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. 2023 Aug;12(16):17149-17170.
doi: 10.1002/cam4.6309. Epub 2023 Aug 22.

Selenium-binding protein 1 inhibits malignant progression and induces apoptosis via distinct mechanisms in non-small cell lung cancer

Ying Zhu  1 , Qiang Pu  2 , Qiongyin Zhang  1 , Yang Liu  1 , Yongfang Ma  1 , Yue Yuan  1 , Lunxu Liu  2 , Wen Zhu  1
Affiliations

Selenium-binding protein 1 inhibits malignant progression and induces apoptosis via distinct mechanisms in non-small cell lung cancer

Ying Zhu et al. Cancer Med. 2023 Aug.

Abstract

Background: Selenium is an essential trace element in the human body. In epidemiological and clinical studies, Se supplementation significantly reduced the incidence of lung cancer in individuals with low baseline Se levels. The significant action of selenium is based on the selenium-containing protein as a mediator. Of note, the previous studies reported that the expression of selenium-binding protein 1 (SELENBP1) was obviously decreased in many human cancer tissues including non-small cell lung cancer (NSCLC). However, its roles in the origin and development of NSCLC are still unclear.

Methods: The expression of SELENBP1 was measured by qRT-PCR, Western blotting and IHC in our collected clinical NSCLC tissues and cell lines. Next, the CCK-8, colony formation, wound-haeling, Millicell, Transwell, FCM assay, and in vivo xenograft model were performed to explore the function of SELENBP1 in NSCLC. The molecular mechanisms of SELENBP1 were investigated by Western blotting or IF assay.

Results: We further identified that the expression of SELENBP1 was significantly decreased in NSCLC tissues in TCGA database and 45 out of 59 collected clinical NSCLC tissues compared with adjacent nontumor tissues, as well as in four NSCLC cell lines compared with normal lung cells. Particularly, we unexpectedly discovered that SELENBP1 was obviously expressed in alveolar type 2 (AT-II) cells for the first time. Then, a series of in vitro experiments uncovered that overexpression of SELENBP1 inhibited the proliferation, migration, and invasion of NSCLC cells, and induced cell apoptosis. Moreover, overexpression of SELENBP1 also inhibited growth and induced apoptosis of NSCLC cells in vivo. Mechanistically, we demonstrated that overexpression of SELENBP1 inhibited the malignant characteristics of NSCLC cells in part via inactivating the PI3K/AKT/mTOR signal pathway. Meanwhile, we found that overexpression of SELENBP1 inducing the apoptosis of NSCLC cells was associated with the activation of caspase-3 signaling pathway under nonhigh level of oxidative stress, but overexpression of SELENBP1 facilitating the cell apoptosis might be related to its combining with GPX1 and colocalizing in the nucleus under high level of oxidative stress.

Conclusions: Our findings highlighted that SELENBP1 was an important tumor suppressor during the origin and development of NSCLC. It may help to discover novel biomarkers or drug therapy targets for NSCLC.

Keywords: GPX1; NSCLC; PI3K/AKT/mTOR pathway; SELENBP1; caspase-3 pathway; tumor suppressor.

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Conflict of interest statement

None.

