# Chelerythrine Inhibits Stemness of Cancer Stem-Like Cells of Osteosarcoma and PI3K/AKT/mTOR Signal

Authors: Zhixing Chen; Hui Yang; Qianlu Zhang; Qiongying Hu; Ziyi Zhao
Journal: Journal of Oncology
 (2022)

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## Abstract

Chelerythrine (CHE) is widely found in many herbs and is the main alkaloid constituent ofToddalia asiatica (L.) LAM. It has been proved to exert remarkable antitumor, antifungal, anti-inflammatory, and antiparasitic effects. In osteosarcoma, CHE is reported to inhibit proliferation and promote apoptosis. However, the effect of CHE on cancer stem-like cells (CSCs), which contribute to metastasis and recurrence in osteosarcoma, is still largely unknown. In this study, we investigated the effects of CHE on the stemness and malignant behaviors of CSCs derived from osteosarcoma cells. CSCs were enriched by culturing in serum-free medium. The effects of CHE on stemness were measured by detecting stemness factors and sphere formation ability. The effects of CHE on chemosensitivity to doxorubicin and MTX were measured by Annexin V-FITC/PI double staining. The effects of CHE on CSC malignancy were measured by performing CCK-8, colony formation, tumor formation in soft agar, migration, and invasion assays. We first enriched CSCs from osteosarcoma cells, which were characterized by upregulated stemness markers, including Oct4, Nanog, and Nestin. The addition of CHE clearly decreased malignant behaviors, including colony formation, tumor formation in soft agar, migration, and invasion. CHE also inhibited stemness and thus induced the failure of sphere formation. Moreover, CHE promoted apoptosis induced by chemo agents, including doxorubicin (DOX) and methotrexate (MTX). After CHE treatment, the protein expression of MMP-2/9 was significantly decreased, potentially inhibiting invasion. CHE also exhibited an inhibitory effect on the phosphorylation of PI3K, AKT, and mTOR, which is an upstream regulatory signaling pathway of MMP-2/9. In summary, CSCs derived from U2OS and MG-63 cells, CHE could inhibit the stemness and malignant behaviors of CSCs potentially by inhibiting the PI3K/AKT/mTOR signaling pathway.


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## Body

## 1. Introduction

Osteosarcoma, as a connective tissue tumor, is the most common primary malignant tumor in bone, originating in mesenchymal tissue. Approximately 80%-90% of osteosarcomas occur in the metaphysis of the long tubular bones of the extremities, with the most common occurring in the distal femur, proximal tibia, and proximal humerus. Osteosarcomas occurring in nonextremities, such as the spine and pelvis, tend to have a poor prognosis. Generally, patients with osteosarcoma have a 5-year survival rate of less than 20%. Despite the use of surgery, radiotherapy, and neoadjuvant chemotherapy, long-term survival for osteosarcoma has not been significantly ameliorated [1]. To date, scientists have successfully isolated tumor stem cells from osteosarcoma [2]. Tumor stem cells have a close bearing on the occurrence, metastasis, treatment resistance and recurrence of tumors, and lung metastasis is the primary cause of death in patients with osteosarcoma. Therefore, tumor stem cells in osteosarcoma prevent osteosarcoma from being cured to a large extent. Osteosarcoma is the most common primary malignant tumor of bone in children and adolescents, accounting for nearly 60% of the common histological subtypes of osteosarcoma in children [3]. In view of its high degree of malignancy, poor prognosis, high possibility of lung metastasis in the early stage, and rapid development, timely ,and early treatment of osteosarcoma is extremely necessary.Despite the poor prognosis, the therapy-resistant cell types in osteosarcoma tumors are poorly understood. The subpopulation of cancer cells with stem/progenitor properties, termed cancer stem-like cells (CSCs), which exhibit stem/progenitor characteristics was explained as the cause of therapy resistance [4]. This subpopulation was characterized by expression of stemness markers, including Nanog, Oct4, and Nestin, formation of spheres in vitro, and tumor-initiating ability in preclinical mouse models [5]. The existence of CSCs derived from osteosarcoma is responsible for the tumor’s drug resistance and high metastatic potential, demonstrating that it is critical to have an in-depth investigation of the strategies to be specifically targeted [6]. In our previous findings, CSCs were successfully enriched from colorectal cancer cells by culturing in serum-free medium [7]. After culture with berberine, a compound extracted from the traditional Chinese medicine Coptis chinensis, CSCs derived from colorectal cancer cells showed decreased stemness and increased chemosensitivity against regular chemo agents. All of these findings indicate that natural compounds may be promising candidates for the use as specific chemo agents against CSCs.Chelerythrine (CHE), also known as chelerythrine chloride or celandine quaternary ammonium base, is a benzphenanthridine type of isoquinoline isolated from plants, such asToddalia asiatica, Chelidonium majus, Macleaya cordata, and Eomecon chionantha Hance. CHE can inhibit cell proliferation and induce cell apoptosis against a variety of tumor cells due to its various pharmacological activities, such as antitumor, antibacterial, antifungal, and anti-inflammatory activities. Chmura et al. [8] confirmed via in vitro experiments that CHE showed cytotoxicity to 9 tumor cells, including MCF7, MCF7ADR, HT29, DaOY, SCC35, SCC61, SQ20B, JSQ3, and LnCap, and had a significant inhibitory effect on tumor growth. In particular, in osteosarcoma cells, CHE was reported to exert suppressive effects on malignancies. Wang et al. reported that CHE treatment inhibited migration and invasion capacities by reducing the mass of actin filaments in the cellular actin filament network [9]. It has also been reported that, in osteosarcoma, CHE treatment activates extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAPK signaling and thus promotes apoptosis in a MEK1-dependent manner [10]. All of these data indicate that CHE acts as an efficient tumor suppressor in osteosarcoma. However, its exact role in CSCs derived from osteosarcoma is still largely unknown.In this study, we sought to evaluate the suppressive effects of CHE on CSCs derived from the osteosarcoma cell lines U2OS and MG-63 and the potential molecular mechanism. We revealed that CHE inhibited CSC malignancies by regulating PI3K/AKT/mTOR signaling and MMP2/9 expression. Moreover, we found that CSCs are significantly more sensitive to CHE than their parental cells, which indicates that CHE may be a promising drug to specifically target CSCs and exert limited side effects.


