Shikonin suppresses proliferation and induces apoptosis in endometrioid endometrial cancer cells via modulating miR-106b/PTEN/AKT/mTOR signaling pathway

Abstract

Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicinal herb, which exerts anticancer effects in various cancers. However, the molecular mechanisms underlying the therapeutic effects of shikonin against endometrioid endometrial cancer (EEC) have not yet been fully elucidated. Herein, we investigated anticancer effects of shikonin on EEC cells and explored the underlying molecular mechanism. We observed that shikonin inhibits proliferation in human EEC cell lines in a dose-dependent manner. Moreover, shikonin-induced apoptosis was characterized by the up-regulation of the pro-apoptotic proteins cleaved-Caspase-3 and Bax, and the down-regulation of the anti-apoptotic protein Bcl-2. Microarray analyses demonstrated that shikonin induces many miRNAs' dysregulation, and miR-106b was one of the miRNAs being most significantly down-regulated. miR-106b was identified to exert procancer effect in various cancers, but in EEC remains unclear. We first confirmed that miR-106b is up-regulated in EEC tissues and cells, and knockdown of miR-106b suppresses proliferation and promotes apoptosis. Meanwhile, our results validated that the restored expression of miR-106b abrogates the antiproliferative and pro-apoptotic effects of shikonin. We also identified that miR-106b targets phosphatase and tensin homolog (PTEN), a tumor suppressor gene, which in turn modulates AKT/mTOR signaling pathway. Our findings indicated that shikonin inhibits proliferation and promotes apoptosis in human EEC cells by modulating the miR-106b/PTEN/AKT/mTOR signaling pathway, suggesting shikonin could act a potential therapeutic agent in the EEC treatment.

Keywords: Shikonin; ant-cancer; endometrioid endometrial cancer; miR-106b/PTEN/AKT/mTOR signaling pathway; microRNAs.

Figure 1. The suppressive effects of shikonin on human EEC cells

(A–D) The human EEC cell lines Ishikawa, HEC-1A, KLE, and RL95-2 were treated with various concentrations of shikonin (0, 1, 2, 4, 8, 10, and 20 μM) for 24 h and the cell viability was measured by the CCK-8 assay. (E) The flow cytometric analysis was used to determine the apoptotic cells in both Ishikawa and HEC-1A cells after treatment with 5 μM of shikonin for 24 h. (F,G) The Ishikawa and HEC-1A cells treated with 5 μM of shikonin for 24 h, the Western blot analysis to detect the expression levels of the apoptosis-related proteins (cleaved-Caspase-3, Bax, and Bcl-2). β-actin was used as an internal control for protein loading. Data are represented as means ± S.D. of three independent experiments (*P<0.05, **P<0.01 compared with blank group).

Figure 2. Shikonin induces the aberrant expression of miRNAs in human EEC cells

(A) The EEC cells were treated with shikonin (5 μM) for 24 h, and then microarray analysis was conducted to identify miRNA expression profiles. The color code is linear within the heat map: blue represents the lowest level of expression and red the highest. (B) The qRT-PCR analysis was performed to determine the miR-106b expression in Ishikawa cells treated with different concentration of shikonin (0, 1, 2, 5, and 10 μM) for 24 h (*P<0.05, **P<0.01 compared with blank group). (C) miR-106b was identified by qRT-PCR analysis in EEC tissues (n=20) and normal endometrial samples (n=20) (**P<0.01 compared with normal endometrial samples). (D) The qRT-PCR analysis was used to detect miR-106b level in EEC cell lines Ishikawa, HEC-1A, KLE, RL95-2, and one normal endometrial cell (ESC) (**P<0.01 compared with ESC). Data are represented as means ± S.D. of three independent experiments.

Figure 3. Knockdown of miR-106b suppresses EEC cells apoptosis

(A,B) The Ishikawa or HEC-1A cells were transfected with miR-106b inhibitor or inhibitor NC, and cell viability was measured using CCK-8 post treatment at 1, 2, 3 and 4 days, respectively. (C) The cytometric analysis used to measure apoptotic cells in Ishikawa and HEC-1A cells treated with miR-106b inhibitor or inhibitor NC. (D,E) After transfection with miR-106b inhibitor or inhibitor NC, the Western blot analysis was used to detect the expression levels of the apoptosis-related proteins (cleaved-Caspase-3, Bax, and Bcl-2). β-actin was used as an internal control for protein loading (**P<0.01 compared with inhibitor NC). Data are represented as means ± S.D. of three independent experiments.

Figure 4. Overexpression of miR-106b attenuates the suppressive effects of shikonin

(A and C) The Ishikawa or HEC-1A cells were transfected with miR-106b mimics or NC, and then treated with shikonin (5 μM) for 24, 48, and 72 h; the CCK-8 was assay used to measure cellular proliferation, respectively. (B and D) After transfection with miR-106b mimics or NC, the apoptotic cells were measured by cytometric analysis in Ishikawa or HEC-1A cells treated with shikonin (5 μM) for 24 h. Data are represented as means ± S.D. of three independent experiments (*P<0.05, **P<0.01 compared with blank group, ^##P<0.01 compared with shikonin + mimics NC group).

Figure 5. PTEN is a target of miR-106b in EEC cells

(A) The PTEN 3′-UTR region containing the wt or mut binding site for miR-106b. (B,C) The Ishikawa or HEC-1A cells transfected with the miR-106b mimic/inhibitor or corresponding NC, and the PTEN expression was measured by Western blot analysis. β-actin was used as an internal control for protein loading. (D) The relative luciferase activity of PTEN wt or mut 3′-UTR in Ishikawa cells after transfection with the miR-106b mimic/inhibitor or corresponding NC. Data are represented as means ± S.D. of three independent experiments (**P<0.01 compared with mimics NC, ^##P<0.01 compared with inhibitor NC).

Figure 6. Overexpression of PTEN inhibits human EEC cell growth

The Ishikawa or HEC-1A cells were transfected with pc-DNA-PTEN or pc-DNA-vector. (A) The PTEN protein expression was measured by Western blot analysis. (B,C) The CCK-8 assay and flow cytometric analysis were used to evaluate the cell viability and apoptosis in Ishikawa and HEC-1A cells, respectively. Data are represented as means ± S.D. of three independent experiments (**P<0.01 compared with blank, ^##P<0.01 compared with pc-DNA-vector).

Figure 7. Shikonin blocks the PTEN/AKT/mTOR signaling pathway via suppressing miR-106b

(A and C) After transfection with or without miR-106b mimic, the Ishikawa or HEC-1A cells were treated with shikonin (5 μM) for 24 h, and then the expression levels of PTEN, AKT, p-AKT, mTOR, and p-mTOR were measured using Western blot analysis. (B and D) Quantitation of PTEN, AKT, p-AKT, mTOR, and p-mTOR ratios relative to controls in Ishikawa or HEC-1A cells, respectively. Data are represented as means ± S.D. of three independent experiments (**P<0.01 compared with blank, ^##P<0.01 compared with shikonin).

Figure 8. The schematic mechanism

The schematic diagram illustrates that shikonin treatment induces the down-regulation of miR-106b in EEC cells, and then results in the up-regualtion of PTEN. Subsequently, up-regualtion of PTEN can antagonize PI3K signal transduction by dephosphorylating the PI3K phosphorylation products (PI3,4,P₂ and PI3,4,5,P₃), leading to the down-regulation of AKT/mTOR, and regulate cell survival and apoptosis.