Figures

FIGURE 1
FIGURE 1
SELENBP1 was downregulated in NSCLC tissues by a comprehensive analysis of TCGA database and collected clinical tissues. (A) The expression levels of SELENBP1 in 483 LUAD tissues (T) and 59 adjacent nontumor tissues (N), 486 LUSC tissues (T), and 50 adjacent nontumor tissues (N; Data were obtained from TCGA database) “*” p < 0.05. T versus N in both LUAD and LUSC, respectively. (B)The relationship between the overall survival (OS) rate of LUAD tissues (N = 240), LUSC tissues (N = 242), and the expression levels of SELENBP1, respectively. (C) The expression of SELENBP1 in 59 paired collected clinical tissues was arranged by log10 C/N. (C) the expression of SELENBP1 in NSCLC tumor tissues. N, the expression of SELENBP1 in paired adjacent nontumor tissues. (D) Total RNA was isolated from collected clinical NSCLC tumor tissues and paired nontumor tissues, and the expression of SELENBP1 was assessed by qRT‐PCR analysis (N = 59). Paired t‐test. (E) The histologic structure of NSCLC tumor tissues and paired adjacent nontumor tissues were evaluated by H&E staining (N = 14). Results of H&E staining were observed under a bright field microscope (×200), Scale bars, 50 μm. (F) The histogram was used to quantify the experimental results of IHC staining. Data were presented as the Mean ± SD, unpaired t‐test. “****” p < 0.001. Adjacent nontumor tissue versus tumor tissues. (G) The expressions of SELENBP1 in collected clinical NSCLC tumor tissues and paired adjacent nontumor tissues were evaluated by IHC staining (N = 14). Results of IHC staining were observed under a bright field microscope (×400), Scale bars, 20 μm. (H) The protein expression of SELENBP1 in collected clinical NSCLC tumor tissues and paired adjacent nontumor tissues were detected by western blotting (N = 6). (I) The serial paraffin sections of the paired adjacent nontumor tissues were subjected to IHC staining with antibodies against SP‐C and SELENBP1 (N = 3). Staining without primary antibody was used as negative controls. All results were observed under a bright field microscope (×400). Scale bars, 20 μm. Images are shown while representative positive stains are indicated by red arrows.
FIGURE 2
FIGURE 2
Overexpression of SELENBP1 inhibited the proliferation, migration, and invasion of NSCLC cells in vitro. (A) The expression of SELENBP1 in four NSCLC cell lines A549, H1299, H358, and SK‐MES‐1 and normal lung cells HBE were measured by qRT‐PCR analysis and western blotting. β‐Actin was used as a control. All data were presented as the mean ± SD, unpaired t‐test, “****” p < 0.001, HBE group versus A549 group, H1299 group, H358 group, and SK‐MES‐1 group, respectively. (B) The expression of SELENBP1 in both A549‐SELENBP1 cells and H1299‐SELENBP1 cells and their control cells were measured by qRT‐PCR and western blotting after Lentivirus infection treatment. The total RNA and protein were extracted from the cells after puromycin screening. All data were presented as the mean ± SD, unpaired t‐test, “****” p < 0.001, A549‐Control group versus A549‐SELENBP1 group, H1299‐Control group versus H1299‐SELENBP1 group. (C) CCK8 assay. (a, c) CCK8 was performed at 0, 24, 48 and 72 h (N = 5). All data were presented as the mean ± SD, unpaired t‐test, “**” p < 0.01, A549‐Control group versus A549‐SELENBP1 group, H1299‐Control group versus H1299‐SELENBP1 group. (b, d) The histogram was used to quantify the cell viability (%) in 72 h. All data were presented as the mean ± SD, unpaired t‐test, “*” p < 0.05. A549‐Control group versus A549‐SELENBP1 group and H1299‐Control group versus H1299‐SELENBP1 group. (D) The number of cell clone in the A549‐SELENBP1 and H1299‐SELENBP1 and the control group were measured by clone forming assay (N = 3). All data were presented as the mean ± SD, unpaired t‐test, “*” p < 0.05, “**” p < 0.01, A549‐Control group versus A549‐SELENBP1 group and H1299‐Control group versus H1299‐SELENBP1 group. (E) The rate of open wounds (%) was measured by wound healing analysis (N = 3), and all data were presented as the mean ± SD, unpaired t‐test, “**” p < 0.01, “***” p < 0.005, A549‐Control group versus A549‐SELENBP1 group and H1299‐Control group versus H1299‐SELENBP1 group. Scale bars, 200 