Polyphyllin I Promotes Autophagic Cell Death and Apoptosis of Colon Cancer Cells via the ROS-Inhibited AKT/mTOR Pathway

Colon cancer is a common malignant tumor of the digestive tract, and it is considered among the biggest killers. Scientific and reasonable treatments can effectively improve the survival rate of patients if performed in the early stages. Polyphyllin I (PPI), a pennogenyl saponin isolated from Paris polyphylla var. yunnanensis, has exhibited strong anti-cancer activities in previous studies. Here, we report that PPI exhibits a cytotoxic effect on colon cancer cells. PPI suppressed cell viability and induced autophagic cell death in SW480 cells after 12 and 24 h, with the IC50 values 4.9 ± 0.1 μmol/L and 3.5 ± 0.2 μmol/L, respectively. Furthermore, we found PPI induced time-concentration-dependent autophagy and apoptosis in SW480 cells. In addition, down-regulated AKT/mTOR activity was found in PPI-treated SW480 cells. Increased levels of ROS might link to autophagy and apoptosis because reducing the level of ROS by antioxidant N-acetylcysteine (NAC) treatment mitigated PPI-induced autophagy and apoptosis. Although we did not know the molecular mechanism of how PPI induced ROS production, this is the first study to show that PPI induces ROS production and down-regulates the AKT/mTOR pathway, which subsequently promotes the autophagic cell death and apoptosis of colon cancer cells. This present study reports PPI as a potential therapeutic agent for colon cancer and reveals its underlying mechanisms of action.


Introduction
Colon cancer is one of the most common malignancies in the world. Because of its high morbidity and mortality (the annual age-standardized colorectal carcinoma incidence rate was 38.7 per 100,000 persons from 2012 to 2016, and the mortality rate was 13.9 per 100,000 persons from 2013 to 2017) [1]), colon cancer is a great health concern in clinical and basic research [2]. Surgery is the primary treatment and results in a cure in approximately 50% of patients when the tumors are localized to the bowel [3,4]. A major problem is recurrence following surgery, which is often the ultimate cause of death. In recent years, adjuvant chemotherapy has become the most preferred treatment plan [5][6][7]. Although adjuvant chemotherapy has an acceptable efficacy and improves survival for patients, the toxicity of chemotherapy drugs to the human body limits its application scope [5]. Therefore, it is imperative to find mild and effective treatment drugs.
The herb Paris polyphylla var. yunnanensis, also called Rhizoma paridis, has been used to treat parotitis, mastitis, and certain malignant tumors for thousands of years in traditional The herb Paris polyphylla var. yunnanensis, also called Rhizoma paridis, has been used to treat parotitis, mastitis, and certain malignant tumors for thousands of years in traditional Chinese medicine [8]. Several steroidal saponins isolated from Paris polyphylla var. yunnanensis possess anti-cancer properties against a variety of cancer cells, including PPI, PPII, PPC, PPD, PP6, and PP7 [8][9][10][11]. PPI is the main bioactive component in Paris polyphylla var. yunnanensis. A growing body of evidence has shown that PPI may exert anticancer effects in various types of cancers [8]. The induction of apoptosis is a classical strategy for anti-tumor therapy. PPI has been evidenced to induce apoptosis via P53 [12], caspase activation, Bcl-2/Bax ratio adjustment, JNK [13], and Wnt [14] pathways. Autophagy also plays a very important role in anti-cancer therapy. Some studies have shown that PPI can induce autophagy by inhibiting the AKT/mTOR signaling pathway [15][16][17]. However, the anti-cancer effects of PPI are still largely unknown in colon cancer. In this work, we aim to investigate the sensitivity of the colon cancer cell line (SW480 cells) to PPI in vitro and further reveal the molecular mechanisms that underlie these processes. Our study could display a new perspective on PPI application in colon cancer treatment, which could be beneficial for colon cancer patients.

