Next Article in Journal
Expression of the Calcium-Binding Protein CALB1 Is Induced and Controls Intracellular Ca2+ Levels in Senescent Cells
Next Article in Special Issue
Breast Cancer with Increased Drug Resistance, Invasion Ability, and Cancer Stem Cell Properties through Metabolism Reprogramming
Previous Article in Journal
The Function of N-Myc Downstream-Regulated Gene 2 (NDRG2) as a Negative Regulator in Tumor Cell Metastasis
Previous Article in Special Issue
Delivery of mGluR5 siRNAs by Iron Oxide Nanocages by Alternating Magnetic Fields for Blocking Proliferation of Metastatic Osteosarcoma Cells
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 23
Issue 16
10.3390/ijms23169368
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Qihui Luo
1,2,†,
Lanlan Jia
1,2,†,
Chao Huang
1,2,
Qi Qi
1,
Asad Jahangir
1,
Yu Xia
1,
Wentao Liu
1,2,
Riyi Shi
3,
Li Tang
2 and
Zhengli Chen
1,2,*
1
Laboratory of Experimental Animal Disease Model, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2
Key Laboratory of Animal Disease and Human Health of Sichuan Province, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
3
Center for Paralysis Research & Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2022, 23(16), 9368; https://doi.org/10.3390/ijms23169368
Submission received: 24 June 2022 / Revised: 14 August 2022 / Accepted: 16 August 2022 / Published: 19 August 2022
(This article belongs to the Special Issue Advance in Targeted Cancer Therapy and Mechanisms of Resistance)
Browse Figures
Versions Notes

Abstract

:
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.

1. 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 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 anti-cancer 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.

2. Results

2.1. 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.

2.2. 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.

2.3. 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.

2.4. 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).
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.

2.5. 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.

2.6. 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 PPI-induced apoptosis in SW480 cells (Figure 9). Taken together, the data above suggest that PPI-induced autophagy and apoptosis are ROS-dependent.

3. 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 previous 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 down-regulated in PPI-treated SW480 cells, suggesting that PPI is a potential therapeutic agent for colon cancer.

4. Materials and Methods

4.1. Chemicals and Reagents

Polyphyllin I (PPI) was purchased from Chengdu Mansite Pharmacetical CO, LTD (Purity ≥ 98%, lot number: MUST—16021905, Chengdu, China), and L-15 medium was purchased from Hyclone (SH30525.01, Logan, UT, USA). FBS was purchased from Gibco (Waltham, MA, USA). DMSO, acridine orange (AO) staining kit and mounting medium were purchased from Solarbio (Beijing, China). MTT and CQ were purchased from Sigma (St Louis, MI, USA). Primary antibodies against β-actin, LC3/Atg8, and mTOR were purchased from Boster (Wuhan, China). Primary antibodies against p-mTOR, AKT, p-AKT, and p53 were purchased from Cell Signaling. ROS assay kits were purchased from Beyotime (Chengdu, China).

4.2. 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% CO2 at 37 °C. Exponentially growing cells were used in the experiments.

4.3. MTT Assay

Cell growth was determined by MTT assay [61]. Briefly, 6 × 103/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.
Cell viability (%) = ODsample − ODblank /ODcontrol − ODblank × 100%
The half-maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism 8.

4.4. DAPI Staining

Next, 5 × 104/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.

4.5. AO Staining

Autophagy was detected by AO staining [62]. Briefly, 5 × 104/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.

4.6. ROS Detection

ROS detection was performed according to the manufacturer’s instructions and published work [63]. Briefly, 5 × 104/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.

4.7. 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.

4.8. 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.

5. Conclusions

In conclusion, we found that PPI induced cell autophagy and apoptosis via the ROS-inhibited 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.

Author Contributions

Conceptualization, Q.L., Q.Q. and Z.C; methodology, Q.L., L.J., C.H., Y.X., L.T. and Z.C.; software, Q.L. and L.J.; validation, R.S. and W.L.; investigation, Q.L., Q.Q., L.J., C.H. and Z.C.; resources, Q.L. and Z.C.; data curation, A.J. and L.J.; writing—original draft preparation, L.J., A.J. and Q.Q.; writing—review and editing, R.S., L.J. and Z.C.; visualization, R.S. and L.T.; supervision, R.S. and Z.C.; project administration, Q.L. and Z.C.; funding acquisition, Z.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from the Sichuan international science and technology innovation cooperation project (2020YFH0148 to Z.C.).