## 2. Materials and Methods

### 2.1. Cell Culture and Treatment

Human osteosarcoma MG-63 and U2OS cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum and 1% antibiotics (penicillin/streptomycin). Cells were maintained in 37°C, 5% CO2, and 95% humidity. Every three days, medium was replaced and cells were passaged.To enrich CSCs from MG-63 or U2OS, cells were suspended and cell concentration was adjusted to1×107 cells per mL. 1×106 cells were seeded in a 6-well plate and cultured in serum-free DMEM/F12 medium supplemented with 20 ng/mL bFGF, 20 ng/mL EGF, and 2% B27. Every three days, medium was half-changed. Briefly, medium was collected and concentrated at 1000 g, 4°C for 5 min. Then supernatant was removed and pelleted spheres were resuspended using fresh medium. After 14 days, formed spheres were imaged using Olympus microscopy X71 (Japan).


### 2.2. CCK-8 Analysis

MG-63 and U2OS cells were seeded in 96-well plates at a density of5×103 cells per well and maintained in DMEM medium supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum and 1% antibiotics (penicillin/streptomycin). 24-h later, 5, 10, 15, 20, 25, 30, 35, or 40 μmol/L CHE were added, respectively, for 24-hour extra treatment. 10 μL of CCK-8 solution were added to each well, after which the cells were incubated for 2 h at 37°C. After incubation, absorbance was measured at 450 nm.


### 2.3. Western Blot

MG-63 and U2OS cells (5×106 cells, respectively) were treated with 15 or 10 μmol/L CHE for 24 h, and then cells were harvested and lysed using animal tissue/cells/bacteria total protein isolation kit (DocSense, Chengdu, China). Cell lysate protein was fractionated via SDS-PAGE at 100 V for 2 h and transferred to an Immobilon-P transfer membrane (Merck Millipore, Burlington, MA, USA) to a nitrocellulose membrane at 45 V for 2 h. The membranes were blocked with bovine serum albumin (Bovogen, Australia), and incubated with primary antibodies against proteins at 4°C overnight. The used of primary antibodies were listed as follows: anti-Oct4 (cat. No.: ab181557), anti-Nanog (cat. No.: ab109250), anti-Nestin (cat. No.: ab105389), anti-beta Actin (cat. No.: ab8227), anti-MMP2 (cat. No.: ab92536), anti-MMP9 (cat. No.: ab76003), anti-PI3K (cat. No.: ab154598), anti-phosphate PI3K (cat. No.: ab182651), anti-pan AKT (cat. No.: ab300473), anti-phosphate AKT (cat. No.: ab38449), anti-mTOR (cat. No.: ab), anti-mTOR (cat. No.: ab134903), and anti-phosphate mTOR (cat. No.: 109268). All primary antibodies were bought from Abcam (Cambridge, England) and diluted at 1 : 1000. The membranes were then washed with TBS-T, and incubated using HRP-conjugated secondary antibody (Jackson Laboratory, Bar Harbor, USA). Chemiluminescence was detected using ECL (Gendepot, Barker, USA) and measured using the ChemiDoc detection system (Bio-Rad, Hercules, CA, USA).


### 2.4. Tumor Formation in Soft Agar

Spheres derived from MG-63 or U2OS were separated and obtained single-cell suspension using SoniConvert® single-cell suspension preparation system (DocSense, Chengdu, China) by following manufacturer’s instruction.1×104 cells for each CSCs were suspended in 0.35% biotechnology-grade agarose (Bio-Rad, Hercules, CA, USA) in DMEM/F12 supplemented with 10 or 15 μmol/L CHE, 20 ng/mL bFGF, 20 ng/mL EGF, and 2% B27 and plated before solidifying on a solid 0.5% agarose with DMEM/F12. Colonies were maintained for 14 days in a 37°C humidified incubator, after which they were stained with methylene blue (Sigma-Aldrich, St. Louis, MO, USA) and counted.