PPI Inhibited the Proliferation of SW480 Cells
To evaluate the cytotoxic effect of PPI on colon cancer, SW480 cells were treated with PPI at concentrations from 1 μmol/L to 5 μmol/L for 12 or 24 h, and a methyl tetrazolium (MTT) assay was applied to test the cell viability. The results showed that PPI significantly inhibited the growth of SW480 cells in a dose-dependent manner, with a 50% inhibitory concentration value of 4.9 ± 0.1 μmol/L after PPI treatment for 12 h and 3.5 ± 0.2 μmol/L for 24 h (Figure 1). Based on the above results, the values of IC50 were used as the intervention concentration for follow-up experiments. The cell viability of SW480 cells treated with different concentrations of PPI was assessed by MTT assay after 12 and 24 h. n = 6, the data are presented as means ± SEM. ** p-value < 0.01; *** p-value < 0.001.

PPI Induced Apoptosis in SW480 Cells
To evaluate whether the decreased cell viability was caused by apoptosis in SW480 cells, DAPI staining was performed after the PPI treatment. During apoptosis, DNA becomes condensed and the cell membrane is compromised; consequently, more DAPI enters the cell and stains it a stronger blue color [18]. We found that the SW480 cells showed a typical apoptosis cell morphology after PPI treatment: the cell volume became smaller; the cell nucleus was solidified; dense blue granular strong fluorescence could be seen in the nucleus; and as the drug concentration increased, the blue fluorescence in the cell Figure 1. PPI inhibited the proliferation of SW480 cells. The cell viability of SW480 cells treated with different concentrations of PPI was assessed by MTT assay after 12 and 24 h. n = 6, the data are presented as means ± SEM. ** p-value < 0.01; *** p-value < 0.001.

PPI Induced Apoptosis in SW480 Cells
To evaluate whether the decreased cell viability was caused by apoptosis in SW480 cells, DAPI staining was performed after the PPI treatment. During apoptosis, DNA becomes condensed and the cell membrane is compromised; consequently, more DAPI enters the cell and stains it a stronger blue color [18]. We found that the SW480 cells showed a typical apoptosis cell morphology after PPI treatment: The cell volume became smaller; the cell nucleus was solidified; dense blue granular strong fluorescence could be seen in the nucleus; and as the drug concentration increased, the blue fluorescence in the cell became stronger ( Figure 2) after both 12 and 24 h. These data demonstrate that PPI decreases the viability of SW480 cells by the induction of apoptosis. became stronger (Figure 2) after both 12 and 24 h. These data demonstrate that PPI decreases the viability of SW480 cells by the induction of apoptosis.

PPI Induced Autophagy in SW480 Cells
To investigate whether PPI could enhance autophagy in SW480 cells, we examined the formation of autophagic vacuoles using acridine orange (AO) staining. An increased formation of AO-labeled autophagic vacuoles was observed after 5 μmol/L PPI treated for 12 h and 3.5 μmol/L PPI treated for 24 h, respectively ( Figure 3A). In addition, we detected levels of LC3-II conversion, a marker of autophagosomes, by Western blot. The ratio of LC3-II to LC3-I increased after PPI treatment in a dose-dependent manner ( Figure 3B), indicating that PPI induced a robust autophagy in SW480 cells.

PPI Induced Autophagy in SW480 Cells
To investigate whether PPI could enhance autophagy in SW480 cells, we examined the formation of autophagic vacuoles using acridine orange (AO) staining. An increased formation of AO-labeled autophagic vacuoles was observed after 5 µmol/L PPI treated for 12 h and 3.5 µmol/L PPI treated for 24 h, respectively ( Figure 3A). In addition, we detected levels of LC3-II conversion, a marker of autophagosomes, by Western blot. The ratio of LC3-II to LC3-I increased after PPI treatment in a dose-dependent manner ( Figure 3B), indicating that PPI induced a robust autophagy in SW480 cells.