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Sichuan Agricultural University (Approval Code: DWYXY-20150124, Approval Date: 9 January 2017).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Source data are provided with this paper and are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Siegel, R.L.; Miller, K.D.; Goding Sauer, A.; Fedewa, S.A.; Butterly, L.F.; Anderson, J.C.; Cercek, A.; Smith, R.A.; Jemal, A. Colorectal cancer statistics 2020. CA Cancer J. Clin. 2020, 70, 145–164. [Google Scholar] [CrossRef] [PubMed]
  2. Benson, A.B.; Venook, A.P.; Al-Hawary, M.M.; Cederquist, L.; Chen, Y.J.; Ciombor, K.K.; Cohen, S.; Cooper, H.S.; Deming, D.; Engstrom, P.F.; et al. NCCN Guidelines Insights: Colon Cancer, Version 2.2018. J. Natl. Compr. Cancer Netw. JNCCN 2018, 16, 359–369. [Google Scholar] [CrossRef]
  3. Labianca, R.; Beretta, G.D.; Kildani, B.; Milesi, L.; Merlin, F.; Mosconi, S.; Pessi, M.A.; Prochilo, T.; Quadri, A.; Gatta, G.; et al. Colon cancer. Crit. Rev. Oncol. Hematol. 2010, 74, 106–133. [Google Scholar] [CrossRef] [PubMed]
  4. Eisenberg, B.; Decosse, J.J.; Harford, F.; Michalek, J. Carcinoma of the colon and rectum: The natural history reviewed in 1704 patients. Cancer 1982, 49, 1131–1134. [Google Scholar] [CrossRef]
  5. Gelibter, A.J.; Caponnetto, S.; Urbano, F.; Emiliani, A.; Scagnoli, S.; Sirgiovanni, G.; Napoli, V.M.; Cortesi, E. Adjuvant chemotherapy in resected colon cancer: When, how and how long? Surg. Oncol. 2019, 30, 100–107. [Google Scholar] [CrossRef] [PubMed]
  6. Alonso, S.; Saltz, L. The Landmark Series: Chemotherapy for Non-Metastatic Colon Cancer. Ann. Surg. Oncol. 2021, 28, 995–1001. [Google Scholar] [CrossRef] [PubMed]
  7. Arredondo, J.; Pastor, E.; Simó, V.; Beltrán, M.; Castañón, C.; Magdaleno, M.C.; Matanza, I.; Notarnicola, M.; Ielpo, B. Neoadjuvant chemotherapy in locally advanced colon cancer: A systematic review. Tech. Coloproctol. 2020, 24, 1001–1015. [Google Scholar] [CrossRef]
  8. Tian, Y.; Gong, G.Y.; Ma, L.L.; Wang, Z.Q.; Song, D.; Fang, M.Y. Anti-cancer effects of Polyphyllin I: An update in 5 years. Chem. Biol. Interact. 2020, 316, 108936. [Google Scholar] [CrossRef]
  9. Yang, C.; Cai, H.; Meng, X. Polyphyllin D induces apoptosis and differentiation in K562 human leukemia cells. Int. Immunopharmacol. 2016, 36, 17–22. [Google Scholar] [CrossRef]
  10. Zhang, C.; Jia, X.; Wang, K.; Bao, J.; Li, P.; Chen, M.; Wan, J.-B.; Su, H.; Mei, Z.; He, C. Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells. PLoS ONE 2016, 11, e0147405. [Google Scholar] [CrossRef]
  11. Deng, D.; Lauren, D.R.; Cooney, J.M.; Jensen, D.J.; Wurms, K.V.; Upritchard, J.E.; Cannon, R.D.; Wang, M.Z.; Li, M.Z. Antifungal saponins from Paris polyphylla Smith. Planta Med. 2008, 74, 1397–1402. [Google Scholar] [CrossRef] [PubMed]
  12. Zhu, X.; Wang, K.; Zhang, K.; Pan, Y.; Zhou, F.; Zhu, L. Polyphyllin I Induces Cell Cycle Arrest and Cell Apoptosis in Human Retinoblastoma Y-79 Cells through Targeting p53. Anti-Cancer Agents Med. Chem. 2018, 18, 875–881. [Google Scholar] [CrossRef]
  13. Liu, J.; Zhang, Y.; Chen, L.; Yu, F.; Li, X.; Dan, T.; Zhao, J.; Zhou, S. Polyphyllin I induces G2/M phase arrest and apoptosis in U251 human glioma cells via mitochondrial dysfunction and the JNK signaling pathway. Acta Biochim. Et Biophys. Sin. 2017, 49, 479–486. [Google Scholar] [CrossRef] [PubMed]
  14. Liang, Y.; Li, X.; He, X.; Qiu, X.; Jin, X.L.; Zhao, X.Y.; Xu, R.Z. Polyphyllin I induces cell cycle arrest and apoptosis in human myeloma cells via modulating β-catenin signaling pathway. Eur. J. Haematol. 2016, 97, 371–378. [Google Scholar] [CrossRef] [PubMed]