### 2.5. Migration and Invasion

Migration of human breast cancer cells was assessed in a transwell chamber assay. Briefly, spheres derived from MG-63 or U2OS were separated and obtained single -cell suspension using SoniConvert® single-cell suspension preparation system (DocSense, Chengdu, China) by following manufacturer’s instruction.1×104 cells were seeded into cell culture inserts containing membranes with 8 μm pores that were placed in wells containing cell culture medium with 2% FBS. 24-hour later, unmigrated cells were removed with cotton swabs, and migrated cells were fixed in cold methanol and stained with Diff-Quik solution. Cells were imaged. Similar results were obtained in three independent experiments.Invasion assay was performed using BD transwell invasion chamber (BD Biosciences, Bedford, MA, USA). Single-cell suspension was prepared as described as before. Matrigel chambers were rehydrated for two hours using 500μL of serum-free DMEM/F12 medium. After rehydration, 750 μL normal growth media containing 2% FBS was added to the bottom of the well. 1.0×104 cells in DMEM/F12 medium with supplements described as before were added to the Matrigel chamber. Chambers were incubated overnight in a cell culture incubator, at 37°C, 5% CO2 atmosphere. 48-hour later, noninvading cells were removed with a cotton swab. Invaded cells were sequentially transferred through a fixative, then 4% paraformaldehyde solution for 15 min at room temperature. After three washed using PBS, invaded cells were allowed to air dry. Membranes were mounted onto slides using immersion oil and covered with a cover slip. Cells were counted using light microscopy at 40× magnification.


### 2.6. Apoptosis Analysis

Annexin-V Apoptosis Detection Kit (Cell Signaling Technology) was used to quantify the levels of apoptosis according to the manufacturer’s instructions. Briefly,1×106 cells were trypsinized and collected by centrifugation at 4°C, 1000 g for 10 min. Then cells were incubated with Annexin V-FITC and propidium iodide. Apoptosis was analyzed by flow cytometry (Beckman, Navios) for the detection of Annexin V-FITC. Data was analyzed using FlowJo (v10). Experiments were performed at least thrice, and statistical analysis was performed with GraphPad Prism.


### 2.7. Animal experiment

All the animal experiments were conducted according to the ethics committee of the Institutional Animal Care and Use Committee of Institute of Chengdu University of Traditional Chinese Medicine (No. 2020QKL-001).Spheres derived from U2OS were separated and obtained single-cell suspension using SoniConvert® single-cell suspension preparation system (DocSense, Chengdu, China) by following manufacturer’s instruction. Collected cells were pretreated with 15μmol/L CHE for 48 h.Eight female nude mice (SPF) were divided into two groups: Mock group (n=4) and CHE group (n=4). Each mouse was subcutaneously injected with 5×105 cells suspension. The tumor growth was measured every five days from day 10, and the length diameter and short diameter were measured by vernier calipers. Tumor volume was calculated (tumor volume=π×lengthdiameter×shortdiameter 2/6). The nude mice were sacrificed 30 days later, and tumor growth curve was drawn.


### 2.8. Statistical Analysis

Statistical analysis was conducted using one-way ANOVA, and statistical significance was set atP<0.05.


## 2.1. Cell Culture and Treatment

Human osteosarcoma MG-63 and U2OS cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum and 1% antibiotics (penicillin/streptomycin). Cells were maintained in 37°C, 5% CO2, and 95% humidity. Every three days, medium was replaced and cells were passaged.To enrich CSCs from MG-63 or U2OS, cells were suspended and cell concentration was adjusted to1×107 cells per mL. 1×106 cells were seeded in a 6-well plate and cultured in serum-free DMEM/F12 medium supplemented with 20 ng/mL bFGF, 20 ng/mL EGF, and 2% B27. Every three days, medium was half-changed. Briefly, medium was collected and concentrated at 1000 g, 4°C for 5 min. Then supernatant was removed and pelleted spheres were resuspended using fresh medium. After 14 days, formed spheres were imaged using Olympus microscopy X71 (Japan).


## 2.2. CCK-8 Analysis

MG-63 and U2OS cells were seeded in 96-well plates at a density of5×103 cells per well and maintained in DMEM medium supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum and 1% antibiotics (penicillin/streptomycin). 24-h later, 5, 10, 15, 20, 25, 30, 35, or 40 μmol/L CHE were added, respectively, for 24-hour extra treatment. 10 μL of CCK-8 solution were added to each well, after which the cells were incubated for 2 h at 37°C. After incubation, absorbance was measured at 450 nm.