Blocking Autophagy Inhibits Apoptosis Induced by PPI
To investigate the role of autophagy in PPI-induced apoptosis, we further tested the cell viability of PPI-treated SW480 cells following the addition of 20 μmol/L of the autophagy inhibitor chloroquine (CQ). First of all, compared with the individual treated group of PPI, the ratio of LC3-II/I was significantly increased in concurrent treatment with CQ and PPI, indicating that the degradation of the autophagosomes was blocked by CQ (Figure 4A-D). Interestingly, when the SW480 cells were treated for 12 h, the cell viability was

Blocking Autophagy Inhibits Apoptosis Induced by PPI
To investigate the role of autophagy in PPI-induced apoptosis, we further tested the cell viability of PPI-treated SW480 cells following the addition of 20 µmol/L of the autophagy inhibitor chloroquine (CQ). First of all, compared with the individual treated group of PPI, the ratio of LC3-II/I was significantly increased in concurrent treatment with CQ and PPI, indicating that the degradation of the autophagosomes was blocked by CQ ( Figure 4A-D). Interestingly, when the SW480 cells were treated for 12 h, the cell viability was decreased after concurrent treatment with CQ and low concentrations of PPI (1 µmol/L and 2 µmol/L) compared with the PPI-only treated groups, while an increased cell viability was found after concurrent treatment with CQ and high concentrations of PPI (5 µmol/L), indicating that PPI exerted autophagic protection at low concentrations and exerted autophagic cell death at high concentrations ( Figure 4E). When the SW480 cells were treated for 24 h, the cell viability was significantly higher after concurrent treatment with CQ and PPI (4-5 µmol/L) ( Figure 4F). decreased after concurrent treatment with CQ and low concentrations of PPI (1 μmol/L and 2 μmol/L) compared with the PPI-only treated groups, while an increased cell viability was found after concurrent treatment with CQ and high concentrations of PPI (5 μmol/L), indicating that PPI exerted autophagic protection at low concentrations and exerted autophagic cell death at high concentrations ( Figure 4E). When the SW480 cells were treated for 24 h, the cell viability was significantly higher after concurrent treatment with CQ and PPI (4-5 μmol/L) ( Figure 4F). The quantification of the ratio of LC3-II to LC3-I. (E,F) Cell viability was evaluated by MTT assay after treatment with PPI and CQ for 12 and 24 h, respectively. n = 3, the data are presented as means ± SEM. * p < 0.05; ** p < 0.01.
DAPI staining also showed that blocking autophagy inhibited PPI-induced apoptosis in SW480 cells. Compared with the individual treated group of PPI ( Figure 5B,C), fewer cells with apoptotic morphology were found in the group which received concurrent treatment with CQ and PPI ( Figure 5). Taken together, the results above indicate that autophagic cell death and apoptosis, are involved in the anti-cancer effect of PPI in SW480 cells.

PPI Induced Autophagy via the AKT/mTOR Pathway in SW480 Cells
To explore the mechanisms underlying PPI-induced autophagy, we examined the effect of PPI on the protein level of phosphorylated mTOR, which is involved in the early triggering of autophagy [19]. Compared to the negative control group, the levels of phosphorylated mTOR decreased in PPI-treated SW480 cells, while the total levels of mTOR remained unchanged, suggesting that PPI suppresses the activity of mTOR ( Figure 6). AKT signaling is involved in the regulation of mTOR activity, and the suppression of AKT decreases mTOR activity and promotes autophagy [20]. Consistent with the change in mTOR, the levels of phosphorylated AKT decreased in PPI-treated SW480 cells, while the total levels of AKT remained unchanged ( Figure 6). The results above indicate that the AKT/mTOR signaling pathway is involved in PPI-induced autophagy.

PPI Induced Autophagy via the AKT/mTOR Pathway in SW480 Cells
To explore the mechanisms underlying PPI-induced autophagy, we examined the effect of PPI on the protein level of phosphorylated mTOR, which is involved in the early triggering of autophagy [19]. Compared to the negative control group, the levels of phosphorylated mTOR decreased in PPI-treated SW480 cells, while the total levels of mTOR remained unchanged, suggesting that PPI suppresses the activity of mTOR ( Figure 6). AKT signaling is involved in the regulation of mTOR activity, and the suppression of AKT decreases mTOR activity and promotes autophagy [20]. Consistent with the change in mTOR, the levels of phosphorylated AKT decreased in PPI-treated SW480 cells, while the total levels of AKT remained unchanged ( Figure 6). The results above indicate that the AKT/mTOR signaling pathway is involved in PPI-induced autophagy.