  15. He, J.; Yu, S.; Guo, C.; Tan, L.; Song, X.; Wang, M.; Wu, J.; Long, Y.; Gong, D.; Zhang, R.; et al. Polyphyllin I induces autophagy and cell cycle arrest via inhibiting PDK1/Akt/mTOR signal and downregulating cyclin B1 in human gastric carcinoma HGC-27 cells. Biomed. Pharmacother. Biomed. Pharmacother. 2019, 117, 109189. [Google Scholar] [CrossRef]
  16. Long, J.; Pi, X. Polyphyllin I Promoted Melanoma Cells Autophagy and Apoptosis via PI3K/Akt/mTOR Signaling Pathway. BioMed Res. Int. 2020, 2020, 5149417. [Google Scholar] [CrossRef]
  17. Tian, Y.; Jia, S.X.; Shi, J.; Gong, G.Y.; Yu, J.W.; Niu, Y.; Yang, C.M.; Ma, X.C.; Fang, M.Y. Polyphyllin I induces apoptosis and autophagy via modulating JNK and mTOR pathways in human acute myeloid leukemia cells. Chem. Biol. Interact. 2019, 311, 108793. [Google Scholar] [CrossRef]
  18. Crowley, L.C.; Marfell, B.J.; Waterhouse, N.J. Analyzing Cell Death by Nuclear Staining with Hoechst 33342. Cold Spring Harb. Protoc. 2016, 2016, 778–781. [Google Scholar] [CrossRef]
  19. Liu, B.; Bao, J.K.; Yang, J.M.; Cheng, Y. Targeting autophagic pathways for cancer drug discovery. Chin. J. Cancer 2013, 32, 113–120. [Google Scholar] [CrossRef]
  20. Guertin, D.A.; Sabatini, D.M. Defining the role of mTOR in cancer. Cancer Cell 2007, 12, 9–22. [Google Scholar] [CrossRef]
  21. Gorrini, C.; Harris, I.S.; Mak, T.W. Modulation of oxidative stress as an anticancer strategy. Nat. Rev. Drug Discov. 2013, 12, 931–947. [Google Scholar] [CrossRef] [PubMed]
  22. Kong, M.; Fan, J.; Dong, A.; Cheng, H.; Xu, R. Effects of polyphyllin I on growth inhibition of human non-small lung cancer cells and in xenograft. Acta Biochim. Et Biophys. Sin. 2010, 42, 827–833. [Google Scholar] [CrossRef] [PubMed]
  23. Zhang, D.; Liu, S.; Liu, Z.; Ma, C.; Jiang, Y.; Sun, C.; Li, K.; Cao, G.; Lin, Z.; Wang, P.; et al. Polyphyllin I induces cell cycle arrest in prostate cancer cells via the upregulation of IL6 and P21 expression. Medicine 2019, 98, e17743. [Google Scholar] [CrossRef]
  24. Zeng, Y.; Zhang, Z.; Wang, W.; You, L.; Dong, X.; Yin, X.; Qu, C.; Ni, J. Underlying mechanisms of apoptosis in HepG2 cells induced by polyphyllin I through Fas death and mitochondrial pathways. Toxicol. Mech. Methods 2020, 30, 397–406. [Google Scholar] [CrossRef] [PubMed]
  25. Yu, S.; Wang, L.; Cao, Z.; Gong, D.; Liang, Q.; Chen, H.; Fu, H.; Wang, W.; Tang, X.; Xie, Z.; et al. Anticancer effect of Polyphyllin Ι in colorectal cancer cells through ROS-dependent autophagy and G2/M arrest mechanisms. Nat. Prod. Res. 2018, 32, 1489–1492. [Google Scholar] [CrossRef] [PubMed]
  26. Lin, L.T.; Uen, W.C.; Choong, C.Y.; Shi, Y.C.; Lee, B.H.; Tai, C.J.; Tai, C.J. Paris Polyphylla Inhibits Colorectal Cancer Cells via Inducing Autophagy and Enhancing the Efficacy of Chemotherapeutic Drug Doxorubicin. Molecules 2019, 24, 2102. [Google Scholar] [CrossRef] [PubMed]
  27. Hippert, M.M.; O’Toole, P.S.; Thorburn, A. Autophagy in cancer: Good, bad, or both? Cancer Res. 2006, 66, 9349–9351. [Google Scholar] [CrossRef]
  28. Liu, Y.; Levine, B. Autosis and autophagic cell death: The dark side of autophagy. Cell Death Differ. 2015, 22, 367–376. [Google Scholar] [CrossRef]
  29. Zhu, X.; Zhou, M.; Liu, G.; Huang, X.; He, W.; Gou, X.; Jiang, T. Autophagy activated by the c-Jun N-terminal kinase-mediated pathway protects human prostate cancer PC3 cells from celecoxib-induced apoptosis. Exp. Ther. Med. 2017, 13, 2348–2354. [Google Scholar] [CrossRef]
  30. Yang, C.; Ma, X.; Wang, Z.; Zeng, X.; Hu, Z.; Ye, Z.; Shen, G. Curcumin induces apoptosis and protective autophagy in castration-resistant prostate cancer cells through iron chelation. Drug Des. Dev. Ther. 2017, 11, 431–439. [Google Scholar] [CrossRef]