## 2.3. Western Blot

MG-63 and U2OS cells (5×106 cells, respectively) were treated with 15 or 10 μmol/L CHE for 24 h, and then cells were harvested and lysed using animal tissue/cells/bacteria total protein isolation kit (DocSense, Chengdu, China). Cell lysate protein was fractionated via SDS-PAGE at 100 V for 2 h and transferred to an Immobilon-P transfer membrane (Merck Millipore, Burlington, MA, USA) to a nitrocellulose membrane at 45 V for 2 h. The membranes were blocked with bovine serum albumin (Bovogen, Australia), and incubated with primary antibodies against proteins at 4°C overnight. The used of primary antibodies were listed as follows: anti-Oct4 (cat. No.: ab181557), anti-Nanog (cat. No.: ab109250), anti-Nestin (cat. No.: ab105389), anti-beta Actin (cat. No.: ab8227), anti-MMP2 (cat. No.: ab92536), anti-MMP9 (cat. No.: ab76003), anti-PI3K (cat. No.: ab154598), anti-phosphate PI3K (cat. No.: ab182651), anti-pan AKT (cat. No.: ab300473), anti-phosphate AKT (cat. No.: ab38449), anti-mTOR (cat. No.: ab), anti-mTOR (cat. No.: ab134903), and anti-phosphate mTOR (cat. No.: 109268). All primary antibodies were bought from Abcam (Cambridge, England) and diluted at 1 : 1000. The membranes were then washed with TBS-T, and incubated using HRP-conjugated secondary antibody (Jackson Laboratory, Bar Harbor, USA). Chemiluminescence was detected using ECL (Gendepot, Barker, USA) and measured using the ChemiDoc detection system (Bio-Rad, Hercules, CA, USA).


## 2.4. Tumor Formation in Soft Agar

Spheres derived from MG-63 or U2OS were separated and obtained single-cell suspension using SoniConvert® single-cell suspension preparation system (DocSense, Chengdu, China) by following manufacturer’s instruction.1×104 cells for each CSCs were suspended in 0.35% biotechnology-grade agarose (Bio-Rad, Hercules, CA, USA) in DMEM/F12 supplemented with 10 or 15 μmol/L CHE, 20 ng/mL bFGF, 20 ng/mL EGF, and 2% B27 and plated before solidifying on a solid 0.5% agarose with DMEM/F12. Colonies were maintained for 14 days in a 37°C humidified incubator, after which they were stained with methylene blue (Sigma-Aldrich, St. Louis, MO, USA) and counted.


## 2.5. Migration and Invasion

Migration of human breast cancer cells was assessed in a transwell chamber assay. Briefly, spheres derived from MG-63 or U2OS were separated and obtained single -cell suspension using SoniConvert® single-cell suspension preparation system (DocSense, Chengdu, China) by following manufacturer’s instruction.1×104 cells were seeded into cell culture inserts containing membranes with 8 μm pores that were placed in wells containing cell culture medium with 2% FBS. 24-hour later, unmigrated cells were removed with cotton swabs, and migrated cells were fixed in cold methanol and stained with Diff-Quik solution. Cells were imaged. Similar results were obtained in three independent experiments.Invasion assay was performed using BD transwell invasion chamber (BD Biosciences, Bedford, MA, USA). Single-cell suspension was prepared as described as before. Matrigel chambers were rehydrated for two hours using 500μL of serum-free DMEM/F12 medium. After rehydration, 750 μL normal growth media containing 2% FBS was added to the bottom of the well. 1.0×104 cells in DMEM/F12 medium with supplements described as before were added to the Matrigel chamber. Chambers were incubated overnight in a cell culture incubator, at 37°C, 5% CO2 atmosphere. 48-hour later, noninvading cells were removed with a cotton swab. Invaded cells were sequentially transferred through a fixative, then 4% paraformaldehyde solution for 15 min at room temperature. After three washed using PBS, invaded cells were allowed to air dry. Membranes were mounted onto slides using immersion oil and covered with a cover slip. Cells were counted using light microscopy at 40× magnification.


## 2.6. Apoptosis Analysis

Annexin-V Apoptosis Detection Kit (Cell Signaling Technology) was used to quantify the levels of apoptosis according to the manufacturer’s instructions. Briefly,1×106 cells were trypsinized and collected by centrifugation at 4°C, 1000 g for 10 min. Then cells were incubated with Annexin V-FITC and propidium iodide. Apoptosis was analyzed by flow cytometry (Beckman, Navios) for the detection of Annexin V-FITC. Data was analyzed using FlowJo (v10). Experiments were performed at least thrice, and statistical analysis was performed with GraphPad Prism.


## 2.7. Animal experiment

All the animal experiments were conducted according to the ethics committee of the Institutional Animal Care and Use Committee of Institute of Chengdu University of Traditional Chinese Medicine (No. 2020QKL-001).Spheres derived from U2OS were separated and obtained single-cell suspension using SoniConvert® single-cell suspension preparation system (DocSense, Chengdu, China) by following manufacturer’s instruction. Collected cells were pretreated with 15μmol/L CHE for 48 h.Eight female nude mice (SPF) were divided into two groups: Mock group (n=4) and CHE group (n=4). Each mouse was subcutaneously injected with 5×105 cells suspension. The tumor growth was measured every five days from day 10, and the length diameter and short diameter were measured by vernier calipers. Tumor volume was calculated (tumor volume=π×lengthdiameter×shortdiameter 2/6). The nude mice were sacrificed 30 days later, and tumor growth curve was drawn.


## 2.8. Statistical Analysis

Statistical analysis was conducted using one-way ANOVA, and statistical significance was set atP<0.05.