μm. (F) The number of migrated cells in A549‐SELENBP1, H1299‐SELENBP1, and control group was measured by Millicell assay (N = 3). And data were presented as the mean ± SD, unpaired t‐test “**” p < 0.01, A549‐Control group versus A549‐SELENBP1 group and H1299‐Control group versus H1299‐SELENBP1 group. Scale bars, 50 μm. (G) The number of invasion cells was measured by Transwell assay (N = 3). The data were presented as the mean ± SD, unpaired t‐test “***” p < 0.005, A549‐Control group versus A549‐SELENBP1 group and H1299‐Control group versus H1299‐SELENBP1 group. Scale bars, 20 μm. All experiments were repeated at least three times.
FIGURE 3
FIGURE 3
Overexpression of SELENBP1 suppressed the proliferation, migration, and invasion of NSCLC cells in part via inactivating the PI3K/AKT signaling pathway in vitro. (A) Western blotting analysis of PI3K/AKT/mTOR pathway markers in A549‐SELENBP1, H1299‐SELENBP1, and control cells. The NSCLC cell lysate was collected and subjected to western blotting analysis with antibodies against members of the PI3K/AKT/mTOR pathway. Levels of β‐Actin were used as loading control. (B) The histogram was used to quantify the experimental results of western blotting in A549‐SELENBP1 and their control cells, and H1299‐SELENBP1 and their control cells. All data were presented as the mean ± SD, unpaired t‐test, “*” p < 0.05, “**” p < 0.01, “****” p < 0.001, A549‐Control group versus A549‐SELENBP1 group and H1299‐Control group versus H1299‐SELENBP1 group. (C) Western blotting analysis of PI3K/mTOR pathway markers in the indicated cells treated with LY294002, MK‐2206, or IGF‐1 at various concentrations. (D) CCK8 was performed at 0, 24, 48 and 72 h in the indicated cells treated with LY294002 (20 μM), MK‐2206 (1 μM), or IGF‐1 (20 μg/mL; N = 4). And all data were presented as the mean ± SD. Multiple groups were compared by using one‐way ANOVA. Two groups were compared using unpaired t‐test “**” p < 0.01. A549‐Control group versus A549‐Control‐LY294002 group, A549‐Control group versus A549‐Control‐MK2206 group, A549‐SELENBP1 group versus A549‐SELENBP1‐IGF‐1 group. H1299‐Control group versus H1299‐Control‐LY294002 group, H1299‐Control group versus H1299‐Control‐MK2206 group, and H1299‐SELENBP1 group versus H1299‐SELENBP1‐IGF‐1 group. (E) The number of migrated cells and the number of invasion cells in indicated cells were measured by Millicell assay and transwell assay, respectively (left). Scale bars, 20 μm. The histogram was used to quantify the experimental results (right), and all data were presented as the mean ± SD. Multiple groups were compared by using one‐way ANOVA. Two groups were compared using unpaired t‐test “**” p < 0.01. “***” p < 0.005. A549‐Control group versus A549‐Control‐LY294002 group, A549‐Control group versus A549‐Control‐MK2206 group, A549‐SELENBP1 group versus A549‐SELENBP1‐IGF‐1 group. H1299‐Control group versus H1299‐Control‐LY294002 group, H1299‐Control group versus H1299‐Control‐MK2206 group, and H1299‐SELENBP1 group versus H1299‐SELENBP1‐IGF‐1 group.
FIGURE 4
FIGURE 4
Overexpression of SELENBP1 inhibiting the growth of NSCLC cells in vivo was associated with the inhibition of PI3K/AKT/mTOR pathway. (A) Establishment of stable SELENBP1 overexpressing A549 cells and control cells tumor xenograft Model. Athymic nude mice (4‐week‐old male, N = 6/point/group) were subcutaneously injected A549‐SELENBP1 cells or A549‐Control cells, respectively. The mice were sacrificed after 29 days, the retrieved tumor samples were presented. (B) Volume of Xenograft tumor was measured once every 3 days after 5 days subcutaneous injection. Data were presented as the mean ± SD, unpaired t‐test, “*” p < 0.05, “**” p < 0.01, A549‐Control tumors group versus A549‐SELENBP1 tumors group. (C) Paraffin sections of the retrieved tumor samples were subjected to H&E staining and IHC staining with antibodies against SELENBP1, Ki‐67. Staining without primary antibody was used as negative controls. Results of H&E were observed under a bright field microscope (×200), Scale bars, 50 μm. Results of IHC were observed under a bright field microscope (×400), Scale bars, 20 μm. The histogram was used to quantify the experimental results of IHC (N = 3). All data were