  31. Lin, N.; Li, Z.; Wang, D.; Zheng, K.; Wu, Y.; Wang, H. Mecambridine induces potent cytotoxic effects, autophagic cell death and modulation of the mTOR/PI3K/Akt signaling pathway in HSC-3 oral squamous cell carcinoma cells. Oncol. Lett. 2018, 15, 292–296. [Google Scholar] [CrossRef] [PubMed]
  32. Yuan, G.J.; Deng, J.J.; Cao, D.D.; Shi, L.; Chen, X.; Lei, J.J.; Xu, X.M. Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells. World J. Gastroenterol. 2017, 23, 5530–5537. [Google Scholar] [CrossRef] [PubMed]
  33. Sun, D.; Zhu, L.; Zhao, Y.; Jiang, Y.; Chen, L.; Yu, Y.; Ouyang, L. Fluoxetine induces autophagic cell death via eEF2K-AMPK-mTOR-ULK complex axis in triple negative breast cancer. Cell Prolif. 2017, 51, e12402. [Google Scholar] [CrossRef] [PubMed]
  34. Shi, Y.M.; Yang, L.; Geng, Y.D.; Zhang, C.; Kong, L.Y. Polyphyllin I induced-apoptosis is enhanced by inhibition of autophagy in human hepatocellular carcinoma cells. Phytomed. Int. J. Phytother. Phytopharm. 2015, 22, 1139–1149. [Google Scholar] [CrossRef]
  35. Ren, G.; Luo, W.; Sun, W.; Niu, Y.; Ma, D.L.; Leung, C.H.; Wang, Y.; Lu, J.J.; Chen, X. Psoralidin induced reactive oxygen species (ROS)-dependent DNA damage and protective autophagy mediated by NOX4 in breast cancer cells. Phytomed. Int. J. Phytother. Phytopharm. 2016, 23, 939–947. [Google Scholar] [CrossRef]
  36. Hu, J.; Cao, X.; Pang, D.; Luo, Q.; Zou, Y.; Feng, B.; Li, L.; Chen, Z.; Huang, C. Tumor grade related expression of neuroglobin is negatively regulated by PPARγ and confers antioxidant activity in glioma progression. Redox Biol. 2017, 12, 682–689. [Google Scholar] [CrossRef]
  37. Chen, X.; Zhao, Y.; Luo, W.; Chen, S.; Lin, F.; Zhang, X.; Fan, S.; Shen, X.; Wang, Y.; Liang, G. Celastrol induces ROS-mediated apoptosis via directly targeting peroxiredoxin-2 in gastric cancer cells. Theranostics 2020, 10, 10290–10308. [Google Scholar] [CrossRef]
  38. Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach? Nat. Rev. Drug Discov. 2009, 8, 579–591. [Google Scholar] [CrossRef]
  39. Sun, Z.L.; Dong, J.L.; Wu, J. Juglanin induces apoptosis and autophagy in human breast cancer progression via ROS/JNK promotion. Biomed. Pharmacother. Biomed. Pharmacother. 2017, 85, 303–312. [Google Scholar] [CrossRef]
  40. Gao, L.; Loveless, J.; Shay, C.; Teng, Y. Targeting ROS-Mediated Crosstalk Between Autophagy and Apoptosis in Cancer. Adv. Exp. Med. Biol. 2020, 1260, 1–12. [Google Scholar]
  41. Zhou, J.; Zhang, L.; Wang, M.; Zhou, L.; Feng, X.; Yu, L.; Lan, J.; Gao, W.; Zhang, C.; Bu, Y.; et al. CPX Targeting DJ-1 Triggers ROS-induced Cell Death and Protective Autophagy in Colorectal Cancer. Theranostics 2019, 9, 5577–5594. [Google Scholar] [CrossRef]
  42. Banskota, S.; Regmi, S.C.; Kim, J.A. NOX1 to NOX2 switch deactivates AMPK and induces invasive phenotype in colon cancer cells through overexpression of MMP-7. Mol. Cancer 2015, 14, 123. [Google Scholar] [CrossRef]
  43. Jeanne, M.; Lallemand-Breitenbach, V.; Ferhi, O.; Koken, M.; Le Bras, M.; Duffort, S.; Peres, L.; Berthier, C.; Soilihi, H.; Raught, B.; et al. PML/RARA oxidation and arsenic binding initiate the antileukemia response of As2O3. Cancer Cell 2010, 18, 88–98. [Google Scholar] [CrossRef]
  44. Alexandre, J.; Hu, Y.; Lu, W.; Pelicano, H.; Huang, P. Novel action of paclitaxel against cancer cells: Bystander effect mediated by reactive oxygen species. Cancer Res. 2007, 67, 3512–3517. [Google Scholar] [CrossRef]
  45. Gao, N.; Rahmani, M.; Dent, P.; Grant, S. 2-Methoxyestradiol-induced apoptosis in human leukemia cells proceeds through a reactive oxygen species and Akt-dependent process. Oncogene 2005, 24, 3797–3809. [Google Scholar] [CrossRef]