## 3. Results

### 3.1. CHE Significantly Decreased Colony Formation, Migration, and Invasion in CSCs Derived from Osteosarcoma Cells

To evaluate the effects of CHE on CSCs, we firstly enriched CSCs from osteosarcoma cells U2OS and MG-63 by being cultured in serum-free medium for 14 days. As it is shown in Figure1(a), formation of spheres was morphologically observed, which is considered as a character of stemness [5]. In addition, as expected, these cells presented a relative higher level of stemness markers (e.g., Oct4, Nanog, and Nestin) [10].Figure 1
Characterization of CSCs enriched from U2OS and MG-63 cells. (a) Cell morphology was observed after being cultured in serum-containing medium or serum-free medium after 14 days. (b and c) Western blot was performed to detect protein levels of Oct4, Nanog, and Nestin in MG-63 and U2OS parental cells (PCs) and CSCs.∗P<0.05 vs. PCs group.
(a)(b)(c)Spheres derived from U2OS or MG-63 were trypsinized and single-cell suspension was collected to evaluate the effects of CHE. By being cultured in different concentration of CHE (ranged from 5-30μmol/L) for 24 h, cell viability was detected and IC30 of U2OS-CSCs is approximately 15 μmol/L, and that of MG-63-CSCs is approximately 10 μmol/L (Figure 2(a)). Notably, osteosarcoma cells present a similar IC30 concentration, indicated that osteosarcoma cells present similar sensitivity to CHE after 24-h treatment compared with corresponding CSCs. Then, we treated CSCs derived from U2OS or MG-63 with 15 μmol/L or 10 μmol/L of CHE, respectively, and colony formation, tumor formation in soft agar, migration, and invasion were further analyzed. As it is shown in Figures 2(b)–2(e), addition of CHE obviously inhibited all these malignant behaviors, respectively.Figure 2
CHE inhibited malignant behaviors in CSCs. (a) After being cultured in 5-30μmol/L of CHE for 24 h, cell viability was performed by performing CCK-8 assay. After being treated with CHE, colony formation (b), tumor formation in soft agar (c), migration (d), or invasion (e) were analyzed.
(a)(b)(c)(d)(e)


### 3.2. CHE Decreased Stemness of Osteosarcoma Cells

CHE was reported to induce apoptosis and reverse chemoresistance in osteosarcoma cell [11], which promoted us to evaluate the effects of CHE on stemness of osteosarcoma. Single-cell suspension of CSCs were seeded with 5-15 μmol/L of CHE and cultured for 14 days. Added CHE obviously decreased number of spheres (Figure 3(a)), potentially via inhibiting sphere formation, but not proliferation. To further confirm the inhibitory effect of CHE on stemness, 48-hour after CHE treatment, stemness markers, including Oct4, Nanog, and Nestin, were detected and results demonstrated that CHE treatment significantly decreased all these stemness markers (Figures 3(b) and 3(c)). Added CHE failed to affect the number of formed spheres (data not shown), which further indicated that CHE affects stemness, but not exert cytotoxicity.Figure 3
CHE inhibited stemness of CSCs. (a). Single-cell suspension of CSCs was cocultured with CHE for 14 days, and then formed spheres were imaged. After being cultured with CHE for 48 h, total protein was extracted from single-cell suspension of CSCs and analyzed by performing western blot to detect stemness marker proteins, including Oct4, Nanog, and Nestin (b and c).
(a)(b)(c)


### 3.3. CHE Induces Chemosensitivity in CSCs

Presence of CSCs was believed to contribute to chemoresistance in osteosarcoma [12]. By considering that CHE treatment significantly decreased stemness without affecting cell viability, the effects of CHE on Doxorubicin (DOX) or Methotrexate (MTX)-induced cell death, which are the first choice of treatment for osteosarcoma clinically [13]. Although addition of CHE failed to obviously increased cell death induced by DOX or MTX in osteosarcoma cells (data not shown), as it is shown in Figure 4(a), addition of CHE significantly increased chemosensitivity to DOX or MTX in CSCs and decreased cell survival rate. To further evaluate the promoting effects of CHE on chemoagent-induced apoptosis, Annexin V-FITC/PI double staining was performed followed by flow cytometric assay. As it is shown in Figure 4(b), expectedly, both DOX and MTX treatments significantly increased proportion of Annexin V+/PI− and Annexin V+/PI+. Addition of CHE significantly increased proportion of both Annexin V+/PI− and Annexin V+/PI+, demonstrated the promoting effects of CHE on chemosensitivity. By considering that CHE slightly affects cell viability, the promoting effects of CHE on apoptosis is mainly via decreasing stemness.Figure 4
CHE enhanced chemoresistance to DOX and MTX in CSCs derived from osteosarcoma cells. (a) After being cultured with or without CHE for 24 h, the cell viability was measured after DOX or MTX treatment.∗P<0.05 vs. no CHE group. (b) After being cocultured using CHE with DOX or MTX, apoptosis was measured by performing Annexin V-FITC/PI double staining followed by flow cytometry assay. ∗P<0.05 vs. DOX group; #P<0.05 vs. MTX group.
(a)(b)


