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. 2020 Mar 31;10(1):5712.
doi: 10.1038/s41598-020-62690-9.

Narciclasine, an isocarbostyril alkaloid, has preferential activity against primary effusion lymphoma

Ramakrishnan Gopalakrishnan  1 , Hittu Matta  2 , Sunju Choi  2 , Preet M Chaudhary  3
Affiliations

Narciclasine, an isocarbostyril alkaloid, has preferential activity against primary effusion lymphoma

Ramakrishnan Gopalakrishnan et al. Sci Rep. .

Abstract

Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin lymphoma associated with infection by Kaposi sarcoma-associated herpes virus (KSHV). PEL is an aggressive disease with extremely poor prognosis when treated with conventional chemotherapy. Narciclasine, a natural product present in Amaryllidaceae family of flowering plants including daffodils, belongs to a class of molecules termed 'isocarbostyril alkaloid'. We have found that narciclasine displays preferential cytotoxicity towards PEL at low nanomolar concentrations and is approximately 10 and 100-fold more potent than its structural analogs lycoricidine and lycorine, respectively. Narciclasine arrested cell-cycle progression at the G1 phase and induced apoptosis in PEL, which is accompanied by activation of caspase-3/7, cleavage of PARP and increase in the surface expression of Annexin-V. Although narciclasine treatment resulted in a marked decrease in the expression of MYC and its direct target genes,time-course experiments revealed that MYC is not a direct target of narciclasine. Narciclasine treatment neither induces the expression of KSHV-RTA/ORF50 nor the production of infectious KSHV virions in PEL. Finally, narciclasine provides dramatic survival advantages to mice in two distinct mouse xenograft models of PEL. In conclusion, our results suggest that narciclasine could be a promising agent for the treatment of PEL.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Narciclasine and its structural analogs have preferential cytotoxicity towards PEL. (A) Chemical structures of narciclasine, lycoricidine, and lycorine. (B) Indicated panel of cell lines were treated with increasing concentrations of narciclasine, lycoricidine, and lycorine for 72 hours. Cell viability was measured using an MTS (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. An arrow represents cell lines with preferential sensitivity to the compounds. The values shown are mean ± SE. (n = 3) of a representative experiment performed in triplicate for three times.
Figure 2
Figure 2
Narciclasine arrested cell-cycle progression. (A) Indicated cell lines were treated with narciclasine (25 nM for 36 hours) or DMSO control, fixed with 70% alcohol followed by staining with Propidium Iodide (PI) and analyzed by flow cytometry. (B) Indicated cell lines were treated with narciclasine or DMSO control were stained with SYTOX Green, a cell-impermeable nuclear dye that stains the nuclei of dead cells, and were examined under a fluorescence microscope and photographed.
Figure 3
Figure 3
Narciclasine induces apoptosis in PEL cells. (A) Indicated cell lines were treated with narciclasine (25 nM for 36 hours) or DMSO control, stained with Annexin V-FITC/propidium iodide, and analyzed for apoptosis by flow cytometry. Data are representative of 3 independent experiments. The gating of cells for analysis were presented in Supplementary Fig. S3. (B) Indicated cell lines were treated with narciclasine (25 nM for 36 hours) or DMSO control, followed by measurement of active caspase-3/7 using Apo-ONE homogeneous assay kit. Data are representative of 2 independent experiments. Statistically significant differences were shown by asterisks (**) at a level of p ≤ 0.01, and (***) at a level of p ≤ 0.001. ns – not significant. (C) BC-1, BC-3, JSC-1 and L428 cell lines were treated with narciclasine (25 nM for 48 hours) or DMSO control, followed by western blotting of whole cell lysates for cleavage of PARP and GAPDH (loading control). Cl – Cleaved; FL – Full Length. Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Fig. S4. Blots are representative of 3 independent experiments.
Figure 4
Figure 4
Narciclasine downregulates MYC. (A) BC-1, BC-3, JSC-1 and L428 cell lines were treated with narciclasine (25 nM for 48 hours) or DMSO control, followed by western blotting of whole cell lysates for MYC and GAPDH (loading control). Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Fig. S5. Blots are representative of at least 3 independent experiments. (B) BC-1 and BC-3 cell lines were treated with narciclasine (50 nM for 24 hours) or DMSO control followed by qRT-PCR analysis of TERT, SLC19A1, MYB and PMM2 mRNA (direct target genes of MYC protein). Real-time PCR reactions were carried out in triplicate and the data were presented as fold change in target gene expression (mean ± SE) from a representative of 2 independent experiments. Statistically significant differences were shown by asterisks (*) at a level of p ≤ 0.05, (**) at a level of p ≤ 0.01, and (***) at a level of p ≤ 0.001.