  46. Li, G.B.; Fu, R.Q.; Shen, H.M.; Zhou, J.; Hu, X.Y.; Liu, Y.X.; Li, Y.N.; Zhang, H.W.; Liu, X.; Zhang, Y.H.; et al. Polyphyllin I induces mitophagic and apoptotic cell death in human breast cancer cells by increasing mitochondrial PINK1 levels. Oncotarget 2017, 8, 10359–10374. [Google Scholar] [CrossRef]
  47. Duan, P.; Hu, C.; Quan, C.; Yu, T.; Huang, W.; Chen, W.; Tang, S.; Shi, Y.; Martin, F.L.; Yang, K. 4-Nonylphenol induces autophagy and attenuates mTOR-p70S6K/4EBP1 signaling by modulating AMPK activation in Sertoli cells. Toxicol. Lett. 2017, 267, 21–31. [Google Scholar] [CrossRef]
  48. Xue, J.F.; Shi, Z.M.; Zou, J.; Li, X.L. Inhibition of PI3K/AKT/mTOR signaling pathway promotes autophagy of articular chondrocytes and attenuates inflammatory response in rats with osteoarthritis. Biomed. Pharmacother. Biomed. Pharmacother. 2017, 89, 1252–1261. [Google Scholar] [CrossRef]
  49. Wang, Z.; Zhou, L.; Zheng, X.; Chen, G.; Pan, R.; Li, J.; Liu, W. Autophagy protects against PI3K/Akt/mTOR-mediated apoptosis of spinal cord neurons after mechanical injury. Neurosci. Lett. 2017, 656, 158–164. [Google Scholar] [CrossRef]
  50. Pant, K.; Saraya, A.; Venugopal, S.K. Oxidative stress plays a key role in butyrate-mediated autophagy via Akt/mTOR pathway in hepatoma cells. Chem. Biol. Interact. 2017, 273, 99–106. [Google Scholar] [CrossRef]
  51. Zhang, L.; Man, S.; Wang, Y.; Liu, J.; Liu, Z.; Yu, P.; Gao, W. Paris Saponin II induced apoptosis via activation of autophagy in human lung cancer cells. Chem. Biol. Interact. 2016, 253, 125–133. [Google Scholar] [CrossRef]
  52. Cheung, J.Y.; Ong, R.C.; Suen, Y.K.; Ooi, V.; Wong, H.N.; Mak, T.C.; Fung, K.P.; Yu, B.; Kong, S.K. Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells. Cancer Lett. 2005, 217, 203–211. [Google Scholar] [CrossRef]
  53. Chen, J.C.; Hsieh, M.J.; Chen, C.J.; Lin, J.T.; Lo, Y.S.; Chuang, Y.C.; Chien, S.Y.; Chen, M.K. Polyphyllin G induce apoptosis and autophagy in human nasopharyngeal cancer cells by modulation of AKT and mitogen-activated protein kinase pathways in vitro and in vivo. Oncotarget 2016. [Google Scholar] [CrossRef]
  54. Zhao, S.; Cheng, L.; Shi, Y.; Li, J.; Yun, Q.; Yang, H. MIEF2 reprograms lipid metabolism to drive progression of ovarian cancer through ROS/AKT/mTOR signaling pathway. Cell Death Dis. 2021, 12, 18. [Google Scholar] [CrossRef]
  55. Shearn, C.T.; Smathers, R.L.; Stewart, B.J.; Fritz, K.S.; Galligan, J.J.; Hail, N., Jr.; Petersen, D.R. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) inhibition by 4-hydroxynonenal leads to increased Akt activation in hepatocytes. Mol. Pharmacol. 2011, 79, 941–952. [Google Scholar] [CrossRef]
  56. Chiarugi, P. PTPs versus PTKs: The redox side of the coin. Free Radic. Res. 2005, 39, 353–364. [Google Scholar] [CrossRef]
  57. Ni, Z.; Sun, W.; Li, R.; Yang, M.; Zhang, F.; Chang, X.; Li, W.; Zhou, Z. Fluorochloridone induces autophagy in TM4 Sertoli cells: Involvement of ROS-mediated AKT-mTOR signaling pathway. Reprod. Biol. Endocrinol. RBE 2021, 19, 64. [Google Scholar] [CrossRef]
  58. Makhov, P.; Golovine, K.; Teper, E.; Kutikov, A.; Mehrazin, R.; Corcoran, A.; Tulin, A.; Uzzo, R.G.; Kolenko, V.M. Piperlongumine promotes autophagy via inhibition of Akt/mTOR signalling and mediates cancer cell death. Br. J. Cancer 2014, 110, 899–907. [Google Scholar] [CrossRef]
  59. Pang, D.; Li, C.; Yang, C.; Zou, Y.; Feng, B.; Li, L.; Liu, W.; Geng, Y.; Luo, Q.; Chen, Z.; et al. Polyphyllin VII Promotes Apoptosis and Autophagic Cell Death via ROS-Inhibited AKT Activity, and Sensitizes Glioma Cells to Temozolomide. Oxid Med Cell Longev 2019, 2019, 1805635. [Google Scholar] [CrossRef]