### 3.4. CHE Decreased the Expression Level of MMP-2 and MMP-9 in CSCs Derived from Osteosarcoma Cells

MMP-2 and MMP-9 are two important members of the MMP family. They are highly expressed in many malignant tumors, and they participate in the degradation and destruction of the ECM and BM and promote tumor metastasis [13]. Moreover, CHE was also demonstrated to be involved in MMP-2 and MMP-9 regulation in hepatocellular carcinoma [14], we then clarified the effect of CHE on MMP-2 and MMP-9 expression. Expectedly, consistent with the effect of CHE on MMP-2 and MMP-9 in hepatocarcinoma cells, 12-h and 24-h CHE treatment significantly decreased MMP-2 and MMP-9 expression levels (Figures 5(a) and 5(b)).Figure 5
CHE decreased MMP-2 and MMP-9. After being treated with CHE for 48 h, total protein was extracted from single-cell suspension of CSCs and analyzed by performing western blot to detect MMP-2 and MMP-9 protein levels (a and b).∗P<0.05 vs. mock group.
(a)(b)


### 3.5. Effects of CHE on the PI3K/AKT/Mammalian Target of Rapamycin (mTOR) Signaling Pathway

The phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signal transduction pathways were reported to be tightly involved in regulating metastasis via modulating MMP-2 and MMP-9 [15]. As it is shown in Figure 6, CHE treatment undetectably affected PI3K, AKT, or mTOR total protein levels in CSCs. After CHE treatment, phosphorylation of PI3K, AKT, and mTOR decreased significantly, indicated that, in CSCs derived from osteosarcoma cells, CHE potentially decreased migration and invasion via inhibiting PI3K/AKT/mTOR pathway and inducing MMP-2, MMP-9 inhibition.Figure 6
CHE inhibited PI3K/AKT/mTOR signaling pathway. After being treated with CHE for 48 h, total protein was extracted from single-cell suspension of CSCs and analyzed by performing western blot to detect PI3K/AKT/mTOR protein levels (a and b).∗P<0.05 vs. mock group.
(a)(b)


### 3.6. Pretreatment of CHE Suppressed Tumor Growth of CSCs Derived from U2OS

To further evaluate the effects of CHE on tumor growth in mice, single-cell suspension of CSCs derived from U2OS was pretreated with 15μmol/L of CHE for 24 h and then injected intraperitoneally (I.P.). As it is shown in Figures 7(a), 7(b), and 7(c), CHE pretreatment significantly decreased tumor growth in nude mice. This indicates that, CHE treatment decreased stemness irreversibly in CSCs and suppressed tumor growth. We then also detected stemness markers, including Oct4, Nanog, and Nestin, in burdened tumors. However, no significant difference between these two groups (Figure 7(d)), potentially due to lose of stemness of CSCs after 4-week’s growth in mice.Figure 7
Tumor growth inhibition in xenograft mice. (a) After being pretreated with or without CHE for 48 h, CSCs were seeded in mice.N=4 for each group. After 30 days, mice were imaged. (b) 10 days after injection, tumor size was measured every five days, and growth curve was drawn. (c) After 30-day growth, tumors were obtained and physiologically analyzed by performing H&E staining. Samples were observed at amplification of 1× or 20×. (d) After 30-day growth, freshly obtained tumors were lysed and expression levels of Oct4, Nanog, and Nestin were analyzed by performing western blot.
(a)(b)(c)(d)


## 3.1. CHE Significantly Decreased Colony Formation, Migration, and Invasion in CSCs Derived from Osteosarcoma Cells

To evaluate the effects of CHE on CSCs, we firstly enriched CSCs from osteosarcoma cells U2OS and MG-63 by being cultured in serum-free medium for 14 days. As it is shown in Figure1(a), formation of spheres was morphologically observed, which is considered as a character of stemness [5]. In addition, as expected, these cells presented a relative higher level of stemness markers (e.g., Oct4, Nanog, and Nestin) [10].Figure 1
Characterization of CSCs enriched from U2OS and MG-63 cells. (a) Cell morphology was observed after being cultured in serum-containing medium or serum-free medium after 14 days. (b and c) Western blot was performed to detect protein levels of Oct4, Nanog, and Nestin in MG-63 and U2OS parental cells (PCs) and CSCs.∗P<0.05 vs. PCs group.
(a)(b)(c)Spheres derived from U2OS or MG-63 were trypsinized and single-cell suspension was collected to evaluate the effects of CHE. By being cultured in different concentration of CHE (ranged from 5-30μmol/L) for 24 h, cell viability was detected and IC30 of U2OS-CSCs is approximately 15 μmol/L, and that of MG-63-CSCs is approximately 10 μmol/L (Figure 2(a)). Notably, osteosarcoma cells present a similar IC30 concentration, indicated that osteosarcoma cells present similar sensitivity to CHE after 24-h treatment compared with corresponding CSCs. Then, we treated CSCs derived from U2OS or MG-63 with 15 μmol/L or 10 μmol/L of CHE, respectively, and colony formation, tumor formation in soft agar, migration, and invasion were further analyzed. As it is shown in Figures 2(b)–2(e), addition of CHE obviously inhibited all these malignant behaviors, respectively.Figure 2
CHE inhibited malignant behaviors in CSCs. (a) After being cultured in 5-30μmol/L of CHE for 24 h, cell viability was performed by performing CCK-8 assay. After being treated with CHE, colony formation (b), tumor formation in soft agar (c), migration (d), or invasion (e) were analyzed.
(a)(b)(c)(d)(e)