PPI Induced Autophagy via the AKT/mTOR Pathway in SW480 Cells
To explore the mechanisms underlying PPI-induced autophagy, we examined the effect of PPI on the protein level of phosphorylated mTOR, which is involved in the early triggering of autophagy [19]. Compared to the negative control group, the levels of phosphorylated mTOR decreased in PPI-treated SW480 cells, while the total levels of mTOR remained unchanged, suggesting that PPI suppresses the activity of mTOR ( Figure 6). AKT signaling is involved in the regulation of mTOR activity, and the suppression of AKT decreases mTOR activity and promotes autophagy [20]. Consistent with the change in mTOR, the levels of phosphorylated AKT decreased in PPI-treated SW480 cells, while the total levels of AKT remained unchanged ( Figure 6). The results above indicate that the AKT/mTOR signaling pathway is involved in PPI-induced autophagy.

PPI Induced Autophagy and Apoptosis via the ROS-Dependent Pathway in SW480 Cells
The regulation of oxidative stress is an important factor in both cancer development and anti-cancer therapies [21]. To further confirm the underlying mechanism of PPI-induced autophagy and apoptosis, the level of ROS was evaluated by DCFH labeling in PPI-treated SW480 cells. We found significantly increased ROS accumulation in SW480 cells after PPI treatment (Figure 7). Pretreatment with the antioxidant NAC for 1 h significantly reduced the ROS accumulation in PPI-treated SW480 cells (Figure 7). Western blotting showed that the ratio of LC3-II/I was significantly decreased after NAC treatment ( Figure 8A,B). Meanwhile, when the SW480 cells were treated with PPI for 12 h, the MMT assay showed that the cell viability was not altered after NAC treatment ( Figure 8C); however, when the cells were treated with PPI for 24 h, the cell viability was significantly increased after NAC treatment ( Figure 8D), indicating that PPI-induced autophagy was involved in ROS accumulation. Furthermore, DAPI staining showed that decreasing ROS inhibited PPIinduced apoptosis in SW480 cells (Figure 9). Taken together, the data above suggest that PPI-induced autophagy and apoptosis are ROS-dependent. h, respectively; (B) the quantification of p-mTOR/mTOR and p-AKT/AKT, respectively. n = 3, the data are presented as means ± SEM. * p < 0.05; ** p < 0.01.

PPI Induced Autophagy and Apoptosis via the ROS-Dependent Pathway in SW480 Cells
The regulation of oxidative stress is an important factor in both cancer development and anti-cancer therapies [21]. To further confirm the underlying mechanism of PPI-induced autophagy and apoptosis, the level of ROS was evaluated by DCFH labeling in PPItreated SW480 cells. We found significantly increased ROS accumulation in SW480 cells after PPI treatment (Figure 7). Pretreatment with the antioxidant NAC for 1 h significantly reduced the ROS accumulation in PPI-treated SW480 cells (Figure 7). Western blotting showed that the ratio of LC3-II/I was significantly decreased after NAC treatment ( Figure  8A,B). Meanwhile, when the SW480 cells were treated with PPI for 12 h, the MMT assay showed that the cell viability was not altered after NAC treatment ( Figure 8C); however, when the cells were treated with PPI for 24 h, the cell viability was significantly increased after NAC treatment ( Figure 8D), indicating that PPI-induced autophagy was involved in ROS accumulation. Furthermore, DAPI staining showed that decreasing ROS inhibited PPI-induced apoptosis in SW480 cells (Figure 9). Taken together, the data above suggest that PPI-induced autophagy and apoptosis are ROS-dependent.