  60. Narayanankutty, A. PI3K/ Akt/ mTOR Pathway as a Therapeutic Target for Colorectal Cancer: A Review of Preclinical and Clinical Evidence. Curr. Drug Targets 2019, 20, 1217–1226. [Google Scholar] [CrossRef]
  61. Zeng, M.; Zhu, L.; Li, L.; Kang, C. miR-378 suppresses the proliferation, migration and invasion of colon cancer cells by inhibiting SDAD1. Cell. Mol. Biol. Lett. 2017, 22, 12. [Google Scholar] [CrossRef]
  62. Kim, K.Y.; Park, K.I.; Kim, S.H.; Yu, S.N.; Park, S.G.; Kim, Y.W.; Seo, Y.K.; Ma, J.Y.; Ahn, S.C. Inhibition of Autophagy Promotes Salinomycin-Induced Apoptosis via Reactive Oxygen Species-Mediated PI3K/AKT/mTOR and ERK/p38 MAPK-Dependent Signaling in Human Prostate Cancer Cells. Int. J. Mol. Sci. 2017, 18, 1088. [Google Scholar] [CrossRef]
  63. Kim, H.; Xue, X. Detection of Total Reactive Oxygen Species in Adherent Cells by 2′,7′-Dichlorodihydrofluorescein Diacetate Staining. J. Vis. Exp. 2020, 160, e60682. [Google Scholar] [CrossRef]
Ijms 23 09368 g001 550
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.
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.
Ijms 23 09368 g001
Ijms 23 09368 g002 550
Figure 2. The effect of PPI on the karyomorphology of SW480 cells (400×). (A,C): The SW480 cells were stained with DAPI after treatment for 12 and 24 h, respectively (note: a: negative control group; b: treated with 1 μmol/L PPI; c: treated with 2 μmol/L PPI; d: treated with 3 μmol/L PPI; e: treated with 4 μmol/L PPI; f: treated with 5 μmol/L PPI); (B,D): The quantitative analysis of the apoptotic cell number. n = 3, the data are presented as means ± SEM. ** p < 0.01. Arrows indicate the apoptotic cells.
Figure 2. The effect of PPI on the karyomorphology of SW480 cells (400×). (A,C): The SW480 cells were stained with DAPI after treatment for 12 and 24 h, respectively (note: a: negative control group; b: treated with 1 μmol/L PPI; c: treated with 2 μmol/L PPI; d: treated with 3 μmol/L PPI; e: treated with 4 μmol/L PPI; f: treated with 5 μmol/L PPI); (B,D): The quantitative analysis of the apoptotic cell number. n = 3, the data are presented as means ± SEM. ** p < 0.01. Arrows indicate the apoptotic cells.
Ijms 23 09368 g002
Ijms 23 09368 g003 550
Figure 3. PPI induced autophagy in SW480 cells. (A) AO staining showed acidic autophagic vesicles (1000×). (B) Autophagic marker LC3 protein was examined by Western blot assay and quantification of the ratio of LC3-II to LC3-I. n = 3, the data are presented as means ± SEM. ** p < 0.01.
Figure 3. PPI induced autophagy in SW480 cells. (A) AO staining showed acidic autophagic vesicles (1000×). (B) Autophagic marker LC3 protein was examined by Western blot assay and quantification of the ratio of LC3-II to LC3-I. n = 3, the data are presented as means ± SEM. ** p < 0.01.
Ijms 23 09368 g003
Ijms 23 09368 g004 550
Figure 4. Blocking autophagy inhibits apoptosis induced by PPI. (A,B) LC3 protein level was examined by Western blot assay after treatment with PPI and CQ for 12 and 24 h, respectively. (C,D) 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.
Figure 4. Blocking autophagy inhibits apoptosis induced by PPI. (A,B) LC3 protein level was examined by Western blot assay after treatment with PPI and CQ for 12 and 24 h, respectively. (C,D) 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.
Ijms 23 09368 g004
Ijms 23 09368 g005 550
Figure 5. The changes in nuclear morphology were tested by DAPI staining after treatment with CQ (1000×). Note: (A): negative control group; (B): treated with 5 μmol/L PPI for 12 h; (C): treated with 3.5 μmol/L PPI for 24 h; (D): only treated with CQ; (E): treated with CQ and 5 μmol/L PPI for 12 h; (F): treated with CQ and 3.5 μmol/L PPI for 24 h. Arrows indicate the apoptotic cells.