## 3.2. CHE Decreased Stemness of Osteosarcoma Cells

CHE was reported to induce apoptosis and reverse chemoresistance in osteosarcoma cell [11], which promoted us to evaluate the effects of CHE on stemness of osteosarcoma. Single-cell suspension of CSCs were seeded with 5-15 μmol/L of CHE and cultured for 14 days. Added CHE obviously decreased number of spheres (Figure 3(a)), potentially via inhibiting sphere formation, but not proliferation. To further confirm the inhibitory effect of CHE on stemness, 48-hour after CHE treatment, stemness markers, including Oct4, Nanog, and Nestin, were detected and results demonstrated that CHE treatment significantly decreased all these stemness markers (Figures 3(b) and 3(c)). Added CHE failed to affect the number of formed spheres (data not shown), which further indicated that CHE affects stemness, but not exert cytotoxicity.Figure 3
CHE inhibited stemness of CSCs. (a). Single-cell suspension of CSCs was cocultured with CHE for 14 days, and then formed spheres were imaged. After being cultured with CHE for 48 h, total protein was extracted from single-cell suspension of CSCs and analyzed by performing western blot to detect stemness marker proteins, including Oct4, Nanog, and Nestin (b and c).
(a)(b)(c)


## 3.3. CHE Induces Chemosensitivity in CSCs

Presence of CSCs was believed to contribute to chemoresistance in osteosarcoma [12]. By considering that CHE treatment significantly decreased stemness without affecting cell viability, the effects of CHE on Doxorubicin (DOX) or Methotrexate (MTX)-induced cell death, which are the first choice of treatment for osteosarcoma clinically [13]. Although addition of CHE failed to obviously increased cell death induced by DOX or MTX in osteosarcoma cells (data not shown), as it is shown in Figure 4(a), addition of CHE significantly increased chemosensitivity to DOX or MTX in CSCs and decreased cell survival rate. To further evaluate the promoting effects of CHE on chemoagent-induced apoptosis, Annexin V-FITC/PI double staining was performed followed by flow cytometric assay. As it is shown in Figure 4(b), expectedly, both DOX and MTX treatments significantly increased proportion of Annexin V+/PI− and Annexin V+/PI+. Addition of CHE significantly increased proportion of both Annexin V+/PI− and Annexin V+/PI+, demonstrated the promoting effects of CHE on chemosensitivity. By considering that CHE slightly affects cell viability, the promoting effects of CHE on apoptosis is mainly via decreasing stemness.Figure 4
CHE enhanced chemoresistance to DOX and MTX in CSCs derived from osteosarcoma cells. (a) After being cultured with or without CHE for 24 h, the cell viability was measured after DOX or MTX treatment.∗P<0.05 vs. no CHE group. (b) After being cocultured using CHE with DOX or MTX, apoptosis was measured by performing Annexin V-FITC/PI double staining followed by flow cytometry assay. ∗P<0.05 vs. DOX group; #P<0.05 vs. MTX group.
(a)(b)


## 3.4. CHE Decreased the Expression Level of MMP-2 and MMP-9 in CSCs Derived from Osteosarcoma Cells

MMP-2 and MMP-9 are two important members of the MMP family. They are highly expressed in many malignant tumors, and they participate in the degradation and destruction of the ECM and BM and promote tumor metastasis [13]. Moreover, CHE was also demonstrated to be involved in MMP-2 and MMP-9 regulation in hepatocellular carcinoma [14], we then clarified the effect of CHE on MMP-2 and MMP-9 expression. Expectedly, consistent with the effect of CHE on MMP-2 and MMP-9 in hepatocarcinoma cells, 12-h and 24-h CHE treatment significantly decreased MMP-2 and MMP-9 expression levels (Figures 5(a) and 5(b)).Figure 5
CHE decreased MMP-2 and MMP-9. After being treated with CHE for 48 h, total protein was extracted from single-cell suspension of CSCs and analyzed by performing western blot to detect MMP-2 and MMP-9 protein levels (a and b).∗P<0.05 vs. mock group.
(a)(b)


## 3.5. Effects of CHE on the PI3K/AKT/Mammalian Target of Rapamycin (mTOR) Signaling Pathway

The phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signal transduction pathways were reported to be tightly involved in regulating metastasis via modulating MMP-2 and MMP-9 [15]. As it is shown in Figure 6, CHE treatment undetectably affected PI3K, AKT, or mTOR total protein levels in CSCs. After CHE treatment, phosphorylation of PI3K, AKT, and mTOR decreased significantly, indicated that, in CSCs derived from osteosarcoma cells, CHE potentially decreased migration and invasion via inhibiting PI3K/AKT/mTOR pathway and inducing MMP-2, MMP-9 inhibition.Figure 6
CHE inhibited PI3K/AKT/mTOR signaling pathway. After being treated with CHE for 48 h, total protein was extracted from single-cell suspension of CSCs and analyzed by performing western blot to detect PI3K/AKT/mTOR protein levels (a and b).∗P<0.05 vs. mock group.
(a)(b)