Discussion
Polyphyllin I (PPI) is an active steroidal saponin isolated from Paris polyphylla var. yunnanensis. Studies have reported that PPI is effective in anti-tumor therapy in models of lung cancer [22], prostate cancer [23], and liver cancer [24]. Some studies have shown that PPI exhibits anti-cancer effects through autophagy and apoptosis in some colorectal cancer cells [25,26], but the effect and mechanism of PPI on human colon cancer is still unclear. In this present study, we found that PPI significantly inhibited SW480 cells' proliferation, which was time-concentration-dependent. PPI promoted the autophagic cell death and apoptosis of SW480 cells. The increased ROS level might be the main reason for this because decreasing the ROS level robustly inhibited the PPI-induced apoptosis in SW480 cells. This is the first study to show that PPI can induce an increase in ROS levels to down-regulate the AKT/mTOR pathway and inhibit SW480 cells' proliferation.
The development of new drugs for autophagy has become a hot topic in drug intervention oncology research, while controversial studies have shown that the role of autophagy in cancer is complex [27,28]. On the one hand, autophagy can act as a temporary protective mechanism for tumor cells to survive in response to certain stresses [29,30]. On the other hand, autophagy can induce cell death (so-called autophagic cell death) [31][32][33]. Shi et al. [34] found that PPI can induce autophagic protection in human lung cancer cells, while Zhang et al. [10] found that PP7 can induce autophagic death in HepG2 cells. There was no evidence to suggest whether PPI-induced autophagy acted as autophagic protection or autophagic death in human colon cancer cells. In our study, we found that the PPI-induced autophagy exerted differential effects on the SW480 cells at differential PPI concentrations and differential treatment timing. When SW480 cells were treated for 12 h, PPI exerted autophagic protection at low concentrations (1 µmol/L and 2 µmol/L) and exerted autophagic cell death at high concentrations (5 µmol/L). After extending the treatment to 24 h, PPI induced autophagic cell death only. Thus, our results suggested that concentration and treatment timing, in particular, should be considered in the application of PPI treatment. Our results were inconsistent with Yu et al.'s finding that PPI-induced autophagy was not related to the PI3k/AKT/mTOR pathway in the colorectal cancer cell line HCT116 [25], suggesting that the mechanism of PPI-induced autophagy might be different in different types of tumors.
In addition to autophagy, ROS also plays a critical role in the progression of cancer. ROS are by-products of cell aerobic metabolism and are involved in regulating a variety of physiological activities in cells [35,36]. Excess ROS is removed by the antioxidants in normal cells, while cancer cells appear to benefit from basally elevated levels of ROS, and slightly elevated ROS may activate proliferation and survival pathways in cancer cells [37,38]. However, recent studies have shown that ROS can be involved as an important medium in the regulation of autophagy [39,40], and oxidative stress from elevated ROS can induce cell apoptosis in cancer cells [37,41]. A recent study showed that various levels of ROS were associated with a differential activation of AMPK, which is important in the apoptosis of colon cancer cells and their progression [42]. Based on these findings, drugs that increase ROS levels may offer avenues for cancer treatment [43][44][45]. We found that ROS levels dramatically increased after PPI treatment in SW480 cells. Reducing the level of ROS by adding the antioxidant NAC can effectively reduce autophagy and apoptosis in PPI-treated SW480 cells. Mitochondria are the major source of ROS. Some studies have shown that PPI might act on mitochondria leading to mitochondrial fission [46] and the depolarization of the mitochondrial membrane potential [13]. These studies provided a possible reason for the increased ROS level, but further study is needed.
The AKT/mTOR pathway plays a crucial role in regulating autophagy in cancer cells [47][48][49][50]. Previous studies have shown that steroidal saponin drugs induce autophagy in cancer cells mainly through this pathway [34,[51][52][53]. The impact of ROS on the AKT/mTOR pathway has been widely investigated, but the conclusions are conflicting. Some studies showed ROS production could activate the AKT/mTOR pathway [54][55][56], whereas others showed ROS inhibited the AKT/mTOR pathway [57,58]. Consistent with our previ-ous study on the effect of PP7 in glioma cells [59], the increased ROS down-regulated AKT/mTORC1 activity in the PPI-treated SW480 cells. Meanwhile, the AKT/mTOR pathway plays a significant role in the initiation and progression events of colorectal cancer, including migration, invasion, and drug resistance, and it is recognized as a striking therapeutic target [60]. We found that AKT and mTORC1 activity were dramatically downregulated in PPI-treated SW480 cells, suggesting that PPI is a potential therapeutic agent for colon cancer.