Figure 5
Figure 5
MYC is not a primary target of narciclasine. BC-1, BC-3, JSC-1 and L428 cell lines were treated with narciclasine (25 nM for 12, 24, 36 and 48 hours) or DMSO control, followed by western blotting of whole cell lysates for PARP, MYC, Caspase-3 and GAPDH (loading control). Cl – Cleaved; FL – Full Length. Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Figs. S6–S8.
Figure 6
Figure 6
Rho inhibitors have no effect on the activity of narciclasine in PEL. Indicated cell lines were treated with 10 μM of Rho pathway inhibitors CCG1423 and Rhosin.HCl in the presence or absence of indicated concentration of narciclasine for 72 hours. Cell viability was measured using an MTS (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The values shown are mean ± SE. (n = 3) of a representative experiment performed in triplicate.
Figure 7
Figure 7
Narciclasine treatment does not induce lytic reactivation of KSHV. (A) Western blotting analysis of KSHV RTA/ORF50 and GAPDH (loading control) in indicated PEL cell lines treated with narciclasine for 48 hours. Expression of RTA in BCBL-TREx-RTA cells treated doxycycline (500 ng/ml for 48 hours) serves as a positive control. Samples were derived from the same experiments, loading controls were from the same blot and the blots were processed in parallel. Original raw blots are presented in Supplementary Fig. S9. Blots are representative of at least 2 independent experiments. (B) Cell free supernatants collected from narciclasine treated PEL cells were used to infect 293A-PAN-Luc cells, which expresses the firefly luciferase gene under the control of KSHV PAN promoter and responds to infection with KSHV. Cell free supernatant from doxycycline treated BCBL-TREx-RTA cells were used as positive control. Data are representative of 2 independent experiments performed in duplicate.
Figure 8
Figure 8
Narciclasine inhibits growth of PEL in an orthotopic cell line xenograft (CLX) model. (A) 6-week old female NOD-Scid-Gamma (NSG) mice were injected intraperitoneally with 1 × 107 BC-1 cells stably expressing firefly luciferase gene (BC-1-Fluc). 5 days after tumor inoculation Bio-luminescence Imaging (BLI) was performed to confirm the presence of tumors in mice. Post confirmation, mice were randomly divided into vehicle control or narciclasine (1 mg/kg b.w. intraperitoneally daily for 12 days) groups. Representative serial BLI images of mice on days 5, 13 and 19 of indicated treatment groups are shown. (B) Tumor burden as measured by relative luminescence measurements of serial BLI imaging from vehicle control and narciclasine treated mice are shown. (C) Body weight gain of mice injected with BC-1-Fluc cells followed by indicated treatments over the period of experiment. Black arrows indicate start (day 5) and stop (day 16) of narciclasine treatment. Asterisks indicate significance (**) at a level of p ≤ 0.01, and (***) at a level of p ≤ 0.001. (D) Survival curves (Kaplan-Meier) of mice bearing orthotopic BC-1-Fluc cells treated with vehicle control and narciclasine (n = 10 in each group). The survival curve was generated in GraphPad Prism 5 software and statistical values for the curves are calculated by log rank (Mantel–Cox) test. (E) Levels of hIL-6 and hIL-10 in plasma of animals on day19 after tumor inoculation in vehicle control or narciclasine treatment groups.
Figure 9
Figure 9
Narciclasine exhibits potent activity in a Patient-Derived Xenograft (PDX) model of PEL. (A) 6-week old female NSG mice were injected intraperitoneally with 1 × 106 UMPEL-1 cells. 4 days after tumor inoculation, mice were randomly divided into vehicle control or narciclasine (1 mg/kg b.w. intraperitoneally daily for 9 days) treatment groups. Gross representative images of mice on day 12 after tumor inoculation of indicated treatment groups were shown. (B) Body weight gain of mice injected with UMPEL-1 cells followed by treatment with vehicle control and narciclasine over the period of experiment. Statistically significant differences are shown by asterisks (***) at a level of p ≤ 0.001. (C) Survival curves (Kaplan-Meier) of mice bearing orthotopic UMPEL-1 cells treated with vehicle control and narciclasine (n = 10 in each group). The survival curve was generated in GraphPad Prism 5 software and statistical values for the curves are calculated by log rank (Mantel–Cox) test. Black arrows indicate start (day 4) and stop (day 12) of narciclasine treatment. (D) Circulating level of hIL-10 on day 12 after tumor inoculation in vehicle control or narciclasine treatment groups.
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