## 3.6. Pretreatment of CHE Suppressed Tumor Growth of CSCs Derived from U2OS

To further evaluate the effects of CHE on tumor growth in mice, single-cell suspension of CSCs derived from U2OS was pretreated with 15μmol/L of CHE for 24 h and then injected intraperitoneally (I.P.). As it is shown in Figures 7(a), 7(b), and 7(c), CHE pretreatment significantly decreased tumor growth in nude mice. This indicates that, CHE treatment decreased stemness irreversibly in CSCs and suppressed tumor growth. We then also detected stemness markers, including Oct4, Nanog, and Nestin, in burdened tumors. However, no significant difference between these two groups (Figure 7(d)), potentially due to lose of stemness of CSCs after 4-week’s growth in mice.Figure 7
Tumor growth inhibition in xenograft mice. (a) After being pretreated with or without CHE for 48 h, CSCs were seeded in mice.N=4 for each group. After 30 days, mice were imaged. (b) 10 days after injection, tumor size was measured every five days, and growth curve was drawn. (c) After 30-day growth, tumors were obtained and physiologically analyzed by performing H&E staining. Samples were observed at amplification of 1× or 20×. (d) After 30-day growth, freshly obtained tumors were lysed and expression levels of Oct4, Nanog, and Nestin were analyzed by performing western blot.
(a)(b)(c)(d)


## 4. Discussion

CHE is known to exert antitumor effects in numerous cancers. Considering that the therapeutic-resistant effects of cancers are mainly due to heterogeneity, it is worth investigating whether CHE exerts antitumor effects on CSCs, which is considered a main cause of recurrence. The present study showed that CHE decreased malignant behaviors, including colony formation, tumor formation in soft agar, migration, and invasion, potentially by inhibiting the stemness of CSCs. Additionally, the effect of CHE was at least partially dependent on the PI3K/AKT/mTOR signaling pathway. Differences were observed in chemoresistance between CHE-treated and CHE-untreated CSCs. These differences in chemosensitivity might be linked to the maintenance of CSC stemness. Furthermore, CHE pretreatment delayed tumor regrowth in mice transplanted with CSCs.Based on current research findings, CHE may induce cell apoptosis through a variety of pathways and mechanisms. In addition to acting on Bcl-2 and Bcl-XL factors in the apoptosis pathway, promoting the release of mitochondrial cytochrome C, inhibiting the activity of protein kinase C as well as the polymerization of tubulin (preventing the progression of mitosis), and rapidly inducing apoptosis through the production of reactive oxygen species, CHE can also induce cell cycle arrest and mitochondria-mediated apoptosis through the caspase-8-dependent KG1a cell pathway [16], balance antiapoptotic and proapoptotic signaling pathways [17], activate P38 and amino-terminal protein kinase pathways [18], and induce cell cycle arrest and mitochondria-mediated apoptosis [19]. Surprisingly, CHE did not exert extra cytotoxicity against CSCs compared to its parental cells in this study. Without inducing obvious cytotoxicity, CHE regulates the PI3K/AKT/mTOR signaling pathway and stemness in CSCs. Subsequently, CHE suppresses malignancies in CSCs in a prosurvival manner.Doxorubicin is widely used to treat malignant tumors, including osteosarcoma [20, 21]. The toxic effects of doxorubicin on malignant cells are as follows: (i) DNA base-pair intercalation; (ii) drug molecules interact with topoisomerase II to form DNA-cleavable complexes; (iii) drug molecules interact with the electron transport chain, which may cause cells to produce superoxide anion radicals [22]. The chemoresistance mechanisms of tumor cells to doxorubicin include: (1) overexpression of membrane-associated efflux pumps and P glycoproteins mediating multidrug resistance; (2) altered expression of topoisomerase II and integrin; (3) altered glutathione levels [23]. MTX is another cancer drug widely used in osteosarcoma chemotherapy. Some evidence suggests that DHFR is critical in regulating chemoresistance to MTX in human osteosarcoma cells [24, 25]. In this study, these two chemo agents were employed to evaluate the effects of CHE treatment on the chemosensitivity of CSCs. Remarkably, CHE treatment significantly decreased cell viability and promoted apoptosis induced by both DOX and MTX. Although the promoting effects of CHE on chemotreatment have been confirmed, the exact molecular mechanism of its promoting effects on chemosensitivity is still largely unknown and is worthy of further investigation.


## 5. Conclusion

The findings of the present study demonstrate that CHE treatment significantly decreased the stemness of CSCs derived from osteosarcoma cells, including U2OS and MG-63 cells. CHE significantly decreased stemness markers, including Oct4, Nanog, and Nestin, and thus suppressed malignant behaviors. The present study also demonstrated that CHE treatment decreased MMP-2/9 protein expression and the PI3K/AKT/mTOR signaling pathway, which might contribute to a decrease in the stemness of CSCs. Moreover, CHE treatment also increased chemosensitivity to DOX and MTX, which are two first-choice chemo agents, in CSCs. These findings may be beneficial to the development of novel antitumor therapeutics by targeting CSCs, which critically contribute to recurrence.Moreover, CHE treatment also increased chemosensitivity to DOX and MTX, which of them are two first-choice chemo agents, in CSCs. These findings may be beneficial to the development of novel antitumor therapeutic strategies by targeting to CSCs, which contributes to recurrence critically.



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*Source: 6435431-2022-09-12.xml*