Cell Culture and Treatments
Human colon cancer SW480 cells were purchased from Boster (Wuhan, Hubei province, China). Cells were cultured in L-15 medium supplemented with 10% FBS in a humidified atmosphere of 5% CO 2 at 37 • C. Exponentially growing cells were used in the experiments.

MTT Assay
Cell growth was determined by MTT assay [61]. Briefly, 6 × 10 3 /well cells were seeded in each well in a 96-well plate for 24 h, then incubated with a fresh medium containing various PPI at 1, 2, 3, 4, and 5 µmol/L, respectively, and with DMSO as a negative control. The blank control was cell-free. The treated cells were incubated for 12 and 24 h, then incubated in fresh DMEM medium containing 1 mg/mL MTT at 37 • C. After an additional 4 h, the supernatants were replaced with DMSO to solubilize the formazan precipitates, then mixed gently by shaking for 10 min. The absorbance (OD value) was measured using an ELISA reader at a wavelength of 570 nm. Each experiment was performed in six replicate wells.

DAPI Staining
Next, 5 × 10 4 /well cells were seeded in each well in a 12-well plate for 24 h, then incubated with fresh medium containing various PPI for 12 and 24 h at 37 • C. Cells were washed with PBS and then fixed with 4% PFA for 30 min. Then, cells were incubated with 0.25% Triton in PBS for 30 min. The mounting medium with DAPI was used to mount the cells. The cells were visualized using a fluorescence microscope. More than 200 cells were counted, then the apoptosis rate was calculated. The experiment was independently repeated three times.

AO Staining
Autophagy was detected by AO staining [62]. Briefly, 5 × 10 4 /well cells were seeded in each well in a 12-well plate for 24 h, then incubated with fresh medium containing various PPI for 24 h at 37 • C. Cells were stained with 1 mg/mL AO in fresh DMEM and incubated for 20 min at 37 • C in the dark. After washing them three times with PBS, the cells were mounted with one drop of the mounting medium. The cells were visualized using a fluorescence microscope.

ROS Detection
ROS detection was performed according to the manufacturer's instructions and published work [63]. Briefly, 5 × 10 4 /well cells were seeded in each well in a 12-well plate for 24 h. The cells were incubated with an IC50 concentration of PPI for 12 h or 24 h; the negative control was incubated without PPI. After washing them three times with PBS, the cells were incubated with 10 µmol/L DCFH-DA (2 ,7 -dichlorofluorodiacetate) in an L-15 medium for 20 min at 37 • C in the dark. The cells were visualized using a fluorescence microscope.

Western Blot Analysis
For Western blot analysis, the PPI-treated cells were washed with PBS and lysed in a fresh 200 µL lysis buffer (with proteinase inhibitor and phosphatase inhibitor) on ice. The cell extracts were homogenized by sonication. The protein concentration of each extract was measured using the BCA Protein Assay kit. Equal amounts of proteins from each group were separated using 10% or 12% SDS-polyacrylamide gels and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in a Tris-buffered saline buffer for 1 h, and then, incubated overnight at 4 • C with primary antibodies (1:400 dilutions for β-actin, LC3, mTOR, JNK1 and p-JNK1; 1:1000 dilutions for p-mTOR, AKT, p-AKT and p53), followed by incubation with the corresponding secondary antibodies (dilution 1:2000) for 1 h at room temperature. Signals were developed using an ECL detection kit. Densitometry measurement of the band intensity was performed with the Image Lab Software (Bio-rad, Hercules, CA, USA). The experiment was independently repeated three times.

Statistical Analysis
All analyses were performed using SPSS version 20.0. The data were recorded as mean ± SD. The statistical significance was determined by one-way ANOVA to compare three or more groups, or by Student's t-test for the comparison between two groups. Differences were considered statistically significant for (*) p < 0.05 and (**) p < 0.01.

Conclusions
In conclusion, we found that PPI induced cell autophagy and apoptosis via the ROSinhibited AKT/mTOR pathway in colon cancer cells. This study provides new insight into the molecular mechanisms of the anti-cancer effect of PPI as a potential therapeutic agent for colon cancer in the future.