Figure 5. The changes in nuclear morphology were tested by DAPI staining after treatment with CQ (1000×). Note: (A): negative control group; (B): treated with 5 μmol/L PPI for 12 h; (C): treated with 3.5 μmol/L PPI for 24 h; (D): only treated with CQ; (E): treated with CQ and 5 μmol/L PPI for 12 h; (F): treated with CQ and 3.5 μmol/L PPI for 24 h. Arrows indicate the apoptotic cells.
Ijms 23 09368 g005
Ijms 23 09368 g006 550
Figure 6. PPI induced autophagy via AKT/mTOR pathway in SW480 cells. (A) AKT/mTOR pathway-related proteins were examined by Western blot assay after treatment with PPI for 12 and 24 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.
Figure 6. PPI induced autophagy via AKT/mTOR pathway in SW480 cells. (A) AKT/mTOR pathway-related proteins were examined by Western blot assay after treatment with PPI for 12 and 24 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.
Ijms 23 09368 g006
Ijms 23 09368 g007 550
Figure 7. Changes in ROS levels in SW480 cells were assayed by DCFH labeling (1000×). (A) Representative images of ROS production in Rosup-positive and -negative control groups (PPI: 0 μmol/L); (B,C) representative images of ROS production in PPI- and NAC + PPI-treated groups; (D) quantification of the DCFH-positive cells. n = 3, the data are presented as means ± SEM. ** p < 0.01.
Figure 7. Changes in ROS levels in SW480 cells were assayed by DCFH labeling (1000×). (A) Representative images of ROS production in Rosup-positive and -negative control groups (PPI: 0 μmol/L); (B,C) representative images of ROS production in PPI- and NAC + PPI-treated groups; (D) quantification of the DCFH-positive cells. n = 3, the data are presented as means ± SEM. ** p < 0.01.
Ijms 23 09368 g007
Ijms 23 09368 g008 550
Figure 8. Effects of ROS on the autophagy and cell activity of SW480 cells induced by PPI. (A,B) LC3 protein level was examined by Western blot assay after treatment with PPI and NAC for 12 and 24 h, respectively; (C,D) 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 NAC for 12 and 24 h, respectively. n = 3, the data are presented as means ± SEM. * p < 0.05; ** p < 0.01.
Figure 8. Effects of ROS on the autophagy and cell activity of SW480 cells induced by PPI. (A,B) LC3 protein level was examined by Western blot assay after treatment with PPI and NAC for 12 and 24 h, respectively; (C,D) 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 NAC for 12 and 24 h, respectively. n = 3, the data are presented as means ± SEM. * p < 0.05; ** p < 0.01.
Ijms 23 09368 g008
Ijms 23 09368 g009 550
Figure 9. The changes in nuclear morphology were tested by DAPI staining after treatment with NAC (1000×). Note: (A): negative control group; (B): treated with 5 μmol/L PPI for 12 h; (C): treated with 3.5 μmol/L PPI for 24 h; (D): only treated with NAC; (E): treated with NAC and 5 μmol/L PPI for 12 h; (F): treated with NAC and 3.5 μmol/L PPI for 24 h. Arrows indicate the apoptotic cells.
Figure 9. The changes in nuclear morphology were tested by DAPI staining after treatment with NAC (1000×). Note: (A): negative control group; (B): treated with 5 μmol/L PPI for 12 h; (C): treated with 3.5 μmol/L PPI for 24 h; (D): only treated with NAC; (E): treated with NAC and 5 μmol/L PPI for 12 h; (F): treated with NAC and 3.5 μmol/L PPI for 24 h. Arrows indicate the apoptotic cells.
Ijms 23 09368 g009
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Luo, Q.; Jia, L.; Huang, C.; Qi, Q.; Jahangir, A.; Xia, Y.; Liu, W.; Shi, R.; Tang, L.; Chen, Z. Polyphyllin I Promotes Autophagic Cell Death and Apoptosis of Colon Cancer Cells via the ROS-Inhibited AKT/mTOR Pathway. Int. J. Mol. Sci. 2022, 23, 9368. https://doi.org/10.3390/ijms23169368