presented as the mean ± SD, unpaired t‐test, “**” p < 0.01, “****” p < 0.001, A549‐Control tumors group versus A549‐SELENBP1 tumors group. (D) Paraffin sections of the retrieved tumor samples were subjected to IHC staining with antibodies against PI3K, p‐PI3K, AKT, p‐AKT, mTOR, and p‐mTOR. Staining without primary antibody was used as negative controls. Results were observed under a bright field microscope (×400). Scale bars, 20 μm. The histogram was used to quantify the experimental results of IHC (N = 3). And all data were presented as the mean ± SD, unpaired t‐test, “*” p < 0.05, “**” p < 0.01, “***” p < 0.005, A549‐Control tumors group versus A549‐SELENBP1 tumors group. (E) The tumor tissues were retrieved, extracted total protein, and subjected to western blotting analysis of key components of PI3K/AKT/mTOR pathway (N = 3/group). Levels of β‐Actin were used as loading control.
FIGURE 5
FIGURE 5
Overexpression of SELENBP1 inducing the apoptosis of NSCLC cells under nonhigh level of oxidative stress was associated with the activation of caspase‐3 signaling pathway in vitro. (A) Flow cytometry analysis was conducted (left), and the ratio of apoptotic cells (%) was calculated (N = 3; right). All data were presented as the mean ± SD, unpaired t‐test, “*” p < 0.05, “**” p < 0.01, A549‐Control group versus A549‐SELENBP1 group and H1299‐Control group versus H1299‐SELENBP1 group. (B) The effect of SELENBP1 overexpressing on the expression of the components of caspase‐3 pathway was measured. A549‐SELENBP1, H1299‐SELENBP1, and their control cells lysate were collected and subjected to western blotting analysis with antibodies against the caspase‐3, cleaved‐caspase‐3, Bcl‐2, and Bax (left). Levels of β‐Actin were used as loading control. The histogram was used to quantify the experimental results of western blotting (N = 3; right). All data were presented as the mean ± SD, unpaired t‐test, “*” p < 0.05, “**” p < 0.01, A549‐Control group versus A549‐SELENBP1 group and H1299‐Control group versus H1299‐SELENBP1 group.
FIGURE 6
FIGURE 6
Overexpression of SELENBP1 promoting the apoptosis of NSCLC cells in vivo was associated with the activation of caspase‐3 signaling pathway. (A) Paraffin sections of the retrieved tumor samples were subjected to IHC staining with antibodies against caspase‐3, cleaved‐caspase‐3, Bcl‐2, and Bax (left). Staining without primary antibody was used as negative controls. Results were observed under a bright field microscope (×400). Scale bars, 20 μm. The histogram was used to quantify the experimental results of IHC (N = 3; right). All data were presented as the mean ± SD, unpaired t‐test, “*” p < 0.05, “**” p < 0.01, “***” p < 0.005, A549‐Control tumors group versus A549‐SELENBP1 tumors group. (B) The expression of caspase‐3, cleaved‐caspase‐3, Bcl‐2, and Bax in mice tumor xenograft model were measured by western blotting (N = 3/group). Levels of β‐Actin were used as loading control. (C) The lever of apoptosis was presented by TUNEL staining (N = 3; left), and the histogram was used to quantify the TUNEL‐positive cells (right). Images were observed under a microscope (×200). Scale bars, 50 μm. TUNEL‐stained cells are in red, and DAPI‐stained nuclei are in blue. And the data were presented as the mean ± SD, unpaired t‐test, “***” p < 0.005, A549‐Control tumors group versus A549‐SELENBP1 tumors group.
FIGURE 7
FIGURE 7
Molecular mechanisms for overexpression of SELENBP1 inhibiting the malignant progression and inducing the apoptosis in NSCLC cells. Overexpression of SELENBP1 decreased the expression of PI3K, p‐PI3K, AKT, p‐AKT, mTOR, p‐mTOR, and Bcl‐2, whereas increased the expression of Bax and caspase‐3. Overexpression of SELENBP1 suppressed the malignant progression of NSCLC cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, overexpression of SELENBP1 inducing the apoptosis of NSCLC cells was associated with the activation of caspase‐3‐dependent axis under nonhigh level of oxidative stress. Furthermore, overexpression of SELENBP1 facilitating the cell apoptosis under high level of oxidative stress was related to its combining with GPX1 and colocalizing in nucleus (ROS, reactive oxygen species).
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