,7-dihydroxy-3,4a,6-trimethyl-1-phenyl- 1,4a-dihydro-4Hbenzofuro[3,2-f ]indazol-4-one (3d) • (R)-8-Acetyl-5,7-dihydroxy-1-(4-methoxyphenyl)- 3,4a,6-trimethyl-1,4a-dihydro4H-benzofuro[3,2-f ]- indazol-4-one (3f) • (R)-8-Acetyl-1-(4-fluorophenyl)-5,7-dihydroxy-3,4a,6- trimethyl-1,4a-dihydro-4Hbenzofuro[3,2-f ]indazol-4- one (3h). • (R)-8-Acetyl-1-(3,4-dichlorophenyl)-5,7-dihydroxy- 3,4a,6-trimethyl-1,4a-dihydro4H-benzofuro[3,2-f ]- indazol-4-one (3j) (Table Presented). • (R)-8-Acetyl-1-(4-chlorophenyl)-5,7-dihydroxy-3,4a,6- trimethyl-1,4a-dihydro4H-benzofuro[3,2-f ]indazol-4- one (3n) Accordingly, the structures of compounds 2b, 3b, 3d, 3f, 3h, 3j, 3l, and 3n in the Supporting Information have been revised. Additionally, the Abstract is revised as follows: Moreover, they induced massive cytoplasmic vacuolization, which was associated with elevated dynamin-dependent endocytosis, a process that has not been reported for usnic acid and indicates a novel mechanism of action of its synthetic derivatives. The authors sincerely apologize for any inconvenience caused by these errors

The pyrazole derivative of usnic acid inhibits the proliferation of pancreatic cancer cells in vitro and in vivo

Abstract Background Pancreatic cancer is one of the leading causes of cancer death in Western societies. Its late diagnosis and resistance to chemotherapies result in a high mortality rate; thus, the development of more effective therapies for the treatment of pancreatic cancer is strongly warranted. Usnic acid (UA) is a secondary metabolite of lichens that shows modest antiproliferative activity toward cancer cells. Recently, we reported the synthesis of a UA pyrazole derivative, named 5, which was more active than the parent compound toward cervical cancer cells. Here, its anticancer potential has been evaluated in detail in other cancer cells, particularly pancreatic cancer cells. Methods The impact of UA and derivative 5 on cell viability, morphology, cell cycle, and death was assessed using the MTT test, electron microscopy, flow cytometry, and immunoblotting, respectively. The calcium ions level was detected fluorometrically. In vivo, the anticancer activity of 5 was evaluated in a murine xenograft model. Results Derivative 5 inhibited the viability of different cancer cells. Noncancerous cells were less sensitive. It induced the release of calcium ions from the endoplasmic reticulum (ER) and ER stress, which was manifested by cell vacuolization. It was accompanied by G0/G1 cell cycle arrest and cell death of pancreatic cancer cells. When applied to nude mice with xenografted pancreatic cancer cells, 5 inhibited tumor growth, with no signs of kidney or liver toxicity. Conclusions UA derivative 5 is superior to UA inhibiting the growth and proliferation of pancreatic cancer cells. ER stress exaggeration is a mechanism underlying the activity of derivative 5

Synthesis of Usnic Acid Derivatives and Evaluation of Their Antiproliferative Activity against Cancer Cells

Usnic acid is a secondary metabolite abundantly found in lichens, for which promising cytotoxic and antitumor potential has been shown. However, knowledge concerning activities of its derivatives is limited. Herein, a series of usnic acid derivatives were synthesized and their antiproliferative potency against cancer cells of different origin was assessed. Some of the synthesized compounds were more active than usnic acid. Compounds 2a and 2b inhibited survival of all tested cancer cell lines in a dose- and time-dependent manner. Their IC50 values after 48 h of treatment were ca. 3 μM for MCF-7 and PC-3 cells and 1 μM for HeLa cells, while 3a and 3b revealed antiproliferative activity only against HeLa cells. All active usnic acid derivatives induced G0/G1 arrest and a drop in the fraction of HeLa cells in the S and G2/M phases. Compounds 2a and 2b decreased the clonogenic potential of the cancer cells evaluated and induced cell cycle arrest at the G0/G1 phase and apoptosis in MCF-7 cells. Moreover, they induced massive cytoplasmic vacuolization, which was associated with elevated dynein-dependent endocytosis, a process that has not been reported for usnic acid and indicates a novel mechanism of action of its synthetic derivatives. This work also shows that naturally occurring usnic acids are promising lead compounds for the synthesis of derivatives with more favorable properties against cancer cells