Identification of natural compounds tubercidin and lycorine HCl against small-cell lung cancer and BCAT1 as a therapeutic target

Abstract

Although small-cell lung cancer (SCLC) accounts for a small fraction of lung cancer cases (~15%), the prognosis of patients with SCLC is poor with an average overall survival period of a few months without treatment. Current treatments include standard chemotherapy, which has minimal efficacy and a newly developed immunotherapy that thus far, benefits a limited number of patients. In the current study, we screened a natural product library and identified 5 natural compounds, in particular tubercidin and lycorine HCl, that display prominent anti-SCLC activities in vitro and in vivo. Subsequent RNA-sequencing and functional validation assays revealed the anti-SCLC mechanisms of these new compounds, and further identified new cellular factors such as BCAT1 as a potential therapeutic target with clinical implication in SCLC patients. Taken together, our study provides promising new directions for fighting this aggressive lung cancer.

Keywords: BCAT1; SCLC; drug screening; lung cancer; natural product.

FIGURE 1

High‐throughput screening and identification of new natural compounds with anti‐SCLC activities. Primary screening results of 756 natural compounds against SCLC, which are arranged in order of inhibition rate. The natural compounds in source plates were delivered at 10 µM (final concentration) to 96‐well plates seeded with SCLC cell line DMS 114 for 72 h treatment; then, cell proliferation was examined using the WST‐1 cell proliferation assays (Roche)

FIGURE 2

Hit natural compounds prominently inhibit SCLC cell growth. (A) The structures of the final 6 hit compounds and their dose‐dependent ‘killing curves’ on SCLC cells. (B, C) The inhibition of DIPG anchorage‐independent growth ability by new natural compounds, using colony formation assays. (D, E) DMS 114 cells were treated with natural compounds or vehicle for 48 h; then, cell apoptosis was measured by Annexin V‐PI staining and flow cytometry analysis. Error bars represent S.D. for 3 independent experiments, **p < 0.01 (vs the vehicle control)

FIGURE 3

New natural compounds displaying effective anti‐SCLC activities in vivo. (A) 6 × 10⁵ DMS 114 cells in 50 µL PBS plus 50 µL growth factor‐depleted Matrigel were injected subcutaneously into the flank of nude mice. When tumours reached 8–10 mm in diameter (~1.5 weeks), the mice were randomly separated into different groups (4 mice per group) and received in situ subcutaneous injection with either vehicle, tubercidin (5 mg/kg) or lycorine HCl (10 mg/kg), 3 days/week. The mice were observed and measured every 2–3 days for the size of palpable tumours for additional 3 weeks. (B, C) At the end of treatments, the tumours were excised for weighing and size comparison, **p < 0.01 (vs the Vehicle control)

FIGURE 4

Transcriptome analysis of new natural compound‐treated SCLC cells. (A) RNA‐sequencing was used to investigate changes in the transcriptome between natural compounds and vehicle‐treated DMS 114 SCLC cells. The significantly altered genes (p < 0.05) are shown in the volcano plot panels. (B) The heat map of commonly altered genes expression by 4 hit natural compounds in SCLC. (C, D) The GO_enrichment (biological process and molecular function) analysis of the commonly changed cellular genes by 4 hit natural compounds in SCLC

FIGURE 5

BCAT1 is required for SCLC cell survival. (A–E) DMS 114 cells were transfected with BCAT1‐siRNA or non‐target control siRNA (n‐siRNA) for 72 h; then, protein expression, cell proliferation and apoptosis were measured by using Western blot, WST‐1 assays and flow cytometry analysis respectively. (F) Cells were transfected with BCAT1‐siRNA with or without tubercidin treatment for 48 h; then, cell apoptosis was measured as above. Error bars represent S.D. for 3 independent experiments, **p < 0.01 (vs the n‐siRNA control or the vehicle control)

FIGURE 6

Clinical relevance of BCAT1 in SCLC. Expression of EIF4G1 in formalin‐fixed paraffin‐embedded (FFPE) SCLC and normal lung tissue arrays were determined using immunohistochemistry (IHC). (A) The IHC images from representative cases. (B–D) The percentage of DAB stained pixels were determined by analysing the raw images with the QuPath software (version 0.2.3). The nested graphs show expressional difference among these groups. **p < 0.01 (vs the normal lung tissue group, the IA group or the T1 group respectively); ns: not significant