AMA Style

Luo Q, Jia L, Huang C, Qi Q, Jahangir A, Xia Y, Liu W, Shi R, Tang L, Chen Z. Polyphyllin I Promotes Autophagic Cell Death and Apoptosis of Colon Cancer Cells via the ROS-Inhibited AKT/mTOR Pathway. International Journal of Molecular Sciences. 2022; 23(16):9368. https://doi.org/10.3390/ijms23169368

Chicago/Turabian Style

Luo, Qihui, Lanlan Jia, Chao Huang, Qi Qi, Asad Jahangir, Yu Xia, Wentao Liu, Riyi Shi, Li Tang, and Zhengli Chen. 2022. "Polyphyllin I Promotes Autophagic Cell Death and Apoptosis of Colon Cancer Cells via the ROS-Inhibited AKT/mTOR Pathway" International Journal of Molecular Sciences 23, no. 16: 9368. https://doi.org/10.3390/ijms23169368

APA Style

Luo, Q., Jia, L., Huang, C., Qi, Q., Jahangir, A., Xia, Y., Liu, W., Shi, R., Tang, L., & Chen, Z. (2022). Polyphyllin I Promotes Autophagic Cell Death and Apoptosis of Colon Cancer Cells via the ROS-Inhibited AKT/mTOR Pathway. International Journal of Molecular Sciences, 23(16), 9368. https://doi.org/10.3390/ijms23169368

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop