FLI-06 Intercepts Notch Signaling And Suppresses The Proliferation And Self-renewal Of Tongue Cancer Cells

Abstract

Purpose: The Notch signaling pathway plays an oncogenic role in tongue squamous cell carcinoma. The aim of this study was to inhibit the proliferation and self-renewal of tongue cancer cells by applying Notch signaling pathway inhibitor FLI-06 (Selleck, USA) and to lay a foundation for the clinically targeted treatment of tongue cancer for the future.

Methods: The mRNA expression level of Notch1 and the overall survival rate of patients with tongue cancer were examined by analyzing the TCGA database. Tongue cancer cells were treated with FLI-06. Cell proliferation, apoptosis, and stem cell self-renewal ability were tested in appropriate ways. A xenograft mouse model was established to observe tumor growth.

Results: From the TCGA data, we demonstrated that patients with high expression of Notch1 had a poor prognosis. We observed that the Notch signaling pathway inhibitor FLI-06 can restrain the activation of the Notch signaling pathway, decrease cell proliferation and induce cell apoptosis in vitro. The xenograft experiment indicated that intraperitoneal injection of FLI-06 inhibited tumor growth and increased cell apoptosis. FLI-06 suppressed both the mRNA and protein expression of Notch receptor and Notch targeted genes. We also observed that FLI-06 suppressed the proliferation of tongue cancer stem cells.

Conclusion: FLI-06 can block the proliferation and self-renewal of tongue cancer cells. It is inferred that this compound, which inhibits the Notch signaling pathway, may serve as a potential targeted drug for the treatment of tongue cancer in the clinic.

Keywords: Notch inhibitor; Notch signaling pathway; cancer stem cells; tongue cancer.

Figure 1

High expression of Notch1 is associated with a poor prognosis in tongue cancer patients. (A) The TCGA database showed that the mRNA expression level of Notch1 in tongue cancer was not different from that in normal tissues; TC represents tongue cancer, and N represents normal tissues. (B) The rate of overall survival in the high Notch1 group was lower than that of the low Notch1 group.

Figure 2

Blocking Notch1 with FLI-06 inhibited tumor growth in vitro. (A, B) After treatment with FLI-06 at different concentrations for 48 h or 72h, the viability of CAL-27 (A) and TCA-8113 (B) cells was measured by the CCK8 assay; CAL-27 and TCA-8113 cells proliferation ability was measured by the CCK8 (C, D) and colony formation (E, F) assays. Representative photomicrographs of the plates are shown in the left panel, and the colony number of each plate is shown in the right panel. Data represent the mean ± S.D. of three independent experiments (** indicates P < 0.01).

Figure 3

FLI-06 induced apoptosis and arrested the cell cycle in CAL-27 cells. (A) Annexin V-PI staining flow cytometry showed that the number of early and late apoptotic cells increased following FLI-06 treatment; (B) The TUNEL assay showed that in the negative control group, TUNEL-positive cells were fewer than that in the FLI-06 treatment groups; (C) The PI staining flow cytometry cycle test demonstrated that in the high concentration group (10 μM FLI-06), the cells were restrained in G0/G1 phase. (D) The expression of the apoptosis-related genes Caspase-3 and Caspase-9 was measured by Western blotting in CAL-27 cells with or without FLI-06 treatment. Data represent the mean ± S.D. of two independent experiments (* indicates P< 0.05 and ** indicates P < 0.01). Abbreviation: n.s, not significant.

Figure 4

FLI-06 inhibited tumor growth in vivo. The changes in body weight (A) and tumor volume (B) of nude mice were monitored during the experiment, and this graph shows no significant change in body weight among the groups, while the tumor volume of the FLI-06-treated group was smaller than that of the control; (C, D) The animals were sacrificed, and the tumors were removed for weighing (C) and photographing (D) The column graph shows that the tumor weight of the FLI-06 treatment group was less than that of the control group, and the photograph of the tumors shows a similar trend; (E, F) The expression of Ki-67 (upper panel) and cleaved Caspase-9 (lower panel) in xenograft tumors using immunohistochemistry (DAB, 400×) (** indicates P < 0.01).

Figure 5

The protein expression of Notch and the activating of the pathway were inhibited by FLI-06. (A) Real-time PCR analysis showed lower expression of Notch target gene mRNA in the FLI-06 treatment group relative to the group without FLI-06 treatment; (B) Western blot analysis showed that the expression of both the full length of Notch1 receptor and NICD1 proteins in the FLI-06 treatment group was decreased compared to that of the control; (C) The protein expression level of HES1 and HEY2, which are Notch signaling pathway targeted genes, were downregulated after treatment with FLI-06. Data represent the mean ± S.D. of three independent experiments(* indicates P < 0.05 and ** indicates P < 0.01).

Figure 6

FLI-06 could inhibit the proliferation of tongue cancer stem cells. (A) FLI-06 was added to CAL-27 cancer stem cells (upper panel) for 10 days and TCA-8113 cancer stem cells for 6 days (lower panel). FLI-06 inhibited the proliferation of stem cells in the high concentration group. Representative images are shown in the left panel (40×), and the statistical column is shown in the right panel. (B) The real-time PCR data demonstrated that the stem cell biomarkers were decreased in the FLI-06 treatment groups. Data represent the mean ± S.D. of three independent experiments (* indicates P < 0.05 and ** indicates P < 0.01).

Figure 7

FLI-06 could block the self-renewal ability of tongue cancer stem cells. FLI-06 was used to treat tongue cancer stem cell spheres. (A) After CAL-27 cancer stem cell spheres were treated with FLI-06 for 8 days, the spheres were broken in the 3 µM concentration group (40×). (B) After TCA-8113 cancer stem cell spheres were treated with FLI-06 for 4 days, the spheres were completely broken in the 2 µM concentration group (40×).